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dissertation entitled

FACTORS CONTROLLING THE TEMPORAL PATTERN OF FEMALE
REPRODUCTIVE BEHAVIOR IN RATS

presented by

LIANG—YO YANG

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FACTORS CONTROLLING THE TEMPORAL PATTERN OF FEMALE
REPRODUCTIVE BEHAVIOR IN RATS

By

Liang-Yo Yang

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Zoology and Neuroscience Program
1996

ABSTRACT

FACTORS CONTROLLING THE TEMPORAL PATTERN OF FEMALE
REPRODUCTIVE BEHAVIOR IN RATS

By

Liang-Yo Yang

When given the opportunity, the female rat regulates the copulatory speed. The
female’s return latencies and percentage exits following sexual contacts provide robust
measures of her temporal copulatory behavior. One possible function of the female’s
pacing behavior is to facilitate successful reproduction. When the female controls the
copulatory speed, the interintromission intervals are longer and fewer intromissions are
sufficient to induce the progestational state of pregnancy. The female’s postejaculatory
refractory period (PER) is important to facilitate or allow sperm transport. If the female
receives intromissions shortly following ejaculation, sperm transport will be stopped until
the next ejaculation. The objectives of the experiments in this dissertation were to
investigate (1) the relation of copulatory stimuli to the female’s PER, (2) the relation among
vaginocervical stimulation, uterine electromyographic (EMG) activity and female temporal
copulatory behavior, and (3) whether the medial preoptic area (MPOA) of hypothalamus is
involved in regulating female pacing behavior.

Female sexual behavior was tested in a two-compartment pacing chamber. Results
indicated that the ejaculation duration and preejaculatory intromission frequency were
positively correlated with the female’s PER. Different hormone replacements also
influenced the female’s PER. No evidence was found to support the idea that the penile
cup formation, the presence of a seminal plug, prostate secretions, or the number of pelvic
thrusts during ejaculation contributed to the f emale’s PER.

Consistent with previous reports, copulation had an immediate effect on uterine
EMG activity. Moreover, the duration of uterine EMG activity associated with ejaculation

was significantly longer than that associated with intromission or mount. This finding

coincides with the fact that the female’s return latency following ejaculation is significantly
longer than that following intromission or mount. Possibly the longer duration of uterine
activity associated with ejaculation contributes to the f emale’s PER.

The MPOA plays an important role in facilitating female sexual motivation in rats.
MPOA lesions significantly increased the female’s latency to return to the male’s chamber
following intromission and ejaculation. This finding suggests that MPOA lesions decrease
female sexual motivation. Moreover, the significant increase of the female’s return
latencies following MPOA lesions parallels the change seen in male sexual behavior
following MPOA lesions. It is suggested that the MPOA plays a similar role in regulating

the temporal copulatory behavior of male and female rats.

To

my wife
Fu-Mei Chen
my daughter
Rena Yang
my parents
Chin-Nan Yang and Dwei Lin
and
my parents-in-law

Tsen-Ken Chen and Jui-Wun l-Isiao

iv

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my major professor Dr. Lynwood
G. Clemens, who always inspired me, encouraged me, and challenged me intellectually
during the past five years. Without his constant support and full trust in my ability, I
would not have completed this project. I would also like to thank my committee members
Dr. Kay E. Holekamp, Dr. Antonio A. Nunez, and Dr. Ralph A. Pax for their guidance
and suggestions on various aspects of my dissertation.

I gratefully acknowledge Dr. Kevin E. McKenna at Northwestern University for
teaching me the electromyographic recording technique and Dr. Ralph A. Pax for giving me
additional advice on this technique. I am grateful for my colleagues Dr. Anthony E.
Ackerman, Mr. Gary M. Lange and Dr. Kevin L. Sinchak for teaching me a variety of
techniques used in the course of these experiments. I also wish to thank Mr. Alan S. Eliott
for teaching me how to use Computer Statistics and Drawing Programs. In addition, I
appreciate Miss Yu-Wen Chung and Dr. Jimmy Fang for their special help and friendship.

Moreover, I would like to thank my parents (Chin-Nan and Dwei) for providing me
with the opportunity and the financial support to complete my dissertation. Further, I
deeply thank my parents-in-law for their constant support. I am also indebted to my
daughter Rena, who is seven months old, for her cooperation in the past seven months.
Finally and most importantly, I would like to give my special thanks and love to my wife
Fu-Mei Chen, who gave me one hundred percent support through both the good and the
bad. Without her thoughtfulness and full support, I would have not been able to complete

my dissertation.

TABLE OF CONTENTS

LIST OF FIGURES ................................................................................ ix
LIST OF ABBREVIATIONS ...................................................................... xv
INTRODUCTION .................................................................................. 1
General Description of Female Rat’s Sexual Behavior ................................ 2
Significance of the Study of Temporal Pattern of Female Sexual Behavior ......... 4
Diversity of Testing Chambers in Studying Female Pacing Behavior .............. 6
Factors Influencing the Temporal Patterns of Female Sexual Behavior ............ 7
Sensory Innervation of Genitalia in Female Rats ....................................... 9
Vaginocervical Stimulation, Uterine Activity and Female Sexual Behavior ....... 10
Relation of MPOA to Female Sexual Behavior ..................................... 1 1
Summary ................................................................................... 1 3
EXPERIMENT 1: RELATION OF INTROMISSIONS TO THE FEMALE’S
POST EJACULATORY REFRACTORY PERIOD IN RATS ....................... 15
ABSTRACT ................................................................................ 1 5
INTRODUCTION ......................................................................... 16
GENERAL METHODS .................................................................. 18
EXPERIMENT 1A: THE TEMPORAL PATTERN OF FEMALE SEXUAL
BEHAVIOR TESTED IN OUR MODIFIED PACING CHAMBER ............... 20
METHOD ................................................................................... 20
RESULTS ................................................................................... 2 1

EXPERIMENT lB: EFFECT OF DIFFERENT NUMBERS OF
INTROMISSIONS ON THE FEMALE TEMPORAL COPULATORY

BEHAVIOR ................................................................................ 28
METHOD .................................................................................. 29
RESULTS .................................................................................. 3 0
DISCUSSION ................................................................................. 3 5

EXPERIMENT 2: FUNCTION OF INT ROMISSIONS IN REIATIONTO
THE INTROMISSION RETURN LATENCY OF FEMALE RATS

DURING PACED SEXUAL BEHAVIOR ............................................ 41
ABSTRACT ............................................................................... 41
INTRODUCTION ..................................................................... 42
GENERAL METHODS .............................................................. 44

EXPERIMENT 2A: RELATION OF MULTIPLE INTROMISSIONS '10
THE FEMALE’S INDIVIDUAL INTROMISSION RETURN

LATENCY ............................................................................. 46
METHOD .................................................................................. 46
RESULTS ............................................................................... 48
EXPERIMENT 2B: INFLUENCE OF DIFFERENT HORMONE
REPLACEMENTS ON THE FEMALE PACING BEHAVIOR ................ 54
METHOD ................................................................................ 55
RESULTS ................................................................................ 56
DISCUSSION ............................................................................... 56
EXPERIMENT 3: INFLUENCE OF MALE RELATED STIMULI ON
FEMALE POST EJACULATORY PERIOD IN RATS .............................. 62
ABSTRACT ............................................................................. 62
INTRODUCTION ...................................................................... 63
GENERAL METHODS ............................................................... 66
EXPERIMENT 3A: EFFECT OF STIMULUS FACTORS ON FEMALE
POSTEJACULATORY REFRACTORY PERIOD ................................... 68
MATERIALS AND METHODS ........................................................ 69
RESULTS ................................................................................ 72

EXPERIMENT 3B: RELATION OF THE EJACULATION DURATION
AND PREEJACULATORY INT ROMISSIONS TO THE FEMALE’S

POSTEJACULATORY REFRACTORY PERI OD .................................... 72
MATERIALS AND METHODS ......................................................... 73
RESULTS ................................................................................... 75
DISCUSSION .................................................................................. 87
EXPERIMENT 4: CORRELATION OF UTERINE ACTIVITY WITH THE
TEMPORAL PATTERN OF FEMALE SEXUAL BEHAVIOR IN RATS ....... 91
ABSTRACT ................................................................................ 91
INTRODUCTION ......................................................................... 92
METHOD ................................................................................... 92
RESULTS ................................................................................... 96
DISCUSSION . ........................................................................... 103
EXPERIMENT 5: SEXUAL MOTIVATION AND THE MEDIAL PREOP'TIC
AREA IN FEMALE RATS ............................................................ 105
ABSTRACT ............................................................................. 1 05
INTRODUCTION ...................................................................... 106
GENERAL METHODS .................................................................. 109
EXPERIMENT 5A: INFLUENCE OF ELECTROLYTIC MPOA LESIONS
ON THE FEMALE PACING BEHAVIOR ........................................ 11 1
METHOD ................................................................................ 1 1 1
RESULTS ................................................................................ 1 13

vii

EXPERIMENT 5B: EFFECT OF CHEMICAL MPOA LESIONS ON

THE FEMALE TEMPORAL COPULATORY BEHAVIOR ...................... 12 1
METHOD ............................................................................. 1 2 1
RESULTS ................................................................................ 1 23
DISCUSSION ......................................................................... 13 1
GENERAL DISCUSSION ..................................................................... 135
Si gnifrcance of Female Temporal Sexual Behavior ................................. 13 5
Stimulus Factors that Affect Female Temporal Copulatory Behavior .............. 136
Relation of Uterine Activity to Female Pacing Behavior ............................ 140
Cenual Neural Correlates of Female Temporal Copulatory Behavior ............. 141
Comparison of MPOA Lesions in Male and Female Rats ............................ 143

Future Directions .......................................................................... 143
Summary and Conclusions ............................................................. 144

LIST OF REFERENCES ....................................................................... 147

viii

Figure l .

Figure 2 .

Figure 3 .

LIST OF FIGURES

Return latency following mounts, intromissions, and
ejaculations for three ejaculatory series (N=67). The
female's return latency following an ejaculation (PER) was
significantly longer than her return latency following an
intromission (IRL) and mount (MRL) for three ejaculatory
series. The IRL was significantly longer than the MRL in
the second and third ejaculatory series. The female's PER
increased significantly as the ejaculatory series increased.
The f emale's IRL increased slightly over three ejaculatory
series but there was no significant increase between two
continuous ejaculatory series. There was no significant
increase in the female's MRL for three ejaculatory series.
Data were analyzed by ANOVA for repeated measures. Bar
represents mean i SEM. ** p<0.01 ........................................ 22

The percentage exits following mounts, intromissions, and

ejaculations for three ejaculatory series. The percentage exit

after ejaculation was significantly higher than that following

intromissions which was higher than that following mounts.

There was no significant difference among three ejaculatory

series for the percentage exit after mounts, intromissions,

and ejaculations. Data were analyzed by ANOVA for

repeated measures. Bar represents mean i SEM.

** p<0.01. N=67 ................................................................ 24

Relation of the number of intromissions prior to ejaculation

and the f emale's return latency following an ejaculation

(PER) for three ejaculatory series. There was a positive

correlation between the number of intromissions received by

the female before ejaculation over three ejaculatory series and

the female's PER. Data were analyzed by Correlation

analysis (r=0.425, p<0.0001). N 2201 ........................................ 26

ix

Figure 4 .

Figure 5 .

Figure 6 .

Effect of intromissions on f emale's latency to return to the
male following an ejaculation (PER, open bar, N211) and
following an intromission without ejaculation (I RL, solid
bar, N=8). ** Return latency after an ejaculation was
significantly shorter for 0-1 intromission group than the
other groups (p<0.01). * Return latency after an ejaculation
was significantly longer for the 24-31 intromission group
than the 5-15 intromission group (p<0.05). # The PER of
females receiving 24-31 intromissions was significantly
longer than those receiving 0-1 or 2-4 intromissions
(p<0.01). $ The female’s IRL was significantly shorter than
her PER in 2-4, 5-15 and 24—31 intromission groups
(p<0.01, paired t test). There was no significant difference
between the f emale’s return latencies following an
ejaculation of 2-4 and 5-15 intromission groups. There was
no significant difference among the female's mean return
latencies following an intromission of the different
intromission groups. All data except comparison of IRL and
PER within the same group were analyzed by ANOVA for
repeated measures followed by SNK post-hoe tests. Bar
represents mean 1 SEM .......................................................... 31

The relation between the number of intromissions preceding

ejaculation and the female's return latency following an

ejaculation (PER). The number of intromissions received by

the female prior to ejaculation in four groups was positively

correlated with her PER. Data were analyzed by Correlation

analysis (120.613, p<0.0001). N=44 .......................................... 33

Four representative portions of individual intromission
return latencies (IRLs) and three PERs of females receiving
three ejaculations are illustrated: (i) the first 4 IRLs (1, 2, 3 ,
4), (ii) 4 IRLs right before and 3 IRLs right after the lst
ejaculation (L-3, L-2, L-l, L, E1, 1, 2, 3), (iii) 3 IRLs right
before and 2 IRLs right after the 2nd ejaculation (L—2, L- 1,
L, E2, 1, 2), and (iv) 3 IRLs right before the 3rd ejaculation
(L-2, L-l, L, E3). Dataare mean _-+_- SEM and analyzed by
one-way ANOVA followed by Fisher's PLSD post-hoc
tests. L: the intromission right before ejaculation. El, E2,
and E3 stand for the lst, 2nd and 3rd ejaculation,
respectively. ** p<0.01, each individual IRL compared
with PER in the same ejaculatory series; #p<0.05,
comparison between adjacent PERs. The number of animals
for each intromission or ejaculation is indicated in the same
sequential order as in Figure 6: S, 9, 7, 7, 8, 8, 7, 9, 11
(E1), 6, 5, 5, 7, 9, 10, 11 (E2), 2, 5, 8, 9, 11, 11 (E3).

// indicates separation of 4 sets of continuous IRLs ........................... 50

Figure 7.

Figure 8.

Figure 9 .

Figure 10.

Figure l 1.

Figure l 2 .

Three representative portions of individual intromission
return latencies (IRLs) of females receiving a large number
of intromissions before ejaculation. These include 3
portions of consecutive IRLs: (i) the first 3 IRLs (1, 2, 3),
(ii) 21RLs right before and 2 IRLs right after the first
prolonged IRL (N-2, N-l, N, N+1, N+2), and (iii)3 IRLs
right before ejaculation (L-2, L—l, L, E). Data are mean :t
SEM and analyzed by one-way ANOVA followed by
Fisher's PLSD post-hoc tests. N: the intromission inducing
the first prolonged IRL. L: the intromission right before
ejaculation. E: ejaculation. ** p<0.01, * p<0.05 compared
with the first prolonged IRL. The number of animals for
each intromission or ejaculation is indicated in the same
sequential order as in Figure 7: 6, 6, 8, 8, 8, 10 (N), 6, 6,
6, 7, 10, 9 (E). // indicates separation of 3 sets of continuous IRLs ....... 52

Comparison of the PER of females receiving two different
hormone treatments. In the first week, the females received

injections of 0.5 pg EB for three consecutive days 24 hours
later followed by an injection of 0.5 mg progesterone. In the

second week, the females were given an injection of 50 pg

EB 48 hours later followed by an injection of 0.5 mg

progesterone. Bar represents mean -_i-_ SEM. Data were

analyzed by paired t test. ** P = 0.0007. N = 14 ............................. 57

compares the ejaculation duration of males between the 4— 18

and the 0-1 intromission Groups (4-18 I and 0-1 I Groups).

The male’s ejaculation duration in the 4— 18 I Group is

significantly longer than that in the 0-1 I Group (t = 4.065, p

= 0.0097, N = 6). Dataare expressed as mean i SEM and

analyzed by paired t-test. ** p<0.01 ............................................ 77

compares the postejaculatory refractory period (PER) of

females between the 4-18 and the 0-1 intromission Groups

(4-18 I and 0-1 I Groups). The PER of females in the 4» 18 I

Group is significantly longer than that in the 0-1 I Group (t =

4.155, p = 0.0089, N = 6). Dataare expressed as mean i

SEM and analyzed by paired t-test.

** p<0.01 .......................................................................... 79

shows the relation of the male’s ejaculation duration (ED) to

the female’s postejaculatory refractory period (PER). There

is a significantly positive correlation between the male’s E)

and the female’s PER (r = 0.814, p<0.0001, N=22) ......................... 81

shows the relation of the number of intromissions prior to

ejaculation to the male’s ejaculation duration. The

preejaculatory intromission frequency is positively correlated

with the male’s ejaculation duration (r = 0.771 , p < 0.0001 ,

N = 22) ............................................................................. 83

Figure 13.

Figure l 4.

Figure l 5.

Figure l 6 .

Figure 1 7 .

The relation of preejaculatory intromission frequency to the

female’s postejaculatory refractory period (PER). The

preejaculatory intromission frequency is significantly

correlated with the female’s PER (r = 0.611, p = 0.002, N =

22) ................................................................................... 85

shows the typical uterine EMG activity associated with a

mount, intromission, and ejaculation. The duration of

uterine EMG activity associated with an ejaculation seemed

longer than that associated with an intromission or a mount.

E ejaculation;I: intromission; M: mount. Amplitude: 1 cm =

1 mV. Time scale (paper speed): 1 cm = 2 seconds. Arrows

indicate the approximate time for dismount .................................... 97

Comparison of the durations of uterine EMG activity

associated with a mount, intromission, and ejaculation. The

duration of uterine EMG activity associated with an

ejaculation was significantly longer than that associated with

a mount or an intromission (** p<0.01). There was no

significant difference found between durations of uterine

EMG activity associated with a mount and an intromission.

Dataare expressed as mean i SEM in seconds and

analyzed by one way ANOVA followed by Fisher’s PLSD

post-hoc tests ...................................................................... 99

Comparison of the female’s return latencies following a

mount (MRL), intromission (IRL), and ejaculation (PER).

The female’s PER was significantly longer than her IRL or

MRL (* p<0.05). Data are expressed as mean -_+_ SEM in

seconds and analyzed by one way ANOVA followed by

Fisher’s PLSD post-hoc tests .................................................. 101

Composite diagrams of largest (shaded area) electrolytic
MPOA lesions. Figures and nomenclature from Paxinos and
Watson (1986). The Bregma coordinates for each figure on
top row from left to right are + 0.20 mm, - 0.30 mm, and -
0.80 mm. The Bregma coordinates for each figure on
bottom row from left to right are - 0.92 mm, and - 1.30 mm.
3V: 3rd ventricle;ac: anterior commissure; aca: anterior
commissure, anterior; AHA: anterior hypothalamic area,
anterior; BSTMPL: bed nucleus of the stria terminalis,
medial division, posterolateral part; BST V: bed nucleus of
stria terminalis, ventral division; f: fornix; HDB: nucleus of
the horizontal limb of the diagonal band of Broca; IA:
lateroanterior hypothalamic nucleus; LH: lateral
hypothalamic area; LPO: lateral preoptic area; MPA: medial
preoptic area; MPO: medial preoptic nucleus; ox: optic
chiasm; PaAP: paraventricular hypothalamic nucleus,
anterior parvocellular part; Pe: peri ventricular hypothalamic
nucleus; PS: parastrial nucleus; SCh: suprachiasmatic
nucleus; SHy: septohypothalarnic nucleus; StHy:
striohypothalarnic nucleus; SO: supraoptic nucleus; VDB:
nucleus of the vertical limb of the diagonal band of Broca .................. 1 15

xii

Figure l 8 .

Figure 19.

Figure 20.

summarizes the f emale’s return latencies following an
intromission (intromission return latency, IRL) before
surgery and two weeks after surgery in the MPOA
electrolytic lesioned group (N = 8) and the MPOA sham-
lesioned group (N = 11). Two Way Repeated Measures
ANOVA revealed a significant group effect [ F( 1, 17) =
19.481, p = 0.0004], surgery effect [F(1, 17) = 16.652, p
= 0.0008], as well as group x surgery interaction [F(1, 17)
= 22.033, p = 0.0002] for the female’s IRL. The SNK post-
hoc tests revealed that the postsurgical IRL of females with
MPOA electrolytic lesions was significantly longer than the
other IRLs (p<0.01). ** p<0.01 .............................................. 117

shows the female’s return latencies following an ejaculation
(postejaculatory refractory period, PER) before surgery and
two weeks after surgery in the MPOA electrolytic lesioned
group (N = 7) and the MPOA sham-lesioned group (N =
11). Two Way Repeated Measures ANOVA revealed a
significant group effect [ F(1, 16) = 9.828, p = 0.0064],
surgery effect [F(1, 16) = 7.868, p = 0.0127], as well as
group x surgery interaction [F( 1, 16) = 11.094, p = 0.0042]
for the female’s PER. The SNK post—hoc tests revealed that
the postsurgical PER of females with MPOA lesions was
significantly longer than the other PERs (p<0.01). **

p<O. 0 1 ............................................................................ 1 l 9

Composite diagrams of largest (shaded area) ibotenic acid
MPOA lesions. Figures and nomenclature from Paxinos and
Watson. The Bregma coordinates for each figure on top row
from left to right are + 0.20 mm, - 0.30 mm, and - 0.80
mm. The Bregma coordinates for each figure on bottom row
from leftto rightare - 1.30 mm, and - 1.80 mm. 3V: 3rd
ventricle; ac: anterior commissure; aca: anterior commissure,
anterior; AHA: anterior hypothalamic area, anterior; AHC:
anterior hypothalamic area, central part; BSTMPL: bed
nucleus of the stria terminalis, medial division, posterolateral
part; BST V: bed nucleus of stria terminalis, ventral division;
f: fomix; HDB: nucleus of the horizontal limb of the
diagonal band of Broca; LA: lateroanterior hypothalamic
nucleus; LH: lateral hypothalamic area; LPO lateral preoptic
area; MPA: medial preoptic area; MPO. medial preoptic
nucleus; opt: optic tract; ox: optic chiasm; PaAP.
paraventricular hypothalamic nucleus, anterior parvocellular
part; Pe: periventricular hypothalamic nucleus; PS: parastrial
nucleus; RCh: retrochiasmatic area; SCh: suprachiasmatic
nucleus; SHy: septohypothalarnic nucleus; StHy:
striohypothalarnic nucleus; SO: supraoptic nucleus; sox:
supraoptic decussation; VDB: nucleus of the vertical limb of
the diagonal band of Broca ..................................................... 125

xiii

Figure 2 1 .

Figure 22.

The f emale’s return latencies following an intromission
(intromission return latency, IRL) before surgery and two
weeks after surgery in the MPOA chemical lesioned group
(N = 6) and the MPOA sham-lesioned group (N = 9). Two
Way Repeated Measures ANOVA revealed a significant
group effect [ F(1, 13) = 12.296, p = 0.0039], surgery
effect [F(1, 13) = 10.510, p = 0.0064], as well as group x
surgery interaction [F(1, 13) = 18.408, p = 0.0009] for the
female’s IRL. The SNK post-hoc tests revealed that the
postsurgical IRL of females with MPOA ibotenic acid
lesions was significantly longer than the other IRLs

(p<0.01). ** p<0.01 ........................................................... 127

shows the female’s return latencies following an ejaculation
(postejaculatory refractory period, PER) before surgery and
two weeks after surgery in the MPOA chemical lesioned
group (N = 6) and the MPOA sham-lesioned group (N = 9).
There were a significant group effect [ F(1, 13) = 39.933, p
< 0.0001], surgery effect [F(1, 13) = 30.108, p = 0.0001],
as well as group x surgery interaction [F(1, 13) = 36.848, p
< 0.0001] for the female’s PER. The SNK post-hoe tests
revealed that the postsurgical PER of females with MPOA
chemical (ibotenic acid) lesions was significantly longer than
the other PERs (p<0.01). ** p<0.01 ......................................... 129

xiv

BC
BCX

IL

EMG
ID

IF

IL

III
[M
IRL

IP

LIST OF ABBREVIATION

approach latency
bulbocavemosus muscle

removal of
bulbocavemosus muscle

intromission latency
estradiol benzoate
ejaculation latency
electromyographic

inner diameter
intromission frequency
intromission latency
interintromission interval
intramuscular

intromission return
latency

intraperitoneal

MPOA

PEI
PER

PX
SCV

SNK
VMN

mount latency

medial preoptic area of
hypothalamus

mount return latency
outer diameter
progesterone

postej aculatory interval

postejaculatory refractory
period

removal of prostate glands
removal of seminal
vesicles as well as

coagulating glands and
ligation of vas def erens

Student-Newman-Keuls

ventromedial nucleus of
hypothalamus

INTRODUCTION

Different species of animals exhibit different patterns of copulatory behavior
(Dewsbury, 1972). In rats, the male displays an intermittent copulatory pattern in which he
delivers multiple intromissions (and/or multiple mounts) prior to ejaculation and achieves
multiple ejaculations before sexual satiety (Dewsbury, 1972; Meisel and Sachs, 1994).
Under a standard test situation, female sexual behavior is tested in a small test chamber
from which she can not exit. The male rat has free access to the female and controls the
timing of copulation. Iordosis behavior is the major measure of female sexual behavior.
However, under test situations where the small testing chamber is divided into the male’s
chamber and an escape chamber available only to the female, the female can regulate the
copulatory speed by exiting the male’s chamber following sexual contacts (Bermant, 1961;
Bermant and Westbrook, 1966; Erskine, 1985, 1987, 1989, 1992; Erskine and Baum,
1982; Erskine etal., 1989; Fadem et al., 1979; Frye and Erskine, 1990; Gilman and Hitt,
1978; Krieger et al., 1976; Lange and Clemens, 1994; Mermelstein and Becker, 1995;
Peirce and Nuttall, 1961a). Under the female-paced conditions, the female’s return
latencies and the percentage exits following sexual contacts, along with lordosis, provide
robust measures of female sexual behavior.

One possible function of the temporal pattern of female sexual behavior is to
facilitate successful pregnancy. When the female rat controls the timing of copulation, the
interintromission intervals increase in length (Erskine, 1985; Erskine et al., 1989; Fadem
and Barfield, 1982; Gilman et al., 1979) and fewer intromissions are sufficient to induce
the progestational state of pregnancy in female rats (Erskine et al., 1989; Gilman et al.,

1979). Since the regulation of the copulatory speed by the female rat facilitates successful
1

2
reproduction, it is important to understand what mechanisms underlie the regulation of the

temporal pattern of female reproductive behavior. The present study aims to investigate (1)
how stimulus factors affect the temporal pattern of female rat’s sexual behavior, especially
the female’s postejaculatory refractory period (PER), (2) how uterine electromyographic
(EMG) activity correlates with each type of sexual event and the temporal pattern of female
sexual behavior, and (3) whether the medial preoptic area of the hypothalamus (MPOA) is

involved in regulating female pacing behavior.

General Description of Female Rat’s Sexual Behavior

The female rat displays a four or five day estrous cycle that is controlled by ovarian
hormones (Carter, 1992; Nelson, 1995). The typiwl four-day estrous cycle consists of
three stages: diestrus (48 hours) (Researchers further divide diestrus into diestrus I and
diestrus II or metestrus and diestrus), proestrus ( 12 hours), and estrus (36 hours) (Carter,
1992; Nelson, 1995). Plasma estradiol levels gradually increase during diestrus,
significantly increase during the morning and early afternoon of proestrus, and return
rapidly to the baseline level during estrus (Smith et al., 1975). The significant increase of
estradiol during the morning and early afternoon of proestrus and the beginning rise of
progesterone during the afternoon of proestrus induce behavioral estrus in female rats
(Smith et al., 1975; Young, 1961). In addition, the dramatic estradiol increase causes a
surge of luteinizing hormone (LH) that in turn triggers ovulation on the evening of
proestrus (Nelson, 1995). The coincidence of behavioral estrus and ovulation induced by
ovarian hormones ensures successful reproduction.

In a standard test situation, female rat’s sexual behavior is tested in a small test
chamber where the male has free access to her and regulates the timing of mating. In
behavioral estrus, the female rat hops and darts to attract the male’s attention and initiate

copulation (Beach, 1976; Madlafousek and Hlinak, 1977; Madlafousek et al., 1976;

3
Nelson, 1995). Consequently, the frequencies of hopping and darting have been used to

measure the female’s proceptivity which refers to “various reactions by the female toward
the male which constitute her assumption of initiative in establishing or maintaining sexual
interaction” (p. 105, Beach, 1976). In addition, the female rat shows lordosis behavior,
the arching of her back and the elevation of her genitalia, in response to the male’s
mounting behavior. The display of lordosis behavior in female rats is necessary for
successful penile insertion that ensures successful insemination (Diakow, 1974; Pfaff et
al., 1978). Therefore, the lordosis quotient [(the number of lordosis responses in the first
10 mounts/ 10) x 100] has been used to measure the female’s receptivity which refers to
the female’s responses essential for successful intravaginal insemination (Beach, 1976) and
has been the primary measure of female sexual behavior in rats (Pfaf f et al., 1994).

In a two—compartment test situation, the latencies for the female to return to the
male’s chamber following sexual contacts and the percentage of times at which the female
exits the male’s chamber after each sexual contact provide robust measures of the temporal
pattern of female sexual behavior. The female's latency to return to the male following a
sexual contact is associated with the type of stimulation she receives from the male. Her
return after an ejaculation (postejaculatory refractory period, PER) is significantly longer
than that following a mount (mount return latency, MRL) or an intromission (intromission
return latency, IRL) (Bermant, 1961; Bermant and Westbrook, 1966; Erskine, 1985, 1989;
Krieger et al., 1976; Lange and Clemens, 1994; Peirce and Nuttall, 1961a). In addition,
her IRL is longer than her MRL (Bermant and Westbrook, 1966; Erskine, 1985; Krieger et
al., 1976). Further, the female’s MRL (Krieger et al., 1976), IRL (Bermant and
Westbrook, 1966; Krieger et al., 1976), and PER (Bermant and Westbrook, 1966; Krieger
et al., 1976) increase with the ejaculatory series. Finally, the female exits the male’s
chamber most frequently following an ejaculation and least frequently following a mount

(Erskine, 1985, 1989;Krieger etal., 1976).

4
Significance of the Study of Temporal Pattern of Female Sexual Behavior

The study of the temporal aspects of female rat’s sexual behavior is important in
several respects. One important function of the temporal pattern of female sexual behavior
is to facilitate successful pregnancy. In rats, the male delivers multiple intromissions
before he ejaculates. These intromissions have at least two major functions. First, multiple
intromissions facilitate sperm transport from the vagina to the uterus. It has been reported
that if the female receives 01 intromission prior to ejaculation, no or few sperm are found
in the uterus 3 hrs following ejaculation and no developing eggs are found in the uterus 2-4
days after copulation (Adler, 1969). In contrast, if the female receives two or more
intromissions before ejaculation, many sperm are found in the uterus 3 hrs following
ejaculation (Adler, 1969). Secondly, multiple intromissions induce the progestational state
of pregnancy that is necessary for blastocyst implantation and successful pregnancy in
female rats (Adler, 1969; Beach, 1965; Chester and Zucker, 1970; Wilson et al., 1965). In
rats, the normal blood progesterone level of the female is not sufficient to support
blastocyst implantation (Hashimoto et al., 1968). However, copulation can induce the
secretion of progesterone to a level sufficient for blastocyst implantation (Adler et al.,
1970). The number of intromissions required to induce the progestational state of
pregnancy depends on the temporal pattern of copulation. In a standard test situation, the
male rat controls the copulatory speed. Under this situation, approximately 10
intromissions are sufficient to induce the progestational state of pregnancy (Adler, 1969;
Chester and Zucker, 1970; Edmonds et al., 1972; Erskine, 1989; Erskine et al., 1989;
Gilman et al., 1979). In contrast, under a test situation where the female rat can escape
from the male following sexual contacts, she regulates the timing of copulation. Under
female-paced situation, the interintromission intervals are lengthened (Erskine, 1985;
Erskine et al., 1989; Fadem and Barfield, 1982; Gilman et al., 1979) and the number of

intromissions required for the induction of the progestational state of pregnancy is reduced

5
(approximately 5 intromissions in female-paced situation) (Erskine, 1989; Erskine et al.,

1989; Gilman et al., 1979).

In addition, one possible function of the female’s postejaculatory refractory period
is to facilitate or allow sperm transport from the vagina to the uterus. Under female-paced
situations, the female rat leaves the male’s chamber and shows a period of sexual inactivity
following ejaculation: the postejaculatory refractory period. The female’s postejaculatory
refractory period is important for sperm transport. Sperm transport will be stopped until
the next ejaculation if the female receives intromissions shortly after ejaculation. This is
supported by the evidence that the number of sperm found in the uterus decreases
significantly if the female receives an intromission within two minutes following ejaculation
(Matthews & Adler, 1977).

Further, the study of female pacing behavior provides a possibility for comparing
male and female sexual behavior under the same measurement scale. Traditionally, the
latency and f requenCy of sexual contacts (mount, intromission, and ejaculation) are used to
measure male rat’s sexual behavior (Meisel & Sachs, 1994). The measures of male sexual
behavior include mount latency (the latency from the introduction of the female to the first
mount), intromission latency (the latency from the introduction of the female to the first
intromission), ejaculation latency (the latency from the first intromission to ejaculation),
mount frequency, intromission frequency, hit rate (intromission frequency / intromission
frequency plus mount frequency), interintromission interval (111, the ejaculation latency
divided by the intromission frequency), and the postejaculatory interval (PEI, the latency
from ejaculation to the next intromission). In contrast, in a standard test situation, the
frequency of lordosis but not the latency of sexual contacts is used to measure female
sexual behavior because the male controls the timing of copulation (For more details, please
see general description of female rat’s sexual behavior in the previous section). However,
under female paced situations, the f emale’s latency to approach the male and her latencies to

return to the male’s chamber following sexual contacts appear comparable to the latency

6
measures of male sexual behavior. For example, the female’s intromission return latency

and postejaculatory refractory period are comparable to the male’s interintromission interval
and postejaculatory interval, respectively.

Finally, the investigation of female pacing behavior facilitates the study of sexual
motivation in female rats. The distinction between female sexual ability and female sexual
desire (sexual motivation) has been elaborated by Wallen (Wallen, 1990). Female sexual
ability is defined as the female’s ability to engage in copulation. Female sexual motivation
refers to “the state underlying sexual initiation and sexual accommodation” (p. 234,
Wallen, 1990). In rats, it has been demonstrated that it is impossible for the female to
allow successful penile insertion without showing the lordosis posture (Diakow, 1974;
Pfaff et al., 1978). Consequently, the display of lordosis behavior becomes the major
measure of female sexual ability (Wallen, 1990). In female-paced situations, the female’s
accessibility and the temporal pattern of her sexual behavior are regulated by her sexual
motivation (Wallen, 1990). In other words, the female’s latencies to approach or return to

the male following sexual contacts can provide measures of her sexual motivation.

Diversity of Testing Chambers in Studying Female Pacing Behavior

Different test situations have been used to measure the f emale's temporal copulatory
behavior (Bermant, 1961; Bermant and Westbrook, 1966; Erskine, 1985, 1989, 1992;
Fadem et al., 1979; Gilman and Hitt, 1978; Krieger et al., 1976; McClintock and Adler,
1978; McClintock and Anisko, 1982; McClintock et al., 1982; Mermelstein and Becker,
1995; Peirce and Nuttall, 1961a). These different test situations can be classified into two
major categories. In one category, the temporal pattern of female sexual behavior is tested
in a small test chamber and usually one male and one female are used. The test situations in
this category can be further divided into three types. In some of the earlier studies, the

females are trained to press a lever in order to get access to a sexually active male (Bermant,

7
1961; Bermant and Westbrook, 1966). In a second type of study, the female is provided

with an escape chamber or platform to which she can retreat (Erskine, 1985, 1989, 1992;
Gilman and Hitt, 1978; Mermelstein and Becker, 1995; Peirce and Nuttall, 1961a). The
male is sharply hit on the nose when he tries to enter the escape chamber or platform. A
third protocol involves tethering the male to one area of the test arena and allowing the
female access to the male (Fadem et al., 1979; Krieger et al., 1976). In this category, the
test situation is simpler and is most suitable for studying the mechanisms regulating the
temporal pattern of female sexual behavior.

In the other category, the temporal pattern of female sexual behavior is tested in a
seminatural environment (McClintock and Adler, 1978; McClintock and Anisko, 1982;
McClintock et al., 1982). A group of intact animals (2 males and 5 females) are placed in a
much larger seminatural testing arena and their mating behavior is videotaped. The female
rat returns to the burrow or stays behind some barrier after she receives sexual contacts.
The time the female stays away from the male following sexual contacts is used to measure
her pacing behavior. Although the seminatural environment is more complicated, this
seminatural testing setup mimics the natural environment and is most appropriate for

studying the evolutionary significance of the mating strategy of females in nature.

Factors Influencing the Temporal Pattern of Female Sexual Behavior

Copulatory stimuli influence the temporal pattern of female rat’s sexual behavior.
In the previous section, it was noted that the female’s PER is significantly longer than her
IRL or MRL (Bermant, 1961; Bermant and Westbrook, 1966; Erskine, 1985, 1989;
Krieger et al., 1976; Peirce and Nuttall, 1961a). Although it has been reported that the
presence of a seminal plug following ejaculation lengthens the female’s PER (Bermant and
Westbrook, 1966), the effect of a seminal plug on the female’s PER is minimal in that

study. The authors have suggested that other factors including the duration of penile

8
insertion, the number of deep thrusts and the tightness with which the male grasps the

female during ejaculation might contribute to the female’s PER. In addition, during
ejaculation, the formation of the penile cup may generate some mechanical stimulation and
contributes to the f emale’s PER. Further, chemical substances released from the prostate
may cause contraction of the vagina, cervix as well as uterus and induces the female’s
PER.

The temporal pattern of female rat’s sexual behavior is affected by gonadal
hormones (Fadem et al., 1979; Gilman and Hitt, 1978). In the presence of estradiol
benzoate (EB), the increases in the progesterone (P) level reduce the return latencies
following both intromissions and ejaculations (Fadem et al., 1979; Gilman and Hitt, 1978).
In addition, increases in progesterone levels at different doses of EB decrease the frequency
with which the female leaves the male's chamber after intromissions (Gilman and Hitt,
1978). Increases in BB do not affect the return latencies following intromissions or
ejaculations nor do they affect the percentage of "exits” with which the female leaves the
male's chamber following intromissions (Gilman and Hitt, 1978).

Different hormone replacement protocols have been used to prime the females into
behavioral estrus in studying female pacing behavior (Bermant, 1961; Bermant and
Westbrook, 1966; Krieger etal., 1976; Lange and Clemens, 1994). Basically these can be

divided into two major categories. In one category, researchers give an injection of a high

dose of estradiol benzoate (50 pg or 100 pg) followed by an injection of progesterone (0.5

or 1 mg) 48 or 72 hours later (Bermant, 1961; Bermant and Westbrook, 1966; Krieger et
al., 1976). In the other category, the females are brought into behavioral estrus by three

consecutive injections of a low dose of EB (0.5 pg) followed by an injection of
progesterone (0.5 mg) 24 hours later (Lange and Clemens, 1994). It has been noticed that

the female’s return latencies following sexual contacts are shorter in the study employing

consecutive injections of low doses of estradiol benzoate followed by an injection of

9
progesterone 24 hours later (Lange and Clemens, 1994). For example, the female’s PER

is about 50 seconds in the study using this low dose of estradiol benzoate protocol (Lange
and Clemens, 1994). In contrast, the females have a PER ranging from 90 seconds to
several minutes in those studies using a high dose of estradiol benzoate followed by an
injection of progesterone 48 or 72 hours later (Bermant, 1961; Bermant and Westbrook,
1966; Krieger et al., 1976). In addition to the difference in hormonal treatments, different
test chambers have been used in the above studies. Whether the difference in the female’s
return latencies in those studies using different hormone replacement protocols results from

the difference in hormone treatments, the difference in testing chambers or both is not clear.

Sensou Innervation of Genitalia in Female Rats

In female rats, the pelvic nerve, pudenda] nerve, and hypogastric nerve convey
sensory stimulation from the external and internal genitalia. It has been reported that the
pelvic nerve carries sensory stimulation from the vagina (Berkley et al., 1990; Berkley et
al., 1993; Komisaruk et al., 1972; Peters et al., 1987), cervix (Berkley et al., 1990;
Berkley et al., 1993; Komisaruk et al., 1972; Peters et al., 1987), uterus (Berkley et al.,
1990; Berkley et al., 1993) and perineal skin (Peters et al., 1987). The hypogastric nerve
responds to the mechanical stimulation of the cervix and three fifths of the uterus near the
cervix (Berkley et al., 1988; Berkley et al., 1993; Peters et al., 198D. The pudenda] nerve
bears sensory stimulation from perineal skin, inner thigh, and clitoral sheath (Peters et al.,
1987).

Fibers with mechanoreceptive fields are found along the vaginal canal to the uterine
body (including the cervix), and more fibers with mechanoreceptive fields are found in the
vaginocervical junction (Berkley et al., 1990). These fibers with mechanoreceptive fields
respond best to the stimuli provided by a probe moving from vaginal opening toward

vaginocervical junction (Berkley et al., 1990). The pelvic nerve fibers with

10
mechanoreceptive fields are significantly more sensitive to the mechanical stimulation of the

cervix and uterus than the hypogastric nerve fibers (Berkley et al., 1993). A functional
analysis of the pelvic nerve fibers and the hypogastric nerve fibers suggests that the former
is closely bound to sensory and behavioral processes associated with copulation and
pregnancy and the latter is closely bound to nociception and conception (Berkley et al.,

1993).
Vaginocervical Stimulation, Uterine Activity and Female Sexual Behavior

In an earlier section, it has been mentioned that vaginocervical stimulation provided
by the male during copulation is associated with the temporal pattern of female sexual
behavior. In addition, vaginocervical stimulau'on provided by a male during copulation or
provided by mechanical manual stimulation has been demonstrated to induce
pseudopregnancy in female rats (Adler, 1969; Beach, 1965; Carlson and De Feo, 1965;
Chester and Zucker, 1970; Edmonds et al., 1972; Erskine, 1989; Erskine et al., 1989;
Gilman et al., 1979; Wilson etal., 1965), and to shorten the period of behavioral estrus in
female rats (Iodder and Zeilmaker, 1976).

I The pelvic nerve is important for induction of pseudopregnancy by copulation or
mechanical vaginocervical stimulation (Carlson and De Feo, 1965) as well as for
abbreviation of behavioral estrus (Lodder and Zeilmaker, 1976). The pelvic nerve and the
pudenda] nerve have also been demonstrated to play an important role in the display of
normal temporal patterns of sexual behavior in female rats (Emery and Whitney, 1985;
Erskine, 1992). The pelvic neurectomy abolishes the induction of pseudopregnancy by
mating or mechanical vaginocervical stimulation (Carlson and De Feo, 1965). In addition,
bilateral transection of the pelvic nerve disrupts the female rat’s pacing behavior (Emery
and Whitney, 1985; Erskine, 1992). The females show no difference between the

percentage exits following mounts and intromissions after transection of the pelvic nerve,

11
whereas control females show a higher percentage of exits following intromissions than

following mounts (Erskine, 1992). Moreover, the number of intromissions and
ejaculations that the male delivers during a constant time and the time that the female spends
with the sexually active males increase significantly after transection of the pelvic nerve
(Emery and Whitney, 1985). Furthermore, anesthetics applied locally to the vagina of
female rats reduce their contact-response intervals following intromissions and ejaculations
(Bermant and Westbrook, 1966). Finally, the female’s return latencies following mounts,
intromissions, and ejaculation are not different from one another after bilateral transection
of the pelvic nerve and pudenda] nerve (Erskine, 1992).

Vaginocervical stimulation plays an important role in the regulation of uterine EMG
activity. Uterine contractions change with the course of copulation (Toner and Adler,
1986). Copulation has both an immediate effect and a delayed effect on uterine EMG
activity (Toner and Adler, 1986). Copulation doubles the frequency of uterine contractions
during active copulation but uterine activity returns to the premating level immediately after
ejaculation, then increases significantly 5 minutes after ejaculation (Toner and Adler,
1986). These results are obtained under the situation where the male controls the
copulatory rate. Although vaginocervical stimulation plays an important role in the control
of uterine EMG activity and the temporal pattern of female sexual behavior, it is not known
how uterine EMG activity is correlated with the temporal pattern of female sexual behavior.
In addition, it is not clear, however, how uterine EMG activity correlates with each type of

sexual contact (mount, intromission, and ejaculation).
Relation of MPOA to Female Sexual Behavior
The MPOA has been reported to play an important role in the control of female

sexual behavior in rats. The display of lordosis behavior in female rats is inhibited by

electrical stimulation of the media] preoptic area of hypothalamus (Moss et al., 1974;

12
Napoli et al., 1972). On the other hand, MPOA lesions have been reported to increase

lordosis frequency in female rats treated with low doses of estradiol benzoate plus
progesterone (Hoshina et al., 1994; Powers and Valenstein, 1972; Whitney, 1986).
Transections made anterior-dorsal to the MPOA also facilitate lordosis responding in female
rats (Yarnanouchi and Arai, 1977). Based on these results, it has been suggested that the
MPOA inhibits female “sexual receptivity” (Powers and Valenstein, 1972). However,
MPOA lesions significantly decrease the time that the female spends with the sexually
active males (Whitney, 1986), suggesting that the MPOA may play a role in facilitating
female sexual behavior.

Vaginocervical stimulation provided by a male rat during copulation, or by a glass
rod, significantly increases c—fos expression in the MPOA of female rats (Pfaus et al.,
1993; Wersinger et al., 1993). In addition, mechanical stimulation of the uterine cervix
induces changes in the electrical activity of neurons in the MPOA (Haskins and Moss,
1983). The above findings strongly suggest that the MPOA may play a role in the
regulation of female sexual behavior. The MPOA has been demonstrated to inhibit female
lordosis responses. It is not known, however, whether the MPOA plays an important role
in the regulation of temporal patterning of female sexual behavior. Since MPOA lesions
have been demonstrated to decrease frequency of sexual contacts and the time that the
female spends with the sexually active males during a constant time test when she can
choose among sexually active males, castrated males and females (Whitney, 1986), the
MPOA lesions are expected to significantly increase the female’s return latencies following
sexual contacts in a two—compartment pacing chamber.

The MPOA plays an important role in the regulation of male rat’s sexual behavior.
It has been reported that copulation significantly induces c-fos expression in the MPOA of
male rats (Baum and Everitt, 1992; Wersinger et al., 1993). In addition, large MPOA
lesions abolish male sexual behavior (Ginton and Merari, 1977; Heimer and Larsson,

1966/ 1967). It is also true that some males with MPOA lesions (some part of MPOA is

13
undamaged) can still copulate although their interintromission interval, ejaculation latency

and postejaculatory interval are prolonged (Ginton and Merari, 1977). Moreover, electrical
stimulation of the MPOA facilitates male rat’s copulatory behavior (Malsbury, 1971). The
above evidence suggests that the MPOA facilitates the temporal pattern of male sexual
behavior in rats. Furthermore, the MPOA regulates the motor responses (lordosis behavior
and mounting behavior) of sexual behavior similarly in both male and female rats. The
MPOA lesions facilitate lordosis behavior in castrated male rats treated with estradiol
benzoate and progesterone (Hennessey et al., 1986; Olster, 1993) just as they do in females
(Hoshina et al., 1994; Powers and Valenstein, 1972; Whitney, 1986). MPOA lesions also
inhibit mounting in ovariectomized females treated with testosterone (Singer, 1968) just as
they do in male rats (Ginton and Merari, 1977; Heimer and Larsson, 1966/1967).
Therefore, MPOA may regulate the female’s temporal copulatory pattern in the same way

as it does in males.

Summary

When given the opportunity, the female rat can control the timing of copulation.
One function of the temporal pattern of female sexual behavior is to facilitate successful
pregnancy. Under female-paced situations, the interintromission intervals are longer and
fewer intromissions are sufficient to elevate the blood progesterone to a level which is
adequate for blastocyst implantation and maintenance of pregnancy. In addition, the
female’s postejaculatory refractory period is important for sperm transport from the vagina
to the uterus. In the past thirty-five years, the temporal pattern of female sexual behavior
has been studied to some extent. However, what mechanisms underlie the female’s
postejaculatory refractory period, how the uterine EMG activity correlates with different
types of sexual contacts and the temporal pattern of female sexual behavior, and whether

the MPOA regulates the temporal pattern of female sexual behavior are not well

14
understood. In the following experiments, several important questions about the temporal

pattern of female rat’s sexual behavior will be investigated including (1) how stimulus
factors affect the female’s postejaculatory refractory period, (2) how intromissions
influence the female’s individual return latencies following intromissions, (3) whether
different hormonal regimes affect the temporal pattern of female sexual behavior, (4) how
the uterine EMG activity correlates with the female pacing behavior, and (5) whether the

MPOA regulates the female temporal copulatory pattern.

EXPERIMENT l: RELATION OF INTROMISSIONS TO THE FEMALE’S
POSTEJACULATORY REFRACTORY PERIOD IN RATS

ABSTRACT

YANG, L. Y. AND L. G. CLEMENS. Relation of intromissions to the female's
postejaculatory refractory period in rats. PHYSIOL BEHAV 59(0) 000-000, 1996.- The
objectives of this study were to investigate the temporal aspects of female sexual behavior
during single and multiple ejaculatory tests. Females were tested in a two-compartment
chamber where they could escape from the male following sexual contacts. In Experiment
1A, correlation analysis showed that the number of intromissions received by the female
over three ejaculatory series was positively correlated with the female's postejaculatory
refractory period (PER). In Experiment 1B, females receiving 24 intromissions before
ejaculation had a PER that did not differ from those receiving 515 (average 10)
intromissions preceding ejaculation. However, if the male ejaculated on the first or second
intromission, the female's PER was significantly shorter than the other groups and did not
differ from her return latency after an intromission without ejaculation. Females receiving
24—31 intromissions preceding ejaculation exhibited the longest PER. Analysis revealed
that the number of intromissions received by females before ejaculation was positively
correlated with the female's PER. We concluded that the male's ejaculatory reflex, seminal
emission, and postejaculatory behavior alone without at least two preceding intromissions
were not sufficient to induce a female's PER comparable to that seen after an ejaculation
during normal copulation. In addition, the number of intromissions received by the female

preceding ejaculation was positively correlated with the female's PER if the range of

15

16

intromission frequency was large enough.

INTRODUCTION

During copulation, the male rat displays an intermittent copulatory pattern that
includes mounts, intromissions, and ejaculations (Dewsbury, 1972; Young, 1961). In a
standard test situation, the male rat has free access to the female and thereby regulates the
copulatory rate. In this situation, the receptive female rat displays soliciting behaviors such
as hopping and darting and when mounted she responds with the lordosis posture to allow
successful penile insertion (Beach, 1976; Young, 1961). This has resulted in the use of
lordosis as the measure of the female sexual behavior. However, when the female is tested
in a situation where she can escape from the male following a sexual contact, she "paces"
the copulatory events (Bermant, 1961; Bermant and Westbrook, 1966; Clemens et al.,
1995; Erskine, 1985, 1989; Erskine and Baum, 1982; Fadem et al., 1979; Gilman and
Hitt, 1978; Krieger et al., 1976; Mermelstein and Becker, 1995; Peirce and Nuttall, 1961a;
Yang and Clemens, 1994, 1995a, 1995b). Following an ejaculation, the female leaves the
male’s chamber for a period of time. This period of female sexual inactivity is referred to
as her postejaculatory refractory period (PER). One important function of the female’s
PER is to allow sperm transport. This is supported by the evidence that sperm transport
will be stopped until the next ejaculation if the female receives an intromission within two
minutes following ejaculation (Matthews and Adler, 1977). On the other hand, multiple
intromissions prior to ejaculation have been reported to facilitate sperm transport (Adler,
1969). However, the relation of multiple intromissions preceding ejaculation to the
female’s PER is poorly understood.

The female's latency to return to the male following a sexual contact is associated
with the type of stimulation she receives from the male. Her return after an ejaculation

(PER) is much longer than that following a mount (MRL) or intromission (IRL) (Bermant,

17
1961; Bermant and Westbrook, 1966; Erskine, 1985, 1989; Krieger et al., 1976; Peirce

and Nuttall, 1961a; Yang and Clemens, 1994). In addition, her IRL is longer than her
MRL (Bermant, 1961; Bermant and Westbrook, 1966; Erskine, 1985, 1989; Krieger et
al., 1976; Peirce and Nuttall, 1961a). The temporal pattern of female rat sexual behavior is
also affected by gonadal hormones (Fadem et al., 1979; Gilman and Hitt, 1978). In the
presence of estradiol benzoate (EB), the increases in the progesterone (P) levels reduce the
return latencies following both intromissions and ejaculations (Fadem et al., 1979; Gilman
and Hitt, 1978). In addition, increases in progesterone levels at different doses of EB
decrease the frequency with which the female leaves the male's chamber after intromissions
(Gilman and Hitt, 1978). Increases in EB alone do not affect the return latencies following
intromissions or ejaculations nor do they affect the percentage of ”exits" with which the
female leaves the male's chamber following intromissions (Gilman and Hitt, 1978).

Different test situations have been used to measure the female's temporal copulatory
behavior (Bermant, 1961; Bermant and Westbrook, 1966; Erskine, 1985, 1989; Erskine
and Baum, 1982; Fadem et al., 1979; Gilman and Hitt, 1978; Krieger et al., 1976;
Mermelstein and Becker, 1995; Peirce and Nuttall, 1961a). Basically these can be divided
into three types. For some of the earlier studies, females were trained to press a lever in
order to get access to a sexually active male (Bermant, 1961; Bermant and Westbrook,
1966). In a second category the female is provided with an escape chamber or platform to
which she can retreat (Erskine, 1985, 1989; Erskine and Baum, 1982; Gilman and Hitt,
1978; Mermelstein and Becker, 1995; Peirce and Nuttall, 1961a). The males are sharply
hit on the nose when they try to enter the escape chamber or platform. A third protocol
involves tethering the male to one area of the test arena and allowing the female access to
the male (Fadem et al., 1979; Krieger et al., 1976).

Although these different pacing paradigms have produced somewhat similar results,
it is still the case that return latencies for the female are considerably longer in some reports

(Bermant, 1961; Bermant and Westbrook, 1966; Krieger et al., 1976; Peirce and Nuttall,

l8
1961a) than in others (Erskine, 1985, 1989). In Experiment 1A, we investigated whether

the temporal aspects of female copulatory behavior tested in our modified pacing paradigm
were similar to or different from those reported by others. In Experiment 18, we examined
whether the postejaculatory refractory period of the female rat was a response to the male's
ejaculatory reflex or whether additional vaginocervical stimulation was necessary. Part of

this work has been presented in abstract form (Yang and Clemens, 1994).

GENERAL METHODS

Animals

Sixty-day-old female and ninety—day—old male Long Evans rats were purchased
from Charles River Laboratories (Wilmington, MA). All females were ovariectomized one
week later upon arrival. Animals were housed in 16:8 hr light-dark cycle with lights off at
17:00-1:00. In general, females were housed three per cage and males were housed singly.
Food and water were available ad lib. The temperature and humidity of the animal room
were maintained at 720 F and 55%, respectively. The air in the animal room was
exchanged 12 times per hour automatically. Ovariectomized females were brought into
estrus by intramuscular (1M) injections of 0.5 pg estradiol benzoate (EB) (Sigma Chemical
Co., St. Louis, MO) dissolved in 0.1 ml sesame oil (Sigma Chemical Co., St. Louis, M0)
for three consecutive days and 24 hours later followed by an IM injection of 0.5 mg
progesterone (P) (Sigma Chemical Co., St. Louis, MO) dissolved in 0.1 ml sesame oil.
All female pacing behaviors were tested 4-7 hours after the injection of progesterone (2-5
hours after the lights off) under the dimly red illumination. All animals were tested once
per week throughout the whole test period unless otherwise specified and had been tested at

least twice in the testing chamber before data were collected. Only those females showing

19
pacing behavior in these preliminary tests were used in the experimental studies. In each

test, a male was always placed in the testing chamber 15 minutes before a female was
introduced. The approximate weights of the female and male rats were 200 - 300 g and
400 - 500 g, respectively, when the tests started.

Pacing Tests

To provide a test situation in which the female controlled the pacing of the
copulation, females were tested in a two-compartment chamber that was divided by a
Plexiglas barrier with 4 holes (3.5~4 cm x 3.5~4 cm) spaced along the bottom of the
barrier. The dimensions of the male's chamber and escape chamber were 35.5 cm x 44.5
cm x 48.3 cm and 21.5 cm x 44.5 cm x 48.3 cm (Length x Width x Height), respectively.
The escape holes were large enough to allow the female to pass through but prevented the

larger male from following her.

Behavioral Measures

Several behavioral measures were recorded in the pacing tests unless otherwise
specified: approach latency (AL), the latency for the female to enter the male's chamber
after the start of the test (she was placed in the escape chamber at the beginning of the test);
intromission latency (IL), the latency from the female's entry into the male's chamber to the
first intromission; mount return latency (MRL), the latency for the female to re-enter the
male's chamber after a mount; intromission return latency (IRL), the latency to return to the
male's chamber following an intromission; postejaculatory refractory period (PER), the
latency to re-enter the male's chamber after an ejaculation. All latencies were measured in
seconds. The frequency with which the female left the male's chamber following a mount,

intromission and ejaculation (percentage exit) was calculated by dividing the number of

20
exits by the number of each type of stimulus. All behavioral data were recorded on a

computer disk.

EXPERIMENT 1A: THE TEMPORAL PATTERN OF FEMALE SEXUAL
BEHAVIOR TESTED IN OUR MODIFIED PACING CHAMBER

The objective of this experiment was to investigate whether the female pacing
behavior tested in our modified pacing paradigm was similar to or different from those
reported previously (Bermant, 1961; Bermant and Westbrook, 1966; Erskine, 1985, 1989;
Krieger et al., 1976; Peirce and Nuttall, 1961a). Although these previous reports of female
pacing behavior conveyed similar information, there were quantitative differences in the
return latencies following a mount, intromission and ejaculation among these reports
(Bermant, 1961; Bermant and Westbrook, 1966; Erskine, 1985, 1989; Krieger et al.,
1976; Peirce and Nuttall, 1961a). For example, the mean return latency following an
ejaculation and an intromission in the first ejaculatory series was significantly shorter in
some studies (Erskine, 1985, 1989) than in other studies (Bermant, 1961; Bermant and
Westbrook, 1966; Krieger et al., 1976; Peirce and Nuttall, 1961a). In addition, the mcan
return latency following an ejaculation increased significantly over consecutive ejaculations
in two studies (Bermant and Westbrook, 1966; Krieger et al., 1976) but it did not do so in
another study (Bermant, 1961). The present experiment was designed to examine the

features of female pacing behavior tested in our modified pacing paradigm.
METHOD

To obtain measures of normal female pacing behavior in our modified pacing
paradigm, 67 male and 67 female Long Evans rats were used in this study. Each female

was tested once with one male during this experiment. Measures of female pacing behavior

21
that included AL, IL, MRL, IRL, PER, and percentage exits following each specific sexual

contact (mount, intromission, and ejaculation) for each female were recorded over three
ejaculatory series. Mean of MRL as well as IRL and mean of percentage exits following
mounts and intromissions for each ejaculatory series were calculated for each female and
used for further analysis. MRL, IRL, PER, and percentage exits following sexual contacts
were analyzed by ANOVA for repeated measures followed by Student-Newman-Keuls
(SNK) post-hoc tests.

RESULTS

The mean latency for the female to enter the male's chamber after the start of the test
(AL) and the mean latency from the female's entry into the male's chamber to the first
intromission (IL) were 4.8 i: 0.4 seconds and 4.7 i 0.5 seconds (mean 3; SEM),
respectively. The results of the female's MRL, IRL, and PER were summarized in Figure
1. ANOVA for repeated measures revealed a significant sexual contact effect [F(2, 198) =
283.7, p<0.0001], ejaculatory series effect [F(2, 396) = 62.7, p<0.0001], as well as
sexual contact x ejaculatory series interaction [F(4, 396) = 28.5, p<0.0001]. The SNK
post-hoe tests revealed that for each ejaculatory series, the PER was significantly longer
than the MRL or IRL (p<0.01). The IRL was significantly longer than the MRL in the
second and the third ejaculatory series (p<0.01) (Figure 1). The SNK post-hoc tests
showed that the PER but not IRL or MRL increased significantly over consecutive
ejaculatory series (p<0.01) (Figure 1).

Percentage exits, the number of times the female left the male's chamber following
a mount, intromission, or ejaculation, were summarized in Figure 2. The percentage exits
following sexual contacts were significantly different among different sexual contacts [F(2,
198) = 193.3, p<0.0001], and did not differ among three ejaculatory series [F(2, 396) =

2.773, p = 0.06]. There was no significant sexual contact x ejaculatory series interaction

T0
i0

Figure l . Return latency following mounts, intromissions, and ejaculations for three
ejaculatory series (N=67). The female's return latency following an ejaculation (PER) was
significantly longer than her return latency following an intromission (IRL) and mount
(MRL) for three ejaculatory series. The IRL was significantly longer than the MRL in the
second and third ejaculatory series. The female's PER increased significantly as the
ejaculatory series increased. The female's IRL increased slightly over three ejaculatory
series but there was no significant increase between two continuous ejaculatory series.
There was no significant increase in the female's MRL for three ejaculatory series. Data
were analyzed by ANOVA for repeated measures. Bar represents mean _-r_-_ SEM.

** p<0.01.

Return latency following each
sexual contact (seconds)

120 -

é

 

E] Mount
Intromission
I Ejaculation

 

Ejaculatory Series

 

 

*
*

 

24

Figure 2 . The percentage exits following mounts, intromissions, and ejaculations for
three ejaculatory series. The percentage exit after ejaculation was significantly higher than
that following intromissions which was higher than that following mounts. There was no
significant difference among three ejaculatory series for the percentage exit after mounts,
intromissions, and ejaculations. Data were analyzed by ANOVA for repeated measures.
Bar represents mean 1'. SEM. ** p<0.01. N=67.

Percentage exit (%)

25

[3 Mount
Intromission
I Ejaculation

*
*

*4-
*4-

 

 

El E2 E3

Ejaculatory series

Figure 3 . Relation of the number of intromissions prior to ejaculation and the female's
return latency following an ejaculation (PER) for three ejaculatory series. There was a
positive correlation between the number of intromissions received by the female before
ejaculation over three ejaculatory series and the female's PER. Data were analyzed by
Correlation analysis (r=0.425, p<0.0001). N=201.

Return latency following an
ejaculation (PER)(seconds)

350

300-

250

200
150

100

27

 

 

 

 

 

 

 

r=0.425 °
p<0.0001
N=201
° 000 o Y=26.94+2.42X
O o a
00 9 c
0080000 0 o o
O O
O
%
0800 0°
5 Q

5101520253035404550

Number of intromissions prior to ejaculation

28
effect [F(4, 396) = 1.875, p = 0.114] in the percentage exits following sexual contacts.

The SNK post-hoc tests showed that for three ejaculatory series, the percentage exit
following ejaculations was significantly higher than that following intromissions (p<0.01)
and the percentage exit following intromissions was significantly higher than that following
mounts (p<0.01) (Figure 2). There was no significant difference among 3 ejaculatory
series for the percentage exits following mounts, intromissions and ejaculations (Figure 2).
Correlation analysis did not show a significant relation between the number of
intromissions received by the female prior to the first ejaculation and the female's PER
(r=0.151, p=0.225). However, there was a significant positive correlation between the
number of intromissions received by the female before ejaculation over three ejaculatory

series and the female's PER (r = 0.425, p<0.0001) (Figure 3).

EXPERIMENT 1B: EFFECT OF DIFFERENT NUMBERS OF
INTROMISSIONS ON THE FEMALE TEMPORAL COPULATORY
BEHAVIOR

The objective of this experiment was to investigate whether different numbers of
intromissions received by the female rat preceding ejaculation affected her PER or whether
the ejaculatory reflex, seminal emission and the male's postejaculatory behavior were, in
and of themselves, sufficient to induce a PER comparable to that seen after an ejaculation
during normal copulation. While it has been reported that the female's PER is much longer
than her MRL and IRL (Bermant, 1961; Bermant and Westbrook, 1966; Erskine, 1985,
1989; Krieger et al., 1976; Peirce and Nuttall, 1961a; Yang and Clemens, 1994, 1995a),
the factors that contribute to this difference have not been elucidated. In this experiment,
we examined the effect of different numbers of intromissions received by the female prior

to ejaculation on her PER.

29
METHOD

In separate biweekly tests, females were allowed to receive different numbers of
intromissions before the male ejaculated: 0—1 intromission (0-1 I Group), 2-4 intromissions
(2-4 I Group), 515 intromissions (5—15 I Group), and 24-31 intromissions (24-31 I
Group). Each female was primed with EB and P once per week and randomly assigned to
test with one male at the beginning of the experiment. Because each female received an
ejaculation from the same male she mated at the beginning of the experiment throughout the
entire test duration, we referred to "each female with the male providing ejaculation during
copulation” as ”female and male partners.” Eighteen females and 9 males (27 additional
stud males were used in the 24—31 intromission group to provide intromissions before the
female was allowed to copulate with her "male partner") were used in this study. Each
male had two "female partners” and was tested with one of two female partners on alternate
weeks.

To obtain baseline measures of normal female pacing behavior (5-15 I Group), 9
females were tested with their "male partners" for one full ejaculatory series in the first
week and another 9 females were tested in the second week. The ejaculation latency and
intromission frequency of the males that were obtained in these tests were used to estimate
when the male was close to ejaculation for the low intromission groups (01 I Group and 2-
41 Group). During the period of week 3 to week 6, the low number of intromissions in
the 0-1 I Group and 2-4 I Group were achieved by pretesting the male with one "female
partner" then replacing her with the experimental female, ”the second female partner,”
when it was estimated that the male was close to ejaculation. During week 7 and 8, the
higher number of intromissions .in 24—31 I Group was achieved by pretesting the female
with 3 stud males, each delivering 6 intromissions without ejaculation, then allowing her to
copulate with her ”male partner" until he ejaculated. Only the PER and IRL of those

females receiving all four different numbers of intromissions prior to ejaculation (0—1 1, 2-4

30
1, 5-151 and 24—31 I) were used for analysis. Mean of IRL was calculated for each female

and used for further analysis. PER and IRL data were analde by ANOVA for repeated
measures followed by Student-Newman-Keuls (SNK) post-hoe tests, respectively.
Comparison of IRL and PER within each group except 0-1 I Group was achieved by paired
t test. In addition, the relation of the number of intromissions prior to ejaculation in the

four different situations to the f emale's PER was analyzed by Correlation analysis.

RESULTS

The mean return latencies following an intromission or ejaculation of females
receiving different numbers of intromissions prior to ejaculation were summarized in
Figure 4. Eleven of 18 females experienced all four intromission conditions preceding
ejaculation during the eight—week experiment. Because only three animals had IRL in the
0—1 I Group due to no intromission or no leave after an intromission, the IRL data of 0—1 I
Group were excluded for IRL analysis. In addition, three animals that did not have IRL in
241 Group due to no exit after an intromission were excluded for IRL analysis. All the
data were represented as mean :1; SEM.

ANOVA for repeated measures showed that there was a significant difference
among the PERs of the four different groups [F(3, 30) = 20.454, p<0.0001]. Student-
Newman-Keuls (SNK) post-hoe tests revealed that the PER of 0-1 I Group was
significantly shorter (p<0.01) than the other groups and that the PER of 24—31 I Group was
significantly longer than that of 5-15 I Group (p<0.05), 2-41 Group (p<0.01) and 0-1 I
Group (p<0.01). However, the PER of females receiving 2-4 intromissions was not
different from that of 5-15 I Group. The paired t test indicated that the female’s IRL was
significantly shorter than her PER in 2-4 I (p<0.01), 5—15 I (p<0.0001) and 24—31 I
(p<0.0001) Groups (Figure 4). The t test showed that there was no difference between the

PER of females receiving 01 intromission and the IRL of females receiving 5-15

31

Figure 4 . Effect of intromissions on female's latency to return to the male following an
ejaculation (PER, open bar, N=ll) and following an intromission without ejaculation
(IRL, solid bar, N=8). ** Return latency after an ejaculation was significantly shorter for
0—1 intromission group than the other groups (p<0.01). * Return latency after an
ejaculation was significantly longer for the 24-31 intromission group than the 5-15
intromission group (p<0.05). # The PER of females receiving 24—31 intromissions was
significantly longer than those receiving 0-1 or 2-4 intromissions (p<0.01). $ The female’s
IRL was significantly shorter than her PER in 2-4, 5-15 and 24—31 intromission groups
(p<0.01, paired t test). There was no significant difference between the female’s retum
latencies following an ejaculation of 2-4 and 5—15 intromission groups. There was no
significant difference among the female's mean return latencies following an intromission
of the different intromission groups. All data except comparison of IRL and PER within
the same group were analyzed by ANOVA for repeated measures followed by SNK post-
hoc tests. Bar represents mean i SEM.

Return latency in seconds

32

80-

at.

70-1

 

60.

50; I

 

40; j

 

30-

20-

au-

 

 

 

 

10- :-

 

 

 

 

 

 

 

 

O- 1 2-4 5- 15 24—3 1

Number of intromissions preceding ejaculation

33

Figure 5 . The relation between the number of intromissions preceding ejaculation and the
female's return latency following an ejaculation (PER). The number of intromissions
received by the female prior to ejaculation in four groups was positively correlated with her
PER. Data were analyzed by Correlation analysis (r=0.613, p<0.0001). N=44.

Return latency following cj aculation

(seconds)

120

100

 

-5

i/‘ 080 8

34

r: 0.613
p <0.0001 o
N = 44 o
o
o
8 o

  

Y=22.50+1.60X
00 o

 

Number of intromissions preceding ejaculation

35
intromissions [t=-0.926, p=0.366] (Figure 4). ANOVA for repeated measures showed

that there was no significant difference among IRLs of three different groups (2-4, 5-15,
and 24-31 I Groups) [F(2, 14) = 2.163, p=0.152] (Figure 4).

Figure 5 showed the relation of the number of intromissions (0-1, 2-4, 5-15 and
24—31) received by the females prior to ejaculation to the female's PER. Correlation
analysis revealed that the number of intromissions received by the females prior to

ejaculation was positively correlated with her PER (r = 0.613, p<0.0001).

DISCUSSION

In rats, the temporal pattern of female sexual behavior has physiological
significance. The progestational state of pregnancy in female rats is more readily induced
when the female controls the copulatory speed. When the female controls the timing of
copulation, the interval between intromissions is longer (Erskine, 1985) and fewer
intromissions are sufficient to induce the progestational state of pregnancy (Edmonds et a]. ,
1972; Erskine et al., 1989; Gilman et al., 1979). In female paced tests (Erskine et al.,
1989; Gilman et al., 1979) or enforced long interintromission interval (Edmonds et al.,
1972), five intromissions are sufficient to trigger the progestational state of pregnancy in
the female rats. However, five intromissions were insufficient to induce pseudopregnancy
(Chester and Zucker, 1970; Edmonds et al., 1972; Erskine et al., 1989; Gilman et al.,
1979) or pregnancy (Erskine et al., 1989) in non-paced female rats. Approximately, 10
intromissions are capable of inducing the progestational state of pregnancy in non-paced
females (Edmonds et al., 1972; Erskine et al., 1989; Gilman et al., 1979).

In the present study, results show that the female's return latency following an
ejaculation (PER) was much longer than her return latency following an intromission (IRL)
or mount (MRL) in each of three ejaculatory series. The return latency following an

intromission (IRL) was significantly longer than the MRL in the second and third

36
ejaculatory series. In general, these results are consistent with previous findings that have

been reported using a variety of pacing test paradigms to measure female pacing behavior
(Bermant, 1961; Bermant and Westbrook, 1966; Erskine, 1985, 1989; Krieger et al.,
1976; Peirce and Nuttall, 1961a). The PER, IRL, and MRL of our results are quite similar
to those of some studies (Erskine, 1985, 1989) but are shorter than those reported by other
research groups (Bermant, 1961; Bermant and Westbrook, 1966; Krieger et al., 1976;
Peirce and Nuttall, 1961a). The MRL in our study did not increase over three ejaculatory
series. This is consistent with the results of two studies (Bermant, 1961; Bermant and
Westbrook, 1966) but different from those reported in another (Krieger et al., 1976). Most
likely these differences in return latencies following specific sexual contacts reflect
differences in the designs of test chambers as well as differences in hormone replacement
protocols. For example, in some studies (Bermant, 1961; Bermant and Westbrook, 1966),
the females were trained to press a lever to obtain access to a male. In this complex test
situation that involves learning, it takes longer for the female to respond after each sexual
contact than in the simpler paradigm used here. In addition, different hormone replacement
protocols are used. In the present study, females receive low doses of EB (0.5 pg) for
three consecutive days and 24 hours later followed by an injection of 0.5 mg progesterone.
Other investigators often give a single injection of 50 or 100 ug EB 48 or 72 hrs prior to an
injection of 0.5 or 1 mg progesterone. Four to eight hours later after the injection of
progesterone, the females were tested for sexual behavior. These differences in the
hormone replacement protocols may contribute to differences in the female's return
latencies.

That the female's IRL is longer than her MRL most likely reflects the occurrence of
penile insertion during an intromission but not during a mount (and/or the summation of
intromission and mounting stimuli during an intromission). However, why the female's
PER is much longer than her IRL is still not clear. Although it has been reported that the

deposit of a seminal plug during ejaculation lengthened the female's PER, the effect of a

37
seminal plug on the female's PER was minimal in that study (Bermant and Westbrook,

1966). The authors suggested that the difference between PER and IRL might result from
the differences between an ejaculation and an intromission including the differences
between the contact durations, the numbers of deep thrusts, and the degree of tightness of
the male's grasp during an ejaculation and an intromission (Bermant and Westbrook,
1966). It is also the case that the male rat emits an ultrasonic (22 kHz) vocalization after an
ejaculation (Barfield and Greyer, 1972; Barfield and Greyer, 1975). Such vocalization
might also contribute to the female's PER. However, the results in Experiment 18
demonstrate that the male's ejaculatory and postejaculatory behavior alone without at least
two preceding intromissions are not sufficient to induce a PER comparable to that seen after
an ejaculation during normal copulation.

In Experiment 1A, we found that the female's PER increased with consecutive
ejaculations. This finding is consistent with some previous reports (Bermant and
Westbrook, 1966; Krieger et al., 1976). In one study, the authors suggested that the
increase in return latencies over consecutive ejaculatory series indicated a decline in the
female's receptivity (Krieger et al., 1976). However, other measures of receptivity did not
change. For example, lordosis quotients ( 100%) remained constant over the 3 ejaculatory
series in their study (Krieger et al., 1976). We also did not observe any lordosis change
over three ejaculatory series in our study. In addition, the MRL in the present study did not
increase over the course of three ejaculations. If the receptivity of the female was
decreasing, we would have expected to see the MRL increase. Given the correlation
between intromission frequency (IF) and PER, it may be more likely that the increase in the
PER during consecutive ejaculatory series results from an increase in the ejaculation
duration which occurs in these cases (Peirce and Nuttall, 1961b). The increase in
ejaculation duration would provide stronger vaginocervical stimulation which might result
in a longer return latency following an ejaculation.

The female's PER was positively correlated with the number of intromissions she

38
received prior to ejaculation but this was only seen when there was a relatively large range

of intromissions (0-31 in this study). In Experiment 1A, the female's PER obtained from
the first ejaculatory series of normal copulation was not correlated with the number of
intromissions she received. In Experiment 1B, however, when females received a large
range of intromissions (0-31), the female's PER was significantly correlated with
intromission frequency before the first ejaculation. In addition, in Experiment 1A, the
female’s PER over three ejaculatory series was positively correlated with the number of
intromissions received by the female prior to ejaculation.

Following at least two preejaculatory intromissions, the ejaculatory reflex itself
provides a more effective stimulus than an intromission in inducing a longer return latency.
The stimulus of the ejaculatory reflex itself becomes more effective as the female receives
more intromissions prior to ejaculation. This finding is supported by the evidence that the
female’s PER is significantly longer than her IRL in all groups except 0-1 intromission
group. In addition, the female’s PER in the 24—31 intromission group is significantly
longer than that in the 2-4 and 5-15 intromission groups while there is no difference among
the female’s IRLs of these three (2-4, 5-15, and 24—31 1) groups (Figure 4). These results
also imply that in addition to the preceding intromissions, some other factors play an
important role in the increasing effectiveness of an ejaculatory reflex in lengthening the
female’s PER. Evidence shows that the male and female contact duration during
ejaculation (“ejaculation duration”) is positively correlated with the preejaculatory
intromission frequency and the female’s PER (Yang and Clemens, 1995b). That is, the
f emale’s PER is directly influenced by the “ejaculation duration” and indirectly regulated by
the preejaculatory intromissions.

Multiple intromissions prior to ejaculation in male rats may have several functions.
It has been proposed that one function of multiple intromissions is to induce the secretion
of progesterone to facilitate blastocyst implantation (Beach, 1965). One group of

researchers extended this hypothesis and postulated that vaginocervical stimulation

39
provided by the multiple intromissions induced the release of pituitary prolactin which in

turn resulted in the secretion of progesterone (Wilson et a1, 1965). This hypothesis was
supported by numerous subsequent reports (Adler et al., 1970; Gorospe and Freeman,
1981; Hashimoto etal., 1968; Smith etal., 1975; Terkel and Sawyer, 1978). In addition,
two groups of investigators proposed that another function of multiple intromissions prior
to ejaculation in male rats was to facilitate the sperm transport through the cervix of the
uterus (Adler, 1969; Chester and Zucker, 1970). This is supported by the finding that no
or very few sperm were present in the uterus 1-3 hours after ejaculation and no developing
eggs were found in the uterus 2-4 days after copulation if the male ejaculated on the first or
second intromission (Adler, 1969). Females receiving 2 or more intromissions prior to
ejaculation contained a large number of sperm in their uteri 1-3 hours after ejaculation and
had developing eggs in their Fallopian tubes and uteri 2-4 days after copulation. Similar
results were also reported by other researchers (Chester and Zucker, 1970). Further,
multiple intromissions of male rats prior to ejaculation may serve to remove seminal plugs
deposited by other competitors under naturalistic situations.

Another potential function of multiple intromissions in male rats may be to prepare
the vaginal passage for the deep penile insertion during ejaculation. At least two
consequences result from the deep penile insertion during ejaculation. The first
consequence is a longer penis-vagina contact duration. It has been reported that an
ejaculation duration is longer than an intromission duration (Erskine et al., 1989; Peirce and
Nuttall, 1961b; Stone and Ferguson, 1940). Further, in Experiment 1B, we found that the
ejaculation duration of males was shorter in the 0—1 intromission group than the other
groups. This shorter ejaculation duration may reflect the possibility that initial
intromissions are relatively shallow. In some cases, the male even ejaculated outside the
female. A second consequence resulting from the deep penile insertion during ejaculation
is a tight deposit of a seminal plug against cervix that, in turn, facilitates sperm transport

from the cervix to the uterus. If the male ejaculates on the first or second intromission, the

40
lack of deep penile penetration may compromise the deposition of a seminal plug against

the cervix with the result that very few or no sperm will be transported to the uterus.
Several studies support this idea (Adler, 1969; Chester and Zucker, 1970; Matthews and
Adler, 1977; Matthews and Adler, 1978). In addition to the studies that examined the
relation of number of intromissions before ejaculation to the sperm transport from the
cervix to the uterus (Adler, 1969; Chester and Zucker, 1970), two other studies
investigated the relation between the position of a seminal plug in the vagina and the
number of sperm transported to the uterus (Matthews and Adler, 1977; Matthews and
Adler, 1978). The authors found that if the seminal plug sealed the cervix tightly, sperm
were transported to the uterus in large numbers. Thus, while multiple intromissions are
correlated with increased sperm transport, a second more proximal mechanism may be the
deeper prolonged intromission that accompanies ejaculation and is itself a result of
preceding intromissions.

In summary, different numbers of intromissions, preceding ejaculation, affect the
female's PER. The number of intromissions received by the female preceding ejaculation is
positively correlated with the female's PER if the range of intromission frequency is large
enough (0-31 in this study). At least two preejaculatory intromissions are required to
induce a PER comparable to the one following the first ejaculation during normal
copulation. Additional intromissions received by the female prior to ejaculation may
lengthen the “ejaculation duration” (the male and female contact duration during ejaculation)
that in turn increases the length of the female’s PER. One potential function of multiple
intromissions in male rats during copulation may be to prepare the vaginal passage for the
deep penile insertion that in turn results in a longer penis-vagina contact duration and a tight
deposit of a seminal plug against the cervix of the uterus. This type of stimulation provided
by deep penile insertion facilitates sperm transport through the cervix of the uterus and

brings about the f emale’s postejaculatory refractory period.

EXPERIMENT 2: FUNCTION OF INTROMISSIONS IN RELATION TO
THE INTROMISSION RETURN LATENCY OF FEMALE RATS
DURING PACED SEXUAL BEHAVIOR

ABSTRACT

YANG, L. Y. AND L. G. CLEMENS. Function of intromissions in relation to the
intromission return latency of female rats during paced sexual behavior. PHYSIOL
BEHAV 60(0):000-000, 1996.- The objectives of this study were to examine how multiple
intromissions affect the temporal pattern of the female rat’s copulatory behavior, in
particular, her latency to return to the male following intromission (intromission return
latency, IRL) and whether different hormone replacement regimens affect the temporal
aspects of female copulatory behavior. Repeated intromissions alone, without ejaculation,
often resulted in prolonged IRLs equal to the postejaculatory refractory period (PER). The
first prolonged IRL occurred most frequently between the 24th and 44th intromission. The
similar pattern of IRLs around the PER and the prolonged IRLs may indicate that the
mechanisms mediating the occurrence of the prolonged IRL are similar to those for the
PER. One possible function of the prolonged IRLs may be to facilitate the male’s
ejaculation after the female has received enough vaginocervical stimulation for the induction
of the progestational state of pregnancy. Finally, females receiving a single dose of 50 pg
estradiol benzoate (EB) followed by an injection of 0.5 mg progesterone (P) 48 hours later
showed a significantly longer PER than those receiving three daily injections of 0.5 pg EB

followed by an injection of 0.5 mg P 24 hours after the last EB injection.

41

42
INTRODUCTION

One function of the temporal pattern of female sexual behavior in the rat is to
facilitate successful pregnancy. Following multiple intromissions, blood progesterone
levels increase significantly in the female to maintain pregnancy (Adler et al., 1970). When
the female is able to control the copulatory speed, the interintromission intervals are longer
(Erskine, 1985; Erskine et al., 1989; Fadem and Barfield, 1982; Gilman et al., 1979) and
fewer intromissions are sufficient to induce the progestational state of pregnancy (Erskine
et al., 1989; Gilman et al., 1979). Following ejaculation, the female rat, if given the
Opportunity, stays away from the male for a longer period of time than she does after an
intromission without ejaculation (Bermant, 1961; Bermant and Westbrook, 1966; Erskine,
1985, 1989; Fadem et al., 1979; Gilman and Hitt, 1978; Krieger et al., 1976; Peirce and
Nuttall, 1961; Yang and Clemens, 1994, 1996a). This period of female sexual inactivity
following ejaculation is referred to as the female’s postejaculatory refractory period (PER),
and its function is related to sperm transport: if the female receives an intromission within 2
minutes following ejaculation, sperm transport will be halted until the next ejaculation
(Matthews and Adler, 1977).

In previous studies, we found that multiple intromissions preceding ejaculation
affected the male’s “ejaculation duration” (the duration of male-female contact during
ejaculation) which in turn influenced the length of the female’s postejaculatory refractory
period (Yang and Clemens, 1995b, 1996a, 1996b). While two preejaculatory
intromissions were sufficient for a normal PER to occur, additional intromissions
lengthened the “ejaculation duration” that, in turn, was associated with an increase in the
female’s PER (Yang and Clemens, 1995b, 1996a, 1996b).

In the present study, we extended this analysis of intromission effects to determine
how multiple intromissions affect the pattern of the female’s individual IRLs over the

course of copulation. In female paced situations, it has been observed that if a long IRL

43
occurs during the course of the third ejaculation, the male often ejaculates on the next

intromission following this longer IRL (Personal observation). Moreover, experimentally
enforced long interintromission intervals have been demonstrated to significantly reduce the
number of intromissions prior to the first ejaculation in male rats (Bermant, 1964; Larsson,
1959). We suggest that, in addition to contributing to the induction of the progestational
state of pregnancy, the female’s timing of intromissions affects the probability of the male’s
ejaculation.

The temporal pattern of female sexual behavior is also affected by gonadal
hormones (Fadem et al., 1979; Gilman and Hitt, 1978). Two hormone replacement
regimens [0.5 pg estradiol benzoate (EB) x 3 + 0.5 mg progesterone (P) 24 hrs after the
last EB injection and 50 or 100 pg EB + 0.5 or 1 mg P 48 or 72 hrs after EB injection] are
often used to study the temporal pattern of female sexual behavior (Bermant, 1961;
Bermant and Westbrook, 1966; Clemens et al., 1995; Krieger et al., 1976; Yang and
Clemens, 1994, 1995a, 1995b, 1996a, 1996b). The general patterning of female sexual
behavior was similar in these reports using these two hormone replacement protocols.
However, the return latencies reported by us (Yang and Clemens, 1996a) were routinely
shorter than those reported by others (Bermant, 1961; Bermant and Westbrook, 1966;
Krieger et al., 1976). For example, in our study the female's return latency following an
ejaculation (Yang and Clemens, 1996a) was 47 seconds, which is significantly shorter than
90 seconds to several minutes reported by others (Bermant, 1961; Bermant and
Westbrook, 1966; Krieger et al., 1976). Whether this reflects the difference in hormone
replacement regimens, or different test chambers is not clear.

In Experiment 2A, we examined how multiple intromissions affect the temporal
pattern of the female rat’s copulatory behavior, in particular, her latency to return to the
male following intromission. Experiment 2B investigated whether different hormone
replacement regimens affect the temporal aspects of female copulatory behavior. Part of

this work has been presented in abstract form (Yang and Clemens, 1994).

44
GENERAL METHODS

Animals

Sixty—day-old female and ninety-day-old male Long Evans rats were purchased
from Charles River Laboratories (Wilmington, MA). All females were ovariectomized one
week after arrival in the laboratory. Animals were housed in 16:8 hr light-dark cycle with
lights off at 17:00-1:00. In general, females were housed three per cage and males were
housed singly. Food and water were available ad lib. Temperature and humidity of the
animal room were maintained at 220 C and 55%, respectively. The air in the animal room
was exchanged 12 times per hour automatically. Ovariectomized females were brought into
estrus by intramuscular (IM) injections of 0.5 pg estradiol benzoate (EB) (Sigma Chemical
Co., St. Louis, MO) dissolved in 0.1 ml sesame oil (Sigma Chemical Co., St. Louis, M0)
for three consecutive days and 24 hours after the last EB injection followed by an IM
injection of 0.5 mg progesterone (Sigma Chemical Co., St. Louis, MO) dissolved in 0.1
ml sesame oil unless otherwise specified. All female pacing behavior was tested 4—7 hours
after the injection of progesterone (2-5 hours after the lights off) under dimly red
illumination. All behavioral tests were ended after the female returned to the male’s
chamber following her PER. Alternatively, the behavioral tests were terminated if the
female did not leave the male’s chamber within 60 seconds following ejaculation. All
animals were tested once per week and had been tested twice in the testing chamber before
data were collected. Males were always placed in the testing chamber 15 minutes before
females were introduced (the test started). The approximate weights of the female and male
rats were 200 - 300 g and 400 - 500 g, respectively, when the tests started.

45
Pacing Test Chamber

To provide a test situation in which the female could control the pacing of the
copulation, females were tested in a two-compartment chamber that was divided by a
Plexiglas barrier with 4 holes (3.5~4 cm x 3.5~4 cm) spaced along the bottom of the
barrier. The dimensions of the male's chamber and escape chamber were 35.5 cm x 44.5
cm x 48.3 cm and 21.5 cm x 44.5 cm x 48.3 cm (Length x Width x Height), respectively.
The escape holes were large enough to allow the female to pass through but prevented the

larger male from following her.

Behavioral Measures

Several behavioral measures were recorded in the pacing tests unless otherwise
specified: approach latency (AL), the latency for the female to enter the male's chamber
after the start of the test (she was placed in the escape chamber at the beginning of the test);
intromission latency (IL), the latency from the female's entry into the male's chamber to the
first intromission; mount return latency (MRL), the latency for the female to re-enter the
male's chamber after a mount; intromission return latency (IRL), the latency to return to the
male's chamber following an intromission; postejaculatory refractory period (PER), the
latency to re-enter the male's chamber after an ejaculation. All latencies were measured in
seconds. The frequency with which the female left the male's chamber following a mount,
intromission, or ejaculation (percentage exit) was calculated by dividing the number of exits
by the number of each type of stimulus. All these behavioral data were recorded on a

computer disk.

46
EXPERIMENT 2A: RELATION OF MULTIPLE INTROMISSIONS TO THE

FEMALE’S INDIVIDUAL INTROMISSION RETURN LATENCY

In previous studies (Yang and Clemens, 1995b, 1996a, 1996b), we demonstrated
that intromissions prior to ejaculation indirectly increased the female’s PER by increasing
the male’s “ejaculation duration” (the duration of male-female contact during ejaculation).
In this experiment, we extended our testing to determine (1) how multiple intromissions
preceding ejaculation affect the pattern of the female’s individual IRLs over three
ejaculatory series, (2) how the female paces her sexual contacts with the male while
receiving a large number of intromissions prior to ejaculation, and (3) whether repeated
intromissions alone, without ejaculation, can induce a prolonged intromission return

latency (prolonged IRL) that is comparable to the PER in length.

METHOD

Procedures

Females were given 2 or 3 tests at weekly intervals. Eleven males and eleven
females plus 30 extra stud males (stud males used to deliver intromissions without
ejaculation during the second and third tests) were used in this study. All females were
tested once per week and had 2 complete pretests before data were collected.

A prolonged female’s return latency following an intromission (prolonged
intromission return latency, prolonged IRL) was defined as a return latency following an
intromission that was equal to or larger than that female's PER following the first
ejaculation during normal copulation. Alternatively, a return latency following an

intromission equal to or larger than 47 seconds (mean PER of 67 females following the

47
first ejaculation during normal copulation under equivalent test conditions, Yang and

Clemens, 1996a) was also regarded as a prolonged IRL only when the female’s PER
following the first ejaculation was larger than 60 seconds and no IRL was equal to or larger
than the female’s first PER.

To obtain baseline behavioral measures of normal female pacing behavior, each of
eleven females was randomly assigned to copulate with one of 11 males for three
ejaculatory series in the first week of this experiment. Because each female received an
ejaculation from the same male she mated in the first week throughout the entire test
duration, we referred to ”each female with the male providing ejaculation during
copulation" as "female and male partners". In a second test, one week later, after each
female received 40 intromissions provided by 6 or 7 stud males, she was allowed to
copulate with her ”male partner" until she received an ejaculation and returned to the male’s
chamber following her PER. Females that did not show a prolonged I RL in the second test
were tested in the third week. After each of these females received 70 intromissions
provided by 12 to 15 stud males, she was allowed to mate with her "male partner" until she
received an ejaculation and returned to the male’s chamber following her PER. Each
individual IRL instead of mean IRL of each female was recorded and used for analysis. In
addition, the number of intromissions and the latency for the first prolonged IRL to occur

during the second and third tests were recorded.

Statistics

Comparison of individual IRLs and PERs over three ejaculatory series during
normal copulation was made by one way ANOVA analysis followed by Fisher’s PLSD
post-hoc tests. Comparison of individual IRLs and PER with the first prolonged IRL
during the second and third tests was made by one-way ANOVA analysis followed by
Fisher's PLSD post-hoe tests. An alpha level of 0.05 was used for all statistical tests.

48
Ideally, we would have used one way Repeated Measures ANOVA to analyze the

individual IRLs and PERs. However, the fact that the female only left the male’s chamber
about 70% of times following intromission (Yang and Clemens, 1996a) resulted in a large
number of missing values that made the use of one way Repeated Measures ANOVA

impossible.

RESULTS

Figure 6 shows the general pattern of IRLs over three ejaculatory series obtained
from 11 females in the first week. The number of intromissions required to achieve
ejaculations varies with each animal. To effectively present these individual IRLs, they
were divided into four sets of continuous IRLS and aligned by the first intromission and
then by each of the three ejaculations. These four sets of continuous IRLs include: (1) the
first four IRLs (1, 2, 3, 4), (2) the four IRLs right before and three IRLs right after the first
ejaculation (L—3, L-2, L—l, L, E1, 1, 2, 3), (3) the three IRLs right before and two IRLs
right after the second ejaculation (L-2, L-l, L, E2, 1, 2), and (4) the three IRLs right
before the third ejaculation (L-2, L-l, L, F3) (Figure 1). L stands for the intromission
right before ejaculation. E1, E2, and E3 represent the first, second, and third ejaculation,
respectively. There was a significant difference among these IRLs and PERs [F(21, 148)
= 11.468, p<0.0001]. The female’s PER was significantly longer than any IRL in the
same ejaculatory series (** p<0.01). All of these IRLs were significantly shorter than any
of these three PERs (p<0.05). The female’s PER increased significantly over three
ejaculatory series (#p<0.05). Although there was a slight increase in the female’s
individual IRLs over three ejaculatory series, this increase was not statistically significant.
Data are mean of individual IRL or PER i SEM and analyzed by one-way ANOVA
analysis followed by Fisher's PLSD post-hoc tests.

49
Ten of 11 females tested in the second and third experimental tests showed at least

one prolonged IRL. Six of these 10 females had multiple prolonged IRLs. In the second
test, eight of 11 females showed at least one prolonged IRL. The three females that did not
show a prolonged IRL were tested in the third test. Eventually, two of them showed a
prolonged IRL. Seven of 10 prolonged IRLS (the first prolonged IRL) were defined under
the criterion: equal to or larger than that female’s PER following the first ejaculation during
normal copulation. The remaining three prolonged IRLS (the first prolonged IRL) were
defined under the criterion: equal to or greater than 47 seconds.

The first prolonged IRL occurred most frequently between the 24th and 44th
intromission (7 out of 10 females). Most of the first prolonged IRLs were achieved
between 12-16 min. after the first intromission (7 out of 10 females). Figure 7 shows the
female’s IRLs at the beginning of the test, around the prolonged IRLs and at the end of the
test in which the female received a large number of intromissions prior to ejaculation, and
showed at least one prolonged IRL. The number of intromissions to achieve the first
prolonged IRL varies with each animal. To effectively present these individual IRLs, the
data points in Figure 7 were aligned by the first IRL, by the first prolonged IRL (N: the
intromission inducing the first prolonged IRL), and by ejaculation (E). Figure 7 includes
three sets of continuous IRLs: (1) the first three IRLs (1, 2, 3), (2) two IRLs right before
and two IRLs right after the first prolonged IRL (N-2, N-l, N, N+1, N+2), and (3) three
IRLs right before ejaculation (L-2, L-l, L, E). N, L, and E represent the intromission
inducing the first prolonged IRL, the intromission right before ejaculation, and the
ejaculation, respectively. Although the IRL increased slightly as the number of
intromissions increased, this was not a statistically significant change. The first prolonged
IRL was significantly longer than the first three IRLS as well as the IRLs just before and
after it but significantly shorter than the PER (** p<0.01, * p<0.05 compared with the first
prolonged IRL). Data are mean individual IRL or PER i SEM and analyzed by one-way

Figure 6 . Four representative portions of individual intromission return latencies (IRLs)
and three PERs of females receiving three ejaculations are illustrated: (i) the first 4 IRLS (1,
2, 3, 4), (ii) 4 IRLS right before and 3 IRLS right after the 1st ejaculation (L-3, L-2, L-1,
L, E1, 1, 2, 3), (iii) 3 IRLs right before and 2 IRLs right after the 2nd ejaculation (L-2, L-
1, L, E2, 1, 2), and (iv) 3 IRLs right before the 3rd ejaculation (L—2, L-l, L, E3). Data are
mean 1'. SEM and analyzed by one-way ANOVA followed by Fisher's PLSD post-hoc
tests. L: the intromission right before ejaculation. E1, E2, and E3 stand for the 1st, 2nd
and 3rd ejaculation, respectively. ** p<0.01, each individual IRL compared with PER in
the same ejaculatory series; #p<0.05, comparison between adjacent PERs. The number of
animals for each intromission or ejaculation is indicated in the same sequential order as in
Figure 6:5, 9, 7, 7, 8, 8, 7, 9, 11 (E1), 6, 5, 5, 7, 9, 10, 11 (E2), 2, 5, 8, 9, 11, 11 (E3).
// indicates separation of 4 sets of continuous IRLs.

Return latency following an intromission
or an ejaculation (seconds)

100-
90:
80-
70;
60:
50.:
40:
3041
20.
10;

 

W

I

l
Ii

51

*-

II II

II I I I I I I I1" I I I I IIII I r I

H

damegganfiaumgjinfilaugfinifl

Sequential order of intromission
over three ejaculatory series

Figure 6 . Four representative portions of individual intromission return latencies (IRLs)
and three PERs of females receiving three ejaculations are illustrated: (i) the first 4 IRLs ( 1,
2, 3, 4), (ii) 4 IRLs right before and 3 IRLs right after the lst ejaculation (L—3, L-2, L-l,
L, E1, 1, 2, 3), (iii) 3 IRLs right before and 2 IRLs right after the 2nd ejaculation (L-2, L-
1, L, E2, 1, 2), and (iv) 3 IRLs right before the 3rd ejaculation (L-2, L-l, L, E3). Data are
mean i SEM and analyzed by one-way ANOVA followed by Fisher's PLSD post-hoc
tests. L: the intromission right before ejaculation. E1, E2, and E3 stand for the lst, 2nd
and 3rd ejaculation, respectively. ** p<0.01, each individual IRL compared with PER in
the same ejaculatory series; #p<0.05, comparison between adjacent PERs. The number of
animals for each intromission or ejaculation is indicated in the same sequential order as in
Figure 6:5, 9, 7, 7, 8, 8, 7, 9, 11 (E1), 6, 5, 5, 7, 9, 10, 11 (E2), 2, 5, 8, 9, 11, 11 (E3).
// indicates separation of 4 sets of continuous lRLs.

Return latency following an intromission
or an ejaculation (seconds)

100-

5‘88
Inlgl

60-.
so:
40-
3o;
20:
10:

 

51

*F-t

Jl Il IL

mNH Div-4 Nq—r

Sequential order of intromission
over three ejaculatory series

Figure 7 . Three representative portions of individual intromission return latencies (IRLs)
of females receiving a large number of intromissions before ejaculation. These include 3
portions of consecutive IRLs: (i) the first 3 IRLs (l, 2, 3), (ii) 2 IRLs right before and 2
IRLs right after the first prolonged IRL (N-2, N-l, N, N+l, N+2), and (iii) 3 IRLs right
before ejaculation (L-2, L-l, L, E). Data are mean -_r-_ SEM and analyzed by one-way
ANOVA followed by Fisher's PLSD post-hoc tests. N: the intromission inducing the first
prolonged IRL. L: the intromission right before ejaculation. E ejaculation. ** p<0.01, *
p<0.05 compared with the first prolonged IRL. The number of animals for each
intromission or ejaculationis indicated in the same sequential order as in Figure 7: 6, 6, 8,
8, 8, 10 (N), 6, 6, 6, 7, 10, 9 (E). // indicates separation of 3 sets of continuous IRLs.

Return latency following an intromission

or an ejaculation (seconds)

E

120

§

N
O

O

888

 

53

I' [I I f I II 'I

1 2 3 N-2N-1 N N+1N+2 L—2 L-l L E

Sequential order of intromission

54
ANOVA analysis [ F(11, 78) = 7.338, p<0.0001] followed by Fisher's PLSD post—hoe

test.

EXPERIMENT 2B: INFLUENCE OF DIFFERENT HORMONE
REPLACEMENTS ON THE FEMALE PACING BEHAVIOR

The objective of this experiment was to investigate whether different hormone
replacement regimens affect the temporal aspects of female sexual behavior tested under the
same test paradigm. The return latencies following a mount, intromission and ejaculation
reported by us (Yang and Clemens, 1996a) were shorter than those reported by the other
investigators (Bermant, 1961; Bermant and Westbrook, 1966; Krieger et al., 1976). For
example, the PER following the first ejaculation reported in our previous study (Yang and
Clemens, 1996a) was 47 seconds, which is much shorter than 90 seconds to several
minutes reported by others (Bermant, 1961; Bermant and Westbrook, 1966; Krieger et al. ,
1976). There are some differences between our pacing behavior tests (Yang and Clemens,
1996a) and those of others (Bermant, 1961; Bermant and Westbrook, 1966; Krieger et al. ,
1976) which may account for these different results. One difference lies in the design of
the pacing test chamber. In our previous study (Yang and Clemens, 1996a), the female can
escape from the male easily from four escape holes along the bottom of the barrier
separating the escape chamber from the male’s chamber. In other studies (Bermant, 1961;
Bermant and Westbrook, 1966; Krieger et al., 1976), females are trained to press a lever to
gain access to the male (Bermant, 1961; Bermant and Westbrook, 1966) or the male is
tethered to half of the test arena (Krieger et al., 1976). Another difference is the hormone
replacement regimens. The hormone replacements used in the above studies (Bermant,
1961; Bermant and Westbrook, 1966; Krieger et al., 1976) were quite similar to one
another. The females were brought into estrus by an injection of a high dose (50 or 100

pg) of EB followed by an injection of 0.5 or 1 mg P 48 or 72 hours after the EB injection.

. 55
However, in our study (Yang and Clemens, 1996a), females were primed with 0.5 pg EB
for three consecutive days followed by an injection of 0.5 mg P 24 hours after the last EB
injection. In the present experiment, we compared the effect of the hormone replacement
regimen we have used (Clemens et al., 1995; Lange and Clemens, 1994; Yang and
Clemens, 1994, 1995a, 1995b, 1996a, 1996b) with the one used by Krieger et al. (Krieger

et al., 1976) on the temporal pattern of female sexual behavior.

METHOD

Procedures

Females were tested for female pacing behavior over a two-week test period in
which two different hormone replacement regimens were used to prime the females for
behavioral testing. Fourteen male and fourteen female Long Evans rats were used in this
experiment. Each of 14 females was randomly assigned to copulate with one of 14 males
in the first week of this experiment. Each female received an ejaculation from the same
male she mated in the first week throughout the entire test duration. In the first week, the
females were injected with 0.5 pg EB for 3 consecutive days followed by an injection of
0.5 mg P 24 hours after the last EB injection (0.5 pg EB x 3 + 0.5 mg P). The females
were tested for female pacing behavior 4—7 hours after the injection of P. In the second
week, the females were brought into estrus by a single injection of 50 pg EB followed by
an injection of 0.5 mg P 48 hours after the EB injection (50 pg EB + 0.5 mg P). The
females were tested for female pacing behavior 4—7 hours after the injection of P. Hormone
treatments were not counterbalanced because the data obtained in our laboratory showed
that repeated testing did not affect female pacing behavior when females were primed with
0.5 pg EB x3 + 0.5 mg P.

Statistics

All behavioral data (AL, IL, MRL, IRL, PER and percentage exits following sexual
contacts) obtained from two different hormone treatments were analyzed by paired t-test.

An alpha level of 0.05 was used for all statistical tests.

RESULTS

Figure 8 compares the PERs obtained from the females treated with two different
hormone replacement regimens. The females receiving 50 pg EB + 0.5 mg P had a
significantly longer PER than those receiving 0.5 pg EB x 3 + 0.5 mg P (t-—--4.448,
p=0.0007). No significant difference was found between these two groups in any of the
other behavioral measures (AL, IL, MRL, IRL, and percentage exit following a mount,

intromission, and ejaculation) recorded in this experiment.

DISCUSSION

In this study, we demonstrate that intromissions alone, without ejaculation, can
induce the female to initiate prolonged latencies between intromissions. Females receiving
a large number of intromissions prior to ejaculation showed one or more IRLs equal to or
exceeding her first PER or the average PER of 47 seconds (Yang and Clemens, 1996a).
Six of these 10 females had multiple prolonged IRLs. The first prolonged IRL occurred
most frequently between the 24th and 44th intromission (7 out of 10 females) and 12 to 16
minutes after the first intromission (7 out of 10 females). The occurrence of prolonged IRL
raises several interesting questions. First, what mechanisms bring about the prolonged

I RL ? Secondly, what function do the prolonged IRLs play?

57

Figure 8 . Comparison of the PER of females receiving two different hormone treatments.

In the first week, the females received injections of 0.5 pg EB for three consecutive days
24 hours later followed by an injection of 0.5 mg progesterone. In the second week, the

females were given an injection of 50 pg EB 48 hours later followed by an injection of 0.5
mg progesterone. Bar represents mean '1: SEM. Data were analyzed by paired t test. ** P =
0.0007. N = 14.

PER (seconds)

80-
70-
60-

,
50-
4o-
30-

20a

10-

 

O

 

I 0.5ngBx3+0.5mgP
I 50ngB+0.5mgP

 

 

 

59
The mechanisms that influence the occrurence of the prolonged IRL may be similar

to those controlling the PER. The pattern of IRLs around the PER and the prolonged IRL
in Figure 6 and Figure 7 strongly supports the idea that similar mechanisms may exist for
the occurrence of the PER and prolonged IRL. In Figure 6, the IRLs were aligned by the
first intromission and then by each of the three ejaculations. The f emale’s return latency is
normally short following intromission before ejaculation and then increases significantly
immediately after ejaculation as a result of the long intromission duration that accompanies
ejaculation (The ejaculatory intromission duration is significantly longer than the non-
ejaculatory intromission duration) (Erskine et al., 1989). Following the postejaculatory
refractory period, the female’s return latency following intromission becomes short again.
In Figure 7, the IRLs were aligned by the first three IRLs, by the first prolonged IRL (N:
the intromission inducing the first prolonged IRL), and by ejaculation (E). The pattern of
IRLs around the first prolonged IRL is similar to that around the PER seen in Figure 6.
The first three IRLs in the test are short. Most of the first prolonged IRL occurs after 24 to
44 intromissions. Following the first prolonged IRL (the N IRL), the N+1 IRL is short as
occurs after the PER. However, the lack of difference between the first prolonged IRL and
the N+2 IRL as well as subsequent IRLs is puzzling. Analysis reveals that this is due
largely to the occurrence of multiple prolonged IRLS by some females (6 of 10). The PER
is associated with an increase in intromission duration during ejaculation. Possibly the
prolonged IRL is also associated with a prolonged intromission duration.

One possible function of the female’s prolonged IRLs may be to induce the male to
ejaculate after the female has received enough vaginocervical stimulation for the induction
of the progestational state of pregnancy. This is supported by several lines of evidence.
First, prolonged IRL.s only occur following a large number of intromissions that exceeds
the number of intromissions necessary for the induction of the progestational state of
pregnancy (approximately 5 in female-paced situation and 10 in non-paced situation)

(Adler, 1969; Chester and Zucker, 1970; Edmonds et al., 1972; Erskine, 1989; Erskine et

60
al., 1989; Gilman et al., 1979). Second, experimentally enforced long interintromission

intervals (1 to 5 minutes) have been reported to facilitate ejaculation by reducing the number
of intromissions prior to the first ejaculation (Bermant, 1964; Larsson, 1959). The mean
of the first prolonged IRL seen in the present study was about 50 seconds (Figure 7) which
would be sufficient to facilitate the male’s ejaculation under normal test conditions.

In Experiment 2B, different hormone replacement regimens were found to affect the
female's PER but not her AL, IL, MRL, IRL and the frequency with which the female left
the male's chamber after each sexual contact. Females receiving 50 pg EB + 0.5 mg P had
a significantly longer PER than those receiving 0.5 pg EB x 3 + 0.5 mg P (Figure 8). The
mean values of PER, IRL and MRL reported in our previous study (Yang and Clemens,
1996a) were shorter than those reported by other researchers (Bermant, 1961; Bermant and
Westbrook, 1966; Krieger et al., 1976). For example, the mean PER and IRL in the first
ejaculatory series reported in our previous study (Yang and Clemens, 1996a) were 47
seconds and 12 seconds, respectively. However, the mean PER and IRL in the first
ejaculatory series reported in other studies (Bermant, 1961; Bermant and Westbrook, 1966;
Krieger et al., 1976) were 90 seconds to several minutes and 40 seconds to over 1 minute,
respectively. We suggest that the quantitative differences among the behavioral measures
of female pacing behavior reported by different groups of researchers may reflect
differences among the test paradigms as well as the hormone replacement regimens.

Increases in P levels but not in EB levels have been demonstrated to reduce the
female’s return latencies following intromissions and ejaculations (Fadem et al., 1979;
Gilman and Hitt, 1978). The present finding that the 50 pg EB + 0.5 mg P trcatment
induced a significantly longer PER in the female than the 0.5 pg EB x 3 + 0.5 mg P
treatment is not consistent with this earlier finding that increases in EB do not affect the
female’s return latency following an ejaculation (Gilman and Hitt, 1978). Differences in
the time of hormone injection and in the dosages used may account for this discrepancy.

First, the time for EB injection was different for the 50 pg EB + 0.5 mg P treatment and

61
the 0.5 pg EB x 3 + 0.5 mg P treatment in the present study. The 50 pg EB was injected

48 hours before P administration, whereas 0.5 pg EB was injected 72, 48, and 24 hours
before P was injected. In contrast, the time for EB injection was the same for different
dosages of EB in the earlier study: EB was always injected 44 hours prior to P injection
(Gilman and Hitt, 1978). Secondly, in the present study, each female received an injection
of 0.5 mg P, a dose that was not examined in the earlier study (Gilman and Hitt, 1978).
Further, the dosage (50 pg EB per animal) used in the present study is double the highest
dosage (90 pg/kg EB, approximately 22.5 pg EB per animal at a body weight of 250 g)
used in the earlier study (Gilman and Hitt, 1978). Finally, the large difference in the EB
dosages (0.5 pg EB x 3 and 50 pg BB) in the present study may also help to explain the
difference in the PER of females treated with these two hormone regimens.

In the present study, the temporal aspects of female rat’s copulatory behavior were
found to vary in relation to hormone replacement regimens and intromission frequency.
Females receiving a high dose of EB plus P show a significantly longer PER than those
receiving three low doses of EB plus P. During copulatory bouts without ejaculation, it
was found that after a large number of intromissions, females began to enforce prolonged
intervals between intromissions. It is suggested that these prolonged intervals may

function to facilitate ejaculation.

EXPERIMENT 3: INFLUENCE OF MALE RELATED STIMULI ON
FEMALE POSTEJACULATORY REFRACTORY PERIOD IN RATS

ABSTRACT

YANG, L. Y. AND L. G. CLEMENS. Influence of male related stimuli on female
postejaculatory refractory period in rats. Physiol. Behav. 60(0) 000-000, 1996.-When
tested in a situation where the female rat can escape from the male during copulation, she
"paces" her sexual contacts with the male. This pacing behavior is influenced by the type
and quality of vaginocervical stimulation the female receives. One possible function of the
female’s return latency following an ejaculation (postejaculatory refractory period, PER) is
to facilitate or allow sperm transport from the vagina to the uterus. The objective of this
study was to investigate the effect of several male related stimuli on the female's PER.
Females were tested in a two-compartment test chamber where they could escape from the
male through one of the four openings along the bottom of the Plexiglas barrier.
Experiment 3A examined the influence of the seminal plug, the penile cup and prostate
secretions on the female's PER. Results showed that neither the seminal plug, the penile
cup nor prostate secretions contributed to the female's PER. In addition, male sexual
performance was not affected by any of the surgical treatments performed in this
experiment. Experiment 3B investigated the relation among the male's ejaculation duration,
preejaculatory intromission frequency, the number of pelvic thrusts during ejaculation, and
the female's PER. Results indicated that both the female’s PER and the male’s ejaculation
duration were found to be significantly shorter in tests where the male ejaculated on the first

or second intromission. Moreover, the male's ejaculation duration, the preejaculatory

62

63
intromission frequency and the female's PER were significantly correlated with one

another. Partial correlation analysis further suggests that preejaculatory intromissions

affect the ejaculation duration which in turn influences the female’s PER.

INTRODUCTION

In test situations where the female rat can escape from the male during copulation,
she regulates the temporal pattern of her sexual contacts with the male (Bermant, 1961;
Bermant and Westbrook, 1966; Clemens et al., 1995; Erskine, 1985, 1987, 1989, 1992;
Erskine and Baum, 1982; Erskine et al., 1989; Fadem et al., 1979; Frye and Erskine,
1990; Gilman and Hitt, 1978; Krieger et al., 1976; McClintock and Adler, 1978;
McClintock and Anisko, 1982; McClintock et al., 1982; Mermelstein and Becker, 1995;
Peirce and Nuttall, 1961a; Yang and Clemens, 1994, 1995a, 1995b, 1996a, 1996c). The
general pattern of this “female pacing behavior” shows that the female's return latency
following an ejaculation, the postejaculatory refractory period (PER), is significantly longer
than her return latency following an intromission (IRL) or a mount (MRL) (Bermant, 1961;
Bermant and Westbrook, 1966; Erskine, 1985; Fadem et al., 1979; Krieger et al., 1976;
Peirce and Nuttall, 1961a; Yang and Clemens, 1994, 1996a). In addition, the female’s
PER increases over consecutive ejaculatory series (Bermant and Westbrook, 1966; Krieger
etal., 1976; Yang and Clemens, 1996a). One possible function of the female’s PER is to
facilitate or allow sperm transport from the vagina to the uterus. If the female receives
intromissions shortly following ejaculation, sperm transport will be halted until the next
ejaculation (Matthews and Adler, 1977). Since the female’s PER is crucial for sperm
transport from the vagina to the uterus, it is important to understand the mechanisms
underlying the f emale’s PER.

Paced coital stimulation is more effective in the induction of the progestational state

of pregnancy in female rats than the same amount of non-paced coital stimulation. A high

64
number of intromissions (approximately 10 in non—paced situation) provided by the male

during nonpaced copulation tests induces the progestational changes necessary for ovum
implantation in the female rats (Adler, 1969; Chester and Zucker, 1970; Edmonds et al.,
1972; Erskine, 1989; Erskine etal., 1989; Gilman et al., 1979). It has also been reported
that the blood progesterone level of the female rat increases significantly after mating if the
female receives a high number of intromissions (9 to 23) during copulation (Adler et al.,
1970). In contrast, the females receiving two or fewer intromissions prior to ejaculation in
nonpaced tests have a significantly lower percentage of pregnancy (Adler, 1969; Wilson et
al., 1965). In addition, 5 intromissions are insufficient to induce pseudopregnancy in non-
paced female rats (Chester and Zucker, 1970; Erskine, 1989; Erskine et al., 1989; Gilman
et al., 1979). However, in female paced tests, five intromissions are adequate to trigger the
progestational state of pregnancy in the female rats (Erskine, 1989; Erskine et al., 1989;
Gilman et al., 1979). Female pacing of sexual behavior (Erskine, 1985; Erskine and
Baum, 1982; Erskine et al., 1989) is more effective in shortening behavioral estrus than the
same amount of non-paced coital stimulation (Blandau et al., 1941; Hardy and DeBold,
1972; Lodder and Zeilmaker, 1976). In female paced situations, ten intromissions are
sufficient to shorten behavioral estrus (Erskine, 1985; Erskine and Baum, 1982; Erskine et
al., 1989). In contrast, at least 25 intromissions are necessary for shortening of behavioral
estrus in non-paced situations (Erskine and Baum, 1982).

Vaginocervical stimulation plays an important role in the regulation of pacing
behavior in female rats. Following bilateral transection of the pelvic nerve and the
pudendal nerve that innervate the vagina, cervix, uterus, perineal skin, inner thigh and
clitoral sheath (Berkley et al., 1990; Berkley et al., 1993; Peters et al., 1987), females do
not distinguish behaviorally among copulatory events; the return latencies following mount,
intromission and ejaculation are not significantly different from one another (Erskine,
1992). Furthermore, anesthetics applied locally to the vagina reduce contact-response

intervals following intromission and ejaculation (Bermant and Westbrook, 1966).

65
The female’s PER is affected by the number of intromissions received prior to

ejaculation (Yang and Clemens, 1994, 1996a). Females receiving 0-1 intromission before
ejaculation have significantly shorter PERs that are not different from her IRLs. The PERs
of females receiving 2-4 intromissions preceding ejaculation do not differ from those
receiving 5-15 (average 10) intromissions. Females receiving 24—31 intromissions prior to
ejaculation have significantly longer PERs than those receiving 5-15 (average 10)
intromissions (Yang and Clemens, 1994, 1996a).

Although it has been demonstrated that at least two preejaculatory intromissions are
necessary for the occurrence of a normal PER following the first ejaculation (Yang and
Clemens, 1994, 1996a), the mechanisms underlying the female’s PER are not well
understood. However, since the female’s pacing is heavily dependent upon vaginocervical
stimulation, it seems likely that some form of sensory stimulation at the time of ejaculation
induces the PER. Several male related stimuli might play a role in the induction of the
PER. First, the deposit of a seminal plug in the vagina during ejaculation may contribute to
the longer return latency following ejaculation. In rats, the ejaculate of the male coagulates
and forms a seminal plug after ejaculation (Brooks, 1990; Luke and Coffey, 1994; Mann,
1981; Price and Williams-Ashman, 1961; Setchell et al., 1994; Wagner and Kistler, 1987;
Walker, 1910a, 1910b; Williams-Ashman et al., 1977; Zorgniotti and Brendler, 1958).
Possibly the seminal plug provides mechanical stimulation that contributes to the female’s
PER. Secondly, the formation of the penile cup during ejaculation may cause mechanical
stimulation resulting in a longer return latency following ejaculation. In addition, chemical
substances released from the prostate may stimulate the vagina, cervix as well as uterus and
consequently cause vagina] and/or uterine responses that alter the female’s return latency
following ejaculation. Finally, the ejaculation duration may contribute to the f emale’s PER.
This is supported by the following evidence. It has been reported that the ejaculation
duration (Peirce and Nuttall, 1961b) and the f emale’s PER (Bermant and Westbrook, 1966;

Krieger et al., 1976; Yang and Clemens, 1996a) increase over the course of copulation. In

66
addition, it was noticed that the ejaculation duration was shorter when the female received

01 intromission before ejaculation (Unpublished observation).

The objective of Experiment 3A was to examine the effect of the seminal plug,
penile cup and prostate secretions on the female's PER. Experiment 38 investigated the
relation among the male’s ejaculation duration, the number of intromissions received prior

to ejaculation, the number of pelvic thrusts during ejaculation, and the female's PER.
GENERAL METHODS

Animals

Sixty-day-old female and ninety-day-old male Long Evans rats were purchased
from Charles River Laboratories (Wilmington, MA). All females were ovariectomized one
week after arrival. Animals were housed in 16:8 hr light-dark cycle with lights off at
17:00. In general, females were housed three per cage and males were housed singly.
Food and water were available ad lib. The temperature and humidity of the animal room
were maintained at 220 C and 55%, respectively. The air in the animal room was
exchanged 12 times per hour automatically. Ovariectomized females were brought into
estrus by intramuscular (IM) injections of 0.5 pg estradiol benzoate (EB) (Sigma Chemical
Co., St. Louis, MO) dissolved in 0.1 ml sesame oil (Sigma Chemical Co., St. Louis, M0)
for three consecutive days and 24 hours later followed by an IM injection of 0.5 mg
progesterone (P) (Sigma Chemical Co., St. Louis, MO) dissolved in 0.1 ml sesame oil.
Female pacing tests were conducted 4—7 hours after the injection of progesterone (2-5 hours
after the lights off) under dimly red illumination. Animals (males and females) were tested
once per week throughout the whole test period and each female had been tested twice in
the testing chamber before data were collected. Those females that did not exit the male’s

chamber at all following sexual contacts were excluded from the following studies. Only

67
those females that showed pacing behavior (over 95% of females) were used in the

following studies. In each test, a male was placed in the testing chamber 15 minutes before
a female was introduced. The approximate weights of the female and male rats were 200 -

300 g and 400 - 500 g, respectively, when testing began.

Pacing Tests

To provide a test situation in which the female could control the pacing of
copulation, females were tested in a two-compartment chamber that was divided by a
Plexiglas barrier with 4 holes (3.5~4 cm x 3.5~4 cm) spaced along the bottom of the
barrier. The dimensions of the male's chamber and escape chamber were 35.5 cm x 44.5
cm x 48.3 cm and 21.5 cm x 44.5 cm x 48.3 cm (Length x Width x Height), respectively.
The escape holes were large enough to allow the female to pass through but prevented the

larger male from following her.

Behavioral Measures

Several behavioral measures were recorded in the pacing tests unless otherwise
specified: approach latency (AL), the latency for the female to enter the male's chamber
after the test started (she was placed in the escape chamber at the beginning of the test);
mount latency (ML), the latency from the f emale’s entry into the male’s chamber to the first
mount; intromission latency (IL), the latency from the female’s entry into the male’s
chamber to the first intromission; mount return latency (MRL), the latency for the female to
re-enter the male's chamber after a mount; intromission return latency (IRL), the latency to
return to the male's chamber following an intromission; postejaculatory refractory period
(PER), the latency to re-enter the male's chamber after an ejaculation. The frequency

(percentage exit) with which the female left the male's chamber following mount,

68
intromission and ejaculation was calculated by dividing the number of exits by the number

of each type of stimulus. All these behavioral data were recorded on a computer disk.

EXPERIMENT 3A: EFFECT OF STIMULUS FACTORS ON FEMALE
POSTEJACULATORY REFRACTORY PERIOD

The objective of this experiment was to examine whether the deposit of a seminal
plug, the formation of a penile cup, or the presence of prostate secretions during ejaculation
influences the female's PER. In rats, the ejaculate of the male forms a seminal plug to seal
the cervix after ejaculation. The formation of a seminal plug involves polymerization of
proteins in the seminal vesicular secretion by an enzyme released from the coagulating
glands (Brooks, 1990; Luke and Coffey, 1994; Mann, 1981; Price and Williams-Ashman,
1961; Setchell et al., 1994; Wagner and Kistler, 1987; Walker, 1910a, 1910b; Williams-
Ashman et al., 1977 ; Zorgniotti and Brendler, 1958). Bilateral removal of seminal vesicles
and coagulating glands prevents the formation of a seminal plug after ejaculation. Penile
cup formation of male rats is observed during ejaculatory reflexes in ex copula tests (Hart
and Odell, 1981). However, when the bulbocavemosus (BC) muscle was removed,
formation of the penile cup was lost during ex copula tests (Hart and Melese-D’Hospital,
1983; Sachs, 1982, 1983). Indirect evidence suggests that removal of the BC muscle
prevents the formation of the penile cup at the time of ejaculation during in copula tests
because the weight of seminal plug attached to the penis is hcavier in the males with BC
removal than in control males (Sachs, 1982). In addition to the deposit of a seminal plug
and the formation of a penile cup, chemical substance(s) released from prostate during
ejaculation might cause contractions of the vagina and uterus and consequently affect the

female’s PER. Removal of the prostate was used to eliminate prostatic fluids.

69
MATERIALS AND METHODS

Procedures

Forty-three male and 43 female Long Evans rats were used in this study. Animals
were randomly assigned into four groups: the control, SCV, SCV+BCX, and SCV+PX
Groups. In the control Group, the males received anesthesia and laparotomy (N=11). In
the SCV males, bilateral seminal vesicles and coagulating glands were removed and the vas
deferens were bilaterally ligated and transected (N=11). In the SCV+BCX Group, in
addition to the SCV treatment, the males received removal of bulbocavemosus muscle
(N=10). In the SCV+PX males, in addition to the SCV treatment, the prostate glands were
also removed (N=1 1).

In a three-week test period (the week before male surgery, one week after male
surgery and two weeks after male surgery), female pacing behavior was tested once per
week. Each female was randomly assigned to copulate with one male before male surgery
and always tested with the same male she mated before male surgery throughout the entire
test period. Therefore, we referred to each female with the male she mated as ”female and
male partners." Before male surgery, each female was allowed to copulate with her male
partner until he ejaculated and then achieved one additional intromission. One week after
male surgery, the male was allowed to have one post surgery experience test. During the
experience test, the female was examined for the presence of a seminal plug in her vagina
immediately following ejaculation. Two weeks after male surgery, each female was tested
with her male partner until she received an ejaculation and one postejaculatory intromission.
The temporal pattern of female sexual behavior was recorded and analyzed before male
surgery and two weeks after male surgery. In addition, male sexual behavior was recorded
before male surgery and two weeks after male surgery including mount frequency (MF), '

intromission frequency (IF), hit rate (number of intromissions / number of intromissions

70
plus number of mounts), mount latency (ML, the latency from the female’s entry into the

male’s chamber to the first mount), intromission latency (IL, the latency from the female’s
entry into the male’s chamber to the first intromission), interintromission interval (111,
ejaculation latency divided by number of intromissions before ejaculation), ejaculation
latency (EL, the latency from the first intromission to ejaculation), and postejaculatory

interval (PEI, the latency from ejaculation to the next intromission).

Surgery

Male rats were anesthetized with intraperitoneal (IP) injection of Sodium
Pentobarbital (Sigma Chemical Co., St. Louis, M0) at a dose of 50 mg per kg body weight
and, if necessary, were given supplemental gas anesthetic, methoxyflurane (Metofane;
Pitman-Moore Inc., Illinois). In the control Group, the males received a 3 to 4 cm midline
incision on the abdomen and the seminal vesicles as well as coagulating glands were
exposed and put back into the abdomen immediately after exposure. In the SCV Group,
the males received a 3 to 4 cm midline incision on the abdomen and the seminal vesicles as
well as coagulating glands were exposed and brought outside the body with fine forceps.
A hemostat was used to hold the lower portion of the seminal vesicles and coagulating
glands and 4—0 silk suture was applied to tie off the tissue below the hemostat. The seminal
vesicles and coagulating glands were removed. In addition, the vas deferens were
bilaterally ligated and transected. After surgery, the incision was sutured with 4—0 plain gut
suture (Ethicon Inc., New Jersey), and antibiotics were applied to the wound before the
skin was closed with wound clips. Each animal was checked and antibiotics were applied
to the incision site daily for 5 days.

In the SCV+BCX Group, in addition to SCV treatment, the bulbocavemosus
muscle was removed. A 2-3 cm midline incision was made in the skin of scrotum

beginning 1 cm posterior to the penile sheath and running caudally. The testes of the

71
animal were pushed up into the body cavity and the incision was opened with the

retractors. In order to expose the penile muscles, the fascia was cut; care was taken not to
damage the bulbouretlrral glands. The bulbocavemosus muscle was then cut away and
removed from the penile bulb. Antibiotics were applied to the surgical area and the skin
around the incision. The incision was closed with wound clips (The procedures for BCX
surgery is adapted from reference Sachs, 1982). Each animal was checked and antibiotics
were applied to the incision site daily for 5 days.

In the SCV+PX Group, in addition to the SCV surgery, the prostate glands of
males were removed. The procedures for this surgery were basically the same as those

described earlier in the SCV treatment.

Statistics:

The data of female’s percentage exits following mount, intromission as well as
ejaculation and the male’s hit rate were transformed by are sine square root before analysis
(Dixon and Massey, 1969). Comparison of the female pacing behavior (AL, MRL, IRL,
PER, percentage exits following mount, intromission and ejaculation) among the four
groups before male surgery and two weeks after male surgery was achieved by Mixed
Factorial ANOVA (Two Way Repeated Measures ANOVA) followed by Tukey’s post-hoc
tests. Comparison of the male sexual behavior (MF, IF, hit rate, ML, IL, 111, EL, and
PEI) among the four groups before male surgery and two weeks after male surgery was
analyzed by Mixed Factorial ANOVA (Two Way Repeated Measures ANOVA) followed
by Tukey’s post-hoe tests. An alpha level of 0.05 was used for all statistical tests.

72
RESULTS

No seminal plug was found in the vagina of females tested with experimental males
one week after the SCV, SCV+BCX, or SCV+PX surgery, nor were significant
differences found among the four treatment groups before male surgery and two weeks
after male surgery in the female’s AL, MRL, IRL, PER, and percentage exits following
mount, intromission, and ejaculation. None of the measures of male sexual behavior
recorded in this experiment changed following any surgical treatment except the
interintromission interval (111) in the SCV+BCX Group. The III of males in the
SCV+BCX Group was significantly longer after male surgery (39.4 :t 5.3 seconds) than
before male surgery (30.0 i 3.0 seconds) (p<0.05).

EXPERIMENT 3B: RELATION OF THE EJACULATION DURATION AND
PREEJACULATORY INTROMISSIONS TO THE F EMALE’S
POSTEJACULATORY REFRACTORY PERIOD

It has been reported that the duration of the male rat’s ejaculatory reflex increases
over successive ejaculatory series during copulation (Peirce and Nuttall, 1961b).
Similarly, the female rat’s PER increases over the course of copulation (Bermant and
Westbrook, 1966; Krieger et al., 1976; Yang and Clemens, 1996a). In our previous
study, we demonstrated that the PER of females receiving 0-1 intromission before
ejaculation was significantly shorter than the other groups (24, 5—15, and 24-31
intromission groups) (Yang and Clemens, 1996a). We also found that the male’s
ejaculation duration was shorter when the female received only 0-1 intromission prior to
ejaculation (Unpublished observation). These findings lend support to the idea that the
male’s ejaculation duration may contribute to the female’s PER. Moreover, it is possible

that the number of pelvic thrusts during ejaculation affects the male’s ejaculation duration

73
and/or the female’s PER. Although preejaculatory intromission frequency is not

significantly correlated with the female’s PER during the first ejaculatory series (Yang and
Clemens, 1996a), preejaculatory intromission frequency was significantly correlated with
the female’s PER if the range of the preejaculatory intromission frequency was large
enough (0-31 in that study) (Yang and Clemens, 1996a). It is possible that the
preejaculatory intromissions may influence the male’s ejaculation duration and/or the
number of pelvic thrusts during ejaculation that in turn affect the female’s PER. The
objective of this experiment was to examine the relation among the ejaculation duration, the
number of intromissions received prior to ejaculation, the number of pelvic thrusts during

ejaculation, and the female's PER.

MATERIALS AND METHODS

Procedures:

In a two-week test period, the pacing behavior of sexually experienced female rats
was recorded on a computer disk and videotaped with a camcorder. Eighteen male and 18
female Long Evans rats were used in this study. In the first week, each female was
randomly assigned to copulate with one male until she received an ejaculation and returned
to the male’s chamber following her PER (the 4—18 I Group). Because each female was
always tested with the same male she mated in the first week, we referred to the female and
the male she mated as “the female and male partners”. In the second week, each male was
allowed to copulate with a stimulus female that was replaced with the “female partner”
(experimental female) when it was estimated that the male was near ejaculation. This
estimate was based on the preejaculatory intromission frequency obtained in the first week
test. To examine whether the male’s ejaculation duration and the female’s PER are

significantly shorter when the female receives 01 intromission prior to ejaculation, only the

74
data of those females that received 0 or 1 intromission prior to ejaculation were recorded

and used for analysis (the 0-1 I Group). Mating treatments were not counterbalanced
because the results obtained in our laboratory indicated that repeated testing did not affect
the temporal pattern of female sexual behavior under the present hormone replacement
regimen.

The male's ejaculation duration was defined as: the duration of male-female contact
during the ejaculatory intromission and was obtained by playing back the videotape 30
times slower with a Hitachi VCR. The male’s ejaculation duration refers specifically to the
time period from the male’s first pelvic thrust to the male’s dismount from the female.
Each ejaculation duration was measured three times during video analysis and the mean
ejaculation duration was calculated and used for analysis. In addition, the number of pelvic
thrusts during ejaculation was determined using the slow play-back of the videotape and
only those animals with clearly identified pelvic thrusts were used for analysis of thrust

frequency.

Statistics:

Comparison of the male’s ejaculation durations between the 4—18 I Group and the
0-1 I Group and comparison of the female’s PERs between the 4—18 I Group and the 0-1 I
Group were achieved by paired t-test. The relations among the male’s ejaculation duration,
the preejaculatory intromission frequency, the number of pelvic thrusts during ejaculation
and the female’s PER were analyzed by Correlation analysis. An alpha level of 0.05 was

used for all statistical tests.

 

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75
RESULTS

In the first week, eighteen females were allowed to copulate for one full ejaculatory
series and received a range of 4 to 18 intromissions prior to ejaculation (the 4—18 I Group).
Two animals were excluded from analysis because one was rejected by the Outlier test and
the other was excluded because of a loss of image focus caused by power failure of the
camcorder. Therefore, data from 16 females were used for statistical analysis. The male’s
ejaculation duration was positively correlated with the female’s PER (r = 0.653, p =
0.0049, N = 16). In addition, the number of intromissions received by the female prior to
ejaculation was positively correlated with the male’s ejaculation duration (r = 0.643, p =
0.0059, N = 16). However, the preejaculatory intromission frequency was not
significantly correlated with the female’s PER (r = 0.270, p = 0.3187, N = 16). This
result replicated our previous finding that the number of preejaculatory intromissions was
not significantly correlated with the female’s PER in the first ejaculatory series (Yang and
Clemens, 1996a). Twelve out of 16 males had pelvic thrusts clearly identified during
ejaculation; thrust frequency ranged from 4 to 7 with a mean of 5.9. There was no
significant correlation between the number of pelvic thrusts during ejaculation and the
female’s PER (r = 0.396, p = 0.2083, N = 12). Nor was thrust frequency during
ejaculation significantly correlated with the male’s ejaculation duration (r = 0.445, p =
0.1512, N = 12) or the preejaculatory intromission frequency (r = -0.197, p = 0.5487).

In the second week, 6 out of 16 females received 0-1 intromission before the male
ejaculated (the 0-1 I Group) and the data of these 6 females were used for analysis. The
data of females (N = 10) receiving more than 2 intromissions prior to ejaculation were not
recorded and excluded from analysis. Figure 9 compares the male’s ejaculation durations
between the 4—18 I Group and the 0-1 I Group. The ejaculation duration of males in the 0-1
I Group was significantly shorter than that of the 4—18 I Group (t = 4.065, p = 0.0097, N
= 6) (Figure 9). Figure 10 compares the f emale’s PERs between the 4—18 I Group and the

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76
0-1 I Group. The PER of females receiving 0-1 intromission prior to ejaculation was

significantly shorter than that of the 4-181 Group (t = 4.155, p = 0.0089, N = 6) (Figure
10). This result replicated our previous finding that the PER of females receiving 01
intromission prior to ejaculation was significantly shorter than the other groups (24, 5- 15,
and 24—31 intromission groups) (Yang and Clemens, 1996a).

When the data from week 1 (the 4—181 Group) were combined with those of week
2 (the 0-1 I Group), the male’s ejaculation duration was positively correlated with the
female’s PER (r = 0.814, p < 0.0001, N = 22) (Figure 11). In addition, the number of
intromissions received by the female prior to ejaculation was positively correlated with the
male’s ejaculation duration (r = 0.771, p < 0.0001, N = 22) (Figure 12). Moreover, the
preejaculatory intromission frequency became significantly correlated with the female’s
PER (r = 0.611, p = 0.002, N = 22) (Figure 13). This is consistent with our previous
finding that preejaculatory intromission frequency was significantly correlated with the
female’s PER if the range of intromission frequency was large enough (0-31 in the
previous study) (Yang and Clemens, 1996a). Furthermore, partial correlation analysis
controlling the ejaculation duration indicated that the correlation between preejaculatory
intromissions and the female’s PER was close to zero (r = -0.0453, N = 22).

The number of pelvic thrusts during ejaculation can be identified in five of the 6
males in the 0-1 I Group; thrust frequency ranged from 6 to 7 with a mean of 6.4. After
combining the data of the4—18I Group and the 0-1 I Group, the number of pelvic thrusts
during ejaculation was not significantly correlated with either the ejaculation duration (r =
0.137, p = 0.6064, N = 17), the preejaculatory intromission frequency (r = -0.304, p =
0.2407, N = 17) or the female’s PER (r = 0.097, p = 0.7169, N = 17).

77

Figure 9 . compares the ejaculation duration of males between the 4—18 and the 0—1
intromission Groups (4— 18 l and 0-1 I Groups). The male’s ejaculation duration in the 4— 18
I Group is significantly longer than that in the 0-1 I Group (t = 4.065, p = 0.0097, N = 6).
Data are expressed as mean 1“. SEM and analyzed by paired t—test. ** p<0.01.

Ejaculation duration (seconds)

1.8 -
1.6 -
1.4 :1
1.2 J

.81
.61
.4-
.2-

 

78

 

 

 

 

 

4—18 0-1

Number of intromissions prior to ejaculation

79

Figure 10. compares the postejaculatory refractory period (PER) of females between the
4—18 and the 0-1 intromission Groups (4—18 I and 0-1 I Groups). The PER of females in
the 4-18 I Group is significantly longer than that in the 0-1 I Group (t = 4.155, p = 0.0089,
N = 6). Dataare expressed as mean i SEM and analyzed by paired t-test. ** p<0.01.

Postejaculatory refractory period (HER)
(seconds)

30:
20+

10-

 

 

 

 

 

 

 

4—18 0‘1

Number of intromissions prior to ejaculation

81

Figure. l l . shows the relation of the male’s ejaculation duration (ED) to the female’s
postejaculatory refractory period (PER). There is a significantly positive correlation
between the male’s ED and the female’s PER (r = 0.814, p<0.0001, N=22).

in) rfirr-r‘lnnl “'1’le (IHJD\

I*fimlfiifll‘.ll

Postejaculatory refractory period (PER)
(seconds)

82

 

 

 

 

Ejaculation duration (seconds)

 

Figure. l 2. shows the relation of the number of intromissions prior to ejaculation to the
male’s ejaculation duration. The preejaculatory intromission frequency is positively
correlated with the male’s ejaculation duration (r = 0.771, p < 0.0001, N = 22).

Ejaculation duration (seconds)

 

 

 

 

 

 

1

-+,.,.,. .,.
10 12 14 16 18 20

2 4 6 8

Number of intromissions prior to ejaculation

85

Figure 13. The relation of preejaculatory intromission frequency to the female’s
postejaculatory refractory period (PER). The preejaculatory intromission frequency is
significantly correlated with the female’s PER (r = 0.611, p = 0.002, N = 22).

x1 (I—‘Iil()

'II(‘I( )I‘V DC‘I'I‘

t()rv ref!

l—‘k entrain-cu Ill

Postejaculatory refractory period (PER)
(seconds)

70

 

 

 

 

r: 0.611

I Tfi

 

 

I ‘ T ' I ' I I ' I T I ' I ' I

2 4 6 8 10 12 14 16 18 20

Number of intromissions prior to ejaculation

post.
stud
con:

mm

mm‘
{Sir
equ
mil

87
DISCUSSION

The principal finding of this study is that the male’s ejaculation duration is
positively correlated with the female’s PER. This finding is consistent with previous
studies showing that the male’s ejaculation duration increased over the course of
consecutive ejaculatory series (Peirce and Nuttall, 1961b) and that the female’s PER
increased with consecutive ejaculatory series (Bermant and Westbrook, 1966; Krieger et
al., 1976; Yang and Clemens, 1996a). Two methods have been used to measure the
male’s ejaculation duration: cinemagraphic analysis of copulation using frame analysis
(Stone and Ferguson, 1940), and a moist contact method to activate electrical recording
equipment (Erskine et al., 1989; Peirce and Nuttall, 1961b). The ejaculation duration of
male rats as revealed by these methods was 1 to 4 seconds (Stone and Ferguson, 1940) and
1 to 2 seconds (Erskine et al., 1989; Peirce and Nuttall, 1961b), respectively, in the first
ejaculatory series. In this study, the male’s ejaculation duration: the time from the first
pelvic thrust to the male’s dismount from the female, is between 1 and 2.1 seconds in the
first ejaculatory series.

The female’s PER appears to be influenced directly by the male’s ejaculation
duration and indirectly by the number of intromissions received prior to ejaculation.
Ejaculation duration was positively correlated with the number of intromissions received
prior to ejaculation and the female’s PER in the first ejaculatory series (the 4—18 I Group)
and after combining the data of the 4—18 I Group and the 0-1 I Group. These findings
support our previous notion that multiple intromissions of male rats prior to ejaculation may
function to prepare the vaginal passage for the deep penile insertion that results in a longer
penis-vagina contact during ejaculation (Yang and Clemens, 1996a). This longer penis-
vagina contact in turn results in a longer return latency following ejaculation in the female.
Although the male’s ejaculation duration is significantly correlated with the female’s PER in

the first ejaculatory series, the number of preejaculatory intromissions is not, which

88
replicates our previous report (Yang and Clemens, 1996a). However, when the range of

number of intromissions received by the female before ejaculation was increased beyond
the normal range seen in the first ejaculatory series, intromission frequency was positively
correlated with the female’s PER (Yang and Clemens, 1996a). After combining the data of
the 4—18 I Group and 0-1 I Group in the present study, the preejaculatory intromission
frequency was positively correlated with the female’s PER indicating that preejaculatory
intromission frequency influences the female’s PER. Further, partial correlation analysis
controlling the ejaculation duration indicated that the correlation between the preejaculatory
intromissions and the female’s PER was close to zero. From these lines of evidence we
suggest that the number of intromissions received prior to ejaculation affects the male’s
ejaculation duration which in turn influences the female’s PER.

Further support for the idea that the male’s ejaculation duration plays an important
role in inducing the female’s PER comes from experimental manipulation of intromission
frequency preceding ejaculation. The male’s ejaculation duration and the f emale’s PER are
significantly shorter when the female receives 0 or 1 intromission prior to ejaculation.
These findings replicate our previous report (Yang and Clemens, 1996a) showing that
when the female received 0 or 1 intromission before the male ejaculated, her PER was
significantly shorter than the other groups (2-4, 5- 15, and 24-31 intromission groups).

The influence of a longer ejaculation duration on the female’s PER probably results
from more intense stimulation due to the longer vagina-penis contact duration that may
result from the deeper penile insertion during ejaculation. The longer vagina-penis contact
associated with ejaculation or a longer PER does not appear to result from more pelvic
thrusts because the number of pelvic thrusts during ejaculation is not significantly
correlated with either the ejaculation duration or the female’s PER. This finding also
implies that the quality of pelvic thrusts may be more important than the frequency of pelvic
thrusts in inducing the female’s PER. Most likely the longer ejaculation duration and/or a

deeper penile insertion generate a more intense stimulus which results in a longer PER.

89
Several lines of evidence support this notion. First, it has been reported that the vaginal

canal close to the cervix is innervated by sensory afferent fibers (Nance et al., 1988).
Secondly, fibers with mechanoreceptive fields that respond to thrusting stimuli are found
along the vagina] canal in female rats (Berkley et al., 1990). In addition, evidence suggests
that more fibers with mechanoreceptive fields are located near the vaginocervical junction
than in other areas of vaginal canal (Berkley et al., 1990). Furthermore, a deeper penile
insertion during ejaculation may simultaneously stimulate pelvic nerve fibers as well as
fibers from the hypogastric nerve (Berkley et al., 1990; Berkley et al., 1993; Peters et al.,
1987). However, a functional analysis of the respective roles played by these two nerves
suggests that “pelvic nerve fibers seem closely tied to sensory and behavioral processes
associated with mating and conception, whereas hypogastric fibers seem closely tied to
pregnancy and nociception” (p. 533, Berkley et al., 1993).

Neither the seminal plug, prostate secretions, nor the penile cup formation appeared
to contribute to the female’s postejaculatory refractory period. In addition, the male’s
sexual performance was not affected by removal of the coagulating glands, seminal
vesicles, prostate glands, or bulbocavemosus muscle. In rats, secretions from the seminal
vesicles constituted a large portion of the male’s ejaculate (Mann, 1964). The enzyme,
transglutanrinase, released from the coagulating glands, plays a major role in the formation
of the copulatory plug (Luke and Coffey, 1994; Mann, 1981; Wagner and Kistler, 1987;
Williams-Ashman, 1984; Williams-Ashman et al., 1977). Bilateral removal of the senrinal
vesicles and coagulating glands, therefore, prevents formation of a copulatory plug after
ejaculation (This was confirmed by the evidence that no seminal plug was found in the
vagina of the females that mated with the males after the SCV, SCV+BCX, and SCV+PX
treatments). However, removal of both sets of tissue did not affect the measures of male
sexual behavior or female pacing behavior including the female’s PER. Extirpation of the
BC muscle presumably eliminates the formation of penile cup during ejaculatory reflexes in

copula tests. Removal of the bulbocavemosus muscle had no effect on the male sexual

90
performance or on the female temporal copulatory behavior including the female’s PER.

Although the prostatic secretions contribute considerable volume to the seminal plasma in
rats, removal of the prostate did not affect sexual performance of either the male or female,
nor did bilateral ligation and transection of vas deferens.

In summary, different types and qualities of vaginocervical stimulation received by
the female affect her pacing behavior. The female’s PER is significantly longer than her
IRL and MRL. Different numbers of intromissions received by the female prior to
ejaculation affect her PER. In the present study, our data show that neither the seminal
plug, prostatic secretions, nor the penile cup contributes to the female’s PER. In addition,
removal of either the prostate glands, bulbocavemosus muscle, coagulating glands, or
seminal vesicles does not affect the measures of male sexual behavior recorded in this
study. The number of pelvic thrusts during ejaculation is not significantly correlated with
either the male’s ejaculation duration, the female’s PER or the preejaculatory intromission
frequency. However, the male’s ejaculation duration is significantly correlated with the
preejaculatory intromission frequency and the female’s PER in the first ejaculatory series
(the 4181 Group) and after combining the data of the 4—18 I Group and the 0-1 I Group.
In contrast, the number of intromissions received prior to ejaculation is significantly
correlated with the female’s PER after combining the data of the 4— 18 I Group and the 0—1 I
Group. Partial correlation analysis controlling the ejaculation duration shows that no
significant correlation exists between preejaculatory intromissions and the female’s PER.
These findings lead us to suggest that the female’s PER is influenced directly by the male’s
ejaculation duration, indirectly by the number of intromissions received by the female prior

to ejaculation and not at all by the amount of thrusting during ejaculation.

EX

lid

alll

Uni
1nd

Ion

dur
cor
the
la

the

EXPERIMENT 4: CORRELATION OF UTERINE ACTIVITY WITH THE
TENIPORAL PATTERN OF FEMALE SEXUAL BEHAVIOR IN
RATS

ABSTRACT

In a standard test situation, it has been reported that copulation has both an
immediate and a delayed effect on uterine contractions. The objective of the present study
was to determine whether uterine activity correlates with different types of sexual contact
and different temporal patterns of female sexual behavior. Electrodes implanted in the
uterus were used to record electromyographic (EMG) activity while females were tested
under a situation where they could escape from the male following sexual contacts. Results
indicated that the duration of uterine activity associated with an ejaculation was significantly
longer than that associated with an intromission or a mount. This finding coincides with
the report that the female’s return latency following an ejaculation is significantly longer
than that following an intromission or a mount and that the intromission duration is longer
during ejaculation than during non-ejaculatory intromissions. In addition, our results were
consistent with the finding that mating has an immediate effect on uterine activity. Possibly
the longer duration of uterine activity associated with ejaculation that reflects a more intense
vaginocervical stimulation contributes to the fast sperm transport following ejaculation and

the longer return latency following ejaculation.

91

92
INTRODUCTION

One function of the multiple intromission pattern of mating in rats is to facilitate
sperm transport following ejaculation (Adler, 1969). When females received 0-1
intromission prior to ejaculation, no or few sperm were found in the uterus 3 hours
following ejaculation. However, larger numbers of sperm were found in the uterus if the
female received 2 or more intromissions prior to ejaculation (Adler, 1969). The
vaginocervical stimulation received by the female rat during copulation increases uterine
contractions. It has been reported that vaginocervical stimulation has both an immediate
and a delayed effect on uterine electrical activity when the male controls the timing of
copulation (Toner and Adler, 1986). Uterine contractions increased significantly during
copulation, returned to the premating level following ejaculation, and increased again 5
minutes after ejaculation (Toner and Adler, 1986).

The objective of this experiment was to examine the relation of uterine
electromyographic (EMG) activity to each sexual contact when the female is able to control
the temporal pattern of female sexual behavior. While previous experiments have shown a
relationship between various types of vaginocervical stimulation and female pacing
behavior, the present study will provide insight into the relation of endogenous responses

of the female to her mating behavior.

METHOD

Animals

Sixty-day-old female and ninety-day-old male Long Evans rats were purchased

from Charles River Laboratories (Wilmington, MA). All females were ovariectorrrized one

week after arrival in the laboratory. Animals were housed in 16:8 hr light-dark cycle with

hth'é
WI?
.110 C
1111‘“
unit
(is:
0.5 r

TIE

in

p

93
lights off at 17:00. Animals (females and males) were housed singly. Food and water

were available ad lib. The temperature and humidity of the animal room were maintained at

220 C and 55%, respectively. The air in the animal room was exchanged 12 times per hour

automatically. Ovariectomized females were brought into estrus by intramuscular (IM)

injections of 0.5 pg estradiol benzoate (EB) dissolved in 0.1 ml sesame oil (Sigma

Chemical Co.) for three consecutive days and 24 hours later followed by an IM injection of
0.5 mg progesterone (P) dissolved in 0.1 ml sesame oil. All female pacing behavior was
tested 4-7 hours after the injection of progesterone (2-5 hours after the lights off) under
dimly red illumination. All animals were tested once per week throughout the whole test
period and had been tested at least twice in the testing chamber before data were collected.
Only those females showing pacing behavior in these preliminary tests were used in the
experimental studies. The approximate weights of the female and male rats were 200 - 300

g and 500 - 600 g, respectively, when the tests started.

Pacing Tests and Behavioral Measures

Females were tested in our standard two-compartment test arena Several
behavioral measures were recorded in the pacing tests: mount return latency (MRL), the
latency for the female to re—enter the male's chamber after a mount; intromission return
latency (IRL), the latency to return to the male's chamber following an intromission;
postejaculatory refractory period (PER), the latency to re-enter the male's chamber after an

ejaculation. All latencies were measured in seconds.

the

“III

mm
silt
by

lubl
sub.
mm

53161

94

Implant of electrodes:

Twenty female rats were implanted with electrodes in the uterus for this study.
Females were anesthetized by injection of mixtures of Ketamine (44 mg/kg) and Xylazine
(10 mg/kg).

A 3 to 4 cm midline incision on the abdomen was made with the scalpel and the
uterus was exposed by muscle retractors. Two mm of Teflon was removed from each tip
of Teflon-coated silver electrodes (0.003 in., bare diameter, Medwire, Mt. Vernon, NY) by
a scalpel. Two electrodes were implanted into the uterus around the uterine bifurcation
(near cervix) one at a time using a 25 gauge needle. The electrode was passed through a 25
gauge needle with the bare tip of the electrode bent backward and the 25 gauge needle along
with the bent electrode wire was inserted into the uterus. The needle was then pulled back
and the bare tip of the microelectrode was left in the uterus. The tips of the electrodes were
3 mm apart. Five mm of Teflon was removed from the free ends of the electrodes and
these were passed through the lumen of a piece of polyethylene tubing (PE-50, ID: 0.58
mm, OD: 0.965 mm) (Clay Adams, Division of Becton Dickinson and Company
Parsippany, New Jersey 07054). The PE tubing was secured to the abdomen muscles near
the operated area via 4—0 gut suture to prevent the electrodes from coming free. The tubing
with the electrode wires was passed subcutaneously around the abdomen, and along the
back of the body to the head. Each of the free ends of electrodes was soldered to a pin of a
miniature IC socket containing subminiature female connectors. A reference Teflon-coated
silver wire with 5 mm bare tip was bent backward and inserted securely into the leg muscle
by the same procedure previously described and passed through a second piece of PE
tubing. The PE tubing was sutured to the adjacent muscle with 4—0 gut suture, then passed
subcutaneously to the head where the free end of the electrode was soldered to a pin of the
miniature IC socket. The miniature IC socket was anchored to the skull by tiny bone

screws and dental acrylic. Antibiotics were applied to the skin around the incision and the

it);

alt
P“

COT

01

lol.

95
incision was closed with wound clips. The animal was injected with 5 mg Amoxicillin

(Amoxi-inject, SmithKline Beecham, West Chester, PA) daily for 4 days starting from the

day of surgery.

Recording of uterine electromyographic (EMG) activity:

One week after the implantation of the electrodes, females were tested with a male.
Prior to copulation, the female’s upper torso was fitted with a cloth harness (Stoelting
50556, Chicago, IL). The connectors of the miniature IC socket on the animal’s head were
attached to the leads on shielded-wires from a commutator (Stoelting 50506) mounted
above the testing arena by means of a small spring. A shielded tether cable was used to
protect the wires from animal’s biting and was tightly attached to the cloth harness. The
commutator, in turn, was connected to a Grass High Performance AC amplifier Model
7P511 (Grass Instrument Co., Quincy, MA 02169). The raw signal was filtered by half-
arnplitude low frequency filter to 10 Hz and half-amplitude high frequency filter to 3 kHz.

The sensitivity was set in the range of 50 to 200 pv/mm.

The female was placed in the pacing chamber and uterine EMG activity was
recorded for 30 minutes before the male was introduced. To prevent the male from
entering the escape chamber, a cloth harness was put on the male’s upper torso. Uterine
EMG activity and the temporal pattern of female sexual behavior were recorded for one
ejaculatory series. Recording was terminated 5 minutes after the ejaculation. The durations
of uterine EMG activity associated with sexual contacts and the female’s return latencies

following sexual contacts were analyzed and reported.

Data analysis:

The duration of uterine activity associated with each type of sexual contact was
recorded and analyzed by one way ANOVA followed by Fisher’s PLSD post-hoe tests.
Comparison of the MRL, IRL, and PER was achieved by one way ANOVA followed by
Fisher’s PLSD post-hoe tests. An alpha level of 0.05 was used for all statistical tests.

RESULTS

Uterine EMG activity of seven females was successfully monitored during
copulation. Uterine activity of two females was recorded at a paper speed of 10 mm/min.,
while in five females, uterine EMG activity was recorded at a paper speed of 5 mm/sec or
10 mm/sec and these data were used for analysis of the duration of uterine activity
associated with different types of sexual contacts.

Typical uterine EMG activity associated with each type of sexual contact recorded at
a paper speed of 5 mm/sec is illustrated in Figure 14. The duration of uterine contractions
associated with mounts, intromissions, and ejaculation is summarized in Figure 15. There
was a significant difference among the durations of uterine contractions associated with
sexual contacts [F(2, 11) = 23.189, p<0.0001]. The duration of uterine activity associated
with an ejaculation was significantly longer than that associated with an intromission or a
mount (p<0.01) (Figure 15). The duration of uterine contractile activity associated with an
intromission was not significantly different from that associated with a mount (Figure 15).
Data are expressed as mean :1; SEM in seconds.

A significant difference existed among the return latencies following a mount,
intromission, and ejaculation [F(2,6) = 5.435, p = 0.045]. The female’s return latency
following an ejaculation was significantly longer than that following an intromission or a

mount (p<0.05) (Figure 16). The female’s return latency following an intromission was

Figure 1 4 . shows the typical uterine EMG activity associated with a mount, intromission,
and ejaculation. The duration of uterine EMG activity associated with an ejaculation
seemed longer than that associated with an intromission or a mount. E ejaculation; I:
intromission; M: mount. Amplitude: 1 cm = 1 mV. Time scale (paper speed): 1 cm = 2
seconds. Arrows indicate the approximate time for dismount.

Figure l 5 . Comparison of the durations of uterine EMG activity associated with a mount,
intromission, and ejaculation. The duration of uterine EMG activity associated with an
ejaculation was significantly longer than that associated with a mount or an intromission
(** p<0.01). There was no significant difference found between durations of uterine EMG
activity associated with a mount and an intromission. Data are expressed as mean i SEM
in seconds and analyzed by one way ANOVA followed by Fisher’s PLSD post-hoc tests.

Duration of uterine activity (seconds)

 

** p<0.01

 

100

 

 

Mount

Intromission

Ejaculation

101

Figure 16. Comparison of the female’s return latencies following a mount (MRL),
intromission (IRL), and ejaculation (PER). The female’s PER was significantly longer
than her IRL or MRL (* p<0.05). Data are expressed as mean i SEM in seconds and
analyzed by one way ANOVA followed by Fisher’s PLSD post-hoc tests.

Return latency following sexual

contacts (seconds)

3501
300:
250i
200:
1503
100:

50‘

 

* p<0.05

 

 

 

102

 

Mount

Intromission

 

Ejaculation

103
not different from that following a mount (Figure 16). Data are expressed as mean i SEM

in seconds.

DISCUSSION

The major finding of the present study is to correlate uterine EMG activity with each
type of sexual contact and the temporal pattern of female rat sexual behavior. The duration
of uterine activity associated with an ejaculation was significantly longer than that
associated with an intromission or a mount. This finding coincides with the result that the
female’s return latency following an ejaculation is significantly longer than that following
an intromission or a mount (Bermant, 1961; Bermant and Westbrook, 1966; Erskine,
1985, 1989; Krieger et al., 1976; Lange and Clemens, 1994; Peirce and Nuttall, 1961a)
and that the intromission duration is significantly longer during ejaculation than during non-
ejaculatory intromissions (Erskine et al., 1989; Peirce and Nuttall, 1961b; Stone and
Ferguson, 1940). Possibly the longer duration of uterine activity associated with
ejaculation that reflects a more intense vaginocervical stimulation contributes to the female’s
PER.

The results of uterine EMG recordings are consistent with previous reports. In the
present studies, few uterine contractions were observed before copulation. This is
consistent with the previous finding that only some uterine contractile activity was observed
during the evening of the proestrus stage (Talo and Karki, 1976) and before copulation
(Toner and Adler, 1986). In the present study, uterine activity increased significantly
during active copulation and returned to the premating level following ejaculation. This is
also consistent with the previous report (Toner and Adler, 1986). In addition, the female’s
return latencies following sexual contacts follow the same pattern reported in other studies.
The female’s return latency following an ejaculation is significantly longer than that

following an intromission or a mount (Bermant, 1961; Bermant and Westbrook, 1966;

104
Erskine, 1985, 1989; Krieger et al., 1976; Lange and Clemens, 1994; Peirce and Nuttall,

1961a). In contrast to our previous report, the female’s return latencies following sexual
contacts lengthened in the present study. This may reflect the fact that the females were
recovering from electrode implants and adjusting to the harness as well as the tethering that
was required during testing.

One possible function of the copulation-induced uterine activity may be to facilitate
sperm transport in three ways. First, the immediate increase of uterine activity at the time
of ejaculation probably facilitates sperm transport from the vagina to the uterus. This is
supported by the evidence that sperm were found in the uterus as soon as 54 seconds
following ejaculation (Hartman and Ball, 1930). Secondly, the immediate increase of
uterine activity in response to active copulation may assist sperm deposited from previous
ejaculation(s) to advance in the uterus at a higher speed. Finally, the delayed increase of
uterine activity following multiple ejaculations (Toner and Adler, 1986) may serve to
facilitate transport of preexisting sperm that were deposited from the previous ejaculation(s)
in the uterus at a higher speed.

In summary, the duration of uterine EMG activity varies with each type of sexual
contact. The duration of uterine contractions associated with an ejaculation was
significantly longer than that associated with an intromission or a mount. Possibly this
longer duration of uterine EMG activity accompanying an ejaculation results from a more
intense stimulus and is responsible for the rapid sperm transport from the vagina to the

uterus and the longer return latency following ejaculation

EXPERIMENT 5: SEXUAL MOTIVATION AND THE MEDIAL PREOPTIC
AREA IN FEMALE RATS

ABSTRACT

Lesions of the media] preoptic area of hypothalamus (MPOA) have been reported to
increase the lordosis response in female rats. The lordosis response of female rats is also
facilitated by transections made anterior-dorsal to the MPOA. Based on these results, it has
been suggested that the MPOA inhibits female rat lordosis behavior. However, MPOA
lesions significantly decrease the time that the female rat spends with the male suggesting
that the MPOA may play a role in enhancing female sexual behavior. The objective of this
study was to investigate the relation of the MPOA to the temporal pattern of female sexual
behavior in rats. In Experiment 5A, we investigated the effect of electrolytic (1.5 mA for
20 seconds) lesions of MPOA on female copulation when the female could regulate the
timing of copulatory events (pacing). Results indicated that MPOA lesions significantly
increased the female’s latency to return to the male after an intromission (IRL) and after an

ejaculation, the postejaculatory refractory period (PER). Experiment 5B examined the

effect of ibotenic acid lesions (0.4 pl of ibotenic acid solution per side at a dose of 10 pg/ pl

0.1 M phosphate buffer, pH 7.40) of MPOA on female pacing behavior. Results for
chemical lesions were similar to those of electrolytic lesions suggesting that the alteration of
timing in lesion females results from loss of MPOA cells and not fibers of passage. It has
been reported that MPOA lesions that spare some part of MPOA significantly lengthened
the male’s interintromission interval (111) and postejaculatory interval (PEI). Our results

with females parallel these effects of WOA lesions in males. Based on these results, we
105

106
suggest that the MPOA plays a similar role in both males and females with regards to

regulating the temporal pattern of copulation and possibly sexual motivation.

INTRODUCTION

Lordosis behavior, hopping, darting and the temporal pattern of copulatory events
form the major components of female rat sexual behavior (Nelson, 1995; Pfaff, 1994). In
a standard test situation where the female rat can not escape from the male during
copulation, lordosis frequency, hopping, and darting serve as the primary measures of
female sexual behavior (Beach, 1976; Nelson, 1995; Pfaff et al., 1994; Young, 1961).
However, when given an opportunity, the female rat actively regulates the timing of
copulatory events (Bermant, 1961; Bermant & Westbrook, 1966; Clemens et al., 1995;
Erskine, 1985, 1987, 1989, 1992; Erskine & Baum, 1982; Erskine et al., 1989; Fadem et
al., 1979; Frye & Erskine, 1990; Gilman & Hitt, 1978; Krieger et al., 1976; Mermelstein
and Becker, 1995; Peirce& Nuttall, 1961a; Yang and Clemens, 1994, 1995a, & b, 1996a,
b, & c). One possible function of the temporal copulatory pattern of female rats is to
facilitate successful pregnancy. This is supported by the evidence that fewer intromissions
are sufficient to induce the progestational state of pregnancy when the female rat controls
the copulatory speed (Erskine et al., 1989; Gilman et al., 1979). Factors including gonadal
hormones (Fadem et al., 1979; Gilman and Hitt, 1978), preejaculatory intromissions (Yang
and Clemens, 1994, 1996a), and the ejaculation duration (Yang and Clemens, 1995b,
1996b) have been reported to influence the temporal copulatory pattern in female rats.
However, what brain regions control the timing of female copulation is poorly understood.

The media] preoptic area of hypothalamus (MPOA) has been reported to play an
important role in the regulation of female sexual behavior in rats. The MPOA lesions
increase the lordosis response in female rats (Hoshina et al., 1994; Powers and Valenstein,

1972; Whitney, 1986). The lordosis response of female rats is also facilitated after

107
transections made anterior-dorsal to the MPOA (Y amanouchi and Arai, 1977). Based on

these results, it has been suggested that the MPOA plays an inhibitory role in the regulation
of female lordosis behavior (Powers and Valenstein, 1972). However, MPOA lesions
dramatically decrease the time that the female spends with the sexually active males when
she can choose among sexually active males, castrated males and females (Whitney, 1986).
This finding suggests that the MPOA may play a role in facilitating female sexual behavior.
It is not known, however, whether the MPOA facilitates or inhibits the temporal pattern of
female sexual behavior. The study of whether and how the MPOA regulates the temporal
pattern of female sexual behavior is important at least in two aspects. First, it provides
further understanding of how the MPOA controls some other aspects of female sexual
behavior in addition to lordosis behavior. Secondly, it elucidates whether the same brain
region regulates the temporal pattern of male and female sexual behavior in a parallel
manner and allows comparison of the role of the same brain region in the male and female
sexual behavior under similar measurement scales.

Vaginocervical stimulation plays an important role in the regulation of the temporal
pattern of female rat sexual behavior. Following bilateral transection of the pelvic nerve
and the pudendal nerve that carry sensory stimulation from the vagina, cervix, uterus,
perineal skin, and clitoral sheath (Berkley et al., 1990; Berkley et al., 1993; Komisaruk et
al., 1972; Peters et al., 1987), females do not distinguish among mounts, intromissions,
and ejaculations; the return latencies following mounts, intromissions and ejaculations are
not significantly different from one another (Erskine, 1992). In addition, anesthetics
applied locally to the vagina of the female rats reduced their contact-response intervals
following intromissions and ejaculations (Westbrook and Bermant, 1966). Based on these

results, it is suggested that vaginocervical stimulation conveyed via the pelvic and pudendal
nerves is important for the temporal pattern of female sexual behavior. Furthermore,
evidence shows that the neurons in MPOA are activated during copulation. Vaginocervical

Stimulation provided by a male rat during copulation or a glass rod by the experimenter

8191

II:

III:
co

m:

108
significantly increases the c-fos expression in the MPOA of female rats (Pfaus et al., 1993;

Wersinger et al., 1993). Mechanical stimulation of uterine cervix excites MPOA neurons
(Haskins and Moss, 1983). That the neurons in the MPOA are activated during copulation
and that vaginocervical stimulation is associated with the temporal copulatory events of
female rats together suggest that the MPOA is a potential candidate in the regulation of the
temporal pattern of female sexual behavior.

The MPOA also plays an important role in the regulation of male rat sexual
behavior. It has been reported that copulation significantly induces c-fos expression in the
MPOA of male rats (Baum and Everitt, 1992; Wersinger et al., 1993). In addition, large
MPOA lesions abolished male sexual behavior (Giantonio et al., 1970; Ginton and Merari,
1977; Heimer and Larsson, 1966/1967; Hendricks and Scheetz, 1973; Lisk, 1968). It is
also true that some males with large MPOA lesions sparing part of MPOA can still copulate
although their interintromission interval (III), ejaculation latency (EL) and postejaculatory
interval (PEI) lengthen (Ginton and Merari, 1977). Further, electrical stimulation of the
MPOA facilitates male rat copulatory behavior (Malsbury, 1971). The above evidence
suggests that the MPOA facilitates the temporal pattern of male sexual behavior in rats.
Whether the MPOA exerts a parallel effect on the temporal pattern of female sexual
behavior is not understood.

The objective of this study was to investigate the role of MPOA in the regulation of
the temporal pattern of female sexual behavior in rats. In addition, we used the data
collected in this study to determine whether the MPOA regulates the temporal aspects of
male and female sexual behavior in a parallel manner. Experiment 5A examined the
influence of electrolytic lesions of MPOA in the regulation of the temporal pattern of female
rat sexual behavior. Experiment 5B investigated the effect of chemical (ibotenic acid)
lesions of MPOA on the female pacing behavior in rats.

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109
GENERAL METHODS

Animals

Sixty-day-old female and ninety-day-old male Long Evans rats were purchased
from Harlan Sprague Dawley, Inc. (Indianapolis, IN). All females were ovariectomized
one week later upon arrival. Animals were housed in 16:8 hr light-dark cycle with lights
off at 17:00. Females were housed three per cage and males were housed singly. Food

and water were available ad lib. The temperature and humidity of the animal room were

maintained at 220 C and 55%, respectively. The air in the animal room was exchanged 12

times per hour automatically. Ovariectomized females were brought into estrus by

intramuscular (IM) injections of 0.5 pg estradiol benzoate (EB) (Sigma Chemical Co., St.

Louis, MO) dissolved in 0.1 ml sesame oil (Sigma Chemical Co., St. Louis, M0) for three
consecutive days and 24 hours later followed by an IM injection of 0.5 mg progesterone
(P) (Sigma Chemical Co., St. Louis, MO) dissolved in 0.1 ml sesame oil. All female
pacing behavior was tested 4-7 hours after the injection of progesterone (2-5 hours after the
lights off) under the dimly red illumination. All animals were tested once per week
throughout the whole test period and had been tested twice in the testing chamber before
data were collected. Females that did not exit the male’s chamber at all during the two
experience tests were excluded from further studies. Only those females (over 95 %)
showing pacing behavior in these preliminary tests were used in the experimental studies.
In each test, a male was always placed in the testing chamber 15 minutes before a female
was introduced. The approximate weights of the female and male rats were 200 - 300 g and

500 - 550 g, respectively, when the tests started.

110
Pacing Tests

To provide a test situation in which the female controlled the pacing of the
copulation, females were tested in a two-compartment chamber which was divided by a
Plexiglas barrier with 4 holes (3.5~4 cm x 3.5~4 cm) spaced along the bottom of the
barrier. The dimensions of the male's chamber and escape chamber were 35.5 cm x 44.5
cm x 48.3 cm and 21.5 cm x 44.5 cm x 48.3 cm (Length x Width x Height), respectively.
The escape holes were large enough to allow the female to pass through but prevented the

larger male from following her.
Behavioral Measures

Several behavioral measures were recorded in the pacing tests: approach latency
(AL), the latency for the female to enter the male's chamber after the start of the test (she
was placed in the escape chamber at the beginning of the test); intromission latency (IL),
the latency from the female's entry into the male's chamber to the first intromission; mount
return latency (MRL), the latency for the female to re-enter the male's chamber after a
mount; intromission return latency (IRL), the latency to return to the male's chamber
following an intromission; postejaculatory refractory period (PER), the latency to re-enter
the male's chamber after an ejaculation. All latencies were measured in seconds. The
frequency with which the female left the male's chamber following a mount, intromission
and ejaculation (percentage exit) was calculated by dividing the number of exits by the
number of each type of stimulus. In addition, the female’s lordosis behavior was recorded
under paced situation and non-paced situation. Under paced situation, the female was
allowed to copulate until she received an ejaculation and returned to the male’s chamber
following her PER. If the female did not leave the male’s chamber following ejaculation,

the test was ended 60 seconds after ejaculation. In addition, if the female did not receive an

1 1 1
ejaculation due to the long return latencies following intromissions within 15 minutes after

the start of the test, the test was terminated. Immediately following paced test, the lordosis
quotient of female rats was obtained by allowing the female to receive 10 mounts under
non-paced situation (The barrier separating the male’s chamber and the escape chamber was
removed). The female lordosis quotient was calculated by the formula: (number of lordosis
during the first 10 mounts / 10) x 100 or (number of lordosis / number of mounts) x 100

when the number of mounts was smaller than 10. All behavioral data were recorded on a

computer disk.

EXPERIMENT 5A: INFLUENCE OF ELECTROLYTIC MPOA LESIONS
ON THE FEMALE PACING BEHAVIOR

The objective of this experiment was to examine the effect of electrolytic lesions of
MPOA on the temporal pattern of female sexual behavior in rats. It has been reported that
the MPOA lesions potentiate the lordosis behavior (Hoshina et al., 1994; Whitney, 1986)
and shorten the time that the female spends with the sexually active males during copulation
(Whitney, 1986). In this experiment, we investigated the influence of electrolytic lesions
(1.5 mA current for 20 seconds) of MPOA on the temporal copulatory pattern of female

rats.

METHOD
Procedure:

Thirty-two male and 32 female Long Evans rats were used in this study. The
MPOA coordinates (B: - 0.7 mm, L: i 0.5 mm, V: - 8.2 mm) were determined empirically.
Animals were randomly assigned into two groups: the MPOA lesioned group and the
control group. In the MPOA lesioned group, females received bilateral electrolytic lesions

(1.5 mA current for 20 seconds) of MPOA. The electrolytic lesions of MPOA were made

1 12
by Tungsten nricroelectrode with 8 degree tapered tip (diameter. 500 pm, catalog #5770, A-

M Systems, Inc., Everett, WA) connected to 3500 Lesion Producing Device (Stoelting
Co., Wood Dale, IL) (N=21). In the control group, the females received anesthesia and
bilateral electrode insertion into the MPOA without passing any current (N=1 1).

In a three-week test period, the temporal pattern of copulation for each female was
tested before surgery, and two weeks after surgery. Each female was randomly assigned to
copulate with one male until she received an ejaculation and returned to the male’s chamber
following her PER in the first week of this experiment. Following the prelesion test,
females received either MPOA lesions (N = 21) or sham lesions (N = 11). Throughout the
rest of the test, each female was tested with the same male she mated in the first week of
this experiment. One week after surgery, the female was allowed to have one post surgery
experience test (without recording data) in female paced situations. Two weeks after
surgery, each female was tested for the temporal pattern of female sexual behavior until she
received an ejaculation and returned to the male’s chamber following her PER. The
temporal pattern of female sexual behavior was recorded and analyzed before surgery and
two weeks after surgery. In the MPOA lesioned group, only those animals with more than

50 % of the MPOA destroyed on both sides were used for analysis.

Histology:

Following postsurgical behavioral tests (two weeks after surgery), all females were
anesthetized with mixtures of Ketamine (44 mg/kg) and Xylazine (10 mg/kg) (Fort Dodge
Laboratories, Inc., Fort Dodge, Iowa) and perfused intracardially with 0.87% saline
followed by 10% formalin. Brains were removed and post-fixed in 10% formalin for

overnight. The brains were then transferred to 20% sucrose solution for cryoprotection

113

until they sank. Frozen coronal sections (50 pm) were taken and the lesioned site was

verified following Nissl staining.

Statistics:

The data of the female’s percentage exits following mount, intromission, and
ejaculation were transformed by arc sine square root before analysis (Dixon and Massey,
1969). The measures of the temporal pattern of female sexual behavior obtained before
surgery and two weeks after surgery were analyzed by Two Way Repeated Measures
ANOVA followed by SNK post-hoc test. An alpha level of 0.05 was used for all statistical

ICSIS.

RESULTS

Electrolytic lesions of the MPOA destroyed an area of approximately 900 pm in the

rostrocaudal direction and were limited to a region under the anterior commissure (Figure
17). Eight of 21 females that had more than 50% of the MPOA destroyed on both sides
were used for analysis. In these 8 cases, large portions of bilateral MPOA and
periventricular hypothalamic nucleus were damaged. In addition, variable amounts of
septohypothalarnic nucleus, parastrial nucleus, bed nucleus of the stria terminalis, lateral
preoptic area, striohypothalamic nucleus, supraoptic nucleus and suprachiasmatic nucleus
were destroyed. The optic chiasm was absent in the brain tissue of the electrolytic MPOA
lesioned animals. This absence of the optic chiasm may not have resulted from the
electrolytic lesions because the MPOA animals did not appear to have difficulty in seeing
during behavioral test.

The mean return latencies following an intromission and an ejaculation in the

114
MPOA lesioned and sham-lesioned groups before surgery and after surgery are

summarized in Figures 18 and 19, respectively. One of 8 MPOA lesioned females with
destruction of both sides of MPOA larger than 50% was excluded from the PER analysis
because the female’s IRL was very long and the test was terminated before the male
ejaculated. All the data are represented as mean i SEM in seconds.

Two Way Repeated Measures ANOVA revealed a significant group effect [ F( 1,
17) = 19.481, p = 0.0004], surgery effect [F(1, 17) = 16.652, p = 0.0008], as well as
group x surgery interaction [F(1, 17) = 22.033, p = 0.0002] for the female’s IRL. The
SNK post-hoe tests revealed that the postsurgical IRL of females with MPOA lesions was
significantly longer than the other IRLs (p<0.01) (Figure 18).

Two Way Repeated Measures ANOVA also revealed a significant group effect [
F(1, 16) = 9.828, p = 0.0064], surgery effect [F(1, 16) = 7.868, p = 0.0127], as well as
group x surgery interaction [F(1, 16) = 11.094, p = 0.0042] for the female’s PER. The
SNK post-hoe tests revealed that the postsurgical PER of females with MPOA lesions was
significantly longer than the other PERs (p<0.01) (Figure 19). The lordosis quotient of
females tested under paced situation was not different from that tested under nonpaced
situation, nor was there a significant difference in the female’s lordosis quotients before
surgery and after surgery. None of the other behavioral measures differed significantly
before surgery and after surgery in the MPOA lesioned and control groups. Thirteen of 21
females receiving bilateral electrolytic MPOA lesions had one or both sides of MPOA
damaged less than 50%. These females appeared to show normal temporal copulatory
behavior.

115

Figure 17. Composite diagrams of largest (shaded area) electrolytic MPOA lesions.
Figures and nomenclature from Paxinos and Watson (1986). The Bregma coordinates for
each figure on top row from left to rightare + 0.20 mm, - 0.30 mm, and - 0.80 mm. The
Bregma coordinates for each figure on bottom row from left to right are - 0.92 mm, and -
1.30 mm. 3V: 3rd ventricle; ac: anterior commissure; aca: anterior commissure, anterior;
AHA: anterior hypothalamic area, anterior; BSTMPL: bed nucleus of the stria terminalis,
medial division, posterolateral part; BST V: bed nucleus of stria terminalis, ventral division;
f: fomix; HDB: nucleus of the horizontal limb of the diagonal band of Broca; LA:
lateroanterior hypothalamic nucleus; LH: lateral hypothalamic area; LPO: lateral preoptic
area; MPA: medial preoptic area; MPO. medial preoptic nucleus; ox: optic chiasm; PaAP.
paraventricular hypothalamic nucleus, anterior parvocellular part; Pe: periventricular
hypothalamic nucleus; PS: parastrial nucleus; SCh: suprachiasmatic nucleus; SHy:
septohypothalarnic nucleus; StHy: striohypothalamic nucleus; SO: supraoptic nucleus;
VDB: nucleus of the vertical limb of the diagonal band of Broca.

\

 

117

Figure 1 8. srunmarizes the female’s return latencies following an intromission
(intromission return latency, IRL) before surgery and two weeks after surgery in the
MPOA electrolytic lesioned group (N = 8) and the MPOA sham-lesioned group (N = 11).
Two Way Repeated Measures ANOVA revealed a significant group effect [ F(1, 17) =
19.481, p = 0.0004], surgery effect [F(1, 17) = 16.652, p = 0.0008], as well as group x
surgery interaction [F(1, 17) = 22.033, p = 0.0002] for the female’s IRL. The SNK post-
hoc tests revealed that the postsurgical IRL of females with MPOA electrolytic lesions was
significantly longer than the other IRLs (p<0.01). ** p<0.01.

Return latency following an

intromission (seconds)

70-

 

 

118

E] MPOA sham-lesioned group
MPOA electrolytic lesioned group

** p<0.01

 

Prelesion

 

 

 

Surgical conditions

 

119

Figure 1 9. shows the female’s return latencies following an ejaculation (postejaculatory
refractory period, PER) before surgery and two weeks after surgery in the MPOA
electrolytic lesioned group (N = 7) and the MPOA sham-lesioned group (N = 11). Two
Way Repeated Measures ANOVA revealed a significant group effect [ F(1, 16) = 9.828, p
= 0.0064], surgery effect [F(1, 16) = 7.868, p = 0.0127], as well as group x surgery
interaction [F(1, 16) = 11.094, p = 0.0042] for the female’s PER. The SNK post-hoe tests
revealed that the postsurgical PER of females with MPOA lesions was significantly longer
than the other PERs (p<0.01). ** p<0.01.

Return latency following an

ejaculation (seconds)

3501
300.3
250-
200-:
150.3
100~

50-

 

E] MPOA sham-lesioned group
MPOA electrolytic lesioned group

** p<0.01

 

 

 

120

 

 

 

Surgical conditions

121
EXPERIMENT 5B: EFFECT OF CHEMICAL MPOA LESIONS ON THE

FEMALE TEMPORAL COPULATORY BEHAVIOR

The objective of this experiment was to examine whether destruction of cell bodies
in the MPOA but sparing the fibers passing through MPOA mimics the effects produced by
electrolytic lesions of MPOA. In Experiment 5A, electrolytic lesions of MPOA
significantly increased the female’s PER and IRL. Both cell bodies in the MPOA and the
fibers passing through that area can be destroyed by electrolytic lesions. The influence of
electrolytic lesions of MPOA in the regulation of the temporal pattern of female sexual
behavior may result from destruction of MPOA cell bodies and/or the fibers passing
through that area. In contrast, chemical lesions can only destroy the cell bodies and spare
the passing fibers (Schwarcz et al., 1979; Schwarcz et al., 1979). In this experiment,
chemical (ibotenic acid) lesions of MPOA were performed in the females and the temporal

pattern of female sexual behavior was recorded before surgery and two weeks after

surgery.

METHOD

Procedure:

Nineteen female and nineteen male Long Evans rats were used in this experiment.
Except for lesion procedures, the procedures in this experiment were the same as those of
Experiment 5A. The MPOA microinjection coordinates (B: - 0.7 mm, L.’ i 0.5 mm, V: -
7.6 mm) were aimed at the top of the MPOA and determined empirically. Animals were
randomly assigned into two groups: the MPOA lesioned group (N = 10) and the control

group (N = 9). In the MPOA lesioned group, the females received bilateral nricroinjection

0f ibotenic acid solution (10 pg ibotenic acid per pl 0.1 M phosphate buffer solution at pH

122
7.40) into MPOA via 1 pl microsyringe connected to a 26 gauge steel needle. A volume of

0.4 pl of ibotenic acid solution was nricroinjected into each site of MPOA via a

microprocessor controlled syringe pump (Model 53100, Stoelting Co., Wood Dale, IL)

over a 10-minute period. The syringe was left in place for another 10 minutes to avoid
backflow of ibotenic acid into the needle track. In the control group, a volume of 0.4 pl

0.1 M phosphate buffer solution (pH 7.40) instead of ibotenic acid solution was
microinjected into each site of MPOA via a microprocessor controlled syringe pump over a
10-minute period. The rest of the procedures were the same as those in the MPOA lesioned
group.

The temporal pattern of female sexual behavior and lordosis responses were tested
before surgery, and two weeks after surgery. In the MPOA lesioned group, only those
females with chemical MPOA lesions destroying more than 50% of MPOA at both sides
were included for analysis. The data obtained before surgery and two weeks after surgery

were used for analysis.

Histology:

Following postsurgical behavioral tests (two weeks after surgery), all females were
anesthetized with mixtures of Ketamine (44 mg/kg) and Xylazine (10 mg/kg) (Fort Dodge
Laboratories, Inc., Fort Dodge, Iowa) and perfused intracardially with 0.87% saline
followed by 10% formalin. Brains were removed and post-fixed in 10% formalin for

Overnight. The brains were then transferred to 20% sucrose solution for cryoprotection

until they sank. Frozen coronal sections (50 pm) were taken and the lesioned site was

Verified following Nissl staining.

123
Statistics:

The data of the female’s percentage exits following mount, intromission, and
ejaculation were transformed by arc sine square root before analysis (Dixon and Massey,
1969). The measures of the temporal pattern of female sexual behavior obtained before
surgery and two weeks after surgery were analyzed by Two Way Repeated Measures
ANOVA followed by SNK post-hoe test. An alpha level of 0.05 was used for all statistical

tests.

RESULTS

The ibotenic acid lesions of MPOA were characterized by disappearance of neurons
and an enlarged third ventricle in tlrionin stained sections. Eight of 10 females had bilateral
ibotenic lesions that were centered in the MPOA at the level of optic chiasm and destroyed

more than 50% of MPOA on both sides. The ibotenic acid lesions damaged an area of

approximately 1,700 pm in the rostrocaudal direction and were limited between the anterior

commissure and the optic chiasm (Figure 20). The lesions included most of MPOA in
these 8 animals. In addition, the lesions also covered variable amounts of the
periventricular hypothalamic nucleus, septohypothalamic nucleus, parastrial nucleus, bed
nucleus of stria terminalis, striohypothalamic nucleus, supraoptic nucleus, suprachiasmauc
nucleus, lateral preoptic area, diagonal band, and the anterior hypothalamus. The lesions
extended anteriorly into part of diagonal band and posteriorly into part of anterior
hypothalamus. Two of these 8 females with MPOA lesions covering the entire MPOA as
Well as anterior commissure and extending laterally to a large portion of lateral preoptic area
did not approach the male during the 15-minute postsurgery test. In contrast, two of 10

IFemales had MPOA lesions less than 50% and were excluded from analysis.

124
Figure 21 summarizes the mean IRLs of the females with MPOA sham lesions and

the females with MPOA ibotenic acid lesions before and after surgery. Two females with
extensive ibotenic acid MPOA lesions including LPO were excluded from the IRL analysis
because the female did not approach the male during 15—minute test period. These two
females showed normal motor activity and moved around in the escape chamber during the
15-minute test period. In addition, while they often rejected the male, they showed normal
lordosis behavior when they were mounted by the male after the barrier was removed. All
the data are represented as mean i SEM in seconds.

Two Way Repeated Measures ANOVA revealed a significant group effect [ F( 1,
13) = 12.296, p = 0.0039], surgery effect [F(1, 13) = 10.510, p = 0.0064], as well as
group x surgery interaction [F(1, 13) = 18.408, p = 0.0009] for the female’s IRL. The
SNK post-hoc tests revealed that the postsurgical IRL of females with MPOA ibotenic acid
lesions was significantly longer than the other IRLs (p<0.01) (Figure 21).

The female’s PERs before and after surgery in the sham group and the MPOA
ibotenic acid group are summarized in Figure 22. The two females with extensive MPOA
ibotenic acid lesions that did not approach the male during the 15-minute test period were
excluded from the PER analysis. There were a significant group effect [ F(1, 13) =
39.933, p< 0.0001], surgery effect [F(1, 13) = 30.108, p = 0.0001], as well as group x
surgery interaction [F( 1, 13) = 36.848, p < 0.0001] for the female’s PER. The SNK post-
hoc tests revealed that the postsurgical PER of females with MPOA ibotenic acid lesions
was significantly longer than the other PERs (p<0.01) (Figure 22). The lordosis quotient
of females tested under the female paced situation was not different from that under the
nonpaced situation. There was no significant difference in the female’s lordosis quotient
before and after surgery. For the other behavioral measures, there was also no significant

difference before and after surgery in the MPOA lesioned and control groups.

125

Figure 20. Composite diagrams of largest (shaded area) ibotenic acid MPOA lesions.
Figures and nomenclature from Paxinos and Watson. The Bregma coordinates for each
figure on top row from left to right are + 0.20 mm, - 0.30 mm, and - 0.80 mm. The
Bregma coordinates for each figure on bottom row from left to right are - 1.30 mm, and -
1.80 mm. 3V: 3rd ventricle; ac: anterior commissure; aca: anterior commissure, anterior;
AHA: anterior hypothalamic area, anterior; AHC: anterior hypothalamic area, central part;
BSTMPL: bed nucleus of the stria terminalis, medial division, posterolateral part; BST V:
bed nucleus of stria terminalis, ventral division; f: fomix; HDB: nucleus of the horizontal
limb of the diagonal band of Broca; LA: lateroanterior hypothalamic nucleus; LH: lateral
hypothalamic area; LPO: lateral preoptic area; MPA: medial preoptic area; MPO medial
preoptic nucleus; opt: optic tract; ox: optic chiasm; PaAP. paraventricular hypothalamic
nucleus, anterior parvocellular part; Pe: periventricular hypothalamic nucleus; PS: parastrial
nucleus; RCh: retrochiasmatic area; SCh: suprachiasmatic nucleus; SHy: septohypothalarnic
nucleus; StHy: striohypothalamic nucleus; SQ supraoptic nucleus; sox: supraoptic
decussation; VDB: nucleus of the vertical limb of the diagonal band of Broca.

126

 

127

Figure 2 1 . The female’s return latencies following an intromission (intromission return
latency, IRL) before surgery and two weeks after surgery in the MPOA chemical lesioned
group (N = 6) and the MPOA sham-lesioned group (N = 9). Two Way Repeated Measures
ANOVA revealed a significant group effect [ F(1, 13) = 12.296, p = 0.0039], surgery
effect [F(1, 13) = 10.510, p = 0.0064], as well as group x surgery interaction [F(1, 13) =
18.408, p = 0.0009] for the female’s IRL. The SNK post-hoc tests revealed that the
postsurgical IRL of females with MPOA ibotenic acid lesions was significantly longer than
the otherIRLs (p<0.01). ** p<0.01.

Return latency following an

intromission (seconds)

100-
90:
so;
70:
604
50.:
4o:
30-
20:

10:

 

128

E] MPOA sham-lesioned group *
MPOA chemical lesioned group

** p<0.01

 

 

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Surgical conditions

129

Figure 22. shows the female’s return latencies following an ejaculation (postejaculatory
refractory period, PER) before surgery and two weeks after surgery in the MPOA chemical
lesioned group (N = 6) and the MPOA sham-lesioned group (N = 9). There were a
significant group effect [ F(1, 13) = 39.933, p < 0.0001], surgery effect [F(1, 13) =
30.108, p = 0.0001], as well as group x surgery interaction [F(1, 13) = 36.848, p <
0.0001] for the female’s PER. The SNK post-hoc tests revealed that the postsurgical PER
of females with MPOA chemical (ibotenic acid) lesions was significantly longer than the
other PERs (p<0.01). ** p<0.01.

Return latency following an
ejaculation (seconds)

130

225 ' [j MPOA sham-lesioned group I a.
200 e MPOA chemical lesioned group

175 - ** p<0-01
150 -
125 .
100 -
75 .
50 ..
25 .

 

 

 

 

 

 

 

 

Prelesion

Surgical conditions

131
DISCUSSION

One of the major findings in this study is that the neurons in the MPOA influence
the temporal pattern of female sexual behavior in rats. In Experiment 5A, electrolytic
lesions of MPOA significantly increased the female’s IRL and PER. This effect appears to
result from destruction of cell bodies not fibers of passage since similar results were seen in
Experiment 5B, using ibotenic acid lesions, which destroyed neurons in the MPOA but
spared fibers of passage (Schwarcz et al., 1979; Schwarcz et al., 1979).

Sexual behavior of female rats has at least three major components: lordosis
behavior, soliciting behavior and the temporal pattern of the female’s approach to the male.
The preoptic area appears to play a role in the occurrence of each of these components. For
lordosis, it has been reported that MPOA lesions significantly increase the lordosis
responding in female rats treated with low doses of estradiol benzoate plus progesterone
(Hoshina et al., 1994; Powers and Valenstein, 1972; Whitney, 1986). In the present
study, the female’s lordosis behavior did not change following MPOA lesions, due to the
fact that the female’s lordosis quotient was already high (around 90% to 100%) before
surgery under the hormone replacement paradigm used in this laboratory (Clemens et al.,
1981; Richmond and Clemens, 1986; Richmond and Clemens, 1988). Based on the
finding that MPOA lesions increase lordosis frequency in female rats treated with low
doses of EB plus P, it has been suggested that the MPOA exerts an inhibitory effect on
female sexual receptivity (lordosis behavior) (Powers and Valenstein, 1972).

As for soliciting behavior, MPOA lesions did not affect the soliciting behavior (the
frequency of hopping, darting and ear wiggling) in female rats under non-paced situations
(Whitney, 1986). This is consistent with the results of a later study (Hoshina et al., 1994).
However, under female-paced situations, MPOA lesions decreased soliciting behavior in

female rats (Whitney, 1986).

132
With regard to the female’s approach to the male, MPOA lesions have been

demonstrated to shorten the time that the female spends with the sexually active males when
she has the opportunity to choose among the sexually active males, castrated males, or
females (Whitney, 1986). In the present study, we found that the MPOA lesions
significantly increase the female’s IRL and PER. This is consistent with the idea that the
MPOA facilitates the f emale’s approach to the male during copulation. Thus, it appears that
the MPOA inhibits the lordosis responding when estrogen is low and facilitates the
initiation of sexual interaction at higher levels of estrogen priming.

The MPOA may be important for facilitating sexual motivation in female rats. A
distinction between sexual desire (sexual motivation) and sexual ability has been proposed
(Wallen, 1990). Female sexual ability refers to the female’s ability to engage in copulation.
Female sexual motivation refers to “the state underlying sexual initiation and sexual
accommodation” (p.234, Wallen, 1990). In female rats, the display of the lordosis
response, the arching of the back and the raising of the hips and genitalia, is necessary for
successful penile insertion (Diakow, 1974; Pfaff et al., 1978). Consequently, occurrence
of the lordosis response has been used to measure the female’s sexual ability. On the other
hand, “female sexual motivation controls female availability and the temporal patterning of
copulation” (p. 235, Wallen, 1990). Within this context, the increased IRL and PER can
be interpreted as a decrease in sexual desire. In the present study, two females with
extensive MPOA ibotenic acid lesions did not approach the male during lS—minute test but
they showed motor activity with no apparent deficit and normal lordosis reflexes when
mounted by a male after the barrier was removed. This suggests that the sexual motivation
of these two females was absent but their sexual ability was unaffected. In male rats, the
interintromission interval is often considered as an index of rearousal rate of his sexual
motivation following a brief refractory period after an intromission (Beach, 1956; Meisel
and Sachs, 1994). Correspondingly, in female rats, the female’s IRL can be a measure for

rearousal rate of her sexual motivation following a brief refractory period after an

133
intromission. Therefore, increases in the female’s IRL as seen in the present study imply a

decrease in her sexual arousal.

Another important finding in this study is that the MPOA regulates the temporal
pattern of male and female sexual behavior in a similar manner. Traditionally, different
measurement scales were used to study the male and female sexual behavior. In male rats,
the frequency of sexual contacts (mount frequency, intromission frequency, and ejaculation
frequency) and the latency of sexual contacts (mount latency, intromission latency,
ejaculation latency, interintromission interval, and postejaculatory interval) were used to
measure male sexual behavior (Meisel and Sachs, 1994). In female rats, the lordosis
response to the male’s mounting behavior was the major measure of female sexual behavior
(Pfaf f et al., 1994). Use of different measurement scales for male and female behavior has
made comparison between sexes difficult. In the present study, we obtain measures for the
female thatare operationally similar to those used in studies of male sexual behavior. The
female’s intromission return latency is equivalent of the male’s interintromission interval.
The female’s postejaculatory refractory period is equivalent of the male’s postejaculatory
interval. The increase of the IRL and PER in female rats following MPOA lesions parallels
the increase of the interintromission interval and postejaculatory interval in male rats that
still copulate following MPOA lesions sparing part of MPOA (Ginton and Merari, 1977).
In addition, two females with MPOA ibotenic acid lesions did not approach the male during
15-minute test period. However, they displayed normal lordosis behavior when they were
mounted by the male after the barrier was removed following 15-minute paced test.
Likewise, males with large MPOA lesions did not copulate after lesion (Ginton and Merari,
1977; Heimer and Larsson, 1966/1967). These data together strongly support the idea that
the MPOA regulates the temporal pattern of male and female sexual behavior in a parallel
manner.

In addition to the temporal copulatory pattern, the MPOA regulates the lordosis

behavior and mounting behavior of sexual behavior similarly in both male and female rats.

134
It is well documented that MPOA lesions facilitate lordosis behavior in female rats

(Hoshina et al., 1994; Powers and Valenstein, 1972; Whitney, 1986). Similarly, it is
demonstrated that MPOA lesions facilitate lordosis in castrated male rats treated with
estradiol benzoate and progesterone (Hennessey et al., 1986; Olster, 1993). In addition,
large MPOA lesions abolished male sexual behavior in male rats (Heimer and Larsson,
1966/ 1967). Mounting behavior is the key feature of male rat sexual behavior. Therefore,
it is doubtless to infer that MPOA lesions decrease mounting in male rats. Likewise,
MPOA lesions have been reported to decrease mounting in female rats treated with
testosterone (Singer, 1968). All the above evidence taken together suggests that the MPOA
regulates the lordosis behavior and mounting behavior of male and female sexual behavior
in a parallel manner.

In summary, we demonstrate that destruction of neurons in the MPOA rather than
destruction of the fibers of passage significantly increases the female’s IRL and PER. This
finding suggests that neurons in the MPOA facilitate the temporal pattern of female rat
sexual behavior. This is also consistent with the previous report that MPOA lesions
significantly decreased the time that the female rat spent with the sexually active males. In
addition, the MPOA regulates the temporal pattern of male and female sexual behavior in a
similar manner. This is supported by the evidence that MPOA lesions significantly
lengthen the III and PEI in male rats that still copulate and dramatically increase the IRL and
PER in female rats. Moreover, in addition to the temporal copulatory pattern, the MPOA
regulates the lordosis response and the mounting behavior of male and female rats in a like
manner. Finally, following MPOA lesions, the dramatic increase in the female’s IRL and
the decrease in the time that the female spent with the sexually active males following
MPOA lesions strongly support the idea that the MPOA plays an important role in

facilitating female sexual motivation in rats.

GENERAL DISCUSSION

The present studies demonstrated that preejaculatory intromissions, the male’s
ejaculation duration, and variation in gonadal hormones affected the temporal pattern of
female rat’s sexual behavior. As intromission frequency prior to ejaculation increased, the
male’s ejaculation duration also increased and this was associated with an increase in the
female’s PER. In addition, uterine EMG activity also changed with the type of stimulus
that the female received during copulation. The duration of the uterine contractions was
longer during ejaculation than during intromission or mount. Finally, we demonstrated that
the MPOA played a role in the regulation of the temporal pattern of female sexual behavior.
Lesions (both electrolytic and chemical) of MPOA significantly increased the female’s
return latencies following intromission and ejaculation. This supports the idea that the

MPOA facilitates female sexual motivation.

Significance of Female Temporal Sexual Behavior

The temporal pattern of female rat’s sexual behavior has both behavioral and
physiological significance. When the female rat regulates the timing of mating, the
c0pulatory speed is slower (Erskine, 1985; Erskine et al., 1989; Fadem and Barfield, 1982;
Gilman et al., 1979) and fewer intromissions are needed to induce the progestational state
of pregnancy (Erskine et al., 1989; Gilman et al., 1979). In female paced situations,
several studies have reported that five intromissions are sufficient to induce secretion of
progesterone to a level sufficient for maintaining successful pregnancy (Erskine, 1989;

Erskine etal., 1989; Gilman etal., 1979). In contrast, in male paced tests, approximately
135

136
10 intromissions were required for the induction of the progestational state of pregnancy

(Adler, 1969; Chester and Zucker, 1970; Edmonds et al., 1972; Erskine, 1989; Erskine et
al., 1989; Gilman et al., 1979). Intronrissions were more effective in shortening the
behavioral estrus of female rats in female paced situations than in nonpaced situations. In
female paced tests, 10 intromissions were adequate to shorten the behavioral estrus
(Erskine, 1985; Erskine and Baum, 1982; Erskine et al., 1989). In contrast, in male paced
situations, shortening of behavioral estrus required at least 25 intromissions (Erskine and

Baum, 1982).

Stimulus Factors that Affect Female Temporal Copulatou Behavior

In the present studies, we demonstrated that several factors affected the female’s
postejaculatory refractory period (PER). These factors include preejaculatory intromissions
and the male’s ejaculation duration.

Different numbers of intromissions received by the female rat prior to ejaculation
affect the female’s PER. In the present experiments, we demonstrated that the PER of
females receiving 0-1 intromission prior to ejaculation was significantly shorter than that in
the other groups (2-4, 5-15, and 24—31 intromission groups) and was not different from
her intromission return latency. The females receiving two intromissions prior to
ejaculation had a PER which was not different from that of females receiving 5—15
intromissions. The females receiving 24—31 intromissions before ejaculation had a
significant longer PER than those in the other groups (0-1, 2-4, and 5-15 intromission
groups). In addition, correlation analysis showed that preejaculatory intromission
frequency was positively correlated with the female’s PER when the range of intromissions
received prior to ejaculation was large (0-31 and 0- 18 in the present studies).

One possible function of multiple intromissions prior to ejaculation may be to

prepare the vagina for a deep penile insertion during ejaculation that consequently induces a

137
longer ejaculation duration. A longer ejaculation duration in turns results in a longer PER.

The male’s ejaculation duration plays an important role in the induction of the female’s
PER. It has been reported that the male’s ejaculation duration increases over multiple
ejaculatory series (Peirce and Nuttall, 1961b). Similarly, the female’s PER increases over
the course of copulation (Bermant and Westbrook, 1966; Krieger et al., 1976). In the
present experiments, it was demonstrated that the preejaculatory intromission frequency,
the male’s ejaculation duration, and the female’s PER were positively correlated with one
another. In addition, the ejaculation duration and the female’s PER were affected by
manipulating the number of intromissions prior to ejaculation. The male’s ejaculation
duration and the female’s PER were significantly decreased if the female received 0—1
intromission prior to ejaculation. Furthermore, partial correlation controlling the ejaculation
duration showed that the correlation between the preejaculatory intromission frequency and
the female’s PER was close to zero. These findings suggest that the preejaculatory
intromissions influence the male’s ejaculation duration that in turn affects the female’s
PER.

The effect of a longer ejaculation duration on the female’s PER may result from
more intense stimulation due to the longer vagina-penis contact duration and/or the deeper
penile insertion rather than the number of pelvic thrusts during ejaculation. First, a longer
ejaculation duration may result in more intense stimulation that in turn induces a longer
PER. One anterograde tracing study shows that the L6 and Sr dorsal root ganglia send
more afferent fibers to the vagina and the cervix (Nance et al., 1988). In addition, along
the vaginal canal in female rats, fibers with mechanoreceptive fields that respond to moving
stimuli are discovered (Berkley et al., 1990). The area near the vaginocervical junction
contains more fibers with mechanoreceptive fields than other areas of vaginal canal
(Berkley et al., 1990). These findings suggest that a longer stimulation of the area near
vaginocervical junction resulting from a longer ejaculation duration can produce more

intense stimulation and induces a longer PER. Secondly, that the number of pelvic thrusts

138
during ejaculation was not significantly correlated with the ejaculation duration and the

female’s PER suggests that a longer ejaculation duration does not result from more pelvic
thrusts and that the quality of thrusting stimuli may be more important than the frequency of
thrusting in inducing the female’s PER. Finally, deeper penile insertion during ejaculation
possibly contributes to a longer ejaculation duration. Deeper penile insertion during
ejaculation can also simultaneously stimulate the pelvic nerve fibers as well as the
hypogastric nerve sending sensory innervation to the vagina, cervix and uterus (Berkley et
al., 1990; Berkley et al., 1993; Peters et al., 1987) and induces more intense stimulation.
However, a functional analysis of the respective roles played by these two nerves suggests
that “pelvic nerve fibers seem closely tied to sensory and behavioral processes associated
with mating and conception, whereas hypogastric fibers seem closely tied to pregnancy and
nociception”(p. 533, Berkley etal., 1993).

Intromissions have been demonstrated to affect the female’s intromission return
latency (IRL) in the present studies. Repeated intromissions alone, without ejaculation,
could induce prolonged IRLs that in length are not different from the female’s PER in the
first ejaculatory series. The first prolonged IRL usually occurred between 24th to 44th
intromission and 12 to 16 minutes after the first intromission. One possible function of
these prolonged IRLs may be to induce the male to ejaculate after the female receives
enough vaginocervical stimulation for induction of the progestational state of pregnancy.
Several observations support this idea. First, the number of intromissions prior to the first
ejaculation have been reduced experimentally by enforcing long interintromission intervals
(1 to 5 minutes) (Bermant, 1964; Larsson, 1959), suggesting that long interintronrission
intervals can facilitate ejaculation. The mean of the first prolonged IRL seen in the present
study (about 50 seconds) would be adequate to facilitate the male’s ejaculation. Secondly,
in the present experiments, the number of intromissions (usually 24 to 44 intromissions)
required for the first prolonged IRL to occur far exceeded the number of intromissions

necessary for inducing the progestational state of pregnancy. Further, in female paced

139
situations, it has been observed that if a prolonged IRL occurs during the third ejaculatory

series, the male will try to get close to the female by protruding his head through the escape
holes of the barrier and usually ejaculates on the next intromission when the female returns
(Unpublished data).

None of the seminal plug, prostate secretions, and the penile cup formation affected
the female’s postejaculatory refractory period. In rats, an enormous portion of the male’s
ejaculate comes from secretions of the seminal vesicles (Mann, 1964). The formation of
the copulatory plug involves polymerization of proteins in the seminal vesicular secretion
by the transglutaminase released from the coagulating glands (Brooks, 1990; Luke and
Coffey, 1994; Mann, 1981; Price and Williams-Ashman, 1961; Setchell et al., 1994;
Wagner and Kistler, 1987; Walker, 1910a, 1910b; Williams-Ashman et al., 1977;
Zorgniotti and Brendler, 1958). Therefore, the formation of a copulatory plug after
ejaculation can be prevented by bilateral removal of the seminal vesicles and coagulating
glands. However, the female’s PER was not affected by removal of both sets of tissue.

The penile cup was observed during ejaculation in ex copula tests (Hart and Odell,
1981). Evidence suggests that the BC muscle is responsible for this flaring of the glans
penis (Hart and Melese-D’Hospital, 1983; Sachs, 1982, 1983). Extirpation of the BC
muscle presumably eliminates the formation of penile cup during ejaculatory reflexes in
copula tests (Sachs, 1982). However, removal of the bulbocavemosus muscle did not
affect the female’s PER and we suggest that the formation of penile cup during ejaculation
does not contribute to the f emale’s PER.

To rule out the possibility that chemical substances released from the prostate may
cause contraction of the vagina, cervix and uterus that in turn contributes to the female’s

PER, we removed the prostate. The female’s PER was not affected by this operation.

140
Relation of Uterine Activity to Female Pacing Behavior

In a standard test situation where the male controls the timing of copulation, it has
been demonstrated that copulation has both an immediate and a delayed effect on the uterine
EMG activity. While little uterine EMG activity was observed before copulation, uterine
EMG activity increased significantly during copulation, then returned to the premating level
for several minutes following ejaculation. Five minutes following the ejaculation,
however, uterine EMG activity again increased even without copulation (Toner and Adler,
1986).

In one of the experiments, we examined how the uterine EMG activity correlates
with different types of sexual contacts and the temporal pattern of female sexual behavior.
While we anticipated an increase in uterine EMG activity during the PER, we did not see it.
However, we found that the duration of uterine EMG activity associated with ejaculation
was significantly longer than that associated with intromission or mount. This finding
coincides with the result that the female’s PER is significantly longer than her IRL or MRL.
It seems likely that the longer duration of uterine EMG activity associated with ejaculation
reflecting a more intense vaginocervical stimulation contributes to the longer return latency
following ejaculation.

We confirmed that copulation had an immediate effect on uterine EMG activity
reported in an earlier study (Toner and Adler, 1986). Before copulation, little uterine EMG
activity was observed. This is consistent with the previous finding that some uterine
activity was observed during the evening of the proestrus stage (Talo and Karki, 1976) and
before copulation (Toner and Adler, 1986). That little uterine activity was seen in the
female’s PER suggests that facilitation of sperm transport from the vagina to the uterus
during the female’s PER results from preventing the disturbance of sperm transport rather

than from the increase of uterine EMG activity. During the active phase of copulation,

141
uterine contractions increased significantly, then returned to the premating level following

ejaculation.

One possible function of the immediate increase and delayed increase of uterine
EMG activity induced by copulation may be to facilitate sperm transport from the vagina to
the uterus and to assist sperm to advance in the uterus at a faster speed. First, the longer
duration of uterine activity associated with ejaculation may facilitate the newly deposited
sperm from the vagina to the uterus. Secondly, the immediate increase of uterine EMG
activity during active phase of copulation and the delayed increase of uterine contractions
following copulation may serve to assist the sperm deposited from previous ejaculation(s)

to advance in the uterus at a higher speed.

Central Neural Correlates of Female Temporal Copulatoryy Behavior

In rats, female sexual behavior is comprised of three major components: lordosis
behavior, soliciting behavior (hopping and darting) and the temporal pattern of the female’s
approach to the male (Erskine, 1989; Nelson, 1995; Pfaff et al., 1994). The brain regions
that regulate lordosis responding have been extensively studied (Pfaff et al., 1994). For
example, the MPOA, ventromedial nucleus of the hypothalamus (VMN), frontal cortex,
olfactory bulbs, and septum are important for the display of lordosis behavior. Several
studies have reported that lesions of the MPOA increase the lordosis response in female rats
treated with low doses of EB plus P (Hoshina et al., 1994; Powers and Valenstein, 1972;
Whitney, 1986). Transections made anterior-dorsal to the MPOA also facilitate lordosis
reflexes (Y amanouchi and Arai, 1977). Lesions of large frontal cortex, olfactory bulb, or
septum facilitate lordosis behavior (Pfaff etal., 1994). In contrast, lesions of VMN inhibit
the display of lordosis reflexes (Pfaf f et al., 1994).

Compared to the studies measuring lordosis responses, brain regulation of the

temporal pattern of female rat’s sexual behavior is poorly understood. Two brain regions

142
have been reported to affect the temporal pattern of female sexual behavior: MPOA and

VMN. MPOA lesions decreased the frequency of sexual contacts and the time that the
female spent with the sexually active males (Whitney, 1986) when she could choose among
sexually active males, castrated males and females. Similarly, VMN lesions decreased the
female sexual contacts with the active males under similar test situations (Emery and Moss,
1984). These findings suggest that the MPOA and VMN may play a role in facilitating
female sexual motivation.

Results of the present study extend the notion that the MPOA plays a role in the
temporal copulatory behavior of female rats. Both electrolytic and chemical lesions of the
MPOA significantly increased the female’s return latencies following intromission and
ejaculation. This finding suggests that the significant increase in the IRL and PER
following MPOA lesions results from destruction of neurons in the MPOA rather than
fibers of passage.

It seems likely that the MPOA plays an important role in facilitating sexual
motivation in female rats. In a recent review, one author emphasized the distinction
between female sexual desire (sexual motivation) and female sexual ability (Wallen, 1990).
Female sexual ability refers to the female’s ability to engage in copulation. Female sexual
motivation refers to “the state underlying sexual initiation and sexual accommodation” (p.
234, Wallen, 1990). It is necessary for the female rat to display lordosis behavior to allow
successful penile insertion (Diakow, 1974; Pfaff et al., 1978). As a result, the lordosis
response has become the major measure of the female’s sexual ability. On the other hand,
the author proposed that the female temporal copulatory behavior was regulated by her
sexual motivation (Wallen, 1990). Consequently, the temporal pattern of female rat sexual
behavior can be used to measure her sexual motivation. Under such circumstances, the
increase in the female’s IRL and PER following MPOA lesions would indicate a decrease
in her sexual motivation. This is consistent with the finding that two females with MPOA

ibotenic acid lesions did not approach the male during the 15-minute test.

143
Comparison of MPOA Lesions in Male and Female Rats

The MPOA appears to regulate the temporal copulatory pattern of male and female
rats in a parallel manner. Following large MPOA lesions, male sexual behavior was
abolished (Giantonio et al., 1970; Ginton and Merari, 1977 ; Heimer and Larsson,
1966/1967; Hendricks and Scheetz, 1973; Lisk, 1968). With MPOA lesions sparing part
of MPOA, some males continued to achieve intromissions and ejaculations. These males
showed a significantly lengthened interintromission interval (111) (Heimer and Larsson,
1966/1967; Ginton and Merari, 1977; Stefanick and Davidson, 1987), ejaculation latency
(EL) (Ginton and Merari, 1977 ; Stefanick and Davidson, 1987) and postejaculatory interval
(PEI) (Ginton and Merari, 1977). In the present experiments, we demonstrated that the
MPOA lesions significantly increased the female’s IRL and PER. Larger MPOA lesions
even abolished the female’s sexual desire. The increase in the female’s IRL and PER
following MPOA lesions parallels the results of males following MPOA lesions. This
comparison uses the same measurement scale to evaluate the role of MPOA in the male and
female sexual behavior and is totally different from the traditional approach comparing
different responses in the male (mounting) and the female (lordosis). These findings in the
present study strongly support the idea that the MPOA regulates the temporal copulatory
pattern of both the male and the female.

Future Directions

In the present experiments, the study of the role of MPOA in the regulation of
female temporal copulatory behavior generated numerous questions about female sexual
behavior. Future research should focus on what brain regions regulate the temporal pattern
of female rat’s sexual behavior, what neurotransmitters are critical for this regulation, and

whether these brain regions function in a similar way in both the male and the female.

144
We have suggested that the MPOA functions to facilitate female sexual motivation.

The next question that needs to be answered is what neurotransmitters in the MPOA are
critical for this function. Evidence suggests that several neurotransmitters are logical
candidates: dopamine and serotonin. In the preliminary studies, dopamine D1 antagonist
(SCH 23390) seems to affect the temporal pattern of female sexual behavior when
administered to the MPOA (unpublished data). Serotonin fibers have been shown in the
MPOA (Yang and Clemens, 1993) and serotonin has been demonstrated to play a role in
the female lordosis behavior (Pfaff et al., 1994) and male sexual behavior (Meisel and
Sachs, 1994).

In addition to the MPOA, VMN lesions have also been shown to decrease the
frequency of coital contacts with sexually active males (Emery and Moss, 1984).
Therefore, the VMN is a good candidate for controlling the temporal pattern of female
sexual behavior. In addition, copulation has been reported to increase c-fos expression in
the VMN of male and female rats (Wersinger et al., 1993). This finding suggests that the
VMN plays a role in both male and female sexual behavior. Since the data collected in the
present experiments strongly support the view that the MPOA regulates the temporal pattern
of male and female sexual behavior in a similar way, it is important to know whether other
brain regions such as VMN control the male and female temporal copulatory pattern in a
similar way. To compare the role of VMN in male and female sexual behavior can be
achieved by investigating the role of VMN in the temporal copulatory behavior in both

sexes using electrolytic and chemical lesion techniques.

Summafl and Conclusions

In the present experiments, we examined the relation of copulatory stimulus factors
to the temporal pattern of female rat sexual behavior. In addition, we investigated the

relation of vaginocervical stimulation and uterine EMG activity to the female’s temporal

145
copulatory pattern. Finally, we investigated whether the MPOA plays a role in the temporal

pattern of female sexual behavior and whether the MPOA controls the temporal pattern of
male and female sexual behavior in a parallel manner.

We demonstrated that the preejaculatory intromissions, the male’s ejaculation
duration and different hormone replacements affected the female rat’s PER. The female’s
PER appeared not to be influenced by the deposit of a seminal plug, the penile cup
formation, prostate secretions and the number of pelvic thrusts during ejaculation. The
male’s ejaculation duration, the preejaculatory intromission frequency and the female’s
PER were significantly correlated with one another. One possible function of multiple
intromissions prior to ejaculation may be to prepare the vagina for a deep penile insertion
during ejaculation. This deep penile insertion during ejaculation results in a longer penis-
vagina contact duration (a longer ejaculation duration). A longer ejaculation duration
causes a longer PER in the female.

The duration of uterine EMG activity associated with ejaculation was significantly
longer than that associated with intromission or mount. This finding coincides with the
result that the female’s PER is significantly longer than her IRL or MRL. It is likely that
the longer duration of uterine EMG activity associated with an ejaculation reflecting a more
intense stimulus contributes to the female’s PER. In addition, we confirmed the previous
finding that copulation resulted in an immediate increase of uterine contractions. The
possible function of the increased uterine activity induced by copulation may serve to
facilitate newly deposited sperm to cross the cervix and to assist sperm deposited from
previous ejaculations to advance in the uterus at a faster speed.

The MPOA is important for facilitating female temporal copulatory behavior and
female sexual motivation in rats. Sexual motivation of female rats regulates their temporal
copulatory pattern during copulation. The MPOA lesions significantly increased the
female’s IRL and PER. Two females with MPOA chemical lesions did not approach the

male during the 15—minute test. These findings strongly support that the MPOA lesions

146
disrupt female pacing behavior and decrease female sexual motivation in rats. Both the

electrolytic and chemical lesions of MPOA significantly increased the female’s IRL and
PER. This finding demonstrated that the effect of MPOA lesions on the female temporal
copulatory pattern and female sexual motivation resulted from destruction of neurons in the
MPOA, not fibers of passage. Finally, that the increase of the female’s IRL and PER
following WOA lesions parallels the increase of the male’s III and PEI following MPOA
lesions strongly supports the view that the MPOA regulates the temporal pattern of male

and female copulatory behavior in a similar manner.

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