.....

PERIPHERAL SEXUAL MORPHOLOGY, SENSITIVITY
AND ANDROGENIZATION= THEIR EFFECTS ON MALE
SEXUAL BEHAVIOR IN RATS AND HAMSTERS

Dissertation for the Degree 'of Ph. D.
MICHIGAN STATE UNIVERSITY
ARCHIE J. VOMACHKA
1976

      
 

fins“? 3’) L113I21‘? RY

This is to certify that the

thesis entitled

Peripheral Sexual Morphology,

Sensitivity and Androgenization:
There Effects on Male Sexual Behavior,
In Rats and Hamsters

presented by

Archie J. Vomachka

has been accepted towards fulfillment
of the requirements for

PH .D. degree in ZOOLOGY

 

 

 

Major professor

June 7, 1976
I)ate

 

0-7639

ABSTRACT

PERIPHERAL SEXUAL MORPHOLOGY, SENSITIVITY AND ANDROGENIZATION:
THEIR EFFECTS ON MALE SEXUAL BEHAVIOR IN RATS AND HAMSTERS.

By

Archie J. Vomachka

Bilateral section of the dorsal nerve of the penis (DNP) in male
rats increased mOunt frequency but eliminated intromission and ejacu-
lation. Unilateral section of the DNP caused a transient decrease in
intromission and ejaculation frequency. Treatment with fluoxymesterone
(FM) in castrated unsectioned males maintained mounting, intromission
and ejaculation, but FM‘was not as effective as testosterone propionate
(TP. The effects of fluoxymesterone on mounting were—sabolished by sec-
tioning thelnfl’suggesting that fluoxymesterone exerts its faciliatory
effect upon mounting via an influence upon genital morphology.

In a second experiment, male and female hamsters castrated on day
1 (day of birth considered day l) were treated neonatally with fluoxy-
mesterone, testosterone or the hormone vehicle. At adulthood, mounting
behavior and lordosis behavior were assessed. 'While the degree of
penile or clitoral virilization did not differ between testosterone
and fluoxymesterone treated animals, behavioral differences occurred.
Neonatal treatment with fluoxymesterone failed to "masculinize" or
"defeminize" behavior. Neonatal treatment with testosterone induced
mounting and intromission. It was concluded from these results and

those of others that fluoxymesterone has little or no central neural effects.

Archie J. Vomachka

A third experiment employed male hamsters castrated on the day of
birth and treated with TP or estradiol benzoate (EB) on days 2, 3 and
4 of life. Neonatal treatment with TP but not EB induced penile viri-
1ization as adults. TP induced sexual behavior in these animals was
assessed prior to and following sectioning of the DNP. Penile desensi-
tization had little effect upon hamster sexual behavior regardless of
the degree of penile virilization. Replacement with either fluoxy-
mesterone or sesame oil caused a decline in sexual behavior but no
statistical differences were observed among sectioned or sham animals
or among neonatal treatment groups. Fluoxymesterone treated animals
in all neonatal groups tended to mount longer than sesame oil treated
animals but this trend was nonsignificant. Penile size or sensitivity
in hamsters appears to have little effect upon the maintenance of

sexual behavior.

PERIPHERAL SEXUAL MORPHOLOGY, SENSITIVITY
AND ANDROGENIZATION: THEIR EFFECTS ON MALE
SEXUAL BEHAVIOR IN RATS AND HAMSTERS

By

,__ 3,\
Archie J? Vomachka

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Zoology

I976

ACKNOWLEDGEMENTS

I wish to thank the members of my guidance committee, Dr. John A.
King, Dr. John R. Shaver and Dr. Harold D. Hafs for their valuable
assistance throughout various portions of my graduate training. A
special word of thanks must go to "The Chief", Dr. Lynwood G. Clemens,
for having faith in my abilities. Thank you for smoothing the rough
spots and developing my academic interest. I could not have done it
without your guidance and understanding. 7

I wish to acknowledge the love and able assistance of my wife, Mary,
who provided the occasional boost_and continued devotion throughout the
last few years. To my fellow graduate students, past and present, the
technicians in the Hormones and Behavior Lab and the cheerful, helpful

secretaries, thank you one and all for the help and good times.

ii

TABLE OF CONTENTS

List of Tables. . . . . . . . .
List of Figures . . . . . . . . . . . .

 

GENERAL INTRODUCTION. . . . . . . . .
EXPERIMENT 1. . . . . .

Introduction . . . . . . . . . . .
EXPERIMENT 1, PART 1. . . . . . . . . .

Methods and Materials. . . . . . .
Results. . . . . . . . . . . . .

Ejaculations. . . . . . . . .
Intromissions .'. . . . .
Mounts. . . . . . . . . . .
Latency Measures. . . . . . .
Histology . . . . . . . . . .

EXPERIMENT1,PART2..........

MEthods and Materials. . . . . . .
Results. . . . . . . . . . . . . .

Ejaculations. . . . . . . .
Intromissions . . . . . . . .
munts I O I O O O O O O O 0

Discussion . . . . . . . . . . .
EXPERIMENT 2. . . . . . . . . . . . . .

Introduction . . . . . . . . . .
Methods and Materials. . . . . .

Subjects. . . . . . . . . . .
Treatment Groups. . . . . . .
Test Procedures . . . . . . .
Morphological Measures. . . .
Statistica1.Analysis. . . . .

Results. . . . . . . . . . . . .

Mounting Behavior of Males. .
'Mounting Behavior of Females.
Lordosis Behavior of Males. .
Lordosis Behavior of Females.
Morphological Measures. . . .

iii

Page
. v
vii
l
. 2
. 4
. 4
. 8
. 8
. 10
12
14
. 17
18
. 18
. 19
O 19
. 19
. 19
. 21
. 27
. 27
. 30
. 30
. 31
. 32
. 33
. 34
. 34
. 34
O 38
. 38
. 38
. 44

Table of Contents cont'd.

Ovarian Histology. . . . . . . . . . . . . .
Discussion. . . . . . . . . . . . . . . . . . . .
EHERIMNT 3 O O O O I O O O O O 0 O O O O O O O O O I

Introduction. . . . . . . . . . . . . . . . .
Methods and Materials . . . . . . . . . . . . .

Subjects . . . . . . . . . . . . . . . . . .
Treatments and Behavior Testing. . . . . .

Morphological Measures . . . . . . . . . . .
Statistical Analysis . . . . . . . . . . . .

Re8u1t8 I O O O O O O O O O O O O O O O O O O 0

Effects of Neonatal Treatments (Tests 1-5)
Effects of Penile Desensitization (Tests 6-8).
Effects of Hormone Manipulation (Tests 9- 16)
Morphology . . . . . . . . . . . . . . . . .

Discussion. . . . . . . . . . . . . . . . . . . .
Conclusion. . . . . . . . . . . . . . . . . . . .

APPENDIX 0 o o o o o o e o o o o o o o o o o o 9 g o .
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . .

iv

Table

1.

2.

A2.

Al. 0

Mean Latency Measures for Male Rats Prior to and Following

LIST OF TABLES

Section of the Dorsal Nerve of the Penis.

Effects of Hormone Manipulation on Mean Latency Measures
(seconds) of Male Rats With or Without Section of the
Dorsal Nerve of the Penis .

Mean mount latency for male hamsters castrated on the day

of birth and treated neonatally with fluoxymesterone,
testosterone or sesame oil (in seconds i s.e.). . . . . . .

Mean amount latency for female hamsters treated neonatally
with fluoxymesterone, testosterone or sesame oil (in

Q

seconds i s.e.) . . . . . . . . . . . . . . . . . . . . .

Morphological measures of male hamsters castrated on the

day of birth and treated neonatally with fluoxymesterone,
testosterone or sesame oil.

Morphological measures of female hamsters treated neonatally

‘with fluoxymesterone, testosterone or sesame oil. . . . . .

Mean anogenital distance, penile bone plus cartilage
lengths (in mm) and penile condition in male hamsters

treated neonatally with TPlOO, TPZS or EB 25. . . . . . . .

Mean latency to first mount (in seconds) for castrated male
hamsters treated neonatally with TP or EB and given various

adult treatments.

Mean head and side mount frequency in castrated male
hamsters treated neonatally with T? or EB and given adult

treatments .

Mean rear mount frequency in castrated male hamsters
treated neonatally with TP or EB and given various adult

treatments.

Mean intromission frequency in castrated male hamsters
treated neonatally with TP or EB and given various adult

treatments.

V

O

Page

15

16

37

41

45

45

60

67

68

69

7O

Table Page

A5.

A6.

A7.

Percentage of male hamsters showing head and side mount,

rear mount or intromission for behavioral tests. Animals

were castrated on day 1, given TPlOO neonatally, and

received various adult treatments. . . . . . . . . . . . . . 71

Percentage of male hamsters showing parameters of male
mating behavior. Animals were castrated on day 1, given
TP25 neonatally and received various adult treatments. . . . 72

Percentage of male hamsters showing parameters of male

mating behavior. Animals were castrated on day 1, given
EBZS neonatally and received various adult treatments. . . . 73

vi

LIST OF FIGURES

Figure

l.

2.

9.

10.

11.

12.

Effects of penile desensitization and hormone replacement

on ejaculation in male rats. . . .

Effects on penile desensitization and hormone replacement

on intromissions per minute active

in male rats. . . . . . .

Effects on penile desensitization and hormone replacement
on mounts without intromission per minute active in male rats.

Effects of neonatal administration
testosterone or sesame oil on mean

in castrated male hamsters . . . .

Effects of neonatal administration
testosterone or sesame oil on mean
castrated male hamsters. . . . . .

Effects of neonatal administration
testosterone or sesame oil on mean
frequencies in male hamsters . - -

Effects of neonatal administration
testosterone or sesame oil on mean
female hamsters. . . . . a a . . .

Paremeters of lordosis behavior in

of fluoxymesterone,
head and side frequencies

of fluoxymesterone,
rear mount frequencies in

o o, o o g o o g o o .10 o

of fluoxymesterone,
head and side mount

of fluoxymesterone,
rear mount frequencies in

castrated male hamsters

treated neonatally with fluoxymesterone, testosterone or

sesame oil . . . . . . . . . . . .

Parameters of lordosis behavior in

female hamsters treated

neonatally with fluoxymesterone, testosterone or sesame oil.

Rear mount frequency and intromission frequency in day 1
castrated male hamsters treated with TPlOO neonatally and

given various adult treatments . .

Rear mount frequency and intromission frequency in day 1
castrated male hamsters treated with TP25 neonatally and

given various adult treatments . .

vii

Page

11

13

35

36

39

40

.42

43

54

55

Figure Page
13. Rear mount frequency and intromission frequency in day 1

castrated male hamsters treated with EB25 neonatally and
given various adult treatments. . . . . . . . . . . . . . . 56

viii

GENERAL INTRODUCTION

While the importance of gonadal hormones for the control of male
'mating behavior is recognized, where gonadal hormones act to control
sexual behavior has been debated extensively. While there is evidence
to indicate that the gonadal steroids stimulate behavior by action on
the central nervous system, there is also reason to suspect gonadal
hormones acting upon genital (Peripheral) structures, exert control
over sexual behavior. The problemthas been summed up most effectively:

Tell me, where is fancy bred,

In the heart or in the head?

In the glands (of which the're legions)

Or is it in the pelvic regions?

(Kohn, 1971)

The following series of experiments examine the role of penile size and
sensitivity in the sexual behavior of rats and hamsters. Experiments
were designed to test hypotheses related to the peripheral control of

sexual behavior.

EXPERIMENT 1

Introduction

Experimental evidence suggests that there are two modes by which
gonadal hormones may facilitate male sexual behavior in the rat; 1)
by direct action upon the central nervous system, 2) by action upon
genital morphology. In support of a central action, it was found that
testosterone implanted into the preoptic area restored mating behavior
in castrated rats when given in quantities far below the amount re-
quired for stimulation of seminal vesicles (Davidson, 1966; Lisk, 1967).
Electrical stimulation of the posterior hypothalamus or lateral pre-
optic area lead to stimulus bound copulation or activation of sexual
behavior (Caggiula & Hoeble, 1966; Caggiula, 1970; Madlafousek, Freund
& Grofova, 1970, Caggiula & Szechtman, 1972). By the same token,
discrete lesions in the medial forebrain area (Hitt, Hendricks, Gins-
berg & Lewis, 1970, Hitt, Byron & Mbdianos, 1973) and medial preoptic
area (Giantbnio, Lund and Gerall, 1970; Singer, 1968) abolished male
sexual behavior in the presence of exogenous androgens.

Experimental decreases in genital sensory feedback or penile
structure in rats, cats, and monkeys reduced or eliminated intromission
and ejaculation behavior but did not cause immediate decreases in
mounting (Beach & Holz, 1946; Carlsson & Larsson, 1964; Adler & Bermant,
1966; Aronson & Cooper, 1966 & 1968 & 1969; Whalen, 1968; Herbert, 1972;
Larsson and Sodersten, 1973). Evidence for hormonal effects upon sexual

behavior via genital morphology was suggested by Beach 5 Levinson (1950)
2

who correlated the decreased size of penile spines following
castration with a decrease in ejaculation and the "tendency to copulate."

Nadler (1969) gave cyproterone acetate, an anti—androgen, pre—
natally to rats and attributed the lack of male copulatory behavior in
the offspring at adulthood to deficient penile development. In addition,
neonatal castration of male rats resulted in a reduction in penile size
and weight. Limited androgen administration neonatally and/or in adult-
hood did not overcome this effect of castration on genital size (Beach
& Holz, 1946; Grady, Phoenix & Young, 1965; Whalen, 1968). Likewise,
neonatal treatment of male rats with gonadotropin antiserum produced
animals with penes of a reduced size and a smaller number of penile
spines than normal control animals (Goldman, Quadagno, Shryne & Gorski,
1972). These neonatal manipulations with antiserum resulted in animals
which mounted a receptive female but failed to intromit or ejaculate.
While these experiments suggest the importance of penile structure in
male sexual behavior, the treatments used to achieve a reduction in
penile development may also have resulted in a reduced central neural
androgenization or virilization. Consequently, we cannot establish
the relative contribution of central and peripheral factors to male
sexual behavior.

In an attempt to circumvent some of these problems, Hart (1972)
gave fluoxymesterone or TP to day 4 castrated male rats and tested these
animals for sexual behavior as adults in response to testosterone treat-
ment. Fluoxymesterone is known to have strong virilizing effects on
genital morphology. While animals treated neonatally with T? show high
levels of mounting, intromission and ejaculation, fluoxymesterone

treated animals showed a marked reduction in ejaculation. However, the

penes of the fluoxymesterone treated animals were not visibly different
from those of the T? treated animals. Hart therefore suggested, that
"the behavioral difference between the groups could not be attributed
to peripheral factors" (p. 845). In a study of adult rats, Beach &
Westbrook (1968) gave fluoxymesterone to non-copulating, castrated males
and although mating behavior was not reinstated, the penile spines and
papillae as well as seminal vesicle morphology were restored to the
level of testosterone treated animals. The data from these experiments
suggest that fluoxymesterone may not have strong effects upon the
central nervous system even though it is a powerful androgen for penile
tissues.

In the present study we provide experiments to determine whether
penile sensory feedback may facilitate mounting, even though it may not
be necessary for this behavior. We surgically sectioned the dorsal
nerve of the penis (DNP) in male rats and tested them for copulatory
behavior. These animals with desensitized penes were placed on various
hormone regimes.‘ Testosterone propionate was used as an androgen with
central neural activity as well as an agent to maintain penile morphology.
Fluoxymesterone was used to provide androgenic stimulation of peripheral
genital structures,presumably in the absence of strong central effects

upon male sexual behavior.
EXPERIMENT 1, PART 1

Methods and Materials
The subjects for this study were 47 male rats of the Long-Evans
strain. These animals were 90-100 days old at the beginning of the

experiment and were housed in groups of 3-5 in stainless steel cages

with food and water available ad_libitum. Lights in the colony were
off from 1100 hr. to 2100 hr.; testing began 1-3 hours after the lights
had gone off. Temperature and humidity were maintained at a constant
level. All rats were castrated under ether anesthesia 2 weeks prior
to the beginning of testing, and were placed on daily injections of
testosterone propionate (TP): SOO'ug in 0.1 cc sesame oil.

At 13-18 days post-castration, animals were selected for the ex-
periment on the basis of sexual performance in a selection test in
which each animal was placed with a sexually receptive stimulus female.
Stimulus females were ovariectomized and brought into sexual receptivity
with exogenous estrogen and progesterone. Animals which failed to
mount the female in 10 minutes or failed to ejaculate within 20 minutes
were excluded from the study. In order to establish a baseline of
sexual activity, two pretests of 20 minutes each (tests 1 and 2) were
given to the experimental animals 4 and 8 days after the selection test.
In each pretest, a sexually receptive female was placed into a 2'x2'
plexiglass arena with the experimental male after the male had been
allowed to adapt to the arena for at least 5 udnutes. All subsequent
testing followed the same procedure.

At the completion of the second pretest, animals were randomly
divided into 2 groups. In one group, a 3-5 mm section of the dorsal
nerve of the penis (DNP) ‘was removed bilaterally from.each animal.
Animals in the remaining group received a sham operation. The opera-
tions were performed under ether anesthesia. The skin was incised and
the penis retracted. The fascia was removed to expose the DNP, which

was then lifted and a section removed. For the sham operated animals,

the DNP was similarly exposed, however, it was neither lifted nor

sectioned.

Animals were allowed a recuperation period of 14-17 days before re-

ceiving 3 postoperative tests (tests 3-5) at 4 day intervals. Upon com-

pletion of the third postoperative test, the 2 groups were subdivided into

a total oftigroups and assigned to the following hormone treatments:

1)

2)

3)

4)

5)

6)

TP Cut, n=8. These animals with DNP section continued to
receive 500 ug TP daily.

TP Sham, n=8. These were Sham Operated animals which received
500 ug TP daily.

Fluoxymesterone Cut, n=8. These animals with DNP section as
well as all of the following groups, received no further TP
administration. Replacement in this group was 500 ug of
fluoxymesterone (Halotestin. Upjohn) daily, suspended in 0.1cc
sesame oil.

Fluoxymesterone Sham, n=8. These Sham operated animals re-
ceived 500 pg fluoxymesterone daily in 0.1cc sesame oil.

011 Cut, n=7. These animals with DNP section received 0.1cc
sesame oil (the hormone vehicle used in this study) daily.

Oil Sham, n=8. These Sham operated animals received 0.1cc

sesame oil daily.

Following the onset of these group assignments, animals were tested

on day 4, 8, 12, 17, 21, 28, 35, 42, 49 and 56 (tests 6-15).

For each 20 minute test, all mounts, intromissions and ejaculations

were recorded on an Esterline-Angus Event Recorder. This provided a

temporal sequence of behaviors and the following measures were derived

from the record:

1)

2)

3)

4)

5)

6)

7)

8)

9)

Mount Latency. The time in seconds from introduction of the
stimulus female at the beginning of the test until the first
mount by the male, up to 1200 seconds for an animal which
failed to mount.

Intromission Latency. The time in seconds from the beginning
of the test until the first intromission response by the male,
or 1200 seconds for animals which did not intromit.
Ejaculation Latency. The time in seconds from the first intro-
mission to ejaculation, or in subsequent ejaculations, time
from first intromission in a series to ejaculation. In animals
which failed to ejaculate, ejaculation latency was taken as
1200 seconds minus the intromission latency.

Mount Frequency. Number of mounts without intromission.
Intromission Frequency. Number of mounts with intromission.
Ejaculation Frequency. Total number of ejaculations in a

20 minute test.

Post-ejaculatory Interval. Time in seconds from ejaculation
to the reinitiation of copulatory activity.

Mounts Per Minute. Mount frequency divided by total test time
(20 minutes) minus total time in post-ejaculatory interval.
Intromissions Per Minute. Intromission frequency divided by

total test time minus total time in post-ejaculatory interval.

Following ejaculation, the male rat enters a post-ejaculatory

interval (PEI) lasting 200-300 seconds. During this period, he shows

no copulatory behavior. Since DNP cut animals rarely ejaculated, they

seldon entered a PEI. Animals with no ejaculations often mounted
regularly throughout the entire 20 minute test, while animals with
multiple ejaculations were sexually active for a much shorter amount
of time. In such a situation, elevated mount frequencies for cut
groups per 20 minute test would reflect a lack of the PEI normally
seen in ejaculating males. To correct for this, comparisons of mount
frequencies and intromission frequencies were made on the basis of
frequencies per minute active. Thus, for animals achieving ejaculation,
the PEI was subtracted from the 20 minute tests, and dividing mount
frequency or intromission frequency by this number gives behaviors per
minute active.

At the completion of testing, all animals were sacrificed and penes
were removed. Penes were fixed in Bouin's solution, divided into glans
and shaft portions and embedded in parafin. The tissue was sectioned
at 10 u and representative sections were mounted on glass slides and

stained for histological examination.

Results

Ejaculations. ‘The mean ejaculation frequencies for each group

 

across all tests are shown in Figure 1. Comparison of ejaculation
frequency scores from the 2 pretests with the ejaculation frequency
scores from the postoperative tests 3-5 showed that sectioning the DNP
nearly abolished ejaculation (Sign Test, p<0.001). In tests 3-5, sham
animals showed a statistically significant increase in ejaculation

frequency over the pretest scores (tests 1 & 2) (Sign Test, p=0.002).

    
   

 

  

 

 

 

 

N H
o
25 P a
c
u R
T E TP SHAM
o c "
2 20 R H
.9.- a A ~
5 IA ‘3
8 ‘.‘ :MP #\ k
s 15 ~“y. O -
I.“ ‘ P \a
a5 ,3 FM SHAM
m' A I
I pit SHAM
1.0 0
IN
E
O‘ o
0.5 ‘-, ’0..-
17).". ..
[A ,~ _ \ FMqCUT
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’.’ ~~ “:' '*-'d«:’ “_-O ‘.‘ '0. .TIE‘CUT
' ~ 01L _cur'\ \
0 "out-:0 h-- a." w

H_q ‘ ‘ ‘“w
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

TEST

 

Figure 1. Effects of penile desensitization and hormone
replacement on ejaculation in male rats.

10

The effect on ejaculation frequency of replacing TP with either
fluoxymesterone or oil in cut and sham groups is also shown in Figure
1, tests 6—15. In all DNP section groups, ejaculation frequency re-
mained low throughout the experiment. The TP Sham group showed no
significant change in ejaculation frequency between tests 6 and 15,
whereas there was a significant decrease in ejaculation frequency be-
tween tests 6 and 15 (Wilcoxon Matched Pairs, Signed - ranks Test,
p<0.01 and p<0.02 respectively) for both the Fluoxymesterone Sham and
Oil Sham groups. At test 6, the scores for the sham groups were not
significantly different, however, by test 15, a statistical difference
was obtained among groups (Kruskal-Wallis, 1 way AOV by ranks, p<0.01).
Ejaculation frequency in the TP Sham group was significantly higher
than in the Fluoxymesterone Sham group (Mann-Whitney U-test, p<0.01);
likewise, the Fluoxymesterone Sham scores were significantly greater
than were those of the Oil Sham group (MWU, p<0.01).

Intromissions. Mean intromission scores per non-PEI minute for

 

each group are shown in Figure 2. DNP section resulted in a significant

decrease in intromission scores in tests 3-5 as compared with tests 1

and 2 (Sign Test, p<0.001). For the sham group, intromission scores

did not differ significantly between pre- and post—operative tests.
Comparison of test 6 scores with test 15 scores showed that both

Fluoxymesterone Sham and Oil Sham animals had significant decreases in

intromission scores over time (Wilcoxon, p<0.002), while TP Sham

animals showed no change in intromission frequency. At test 15, TP,

Fluoxymesterone, and Oil Sham animals were significantly different on

intromission measures (K-W, p<0.01), whereas, in test 6, these groups

11

 

 

 

 

 

H
D
m A III
2 c 8
t3 1’ E
< c
I; 8 2
Z S g
2 H
a III E
a.
O o
S E
R
A
2 T
0
g N
IL
0
g ',.o"'s
H
34 5 6 7 8 9101112131415

Figure 2. Effects of penile desensitization and hormone replacement
on intromissions per minute active in male rats.

12

were not different from each other. Onttest 15, TP Sham animals
achieved intromission at a higher rate than the Fluoxymesterone Sham
animals (MWU, p<0.05) and likewise, Fluoxymesterone Sham animals had
a significantly higher intromissions-per-minute score than did the
Oil Sham animals (MWU, p=0.002). Intromission scores for the cut
groups remained low throughout tests 6—15.

Mounts. Mean mount frequency per non-PEI minute for each group
is shown in Figure 3. Mount scores in the DNP section groups showed a
significant increase between pre- and post-operative tests (Sign Test,
p-0.034), however, sham groups showed no change when comparing tests
1 and 2 with tests 3-5.

In Fluoxymesterone Sham and Fluoxymesterone Cut groups, mount
scores decreased significantly between tests 6 and 15 (Wilcoxon,
p<0.05 and p<0.002, respectively), as did Oil Sham and Oil Cut groups
(Wilcoxon, p<0.002 and p=0.002, respectively). All TP animals remained
unchanged over time. Although the 6 groups did not differ significantly
at test 6, a statistically significant difference in mount scores was
seen among the 6 groups in test 15 (K-W, p<0.01). In test 15, TP Sham
animals had higher mount scores than did the Fluoxymesterone Sham
animals (MWU, p<0.05) and Fluoxymesterone Sham animals had significantly
higher mount scores than the Oil Sham group (MWU, p<0.02). In the cut
groups, TP Cut animals achieved significantly higher mount scores at
test 15 than did the Fluoxymesterone Cut animals (MWU, p<0.002),
however, Fluoxymesterone Cut animals were not different from Oil Cut

animals.

13

 

 

 

 

 

 

 

 

 

an I
R a. 8
Lu ~ .
2 I' P 3’ "s, 5‘
5 C I" "' 0
< l1! N
" E
IL" 0 C
3 *4: R H
g 15 . " a N
P 1" G
1.0 h 1M E
(I)
p.
Z
8 05 b
2
5 O l J L A l
g 1 2 3 4
TEST

   

,, OIL SHAM
I

x
on. cur-g 5M. ’

o I- ‘w "am-"W”—

 

 

. J L I J l j I l L

6 789101112131415
TEST

 

Figure 3. Effects of penile desensitization and hormone
replacement on mounts without intromission per
minute active in male rats.

l4

Latengy Measures. Results of dorsal nerve section on latency
measures can be seen in Table l. Sectioning the DNP caused a signifi-
cant increase in ejaculation latency (Sign Test, p=0.002) with no
change in mount latency or intromission latency for pre- and post-
operative scores. In sham operated animals, a statistically significant
decrease in ejaculation latency was observed (Sign test, p<0.034) with
no changes in mount latency or intromission latency between these same
tests.

Latency scores for tests 6 and 15 can be seen in Table 2. Following
the change in hormones in test 6, mount latency and intromission latency
were not significantly different among the 6 groups, although ejaculation
latency did differ among the groups (Kruskal-Wallis, p<0.02). By test
15 a significant difference for all measures was noted among groups
(KPW; EL, IL, & ML p<0.001). Comparison of test 6 latency scores with
test 15 scores within groups gives an indication of changes over time.

TP Sham animals showed no change in mount latency or intromission latency
measures but did exhibit significant increases in ejaculation latency
(Wilcoxon, p<0.05): TP Cut animals showed no change in mount latency

or ejaculation latency but did increase in intromission latency (W.,
p<0.05) in a comparison between the 6th and final test. Fluoxymesterone
Cut and Sham animals as well as Oil Cut and Sham animals all exhibited
statistically significant increases in mount latency, intromission
latency and ejaculation latency between tests 6 and 15 (W., all p

values<0.02).

15

Table 1. Mean Latency Measures for Male Rats Prior to and Following
Section of the Dorsal Nerve of the Penis.

Test 2 Test 5
Mount Cut M 18.3 9.5
Latency (sec.) SE i 3.7 i 1.7
Sham M 10.1 6.5
SE i 2.3 i 0.6
Intromission Cut M 56.0 187.9
Latency (sec.) SE $17.7 $73.4
Sham M 34.1 29.8
SE :12.3 i 9.4
Ejaculation Cut M 518. 0 1047 . O
Patency (sec.) SE $71.6 $53.5
Sham M 460.9 356.8
SE $75.3 $70.2

 

 

 

 

 

 

 

 

16

 

 

 

 

 

 

 

 

 

 

 

 

 

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no sues mood can: mo Amoaououv sensuous unsound nous so soauoaonfiomz oaoahom mo uuomwwm

m.~m o.mm n.5nH e.coa o.mo m.H um
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m.mm ~.nu ~.m n.~w m.m H.H mm
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coauoasoonu doammaaouuoH uwooz aoaumflsomfim scammwaouuoH ammo:

n.— uooa o omen.

.N manofi

 

17

Between group comparisons of test 15, latency scores revealed that
although Fluoxymesterone Cut animals were not different from Oil Cut or
Sham animals in ejaculation latency, intromission latency or mount
latency, Fluoxymesterone Sham animals showed significantly shorter
latencies in all measures than did Fluoxymesterone Cut, Oil Cut or Oil
Sham.animals (Mann Whitney U, p<0.02 in all comparisons). Scores of
Fluoxymesterone Sham animals compared to the TP Sham animals at test 15
showed that the TP group had a significantly shorter mount latency (MWU,
p<0.05) and intromission latency (MWU, p<0.05) but that TP and Fluoxy-
mesterone Sham animals did not differ with respect to ejaculation latency.
On test 15, TP Cut animals had a shorter mount latency than the Fluoxy-
mesterone Sham animals (MWU, p<0.05) while intromission latency did not
differ between these groups. Fluoxymesterone Sham animals had a signifi-
cantly shorter ejaculation latency than did the TP Cut animals on test
15 (MWU, p<0.05).

Histology. Microscopic examination of cross sections of the glans
penis confirmed earlier observations (Beach and Levinson, 1950; Beach
and Westbrook, 1968) in that penes of animals treated with TP and
Fluoxymesterone were indistinguishable, while those animals treated
with daily sesame oil showed a marked regression of papillae and a nearly
complete lack of penile spines. Within hormone treatments, no difference
could be seen between DNP Cut and Sham animals. Although a large
difference in behavior was seen between cut and sham animals, no histo-
logical differences were evident upon examination of the shaft.

The following study was run to assess the effects of partial penile

desensitization on male sexual behavior in rats.

18

EXPERIMENT 1, PART 2

Methods and Materials

 

Subjects for this experiment were 19 male rats of the Long-Evans
strain, approximately 120 days old at the beginning of the study. They
were housed under the same conditions as were those in Experiment 1,
Part 1. All animals were castrated as adults and given TP replacement
of 500 ug daily. Behavioral measures and conditions were identical to
those in Experiment 1, Part 1.

A selection test was given two weeks after castration. At 4 and 8
days after the selection test, animals were given a 20 minute pretest
with a sexually receptive female to establish a baseline of copulatory
behavior. Following a second pretest 1 day later, animals were randomly
assigned to a cut or sham group.

DNP sectioned animals were divided into 2 groups of 6 animals each.
One group received a surgical section of the left dorsal nerve of the
penis; in the second group, the right dorsal nerve of the penis was
sectioned. Following a 2 week recuperation period, all animals were
tested 3 times at 4 day intervals (Tests 3-5). After test 5, surgery
was again performed to section the remaining nerve. Following a two
week recovery period, the males were given the final 3 tests at 4 day
intervals (Tests 6-8). Sham animals received 2 sham operations and
were tested similarly. Since unilateral sectioning of the left and
right produced scores which were not different, the data were pooled

for statistical analysis.

19

Results

Ejaculations. The effects of cutting one of the DNP and then the
other on ejaculation frequency are shown in Figure 4. Surgical section
of one nerve caused a decrease in ejaculation frequency in test 3, when
compared to pretest levels. However, this decrease was followed by a
recovery in test 5. Ejaculation frequency for DNP section animals and
sham animals was not significantly different in test 1 and test 5.
However, recOvery of ejaculation frequency was not seen following sec-
tioning 0f the second nerve. Test 8 scores for ejaculation frequency
were significantly lower in the sectioned animals than in the sham
operated animals (MWU, p<0.002). Within groups, comparing test 1 with
test 8, sectioned animals showed a significant decrease in ejaculation
frequency (Wilcoxon, p<0.002), while scores for the sham animals
showed no change between tests 1 and 8.

Intromissions- Intromission scores on tests 1-8 are shown in

 

Figure 4. Unilateral sectioning caused a decrease in intromission
scores, but intromission frequency returned to the level of the Sham
group by test 5. Bilateral sectioning caused a sustained decrease in
intromissions-per-minute, as seen in Experiment 1. Sectioned animals
had significantly lower intromission-per-minute scores than did the
sham animals on test 8 (MWU, p<0.002). Sectioned animals showed a
significant decrease in intromissions—per-minute on test 8 as compared
to the pretest score on test 1 (Wilcoxon, p<0.002). Sham animals did
not change in intromissions-per-minute between tests 1 and 8.

Mounts. Mount scores for all tests are given in Figure 4.

Sectioning of one of the dorsal nerves had no effect on mount scores

20

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SHAM
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SECOND DNP CUT OR SHAM

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IHHIIIHIIHHH ”memmwwtnmtunfimfim
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m. m m o m m w m. a o m m m
whDZ=2 mun. 9.2302 3.32.5. mum wszmgomhz. mZO_._.<._DU<—.u

TEST

Effects of partial and complete penile desensitization
on mounting, intromissions and ejaculations in the male rat.

Figure 4.

21

when compared to sham animals on test 5. However, following the second
nerve section, a significant increase in mounts-per-minute was noted
on test 8 (MWU, p<0.01). Sectioned animals showed a significant in-
crease in mounts-per-minute between tests 1 and 8 (Wilcoxon, p<0.002),

but sham animals had scores on tests 1 and 8 which did not differ.

Discussion

 

Androgenic steroids and their metabolites vregulate'male sexual
behavior by action at various sites. In the adult male rat, evidence
indicates that the central nervous system, particularly the medial
preoptic area (MPOA) and its connections represent a major site of
androgen action. Intracerebral implants of testosterone into the MPOA
will reinstate mating behavior in castrated animals (Davidson, 1966;
Lisk, 1967; Christensen & Clemens, 1974). However, minute quantities
of testosterone in the brain enter the peripheral circulation in amounts
high enough to cause stimulation of penile tissue and growth of penile
spines and papillae (Kierniesky & Gerall, 1973). While this penile
stimulation might be regarded as only a side effect of the presence of
central androgen, it renders conclusions based on these findings diffi-
cult to interpret in terms of strict central control of male behavior.

Intracerebral implants of estrogen, the aromatized metabolite of
testosterone, are also very effective in restoring male behavior in male
rats, however, no penile virilization is seen with estrogen implants
(Christensen & Clemens, 1974). Stimulation of behavior without concom-
mitent penile stimulation, ejaculatory maintenance by systematic
injections of EB (Sgdersten, 1973), studies of estrogen

uptake (Whalen & Littge, 1970) and in_vitro studies of brain tissue

22

metabolism.of steroids (Naftolin, Ryan & Petro, 1971 a,b) have been
noted. These findings have led to the hypothesis that estrogenic
metabolites of androgens may be the centrally active compounds respon-
sible for male mating behavior.

It has been suggested that testosterone's action may be achieved by
its metabolic products; So reduced androgens appear necessary for morpho-
logical maintenance of penile and sexual accessory tissues, while estro-
genic metabolites or their precursors function centrally (Parrott, 1974).
That androgens may influence sexual behavior at a peripheral locus has
been suggested by the finding that the administration of presumably peri-
pherally active non aromatizable androgens such as dihydro—testosterone
(DHT) presents or slows down the loss of ejaculatory performance in
castrated male rats (Parrott, 1974 a,b). Although androgen metabolites
such as DHT will stimulate penile and sexual accessory tissues, DHT,
even in large doses will not reinstate male mating behavior (McDonald
et a1. 1970; Whalen & Luttge, 1971). Brain implants of DHT are also in-
effective (Johnston & Davidson, 1972).

Recent evidence has shown that administration of estrogen, in com-
bination with DHT or fluoxymesterone, to castrated male rats will induce
the full complement of male sexual behavior (Baum.& Vreeburg, 1973;
Larsson, Sgdersten & Beyer, l9733,b; Feder, Naftolin & Ryan, 1974;
Johnson & Tieffer, 1974). Presumably, such treatment provides a peri-
pherally active androgen for appropriate stimulation and androgen main-
tenance of the genitalia, coupled with activation of central neural
tissues which mediate sexual behavior.

Studies of male sexual behavior utilizing estroge, DHT combination

indicate that the administration of EB + DHT to castrated male

23

rats with the DNP sectioned yielded results similar to those of intact
rats (Clemens & Vomachka, unpublished observations). However, upon
withdrawl of estrogen treatment, animals with desensitized penes still
receiving DHT showed a more rapid decline in mounting behavior than
intact animals receiving only DHT. The more rapid decline in rats

with desensitized penes suggests an important behavioral role of penile
tissues.

In the present study, fluoxymesterone administration after TP
withdrawl produced 3 effect similar to that seen with EB + DHT following
desensitization. Animals with desensitized penes showed a more marked
decline in behaviors than animals with intact penile afferent nerves.
While these experiments suggest a synergistic influence of brain and
genital mechanisms, it is apparent that genital influences are neither
necessary nor sufficient for mounting behavior. Likewise, treatment
with testosterone propionate maintained mount frequencies at a high
level even after section of the DNP. These findings with TP and
fluoxymesterone suggest that sensory feedback from the penis may have
a facilitative influence upon behavior, but in the presence of centrally
active steroids, this influence is masked.

Studies of the influence of other sensory processes on sexual
behavior have suggested that sensory effects are additive in terms of
their influence on sexual behavior and that like penile sensitivity,
the other sensory channels are not individually essential for the
initiation of mating (Beach, 1942, Adler & Bermant, 1966; Carlsson &
Larsson, 1964). For example, the effects of deprivation of different

sensory modalities, including blinding, anaemia and deafening, on male

24

sexual behavior were explored in a classic study by Beach (1942). Loss
of each modality individually does not eliminate copulation in sexually
vigorous males. However, elimination of two or more of these did cause
a reduction in copulation. Given the additive nature of sensory stimu-
lation, it might be predicted that loss of penile sensations combined
with loss of other sensory modalities would result in decreased sexual
behavior.

The question remains as to how penile afferent sensations, sup-
ported by androgenic agents, mdght exert an effect on sexual arousal.
An analogy may be drawn to the effects of intromissions on ejaculation.
It has been suggested that the input of information from each.intro-
mission adds to the general excitatory state of the animal's central
nervous system or maintains the excitatory state until the ejaculatory
‘mechanism and ejaculation occurs (Beach, 1956: Sachs & Barfield, 1974).
An animals readiness to mount a receptive female may also be influenced
by penile stimulation in a similar manner. Day to day stimuli from the
fluoxymesterone treated penis, whether from contact with the penile
sheath, grooming, copulation, or non-specific tactile input, may in-
crease a central excitatory state. This state then becomes manifest
in the form of mounting when the proper stimulus condition is met. In
the normal animal with a high androgen titer in the CNS, these penile
contributions to sexual arousal, seen as shorter latencyand higher
frequency measures, are masked and only act to augment a highly
stimulated motivational mechanism. In the present preparation, with
reduced central androgenization but with selective androgenic support
of penile tissue, a possible contribution of penile stimuli to behavior-

al maintenance becomes evident.

25

While fluoxymesterone treatment maintains penile morphology and
facilitates mounting, the effects of FM on penile sensitivity are not
known. Beach and Levinson (1950) postulated a sensory role for penile
spines and papillae. However, in a series of neurophysiological ex-
periments, Cooper and Aronson (1974) found that genital sensory fields
and sensory thresholds were not different between intact and castrated
cats. Assuming that this finding in another species applies to rats
as well, where might androgen act to facilitate penile sensory processes?
One site of androgen action could be at the level of the spinal cord.

Possibly fluoxymesterone maintained penile input to the CNS through
spinal pathways to a greater degree than that seen in oil treated
animals or animals with the DNP severed. Hart (1967, 1968, 1973) has
investigated the decrease in sexual reflexes in spinal male rats fol-
lowing castration. Such decreases may reflect changes in the neural
functioning of the spinal cord involved in the transfer of information
from the genitalia to the CNS. Cooper and Aronson (1974) report that
castration alters central processing of genital sensory input, in that
stimulation of penile afferent nerves of castrates and non castrates
resulted in differences in various measures of neocortical activity.
The "thalamus or cortex or other sensory relay or integrative centers"
were proposed as possible areas involved in the observed differences
between castrates and non-castrates. In this light, the spinal path-
ways, devoid of androgen stimulation could conceivably alter penile
afferent sensations and thereby alter behavior.

Of course, the possibility remains that fluoxymesterone is

actually acting at other central neural sites to maintain sexual

26

behavior. Although this seems unlikely, the following experiment
was designed to test the central neural androgenicity of fluoxymesterone

in the developing animal.

EXPERIMENT 2

Introduction

Androgenic steroids which stimulate growth of genital and sexual
accessory tissues do not always stimulate male sexual behavior. Di—
hydrotestosterone (DHT) is one such peripherally active compound. This
steroid has been shown to have little influence upon behavior but is a
powerful virilizing agent peripherally (Feder, 1971; Johnston and
Davidson, 1972; Parrott, 1974 a,b, and c, 1975; Christensen, Coniglio,
Paup, and Clemens, 1973; Feder, Naftolin and Ryan, 1974). Another such
compound is fluoxymesterone which was originally administered clinically
to humans (Reilly and Gordon, 1961). When given to stimulate growth
in boys of short stature, a concomitant phallic enlargement was noted
without "evidence of stimulation of libido or masturbation". In rats
fluoxymesterone would not restore the loss of mating in long term
castrated males but did stimulate the normal size of seminal vesicles
(Beach and westbrook, 1968). Likewise, the penile epithelium, which
generates and becomes smooth following castration, was maintained
with papillae and cornified penile spines by fluoxymesterone.

Johnson and Tiefer (1974) also found that fluoxymesterone, given
to castrated male rats, did not maintain mounting, intromission or
ejaculation any better than did sesame oil. However, combination of

estradiol benzoate (EB) treatment with fluoxymesterone was highly

27

28

effective in maintaining sexual behavior at a level above that achieved
with either EB or fluoxymesterone alone. Similar findings with DHT

in combination with EB (Baum and Vreeburg, 1973; Larsson, Sgdersten

and Beyer, 1973 a and b; Feder, Naftolin and Ryan, 1974) have strength-
ened the hypothesis that sexual behavior is the result of both central

neural activation of behavior (by estrogenic steroids) and maintenance

of genital tissues (by peripherally active steriods).

In an experiment designed to determine the extent to which penile
development is influential in the development of male sexual behavior,
Hart (1972) administered either testosterone propionate (TP) or
fluoxymesterone to neonatal male rats and tested their sexual behavior
and sexual reflexes as adults. Comparison between the sexual behavior
of animals which had been castrated on day 4 and received TP on days
5, 7, 9, and 11 and those which were castrated and received fluoxy-
mesterone showed that fluoxymesterone animals achieved fewer ejacu-
lations. While the fluoxymesterone treated animals did mount and
intromit, vehicle control animals achieved significantly fewer intro-
missions. Since gross penile development and reflexes were not
different between the androgen treated groups, Hart could not attribute
the behavioral differences observed to peripheral factors. The results
support the concept that ejaculation behavior requires the influence
of androgen during neonatal development (Hart, 1972).

That Hart could not find differences between testosterone and
fluoxymesterone treatment neonatally on mounting and intromission does
not mean that these behaviors develop independent of steroid influences.

Lack of differences may have been the result of the nature of the

29

animal with which he was working. In the rat, testicular differentiation
occurs on about day 13 (post coital age), while behavioral sexual differ-
entiation occurs between days 18 and 27 (see Clemens, 1973). When
castrated on day 4 of postnatal life (assuming that birth occurs on day
21 post coital) neonatal male rats have already been exposed to their

own endogenous androgens for at least 7 days. Conclusions at to the
"organization" of mounting and intromission based on post natal hormone
administration to rats are tenuous at best.

Since the hamster has a relatively short gestation period (16 days)
it offers a better model for studying androgenic control of behavioral
development. While testicular differentiation occurs at about 11.5 days
post coital, behavioral differentiation appears to be entirely post
natal, beginning at 16 days post coital age (Nucci and Beach, 1971;
Clemens, 1973). The male hamster castrated on the day of birth (day 1)
will not show mounting, intromission, or ejaculation as an adult even
when given large doses of androgen as an adult. On the other hand,
the female or Day 1 castrated male hamster which receives gonadal
hormones during the first few days of life will show mounting and sup-
pressed lordosis behavior at adulthood (Carter, Clemens and Hoekema,
1972; Paup, Coniglio, and Clemens, 1972, 1974; Coniglio, Paup, and
Clemens, 1973; Coniglio, 1973). Consequently, the estent to which
neural or genital virilization influence behavioral sexual differen-
tiation and later behavior may be more easily examined in the hamster.

The following experiment:

1. tests the virilizing effects of fluoxymesterone upon behavior

and genital tissue when administered to day 1 castrate male or

30

female hamsters.
2. tests two alternate hypotheses regarding the mechanism of
androgen influence upon development and the control of mating:
a) Does treatment neonatally with fluoxymesterone induce a
behavioral differentiation similar to that seen with testos-
terone,
and
b) is male sexual behavior in the hamster programmed prior
to the neonatal organization of peripheral tissues.
3. provides comparative data on the development of sexual
behavior in rodents.
The timing of behavioral sexual differentiation in the hamster makes
it a more suitable subject than the rat to study the androgenicity
of certain compounds (meachka, Paup, Coniglio, McManus and Clemens,
1974), the control of sexual differentiation (Clemens, 1973) or the
extent to which sexual behavior is controlled by central or peripheral

stimuli (see Experiment 3).

Methods and Materials

 

Subjects

The subjects of this experiment were 27 male and 36 female hamsters
(Mesocricetus auratus) born and raised in the Hormones and Behavior
Laboratory at Michigan State University. All animals were given

food and water §g_libitum and were maintained on a reversed day-night

31

light cycle of 14 hours light, 10 hours dark with lights out at

11:00 hours. All animals were weaned at 21 days of age and housed in
unisexual groups of 2-6 animals. Cages used were plexiglass with
removable stainless steel tops with dimensions of 26X20X15 cm or

35X3OX17 cm depending upon the size of the group.

Treatment Groups

 

0n the day of birth (day 1), males were castrated through a mid-
ventral incision using hypothermal anesthesia. The incisions were
sutured and sealed with flexible collodion and the pups were returned
to their mothers. On days 2, 3, and 4 of postnatal life, all animals
in each litter received a subcutaneous injection of one of the follow-
ing substances: fluoxymesterone (lOOug/day) suspended in sesame oil,
testosterone (lOOug/day) free alcohol form, or the hormone vehicle,
sesame oil (0.03 cc/day). Subcutaneous injections were administered
under the skin of the back with needle entry in the posterior back
region and the bolus of hormone being deposited under the skin of the
neck. Upon withdrawl of the needle, puncture sites were sealed with
flexible collodion and animals were inspected for leadage of hormone
before being returned to their mothers. At 60 days of age, all
animals were earpunched for identification and females were ovariect-
omized under ether anesthesia. Ovaries were fixed in Bouin's solution,
embedded in paraffin, sectioned and stained with hematoxylon-eosin

for histological examination.

32

Test Procedures

 

MOunting Behavior:

At approximately 75 days of age, all animals began weekly testing
for mounting behavior. Testing (1300-1700 hr.) was conducted in an
air conditiOned annex to the animal quarters which were dimmly illumi-
nated. Observation arenas were 10 gal. aquaria with Sani-cellr
(ground corn cobs) covering the floor. All mounting behaviors during
a 10 minute test were recorded on an Esterline-Angus even recorder.
Tests followed a 3 minute adaptation period during which the experi-
mental animal was placed alone in the observation arena. After the
adaptation period, a sexually receptive stimulus female was introduced
into the arena and behaviors were recorded. Receptivity was induced
by administration of 12ug EB for 3 days and 0.5 mg progesterone to
ovarectomized females on the morning of testing. Stimulus females were
proven receptive prior to experimental use by placing themwwith vigorous
nonexperimental males.

Male and female experimental hamsters were tested weekly for 5 con-
secutive weeks. Following the first test for mounting (pretest) daily
administration of 300ug TP was used to induce mounting. 'All mounts and
intromissions were recorded and mounts without intromission were further
divided into catagories as to whether they were directed to the head
or side, or to the rear of the receptive stimulus female. A mount
‘was scored as such only if it was accompanied by a clasp and pelvic
thrusting. In addition, the length of time to the first mount and/or intro-
mission was measured (mount latency and intromission latency). If no

sexual behavior was observed during a particular test, a latency

33

measure of 600 seconds (the length of the test) was assigned for
that test. Following the final test for mounting, TP administration

ceased. _

Lordosis Behavior:

At approximately 30 and 44 days following the fifth test for
mounting behavior, 10 minute tests for lordosis behavior were given to
all experimental animals. Lordosis tests were preceded by daily admini-
stration of 6 ug EB for 3 days and an injection of 0.5 mg progesterone
on day 4, 4-6 hours prior to testing. Testing occurred between 1300 and
1700 hours. Measurement of lordosis behavior entailed placing the
experimental animal into an observation arena (10 gal. aquarium) with a
sexually vigorous indicator male and recording the lordosis behavior
of the experimental animal. Indicator males which did not mount were
replaced. Measures derived from the Esterline-Angus recorder record
included the frequency of lordosis exhibited, the total amount of
time spent in lordosis and the duration of the longest lordosis re-
sponse during each 10 minute test. A mean for each of these measures

was taken for each animal.

Morphological Measures

 

Approximately one month after the final lordosis test, all
animals were sacrificed and inspected for the presence of testicular
or ovarian tissue. If gonadal tissue remained, the data from that
particular animal were eliminated from the study. Anogenital distance
was measured in all animals. Penile or clitoral bone and cartilage

was dissected out and the total length of these was measured.

34

Statistical Analysis

 

The data were evaluated using one way analysis of variance and
further comparisons were made using the Student-Newman-Keuls test for

multiple comparisons among means (Sokal and Rohlf, 1969).

Results

MountingZBehavior of Males

Twenty eight days of TP administration induced high levels of
mounting in males treated neonatally with testosterone (Fig. 5 and 6,
Table 3) but not in animals treated neonatally with fluoxymesterone
or sesame oil. By test 4 (28 days) analysis of variance revealed a
significant difference among groups for mount latency, head and side
mount frequency, and rear mount frequency. While animals treated neo-
natally with T showed significantly higher head and side mount scores
and rear mount scores than the other groups (p<0.05), animals treated
neonatally with fluoxymesterone or sesame oil did not differ statistic-
ally (F-6.58, df=2,24, p=0.005; F=ll.9l, df=2,24, p<0.01; and F=3.64,
df-2,24, p<0.05 respectively). Similarly, fluoxymesterone and sesame
oil treated animals showed significantly longer latencies to the first
mount than did the neonatal testosterone group (p<0.05). Fluoxymesterone
and sesame oil groups did not differ in mount latency. The only group
to show any intromission patterns was the neonatal testosterone

group (mean intromission frequency - 1.8 on test 4).

35

 

 

TEST

 

 

s m o
nuanbm anOW aaIs cm (was NVSW

Figure 5. Effects of neonatal administration of fluoxymesterone,
testosterone or sesame oil on mean head and side
frequencies in castrated male hamsters.

36

 

 

 

2
TEST

 

 

Pro

 

ADNIODJUi .lNflOW 8V3! NVIW

Figure 6. Effects of neonatal administration of fluoxymesterone,

testosterone or sesame oil on mean rear mount frequencies
in castrated male hamsters.

37

Table 3. Mean mount latency for male hamsters castrated on the day
of birth and treated neonatally with fluoxymesterone,
testosterone or sesame oil (in seconds i s.e.).

 

 

 

 

 

 

Neonatal Treatment Weekly Tests
(d2-3-4) Pretest 1 . ' 2 3 4
Fluoxymesterone 600 600 600 573 458
(100 ug/day) i0 i0 i0 :20 £73
Testosterone 600 389 p 310 261 106
(100 113/ day) :0 :80 :85 :86 :50
Sesame Oil 600 600 454 389 373

 

(0.03 cc/day) i0 i0 . :74 :84 £92

 

 

38

Mounting Behavior of Females

At 28 days of daily TP treatment, females treated neonatally with
testosterone showed higher head and side mount and rear mount fre-
quencies than fluoxymesterone or sesame oil treated females (p<0.05).
Fluoxymesterone and sesame oil treated females were not different from
each other in mount frequencies (Fig. 7&8). Fluoxymesterone and sesame
1011 groups did not differ from each other in latency to the first
mount (Table 4). However, both groups took a longer time to the first
mount than did the testosterone treated females, (H&SMF, Fb8.83, df-2,33,
p<0.01; RMF, Fb15.49, df-2,33, p<0.001; and ML, Fb7.05, df-2,33, p<
0.005). Intromission patterns were not seen in any of the treatment

groups.

Lordosis Behavior of Males

Testosterone treated animals showed more lordoses than did
fluoxymesterone treated animals (F-4.24, df-2,23, p<0.05) but the
treated animals were not different from sesame oil animals (Figure 9).
Likewise, fluoxymesterone and sesame oil animals were not different

from each other in lordosis frequency.

Lordosis Behavior of Females

Females treated neonatally with testosterone showed more lordoses
than sesame oil or fluoxymesterone treated animals (Figure 10) (L.F.
F-5.20, df-2,33, p<0.025, and Longest L F-4.53, df-2,33, p<0.025)
while sesame oil and fluoxymesterone animals were not significantly
different. In contrast to lordosis frequency, the mean longest

lordosis for testosterone

39

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5
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.—
F
O
b
O.

      

2 ‘0 °
ADNJOOSIH lNflOW 301$ ONV Oval-l NVSW

Figure 7. Effects of neonatal administration of fluoxymesterone,
testosterone or sesame oil on mean head and side mount
frequencies in female hamsters.

4O

 

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~ 9.
2
IL
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Figure 8. Effects of neonatal administration of fluoxymesterone,
testosterone or sesame oil on mean rear mount frequencies
in female hamsters.

41

Table 4. Mean mount latency for female hamsters treated neonatally
with fluoxymesterone, testosterone or sesame oil
(in seconds:ts.e.)

 

 

 

 

 

 

Neonatal Treatment Weekly Tests
(d_2-3-4) Pretest 1 2 3 4
Fluoxymesterone 600 600 571 ‘ 523 445
(100 ug/day) i0 i0 , :29 .t49 :60
Testosterone 461 170 123 49 132
(100 ug/day) 161 :66 :55 :10 :53
Sesame 011 550 600 535 473 457

(0.03 cc/day) :50 i0 :76 :76 £75

 

 

 

42

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auv nouvana 5500301 111101 uvaw

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Figure 9. Parameters of lordosis behavior in castrated male
hamsters treated neonatally with fluoxymesterone,
testosterone or sesame oil.

43

sonooas NI (OiOVHS) SIsoozIO'I 1539N01 nvaw
an Mouvana SIsoaIIOI 1v101 nvaw

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0 n v 8

 

 

 

 

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T

 

 

 

 

NEONATAL TREATMENT

 

 

 

 

 

 

 

In Q ('5 N '-

ADNiflOBIH SISOOUO'I NVJW

Figure 10. Parameters of lordosis behavior in female hamsters
treated neonatally with fluoxymesterone, testosterone
or sesame oil.

44

treated females was shorter than that of either fluoxymesterone or

sesame oil treated females.

Morphological Measures

 

Males:

While anogenital distance did not vary significantly among the
three groups, analysis of variance did reveal a significant difference
among treatments in length of penile bone and cartilage (F=58.63,
df-2,24, p<0.01). Neonatal fluoxymesterone and testosterone treatment
caused a significant increase in penile length when compared to sesame
oil treated animals (Table 5) (P<0.05). Fluoxymesterone and testos-

terone treated males did not differ from each other significantly.

Females:

Neonatal treatment with fluoxymesterone on testosterone resulted
in a longer anogenital distance than did sesame oil treatement
(Fh69.5, df=2,33, p<0.001); fluoxymesterone and testosterone treated
animals were not different from each other (Table 6). Fluoxymesterone
and testosterone treated females, while not different from each other,
showed greater clitoral virilization than did sesame oil treated

females (F=58.4l, df=2,30, p<0.001).

Ovarian Histology

 

None of the neonatal treatments, fluoxymesterone, testosterone,
or sesame oil disrupted ovarian function. Ovaries possessed.corpora-

lutea and developing follicles, and appeared normal.

45

Table 5. Morphological measures of male hamsters castrated on the
day of birth and treated neonatally with fluoxymesterone,

testosterone or sesame oil.

 

 

Neonatal treatment
(d 2-3-4)

AeG distance
mean length in mm

Penile bone and
cartilage mean

 

 

 

(i s.e.) length in mm ($s.e.)
Fluoxymesterone 16.0 4.6
(100 Ug/day) $0.4 $0.0
Testosterone 15.2 4.5
;(100 ug/day) $0.4 $0.0
Sesame Oil 14.7 3.4
'(0.03 cc/day) $0.2 $0.0

 

 

 

Table 6. ‘Morphological measures of female hamsters treated neonatally
with fluoxymesterone, testosterone or sesame oil.

 

 

Neonatal treatment
. (d 2-3-4)

A-G distance
mean length in mm

Clitoral bone and
cartilage mean

 

 

 

 

($s.e.) length in mm ($s.e.)
Fluoxymesterone 10.2 4.0
1 (100 ug/day) $0.2 $0.0
Testosterone , 10.3 3.9
(100 ug/day) $0.2 $0.0
Sesame Oil 7.2 3.0
(0.03 cc/day) $0.2 $0.0

 

 

 

 

46

Discussion

 

While neonatal administration of fluoxymesterone or testosterone
to castrated male and female hamsters induced similar patterns of
penile and clitoral growth in response to androgen at adulthood, only
testosterone achieved significant masculinizing influence upon behavior.
Fluoxymesterone neither "masculinized" nor "defeminized" behavior.
These findings as well as those of others (Reilly and Gordon, 1961;
Beach and westbrook, 1968; Johnson and Tiefer, 1974; Hart, 1972)
strengthen further the idea that fluoxymesterone is a peripherally
acting androgen with little or no central neural actions.

The absence of mounting and intromission in fluoxymesterone treated
animals in spite of marked genital virilization agrees with other work
in which a dissociation of sexual behavior and genital virilization was
found (Coniglio, 1973; Paup et a1., 1974). The fact that the present
results with hamsters do not agree entirely with those Hart obtained
with the rat may be attributable to species or dosage differences.
Indeed, male rats castrated neonatally normally show male sexual be-
havior (Larsson, 1966) when given androgen as adults, while hamsters
do not.

Although the neonatal dosages of hormone given did not induce
ejaculation in any group, mounting and intromission were induced by
testosterone given neonatally. This suggests that in the hamster,
mounting and intromission are not organized independent of androgen as

has been speculated for the rat (Beach and Holz, 1946; Whalen, 1968).

47

Whalen (1968) argued that in the rat, the reduced incidence of intro-
mission in males castrated neonatally reflects inadequate penile
development. Similar conclusions have been reached by Nadler (1969)
and Goldman et a1., (1972). Loss of intromission after various mani-
pulations including removal of sections of penile bone (Beach and Holz,
1946), the glans penis (Whalen, 1961) anesthetization of the penis
(Carlsson and Larsson, 1964; Adler and Bermant, 1966) and removal of
the dorsal nerves of the penis (Larsson and Sgdersten, 1973) lend
credence to the hypothesis that successful intromission is a function of
an adequate phallus. Although the phalli of the testosterone and
fluoxymesterone groups in this as well as in Hart's study (1972) were
not different, intromission behavior always occurred with greater
frequency in the testosterone treated groups.

It is proposed that testosterone, prenatally in the rat, and
postnatally in the hamster programs the CNS for mounting. FM on the
other hand lacks this effect, but shares with T the ability to virilize
the genital morphology. The differences in T & FM in intromission
scores reflect the differential effect of these hormones on mounting
behavior. Since intromissions cannot occur without a mount, an androgen
which does not program the CNS for mounting cannot induce intromissions.

In the present study, higher doses of testosterone neonatally or
more prolonged administration might have induced adult ejaculations.

It is doubtful that increased fluoxymesterone would have, since
ejaculation was not induced in rats with much higher doses of fluoxy-

mesterone .

EXPERIMENT 3

Introduction

 

Evidence that sensory feedback from the penis is necessary for
intromission and ejaculation has accumulated for several species
(Adler and Bermant, 1966; Whalen, 1968; Aronson and Cooper, 1966,
1968; Herbert, 1973; Larsson and Sgdersten, 1973). However, manipu-
lations which result in changes in penile sensitivity, size, and
structure of the penis do not have as profound an effect on mounting
as they do on intromission (Beach and Holz, 1946; Adler and Bermont,
1966; Whalen, 1968; Nadler, 1969; Goldman et a1., 1972).

The fact that penile sensitivity has little influence on mounting
.is not entirely clear. Beach and Levinson (1950) postulated that the
loss of mounting behavior subsequent to castration in rats was
correlated with a loss in penile sensitivity. Even though electro-
physiological research suggests that castration does not cause a
decrease in penile sensitivity in cats (Cooper and Aronson, 1974), it
has been shown that decreases in peripheral sensitivity produced by
local anesthesia or nerve section can lead to decreases in mounting
over time (Carlsson and Larsson, 1964; Aronson and Cooper, 1968;
Herbert, 1973). Experiment 1 of this thesis showed that penile sensory
feedback may have a facilitatory effect upon mounting as well as
intromission and ejaculation in rats. In order to extend the work of

Experiment 1 and to entend the comparative basis for theories of

48

49

the peripheral influence on male mating behavior, the hamster was
chosen for further work. The questions asked in this experiment are:

1) Does penile sensory feedback facilitate male mating

behavior in the hamster?

2) Does sexual behavior vary as a function of penile

size in the hamster?

Sexual differentiation of male mating behavior in hamsters is
presumably under the control of gonadal steroids present during some
"critical period" in development (Clemens, 1973; Eaton, 1970; Coniglio,
1973). The presence of estrogens or androgens which can be aromatized
to estrogens during the period of behavioral sexual differentiation
leads to an animal which will show mounting as an adult. In the.
absence of such androgen or estrogen the female behavior pattern of
receptivity (lordosis) but not mounting behavior are evident at
adulthood. Gonadal steroids also affect the growth potential of the
genital tissue of the male. The presence of androgens during develop-
ment results in a phallus which will respond with growth to pubertal
increases or exogenous administration of androgens at adulthood
(Bruner and Witschi, 1946; Beach, Noble, and Orndoff, 1969; Coniglio,
1973). However, estrogens, which will cause behavioral masculinization
do not increase the potential for genital virilization (Coniglio, 1973,
Paup, Coniglio and Clemens, 1974). This dissociation of behavioral

and morphological development with estrogens and the concomitant

50

behavioral and morphological virilization with androgens can be used
to create an experimental model to study the influence of penile
sensory feedback on behavior. Neonatally androgenized and estrogenized
animals have a similar potential for mounting but a different potential
for phallic growth when given T? as adults.

It was presumed that sensory feedback from the penis in neonatally
androgenized hamsters would be greater than the sensory input from
the unvirilized neonatal estrogenized animal., The present experiment
attempted to.reveal differential feedback effects on the maintenance
of mounting behavior. Fluoxymesterone maintains penile size and
integrity but not sexual behavior while section: (DNP) 0f the dorsal nerve

of the penis provided a presumably complete reduction in penile sensations.

Methods and Materials

Subjects

The subjects of this study were 107 male golden hamsters (Meso-

 

cricetus auratus) born and raised in the Hormones and Behavior
Laboratory at Michigan State University. Animals were maintained as

outlined in Experiment 2.

Treatments and Behavior Testing_

 

All males were castrated on the day of birth (day 1) using the
procedures described in Experiment 2. On days 2, 3, and 4 of life all
animals received subcutaneous injections of one of the following
hormones: testosterone propionate, either lOOug or 25ug per day (TP
100 or TP25) or estradiol benzoate 25 ug/day (E825). Injections were

administered under the same procedural paradigm as Experiment 2.

51

At 60 days of age all animals were earpunched for identification.
When the animals were 75 days of age they entered a weekly testing
schedule for mounting behavior with test procedures and measures
identical to those in Experiment 2. The first 5 weekly tests were the
same as those in Experiment 2, namely, a 10 minute pretest prior to
T? treatment, daily injections of 300ug TP and 10 minute tests at 7 day
intervals for 28 days.

Following the 5th test for mounting behavior, animals from each of
the neonatal treatment groups were randomly assigned to one of two
groups; to be called Cut or Sham. Males in the Cut groups were sub-
jected to bilateral removal of a 2-4 mm section of the dorsal nerve
of the penis (DNP). Procedures used were the same as in Experiment 1.

Animals were allowed a recuperation period of 10 days with daily
TP injections before receiving 3 weekly postoperative tests (tests 6-8).
Following the third postoperative test, TP injections were terminated
and animals from cut and sham groups were assigned to one of the fol-
lowing two hormone conditions. Condition 1 consisted of daily admini-
stration of 300ug fluoxymesterone. Condition 2 was no exogenous
hormone at all but daily injections of the sesame oil vehicle (0.05cc).
All animals were then tested for mounting at weekly intervals for 8
weeks (tests 9-16). Behavioral measures were the same as those taken
for male behavior in Experiment 2. The final groups for data analysis
are as follows:

TP IOOEg neonatal

 

1) FM CUT: DNP section; fluoxymesterone for the final 8 tests.
n-9

52
2) FM SHAM: sham operated males; fluoxymesterone for the final
8 tests. n=11
3) OIL CUT: DNP section; sesame oil for the final 8 tests. n=10

4) OIL SHAM} sham operated; oil vehicle for the final 8 tests.
n=lO .

TP 25 pg neonatal

 

1) FM CUT n=8

2) FM SHAM n=6

3) OIL CUT n-IO
4) OIL SHAM n=10
EB 25 uggneonatal

1) FM CUT n=9

2) FM SHAM n=9

3) OIL CUT n=8

4) OIL SHAM n=7

These 12 final groups were sacrificed 1-2 days after the completion

of test 16.

Morphological Measures

At sacrifice all animals were inspected for completion of gonadect-
omy. Any evidence of remaining testicular tissue eliminated the
animals from.the experiment. Ana-genital distances were measured in
all animals. Penile bone and cartilage were removed as a unit and
measured in all animals. Portions of the glans penis‘were fixed in
Bouin's solution for histological examination. Penile glans tissues
were embedded in paraffin, sectioned at 10 u, stained with hematoxylin-

eosin and examined for the presence of spines and/or papillae.

53

Statistical Analysis

Data.were evaluated using one way analysis of variance. Further
comparisons were made using the Student-Newman—Keuls test for multiple
comparisons among means (Sokol and Rohlf, 1969) or Mann-Whitney U-test.

Although not listed in results section,S-N-K and MPW-U p<0.05.

Beagles.

Results of neonatal treatment (Tests 1-5), penile desensitization
(Tests'6—8) and hormone manipulation (Tests 9-16) on parameters of
male mating behavior will be considered separately. Appendix Tables
Al-A4 give means $ standard errors for various copulatory behaviors.
(Tables A1, mean latency to first mount; Table A2, mean head and size
mount frequency. Table A3, mean rear mount frequency, and Table A4
mean intromission frequency. Tables A5-A7 list the percentage of
animals exhibiting either head and side mounts, rear mounts or intro-

missions for each neonatal treatment group.)

Effects of Neonatal Treatments (Tests 1-5)

Twenty eight days of TP administration in adulthood caused an in-
crease in mating behaviors in all groups (Table A l and Figures 11-13,
tests 1-5). Analysis of variance of scores from test 5 revealed no
differences within any of the neonatal treatment groups for mean rear
mount frequency, latency to mount or mean intromission frequency.
Comparison of test 5 scores among the 3 neonatal treatment groups
revealed no differences in rear mount frequency, however TP 100 and TP
25 animals achieved significantly more intromissions than did neo-

natally EB 25 treated animals (f-4.730, df-2,33, p<0.025; S-N-K p<0.05).

54

10}
FM CUT CW.
0". CUT a-v-u

/‘ FM SHAM 0—0
W“ on SHAM ._.

MEAN INTROMISSION FREQ.
on

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67891011‘23T4'5T6
TEST

 

 

 

_.
up
up
#-
“I'

Figure 11. Rear mount frequency and intromission frequency in day l
castrated male hamsters treated with TPlOO neonatally
and given various adult treatments.

55

 

 

 

 

   

1 FM cur .............
o to on cur ...........
“ on SHAM ._..
3 .._...
g s . ./ \
o .
E.
z D
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I I I I I I I I I I I I

 

 

 

 

78910111213141516

...
”I
a.
ub-
u.-
0

TEST

Figure 12. Rear mount frequency and intromission frequency in day l
castrated male hamsters treated with TP25 neonatally and
given various adult treatments.

56

 

 

 

 

 

 

 

 

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Figure 13. Rear mount frequency and intromission frequency in day l
castrated male hamsters treated with EB25 neonatally and
given various adult treatments.

57

Ejaculations were infrequent in all neonatal treatment groups. Only
7.5% of TP 100 animals, 18% of T? 25 animals and 0% of EB 25 animals

achieved ejaculation on test 5.

Effects of Penile Desensitization (Tests 6-8)

For the 4 groups treated neonatally with TP 100, fluoxymesterone
sectioned animals had significantly more mounts on test 8 than the
other 3 groups (p<0.05) while those groups did not differ from each
other (Figure 11). Analysis of latency measures showed that the TP 100
sectioned groups had shorter latencies to mount (Table A1) than sham
animals (RMF f=4.752, df=3,36, p<0.01 and ML F=5.346, df=3,37, p<0.005).
Mean intromission frequency on test 8 did not differ statistically
among the 4 groups of TP 100 animals. Animals treated neonatally with
T? 25 (Figure 12) did not differ among groups on test 8 in rear mount
frequency. While fluoxymesterone sham and oil sham groups did not
differ in intromissions, fluoxymesterone sham animals did intromit more
than cut groups (F=3.509, df=3,35, p<0.05). The 4 groups of animals
treated neonatally with EB 25 were not different from each other in
either mount latency, rear mount frequency or intromission frequency
on test 8.

As can be seen in appendix Tables A5-A7, a considerable variation
in the number of animals mounting occurred on each test. To compensate
for animals which did not mount on test 8, analysis of mount positive
animal scores was run:

Mount + rear mount frequency = RMF X 1
Z mounting.

 

When only animals which mounted were considered, no differences on

58

test 8 rear mount scores were observed for animals receiving neonatal

TP 100, TP 25 or EB 25.

Effects of Hormone Manipulation_(Tests 9-16)

 

Following withdrawal of daily TP treatment and substitution
of either daily fluoxymesterone or sesame oil, a marked decline in
mounting and intromission was observed in all 3 neonatal treatment
groups (Table A1, Figure 11-13, Tests 9-16). Analysis of test 9 scores
for rear mount frequency or intromission frequency gave results identi-
cal to those of test 8 without consistent differences among treatment
conditions in each neonatal treatment group. By test 16, mounts and
intromissions were at a very low level in all groups in each neonatal
treatment. Analysis of variance revealed no differences in rear mount
or intromission frequency within neonatal treatment groups. Likewise,
analysis among neonatal treatment groups revealed no differences. How-
ever, there was a trend that within each neonatal treatment group,
fluoxymesterone treated animals showed more mounts and intromissions than
oil treated animals but this was not statistically significant. By test
16, no oil treated animal in any group exhibited intromissions. The
only oil treated animals to show rear mounting on test 16 were one

animal in TP 100 oil cut group and one animal in TP 100 oil sham.

Morphology

Measures for anogenital (AG) distances and penile bone plus
cartilage were analyzed for differences both within, each neonatal
treatment group and among neonatal treatments. Results of these

comparisons will be considered seperately.

59

Within Neonatal Treatments:

NEONATAL TP 100 GROUP. (Table 7) For AG distance, oil cut, oil
sham and fluoxymesterone cut animals did not differ statistically.
Fluoxymesterone sham animals exhibited significantly longer AG distances
than any other group (AG F=8.792, df=3,35 p<0.001). Mean bone and
cartilage lengths for oil out and oil sham animals did not differ from
each other. Oil cut animals had significantly smaller penile bone and
cartilage lengths than did fluoxymesterone treated animals (B & C
F53.437, df=3,36, p<0.05). Oil sham, fluoxymesterone cut and sham
groups did not differ from each other.

NEONATAL TP 25 GROUP. (Table 7) Within the neonatal TP 25 group
oil sham.and oil cut animals did not differ in AC distance, however
both groups had significantly shorter AG distances than did the fluo-
xymesterone cut group (F=l4.531, df=3,30, p<0.001). Fluoxymesterone
cut animals had significantly shorter AG distances than did the fluo-
xymesterone sham group (p<0.05). Fluoxymesterone sham animals showed
greater penile virilization than did any of the other groups (F-6.350,
df=3,30, p<0.005) which did not differ from.each other.

NEONATAL EB 25 GROUP. (Table 7) For the neonatal EB 25 treatment
group, bone and cartilage lengths were uniformly small for the 4
treatment conditions. Analysis of the AG distances revealed that the
oil sham group had significantly shorter AG distances than the other
3 groups (Fh5.016, df-3,29, p<0.01) which did not differ from eadh

other.

- ‘(llllll

60

 

 

 

 

 

 

 

 

Table 7. Mean anogenital distance, penile bone plus cartilage lengths
(in mm) and penile condition in male hamsters treated neo-
natally with TPlOO, TP25 or EB 25.
AG distance Bone + Cartilage Penile spines and
$ s.e. length $ s.e. papillae at sacrifice
‘TP100
FM Cut 15.9 0.6 5.5 0.1 +
Oil Cut 15.0 0.3 5.2 0.0 0
FM Sham 18.2 0.6 5.6 0.0 +
011 Sham 15.7 0.4 5.4 0.0 0
TP25
FM Cut 18.0 0.6 5.4 0.0 +
011 Cut 16.3 0.7 ’5.3 0.1 0
FM Sham 19.5 0.5 5.8 0.0 0
Oil Sham 5.4 0.3 5.5 0.0 O
(EB25
FM Cut 15.5 0.7 3.5 0.2 +
Oil Cut 14.7 0.3 3.3 0.1 0
FM Sham. 14.8 0.5 3.7 0.0 +
Oil Sham 12.8 0.2 3.4 0.0 O

 

 

 

1 I__‘ .IIIIII I II III lull l Il'lll I." I I‘ll:

61

Among Neonatal Treatments:

Comparing neonatal TP 100, TP 25 and EB 25 animals revealed that
EB 25 animals, whether from fluoxymesterone or sesame 011 groups, had
significantly shorter AG distances than either of the TP groups
(fluoxymesterone Fe16.760, df=2,50, p<0.001) (sesame oil F=9.902,
dfs2,50, p<0.001). TP 100 and TP 25 animals did not differ from each
other. Mean length of penile bone and cartilage across neonatal treat-
ment groups also revealed highly significant differences whether the
final hormone condition had been fluoxymesterone (F5199.966, df-2,51,
p<0.001) or sesame oil (F=230.337, df=2,48, p<0.001). Neonatal EB 25
treated animals showed significantly less penile virilization than
either neonatal TP treated group regardless of final hormone condition
.(p<0.05). TPlOO and TP 25 induced the same amount of penile virili-
zation.

Penile Spines and Papillae:

Because of the procedures used for removal of penile bone and
cartilage, glans penis tissue was too damaged for quantitative measure-
ment of penile spines and papillae. However, histological examination
of portions of the glans revealed that all fluoxymesterone treated
animals possessed penile spines and papillae while sesame oil treated
groups had none (see Table 7). Microscopic examination did reveal
subtle differences among the neonatal treatment groups after adult
fluoxymesterone administration. Neonatal TP groups, whether TP 100 or
TP 25, exhibited many long spines and deep, regular papillae. Neonatal

EB 25 animals had spines and papillae which were more irregular and

62

misshapen than those of neonatal TP animals. The general impression
is that fluoxymesterone treatment of neonatal EB animals induced fewer

and smaller spines.

Discussion

 

The present study demonstrates that penile desensitization yields
no clear evidence of an effect of genital sensations on male sexual
behavior in hamsters. Sectioning the DNP caused no consistent loss of
intromission as has been reported for rats (Experiment 1; Larsson and
Sgdersten, 1973), cats (Aronson and Cooper, 1968) and monkeys (Herbert,
1973). Thus for the hamster although the 3 neonatal hormone treatments
used in the present study induced different degrees of penile viriliza-
tion, few behavioral differences were observed. This was contrary to
the prediction based upon data on penile feedback effects in rats. TP
administration in adulthood induced similar mounting levels in neo-
natally TP or EB treated animals but intromissions were fewer in the neo-
natal EB group. This dissociation of behavioral and genital virilization
has been previously reported (Coniglio, 1973; Paup et a1., 1974).

The general decline in mating behaviors for all groups when TP
treatment was withdrawn and sesame oil or fluoxymesterone was sub-
stituted is consistent with results reported by other workers.
(Christensen, Coniglio, Paup & Clemens, 1973). Although fluoxymesterone
has not been used previously in hamsters the decline in sexual response
observed was similar to that seen in male rats (Johnson and Tiefer,
1975; Experiment 1). The trend for animals treated with peripheral
androgens to continue mounting longer than non-treated castrated

animals has been previously reported in rats (Parrott, 1974b).

63

However, fluoxymesterone does not have dramatic behavioral effects on
castrated male hamsters suggesting that penile integrity is not as
important for maintenance of sexual behavior in hamsters as it is in
rats. The general decline following the hormone change was not cor-
related with differences in penile size. Fluoxymesterone did maintain
penile integrity in the form of penile spines and papillae.

Since the role of penile sensations in the control of male mating
behavior seems to be different for rats and hamsters, a consideration
of possible reasons for this apparent difference is in order. That
section of the DNP in hamsters was less effective than DNP section
of rats in blocking intromissions may reflect anatomical species dif-
ferences. The DNP of rats branches extensively as it approaches the
distal end of the penis (Calaresu, 1970). The position of the nerve
cut along the penile shaft was made as close to the proximal end of the
penis as possible to avoid this branching. While the branching pat-
tern of the hamster's DNP has not been studied, it is possible that
the hamster has more extensive branching at the proximal end of the
penile shaft. This would result in incomplete desensitization and
would account for the persistence of intromission in nerve cut groups.
Likewise, if both cut and sham groups continued to receive penile
sensations through undamaged portions of the nerves, the similar
behavioral decline which was seen would not be surprising. Experi-
ment 1 with rats and work by Herbert (1973) in monkeys have shown that
partial desensitization does not eliminate intromission. Further

anatomical work would help us understand this problem.

64

Differences in behavioral decline that were seen with the rats in
Experiment 1 and not in the present experiment may have been related
to the level of masculinization in these hamsters. Male mating be-
havior in rodents might be viewed as a continuum with sexual arousal
at one end and ejaculation at the other. An animal must be sexually
aroused in order to mount a receptive female. Intromission cannot occur
without a mount and intromission must preceed ejaculation. In the
present study the experimental model used was at the lower end of the
continuum. The specific neonatal treatments used in this experiment
resulted in males which were sexually aroused, mounted, intromitted
occasionally and seldom ejaculated. Experiment 1 employed experimental
animals which encompassed the whole range of the continuum. If the
hamsters in the present study had also shown the whole range of behav-
iors, more consistent differences among groups may have resulted.

Another possible explanation for the discrepancy seen between rats
and hamsters is the difference in mating pattern. In the rat, a basic
unit of sexual behavior is the mount bout. The mount bout is defined
as a sequence of mounts with or without intromission, uninterrupted by
any behavior other than genital autogrooming. A series of mount bouts
leads to ejaculation. Reducing genital sensations with local anesthetic
in rats increases mount frequency, mount bout length and the number of
mounts per mount bout (Sachs and Barfield, 1970). In Experiment 1,
continued copulation in the fluoxymesterone treated animals may have
been related to this phenomenon instead of genital feedback. The male
hamster shows a somewhat different pattern with mount bouts interrupted

only by ejaculation. Our inability to find differences between cut and

65

sham animals may be related to this pattern since these hamsters were
not programmed for ejaculation.

The discrepancy seen between the results of Experiments 1 and 3
with rats and hamsters emphasizes the need for comparative work. Pre-
dictions based upon findings with the rat did not hold for the hamster.
Likewise behavior seen in these hamsters following nerve section ap-
peared to be different from work with cats and monekys. Further com-
parisons between species will add more to our knowledge of the control

male sexual behavior.

Conclusion

 

In the rat, penile sensations are involved in the expression of
intromission and ejaculation. However, previous experiments have
shown little effect of these sensations on mounting behavior. In
Experiment 1 we were able to demonstrate for the first time that sensory
feedback from the penis is involved to a degree in the maintenance of
mounting.

Other investigations have tried to show that penile virilization
at some critical period in development is necessary for later male
behavior. Contrary to this position, Experiment 2 showed that fluoxy-
mesterone treated hamsters which had the capacity for penile growth at
adulthood exhibited no capacity for male behavior. These results con-
firmed and extended those of others in establishing that penile virili-
zation during development is not sufficient to bring about adult sexual
behavior.

Penile sensations in adult hamsters seem to be less important for

male mating behavior than in rats. In Experiment 3 reduction of penile

66

sensory feedback in male hamsters caused little interruption of intro-
mission behavior. The decline in behaviors seen with fluoxymesterone
was not related to genital size or sensitivity.

Clearly, penile sensations via the dorsal nerve of the penis are
not among the most important sensory mediators of male mating behavior.
Whether sensations from deep pressure receptors, or other perigenital
nerve endings are involved to a greater degree is not known. Further
work along these lines may lead to other conclusions. The old central
vs. peripheral controversy is little more than academic. That central
neural stimulation and maintenance of behavior has far more import than
peripheral sensation has been shown many times. The present series of
experiments which was designed to explore the genital feedback effects
on behavior lends little support to the peripheral side of the central-

peripheral debate.

APPENDIX

67

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

HN NNH HN NNH HN NN NHH NNH NN NN HN HN NN NNH. «N o

NN« NH« N«« .N«N, NNN NNNII NNN NNH NNH N«H N«H NHH NNH NNN NNN NNN swam HHo
NNH NNH NN HN NN NN HN NN NN NN NN oN NN HN HN o

NNN NNN NNN NNH, NNN NHH. NNH. No NNH NNH N«H NNNIINNH NNH NNN NNN swam 2N
NN NN NN NN NN NN om NN NNH oN NN NN NN NN NN NN

NNN H«N NN« NN« «««. NNN, NNN NNN NHN oNN NNN .NNN NHN NNN «N« NNN use HHo
NN NN NN NN NN NN NN HN NN «N NN NN NN NN NN o

NN« NNN NN« No« «NN NHN HH« NNN N«N NNN HNH NNH NNN NNH NNN NNN one am
N« HN NN NN «N NN NN NN «N NN NN NN NN NN «N on

NNN N«« HN« NNN N«N. NN «N Na NNH «NH NN N«H N«H «NH NNN H«N swam HHo
oN NN NN NN NN NN NN NN NN NH NH «N NN NN NN NN

NN« HNN NNN HHN «NN NHN NNH, N«H NNH «N NN NNH HNH «NH NNN N«N aNNN ea
c o o o o NHH No «N HHH NNH NHH HNH NN NNN NN a

CNN NNN NNN NNN NONI. NH« .NN« NNH NNN NNN HNN «NN NNN NNN NN« NNN use HHo
HN NN NN NN NN NN HN NN NN NN NN NN NN N NNH «N

NNN NNN NNN NHN‘ NNN NNN N«H NNH NNH NNH NNH NNH «NH NN HNN NHN use 2N
NN «N o HN NN NN NN NN NN NN NN NN «N NN ea c

NN« NNN NNN «N« omet HN« NNN NN« NNN NNN «N« NNN NN« NNN NNN NNN swam HHo
NN NN «N NN «N NN NN NN NN NN NN Hm NN NN NN NN

N«N, NN« «N« H«NI, NN« NN« NN« NNN N«« «NN NNN NNN ««N NNN NNN NN« aNNN za
NN NN «N NN NN HN NN NN NN NN NN HN NN NN NN «

NNN NN« NNN. No« «HN HNN «NN NNN NNN NNH NNH NNN N«H NNH NNH NNN .uao HHo
NN NN NN HN NN NN NN NN NN NN HN NN NH NH «N «N

HNN NNN NNN NNN NNN NHH oN NNH NN NNH NNH N«H «N NN NNN NHN use an
NH NH «H NH NH HH oH N N N N N « N N H guano

NNNN
.mudguwwuu HHH—flu 30.?“ri fl0>fiw van mm HO 9H Sag hHHflufldOOd
vmuwwuu anon—mam..— OHQE vmufluummu Rom Amvfioowm adv Ugo:— umHHH OH hugumfi so: .H< Quads

EB 25

TP25

TP 100

Mean head and side mount frequency in castrated male hamsters treated

neonatally with TP or EB and given various adult treatments.

Table A2.

68

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

so moorxoonnmoomo‘ PTMHHOOHHONOQ
H .oooooro o at. o o o o so to o coo
filcawIwIc>c>~Ic>rICPc>c>c>c>c>c>oIoIc>c>¢>0IFIFI
I I ‘ .
.n cuconncouncncucn~o~o INIn<DIOIxune4v4<n<o~cvq
H 00 o. .0900. 0000 so 00 0000
rIc>NIc>c>c>c>c>eINIc>c>c>c>NIc>c>c>c>c>vsoaktc:
-o curqcaxor4\o c>ux OJGDGDQ’Flh-Q'OIW)C>FTF-
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u -H ~H -H -H
:5 EoEoEoEanES
TP 100 TP 25 EB 25

 

Mean rear mount frequency in castrated male hamsters treated
neonatally with TP or EB and given various adult treatments.

Table A3.

69

 

 

TEST

ll 12 13 14 15 16

10

Group

 

4.4
1.7

2.0
1.4

3.8 3.1
1.6

4.6

6.2

10.7 7.6

3.4 7.6 13.6
2.4 2.2 2.8

0.6 3.1 7.1 4.6 4.8
0.4 1.8 3.6 1.4 1.4

FM Cut

2.3 1.8

U.

2.3

3.1 2.0

 

UoJ

A

0.4

U78:

4.0
0.6

1.6

6.4 6.]

5.2' 6}U 6.2

2.47 410T’2.0

3T2

0

Oil Cut

 

0.3

0.3

0.5

1.9 3.1

1.9 2.4 2.2

1.5 0.8 1.3 0.7

1.2 2.6 2:4’

1.0 1.7 2.0

1.2 1.0 ”1.8 1.2

NH

0.7» 0.2
0.5 0.2
1.27’TI.4
0.8

.2
1.7
4.0
2.3
3.5
1.5

1.1
1.2 0.6
3.0 518
1.7 2.9
7.6 5.9
3.4 12.9

0.7 0.6 '1.2 0.9

0
0

FM Sham
Oil Sham 0

 

U

1.3 2.9 4213
0.6 1.5 1.6

0.4 1.1 1.9 3.7

 

1.4

0.4 0.7 1.3 1.7
0.1 2.8 2.1 3.5 4.8
0.1 4.4 1.6 1.5 1.7

O

 

1.4 2.5 1.1 1.5
1.2 0.9

0.9

4.8

6.5 5.5 9.4
3.2 2.0 3.1

3.5 5.7 4.2

PM Cut

1.5

2.5

 

1.2
0.7

6.0, 0.3

2.5 4.8
2.1 3.3

0.7 1.7 3.0 3.4

0.5 0.5 0.8 1.1

0
Oil Sham 091 1.1 2.8 4.6 5.2

O

0.2

0

Oil Cut

2.4 0.3

8.4 11.5
2.2 2.6

11.7 14.1

2.9 3.1 3.3

0.1

 

1.3

0.1
0.1
0.1
0.1
2.2
2.1

4.0 0.7 1.3

5.8
2.2

9.8 7.8 9.1

0
0

FM Sham

1.2 1.1

0.6

1.6

1.8 1.5 2.2

 

0.3
0.2

2.6

1.4

5.8
1.6

5.8 9.7 8.7

3.4 3.8

1.1 2.7 2.7

0.1 0.6 1.0 1.2 1.7

 

1.9 2.2 3.1
2.1

1.2

3.1
1.7
1.4
0.8

2.7

7.1 5.9

4.2 4.2

3.1 3.6 4.4
1.6 1.8 2.1

1.2 5.6 4.7 5.1

O
0

FM Cut

101

1.8

0.8 1.6 2.6 2.1
0.2 2.0 0.9 3.6 1.9

 

3.4

4.2 1.0
1.7 0.7

5.0 3.1

2.7 2.9 4.4
1.4 1.3 2.2

011 Cut

2.1

0.2 1.5 0.6 1.4 1.0

2.5
1.6

1.4

0.9

3.8
1.5
1.1

1.5 2.5
0.8

0.6

5.1 4.0 5.3 5.1

0 1.9 4.3 5.9 6.6
0

Oil Sham 0

FM Sham

1.9

1.9 1.4

7.0 12.0
3.2 8.0

1.6 1.3 1.8

1.1 2.7 2.3 2.5

 

5.4 3.4 0.6
2.8

3.2

7.0
5.4

8.4 6.9 8.4

1.0 3.8 5.7 5.7

0.4

0.8

3.0 2.4 5.0

0.8 1.7 2.7 1.7

0

 

 

 

Mean intromission frequency in castrated male hamsters treated

Table A4.

neonatally with T? or EB and given various adult treatments.

70

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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~c IdCD~¢\O~¢~3<h<fi-¢l\Inln<Dln¢3<3¢fi¢fi\DIdlnCDvfiI4
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0‘ Ftaaospd F«F4 vwasoan+ \OIn\O\D .
.000 00 0... 0...
F4c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>
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0.
F4 c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c>c:
a E
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H -H -H -H -H -H
sEoEoEoEoEo-Es
TP 100 TP 25 EB 25

 

71

 

 

 

 

 

 

 

O OH ON OH ON Om O H.O n.ne N.- s.eo n.OO OH
O OH OH OH OH Om O H.O m.m~ H.HH m.mm e.e¢ nH
O O O OH ON on O «.mH «.mm ~.- e.¢¢ e.ce «H
O OH ON O OH OH O «.mH n.m¢ m.mm O.mn s.eo OH
O ON on O on On O m.n~ O.mO ~.N~ O.nm c.e< NH
O O< Om OH ON Om H.O m.em n.9m H.HH 0.0w O.mm HH
ON ow Om OH on on O m.en m.<m O.mm 0.00 w.- OH
ON OQ ON OH ON ow ¢.Om O.mo m.cm m.mm O.mn 0.00 O
ON on Oc OH ON Oc «.Om O.mO ~.mo ¢.ec OOH OOH O
ON oq oq ON Om ow ¢.Om O.mO n.~n ¢.<¢ OOH 0.00 5
ON oe Om OH ON on e.cm O.mO m.~m ~.- 0.0m 0.0w O
on ow Om OH on OO c.Om O.me O.Hw O.mm s.eo 0.0m O
OH Om Om OH OQ OO m.¢m O.mO O.Hm n.OO OOH OOH ¢
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O O O O O on O O «.mH HHHH ~.NN ~.N~ H
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.H kmv do vmumuummu mama «HwaHa< .mumou Hau0H>mnoo now :onmHaouucH
no unaoa.umou .uanoavmva com com: wcHsonm muoumams uHma mo ammunmunmm .m< mHomH.

 

72

 

 

 

 

 

 

 

O O OH OH ON ON O O O O m.NH ON OH
O OH on O OH ON O O O O ON n.Nm OH
O O on OH ON ON O O O Om on On OH
O ON OO OH ON Om O O O O.NH ON ON OH
O OO OO Om OO OO O O O mN m.NO Om NH
Om Om OOH OO OO OO O m.mm m.mm ON m.NO ON HH
on em Om OO OO OO O N.OH Om ON Om m.NO OH
OO Om Om ON Om Om O N.OO OOH m.NH m.NO m.NO m
Om ON Om ON ON .OO O m.mm N.OO ON OOH ON O
ON Om Om Om OOH OOH O on N.OO O O.NO m.Nw N
OO OOH Om Om Om OOH O on N.OO O n.NO OOH O
O.NN O.NN m.OO ON Om Om m.mm m.mm m.mm H.Nm N.OO m.mw O
OO ON Om OO Om OOH N.OH Om N.OO Om mN O.NO q
H.Nm H.Nm OOH om OO Om O O Om m.Nm mN OOH O
OH On On OH ON ON .O N.OH m.mm m.NH Om Om N
O OH O O O OH O O O O m.NH mN H
H 2m Swan H 2% ZOOM H 2m Swan H xx imam umoa
OH»: aosm HHO OHIc swam 2h Ona uao HHO Oua uso ah
.muawaumouu uHavm m50Hua>
Ow>Hmumu can NHHmuaaom: mth amwa .H Now do vuumuummu who: mHmaH:<
.u0H>m:wn waHuma mHma mo mumumamuwm wafisonm mumumao: mHma mo ownusouumm .O< «Hams

 

73

 

 

 

 

 

 

 

O O 0.0N O 0.00 OO O O H.HH O m.OO m.OO OH
O 0.0N 0.0N O O.NO O.NO O O H.HH O N.NN N.NN OH
O 0.0N m.Ne O.NH O.NO O.NO O O m.OO O 0.00 N.NN OH
O O.NO ¢.HN O.NH O.NO ON O O m.OO O N.NN 0.00 OH
m.OH. m.NO N.OO O O.NO ON O m.OO m.OO O m.OO 0.00 NH
m.OH H.NO N.OO O.NO ON O.NO O m.OO O.¢O O N.NN N.OO HH
m.OO N.OO N.OO O.NH OO ON O m.OO N.OO O N.NN O.NN OH
0.0N H.NO O.HN O.NH O.NO O.NO H.HH N.OO N.OO H.HH 0.00 O.NN m
0.0N H.NO N.OO N.OH m.OO m.OO N.NN 0.00 0.00 H.HH 0.00 N.OO O
0.0N N.OO N.OO ON O.NO OOH N.NN N.NN 0.00 H.HH N.OO m.OO N
O.NO O.HN N.OO O ON O.NO H.HH 0.00 N.OO O O.NN 0.00 O
0.0N N.OO N.OO O.NO ON O.NO N.NN 0.00 0.00 m.OO O.NN O.NN O
m.OH ¢.HN N.OO OO ON O.NO e.ee O.e< 0.00 H.HH O.ee O.NN O
m.OO O.HN N.OO m.NO H.NO O.NO N.NN N.NN 0.00 N.NN O.NN O.NN n
O 0.0N H.NO O.NH O.NO ON H.HH m.OO 0.00 O N.OH 0.00 N
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