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‘ This is to certify that the
thesis entitled .

PHYSIOLOGICAL AND BIOCHEMICAL PARAMETERS OF
GONADOTROPIN INDUCED OVULATION IN THE
NONHUMAN PRIMATE

presented by

Jon M.R. Rawson

has been accepted towards fulfillment
of the requirements for

Ph.D. Physiology

degree in

 

Major professor i

 

0-7639

 

ABSTRACT
PHYSIOLOGICAL AND BIOCHEMICAL PARAMETERS OF

GONADOTROPIN INDUCED OVULATION IN THE
NONHUMAN PRIMATE

BY

Jon M.R. Rawson

These studies were conducted using two species of

nonhuman primates (Macaca fascicularis and Saimiri sciureus)

 

as models to evaluate the effects of systemic administration
of gonadotropins on ovulation induction, to develop a tech-
nique which would allow the determination of the effects of
local administration of gonadotrOpins on the ovarian follicle,
to assess the ovulation—inducing properties of gonadotropins
from primate and nonprimate sources when administered at

the follicular level, and to analyze the ability of proposed
biochemical intermediates of LH action to induce ovulation
when administered at the follicular level.

In the Saimiri sciureus Human Chorionic Gonadotropin

 

(HCG) was able to stimulate ovulation in 70% of females
without administration of Follicle Stimulating Hormone (FSH)

eight days following a previously synchronized ovulation.

Jon M.R. Rawson

There was a trend towards a second increase in ovulatory
response rate around days 14 to 18 following synchronization,
which was in agreement with the concept of a 7 to 9 day
cycle length for this species.

In 458 menstrual cycles from a colony of 28 female
Macaca fascicularis, the average cycle length was 30.9 i 0.21
days; with an average menstrual flow duration of 2.6 i 0.1
days. Of the 51 cycles where ovulation could be accurately
timed the follicular phase length was 14.7 i 0.27 days.

Four daily intramuscular injections of 1 mg of FSH
were superior to either two or three daily injections for
producing normal appearing follicular growth in the squirrel
monkey. In the macaque, FSH (5 mg/day for 5 days) induced
morphologically normal follicular growth in only 1 of 20
animals with the others exhibiting a high incidence of
cystic and hemorrhagic follicles.

Filter paper disks (2 mm diameter) saturated with either
FSH or Luteinizing Hormone (LH) had no effect on the
occurrence of ovulation in the macaque when administered
directly to the follicular surface. Subsequent experiments
suggested that the one hour interval, necessitated by
anesthetic limitations, was not sufficient to allow an
effect.

By using laparoscopy, a technique was developed for
injection of substances directly into the follicular antrum.
Injection of saline did not result in ovulation, and

injection of India ink revealed no observable leakage from

Jon M.R. Rawson

the injection site. The ovulation rate following intra-
follicular injection of LH into squirrel monkeys who were
not pretreated with FSH, while higher than that of females
receiving intrafollicular saline, FSH pretreatment only, or
the FSH pretreatment followed by intrafollicular saline,
does not compare with the rates of animals receiving an FSH
pretreatment designed to supply follicles at a preovulatory
stage. Furthermore, there was no difference in ovulation
rate among animals pretreated with FSH and then given
systemic HCG, intrafollicular HCG or intrafollicular LH.
Intrafollicular injection of HCG in the Macaca

fascicularis pretreated with FSH neither induced ovulation

 

nor caused a rise in the peripheral plasma progesterone
levels. However, this treatment caused a significant
lengthening of the menstrual cycle, with a return to nor-
malcy in the immediate post-treatment cycle.
Intrafollicular injection of prostaglandin an (PGFZQ)

induced ovulation in FSH primed squirrel monkeys at a rate

similar to that following LH. Neither PGE cyclic

2' .
3',5' AMP, or dibutyryl cyclic 3',5'AMP caused ovulation
in a significant number of animals when administered into

the ovarian follicle.

PHYSIOLOGICAL AND BIOCHEMICAL PARAMETERS OF
GONAODTROPIN INDUCED OVULATION IN THE
NONHUMAN PRIMATE

BY

Jon M.R. Rawson

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Physiology

1975

To those less fortunate

ACKNOWLEDGMENTS

During the course of my graduate program, several
individuals have distinguished themselves as worthy of
special recognition for their assistance in making that
program successful. Dr. W.R. Dukelow, as well as accepting
me into his laboratory and providing the facilities for
this research, has given his guidance and support for which
I am grateful. Drs. J.M. Rawson, J.R. Toepfer, W.R. Dukelow
and G.D. Riegle have served as models of professional
excellence upon which many of my ethical values have been
based. Drs. R.M. Harrison, D.E. Wildt and M.R. Bixby
deserve special thanks for their hours spent advising and
assisting these studies. To Drs. R. Bernard, J. Britt,

L. Clemens, J. King and J. Meites I would like to express
my appreciation for their time spent as a guidance com-
mittee. Sincere appreciation is also extended to three
individuals who have played a greater role in my graduate
career than they realize, Amylou Davis, Pat Smith and
Bonnie Cleeves.

Finally, to my parents, Dr. and Mrs. Jesse M. Rawson
and my wife Mary Frances I owe more than can be expressed,

for without them I would not be.

LIST OF TABLES . . .
LIST OF FIGURES . .
INTRODUCTION . . .

LITERATURE REVIEW .

Reproductive Cycle

Macaca spp.

 

Saimiri sciureus

TABLE OF

CONTENTS

Characteristics

 

Ovulation Induction and Follicular
Physiology of the Mammalian Ovarian Follicle
Follicle Growth

Theories of Ovulation

Growth.

Involvement of Prostaglandins in Ovulation
Involvement of Cyclic AMP in Ovulation
Mediatiation of Gonadotropic Action by

cAMP and/or Prostaglandins .

Intrafollicular Injection Techniques

MATERIALS AND METHODS

Experimental
Induction of
Cyclicity in

Units
Follicle Growth
Saimiri sciureus

 

Laparoscopic
Experimental
Progesterone

Techniques
Compounds
Assay

Statistical Analysis and Resource Allocation

iv

Page
vi

vii

35

35
36
37
37
41
41
42

RESULTS 0 O O O O O O O O O O I O I I

Macaca fascicularis . . . . . . .
Induction of Follicular Growth .
Local Administration Experiments

GonadotrOpin Studies . . . .

 

Studies Using Biochemical Intermediates

DISCUSSION . . . . . . . . . . . . .
SUMMARY AND CONCLUSIONS . . . . . . .
REFERENCES . . . . . . . . . . . . .
APPENDICES . . . . . . . . . . . . .

I. PORCINE FOLLICLE CULTURE IN

II. LAPAROSCOPY IN THE RABBIT .

VITRO

III. INDUCTION OF FOLLICLE GROWTH IN

AN IMMATURE SAIMIRI SCIUREUS

 

IV. PUBLICATIONS BY THE AUTHOR

V. VITA O O O O O O O O I O O

Page
44
45
52
53
53
69
73
81
84
96
98

102

105
108

112

Table

1.

LIST OF TABLES

Ovulatory Response of Saimiri sciureus to
500 IU HCG at Varying Intervals Following
Synchronization . . . . . . . . . . . . . .

 

Individual Mean Cycle Lengths and Menstrual
Flow Duration for Macaca fascicularis Used
in This Study . . . . . . . . . . . . . .

 

Effects of FSH and Intrafollicular HCG
in Macaca fascicularis . . . . . . . . . .

 

Effects of Local Administration of Ovarian
Disks in the Macaque . . . . . . . . . .

Response of Saimiri sciureus to Intra-
follicular Injections at Laparotomy . . . .

 

Comparison Between Intrafollicular Injection

Technique at Laparotomy and at Laparoscopy.

Response of Saimiri sciureus to Primate
and Nonprimate Gonadotropin Sources . . . .

 

Effects of Exogenous Gonadotropin Adminis-
trations on the Length of the Macaca
fascicularis Menstrual Cycle . . . . . . .

 

Effects of Intrafollicular Injections of
Biochemical Intermediates in the Saimiri
sciureus . . . . . . . . . . . . . . . . .

 

vi

Page

44

46

54

56

58

62

64

66

72

LIST OF FIGURES

Figure Page

1. Variation of Menstrual Flow and Cycle
Lengths of Macaca fascicularis During
the Year 0 O O O O O I O O O O O O O I O I O 48

 

2. Relationship Between Follicular Phase
Length and Menstrual Cycle Length of
Macaca fascicularis . . . . . . . . . . . . 51

 

3a. Mature Follicle on Ovary of Saimiri
sciureus Following 4 days of FSH . . . . . 60

3b. Mature Follicle on Ovary of Saimiri
sciureus Following Intrafollicular
Injection of India Ink . . . . . . . . . . 61

4. Plasma Progesterone Levels in Macaca
fascicularis . . . . . . . . . . . . . . . 68

 

5. Plasma Progesterone Levels Following
Intrafollicular Injection of HCG . . . . . 71

vii

INTRODUCTION

Regulation of mammalian reproduction offers a rich
Opportunity for the maintenance of the quality of life in
our environment. Methods of increasing the fertility of
food producing species must be found which have no side
effects on the consumer. Similarly, adequate procedures
for controlling human fertility must be developed, which
are more desirable than the present methods.

Control of ovulation offers one of the best opportu-
nities for this biphasic effect on fertility, in that its
occurrence can be either increased or decreased as desired.
However, the present methods for ovulation control do not
satisfy many of the necessary conditions for widespread
usage, since they are too costly or ineffective for the
majority of the world's population, and in short supply
relative to the overall need. In addition, many produce
side effects in some of the users.

Various nonhuman primates have been studied for their
applicability as models for the control of human repro-

duction. As early as 1897, Heape had studied the menstrual

2

cycle lengths of two species of Macaques and found that
their cycles closely resembled that of the human female.
Hartman's studies in the 1930's pioneered our knowledge of
the macaque menstrual cycle, and contributed much of the
background for our present studies. Regardless, the true
value of these species as models of human reproductive
physiology will only be realized when we have a complete
understanding of the complex interactions occurring during
their reproductive cycles.

Within the past decade another species of nonhuman

primate, the squirrel monkey (Saimiri sciureus) has become

 

popular as a research animal. This species is easier to
handle than the larger macaques and can be maintained for
a fraction of the cost. These facts, coupled with their
availability, have caused the squirrel monkey to become
the second most widely used nonhuman primate in biomedical
research. Despite this wide usage, literature estimates
of the reproductive cycle length of the females of this
species vary from 7 to 54 days. Understandably, documen-
tation of their actual cycle length would play a significant
role in the definition of the value of this species as a
model of human reproductive function.

One of the common methods for studying the physiology
of the ovary and its components has involved exogenous
treatments with gonadotrOpins from various sources. His-
torically, the response of the primate species to such

treatments has been less than satisfactory, an observation

3
attributed to a species specificity for the various hormones

(van Wagenen, 1968). Clearly, as the use of gonadotropic
hormones is increased both in the scientific and the clinical
fields, efforts must be made to assess the normality of
induced ovulations. Further, experiments employing systemic
gonadotropin treatments fail to reveal the actual effects
of these hormones at the level of the ovarian follicle, since
plasma protein binding, molecular modification in the cir—
culatory system, the presence of a countercurrent
multiplication in the ovarian vasculature or simply an
uneven distribution of ovarian blood flow may act to alter
the actual effects of these substances on the ovarian
follicle.

There has recently been a number of investigators
who have implicated prostaglandins or cyclic AMP as mediators
in the ovulation—inducing activity of LH. Such studies have
usually involved the systemic administration of these com—
pounds or their various inhibitors while subsequently
noting the effect upon ovulation. Other studies have
demonstrated the effects of LH sources on ovarian levels
of these compounds. Results of such studies are not
sufficient to conclusively describe the biochemical sequence
of events leading to and culminating in rupture of the
mature ovarian follicle. Further, few studies have been
reported using primate models for the study of follicular
physiology. Because of the importance of an understanding

of the physiology of the primate ovarian follicle, the

4

present studies were undertaken with the following

objectives:

1.

To analyze the responsiveness of the Saimiri
sciureus ovary to systemic HCG following a
previously synchronized ovulation, in order to
further define the reproductive cycle length of
this species.

To evaluate important cyclic ovarian phenomena

occurring in a stable colony of Macaca fascicularis.

 

To examine the ability of exogenous FSH to dupli-
cate the induction of follicular growth and normal
morphological development in these species.

To develop a technique which would allow the eval-
uation of the ovulation-inducing effects of
substances when administered directly to the
ovarian follicle.

To determine the efficacy of locally administered

gonadotrOpins for ovulation induction in the

‘squirrel monkey.

To assess the ovulation-inducing capabilities of
locally administered gonadotropin sources from
primate and nonprimate species, as well as the
effects of several biochemical intermediates of

LH action.

LITERATURE REVI EW

A thorough understanding of the ovulatory process in
the human is necessary in order to facilitate the intro-
duction of new biomedical therapies designed for the
alleviation of many fertility problems presently encountered.
It is evident that the basic research required to provide
this understanding can not employ human subjects and,
therefore, an attempt must be made to find an acceptable
model for this system. The nonhuman primate presents one
such possible model. Unfortunately, the available primate
literature is not extensive due to the rarity and the value
of such species.

In the following discussion of pertinent literature,
references to experiments with primates (especially the
genera Macaca and Saimiri) are included where possible.
Other references have been included which cite work in
nonprimate species which provide the foundation for the

present studies in the nonhuman primate.

6
Reproductive Cycle Characteristics

Macaca spp.

 

The burgeoning use of nonhuman primate species for
research purposes has resulted in a great wealth of data
regarding the reproductive cycles of the more commonly
used species such as the various members of the genus Macaca.
In 1973, this author reviewed the literature pertaining to
the reproductive cycle characteristics and discussed the

normality of the colony of Macaca fascicularis used in the

 

present experiments (Rawson, 1973). Recently, Butler (1974)
presented a review of this area which compared various
reproductive parameters among all of the Families of the

Order Primates.

Saimiri sciureus

 

One of the earliest reported attempts at the definition
of the squirrel monkey reproductive cycle was reported in
a technical note by Denniston (1964), who suggested a 25.2
day cycle based on vaginal cornification. He noted the
occasional presence of erythrocytes in vaginal smears but
was unable to correlate these cycles with an increase in
sexual receptivity or attractiveness to the males. In
1967, Lang utilized observations of the external genitalia,
presence of a vaginal plug and presence of sperm in vaginal
smears as well as vaginal cytology to describe a 12.5 day
estrous cycle. Rosenblum, Nathan, Nelson, and Kaufman

(1967) detected a cyclic appearance of erythrocytes in the

 

 

? F349"; ;. n if. .

aid; wrai

7

vaginal smears of 11 of 15 squirrel monkeys and, along
with data on the presence of sperm in vaginal smears,
defined a seven day estrous cycle. In 1968 Costellanos
and McCombs described a cyclic swelling of the external
genitalia and a prominent vulvar hyperemia which suggested
a cycle length of 12 days. No evidence of menstruation
was detected by this study. In a similar study Hutchinson
(1970) found no evidence of erythrocytes in the vaginal
smears of 26 female squirrel monkeys. Using vaginal
cytology, he described a 12.3 day cycle. In 1970,
Srivastava, Cavazos, and Lucas found no evidence of cyclic
menstrual flow or swelling of the external genitalia.

They observed a vulvar plug of desquamated vaginal cells
which appeared in a cyclic fashion. This, combined with
cytological data, suggested an 18 to 20 day estrous cycle.
Another study of this type (Gould et al., 1973) reported
an 8 day cycle in the percentage of cornified cells in the
vaginal smear.

Studies up to this time had made no attempt at direct
ovarian observation. Harrison and Dukelow (1974) reported
laparOSCOpic evidence suggesting a nine to eleven day cycle
length. Their procedure involved serial laparoscopies at
2 day intervals beginning 5 days after an artificially
induced ovulation. Subsequently, ovulation was diagnosed
in 4 of the 6 animals at an interval of between 9 and 11

days.

8
Ovulation Induction and Follicular Growth

 

In 1973, van Wagenen and Simpson published a text on
the subject of ovulation induction in the macaque which
included an extensive review of the early experiments in
this area. The consistent result of such early studies
using heterologous pituitary gonadotropins was successful
follicular growth with only a few reports of ovulation and
corpus luteum formation. Administration of anterior
pituitary extracts with HCG supplementation was found to
cause luteinization of the thecal layers but again, without
ovulation (Engle, 1933, 1934; Engle and Hamburger, 1935).
Hartman (1938, 1942, 1943) observed marked follicular
development in response to equine gonadotropin, partially
purified pituitary extracts, or transplantation of heter—
ologous pituitaries but only sporadic ovulations were seen.
The majority of these early studies stated that, while
equine gonadotropin resulted in follicular development, it
rarely caused ovulation, and that HCG given alone had
doubtful beneficial effects (Engle and Hamburger, 1935;
Hartman, 1938; Sielger and Fein, 1939; and van Wagenen and
Cole, 1938).

In 1956, Knobil, Morse and Greep suggested a species
specificity of monkey pituitary growth hormone for the
repair of the metabolic abnormalities observed in hypo-
physectomized rhesus monkeys since beef growth hormone
failed in this respect. The following year van Wagenen

and Simpson (1957) demonstrated the successful induction

9

of multiple ovulation in the immature and adult rhesus

monkey using a follicle stimulating substance prepared from
rhesus monkey pituitaries, followed by a mixture of this
substance with human Chorionic gonadotrOpin. They suggested
that the primate source of the gonadotropin may have accounted
for their repeated successful experimental ovulations, and
inferred that a species specificity existed in primates

for the ovulating hormone.

Knobil, Kostyo and Greep (1959) attempted to induce
ovulation in a group of 13 hypophysectomized rhesus monkeys,
and found that follicle stimulation was readily accomplished
with FSH preparations of non-primate origin and further,
that excessive follicle stimulation achieved too rapidly
inhibited ovulation in response to subsequent doses of
Chorionic gonadotropin. Their recommendation was that a
gradual stimulation of follicles with FSH followed by large
doses of HCG in a time span corresponding to the follicular
phase of the menstrual cycle was most likely to result in
ovulation.

The successful induction of ovulation in the rhesus
monkey using human urinary gonadotropins was reported in
1962 by Simpson and van Wagenen. This was a significant
accomplishment as these hormones are a more readily
available source of gonadotropic activity than either human
or monkey pituitaries. Their regime included five days of
human menOpausal gonadotropin (HMG) followed by 3 to 4
days of HMG + HCG. This produced from 3 to 6 ovulations

per ovary in four adult females.

10
Dede and Plentl (1966) subjected a group of rhesus

monkeys to 8 to 10 days of HMG with the addition of 2000
units of HCG to the HMG for 2 more days. Although they do
not state the total number of cycles tested, they did
report that ovulation was induced in 31 cycles, with five
pregnancies resulting.

Administration of 3 mg/kg clomiphene citrate by
gastric intubation, was observed by Valerio and Courtney
(1968) to increase the incidence of pregnancy from 15%
in a group of 30 infertile control females, to 56% in 27
infertile clomiphene females. This was assumed to have been
due to a correspondingly increased ovulation rate.

In 1968, van Wagenen suggested that successful
ovulation induction in the nonhuman primate was possible
with macaque pituitary gonadotropin, human pituitary gonado-
tropin, or human menopausal gonadotropin when combined with
human Chorionic gonadotropin. It was further stated that
pituitary preparations from other species (specifically
ovine) gave sporadic and inconsistent ovulations.

Results of a study designed to compare the relative
advantages of the various methods of ovulation induction
(Wan and Balin, 1969) suggested that while 60% of the
treated cycles were ovulatory in response to intramuscular
HMG (75 IU for 5 to 8 days) followed by HMG (35.5 IU) and
HCG (2000 IU for 3 days), 46% of these produced multiple

ovulations. In addition they found that 59% of clomiphene

11

treated cycles were ovulatory and that all clomiphene in-
duced ovulations were single.

Using clomiphene in Macaca fascicularis, Mahoney (1970)

 

found ovarian enlargement with 5 of 14 cycles being ovulatory.
Wan and Balin (1971) reported that 94% of their rhesus
monkeys treated with clomiphene responded with ovulation in
59% of the treated cycles. The clomiphene cycles were

said to be characterized by normal ovarian size and appear-
ance with single-site ovulations in every instance where
ovulation occurred. Using an HMG/HCG induction regime,

the same authors reported a total of six instances of ovarian
hyperstimulation, noted by exaggerated cystic enlargements.

In 1971, Breckwoldt, Bettendorf, and Garcia published
a study of the effects of various FSH/LH ratios in
amenorrheic or hypophysectomized rhesus monkeys. They
concluded that the minimum dose of LH required for full
follicular maturation was 35 IU/day for 10 to 12 days in
amenorrheic animals and 75 IU/day in hypophysectomized
animals. Further, they suggested that ovarian hyperstimu-
lation might be avoided by reducing the dose of FSH during
follicular develOpment, and using purified human pituitary
LH for the induction of ovulation.

Ovadia, McArthur, Smith, and Bashir-Farahmand (1971)
suggested that each animal should be considered individually
when inducing ovulation, and by monitoring several clinical
signs (total urinary estrogens, cervical mucus ferning,

vaginal smear, and sex skin coloration) they developed a

12

method involving "staircase" increases in HMG until the
total urinary estrogens had increased to 6 ug/24 hours
following which they withheld treatments for 2 days and
gave a single injection of 500 IU of HCG to simulate the LH
surge. Using this technique they reported the reliable
induction of single ovulations.

A much lower ovulation rate was obtained by Batta
and Brackett (1974), who found that only 2 of 6 animals
receiving PMS (900 IU over 6 to 8 days) followed by HCG
(2000 IU or 8000 IU) responded with ovulation. The addition
of 5 mg of prostaglandin E1 or E2 on the day following the
last HCG injection, however, was sufficient to increase the
observed ovulatory response to 100 % (18/18).

The first report of ovulation induction in a New
World primate species was published in 1967 by Bennett who
used a five day administration of pregnant mares serum
gonadotrOpin (PMSG) followed by 4 days of a combination of
PMSG and HCG. Superovulation (5.3 ovulations per ovary)
resulted from this regime. In 1970, Dukelow extended the
observations of Bennett with a study which compared the
follicle stimulating ability of FSH sources in the squirrel
monkey. His results indicated that 4 single daily injec-
tions of PMSG alone were not sufficient to cause large
numbers of follicles to develop. The optimal procedure
indicated by this study involved a pretreatment with pro—
gesterone for 5 days, to simulate the animal's luteal

phase, followed by 4 days of FSH and an injection of 500 IU

13
of HCG. With this procedure an ovulation rate of 56%

was reported, 75% of which were single ovulations. The
ovulation latency ranged from 10 to 12 hours following
the HCG stimulus.

In 1971, Fajer and Bechini noted a decline in ovarian
venous progesterone concentrations in the squirrel monkey,
within 5 days of an ovulation induction regime. This
suggested that either these induced ovulations were abnormal
or that indeed this may relate to an inherently short cycle
length.

The observations of Dukelow (1970) were extended in
1973 by Harrison who used the technique of laparoscopy.
This method allowed more frequent observation of ovarian
condition. Harrison's experiments confirmed the previous
reports of an approximate 60% ovulation rate following
the use of a Progesterone-FSH-HCG regime for ovulation
induction in the squirrel monkey. Further, it was dis-
covered that an identical ratio was found when the
progesterone pretreatment was deleted. Ovulation occurred
in a range from 6 to 14 hours after the HCG.

In the absence of any primate gonadotropin source,
Gould, Cline and Williams (1973) reported induction of
ovulation in 12 of 16 S. sciureii approximately 45 hours
after the injection of 100 IU of PMSG. Recently laparo-
scopic techniques were used to define ovulatory morphology

of the Saimiri sciureus following the standard FSH—HCG

 

regime for that species (Harrison & Dukelow, 1974). These

14
authors defined the preovulatory morphological development

of the ovarian follicle, and reported that laparosc0py

made diagnosis of ovulation in this species a relatively
simple task. Spontaneous ovulation was also observed

during this study and there were no morphological differences

detected between natural and induced ovulations.

Physiology of the Mammalian Ovarian Follicle

 

The studies reviewed in the preceeding section have
dealt with the induction of ovulation in the nonhuman
primate through administration of gonadotropic hormones
from various sources. The consensus View favors the
necessity of gonadotropins having both follicle stimulating
and luteinizing activity, the latter of which should be
from a primate source. In order to understand the individual
roles of FSH and LH sources in the processes leading to
oocyte maturation and ovulation, specific histological and
physiological studies are required which have not pre-
viously been feasible with primates. The following
discussion will focus on the current knowledge of the
biochemical and physiological events occurring in the
mammalian ovarian follicle from the start of its pre-

ovulatory growth through actual ovulatory rupture.

Follicle Growth

 

Recently, Ryle (1972), using in vitro culture methods,

corroborated what had been an accepted principle of

15

gonadotropic action -- that FSH was necessary in culture
to maintain the growth rate of one and two cell layer
follicles. Goldenberg et al. (1973) found that in the
hypophysectomized female rat, PMSG or a combination of
FSH and HCG were necessary for the optimal uptake of 3H—HCG
by the ovarian tissue in yiyg. Although FSH or HCG alone
was able to increase the ovarian uptake of the labeled HCG,
they found that the maximum stimulation occurred only fol—
lowing treatment which resulted in ovulation and formation
of corpora lutea. They suggested that the effect of FSH
on 3H-HCG uptake was dependent on its role in stimulating
the maturation of the follicle as a prelude to ovulation.
This concept is supported by Channing and Kammerman (1974)
whose studies show a 10 to 1000 fold greater affinity of
large follicles to HCG than is exhibited by smaller follicles.
That endogenous FSH is not essential for the induction
of ovulation in the preovulatory follicle was shown by
Schwartz et al. (1973) when administration of an FSH
antiserum failed to inhibit ovulation or estrogen secretion
in the female rat. A necessity of FSH for follicular
growth, however, was supported by the observation that
treatment with this antiserum exerted a negative effect on
follicle growth and development in subsequent cycles.
Seleznik et a1. (1974) found that one of the roles of
FSH in the rat may be to stimulate the maturation of the

follicular granulosa cells thus inducing or activating

16

receptors for LH (HCG). This idea is based on their experi-
ments which illustrated an increased binding of HCG following
FSH administration.

Follicular stimulating activity has been shown to be
essential to ovulation induction in the hypophysectomized
immature female rat by Nuti et a1. (1974) who demonstrated
that while the administration of 2 ug FSH/day for 4 days
had no effect on serum progesterone levels, this priming
was essential for an ovulating hormone to be effective.

That FSH alone has little effect on the preovulatory
follicle was recently illustrated by Lipner et a1. (1974).
Their experiments used purified FSH as well as a highly
specific LH antiserum in hypophysectomized, immature female
rats to insure that the animals were totally devoid of LH
influence. Their results indicate that LH-free FSH has
minimal intrinsic ovulating ability.

Inferences that the actions of various gonadotrOpins
are similar in the primate species must presently be
considered speculative, due largely to the paucity of data
in these species. Reviewing the clinical literature relating
to human ovarian follicular morphology following spontaneous
or pharmacologically induced suppression of pituitary
function, Ross (1973) stated that the literature supports
the concept that pituitary gonadotropic stimulation is
required for advancement of follicular development beyond

the primary follicle stage.

17
Another recent review (Koering, 1974) showed that

follicular development appears to be similar in most primate
species. This author cites the continuous development and
atresia of ovarian follicles in the prepubertal monkey

(in the absence of ovulation) as evidence that the gonado—

tropic stimulus necessary for further maturation is lacking.

Theories of Ovulation

 

Espey in 1974 reviewed the modern theories of the
mechanism of ovulation. He points out that until the early
1960's there existed two general schools of thought, one
of which suggested an active rupturing process via an
increased intrafollicular pressure. The second theory
pr0posed that ovulation was a passive process brought about
by the enzymatic degradation of the follicular wall or
possibly by necrotic changes in the follicular tissue.

The presence of smooth muscle fibers in the ovary lead
to the hypothesis as early as 1858 (Rouget, 1858) that
follicular rupture was caused by vascular congestion due
to a restriction of venous return following constriction
of these elements. This theory was supported by the study
of Guttmacher and Guttmacher (1921) who demonstrated con—
tractions in the wall of sow tertiary follicles that
appeared similar to smooth muscle contractions. Aside from
direct evidence, one of the most frequent arguments in favor

of this concept prior to 1960, was based on the inferences

18

made following observation of the dynamic nature of the
rupture of the follicular wall (Kelly, 1931).

In 1947, Claesson published a study in which he was
unable to show the presence of smooth muscle cells in the
follicular wall of the cow, pig, rabbit or guinea pig.
Further ultrastructural studies have substantiated this
lack of smooth muscle in the rabbit ovarian follicle
(Espey, 1967). In addition, several of the classical
stimulants of smooth muscle activity have been found to have
no effect on the activity of sow follicles (Espey, 1964).

Despite evidence to the contrary, there has recently
been revival of interest in the role of smooth muscle cells
in ovulation. One recent report cites the demonstration
of an in_zitrg spontaneous contractile activity of the
ovary (Palti and Freund, 1973), while the electron micro-
graphs of another have been reported to show myofibrils in
the thecal layer of the ovarian follicles of several
mammalian species (O'Shea, 1970; Okamura et al., 1972).

In addition, Virutamasen et a1. (1971) were able to demon-
strate significant effects of several adrenergic drugs on
the occurrence of ovulation in the rabbit. These effects
were attributed to actions on ovarian smooth muscle con-
tractile activity. The same authors have subsequently

demonstrated effects of prostaglandins on both the in vivo

 

and the in vitro contractile activity of the rabbit ovary

(Virutamasen et al., 1972). Regardless, this question is

19
still being disputed and will be settled only when new

evidence is presented.

Increases in intrafollicular pressure have also been
postulated to arise from other sources. Zachariae in 1958
was able to show an increase in the permeability to Evans
Blue of the blood-follicle barrier in rabbits after an
ovulatory stimulus. This investigator concluded that ovu-
lation was due to a breakdown of intrafollicular
mucopolysaccharides, thus causing an increase in the intra-
follicular osmotic pressure and a resultant increase in
follicular pressure sufficient to reach the rupture point.

Regardless of source, increased intrafollicular
pressure was eliminated as a cause of ovulation by three
separate laboratories by the demonstration that intra-
follicular pressure did not increase prior to or during
ovulation in the rat (Blandau and Rumery, 1963) or rabbit
(Espey and Lipner, 1963; Rondell, 1964). These studies
demonstrated a slight fall in pressure as rupture approached.
The absence of a pressure increase prior to follicle rupture
is indirect evidence favoring some type of degradation of
the follicular wall, since it seems unlikely that the
contraction of myofibrils, the venous constriction or an
increase in osmotic pressure could occur without a cor—
responding increase in follicular pressure.

The theory relating ovulation to a sequential degrada—
tion of the layers of the follicle was postulated in 1916

by Schocket. Corroborating evidence was reported by Rugh

20
(1935) when, working with frogs, he found that a combination

of pepsin and hydrochloric acid would initiate follicle
rupture.

Working with rabbits, Espey and Lipner (1965) identi-
fied several enzymes which would cause morphological changes
similar to normal swelling, stigma formation, and ovulation
when injected into the mature follicle. The most effective
of these preparations were nagarse, pronase and a bac-
terial collagenase.

Rodbard (1968) warned against the assumption that
intrafollicular administration of enzymes was causing
ovulation through a degradation of the follicular wall and
suggested that this treatment would also raise the intra-
follicular osmotic pressure. Subsequently, Rondell (1970)
showed, using a mathematical evaluation of the relationships
between the wall tension and luminal pressure of thin-walled
spheres, that it is possible to increase follicular volume
(via osmotic or secretory phenomena) to the breaking point
without measurably increasing the intrafollicular pressure.

Another observation of importance was the histological
demonstration that the collagen network in the apex of the
rabbit follicle becomes dissociated as ovulation approaches
(Espey, 1967b). Espey (1974) has provided evidence that
this breakdown of the framework of the follicle is caused
by collagenolytic or proteolytic enzymes in response to
gonadotropic stimulation. Others believe that these

degradative changes in the follicular wall may be due to

21
necrotic changes, such as an interruption of the vascular

supply to this area (Blandau, 1966).

It is obvious that the exact nature of the stimulus
for follicular rupture remains to be elucidated. It is
conceivable that the ovulatory mechanism may vary among
species, and that this mechanism may actually reflect a

combination of physiological events.

Involvement of Prostaglandins in Ovulation

 

With the indication that the prostaglandins might be
important mediators of luteinizing hormone action on the
ovary (Kuehl, et al., 1970), an intense investigation into
the role of these potent physiological compounds in the
process of ovulation began. In 1971, Coutinho and Maia
reported a series of rapid changes in human intraovarian
pressure following the administration of prostaglandin an
(PGFZG). Such pressure fluctuations were attributed to
muscular contractions and were not observed following
administration of PGE2. Similar results in the rabbit were
noted the following year by Virutamasen, Wright and Wallach
(1972). The latter report detected an increased level of
ovarian contractility following PGFZa administration either

in vivo or in vitro. This effect was inhibited by PGEZ.

 

A direct role in follicle rupture was theorized by
Armstrong et al. (1972) following experiments in which
Indomethacin, a potent inhibitor of prostaglandin synthesis,

was shown to effectively block LH-induced ovulation in the

22
rabbit. These authors did not observe a prevention of

luteinization in those follicles where ovulation was
blocked.

Working with rhesus monkey granulosa cell cultures,
Channing (1972a) described a stimulation of progestin
secretion and morphological luteinization by several prosta-
glandins with the order of effectiveness being PGEZ or
PGEl much greater than PGAl, which was greater than PGFZd'
Channing (1972b) also reported that prostaglandin inhibitors
could block the stimulatory action of gonadotropins on
luteinization and progesterone secretion. From these obser-
vations, she concluded that prostaglandins may be an
intermediate in the action of LH or HCG upon granulosa cell
luteinization in the primate.

Similar studies with the rat caused Tsafriri et al.
(1972a) to agree with the concept of prostaglandins being
intermediates of LH action. They reported that PGE2 would
induce ovulation in adult rats whose endogenous ovulatory
surge of LH had been blocked with sodium pentabarbital.
Treatment of animals with indomethacin was observed to
inhibit follicular rupture but no ovum maturation following
an ovulatory dose of LH. Additionally PGE2 was capable of
inducing ovum maturation, although not essential for
this process. Another study reported by the same authors
(Tsafriri et al., 1972b) attempted to ascertain the effect
of LH on the maturational development of the follicle-

enclosed rat oocyte. Their results indicated that the

23
addition of either LH, HCG, FSH or PGE2 to an in vitro

culture system would induce completion of the first meiotic
division. A further observation was that cyanoketone, an
inhibitor of steroid synthesis, would not inhibit the
maturation-inducing action of LH. From such studies these
authors proposed that the action of LH on oocyte maturation
possibly involved the prostaglandins.

Further support for the concept that prostaglandins
play a role in the process of ovulation came in 1973 when
it was noted that ovulatory stimulation in the rabbit would
produce a marked increase in the follicular levels of
prostaglandins E and F (Yang et al., 1973). Also reported
was the ability of intravenous indomethacin to inhibit the
follicular prostaglandin increases. In a continuation of
this study, Yang et a1. (1974) demonstrated that these
increases were limited to the follicles that actually
ovulate.

Labhsetwar (1973) suggested that PGFZa treatment may
cause an estrogen positive feedback which stimulates the
release of the ovulating hormone through the hypothalamo-
pituitary system.

Richman et a1. (1974) used the procedure of infusing

PGE or PGFZa into the ovarian arteries of rabbits seven

2
hours following an intravenous injection of HCG. Animals
receiving PGFZa infusion responded with ovulation at the

same rate as the control animals who received HCG only.

Treatment with PGE2, on the other hand, decreased the

24
number of follicles rupturing, an effect attributed by

these authors to a direct effect of the prostaglandins
on the ovary.

In mice, the injection of an antiserum specific for
PGFZa caused an 80% decrease in the number of animals
exhibiting spontaneous ovulations (Lau, et al., 1974).

When PGFZa was administered along with the anti—PGFZa

the ovulation percentage was increased by 50%. In other
studies in the mouse, PGEZ was shown to reverse the inhi-
bition of ovulation seen following systemic indomethacin,
while PGFZG was only partially effective (Saksena et al.,
1974). These experiments indicated that both the E and

the F series of prostaglandins were probably involved in
ovulation. The blocking of ovulation by indomethacin was
attributed to an interference with LH action at the ovarian
level as well as to its inhibition of PG synthesis.

Utilizing the Ovarian Ascorbic Acid Depletion bioassay
for LH activity, Sato et a1. (1974) found a response di-
chotomy following prostaglandin treatment. On the one
hand, either PGEl, E2 or F2a in hypophysectomized immature
rats would produce the significant depletion of ovarian
ascorbic acid characteristic of compounds with LH-like
activity. However, when given to PMS pretreated animals,
these prostaglandins were not able to mimic the ovulation-
inducing capabilities of HCG.

Another possible site of the ovulation-inducing action

of systemic prostaglandins is the hypothalamo-hypophyseal

25
axis. A recent study of Harms et a1. (1974) suggests that,

at this level, PGE2 may have a specific effect in stimulating
the release of LH by the adenohypOphysis. Prostaglandins
E1, Fla and F2a were ineffective in causing LH release.
Similar effects of prostaglandins may be expected
in the nonhuman primate. Batta and Brackett (1974)
demonstrated that systemic administration of prostaglandins
of the E series to rhesus macaques could complement PMS
and HCG treatments and result in a more consistent induction
of ovulation than is possible through gonadotrOpin therapy
alone. The mode of action was not defined by these authors
nor by Shaikh and Klaiber (1974), who indicated that
sequential treatment with estradiol and PGFZd would cause

a shortened menstrual cycle length in the rhesus.

Involvement of Cyclic AMP in Ovulation

 

In 1960, Haynes et a1. implicated cyclic 3',5' adenosine
monophosphate (cAMP) as a mediator in the action of adreno-
corticotropic hormone on the adrenal cortex. Shortly
thereafter, Marsh and Savard (1966) found that the addition
of cAMP to slices of bovine corpora lutea in vitro would
significantly stimulate steroid synthesis. They also
observed that the effects of cAMP closely resembled the
ability of luteinizing hormone to stimulate the utilization
of acetate—l-Cl“ and cholesterol-7—H3 for progesterone

synthesis. They concluded that this study was evidence

26
that cAMP was a mediator of the steroidogenic action of

LH in the bovine corpus luteum.

Although at first appearing contradictory, the
reported inability of either cAMP or its dibutyryl deriva-
tive to initiate luteinization in cultured rabbit follicles
was attributed to inadequate penetration of the granulosa
cells (Keyes, et al., 1972). This observation was supported
by Tsafriri et a1. (1972 a, b), who reported that while the
addition of LB or HCG to a culture medium including ex-
planted rat follicles would stimulate meiotic division,
such stimulation did not occur with either cAMP or dibutyryl
cAMP. However, it was found that when dibutyryl cAMP was
administered directly into the follicle, meiotic activity
would resume. Thus, incorporating their earlier results,
these authors prOposed that the action of luteinizing hormone
on oocyte maturation involved the mediation of the adenyl
cyclase/CAMP system and possibly of the prostaglandins.

The relationship of cAMP to the process of ovulation
was suggested by Marsh et a1. (1972) following studies
indicating that there was a steady decline in the accumu-
lation of this nucleotide in the rabbit tertiary follicle
between the ovulatory stimulus and actual rupture.

The poor response of the follicle to cAMP demonstrated
by Keyes et al. in 1972 was improved in 1974 by Miller and
Keyes simply by eversion of the follicle to expose the
granulosa cells to the culture medium. Following incubation

with LH or dibutyryl cAMP and autotransplantation, corpora

27
lutea were seen to develop, which was a response not

observed in follicles incubated in a control media.
Extension of the hypothesized intermediate role of
cAMP in the action of LH to a primate species has been re—
ported by Channing (1974). Her studies indicate that a two
day in_zitrg culture of granulosa cells from the follicles
of monkeys in a preovulatory state, a midfollicular state
or a gonadotropin (HMG or PMSG) treated state, would cause
luteinization and progestin secretion if LH or dibutyryl

cAMP was present in the culture media.

Mediation of Gonadotropic Action by cAMP and/or
Prostaglandins

 

 

In 1965, Savard et al. reported that 0.02 M cAMP
would cause a stimulation of progesterone synthesis from
slices of bovine corpus luteum that was comparable to that
produced by saturating amounts of luteinizing hormone. The
following year, the same laboratory demonstrated the rapid
accumulation of cAMP in response to the addition of
luteinizing hormone to the incubated slices from bovine
corpora lutea (Marsh et al., 1966). This response was not
seen following the addition of substances shown to be
ineffective in stimulating progesterone synthesis (hydrogen
peroxide inactivated LH, prolactin, ACTH, epinepherine, or
glucagon). These authors also observed a marked accumulation
of this nucleotide in incubated slices of human corpora

lutea following the addition of HCG. The increased cAMP

28
brought about by LH precedes the increase in progesterone

synthesis which is additional evidence of a role of this
nucleotide in the steroidogenic action of LH.

Further complication of the issue was demonstrated in
1970 by Kuehl et al., from a study of the kinetics involved
in the stimulation of ovarian cAMP accumulation by LH,

PGEl and PGE2. Their evidence suggested that prostaglandins
stimulated adenyl cyclase as their role in hormone action,
and that the prostaglandin receptor was an essential com-
ponent of the LH effect.

In 1971 these observations were extended to the tertiary
follicle of the proestrous rat, which, in a culture system,
was also seen to respond to the addition of LH or PGEZ
with the production of cAMP (Lamprecht et al., 1971). A
study by Marsh et a1. (1971) showed that the follicle of the
rabbit could synthesize cAMP and that its accumulation is
greatly enhanced by luteinizing hormone. Employing a
prostaglandin antagonist, Ellsworth and Armstrong (1974)
stated that this inclusion in a culture system prevented the
luteinization of rat ovarian follicles in response to the
luteinizing agents LH, PGE2 or dibutyryl cAMP. They con-
cluded that LH and PGE2 have a common mode of action via
CAMP.

Similar conclusions were drawn from a study of LH and
FSH-induced luteinization of granulosa cells harvested from
rhesus monkey preovulatory follicles (Channing & Crisp,

1972). These workers stated that, as in other species,

29

gonadotropin-induced luteinization of primate granulosa
cells was mediated by cAMP with the possible involvement of
prostaglandins. Also working with a primate species, Wilkes
et a1. (1972) demonstrated the active synthesis of PGFZG by
ovarian tissue in 31359. This response increased following
the addition of LH to the culture system.

In 1973, Yang et a1. indicated that systemic adminis-
tration of HCG or LH would stimulate follicular levels of
both PCP and PGE. These increases were due to increased
synthesis of prostaglandins since the increases were
abolished following pretreatment with indomethacin. They
concluded that increasing levels of prostaglandins within
the Graafian follicle are associated with the normal
physiological process of ovulation. This conclusion is
substantiated by a report indicating that various prosta-
glandins can stimulate ovarian cAMP accumulation in a manner
similar to LH (Mason et al., 1973).

It has been suggested, however, that prostaglandins
may not be mediators of gonadotropic action upon cAMP
formation in some systems (Kolena & Channing, 1972).
Evidence against the theory that PGEZ is an obligatory
intermediate of LH action on the ovary was presented by
Lamprecht et a1. (1973) who found an additive effect of
maximally stimulatory levels of LH and PGE2 on cAMP
formation in whole ovary cultures. These authors observed
a refractory state following LH incubation during which

the ovaries could no longer be stimulated by LH yet

30

remained fully responsive to PGEZ. In another study
reported the same year, incubation of juvenile rat ovaries
with LH caused an increased cAMP accumulation without
increasing the activity of prostaglandin synthetase. In-
hibitors of this synthetase activity were also found to
abolish the prostaglandin-induced increases in cAMP for-
mation in vitro, without preventing the stimulatory effect
of LH on formation of this nucleotide (Zor et al., 1973).
These authors argue against the hypothesis that prosta-
glandins of the E series are involved as obligatory
intermediates of the cAMP dependent actions of LH on the
ovary.

Recently, LeMaire and Marsh (1975) have attempted to
reconcile the contradictory proposals regarding the inter-
relationship of these substances with ovulation. They find
progressively increasing levels of both PGE and PGF as
ovulation progresses in the face of a declining cAMP
synthesis. Current speculation involves the relationship
of prostaglandins to follicle rupture rather than to ovum

maturation or to luteinization.

Intrafollicular Injection Techniques

 

The first procedure reported for the in vivo monitoring

 

of intrafollicular phenomena was the microcannulation
technique used by Espey and Lipner (1963) to record pressure
changes inside the rabbit follicle. Their procedure

employed capillary glass tubing with a small diameter tip.

31
These authors modified this technique in 1965 for the

intrafollicular injection of various enzymatic solutions.
Local administration of collagenase, pronase or nagarse in
l microliter volumes induced follicle rupture in a manner
similar to that produced by mating. There was a total
absence of effect with injections of up to five microliters
of saline. These relatively large volumes of saline would
neither induce nor inhibit ovulation in coitally stimulated
females and was good evidence that the observed effects
were not artifacts of the technique.

Blanchette (1966) reported the use of a similar
technique for the study of the morphological differentiation
of follicular granulosa cells into luteal cells. In this
study, injections were made through either drawn capillary
pipettes or 30 gauge needles. Leakage of the injected
substance was prevented by withdrawing the needles slowly,
approximately a minute following the completion of the
injection. Injection of volumes of one to five ul were
studied with the adoption of one ul as the standard. The
results of this study demonstrated that the intrafollicular
administration of 8 ug of purified ovine LH (NIH-LH-S7) or
1 IU of HCG (APL-Ayerst) would cause the cytological events
normally occurring in follicular granulosa cells undergoing
differentiation into lutein cells.

Ferrando and Nalbandov (1969) reported the successful
inhibition of ovulation from alternate follicles on one

ovary in the rabbit following the injection of an adrenergic

32
blocking drug, dibenzyline. They used 27 gauge needles

and confirmed the absence of effect of saline intra-
follicular injections.

The presence of corpora lutea five days following
intrafollicular administration of as little as 0.1 ng of LH
was reported by LeMaire et a1. (1972). When 0.4 u mole of
cAMP or dibutyryl cAMP was administered concurrently with
the LH a marked decline in corpora lutea production was
observed. Furthermore, intrafollicular administration of
25 ug of phosphodiesterase, which was thought to decrease
the endogenous levels of cAMP, would also produce luteini-
zation. These authors concluded that the normal function
of cAMP might be to inhibit premature luteinization. Their
technique had utilized a "very fine" needle to deliver one
pl volumes from a ten pl Hamilton syringe. Methylene blue
was added to the solutions to monitor follicular leakage
which was rarely observed.

Although the preceeding report made no mention of
ovulation, the production of corpora lutea can be considered
as presumptive evidence in its favor. Follicle rupture
in the rabbit was demonstrated in response to intrafollicular
administration of either 10 ng LH or 100 ng FSH by Jones a
and Nalbandov in 1972. Their technique involved entry into
the follicle through the ovarian stroma to facilitate tissue
sealing. Follicle locations were sketched for subsequent
identification. The ovaries were re-exposed following 18

to 24 hours, and the number of follicle rupture points were

33

observed and counted. Luteinization was invariably seen
following the injection of subovulatory doses of LH or FSH,
and these luteal structures were competent in progesterone
production. This technique produced additional evidence
implicating the sympathetic nervous system in the process
of ovulation, following the demonstration that the alpha
adrenergic blocking drug, phentolamine, would inhibit ovu-
lation. It was further shown that this blockade could be
overcome by catecholamines, cAMP, or a mixture of FSH and
LH. The theory was proposed that the effect of LH was to
stimulate the cAMP system which then may activate synthesis
of the catecholamines and, in turn, activate an ovarian
collagenase resulting in ovulation (Nalbandov et al., 1973).

The ability of cAMP to induce ovulation when adminis-
tered locally was disputed by Das and Talwar (1974). No
ovulations were observed following injection of from 5 to
500 ug of cAMP even though LH would induce ovulation when
given in this manner. Their results indicated an inability
of cAMP to mediate the ovulation—inducing activity of LH.
This conclusion may not be comparable to the other studies
involving intrafollicular injections, however, as they made
no mention of administering their compounds into the ovarian
follicle. Rather their substances were injected directly
"into one of the ovaries."

Prostaglandins E2 and an were administered via intra-
follicular injection by Moon and Armstrong (1974), resulting

in their implication of the latter in the process of

34

ovulation, possibly by its action on the follicular contrac-
tile process. Further evidence of the involvement of
prostaglandins with follicular rupture was the report that
antisera to PGFZa would consistently inhibit ovulation

(Moon and Armstrong, 1974). PGEZ antisera was also inhibi-
tory, but to a lesser extent, while intrafollicular
injections of indomethacin, an inhibitor of prostaglandin
synthesis, proved 100% effective in blocking LH induced

ovulation in the rabbit.

MATERIALS AND METHODS

Experimental Units

 

The nonhuman primates used in this study were from the
permanent colony maintained at the Endocrine Research Unit
on the campus of Michigan State University. This colony is

made up of two species, Macaca fascicularis and Saimiri

 

sciureus, which are used exclusively in non—terminal studies

 

of various physiological aspects of primate reproduction.
Their environmental conditions include a 12 hour light-dark
cycle, a temperature maintained between 21° and 26°C, and
fluctuations in relative humidity from approximately 40 to
60%. All animals received water ad libitum and a com-
mercially prepared monkey diet (Wayne Co.).

The macaques were housed individually in 48-inch
double unit stainless steel cages. Cages were equipped
with slotted floors, a back mounted perch, and a squeeze-
back apparatus to facilitate animal handling. The squirrel
monkeys were kept either in a 280 cubic foot community type
cage or were kept as small groups in the same type of cage

as that used for the macaques. The cages were washed

35

36

daily, at which time a visual inspection of the macaque

cages was made for the observation of menses.

Induction of Follicle Growth

 

Since ovulation served as the endpoint for the majority
of these studies, it was necessary to have a procedure for
the reliable production of mature follicles. Such a tech—
nique was first described in the squirrel monkey by Dukelow
in 1970. It involves four daily injections of 1 mg follicle
stimulating hormone (FSH-P), Armour Baldwin Laboratories,
Omaha, Nebraska), followed by an ovulation-inducing injec-
tion of human Chorionic gonadotrOpin (HCG, 500 IU, A.P.L.,
Ayerst Co.). This regime will yield ovulation approxi-
mately 6 to 14 hours after the HCG injection in 60% of
the animals (Harrison, 1973).

Although the production of ovulation in the macaques
has been less reliable than in the saimiri, extrapolation
of the effective dose level suggested that 5 mg/day would
be sufficient for the macaque. For an ovulation induction
system in the rabbit, this dosage has been shown to be
equipotent with 400 IU PMSG (Kennelly and Foote, 1965).

This level is higher than that necessary for the stimulation
of mature follicular growth in the cynomolgus macaque
(Jainudeen and Hafez, 1973). Assessment of follicular
growth and quantification of ovulation sites was made
possible through visual observation of the ovaries with

the laparosc0pe.

37

Cyclicity in Saimiri sciureus

 

To detect the presence of a cyclical pattern of ovarian
response to exogenous gonadotropins, a total of 70 female
squirrel monkeys were treated with the regime consisting of
4 daily FSH administrations followed by an ovulation inducing
treatment on day 5. Without further FSH treatments, the
animals received intramuscular injections of 500 IU HCG at
two day intervals beginning 5 to 17 days following the
synchronized ovulation. Subsequent laparoscopies were
performed to determine the number of animals ovulating at

each interval.

Laparoscopic Techniques

 

Surgical anesthesia was produced in the squirrel monkey
by intraperitoneal injection of 16.2 mg sodium pentobarbital
(Halatal, Jensen-Salsberry Laboratories, Richardson-Merrill
Inc., Kansas City, Mo.). In the macaque, intramuscular
administration of 15 mg of phencyclidine-HCl (Sernylan,
Bio-Ceutic Laboratories) produced adequate anesthesia for
up to 45 minutes. The animals were placed on a tilted
surgical table with their feet elevated and secured with
adjustable leg straps. The abdominal region was shaved
and prepared with benzalkonium chloride (Zephiran, Winthrop,
New York). Insertion of the trocar-cannula was made through
a 5 mm periumbilical incision following which the trocar
was removed and replaced by the laparosc0pe. The laparo-

scope used in these studies was a 135 degree pediatric

38
model (Richard Wolf Co., Knittlingen, W. Germany) in

conjunction with a Wolf model 4000 light source and flexible
fiber optic cable. Transillumination of the abdominal wall
insured proper placement of a Verres-cannula which has been
found to serve adequately as a probe for intra-abdominal
manipulations. A COz-produced pneumoperitoneum facilitated
observation. When experiments were expected to last longer
than 30 minutes, uretheral catheterization was performed to
prevent obstruction of the procedure by the urinary bladder.

For laparoscopic photography a Canon TL 35 mm single
lens reflex camera was utilized since this allowed simul-
taneous observation and photography. Shutter speeds ranging
from 1/2 to 1/8 of a second were found to produce the most
desirable exposures with Ektachrome DHB film (ASA 120).

When the procedure was completed, the laparoscope was
withdrawn, leaving the cannula in place to allow ready

escape of the CO Following removal of the cannula and

2.
probe, nitrofurazone powder (Furacin, Eaton Labs) was
applied as a hemostatic and the incision was closed with
subcutaneous suturing. Topical Furacin ointment and systemic
penicillin administrations were used to combat infection.
Studies involving local ovarian application of com-
pounds required the laparosc0pic placement of disks of 2 mm
in diameter on the surface of the ovary. For these studies,
a 3 mm laparoscopic forceps with a fiberglass cannula was

substituted for the Verres cannula. This forceps allowed

placement and recovery of the disks without requiring major

39

abdominal surgery. Disks were left in place for one hour
except in cases where anesthetic limitations required early
removal. Such cases were not considered to be completed
experiments and were not included in the results.

The ovarian disks were made from either (a) #42 filter
paper (Whatman Co.) or (b) Gelfoam (Upjohn Co., Kalamazoo).
Preliminary comparisons revealed that, although the Gelfoam
disks would hold more liquid than would the filter paper,
they were less stationary when on the ovarian surface, and
therefore all subsequent studies were carried out using
the filter paper disks. Identical disks were used in the
in yitrg experiment to be described later (Appendix I).

A technique for injecting substances directly into the
ovarian follicle of the squirrel monkey was developed.
Initially, compounds were injected with a 10 microliter
Hamilton syringe equipped with a Chaney adaptor and custom
made 37 gauge needles (Hamilton Co., Whittier, Cal.).
Follicles were randomly chosen and entry was made at the
base of the transilluminated follicular dome. Control and
experimental injections were made in 0.2 ul volumes into
contralateral ovaries. The needle was allowed to remain in
the follicle for approximately 30 seconds and was then
slowly withdrawn. This procedure prevented any obvious
flow of fluid from the follicle. Follicular location was
carefully mapped with notations referring to the normal

orientation of the ovary in the animal. Subsequent

40

laparosc0pic examinations were performed using observer-
blind analysis of the ovarian surface.

Adaptation of the intrafollicular injection technique
to laparosc0py required a syringe that could be manipulated
with one hand and, therefore, the Gilmont Micrometer Syringe
was used. This syringe could more accurately deliver the
small volumes necessary for these studies. At this time
the injection volume was increased to 1 ul to further increase
delivery precision. As the 37 gauge needles bent frequently,
32 gauge needles were substituted with no detrimental effects
and with the added benefit of less frequent clogging. Twenty-
four gauge needles (% inch long) were inserted with laparo-
sc0pic guidance, to serve as cannulae for the fine injection
needles. As needle clogging occasionally occurred, patency
was tested prior to entry into the abdomen and again
immediately before follicular penetration. Experimental
injections were accompanied by vehicle injections into
contralateral ovaries using separate syringes and needles.
Some follicles were marked with either India ink or Trypan
Blue (0.05%) to assess the reliability of the delivery
technique, as well as to check for follicular leakage.

Laparoscopic examinations were carried out at intervals
sufficient to detect any post-ovulatory changes (12 to 36
hours). Ovulation was diagnosed using the criteria pre-
viously described for the squirrel monkey (Harrison &
Dukelow, 1974) and for the cynomolgus monkey (Jewett &

Dukelow, 1972; Rawson & Dukelow, 1973; Dukelow, 1975).

41

Experimental Compounds

 

All substances for the ovarian disk or intrafollicular
injection studies were kept frozen and thawed shortly prior
to use. The gonadotrOpins used were luteinizing hormone
(NIH-ovine-LH-SlB) and human Chorionic gonadotrOpin (A.P.L.,
Ayerst Co.). The LH was dissolved with sterile saline
while the HCG was prepared using the commercially supplied
diluent. Prostaglandins E2 and an (The Upjohn Company)
were prepared by initial dissolution in 95% ethanol with
subsequent dilution by sterile saline containing sodium
carbonate resulting in a final PG concentration of 10 mg/ml.
Cyclic 3',5' AMP and its dibutyryl derivative (Cal Biochem,
Los Angeles) were dissolved in sterile saline. Dilution
was such that 0.6 uM of the nucleotide could be delivered
in 1 ul volume.

The pH of all solutions was between 6.8 and 7.1 and

the described vehicles were used as controls in all cases.

Progesterone Assay

 

The assay of peripheral plasma progesterone has been
described previously (Louis et al., 1973). Basically, the
procedure involved the extraction of 200 pl of duplicate
samples of plasma with 2 ml benzene-hexane (1:2). Following
removal of organic solvent, 200 pl of rabbit antiprogesterone
(prepared against 6 B-succinylprogesterone conjugated to
bovine serum albumin and supplied by Dr. G.D. Niswender,

Colorado State Univ.) was added to the aqueous phase and

42

samples were incubated at room temperature. Next, 3H-
progesterone (approximately 24,000 CPM) was added to each
sample, and the tube contents were mixed, and incubated at
4 for 4 to 20 hours. Dextran-coated charcoal was then
added to each sample, while maintaining them in an ice bath.
The samples were then mixed and centrifuged (2500 g for
10 min.). This step effectively separates the bound
progesterone from the free. A liquid scintillation spec—
trometer was then used to measure the amount of 3H-
progesterone bound to the antibody in 0.5 m1 of the super-
natant.

Previous studies (Louis et al., 1973) have demonstrated
that the efficiency of the extraction procedure is 80 i 1%
while the sensitivity of the assay is less than 25 pg of

progesterone.

Statistical Analysis and Resource Allocation

 

The studies contained in this thesis have dealt with
two types of data; quantitative data and binomial data.
In experiments where the data generated fit a continuous,
numerical scale (quantitative data), statistical analysis
was by analysis of variance. Such data are exemplified
by the analysis of the cycle characteristics of the Macaca

fascicularis. In studies where the data involved the

 

presence or absence of an animal’s response (binomial data),
Chi-Square, 2 x 2 contingency tables were used for analysis

(Pearson and Hartley, 1958). Examples of these data include

43
the experiments designed to assess the ovulation inducing

ability of locally administered compounds. Other experi-
ments generating binomial data involved the need to compare
treatment effects to a hypothetical probability of event
occurrence, as well as the evaluation of the difference
between sample percentages. These experiments were analyzed
using the procedures for handling such data which were
described by Goldstein (Chapter 3, 1964). For all statis-
tical considerations in this thesis, an alpha of 0.05 was
presumed to reflect significant differences.

Recommended animal numbers for binomial experiments
were based on the probability of the occurrence of the
event being observed. In this case, that event referred
to the presence of ovulation, which occurs in approximately

60% of the Saimiri sciureus submitted to an ovulation

 

induction regime (Dukelow, 1970; Harrison, 1973). The
formula for determining recommended numbers is given by
Goldstein (p. 94, 1964) to be np = 5, where p represents
the probability of the occurrence of an event, and n refers
to the animal numbers. If the value of 50% is assigned to
p (making the estimate more conservative), the resultant n
equals 10 animals. Accordingly, the majority of these
experiments were carried out using 10 animals in each

group.

RESULTS

In order to define the reproductive cycle length of
the squirrel monkey, an experiment was undertaken to test
the ovarian responsiveness of animals synchronized by an
ovulation inducing treatment, to a subsequent dose of HCG
(500 IU). The results are displayed in Table l. The

greatest ovulatory response (70%) was in the group of

TABLE 1

Ovulatory Response of Saimiri sciureus to 500 IU HCG
at Varying Intervals Following Synchronization

 

 

 

 

 

Daya
6 8 10 12 14 16 18
Animals ovulating/
total 3/10 7/10 2/10 0/10 4/10 1/10 4/10

 

aInterval between bynchronization and subsequent ovulation
induction (in days)

animals induced to ovulate eight days following their pre-

viously synchronized ovulation. When these responses are

compared to the expected level (60%) for this species

44

45
(Dukelow, 1970; Harrison, 1973), the responses observed on

days 10, 12 and 16 are significantly lower (p < 0.05) while
the remaining responses are not different. It is interesting
to note that eight days following a synchronized ovulation,
there appears to be no requirement for exogenous FSH in

order to stimulate levels of ovulation similar to those

seen following the standard regime which includes 4 daily

FSH treatments.

Macaca fascicularis

 

When studying Old World primate species such as the

Macaca fascicularis it is much easier to discern reproductive

 

cycle parameters as these animals exhibit regular menstrual
cycles. During the course of a four year study (January
1970 to December 1973), several such parameters were

recorded for a colony of 28 female M. fascicularis. Table 2

 

presents the mean duration of menstrual flow and the cycle
lengths for each individual female. As illustrated by these
data the mean cycle length for this colony was 30.9 i 0.21
days, while the mean duration of menstrual flow was 2.6 i 0.1
days. The median cycle length for this period was 30 days,
while the modal interval was 29 days. It should be noted
that the low numbers of cycles from several of the females
can be attributed to pregnancies, lactation, experimental
treatments or amenorrheic conditions.

Although there was apparently no effect of season on

the duration of menstrual flow (Figure 1) there was a

46

TABLE 2

Individual Mean Cycle Lengths and Menstrual Flow Duration
for Macaca fascicularis Used in This Study

 

 

 

 

No. of Mean Cycle Mean Flow

Animal Cycles Length (S.E.) Duration (S.E.)

7 4 28.8 (1.5) 1.2 (0.1)

8 3 27.7 (0.3) 1.4 (0.3)
9 32 34.4 (1.0) 3.2 (0.2)
10 1 27.0 2.0

11 20 30.6 (0.9) 4.1 (0.4)
12 29 32.5 (0.6) 4.2 (0.5)
l4 19 30.4 (0.6) 2.0 (0.2)
15 7 33.7 (0.6) 1.5 (0.2)
16 1 29.0 3.0

17 12 32.3 (2.9) 2.1 (0.2)
18 9 31.4 (1.7) 2.0 (0.3)
25 14 29.1 (1.5) 1.5 (0.2)
26 1 33.0 1.0

27 7 35.1 (1.7) 2.4 (0.3)
31 26 29.1 (0.6) 3.2 (0.4)
39 19 36.2 (0.8) 2.8 (0.2)
40 24 30.3 (0.8) 3.0 (0.2)
41 27 32.6 (0.9) 2.0 (0.2)
42 29 29.3 (0.8) 2.9 (0.2)
43 35 28.5 (0.4) 2.8 (0.2)
44 36 29.4 (0.5) 3.7 (0.2)
45 1 29.0 1.0

46 2 30.5 (2.5) 1.5 (0.5)
51 21 29.4 (0.3) 3.0 (0.2)
52 11 31.7 (1.2) 2.0 (0.3)
53 27 30.3 (0.8) 2.0 (0.1)
54 22 31.4 (1.2) 2.1 (0.2)
55 19 29.1 (0.9) 1.8 (0.2)

 

 

28 458 30.9 (0.21) 2.6 (0.1)

47

FIGURE 1

Variation of Menstrual Flow and Cycle Lengths of
Macaca fascicularis During the Year

 

 

Cycle Length (Days)

33

32

31

30

29

 

48

FIGURE 1

 

 

 

 

III!
1 3

Illll
57 9

Lunar Months

I I I I
11 13

Menstrual Flow (Days)

49

significant variation in cycle length during the year, the
longest cycles occurring during the fifth and sixth lunar
months while the shortest were seen during the third and
eleventh months.

During 104 menstrual cycles in this species, laparo-
scopic examinations allowed the definition of ovulatory
status. In 93 of these cycles (89.4%), the ovarian mor-
phology indicated that ovulation had taken place. The
other 11 cycles (10.6%) were anovulatory. Ovulation was
observed on the left ovary in 62.3% of the cases and in
the right ovary in 37.7% of the cases. During 27 pairs
of consecutive cycles, the second ovulation occurred on
the contralateral ovary 70.4% of the time, and on the
ipsilateral ovary 29.6% of the time. In 51 cycles
laparoscopy was utilized to define the precise time of
ovulation. Figure 2 reveals the relationship between the
day of ovulation and the length of the menstrual cycle.

If we disregard the top three points, which represent only
one cycle each, we can begin to see a pattern with the cycles
ranging from 28 to 34 days in length. There is, however,

no significant relationship between these two variables.

Twin ovulations were infrequent with contralateral
twin ovulations noted only three times while twin ovula-
tions on the same ovary were seen only once. Actual
follicular rupture was witnessed on four separate occasions.
In three of these ovulations, the site of follicular

rupture was located near the apex of the ovarian stigma.

50

FIGURE 2

Relationship Between Follicular Phase Length and
Menstrual Cycle Length of Macaca fascicularis

 

 

Cycle Length

40

30

24

<24

 

51

FIGURE 2

9 (1)

0(1)

0 (1)

0(2)
0 (l)
e (4)
o (8)
g (5)
.(5)
o (4)
0(6)

0(4)

. <2)

. (l)

g (6)

 

I I I I I I I I I
12 l4 16 18 20

Day of Ovulation

52
The fourth rupture occurred from the side of the stigma,

near the base of the follicle. Considerable bleeding was
noted from the ovulation site at the time of rupture in
three of these cases, making the cumulus mass take on a

hemorrhagic appearance.

Induction of Follicular Growth

 

Preliminary trials of local gonadotropin application
in squirrel monkeys utilized animals with no pretreatment,
2 days of FSH pretreatment (1 mg/day), or 3 days of FSH
pretreatment. In five animals receiving no pretreatment
there was only one follicle observed which had the appearance
of a preovulatory follicle. Following two days of FSH,
four of seven animals (57%) were observed to have normal
preovulatory follicular development on the fifth day after
start of the treatments. Addition of a third FSH adminis-
tration did not increase the number of animals exhibiting
normal follicular development (six out of twelve). Four
days of FSH pretreatment, however, significantly (p < 0.05)
increased the number of animals responding with normal pre-
ovulatory development (50 out of 64, or 78%). Thus, the
four day FSH regime was adOpted for all studies.

In the macaques a regime consisting of 5 mg FSH/day
was administered for each of the first five days of the
animals menstrual cycle. Of twenty animals receiving this
treatment, only one exhibited the normal, preovulatory

follicular development characteristic of this species

53
(Table 3). The most common response included the develop—

ment of multiple follicles in 15 (75%) of these animals.
Other abnormal occurrences included the presence of large
follicles appearing to be filled with fluid, therefore
referred to as cystic, in three of these animals and the
development in two animals of follicles having the appear-
ance of containing coagulated blood. The latter type
follicles are frequently observed in the rabbit following
FSH administration and have been referred to as "blood"
follicles. This treatment had no significant effect on
the menstrual cycle length of either the treated cycle or

the post-treatment cycle in these animals.

Local Administration Experiments

Gonadotropin Studies

 

At the inception of this project it was decided that
the technique of local administration of substances to the
ovarian follicle which involved the least amount of experi-
mental manipulation would be advantageous. We, therefore,
elected to administer purified gonadotropins to the ovarian
surface with the laparoscopic procedures previously described.
Table 4 illustrates the results of experiments in which
2 mm hormonal saturated disks were placed on the ovarian
surface in the macaque. These data reveal no apparent
effects of either FSH or LH on the occurrence of ovulation.
The two ovulations in the FSH treated group were thought

to represent chance observations since they were not

54

 

 

sm\sm sm\sm 0 ++ wmamnuase = mmm mm
mm\mm Nm\om ++ maanuflsa = mmm an
wamfluase = came as
mm\am mm\mm + ++ noon = mmm as
mmxmm mm\mm 0 ++ wamnunss = mmm ma
mmxvs mm\om o o msaflunss = mmm Na
mmxmm am\ss + 0 season = mmm mm
~m\mm mm\om o + sum>o emmnmacm = mmm an
smxmm am\am o o mmsanunse owom EH +
mmm a
Nm\vm mm\mm Hood emu mm
mm\mm m~\m~ noon mmm Hm
mm\om mmxmm noon mmm Ha
om\om omxnm mmnanuase mmm mm
~m\am mm\~s maafluase mmm mm
m~\m~ am\nm mmaanuass mmm as
om\mm om\mm mmamwuans mmm ow
mmaasuase name NH
OHONU mmHoNU coHumNH mGOHuwufl cowumHs>O
Dxmz cannons lumasomm> Icflmusq
mmwao>o co uommmm 00m EH mo muommmm Damemoam>mo Dcmfiummna HmEflcd
umssoflaaom

 

 

mHHMHDOHUmMM MUMOMZ CH 003 HMHSOHHHOMMHfiQH UQM mmh MO mHUTMMW

m mqmfiB

55

numcma oaomo monum>m msofl>mum\m>mp as mnpmcma maomum

DHQDMH cH :mmaoflaaom pooHn= mHQEommu .UOOHD Umumadmmoo :«mucoo o» “momma mmHoHHHomm
.mmaoflaaom oaumwo mcHHnEmmmH .pmaaflm madam can .mmHMH Hmmmmm mmaoflaaomm
mum>o some so maoflaaom mco ODGH pmuomflcw comp

maco mum>o mco co maoflaaow wco oucfl pmuomwcfl wumo

mmp\mE m .mwmp ma

Amaowaaom Umummnu mo coflumnoHoo HampsH ..m.flv =coflumNficflmusa Hmowmoaonmnoez ucmummmdm

 

 

 

 

om\om om\ma ++ maanuase . mmm mm
mom nmumm

mm\mm mmxmm o + msme a «Hanuasa = mmm Hm
mmxmm mm\mm + + mmaafluass mom EH +

mmn as
wHONU meONU cofiumuw mcoflumufl coaumas>o
uxmz pmpmmue lunasomm> Icflmusq
mmaomu co pommwm 00m mH mo muomwmm DcwEmon>mo ucmfiummue HmEHC¢

smasoflaaom

 

A.o.ucoov m mamas

56

coaumnoaoo ammusa CH mmmmmnocw mo mcowumoam>m

m>nuommnsmn

 

 

 

 

mmaomo HmEhoc map mcflnsp
©m>nmmno mam>ma o>onm coflpmNflumHsomm> ca mommmuocfl mo coflumsam>o o>Huommndmm
Awmmv m\m Awmmv m\m “wave m\s mxo ma
Awomv oaxm oa\o Awomv oaxm lwomc oa\m mmm
acoflumuflcwwusq mcoflumNHHmasomm>
muomwwm oz coHumnmuad HMOHmoHosmnoz cofipmas>o ucmfiummne

 

 

magnum: mnu Ga mxmflo CMHHM>O mo coaumuumwcflapfi Hmooq mo muommmm

v mqmde

57
repeatable. Subjective evaluations of the degree of

vascularity and luteal coloration were also recorded. These
observations suggest that LH administered by the topical
ovarian disk may have had an effect to increase vasculari-
zation over that seen in a normal cycle.

Due to the lack of effects of these treatments, it was
hypothesized that either the ovarian membrane was imper-
meable to the gonadotropin or that the one hour exposure
was not sufficient to allow an effect. In vitro porcine
follicle culture experiments suggested the latter to be
the case (Appendix I) and, therefore, it was decided that
another technique for local administration of these com-
pounds was needed. It was on this basis that the technique
of intrafollicular injection was developed.

Initial experiments using the 10 ul Hamilton syringe
were done at laparotomy in the unprimed squirrel monkey.
Ten ng of purified LH were injected into all externally
observable antral follicles (2 to 4 per animal) with the
procedure previously described. Control animals received
saline injections. The results indicate that while this
treatment appears to cause a luteal-like coloration of the
injected follicle, in only one case did this cause ovulation
(Table 5). The follicle that ovulated in response to this
treatment was the largest follicle observed in any of the
animals and closely approximated in size the follicles seen
in FSH primed females. It was assumed to be a natural

preovulatory follicle. When given the FSH pretreatment,

58

coflumas>o mo mocmmnm may CH mwaoflaaom
powwow» on» NO coflumNflchusa no coHumeHmHsomm> ca mmmcmno pcmummmmm

 

 

 

 

 

 

Ha\m Awomc qu HH\4 Awomv m\m mg EH +
unmebmmupmnm mmm
ma\va Awooav m\m mH\H Awomv m\H mg HMHSUMHHOMMHucH
m\o m\o m\o m\o mcHHmm
ansoHHHOMmHucH
mmcommmm va mmcommom oncommmm va mmcommmm
maofiaaom Hmeflca mHoHHHom HmEHcd
mmcowpmuouad Hmoflmoaonmnoz coflumas>o ucwEummHB

 

 

 

>EODOHMQMA um mcofluommcH HmasoflaaommnucH on msmhsflom HHHEHmm mo mmcommmm

m mqmdh.

59
these animals exhibited a marked but non—significant

increase in ovulation rate.

Adaptation of this procedure to be used with laparo—
scopy required only a few minor technical adjustments as
described earlier. In order to insure the accuracy of
delivery of this procedure, five squirrel monkeys received
intrafollicllar injections of India ink. This substance
was seen to rapidly disperse following injection (Figure 3a,b)
and would clearly define the follicular boundaries. Blood
loss can be seen from the site of follicular puncture,
probably from the extrafollicular capillary network as no
ink leakage was observable. Injection of LH dissolved into
0.05% Trypan Blue in saline resulted in ovulation from two
of five animals treated. Similar injections of the vehicle
alone failed to cause follicular rupture. These injections
of dye markers demonstrated that the ovarian mapping would
allow accurate identification of treated follicles since in
the laparoscopic position the ovaries had a characteristic
orientation in the abdominal cavity.

Table 6 shows the results obtained when 10 ng of
purified LH were injected into the ovarian follicle using
laparoscopy. These results are not different from those
seen when the injections were made into exteriorized ovaries
under a dissecting microscope.

In order to test the ability of an intrafollicular
injection of a primate LH source to induce ovulation in the

squirrel monkey, this procedure was repeated with the

60

 

 

Figure 3a. Mature follicle on ovary of Saimiri
sciureus following 4 days of FSH

61

 

 

Figure 3b. Mature follicle on ovary of Saimiri
sciureus following intrafollicuIar
1nject10n of India ink. Bleeding
can be observed from the site of
injection without loss of the dye.

 

62

cowumad>o mo mocmmnm on» CH mmHOAHHom

pmummnu on» no coflpmnwcfimusa no coflumNHHMHsomm> EH mmwnmno pcmummmdm

 

 

 

 

 

 

HH\m Awomc m\s Haxq Amomc m\m Assououmamnv
ma mH +
unwaummuumum mmm
Ha\m Amomc sxm Ha\m “some m\m AEEoomoumquv
ma EH +
ucwspmmuumnm mmm
wmcomwmm va mmcomwwm mmcomnmm Amy mmcommmm
maoflaaom Hmeficd maoflaaom Hmeflcd
mmGOEDmHmuad HMUHmoaonmnoz noflumas>o ucmEummna

 

 

Emoomoummmq um can >EODOHMQMA um
msqflcsome cofluommcH amaonHH0mmnucH com3umm confinmmEou

m mqmdfi

63
injection of 0.5 IU of HCG. It can be seen from Table 7

that this procedure resulted in the same ovulation rate
observed previously with the ovine LH. Further, both the
ovulation rates and the levels of morphological alterations
following the intrafollicular injection of either 10 ng

of ovine LH or 0.5 IU of HCG were significantly greater than
the respective levels seen following intrafollicular saline
injections. Support of the concept of a species-specific
requirement of a gonadotropin source to induce ovulation
was demonstrated by the inability of systemic ovine LH to
stimulate ovulation in the FSH pretreated squirrel monkey,
since systemic LH from a primate source, HCG, was effective
in stimulating ovulation in these animals.

As this procedure had proven to be satisfactory for the
local administration of compounds to the ovary in the
squirrel monkey, and as a frequent effect of the injection
of an LH source was the appearance of a luteal coloration
of the follicle, it was decided to use this technique in
the macaques where menstrual cyclicity and circulating
progesterone levels could be monitored.

The results of either FSH administration alone or FSH
administration followed by intrafollicular injection of 2
IU of HCG are presented in Table 3. Analysis of Variance
revealed no significant differences between the lengths of
treated cycles and the previous mean cycle lengths of 8
animals receiving 5 daily, intramuscular injections of 5 mg

FSH. Neither did FSH alone have any effect on the length

64

U pomeE msoum EOHM Amo.o v my mocmHmMMHp unmoHMHcmHmp
msoum Houunooo
mHQMOHammm uozn

:oHumas>o mo wocmmnm may CH mmHoHHHOE
woumwnu may mo GOHDMNHchusH Ho :oHEMNHHMHsomw> :H mmmnmno ucmnmmmdm

 

om\a AEHEV HH\E om\m HEEHV HH\m mom EH +
ucmfiumwnumum mmm Hmuoe

-\m Asses HH\E m~\s Hmmmc HH\E EH EH +
unmeummuumum mmm Hmuoe

mmxo mm\o mm\o mcHHmm EH +
m~\o ucmsummuumua mmE

.<.z .E.z .«.z m\o Ame my EH

mcH>o HaasomssmmunH +
ucmEummHumHm mmE

 

 

 

 

 

.a.z .<.z n.<.z AEOHV oH\H EHco ucmsnmmuumua mmE
mmcommmm Amv mmnomnmm wmcommmm va mmcommmm
mHoHHHom HmEHH¢ mHOHHHom HmEHcd
mmcowumnmuaa HMUHmoHoanoz cowumas>o unmeummne

 

 

 

mmousom aflmouuopmcow mumEHHmcoz can mumEHHm ou msmHDHom HHHEHmm mo mmcommmm

h mnmdfi

65
of subsequent cycles. When HCG was injected into FSH-

stimulated follicles, there was no induction of ovulation
observed except in one animal whose ovulation occurred four
days following the treatment making any correlation between
injection and effect questionable. There was, however, a
significant increase in the menstrual cycle length of the
animals receiving intrafollicular HCG when compared to the
group mean of their previous cycle lengths. Table 8
illustrates these effects of treatment on menstrual cyclic-
ity. Furthermore, the injection of HCG into one follicle

in each ovary (two follicles per animal) did not signifi-
cantly increase the cycle length over that seen in animals
having only one treated follicle. As observed previously

in the squirrel monkey, the apparent increases in follicular
vascularization and luteal coloration were also quite evident
following this treatment (Table 3).

These results suggested the possibility that the intra-
follicular gonadotropin administration was causing follicular
luteinization (in the macaques in the absence of ovulation)
which may then be lengthening the cycle. To test this
hypothesis, peripheral plasma levels of progesterone were
assayed.

Figure 4 illustrates the plasma progesterone levels

in the normal cycle of six Macaca fascicularis females.

 

During these cycles the animals were laparoscoped 4 to 10
times each (mean = 6.3), and the cycles were adjusted to

the day of ovulation (mean = 14.6). Following intrafollicular

66

“no.0 v my umem mmocmHmMMHp unmowmwcmHmm

 

 

N.Hm\m.mm m~.Hm\mm.mv HH com EH + mmE Hmuoe
mm.om\m.mm mm.om\m.sv m Enm>o HmE mHOHHHoH
mco oucH DH m
mom EH + mmE
m.mm\o.om ~.~m\s.os m Hmchm HmE mHoHHHoH
0:0 oucH DH m
00m EH 1. mmm
H.om\n.om H.om\m.Hm E Ammo HmE as m\msme my
EHco mmE
numcmq waomu ucmsqmmndm gumcmq macho pmummne z unmfiummne

 

 

mHomu stnumnmz mHHMHsoHommm women:

map mo numcmq may no mGOHpmuuchHEU< :Hmonuopmaow msocmmoxm mo muowmmm

m maHmfiuH.

67

FIGURE 4

Plasma Progesterone Levels in Macaca
fascicularis x i S.E. (in 6 animals)

 

68

man maomo
Am.vav

vH+ 5+ o MI CHI

 

___—____LP____—_—___—L___

 

 

v MmDUHm

IIIIT

IIFII‘IITII

 

«H

ma

ma

Im/bu

69
injection of 2 IU of HCG the plasma progesterone levels

of six macaques were not different (Figure 5) from the

normal follicular plasma levels seen in this species.

Studies Using Biochemical Intermediates

 

The success of the laparosc0pic intrafollicular in-
jection technique in the squirrel monkey allowed utilization
of this procedure for the evaluation of the ovulation-
inducing ability of several biochemical compounds which are
currently thought to serve as mediators of the follicular
rupture inducing properties of luteinizing hormone. Table 9
demonstrates the results obtained with several of these
compounds. From these data it can be seen that the intra-
follicular administration of PGFZQ induced ovulation in a
significantly higher proportion of treated animals than the
control injections. This treatment also resulted in the
frequent observation of an increased level of follicular
vascularity. PGEZ did not stimulate follicular rupture in
a significant number of animals nor did it cause the pro-
found alterations of follicular morphology seen following
PGFZa' Similarly, neither cyclic AMP nor dibutyryl cyclic
AMP caused ovulation of the mature ovarian follicle in this
animal. Dibutyryl cAMP did appear to induce a luteal
coloration of the treated follicles in a high proportion
of the animals. The majority of the ovulations observed
following prostaglandin treatments occurred within 24 hours

of the treatment.

70

FIGURE 5

Plasma Progesterone Levels Following Intrafollicular
Injection of HCG (2 IU) in Macaca fascicularis
x i S.E. (in 6 animals)

 

71

 

 

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DISCUSSION

The cynomolgus macaques used in these studies were
members of a colony being observed for the assessment of
normal reproduction of this species. Four years of data
analysis has demonstrated that the mean menstrual cycle
length of the members of this colony is 30.9 days (i 0.21
days). This value agrees with those reported by Fujiwara
et a1. (1969), Mahoney (1970) and MacDonald (1971). There-
fore, Macaca fascicularis has a longer cycle than either

its well-studied relative, Macaca mulatta, or the human

 

female.

Laparoscopy has been shown to be an effective technique
for the prediction and timing of ovulation in the nonhuman
primate, and the criteria for morphological definition of
ovulation has been well documented (Rawson & Dukelow, 1973;
Harrison & Dukelow, 1974; Dukelow, 1975). Additionally,
it has been shown that frequent laparoscopic observations
do not impose adverse effects on either reproductive cy-
clicity or the occurrence of ovulation (Rawson, 1973;

Rawson & Dukelow, 1973). Further evidence that laparo-

scopy has little effect on the endocrine cyclicity of the
73

74
macaque, is the present observation that progesterone

profiles in 6 animals receiving an average of 6.5 laparo—
sc0pic examinations per cycle are similar to those seen in
untreated animals. The present study utilized laparoscopy
to demonstrate that at least 90% of all menstrual cycles
observed were ovulatory, with the left ovary ovulating more
frequently than the right.

The lack of a significant relationship between the day
of ovulation (i.e., the length of the follicular phase) and
the length of the menstrual cycle suggests that the luteal
phase length of this species is no more consistent than is
its follicular phase length, since we would expect a linear
plot with a positive slope if the luteal phase length was
more regular than the follicular phase length.

The occurrence of a peak ovulatory response of the
squirrel monkey eight days following their previous ovu-
lation is in agreement with the concept of a 7 to 9 day
cycle length in this species. This interval agrees with
the indirect evidence presented by Rosenblum et a1. (1967)
and Gould et a1. (1973), as well as the previous laparo-
sc0pic observations of Harrison and Dukelow (1974). The
present study demonstrates that HCG, when administered at
the appropriate interval to nonprimed animals, will induce
the same ovulation rate seen following the standard regime
including FSH. If this is the case, one would expect to
see a return to this level of ovulation at periodic 8 to 9

day intervals. The lack of a clear return to such levels

75

on days 16 to 18 of this study may represent interanimal
variation as the effect of the synchronizing ovulatory

regime becomes further away and the animals begin to exhibit
the cycle lengths characteristic of each individual. Regard-
less, although a full recovery is not observed, there is a
trend towards an increased response at day 18.

Laparoscopic evaluations of follicular develOpment
following FSH administration to the squirrel monkey,
demonstrated that neither 2 nor 3 days of FSH pretreatment
were sufficient to induce normal follicular development,
while 4 days proved adequate. It was also shown that FSH
pretreatment would not stimulate ovulation alone, making
this a good model for the studyof ovulation induction.

The superiority of the 4 day FSH treatment lent further
support to the ovulation-induction regime of Dukelow (1970).

Unlike the case in the squirrel monkey, where the FSH
regime was begun on a randomly selected day in the cycle,
the FSH was administered beginning on day 1 of the macaques
menstrual cycle. This allowed differentiation of the
treatment effects from spontaneous follicular growth and
ovulation. The inability of this regime to stimulate normal
appearing follicular growth was unexpected. Unfortunately,
our animal numbers were too low to allow the establishment
of the cause for this inactivity, an important difference
between the ovarian physiology of the macaque and Saimiri.

The lack of local effect of gonadotropin saturated

disks is believed due to the short period of contact with

76

the ovary. Aratei (1972) proposed a theoretical analysis
of the permeability of the ovarian follicular membranes and
suggested that the transfer of a protein across these mem-
branes was time dependent. This concept would suggest that
if the disks could be held in place for a longer interval
the expected effects might be seen. The results of an
experiment utilizing in yitrg culture of porcine follicles
(Appendix I) are supportive of this hypothesis.

Injection directly into the ovarian follicle was a
method which resulted in exposure of the follicle to gonado-
trOpin sufficient to produce an effect. The ovulation
rate in non-pretreated squirrel monkeys following intra-
follicular LH; while higher than females receiving
intrafollicular saline, FSH pretreatment only, or the FSH
pretreatment followed by intrafollicular saline; did not
compare with the rates of animals receiving an FSH pre—
treatment followed by intrafollicular administration of LH.
This is further evidence of the necessity of both FSH and
LH as synergists for the induction of ovulation in the
primate.

The inability of saline injections to cause mechani-
cally induced follicle rupture is consistent with the
reports of earlier advocates of similar techniques (Espey
& Lipner, 1963; Blanchette, 1966; Ferrando & Nalbandov,
1969) all of whom utilized either greater injection volumes
or larger gauge needles than the present studies. In-

jection of an ink marker demonstrated that injected

77
substances do enter the follicular antrum and are trapped

there without escape. Thus, this system allows the evalu-
ation of the effect of various substances on ovulation
when applied directly at the follicular level.

The sufficiency of ovine-LH for ovulation induction
in the squirrel monkey administered by this method suggests
two possible conclusions. The first, that this species does
not have a specific requirement for an LH source of primate
origin. This is refuted by the inability of systemic ovine-
LH to stimulate ovulation in similar animals. Also, this
hypothesis ignores the previous experience with ovulation
induction in the nonhuman primate. The second hypothesis
is that this species-specificity is at an extrafollicular
level, possibly relating to binding in the circulatory
system or some type of reactivity at the blood-follicle
barrier.

As stated previously, the poor response of the macaque
to this procedure demonstrates an important difference
between these two species. An effect was definitely
indicated by the significant increase in the length of the
menstrual cycles, but there did not appear to be the increase
in circulating progesterone levels that would be expected
if luteinization were being induced by the procedure. It
is possible that the poor response observed in macaques
relates to an inappropriate stimulation of follicular
growth seen following FSH. This would agree with the

conclusion of Knobil et a1. (1959), who suggested that

78
excessive follicle stimulation achieved too rapidly would

inhibit ovulation in reSponse to a subsequent ovulating
dose of Chorionic gonadotropin.

Whether follicular rupture results from a degradation
of the wall of the follicle or from the contraction of
smooth muscle elements, the initial stimulus is known to
be LH. The mechanism of this action of LH at the ovarian
level has recently been an area of extensive investigation.
In 1970, it was suggested that prostaglandins might be
obligatory intermediates of the action of LH on the mam-
malian ovary (Kuehl, et al., 1970). Subsequently,
Armstrong et al. (1972) demonstrated a role of these com-
pounds in ovulation, when an inhibitor of prostaglandin
synthesis (indomethacin) was shown to inhibit follicular
rupture in the rabbit. The present studies have shown
that PGFZa administered directly into the ovarian follicle
of the squirrel monkey mimicked the induction of follic-
ular rupture observed following administration of an LH
source. This is evidence corroborating the possibility of
an intermediary role of PGFZa in the LH induction of
ovulation in the nonhuman primate. This concept is sup-
ported recently by studies in the mouse (Lau, et al., 1974)
and in the rabbit (Richman, et al., 1974). Although PGE2
caused an increase in the ovulation rate following intra-
follicular administration, it was unable to stimulate

significantly greater levels than the control values.

79
This partial effect of PGE2 has also been observed by

Moon and Armstrong (1974) who demonstrated that while PGFZG
antiserum effectively inhibited ovulation in the rabbit,
anti-PGEZ was effective in only 43% of the animals. In the
mouse Saksena et a1. (1974) found PGE2 to be more effective
than PGde in the reversal of an indomethacin blockade of
ovulation. The latter experiment, however, was conducted
with systemic administration of the drugs and may have
represented a different level of activity.

The demonstration that cAMP caused an LH-like stimu-
lation of progesterone synthesis by slices from bovine
corpora lutea (Savard et al., 1965) was strong evidence that
cAMP served as an intermediate in the action of this
hormone. That prostaglandins would stimulate cAMP accumu-
lation by the ovaries suggested that prostaglandins may
also be implicated in this hormone action (Kuehl, et al.,
1970). There is good evidence supporting the role of
cAMP in the LH-stimulation of luteinization (Marsh & Savard,
1966; Miller and Keyes, 1974; Channing, 1974). There is,
however, little direct evidence supporting a role of cAMP
in the process of ovulation. The experiments reported
here refute the proposed intermediary role of cAMP in LH-
induced ovulation in the primate. Our results concur with
the observations of LeMaire et a1. (1972) who demonstrated
an inhibition of LH-induced ovulation in the rabbit by the

intrafollicular administration of either cAMP or dibutyryl

80
cAMP. These authors proposed that the intrafollicular

cAMP may act normally to inhibit luteinization of the
follicle.

The present experiments have involved the local
administration of various substances to the primate ovarian
follicle to evaluate the direct effect of each compound.
The results suggest that prostaglandins (especially PGFZQ)
may be involved in the biochemical sequence of events
culminating in follicular rupture. Although cAMP did not
appear to be involved in this process, there can be no
doubt that it is present and functional in the ovarian
follicle. The present studies support the theory prOposed
by LeMaire that the ovulation-inducing properties of LH may
be mediated by prostaglandins while cAMP may serve as an
intermediate for the action of LH on oocyte maturation or
luteinization.

The laparoscopic technique developed and used in these
studies offers a method for evaluating the influence of
various chemical compounds on the processes involved in
ovulation in the nonhuman primate without submitting the
animal to the stress of a major surgical procedure. Further
use of this technique will increase our understanding of

the follicular mechanisms involved in ovulation.

SUMMARY AND CONCLUS I ONS

Several parameters of reproductive function were
studied in two species of nonhuman primates, with an
emphasis on the development of a primate model for the
study of follicular physiology. The results of the present
experiments indicate the following conclusions.

In the squirrel monkey, ovulation can be induced with
HCG in the absence of an FSH pretreatment at 7 to 9 day
intervals following synchronization. There is a trend
toward another increase in ovulation rate between 16 and
18 days following synchronization which would agree with
the concept of a short reproductive cycle length (7 to 9
days) for this species.

Analysis of data on the reproductive cycle of the

Macaca fascicularis, collected over a four year span, is

 

in accord with previous observations of a 30 day menstrual
cycle, having a 14 day follicular phase. Almost 90% of

the cycles studied were shown to be ovulatory, with more
ovulations occurring on the left ovary than on the right.
Follicle rupture was observed in four Cases, three of which

were accompanied by bleeding from the site of ovulation.
81

82
Treatment of the Saimiri sciureii with 4 daily, 1 mg

 

injections of FSH was shown to result in apparently normal
follicular development. Similar treatment (5 days, 5 mg/day)

in the Macaca fascicularis did not induce normal appearing

 

follicular growth, as there was a high incidence of cystic
and hemorrhagic follicles.

A technique was developed for the assessment of the
ability of various compounds to mimic the ovulation-inducing
properties of luteinizing hormone. This procedure involved
the injection of these substances directly into the antrum
of the preovulatory follicle. When administered in this
manner, ovine LH was shown to stimulate an ovulation rate
similar to that seen following systemic administration of
human Chorionic gonadotrOpin. Intrafollicular injections
of an LH from a primate source (HCG) did not cause a higher
ovulation rate than an LH from a nonprimate source, which
suggested that the reported species specificity of response
of the primates to LH was at a suprafollicular level.

Intrafollicular injections of PGFZG were able to induce
ovulation rates that were similar to those seen following
LH administration. This is in agreement with the proposed
mediation of this prostaglandin in the ovulation-inducing
mechanism of LH. Prostaglandins E2, cyclic 3',5' AMP, and
dibutyryl cyclic 3',5' AMP were unable to stimulate such
levels.

Intrafollicular injection of HCG in the FSH-primed

macaque would not induce ovulation, apparently due to the

83
abnormal follicular development induced by the pretreatment.

Neither did this treatment have any effect on the plasma
progesterone levels of this species. Menstrual cycle
lengths of these animals were, however, significantly longer
than their previous cycles with a return to normalcy during

the first post-treatment cycle.

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APPENDICES

APPENDIX I

 

II III! 5‘!

"u

APPENDIX I

PORCINE FOLLICLE CULTURE IN VITRO

Following the observed lack of effects of gonadotropin
saturated filter paper disks on the ovarian follicle in the
macaque, it was decided to investigate the possibility that
this may have been due to the short exposure interval
necessitated by the anesthetic limitations. Since oocyte
maturation had been shown to be a more sensitive index of
gonadotrOpin stimulation than either ovulation or corpus
luteum formation (Vermeiden & Zeilmaker, 1974), experiments
were designed using porcine follicles in culture for various
intervals with the LH saturated disks. The maturation of
the oocyte was used as a sign of gonadotropin activity.

The ovaries were obtained at slaughter; individual
follicles were dissected free and placed on stainless steel
wire grids in sterile petri dishes. Only those follicles
protruding from the ovarian surface and greater than three
mm in diameter were used. Follicles were randomly assigned

to one of four groups as follows:

98

99
El - Follicles in contact with an LH saturated ovarian

disk for one hour

C1 - Follicles in contact with saline-saturated disk
for 1 hour

E24 - Follicles in contact with LH-saturated ovarian
disk for 24 hours.

C24 - Follicles in contact with saline-saturated
ovarian disk for 24 hours.

All four of the groups were cultured for 24 hours in
media consisting of 85% Hams F10 and 15% fetal calf serum,
with 100 IU of penicillin per milliliter of medium. The
environment was maintained at 37 C and contained a mixture

of 5% CO2 in the air.

Following culture, the follicles were opened, the
oocytes recovered and placed on clean slides. The oocytes
were then fixed with 10% formalin and stained with either
aceto-orecin or lacmoid. The slides were scored under a
light microsc0pe on a scorer-blind basis. Oocytes were
considered to be immature if there were no distinct devel-
0pmental characteristics, or if a germinal vessicle was
discernible. Only those containing mitotic figures or
polar bodies were classified as undergoing maturation. Any
estimate of oocyte maturation obtained by this technique
will be conservative and will, therefore, have a greater
chance of reflecting real differences.

The percentages of oocytes that did not exhibit signs

of maturation were as follows: C1, 100% (N = 10); C24, 100%

100
(N = 10); E1, 74% (N = 19); 224, 18% (N = 17). Analysis

reveals that the group cultured with the LH disk for 1 hour
had a significantly greater number of follicle-enclosed
oocytes revealing no signs of maturation than did the
follicles cultured with the LH disk present for 24 hours.
These data suggest that the follicle wall may impose a
slowly permeable barrier to LH and that 1 hour exposure to
LH in the foregoing manner is not sufficient to induce ovum

maturation.

APPENDIX I I

APPENDIX II

LAPAROSCOPY IN THE RABBIT

During the course of these studies, the technique
of laparoscopy was developed to allow frequent examination
and manipulation of the reproductive tract of the rabbit.
This technique allows the serial observation of ovarian,
oviductal and uterine phenomena in the same rabbit. A
description of the technique and an example of its use is

illustrated in the following paper.

102

_...._ .,."-,."' ”7 ~.’ ' ‘-

J. Reprod. Fert. (3.974) 38: 97-103.

HORMONAL INFLUENCES ON THE TIME OF
OVULATION IN THE RABBIT AS DETERMINED

BY LAPAROSCOPY

BY

S. Fujimoto, J. M.R. Rawson 8: W. R. Dukelow

 

 

Endocrine Research Unit
Michigan State University
East Lansing, Michigan

J. Reprod. Fm. (1974) 38, 97—103

HORMONAL INFLUENCES ON THE TIME OF
OVULATION IN THE RABBIT AS DETERMINED
BY LAPAROSCOPY

S. FUJIMOTO,‘ J. M. R. RAWSON AND W. R. DUKELOW

Endocrine Research Unit, Michigan State University,
East Lansing, Michigan 48823, U.S.A.1’

(Received 30th July 1973)

Summary. The technique of laparoscopy was modified for use in the
rabbit and was found to facilitate continuous observation of follicular
development and ovulation. The timing of ovulation was studied and
suggests that 100 i.u. HCG, whether used in a superovulation regimen or
solely as an ovulatory stimulus, causes a greater ovulation rate than does
a mating stimulus. Follicular rupture was observed and frequently
occurred in the smaller follicles first. Laparoscopy in pregnant rabbits
suggests that quantification of preimplantation blastocysts is not only
possible but also very accurate and is without deleterious efl‘ects on the
developing embryos. These results suggest that the laparoscopic tech-
nique may be a valuable asset to the study of reproductive phenomena
in the rabbit.

INTRODUCTION

The recent application of laparoscopy to ovulation examination, ovarian
biopsy, oviducal ligation and clinical diagnosis of various reproductive dis-
orders reflects the pioneering efforts of Semm (1969) and Balin, Wan & Rajan
(1969). Previous reports have shown that ovulation in the rabbit occurs as
early as 8 hr (Hill, Allen & Kramer, 1935) and as late as 13} hr (Walton &
Hammond, 1929) after mating. By contrast, Harper (1963) reported that no
ovulation occurred by 10 hr but that 50% of the ovulations occurred between
10% and 101i hr and 100% by 14- hr after an intravenous injection of 25 to
50 i.u. LH.

Laparoscopic observation of ovulation in rabbits has not been reported and,
with the exception of the excellent cinematographic observations by Blandau
(1955), few have been able to predict ovulation accurately and perform serial
observation of follicular morphology near ovulation.

The present paper describes the adaptation of laparoscopy to the observation
and timing of follicular development and ovulation in the rabbit.

‘Present address: The Population Council, Biomedical Division, The Rockefeller University,
New York, NY. 10021, U.S.A.

1‘ Address for reprint requests.

97

98 S. Fujimoto et a1.

MATERIALS AND METHODS

Twenty-one, virgin, adult, female rabbits of mixed breeding and 3-4 to 4-6 kg
in body weight were used. They were separately caged for at least 3 weeks
before use to preclude the development of pseudopregnancy.

All rabbits were assigned to one of three treatments: Group 1, natural mating.
Six does were mated two or three times to males of proven fertility. No exo-
genous gonadotrophins were given; Group II, HCG-induced ovulation. Five does
were injected intravenously with 100 i.u. HCG (A.P.L., Ayerst Laboratories);
Group III, PMSG-HCG induced ovulation. Ten does were injected intravenously

 

TEXT-FIG. l. Diagrammatic representation of the ovarian suspension technique used to
facilitate ovarian observation during laparoscopy in the rabbit (01 = ovarian ligament;
= oviduct; 0v = ovary; ut = uterus).

with 100 i.u. HCG 4 days after priming with 100 i.u. PMSG (Lilly Labora-
tories) intramuscularly.

The first laparoscopic examinations were made 8 to 9 hr after the ovulatory
stimulus (mating or HCG) under sodium pentobarbitone anaesthesia.

The laparoscope was a Wolf paediatric 135° model. The laparoscopic
equipment and its use in other species has been previously reported (Dukelow,
Jarosz, Jewett & Harrison, 1971; Jewett & Dukelow, 1972, 1973; Rawson &
Dukelow, 1973a, b). Abdominal distension by 5 ‘X, COz/air was required in the
rabbit.

For assistance in locating the ovaries, 3-0 gut sutures were threaded through
the abdominal wall around the ovarian ligament and back through the wall
where they were loosely tied (Text-fig. 1). Manual elevation of the external
portion of the sutures made suspension and observation of the ovaries a simple
procedure (Pl. 1, Fig. 1).

Ovulation and laparoscopy in the rabbit 99

Serial observations were continued at intervals of l or 2 hr. Photographs
were taken with a Canon-TL, 35 mm camera/Ektachrome EHB film at l to
I see exposure.

The laparoscopic technique described herein was subsequently used in four
does from Group I for the counting of the potential implantation sites on Day 6
as well as the observation of the uterine vascularization associated with the site
of implantation.

For ease of classification of developing follicles, five types were assigned to
an alphabetical system: Type A follicles were large dome-shaped structures
with a haemorrhagic appearance at the apex. Type B referred to a large
dome-shaped structure without the haemorrhagic appearance. Type C was
smaller, but still elevated above the ovarian surface while Type D was not
elevated from the ovarian surface. Type E was used to refer to a follicle not
present at the previous observation.

RESULTS

The total number of follicles, postovulatory follicles and percentage ovulation
for the three groups are shown in Tables 1, 2 and 3, respectively. They are
classified according to the time interval after ovulatory stimulus.

In Group I (Table l), the initiation of ovulation was delayed and the

Table 1. Ovulation in rabbits“ subjected to
natural mating

 

 

Tm “‘53:“ 7 al f M" "f y
mating at no. a tooula 0
up???” flunk: ”39,115,130 ovulation

8 to 8} 74 1 1-4
10 to 10; 86 6 7-0
12 to 12; 94 14 14-9
14 to 141 99 25 25-3

 

 

 

 

"' Group 1, six does.

percentage ovulation at 14 to 14-} hr was 25-3 %, that is, one half that of the other
two groups (Text-fig. 2). The mean number of ovulation points per animal, 4'2,
was also low.

In Group II, twenty laparoscopic examinations were carried out in five
females between 8 and 144} hr after the HCG injection (Table 2). In this group,
ovulation started later than in Group 111, but as illustrated in Text-fig. 2, both
groups ovulated at a similar rate at 14- to 14-) hr. The number of ovulation
points per animal at 8 to 8} hr and 14 to 141} hr were, 0 and 4-8, respectively.

In Group III, forty-three laparoscopic examinations were performed in ten
does between 8 and 14% hr after the HCG injection (Table 3). Ovulations were
observed at 8 to 81 hr in four out of nine does (44 "/0) and at 9 to 9} hr in six out
of ten (60 %). At 10 to 10} hr, ovulation had occurred in all ten does. Ovulation
rates increased from 69% at 8 to 85 hr, to 529% at 14 to 14-} hr. The mean

100 S. Fujimoto et al.

numbers of ovulation points per animal increased from 0-9 at 8 to 8) hr to
10-8 at 14 to 14} hr. Superovulation occurred in two animals with nineteen and
twenty-three ovulations, respectively.

The results shown above demonstrate a large variation in the time of onset,
the duration of ovulation and ovulation rates between groups.

We also observed the moment of rupture of ten follicles in two animals of
Group I and three animals of Group III. Preovulatory follicles representing

.//

1 1 1
8 10 l2 l4

‘7: ovulation

 

Time after ovulatory stimulus (hr)
TEXT-FIG. 2. Ovulation rate at various times after an ovulatory stimulus of mating (O),
HCG alone (x) or PMSG and HCG (I) in rabbits.

various stages of development are illustrated in P1. 1, Figs 2 and 3(a). Post-
ovulatory sites are visible in P1. 1, Figs 3(b) and 4. Occasionally haemorrhagic
follicles attained a greater volume than the normally developing mature
follicles. Plate 1, Fig. 5 illustrates a cumulus mass adhering to the ovarian sur-
face shortly following follicular rupture. Later (Pl. 1, Fig. 6), the cumulus mass
could be seen on the fimbria.

Although a small non-protruding follicle was not defined as a mature follicle

 

EXPLANATION OF PLATE 1

FIG. 1. Elevation of the external portion of sutures laparoscopically placed around the
ovarian ligaments to facilitate suspension and observation of the ovaries in a rabbit.

FIG. 2. Preovulatory (Type A) follicle. Note the ease of observation of the entire ovarian
surface made possible by the suspension procedure.

F10. 3. Several preovulatory (a) and postovulatory (b) follicles present on the same ovary.
FIG. 4. Large immediately postovulatory follicle.

F10. 5. Cumulus mass adhering to a postovulatory site.

F10. 6. Cumulus mass on fimbria.

 

I’I. \Tl-l l

 

I'l/u’iug [1.1001

 

Ovulation and laparoscopy in the rabbit 101

Table 2. Ovulation in rabbits“ subjected to
induced ovulation with 100 i.u. HCG

 

 

Tamas“? Total .no- of ”mg %_
8 to 8} 34 o o
10 to 10} 43 5 11-7
12 to 12} 44 15 34-1
14 to 141 46 24 52-2

 

 

 

 

" Group 11, five does.

by Walton & Hammond (1929), we observed laparoscopically that in many
animals this type of follicle had developed into a large mature follicle during the
serial examinations ranging from 8 to 144 hr after the induction of ovulation.

As previously described by Walton & Hammond (1929), and Hill et al.
(1935) , the major developments of follicles about to ovulate take place about 2 hr
before rupture. In the present studies, the blood supply increased remarkably
on the follicular surface and extravasation of blood was observed during the
last 30 min before rupture.

Laparoscopy of four pregnant rabbits on Day 6 revealed a total of twenty-
two visible blastocysts, equally distributed in the uterine horns. Immediately

Table 3. Ovulation in rabbits“ subjected to induced
ovulation with 100 i.u. PMSG and 100 i.u. HCG

 

 

 

 

 

 

 

Time between 1%. 9f
H00 and No. of Total .no. of 1,03qu % .
14”,“pr does follicle: jbllicle: ovulatwn
(1")
8 to 8} 9 116 8 6'9
9 to 9} 5 66 10 15.2
10 to 10* 9 128 43 33-7
11 to 11} 4 61 25 41-0
12 to 12} 7 110 49 44-6
13 to 13} 4 65 34 52-3
14 to 14} 5 102 54 52-9
* Group 111.

after laparoscopy, the animals were killed and the blastocysts flushed from the
horns. Twenty-three blastocysts were recovered, verifying the applicability of
laparoscopy to achieving a close estimate of the number of Day 6 blastocysts.

DISCUSSION

The present findings are in general agreement with the laparotomy—autopsy
data of Walton & Hammond (1929) and Harper (1961, 1963), and the cine-
micrographic observations of Hill et al. (1935).

Harper (1963) reported that no ovulation was observed by 10 hr, 50% ovula-

 

102 S. Fujimoto et al.
tion was observed between 10) and 10% hr and 100% by 14 hr after the HCG

injection.

The earlier onset of ovulation observed at 8 to 8) hr after HCG injection in
the present Group III was probably due to the regimen of ovulation induction
(PMSG 100 i.u., HCG 100 i.u.).

The ovulation rates in the present studies were lower than the percentage
ovulation in the time periods between injection of LH and autopsy reported by
Harper (1963). The mean number of total follicles in Group I and Group II,
however, did not differ from his data. Our data do indicate an increase in the
slope of the ovulation curve (Text-fig. 2) for animals receiving HCG, indicating
earlier ovulation. This was of greatest magnitude in animals subjected to a
superovulation regimen, a factor to be considered in reproduction assay tech-
niques involving timed ovulations.

An interesting observation on the development of the follicle towards ovula~
tion was that often the Type B and C follicles at one examination period had
ovulated before those follicles with a Type A appearance at the initial examina-
tion. Whether this is due to follicular atresia of the Type A follicles is not
known.

The results of the laparoscopic examination of the pregnant rabbits reveal a
high degree of accuracy in the quantification of potential implantation sites.
These observations also suggest that the anaesthesia and laparoscopic tech-
nique had no effect on the fertilizability of the ovum since the pregnant animals
had been previously subjected to laparoscopy at the time of ovulation. These
results are similar to those reported after laparoscopy in non-human primates
(Rawson & Dukelow, 1973a).

A significant finding of this study relates to the relative ease of the technical
manipulation required for laparoscopy in the rabbit. With the suspension of the
ovarian ligament, the entire ovary easily came into view and could be quickly
and easily observed. The results serve to emphasize the usefulness of the
laparoscopic techniques for studies necessitating ovulation detection and timing.
diagnosis of pregnancy and enumeration of CL and for studies in which oviducal
and uterine manipulations such as ligations or sampling are indicated.

ACKNOWLEDGMENTS

The authors wish to express their appreciation to Dr Nahid Pahlavan for her
excellent assistance. This work was supported by NIH Contract No. 70-2061
and NIH Research Career Development Award N0. 1-K4~HD36, 306. Michi-
gan Agricultural Experiment Station Journal Article Number 6496.

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RAWSON, J. M. R. & DUKELOW, W. R. (1973b) Observation of ovulation in Macaca fascicularis. J.
Reprod. Fert. 34, 187.

SEMI, K. (1969) Gynaecological pelviscopy and its instrumentarium. Acta euro/2. fertil. l, 81.

WALTON, A. & HAMMONDJ. (1929) Observations on ovulation in the rabbit. Br. J. exp. Biol. 6, 190.

1935) Cinematographic studies of rabbit ovulation. Anat.

103
Subsequently, the technique was employed to follow

the complete pregnancy in this species. The animal
involved had been mated for the recovery of capacitated
spermatozoa and ovulation was confirmed by laparotomy on
day l of pregnancy. On day 5 the sutures were placed and
eight corpora lutea were observed on the ovaries. By day
15 the sutures were gone, probably due to the animal's
metal cage, and 2 large corpora lutea were observed on the
left ovary. The right uterine horn contained two large
vascular fetal swellings. Similar observations were made
on day 25 of pregnancy except the degree of swelling and
uterine vascularization had increased. On day 32 (found
on the morning of day 33), the animal delivered two normal-
appearing infants which were dead when found.

This observation suggests that laparoscopy had little
effect on pregnancy in the rabbit even though performed at
several stages in the gestation. This further illustrates

the utility of laparoscopy as a research tool.

APPENDIX I I I

APPENDIX III

INDUCTION OF FOLLICLE GROWTH IN AN
IMMATURE SAIMIRI SCIUREUS

 

In 1974, a female Saimiri sciureus was brought into

 

the primate colony at the Endocrine Research Unit and
immediately began to exhibit traits frequently observed in
immature animals. She was considerably smaller than usual
and spent a great deal of time riding the other females.
Laparoscopic examinations revealed the presence of an
extremely small reproductive tract with ovaries appearing
devoid of follicular growth. Such characteristics suggested
that this animal was pre—pubertal. Four days of intra-
muscular FSH treatments (1 mg/day) produced negligible
follicular development in this animal. Following a three
month interval, this FSH regime was repeated with the
addition of 500 IU of HCG on the fifth day. The follicular
response was markedly increased with large, vascular
follicles being present on both ovaries, although no ovu-
lations were seen. Four weeks later, the initial FSH only

regime was again administered without observable effects.

105

106

This was considered to suggest the possibility of a role
of LH in the growth and deve10pment of follicles in the

immature monkey.

APPENDIX IV

 

Papers:

APPENDIX IV

PUBLICATIONS BY THE AUTHOR

Natural and artificial control of ovulation in
nonhuman primates. W.R. Dukelow, R.M. Harrison,
Jon M.R. Rawson and M.P. Johnson. Medical
Primatology, 1972.

 

Follicular development and ovulation in Macaca
fascicularis, Saimiri sciureus and Galago
genegalensis. W.R. Dukelow, D.A. Jewett and
Jon M.R. Rawson, American Journal of Physical
Anthropology, 38:207F209, 1973.

 

 

Effect of laparoscopy and anesthesia on ovula-
tion, conception, gestation and lactation in

a yacaca fascicularis. Jon M.R. Rawson and
W.R. Dukelow. Laboratory Primate Newsletter,
12:4-5, 1973.

Observation of ovulation in the Macaca fascicu-
laris. Jon M.R. Rawson and W.R. Dukelow.
Journal of Reproduction and Fertility, 34:
187-191, 1973.

 

Ovulation in Macaca fascicularis. Jon M.R.
Rawson. M.S. Thesis, Michigan State University,
1973.

Megestrol Acetate: II. Effects on ovulation
in nonhuman primates as determined by laparo-
scopy. R.H. Harrison, Jon M.R. Rawson and
W.R. Dukelow. Fertility and Sterility, 25:
51-56, 1974.

 

108

Abstracts:

109

Hormonal influences on the time of ovulation
in the rabbit as determined by laparoscopy.

S. Fujimoto, Jon M.R. Rawson and W.R. Dukelow.
Journal of Reproduction and Fertility, 38:
97-103, 1974. '

 

Effects of intrafollicular administration of
luteinizing hormone in the nonhuman primate
using laparoscopy. Jon M.R. Rawson and W.R.
Dukelow. Advances in Medical Primatology,
1975 (accepted).

 

The environmental enemy -- US. Jon M.R. Rawson;
presented at First National Biological Congress,
November 8, 1970, Detroit, Michigan.

The effect of bone marrow depletion on blood
volume of the rat. Jon M.R. Rawson and J.R.
Toepfer; presented at Regional Meeting of
American Institute of Biological Sciences,
1971, Erie, Pennsylvania.

Ovulation morphology in nonhuman primates.

Jon M.R. Rawson and W.R. Dukelow; presented at
Michigan Academy of Science, Art and Letters,
March 24, 1972, East Lansing, Michigan.

Comparative ovulation in nonhuman primates.
W.R. Dukelow and Jon M.R. Rawson; presented
at Federation of American Societies for Ex-
perimental Biology, Atlantic City, New Jersey,
1972 (Fed. Proc. 31:277).

 

Ovulation-menstrual cycle relationships in
Macaca fascicularis. Jon M.R. Rawson, W.R.
Dukelow and T.J. Kuehl; presented at Federation
of American Societies for Experimental Biology,
Atlantic City, New Jersey, 1974 (Fed. Proc.
32:283).

 

 

Clomiphene citrate: Effects on ovulation and
menstrual cyclicity on the nonhuman primate.
Jon M.R. Rawson and W.R. Dukelow; presented at
the American Society for Pharmacology and
Experimental Therapeutics, East Lansing, Mich-
igan, Fall 1973 (The Pharmacologist 15:256).

 

Laparoscopic techniques for reproductive
physiology studies in nonhuman primates and
other species. Jon M.R. Rawson and W.R.
Dukelow; presented at Federation of American
Societies for Experimental Biology, Atlantic
City, New Jersey, 1974 (Fed. Proc. 33:281).

 

110

Lack of a species-specific response of Saimiri
sciureus to gonadotropins at the follicular
level. Jon M.R. Rawson, James P. Mahone, and
W.R. Dukelow; presented at Federation of
American Societies for Experimental Biology,
Atlantic City, New Jersey, 1975 (Fed. Proc.
34:544).

APPENDIX V

 

Name:

Born:

Formal Education:

Degrees Received:

Awards and Honors:

APPENDIX V

VITA

Jon M.R. Rawson

May 8, 1949
Lansing, Michigan

University High School
Carbondale, Illinois 1963-67

Southern Illinois University
Carbondale, Illinois 1967-68

Youngstown State University
Youngstown, Ohio 1968-71

Michigan State University
East Lansing, Michigan 1971-75

Bachelor of Science
Youngstown State University, 1971

Master of Science
Michigan State University, 1973

Michigan State University Sigma Xi
Graduate Award for Meritorious Research
(1974)

Physiology Graduate Merit Scholarship
Award (1974)

Phi Kappa Phi Scholastic Honorary

112

Society
Memberships:

113

Outstanding Graduating Biology Major
Youngstown State University (1971)

Invited Speaker at First National

Biological Congress, Detroit, Michigan
(1970)

Who's Who Among Students of American
Colleges and Universities (1971)

Omicron Lambda (Honorary Biology
Fraternity) (1970, 1971)

American Physiological Society

Sigma Xi

Society for the Study of Reproduction
International Primatological Society

 

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