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THE EFFECT OF ESTRADIOL ON FSH INDUCTION OF FSH RECEPTORS IN

GRANULOSA CELLS OF THE RAT

BY

SHARON ANN TONETTA

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Animal Science

1984

ABSTRACT

THE EFFECT OF ESTRADIOL ON FSH INDUCTION OF FSH RECEPTORS
IN GRANULOSA CELLS OF THE RAT

BY

Sharon Ann Tonetta

I examined whether estradiol is required for FSH to increase its
own receptor in granulosa cells of the rat ovary and promotes
follicular development. First, I characterized granulosa cells
receptors for estradiol and compared these results (Scatchard analyses,
sucrose density gradient analysis, effects of temperature and emzymes,
steroid specificity and translocation of receptor) to that known for
the uterus. Next, I blocked the synergistic effects of estradiol with
FSH by pharmacologically inhibiting estradiol synthesis (cyanoketone)
or estradiol binding to its receptor (CI628) in granulosa cells.
Hypophysectomized rats were divided into five groups: 1) saline, 2)
CI628 or cyanoketone, 3) FSH, 4) CI628 or cyanoketone--then FSH, and 5)
CI628 or cyanoketone plus estradiol--then FSH. Animals were
decapitated at 0, 6, 12, or 24 h post-FSH injection and granulosa
receptors for FSH and LH and nuclear receptors for estradiol were
measured. Finallly, I examined whether estradiol affects the
FSH-induced cAMP-adenylate response system. Hypophysectomized. rats
were divided into five groups as described. Twelve h after the initial

FSH injection, rats were injected with FSH. Animals were killed 60 min

Sharon Ann Tonetta
later and granulosa CAMP measured. Granulosa estradiol receptors were
identified, characterized, and found similar to those described in the
uterus. After administration of CI628 or cyanoketone, LH receptors
were unchanged unless estradiol was administered comcomitantly.
Numbers of receptors for FSH and estradiol were similar in saline-,
CI628- and cyanoketone-treated animals. In group 3, FSH and estradiol
receptors increased 6-fold (p<0.01) over controls. In group 4, CI628
or cyanoketone administration prevented the FSH-induced increase in FSH
receptors at all times. Namber of estradiol receptors were similar to
controls. Estradiol administration with CI628 or cyanoketone (group 5)
prior to FSH reversed the inhibitory effects of the drugs on FSH and
estradiol receptors. Progesterone ,testosterone, R5020 and DHT
partially reversed inhibitory’ effects of cyanoketone prior to FSH.
Estradiol appears to be required for maximal stimulation of FSH
receptors after FSH injection. The interaction of estradiol and FSH
appears to be past the cAMP-adenylate cyclase system since treatment
with CI628 prior to FSH had no effect on FSH-stimulated cAMP
production. Thus, estradiol may be required for FSH to increase its

own receptor and, in turn, promote follicular growth.

To my parents

ii

ACKNOWLEDGEMENTS

I would like to thank the members of my committee, Dr. Roy Fogwell,
Dr. William Smith, Dr. Edward Convey and Dr. Scott Walsh for their
advice and cooperation during my program. I am especially grateful to
my major professor, Dr. James Ireland, not only for his expert guidance
and assistance, but for his friendship.

My special thanks to Barbara Townsend, Leon Spicer, Andrea Curato
and Randall Grimes for their technical assistance in these studies. I
would like to especially thank Dr. Vasantha Padmanabhan and Dr. Kwanyee
Leung for their encouragement and understanding. Also, I would like to
thank my fellow graduate students for their invaluable moral support
and advise.

Finally, the encouragement, motivation and help of my husband, Dr.

Vincent Hylka, and my parents helped make this dissertation a reality.

iii

TABLE

DEDICATION . . . . . . . . . . . .
ACKNOWLEDGEMENTS . . . . . . . . .
LIST OF TABLES . . . . . . . . . .
LIST OF FIGURES. . .

LIST OF ABBREVIATIONS. . . . . .
INTRODUCTION . . . . . . . . . . .
REVIEW OF LITERATURE . . . . . . .
CHAPTER I. ESTROGEN RECEPTORS
Introduction . . . . . . . .
Materials and Methods. . . .

Results 0 O O O O O O O O O 0
Discussion . . . . . . . . . .

CHAPTER II.

0 O

O

O

O O

O O
O

O I

IN GRANULOSA CELLS OF

IN ESE RECEPTORS IN GRANULOSA

Introduction . . . . . . . . .
Materials and Methods. . . . .
Results. . . . . . . . . . . .
Discussion . . . . . . . . . .

CHAPTER III.
RECEPTORS FOR ESE IN

Introduction . . . . . . . . .
Materials and Methods. . . . .
Results. . . . . . . . . . . .
Discussion . . . . . . . . . .
GENERAL DISCUSSION . . . . . . . .
SUMMARY AND CONCLUSIONS. . . .

”mu 0 O 0 O O O O O O O O O O

C1628 INHIBITS ESE-INDUCED

iv,

OF CONTENTS

0 O O O O O O O O O O 0

EFFECT OF CYANOKEYTONE ON ESE-INDUCED INCREASES

CELLS OF THE RAT . . .

O O O O O O O O O O O
O O O O O O O O O O O O
O O O O O O O O O O O O

INCREASES IN
OVARY

OF THE RAT. . . . . .

. ii

.iii

OVi

OVii

o 014

. l6
. 22
. 35

. 38

. 45

. 77

.87

LIST OF REFEREMES .

O 91

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

10

11

12

13

LIST OF TABLES

Estradiol binding to residual tissue from granulosa
C2113 and uteri o o o o o o o o o o o o o o o o o o o o 26
Binding specificity of nuclear and cytoplasmic

receptors 0 O O O O I O O O O O O I O O O O O O O O O O 31

Effects of various preparations of FSH on FSH and
LH receptors in granulosa cells of hypophysectomized
imature rats 0 O O O O O O O O O O O O O O O O O O 0 O 42

Effect of various doses of cyanoketone plus
FSH on ovarian and uterine weights . . . . . . . . . . .47

Effect of cyanoketone on estradiol binding to
cytosol of granulosa cells . . . . . . . . . . . . . .

Effect of cyanoketone on FSR induction of LH
binding sites on granulosa cells . . . . . . . . . . .

Effect of cyanoketone, FSH and steroids on
ovarian and uterine weights, and levels of
progesterone and testosterone in serum . . . . . . . . .53

Estradiol binding to granulosa cells with and
without preincubation with C1628 . . . . . . . . . . .

Time course of hFSH stimulation of cAMP content
in granulosa cells of rats . . . . . . . . . . . . . . .65

Effect of various doses of CI628 and FSH on
ovarian and uterine weights. . . . . . . . . . . . . . .68

Effect of C1628 on FSH induction of LH binding

sites on granulosa cells . . . . . . . . . . . . . . . .71
Effect of CI628 on FSH induction of FSH and

nuclear estradiol receptors in granulosa cells

0f rats. 0 O O O O O O O O O O O I O O O O O O O I O O 073
Effect of CI628 and FSH on ovarian and uterine

weights and levels of progesterone and

testosterone in serum. . . . . . . . . . . . . . . . . .74
Effect of CI628 on FSH stimulation of cAMP in

granulosa cells 0 O O O O O O O O I O O O 0 O O O O O O O 76

vi

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

10

11

12

13

LIST OF FIGURES

Estradiol binding to partially purified nuclei of
granulosa cells and uteri . . . . . . . . . . . . .

Effect of dithiothreitol on binding of 3H-E

to nuclei of granulosa cells. . . . . . . .123.

Effect of sodium molybdate on binding of 3H-E17B
to nuclei of granulosa cells. . . . . . . . . . .
Estradiol binding to cytosol from granulosa

cells and uteri O O O O O O O O O O O O O O O I O 0

Optimum time of incubation for nuclear and cytosol
assays for estradiol receptor . . . . . . . . . . .

Sucrose density gradient sedimentation profile
for nuclear and cytosolic estrogen receptors from
granulosa cells and nuclear receptors from uteri. .

Effect of temperature and various enzymes on
nuclear and cytoplasmic receptors . . . . . . . . .

Translocation of receptor . . . . . . . . . . . . .

Effect of various dossg of cyanoketone on
specific binding of 1-oFSH to granulosa
cells of rats . . . . . . . . . . . . . . . . . . .

Effect of cyanoketone on FSH induction of
FSR and estradiol binding sites on granulosa
cells of rats . . . . . . . . . . . . . . . . . . .

Effect of estrogfg, progestin, or androgen
replacement on I-oFSH binding in granulosa
cells of cyanoketone-treated rats . . . . . . . . .

Effect of estrogen, progestin, or androgen
replacement on H-E binding in granulosa
cells of cyanoketone-treated rats . . . . . . . . .

Effect of VIESOUS doses of CI628 on specific
1

binding of ~0FSH to granulosa cells and
H-E17B to nuclei of granulosa cells. . . . . . . .

vii.

.23

.24

.25

.27

.28

.46

.48

.67

Figure
Figure
Figure

Figure

14

15

16

17

viii

Effect of C1628 on FSH induction of FSH and

estradiol binding sites on granulosa cells.

Effect of C1628 on hFSH induction of FSH and

nuclear estradiol receptors . .

Model for interaction of estradiol and FSH

during early follicular development . .

Model for interaction of estradiol and FSH

during follicular development .

.72

.83

.85

ACS. . . . .
ADH. . . . .
BSA. . . . .
CAMP . . . .
CI628. . . .

cm. 0 O O O
cyanoketone.

DES. . . . .
DHT. . . . .

hazzz:
h. . . . . .

hC G. O O O 0
LE 0 O O O 0

PBS. . . . .
R5020. . . .
s.c. . . . .
T. . . . . .
TED. . . . .
ul . . . . .

LIST OF ABBREVIATIONS

.aqueous counting scintillant

.alcohol dehydrogenase

.bovine serum albumin

.cyclic 3', 5', adenosine monophosphate
.lr4-pyrrolidino-ethoxy-phenyl-4-methoxy-
d—nitrostilbene

.counts per minute
.Z-Q-cyano-4,4,17¢rtri-methylandrost-S-en-l7B-ol-3-one
.diethylstilbestrol
.dihydrotestosterone

.estradiol

.follicle stimulating hormone

.hour(s)

.human chorionic gonadotropin
.1uteinizing hormone

.milligram(s)

.minute(s)

.milliliter(s)

.millimolar

.sodium thiocyanate

.progesterone

.phosphate buffered saline
.promegestrone

.subcutaneously

.testosterone

.Tris-EDTA

.microliter(s)

ix

INTRODUCTION

Gonadotropins from the pituitary gland interact with ovarian
steroids to stimulate growth of follicles, ovulation, and formation of
corpora lutea.

Follicle stimulating 'hormone (FSH) and luteinizing hormone (LH)
interact with their receptors in follicular tissue to increase numbers
of receptors for FSH and LH and promote steroidogenesis. FSH
stimulates production of progesterone and estradiol in granulosa cells
while LH stimulates androgen production in thecal cells. The
androgens, androstenedione and testosterone, are aromatized to
estradiol by the aromatase enzymes in granulosa cells which are under
the control of FSH.

Although androgens appear to be associated with atresia of
follicles, estradiol promotes the formation of preantral follicles and
renders the ovaries more responsive to gonadotropins, thus further
promoting follicular development.

Estradiol has no effect on content of FSH receptors in granulosa
cells; however, FSH can increase the number of receptors for L8 and
FSH. Moreover, estradiol-priming prior to administration of FSH
increases levels of receptors for FSH and LH more expediently than with

FSH alone. However, androgens prevent hormonal induction of receptors

for 111 in antral follicles. This suggests that differentiation of
granulosa cells is a hormonally-regulated event involving synergism of
estradiol and gonadotropins.

Since FSH increases endogenous levels of estradiol and estradiol
can enhance gonadotropin action, a synergistic action of estradiol and
FSH may be required for follicular maturation.

The first objective of my research was to determine if granulosa
cells have receptors for estradiol. The second objective was to
determine if estradiol is required for FSH action. The third objective
was to attempt to determine if the interaction of estradiol and FSH
occurs on the cAMP-adenylate cyclase response system of granulosa
cells. In the following review of the literature, a brief history is
given of the research on ovarian function and an overview of the roles

that estradiol, LH and FSH have on follicular development.

LITERATURE REVIEW

Although the ovarian follicle was identified by de Graaf in 1672
and ovum described by von Baer in 1827, it was not until the early
1900's that the reproductive organs were identified as having an
endocrine function (Asdell, 1969). At first, phases of the female
cycle were described and ovarian follicles and corpora lutea identified
(Heape, 1990; Marshall, 1903; Ancel and Bouin, 1909). Next, crude
extracts of gonads were injected into animals, but results were
discouraging as uterine weight increased only occassionally. However,
in 1917, Stockard and Papunicolaou published their results from guinea
pigs showing that changes in vaginal epithelium corresponded with
changes in days of the estrous cycle.

Through the use of vaginal smears, Allen and Daisy (1923) isolated
and identified a follicular hormone, estrogen. Isolation of estrogens
was quickly followed by that of progesterone from corpora lutea of pigs
by Corner and Allen (1929). About this time, testosterone, considered
the male hormone, was isolated by Gallagher and Koch (1929).

The interaction of substances from the gonads with those from the
pituitary in the control of follicular growth was suggested as early as
1905 (Bellerby, 1929). Injection of extracts from the anterior
pituitary induced follicular growth and ovulation. Pituitary extracts
were semi-purified and found to contain two hormones, follicle

stimulating hormone and luteinizing hormone (Fevold g _a_l_., 1931;

 

4

Evans £3 313, 1936; Fevold, 1939; 1943). Due to the purity of these
hormones, however, some results from early experiments are
questionable. For example, using intact female rats, injections of FSH
markedly increased ovarian weight (Fevold 33 31., 1933), however, more
recent studies demonstrate that FSH alone has little effect on ovarian
weight (Dorrington and Armstrong, 1979). LR in large quantities
produces pseudolutein bodies in follicles by hypophysectomized rats and
causes ovulation (Creep 33 31., 1942; Fraenkel-Conrat £5 31., 1943).
Generally, LH augments FSH stimulation of ovarian weight in intact and
hypophysectomized rats, and this is associated with an increase in
estrogen production and follicular growth (Simpson 35 21,, 1941; Bates
and Schooley, 1942; Creep e£_313, 1942).

FSH plays an important role in follicular development. Although
administration of estrogen alone promotes growth of preantral
follicles, FSH is required for preantral follicles to form antral
follicles (Lane and Creep, 1935; Simpson 25.31., 1941). Further, FSH
administration after estrogen pretreatment causes more expedient
differentiation of preantral follicles into mature follicles
(Goldenberg 35. 21,, 1972a,b; Richards 55_ 313, 1976; Richards and
Kersey, 1977).

Most of the actions of ovarian and pituitary hormones on follicular
growth were elucidated after development of the technique of
hypophysectomy, parapharangeal surgical removal of the pituitary gland,
by P.E. Smith (1930). Hypophysectomy results in immediate atrophy of
the gonads. This was reversed with either pituitary' implants or

injections of extracts from the pituitary (Smith, 1930).

5

The hypophysectomized rat continues to be a good model for testing
the effects of steroids and gonadotropins, either alone or in
combinations, on ovarian function. This model has been used to study
the effects of hormones on numbers of receptors for gonadotropins and
steroids, changes in levels of cAMP and protein kinases, and mechanisms
of hormone action in the ovary.

After hypophysectomy, administration of estradiol increases mitotic
activity (Bullough, 1942) and prevents atrophy of ovaries (Williams,
1940; 1944; 1945; Pencharz, 1940). Estradiol also increases ovarian
and uterine weights and renders ovaries more responsive to injections
of gonadotrOpins (Williams, 1940; 1944; 1945; Pencharz, 1940).
Although estradiol increases mitotic activity and ovarian weight,
estradiol alone promotes development of only preantral follicles, with
little effect on antrum formation (Lane and Greep, 1935; Simpson it.
‘21., 1941; Richards 35 31., 1978).

Administration of estradiol or progesterone during follicular
development causes atresia in monkeys (Clark 35 11., 1981). Also,
estradiol implanted into one ovary of rats can decrease the number of
ovulations from that ovary without any effect on the other ovary
(Dierschke 32 31., 1983). This suggests a direct inhibitory effect of
estradiol on ovulation. The mechanism by which estradiol causes these
inhibitions is unknown.

Estradiol can have inhibitory effects on steroidogenesis. 1n
culture, estradiol inhibited progesterone secretion by granulosa cells
from small (Thanki and Channing, 1976) and medium (Schomber £5 11:,

1976) but not large preovulatory follicles (Goldenberg 23.313, 1972a;

Schomberg 3£H31., 1976; Haney 25.2l3' 1978). Fortune and Hansel (1979)
demonstrated that estradiol inhibits progesterone secretion by bovine
granulosa cells, however, if LR is added to the culture, estradiol has
no effect.

In intact animals, estradiol has no effect on progesterone
production (Leung 35_ 213, 1978; Leung and Armstrong, 1979), but
inhibits androgen production (Leung 35 21., 1978; Leung and Armstrong,
1979; Leung e£_al,, 1979). This inhibition by estradiol on androgen
synthesis appears to be a direct effect on the ovary (Leung _e£ 11.,
1979). Estradiol implanted in one ovary can inhibit androgen
production without any effect on the contralateral ovary (Leung 5£H§1.,
1979).

In hypophysectomized animals, estradiol prevents LH-stimulated
progesterone production (Leung gtflal., 1978; Leung 35.31., 1979; Leung
and Armstrong, 1979) however this inhibitory effect by estradiol can be
prevented by administering FSH and LE suggesting an interaction of
estradiol with gonadotropins (Leung st‘al., 1979).

Administration of testosterone to rats causes atresia of follicles
(Payne and. Runser, 1958; Schreiber and. Ross, 1976; Farookhi, 1980)
unless FSH is administered (Ireland and Richards, 1978). Then, growth
of follicles occurs. Dihydrotestosterone (DHT), a nonmetabolizable
androgen, prevents the induction of receptors for LH in antral
follicles after FSH administration (Farookhi, 1980). Androgens do,
however, stimulate progesterone synthesis in granulosa cells, but not
as well as FSH (Hillier st 31., 1977; Lucky SENEl" 1977).

Although progesterone can have an inhibitory effect on follicular

7

development in primates (Goodman Si _a_l_., 1977; Goodman and Hodgens,
1977), hamsters (Greenwald, 1977; Moore and Greenwald, 1974) and rats
(Kalra and Kalra, 1974; Schreiber E£“21., 1980; Schreiber st 31., 1981;
Schreiber 35 31., 1982), Richards and Bogovich (1982) have shown that
elevated progesterone in rats can enhance responses of small antral
follicles to subtle changes in LH. The increased progesterone levels
override the need for a sustained rise in LH and allow small antral
follicles to grow and develop when LH is low as during most of an
estrous cycle and early pregnancy. Thus, the exact role of
progesterone in follicular development is controversial.

Rats injected with progesterone have decreased levels of
gonadotropins (Beattie and Corbin, 1975; Goodman, 1978; Richards SE.
31., 1980; Taya £3 21., 1981), inhibition of FSH-induced increases in
LH receptors (Schreiber egual., 1982) and decreased levels of androgens
and estradiol (Kalra and Kalra, 1974; Saidapur and Greenwald, 1979;
Richards 2?. 31., 1980; Schreiber e_t_ §_1_., 1980; Taya 31; 11:, 1981;
Richards and Bogovich, 1982). Progesterone depresses FSH induction of
the aromatase enzyme system in granulosa cells thus preventing
conversion of androgens to estradiol (Schreiber st 31., 1981; Fortune
and Vincent, 1983). Besides regulating estradiol synthesis,
progesterone inhibits estradiol action by preventing retention of the
receptor-hormone complex in the nucleus which is necessary for hormone
action (Evans it. 31., 1980; Okulicz e_t_ fl” 1981; Leavett 35 11.,
1982).

Estradiol, progesterone, and testosterone are in follicular fluid

of follicles. Cystic and atretic follicles have a higher proportion of

8

androgens and progesterone to estradiol whereas healthy follicles have
a larger concentration of estradiol (Short and London, 1961; Short,
1962; Ireland and Roche, 1983).

Estradiol, testosterone and progesterone in conjunction with the
gonadotropins stimulate follicular growth. Testosterone plus FSH
stimulates follicular development. FSH apparently increases aromatase
activity' which converts testosterone to estradiol. However,
testosterone alone inhibits follicular maturation by causing atresia
(Payne and Runser, 1958). Progesterone plus hCG administered to intact
rats promotes follicular growth, however an interaction of hCG and/or
progesterone with FSH and estradiol present in the ovary can not be
ruled out. Progesterone alone stimulates development of small antral
follicles in rats, however, this hormone has a negative effect on
induction by FSH of the aromatase system and estradiol synthesis. As
levels of estradiol are high in non-atretic, healthy follicles,
progesterone ultimately' would decrease follicular' growth. Although
estradiol inhibits progesterone production in cultures of granulosa
cells and hypophysectomized rats, it has little effect in _vizg.
Estradiol does decrease levels of androgens, however androgens in large
amounts cause atresia. Therefore, estradiol either alone or in
conjunction with gonadotropins generally promotes follicular growth and
maturation.

Because estradiol is a steroid, it is capable of moving freely
through cell membranes. Thus, estradiol is found in non-target and
target tissues. However, in target tissues such as ovaries and uteri,

estradiol is retained and is found in higher concentrations than in

9

non-target tissues (Jensen and Jacobson, 1962; Stone e_d 21., 1963;
Stone and Baggett, 1965; Jensen ggnal., 1966; Terenius, 1966; Jensen SE
313, 1967). Binding of tritiated estradiol to subcellular fractions of
the rat uterus was competitive only in the soluble and
nuclear-myofibrillar fractions (Noteboom and Gorski, 1965) and the
tritiated steroid was released when fractions were treated with
proteases. This suggested that the hormone was bound to proteins in
the cytosol and nucleus (Toft and Gorski, 1966; Jensen 35.31,, 1971).

It is now known from studies with uteri that estradiol binds to its
receptor in the cytoplasm. This receptor then undergoes a
transformation, and the receptor is rapidly translocated into the
nucleus (Shayamala and Gorski, 1969; Jensen E£.2l;i.1969§1 Jensen gt
al.,1969b; Jensen st 31., 1971; Jensen e_t_ g” 1972; Williams and
Gorski, 1972; thides at 353* 1975; Notides and Nielsen, 1975). The
nuclear receptor binds to specific acceptor sites on chromosomes and
there is an increase in mRNA synthesis (Aizawa and Mueller, 1961;
Gorski and Nicolette, 1963; Hamilton st’al., 1965; Means and Hamilton,
1966; Billing e_t_ 31., 1969; Luck and Hamilton, 1972; Mohla _e_t_ 31.,
1972; Yamamoto and Alberts, 1975; Ron and Spelsberg, 1982). The amount
of growth of tissue has been associated with the level of estradiol
receptors in the nucleus (Jensen 55.31., 1968; Anderson EE.2£" 1975).
After nuclear action, receptors from the nucleus can be recycled back
into the cytoplasm (Kassis and Cbrski, 1981).

Although early studies have established a single binding site for
estradiol in the cytosol and nucleus of the uterus, recently, studies

have demonstrated two binding sites for estradiol in the uterus (Clark

10
e_t_ a_1_., 1978). The type I site in the cytosol has a dissociation
constant (Rd) similar to the classical uterine estradiol receptor (lnM)
with a limited binding capacity (RovlpM). This receptor is
translocated to the nucleus. The type II site has a lower Kd, 4-fold
more binding sites and is not translocated from the cytoplasm into the
nucleus. The function of the type II binding sites is unknown.

Two binding sites for estradiol have been identified in the nucleus
of uteri (Markaverich and Clark, 1979). As in the cytosol, the type 1
site is similar to the classical uterine receptor (Kd- lnM, Ro-
lpM/uterus). The type 11 site persists after levels of type 1 sites
are lower and is thought to be associated with long term uterine growth
(Clark and Markaverich, 1981; Markaverich SE 31., 1981; Clark 23 31.,
1982).

Two distinct high affinity, low capacity binding proteins have been
demonstrated in the cytoplasm and nucleus of chick oviduct and human
uteri (Smith 35 21., 1979) and the cytosol of rat uteri (Erickson 25
31.,1978). The importance of these two receptors biologically remains
to be elucidated.

Receptors for estrogen have been identified and characterized in.
uteri and ovarian homogenates (Saiddudin and Zassenhaus, 1977).
However, only binding of estrogen has been demonstrated in granulosa
cells which both synthesize and respond to estrogens (Fortune land
Armstrong, 1978). Whether a single, specific, high affinity receptor
for estradiol is present in granulosa cells has not been determined.

Follicular development is under the control of estradiol and the

gonadotrOpins. Estradiol increases responsiveness of follicles to

11 '

gonadotropins (Pencharz, 1940; Williams, 1945), increases proliferation
of granulosa cells (Rao g 31., 1978; Coldenberg _e_t_ 11;, 1972a) and
induces follicular growth of preantral follicles. FSH alone promotes
estrogen production and formation of antra in ovarian follicles (Moon
25.2if’ 1975; Zeleznik 35 31., 1974). However, estrogen priming prior
to FSH increases ovarian and uterine weights and promotes follicular
growth above that with FSH alone (Lane and Creep, 1935; Simpson 52.31.,
1941; Richards 35,21., 1976). As FSH increases estradiol production
and estradiol, in turn, can further enhance FSH action, there appears
to be an interaction between estradiol and FSH. during follicular
development.

Estradiol and FSH have a synergistic effect on induction of
receptors for estradiol and gonadotropins in granulosa cells.
Administration of estradiol alone increases numbers of receptors for
estradiol and gonadotropins per ovary but has no effect on numbers of
receptors for FSH or LH per granulosa cell (Louvet and Vaitukaitis,
1976; Richards and Midgley, 1976; Richards, 1978; Richards and Kersey,
1979). Administration of FSH alone increases receptors for estradiol
and FSH but has little effect on numbers of receptors for ‘LH in
granulosa cells (Richards 35.21., 1976). However, pretreatment with
estradiol prior to FSH causes a significant increase in numbers of
receptors for estradiol, FSH and I}! per granulosa cell compared to
animals receiving FSH alone (Richards and Midgley, 1976; Richards st
31., 1976; Richards and Kersey, 1979). The interaction of estradiol
and FSH on induction of receptors for FSH was shown to be time

dependent by Ireland and Richards (1978). Estradiol priming 12 or 24 h

12
prior to FSH significantly increased numbers of receptors for' FSH
compared to levels after FSH alone.

Estradiol also enhances FSH stimulation of cAMP accumulation and
binding sites in granulosa cells (Richards, 1978; Richards it. £.,
1976; 1979). Although estradiol alone had no effect on cAMP
accumulation or number of binding sites for cAMP, it enhances the
ability of FSH to increase production of cAMP 2-fold and increases
numbers of cAMP binding proteins 10-20 fold compared to FSH alone.
Further, the increase in cAMP accumulation occurs without a change in
number of FSH receptors per granulosa cell (Richards, 1978; Richards 35
31., 1979). Thus, estradiol appears to enhance FSH induction of cAMP
production independent of increases in numbers of FSH receptors.

Since estradiol priming prior to FSH increases ovarian and uterine
weights, follicular growth, numbers of estrogen and gonadotropin
receptors per granulosa cell and cAMP accumulation over an injection of
FSH alone, and FSH increases levels of estradiol, estrogen may have a
role in FSH action. Estradiol synthesis or action through its receptor
may be required for FSH to increase its own receptor or increase cAMP
accumulation in granulosa cells. Because growth of preovulatory
follicles requires the presence of estradiol plus FSH, control lof
synthesis of estradiol may control development of antral follicles.

Although the theca cells were originally considered the site of
synthesis of estradiol (Allen and Doisy, 1923; Corner, 1938), only
after the unique studies of Falck (1959) using autotransplants of cells
from the ovary of rats to the eye chamber was there evidence that

estrogen biosynthesis requires the interplay of at least two cell

13 '

types, theca and granulosa. The theca cells are the major source of
androgens which are required for synthesis of estrogen (Erickson and
Ryan, 1976; Fortune and Armstrong, 1977; Moor, 1977; Tsang 55 g”
1979). The androgens are released from the theca cells and diffuse
into granulosa cells. The conversion of androgens to estrogens occurs
in granulosa cells and is under the control of the aromatase enzyme
system. FSH can increase estradiol accumulation by granulosa cells by
increasing aromatase activity (Moon E£.§Qr: 1975; Dorrington £5,313,
1975; Erickson and Hsueh, 1978; Daniel and Armstrong, 1980; Adashi and
Hsueh, 1982). Furthermore, estradiol augments FSH stimulation, of
aromatase activity (Watson and Howson, 1977; Adashi and Hsueh, 1982;
Veldhuis g£_al:, 1982).

Follicular growth of preovulatory follicles depends on the
interaction of estradiol and FSH. The FSH-cAMP response system in
small antral follicles can maintain some aromatase activity, however,
with increased estradiol production, this activity is increased.
Acting via its receptor, estradiol further modifies granulosa cell
function. It enhances the ability of FSH to stimulate cAMP production
and increases cAMP binding sites and responsiveness of granulosa cells
to gonadotropins. Thus, it appears that once estradiol production
begins, it assumes an important role in follicular development.

In this dissertation, I attempted to establish if a single class of
high affinity receptors for estradiol are present in granulosa cells
and if synthesis of estrogens or action. of estrogens through the

receptor is required for follicular deve10pment.

CHAPTER I

IDENTIFICATION OF

ESTROGEN RECEPTORS IN GRANULOSA CELLS OF INMATURE RATS

Introduction

 

Estradol plays a major ’role in ovarian follicular growth and
development. Estradiol directly increases ovarian weight and causes a
proliferation of granulosa cells (Bradbury, 1961). Estrogen also
synergizes with gonadotropins to cause maturation of follicles in
preparation for ovulation (Richards 55 31., 1976; Ireland and Richards,
1978).

In developing follicles, granulosa cells both synthesize and
respond to estrogens (Fortune and Armstrong, 1978). Estrogen receptors
are found in the corpus luteum of various species (Yuh and Reyes, 1979;
Richards, 1974) and in whole ovarian homogenates (Saiddudin and
Zassenhaus, 1977). Although binding of estrogen has been demonstrated
in granulosa cells (Richards, 1975), the specific receptor for estrogen
in granulosa cells has not been fully characterized.

In this study, 1 characterized estrogen receptors in granulosa

cells of rats. The properties of these receptors were compared to

14.

15

those of uterine estrogen receptors.

Materials and Methods

 

Materials

The following reagents were used: (2,4,6,7,l6,17-3H)-estradiol-17-B
(137.1 Ci/mmol: 3H-EUB); New England Nuclear; sodium thiocyanate
(NaSCN, analytical grade), Trizma-HCl (reagent grade), diethylstil-
bestrol (DES), estradiol-17B (E173), testosterone, progesterone,
androstenedione, corticosterone, cortisone, ZOB-hydroxy- pregnenolone,
l7 -hydroxy-progesterone, estrone, sucrose (Grade 1), DNA from calf
thymus, DNase from bovine pancreas, RNase from bovine pancreas,
protease, and charcoal (Norit A); Sigma Chemical Co.; Dextran T-70:
Pharmacia; aqueous counting scintillant (AC8): Amersham; propylene
glycol: J.T. Baker Chemical Co..

Animals

Immature (21-day-old) female Sprague Dawley rats were obtained from
Spartan Research Animals (Haslett, Michigan), exposed to 12L:12D
(24°C), and given food and water ad libitum. Rats were decapitated

between 22 and 28 days of age.

Tissue preparation

 

Ovaries and uteri were dissected, trimmed of fat, and placed in TED
buffer (10 mM Tris, 1.5 mM EDTA, pH 7.4) on ice.

For each Scatchard analysis, granulosa cells were obtained from
ovaries of 100 rats according to the method of Zeleznik 32.213, (1974);
gentle pressure was applied to follicles and expressed cells were

collected after centrifugation at 800 x g (20 min, 4°C). Cells were

16,,

l7'

washed three times in TED ( 2 ml/wash), homogenized in a glass Dounce
homogenizer ( 2 ml TED), and centrifuged at 4000 x g for 20 min at 4°C.
Pellets were washed 3 times in 2 ml TED buffer, then resuspended in TED
(granulosa cells from 2 ovaries/tube, 0.1 ml volume) for the estradiol
nuclear receptor assay. The supernatant was further centrifuged at
106,000 x g (Type 30 rotor, Beckman) for 1 h (4°C) to obtain a cytosol
preparation for the cytosol receptor assay.

Uterine tissue from 50 rats was homogenized in ice-cold TED buffer
(2 uteri/5 ml) using a Servall omni-mixer 8-1515. The homogenate was
centrifuged at 4000 x g for 20 min at 4°C. Pellets were washed 3 more
times with buffer then resuspended in TED for the nuclear receptor
assay. The supernatant was further centrifuged at 106,000 x g for 1 h.
After centrifugation, the supernatant was removed using a Pasteur
pipette, taking care to exclude the fat layer floating on top, and used
in the estradiol cytosol receptor assay.

Exchange assays for Scatchard analysis

 

Two assays for estradiol nuclear receptors were used. The method
of Anderson 3£_al. (1972) involved incubation of nuclei from granulosa

cells at 37°C for 1 h in TED buffer containing various concentrations

of 3H-E17B (1 to 20 nM). Total reaction volume was 0.5 ml. After

incubation, nuclear samples were washed 3 times with TED then extracted
(2 times) with 1 m1 of methanol. Extracts were placed in scintillation
vials, dried, and reconstituted in 10 ml of ACS. The method of Sica g£_
31. (1981) involved incubation of nuclear preparations from granulosa

cells in TED buffer with various concentrations of 3H-E (1 to 20 nM)

173
at 4°C. Total volume equaled 0.25 ml. After 1 h, 0.05 ml of n30

18'

buffer containing 3 14 NaSCN which solubilizes the nuclear estradiol
binding site was added to each tube for a final volume of 0.3 ml (final
NaSCN concentration - 0.5 M). After overnight incubation at 4°C, 0.3
ml of Dextran-coated charcoal (DCC; 12 charcoal, 0.052 Dextran in TED)
was added to separate free from bound estradiol in the nuclear extract.
Fifteen min after adding DCC, samples were centrifuged at 8,000 x g for
10 min to precipitate the charcoal and nuclei. The supernatant was
then removed and placed in scintillation vials containing 10 ml of ACS.
Radioactivity was quantified in an Isocap/300 6872 Liquid Scintillation
System (Searle Analytic, Inc.). Specific binding was calculated as the
difference in counts bound in the presence (non-specific) or absence
(total) of a SOC-fold excess of DES.

The two methods utilized for the estradiol cytosol receptor assay
were similar. The method of Richards (1974) utilized TED buffer with a
24 h incubation, while the method of Sica g 11;. (1981) required
addition of 0.5 M NaSCN to the TED buffer and incubation for 3 h.
Aliquots of cytosol (0.2 ml) were added to tubes containing 1 to 20 nM
3H-EUB. To determine non-specific binding, 500-fold excess DES was
added to a parallel set of tubes. After incubation for 3 h (Sica _e_E
'21., 1981) or 24 h (Richards, 1974) at 4°C, 1 ml of DCC was added to
each tube. The suspension was incubated for 15 min (4°C) and was
centrifuged at 8,000 x g for 10 min. The supernatant was added to
scintillation vials along with 10 ml of ACS and counted.

Because Scatchard analysis of estradiol binding to uterine nuclei
or granulosa and uterine cytosol using the method of Anderson st 31.

(1972) or Sica ES 31. (1981) gave similar results, the method of Sica

19'
E£.Elf (1981) was used for all characterizations and assays.
Exchange assays for saturation analysis

3

Nuclear or cytosol preparations were added to 19 nM H-E with or

17B
without SOC-fold DES plus 0.5 M NaSCN-Tris. After incubation for 16-24
h (nuclear assay) (4°C) or 3 h (cytosol assay), DCC was'added (0.3
ml-nuclear assay; 1. ml-cytosol assay). Tubes were further incubated
for 15 min then centrifuged for 10 min at 3000 x g (4°C). Supernatant
was removed, placed in 'vials containing 10 ml ACS, then counted.
Amount of tissue in samples was estimated by measuring DNA (for nuclear
receptor assay; Burton [1956]) or protein content (for cytosol receptor
assay; Lowry S£.Elf[1951])'

Sucrose density gradient

Aliquots (0.2ml) of either cytosol or nuclear preparations (4 rats
total) were layered onto 4.8 ml of 5-202 linear sucrose density
gradient (cellulose nitrate tubes) made in TED-0.5 M NaSCN buffer
(4°C). Gradients were centrifuged in a Beckman L8-70 ultracentrifuge
using a sw 50.1 rotor at 175,000 x g for 24 h (4°C). Alcohol
dehydrogenase (ADH) from yeast and bovine serum albumin (BSA) were used
as standards to estimate sedimentation constants (S). Individual
fractions were obtained by puncturing tubes through the bottom and
collecting fractions of approximately 0.3 ml in a series of tubes.
Excess DES was used to demonstrate specificity of binding. Fractions
were assayed for either nuclear or cytosol receptor as stated above

(Sica £5.2l!’ 1981).

20‘

Steroid specificity

 

Cytosol or nuclear preparations. (2 ovaries/tube) were added to

tubes containing 19 nM 3H-E Excess (lOOO-fold) of DES,

l7B° E17B’

ltestosterone, progesterone, androstenedione, corticosterone, cortisone,
ZOE-hydroxy-pregnenolone, 17 -hydroxy-progesterone, or estrone were
added to determine binding specificity. Assays were completed as
stated above.

Effects of treatment gf_receptors with various enzymes

 

Nuclei were prepared and preincubated with 3H-E17B with or without
DES for 1 h at 4°C. Then 0.6 mg/ml RNase, 1.2 mg/ml protease, or 0.75

mg/ml DNase (Saiddudin and Zassenhaus, 1977) was added to the

incubation mixture. Control tubes contained only 3H-E with or

173
without DES. Nuclei were incubated for an additional 30 min at 37°C

and number of nuclear estradiol binding sites were determined by the
method of Sica sgual. (1981).

For cytosol, each tube contained cytosol and 3 ng 3H-E with or

17B
without DES (300 ng). After pre-incubation for 1 h on ice, one of the
following enzymes was added: 0.9 mg/ml RNase, 1.8 mg/ml protease, or

0.37 mg/ml DNase (Saiddudin and Zassenhaus, 1977). Controls had only
3H-E17B with or without DES. Tubes were then incubated for an
additional 30 min at 25°C and processed by the method of Sica eg‘al.
(1981).

Translocation 2£_receptor

 

Rats were injected subcutaneously with 4 mg of estradiol in
propylene glycol (4 mg/rat). At 0, 15, 30, 45, 60, and 120 min, rats

(n-12 or 12/interval) were ‘killed and cytosol. and. nuclear’ estrogen

21
receptors in granulosa cells were measured. Nuclear and cytosol

preparations were added to tubes containing 19 nM 3H-E Assays were

173'

performed as stated above.

Statistical analysis

 

Scatchard plots (Scatchard, 1949) were analyzed 'by linear
regression. Curvilinear Scatchard plots were analyzed by the computer
program ISIS-59 developed by Thakur ‘25 '31. (1980) for NICHD,
Biophysical Endocrinology Section. Each Scatchard was repeated 3

times. Other experiments were repeated 2 times.

Results

Characterization of binding assays

 

In the nuclear assay, the method of Anderson ggual. (1972) resulted
in a curvilinear Scatchard (see insert, Figure 1). The dissociation
constant (Rd) of the higher affinity binding component was 3.4 x lO-IOM
with a binding capacity (R0) of 133 pM. The lower affinity site had a
Kd of 4.9 x 10-9M and a R0 of 872 pM. Two component Scatchard plots
were not found for uterine nuclei or for granulosa cell or uterine
cytosol using this method (data not shown). Use of dithiothreitol as a
reducing agent which prevents binding to type 11 sites (0.1 mM,
[Markaverich e£_ El)! 1981]; Figure 2) or sodium molybdate which
minimizes denaturation and loss of receptor (100 mM, [Krozowski and
Murphy, 1981]; Figure 3) in TED buffer during nuclear exchange assays
gave similar curvilinear results (e.g., the Rd 01‘- the high affinity

lOM

component was 1.5 x 10- ). However, the lower affinity binding site

‘8 to 10‘7M).

was in the range of a binding protein in serum (Kd - 10

As shown in Figure 1A, a single class of high affinity (Rd 8 1.9 x
IO-IOM) binding sites for estradiol with a R0 of 80 pM (1.4 fmol/ug
DNA) was found in NaSCN extracts of granulosa cell nuclei. After
extraction of nuclei with NaSCN, no specific binding of estradiol was
observed in the remaining nuclear pellet (Table 1). When estrogen
receptors from rat uteri were prepared and treated in a fashion
identical to that already described, estradiol binding sites with a Kd
similar to that of receptors of granulosa cell nuclei (Rd - 2.5 x

-1

10 0M) was observed. .A binding capacity of 63 pM (5.0 fmol/ug DNA)

22,

Figure 1.

23'

  
  
     

 

 

 

 

 

K023.4xld'°M
L /F 0.02 -9
0.4 . Kd=4.9xlo M
\ 0.01 l L Ro*872 DM
8 0.3 - \0\ 200 600 I000
/F \ pM BOUND
02 — \.
Kd=l.9xlo"°M Noe.
0.: e R0: 80 pM \.
l I L l l 1 L\A
IO 20 30 4O 50 60 70 BO
pM BOUND
C14»h E3
8/ 0-3 ' Kd =2.5 x I0"°M
F R03 63 pM
0.2 \o
“-
’\.
O.l -
l l l l l . 44, J
IO 20 30 40 . 50 so 70 so
pM BOUND

ESTRADIOL BINDING TO PARTIALLY PURIFIED NUCLEI OF GRANULOSA

CELLS AND UTERI. Ovaries (A) and uteri (B) from 100 rats
were removed and granulosa cells expressed. Nuclear
exchange assays were conducted according to the method of
Anderson _e_t_:__a_l. (1972)(A insert) or Sica if}; (1981).

24'

0.11-

Ko:2.46x1O-9M
Roszsz 9M
.1

01M?-

0.07 -'

B”:

0.05 -

 

CITI-

A“
gKa:1.ssx10'7M
I I l I R0=2168 old

I I [—

200 600 1000 1400 1800 2200 2600

DM BOUND

 

 

Figure 2. EFFECT OF DITHIOTHREITOL ON BINDING OF 3H-E17B TO GRANULOSA
CELL NUCLEI. Ovaries from 100 rats were removed and gran-
ulosa cells expressed into a buffer containing 0.1 M
dithiothreitol, 10 mM Tris-HCl, and 1.5 mM EDTA, pH 7.4.

8”:

Figure 3.

25'

    

 

 

0.08 d
0.06 -
d
(104 .
..
V6 -
K0: 1.48x10 ‘OM
0.02 .. R0: 270 pM
- O
Ka:1.4sx 10”»: ‘
80:2725 9M
1 I I l I l T
400 800 1 200 1 600 2000 2400 2800
pM BOUND
EFFECT OF SODIUM MOLYBDATE ON BINDING OF 3H-EUB T0 NUCLEI

OF GRANULOSA CELLS. Ovaries from 100 rats were removed and
granulosa cells were expressed into a buffer containing 100

mM sodium molybdate, 10 mM Tris-HCl, and 1.5 mM EDTA, pH
7.4.

26'
was demonstrated for uterine nuclei (Figure 13) using the method of

Sica e£_al, (1981).

TABLE 1

ESTRADIOL BINDING TO RESIDUAL TISSUE FROM GRANULOSA.
CELLS AND UTERI

 

cpm in
Tissue nuclear extract 2 of total binding
granulosa residual 475 2.40%
uterine residual 570 0.29%

 

Data are expressed as means.

As shown in Figure 4, Scatchard analysis of estradiol binding to
cytosol of granulosa cells and uteri using the method of Sica 35.21.
(1981) also resulted in a single class of binding sites with a K a 3.5

d
x 10-10M (Ro - 45 pM, 0.8 fmol/ug DNA) for granulosa cells and a K =

d
1.4 x 10-10M for uterine tissue (R0 = 20 pM, 1.6 fmol/ug DNA).

At 37°C, a steady state was achieved with nuclear exchange assays
by 45 min, and binding was stable until 90 min (Figure 5). For the
NaSCN extraction at 0-4OC, steady state was achieved at 16 h. Similar
studies were performed for cytosol assays. Utilizing the method of
Richards (1974), steady state occurred after 16 h. With the method of
Sica ££_213 (1981), steady state occurred after 2.5 h.

Sucrose density gradient analysis

Estradiol was specifically bound by a macromolecule which

sedimented in approximately the 58 region in NaSCN-treated nuclear

Figure 4.

27

 

 

 

 

I— A
I4 .
.2 N
B/ IO - \ §d=355xIOmM
F 8 — 0" pM
(xIO-Z) 6 - \.
4 — \'
2 _ \
I I I I
9 IS 27 36 45
pM BOUND
B
I4
'2 -\\ ..
_ Kd=I.4xIO M
B/F ': __ \, Ro=20 pM
(“0.2) 6 -
4 _.
2 _.
I I l I
4 8 I2 I6 20
pM BOUND

ESTRADIOL BINDING TO CYTOSOL FROM GRANULOSA CELLS AND
UTERI. Ovaries and uteri were removed from 100 rats and
granulosa cells were expressed. Estradiol binding to
cytosol suSpensions of granulosa cells of ovary (A) or
uteri (B) were measured.

ll. llllllll ILQC: \
Ihccc AUAUO

 

28

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mwoMNMm—woepfiwdmwz My“: poumosocfi one: mHHoo mmofiscmcm sore mcoaumemeoco Homoamo new

umoaosc mango. .Ao Q<mhmm mom mw<mm< AOmOHyu az< mcmauzz mom onh<m=UzH mo mzHH zathmo

 

 

 

 

 

 

 

 

mmDOI wmnOI
V a N p NV 00 up 9
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no no
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000 COD
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32a

fit

the

So:

3r:

SIM

6X5

m

29 '

preparations of granulosa and uterine cells (Figure, 6). Estrogen
receptors prepared from cytosol sedimented as an 88 form with a much
smaller peak at approximately SS. Since the smaller peak coincided
with the nuclear peak, the SS in the cytosol could be due to a small
amount of nuclear receptor contamination rather than disaggregation of
the cytosol receptor which. appears at 48 (DeSombre .EE..2£R! 1969;
Notides and Nielson, 1975) as previously demonstrated (Puca and
Bresciani, 1970).

Steroid specificity

 

Specificity of binding of estradiol to estrogen receptors in the
nucleus and cytosol was examined (Table 2). Maximum competition
occurred with estradiol and DES. Estrone was able to compete for
receptor, but to a lesser extent than E173 or DES- All other hormones
examined had little effect on binding of estradiol to its nuclear or
cytosolic binding site.

Effects of temperature and enzyme treatments 23_binding of
estradiol-

Estrogen receptors in granulosa cells were shown to be proteins and
heat labile (Figure 7). After preincubation of receptor at 37°C,
specific binding of estrogen was very low ((10% in nucleus; <52 in

cytosol). As incubations of assays after enzyme treatments are at

37°C, receptors were pre-incubated with 3H-E17B to prevent degradation

due to temperature. Treatment with protease greatly diminished ((30%)
Iestrogen. binding in. both. nuclear and cytosol preparations, whereas

RNase and DNase had little effect.

 

30'

 

 

_ ADH BSA
Ho — R i l
u [I \\ o GRANULOSA NUCLEAR EXTRACT
90 A] \ E1 UTERINE NUCLEAR EXTRACT
I! \‘ A GRANULOSA CYTOSOL
N
O
3: 70
E
U 50
30
IO . .
l l l 1 l l l l l l l .0. 1
l23456789l0l|l2l3l4l5
bottom FRACTION top
Figure 6. sucaosz DENSITY GRADIENT SEDIMENTATION PROFILE FOR NUCLEAR

AND CYTOSOLIC ESTROGEN RECEPTORS FROM GRANULOSA CELLS AND
NUCLEAR RECEPTORS FROM UTERI. Crude nuclear and cytosol
preparations from granulosa cells were extracted with 10 mM
Tris-1.5mM EMA-0.5 M NaSCN. Uterine nuclear extractions
were used as a control. All data are expressed as specific
binding. Arrows indicate the fractions where -BSA (4.4
S) or H-ADH (7.4 S), markers for molecular weight, were
collected.

31'

TABLE 2

BINDING SPECIFICITY OF NUCLEAR AND CYTOPLASMIC RECEPTORS

 

Specifically Bound Specifically Bound
in Nucleus in Cytoplasm
Competing Steroid (2 control) (Z control)
Control 100 100
Cortisone 98 103
Corticosterone 100 101
Progesterone 97 99
ZOE-OH-pregnenolone 110 102
l7drOH-progesterone . 92 9S
Testosterone 110 101
Androstenedione 104 105
DES 9 12
Estradiol 10 8
Estrone 22 25

Nuclear exchange assays were as described in Appendix. Suspen-
ions of nuclei or cytosol from granulosa cells were added to 19.9 nM
3‘3173 4- 19.9 in! of cortisone, corticosterone, progesterone, 20-D-

hydroxpregnenolone, l7-drhydroxyprogesterone, testosterone, andro-
stenedione, DES, estradiol, or estrone. Data are expressed as per-
cent of specific binding.

SPECIFIC BINDING
96 OF CONTROL

Figure 7.

32’

—J'—
lOO——1_—1 '_I
80-
60- 33'
<1
E
40".1 .1 O
8 e E
I—gggo H33
20—Zoo<?\ Zoo 0
0223.0 022 re
0010 000: m
m
a

 

 

 

 

 

 

 

 

 

 

CYTOSOL

z
c
o
r.
'1!

EFFECT OF TEMPERATURE AND VARIOUS ENZYMES ON NUCLEAR AND
CYTOPLASMIC RECEPTO S. Nuclei and cytosol were prepared
and incubated w th 3‘5173. After extraction, nuclei were
incubated with “-5173 with or without DES identical to the
receptor assay except 0.6 mg/ml RNase, 1.2 mg/ml protease,
or 0.75 mg/ml DNAse was also added. Nuclei were then
incubated for 30 min at 37°C, t en processed. For cytosol,
tubes contained cytosol, 3 ng H'El7B : DES (300 ng) plus
one of the following enzymes: 0.9 mg/ml RNAse, 1.8 mg/ml
protease, or 0.37 mg/ml DNAse. For heat lability, nuclear
and cytosol preparations were incubated at 37°C for 1 h
prior to the exchange assays.

33
Translocation of receptor

 

As shown in Figure 8, cytosol receptors appeared to be translocated

into the nucleus by 15 min after a 4 mg injection of E173 fi_n vivo.

 

Nuclear receptor levels continued to increase until 45 min post-
injection, when they reached a plateau. At 120 min, numbers of nuclear
receptors were still high with cytosol receptors starting to rise
slightly. Basal levels of cytosol and nuclear receptors were not

restored by 2 h.

34'

    
   

 

 

:I
000
(“5' 5 r- H was:
a O--O CYTOSOL
rs 4000 — K.
7- CLC,
L” - “No-
i 3000 - ‘~
co ““0-‘
8 2000 r— ~‘so....//L--C)
.J
:3
o 1000 —
Lu
.1
O l l L I L 71,—]
2 0 15 30 45 60 120
TIME (min)
Figure 8. TRANSLOCATION OF THE ESTROGEN RECEPTOR. Rats were injected

subcutaneously with 4 mg of estradiol. At 0, 15, 39, 45,
60, and 120 min after injection, rats ‘were killed and
cytosol and nuclear estrogen re eptors measured. All data
were expressed as molecules of ( H)-estradiol/cell, using 6
pg DNA/cell.

Discussion

 

Reports of multiple binding sites for estradiol in the nuclear and
cytosol preparations of uteri are well documented (Erickson fl a_l.,
1978;. Clark e_t_ 31:, 1978, Smith £5 5%., 1979; Gibbons e_t '_a_l_:_., 1979).
Due to these studies, I utilized various methods to examine estrogen
receptors in granulosa cells. As demonstrated in Figure 1 (insert), a
curvilinear Scatchard plot for estradiol binding to granulosa but not
uterine nuclei was observed when I used the method of Anderson $5.2lf
(1972). This could have been the result of another class of binding
sites for estradiol in the granulosa cell nucleus. However, a curvi-
linear Scatchard plot was not observed when I examined estradiol
binding in NaSCN nuclear extracts of granulosa cells. In addition, no
binding of estradiol occurred to the nuclear pellet of granulosa cells
after NaSCN extraction. Thus, it is unlikely that a second class of
3173 binding sites exist in nuclei of granulosa cells. Also, de Boer
and NOtides (1981a, 1981b) suggest that high concentrations of steroids
which would be present in granulosa but not in uterine cells interact
with hydrophylic sites on the receptor, exerting a non-specific
detergent action on the receptor. Perhaps NaSCN minimizes this effect.
Binding of estradiol to the nuclear receptor may be altered depending
upon whether the nuclear receptor is bound to chromatin or is free
(Yamamoto and Alberts, 1975; de Boer and Notides, 1981b). NaSCN
extraction may eliminate this interference. Although the reason for
curvilinear Scatchards when the method of Anderson (1972) is used is

not known, alterations in binding of E1713 to its nuclear receptor in

35.

:4

OI.

ark

I11.

ho

re

1"

’o'v

36
granulosa cells which were reflected in Scatchard plots (Figure 1), may
be eliminated when nuclear exchange assays are done with solubilized
receptor preparations at cold temperatures.

Utilizing the NaSCN extraction, the estrogen receptor in granulosa
cells had a high affinity, low capacity for estradiol. The binding
affinities of receptors in the nucleus and cytosol were in the range of
those reported for rat uteri (Kc1 - 2 to 5 add-lord; [Saiddudin and
Zassenhaus, 1977; Anderson gal” 1972; Sica fi£n 1981; Pavlik and
Rutledge, 1980]), rat endometrium (Kcl a 3 to 8 x 10-10M; [Farookhi and
Sonnenschein, 1976]), and hypothalamus and pituitary (Kd - 2 x 10-10M;
[Kelner and Peck, 1981]). Saiddudin and Zassenhaus (1977) examined
nuclear and cytosol estrogen receptors in ovarian and uterine
homogenates from immature rats. The affinities of the cytosol estrogen
receptors in the ovary (Kd =- 4.13 x IO-IOM) and uterus (Kd - 1.22 x
lO-IOM) were very similar to the cytosol estrogen receptors of
granulosa cells. However, the binding affinity of estrogen nuclear
receptors from whole ovarian homogenates (Kd =- 1.26 x 10‘9M) was found
to be much higher when only granulosa cells were used (Kd = 1.9 x

10-10

M).

Estrogen receptors in granulosa cells have biochemical
characteristics similar to uterine estrogen receptors. Sucrose density
gradient analysis has been previously used to characterize estrogen
receptor complexes. The radioactive hormone in the cytosol sediments
as a discrete band with a coefficient of 88 (Puca stab, 1971; Notides

g 31:, 1972) and the estradiol-receptor complex in the nucleus

sediments at SS (DeSombre e_t 11:, 1969; Jensen g 31., 1968). The

37'

'results in this study were similar to those previously described for
estrogen receptors in uteri and other tissues (Saiddudin and
Zassenhaus, 1977; Notides and Nielsen 1975; DeSombre st 31., 1969; Puca
and Bresciani, 1970). Binding specificity of estradiol to its receptor
in granulosa cells is similar to that in ovarian homogenates (Saiddudin
and Zassenhaus, 1977), corpora lutea (Yuh and Keys, 1979; Richards,
1974), and uteri (Cidlowski and Muldon, 1978; Richards, 1980). Also,
estradiol receptor in granulosa cells was found to be proteinaceous and
heat labile as demonstrated for estrogen receptors in ovarian
homogenates (Saiddudin and Zassenhaus, 1977) and uterine preparations
(Krozowski and Murphy, 1981; Cidlowski and Muldon, 1978). Finally,
translocathmn of cytosol estrogen receptors into the nucleus after a
bolus injection of estradiol was demonstrated in granulosa cells
(Figure 8). Results were comparable to those found in uterine (Jensen
3£Hg£., 1968) and ovarian (Saiddudin and Zassenhaus, 1977) tissue.

In conclusion, receptor sites specific for estradiol are present in
nuclei and cytosol of granulosa cells from immature rats. Thus, many
of the effects of gonadotropins, which stimulate estrogen synthesis, or
of estradiol on ovarian follicular growth and development (Richards,
1980) may be mediated via the estrogen receptor in granulosa cells.

I next examined the affect of blocking steroidogenesis on the

ability of FSH to increase receptors for FSH and estradiol.

CHAPTERII

EFFECT OF CYANOKETONB OI FSH-INDUCTION OF FSH RECEPTORS

INQARULOSACELISOPTHBRAT

Introduction

 

Although estradiol increases numbers of granulosa cells in ovaries,
estradiol alone does not increase number of receptors for FSH per
granulosa cell. FSH, however, can increase number of receptors for LH
and FSH (Richards e_t. _a_l_., 1976). Moreover, estradiol priming followed
by administration of FSH increases levels of FSH receptors more
Iquickly than FSH alone (Richards it. £.,1976). Since FSH alone can
increase production of estradiol (Moon ESE" 1975; Zeleznik e_t_a_1..,
1974; Armstrong 311:, 1979; Richards 25%" 1982) and estradiol can
enhance FSH action (Pencharz, 1940; Williams, 1945), a synergistic
action of estradiol and FSH may be required for maturation of ovarian
follicles. As an injection of estradiol and/or FSH increases numbers
of nuclear estradiol receptors in granulosa cells (Richards, 1975) and
uterine and ovarian weights (Pencharz, 1040; Williams, 1945), these
effects of FSH are probably mediated via an FSH-induced increase in
estradiol. I hypothesize that an increase in intraovarian levels of

estradiol is required before FSH can increase its own receptor. I

38'

39 '
attempted to test this hypothesis through the use of cyanoketone
(2&rcyano-4, 4, 121-tri-methylandrost-S-en-l7B-ol-3-one), a. potent
antagonist to steroidogenesis which blocks the, conversion. of
pregnenolone to progesterone. Cyanoketone also binds to the cytosolic
receptor for estrogen thus blocking estrogen action (Wolfson EE.E$}9
1983).

Rather than attempt to determine the direct effects of cyanoketone
on intraovarian production of estradiol, I examined whether cyanoketone
pretreatment prevented FSH-induced increases in numbers of receptors
for FSH and estradiol in granulosa cells as well as ovarian and uterine

weights.

Materials and Methods

 

Materials
The following reagents were used: (2, 4, 6, 7, 16, 17‘3H)-

estradiol-17B (136.1 Ci/mmol; 3H-E lZSIOdi-ne (1M5 30). promeg-

173)’
estrone (R5020, NLP-004B, lot 1461-129), New England Nuclear; ovine FSH
(Sl390-2BR,100x FSH 310,0.056 units LH-Sl9/mg, oFSH), human chorionic
gonadotropin (CR-119,11600 IU/mg, hCG); sodium thiocyanate (NaSCN,
analytical grade), chloramine-T, Trizma-HCl (reagent grade), ethylene-
diamine tetraacetic acid (EDTA, Sigma grade), calf thymus DNA, diethyl-
stilbestrol (DES), estradiol-17B (E173), testosterone, progesterone,
dihydrotestosterone (DHT), charcoal (Norit A), Sigma Chemical Co.;
Dextran T-70, Parmacia; aqueous counting scintillant (AC8), Amersham;
propylene glycol, J.T. Baker Chemical Co.; sodium metabisulfite
(reagent grade), Matheson, Coleman. and Bell; Bio-Gel P-60 (100-200
mesh), Bio Rad Laboratories; 2 -cyano-4, 4, 17 -trimethylandrost-
5-en-l7B-ol-3-one (cyanoketone, R-lOB-UH), Sterling-Winthrop Research
Institute (Renssilaer, N.Y.).

Animals

Immature female Sprague-Dawley rats, hypophysectomized on day 24 of
age, were obtained from Hormone Assay Labs (Chicago, Illinois). Rats
were housed under 12L=120 (24°C): and given food and water ad_1ibitum.

Rats were decapitated between 26 and 30 days of age.

40,

41'

Injection regime

 

Cyanoketone (0.05 mg, dose obtained from dose response), progester-
one (2 mg), testosterone (2 mg), estradiol-178 (2 mg), R5020 (2 mg), or
DHT (2 mg) dissolved in 0.2 ml pr0pylene glycol were injected
subcutaneously (s.c.) into rats (20 rats/treatment group, each
experiment repeated twdce). Twenty-four hours later, FSH (200 ug/0.2
ml PBS) was administered s.c. with or without cyanoketone, estradiol-
17B, progesterone, testosterone, DHT, or R5020. Animals were then
decapitated 12 or 24 h later.

The dose and preparation of FSH chosen in my study (200 ug/O.2 ml
PBS) provided maximum increase in FSH receptor with little change in
number of LH receptors (Table 3).

Tissue preparation

 

Ovaries and uteri were dissected, trimmed of fat, and weighed on a
Mettler balance. After weighing, ovaries were placed in 0.01 M PBS
buffer (pH 7.0) on ice.

Granulosa cells were obtained from rat ovaries (2 ovaries/tube)
according to the method of Zeleznik 55 ii“ (1974); gentle pressure was
applied to follicles to express cells. After centrifugation at 800 x g
(20 min, 4°C), granulosa cells were washed three times in PBS buffer (2
ml/tube/wash). Cells were then resuspended in PBS buffer (2 ovaries
/tube, 1 ml volume) and aliquots of 0.1 ml were used for FSH and LH
receptor assays.

For estradiol receptor assays, granulosa cells were washed three
times in TED buffer (1 mM Tris, 1.5 mM EDTA, pH 7.4), homogenized in a

glass Dounce homogenizer, and centrifuged at 4,000 x g for 20 min at

42

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43 '
4°C. Supernatant was further centrifuged at 106,000 x g (type 30
rotor, Beckman) for 1 h (4°C) to obtain a cytosol preparation. Pellets
were washed 3 times in TED buffer and resuspended in 0.5 ml TED for the

nuclear receptor assay.

Measurement of specific FSH and LE binding sites

 

 

 

Ovine FSH (10 ug/lO ul) or hCG (5 ug/10 ul) was iodinated in the
presence of chloramine-T and purified by gel filtration on a column of
Bio-Gel P-60 as described previously (Spicer‘gg'al., 1981). Specific
activity of radioactive oFSB and hCG was 24 cpm/pg and 19 cpm/pg,
respectively.

The procedures used to measure specific binding sites i2_zi££2_have
been described previously (Spicer st 21., 1981). Briefly, aliquots of
resuspended cells were incubated in triplicate at 27°C for 24 h with
radioactive oFSH or hCG (200,000 cpm) in the presence or absence of
unlabeled crude preparation of oFSH (kindly provided by Abbott
Laboratories, Chicago, IL, 4 mg/ml, 0.02 ml/aliquot) or hCG (1 mg/ml,
0.02 ml aliquot). After incubation, samples were washed twice with PBS
(4°C), centrifuged at 3,000 x g for 10 min (4°C), and radioactivity

counted in a gamma counter. Specific binding, expressed as cpm 1251-

oFSI-I or 125I-hCG/ug DNA, was calculated as the difference in counts
bound in the presence (non-specific) or absence (total) of an excess of
unlabeled hormone. DNA values were determined by the diphenylamine

method of Burton (1956).

Estradiol receptor assays

 

I have previously identified the presence of cytosolic and nuclear

estradiol receptor sites for rat granulosa cells (Chapter I). Assays

44'

for estrogen receptors were performed as described in Chapter I (also
see Appendix).

Radioimmunoassays for progesterone and testosterone

Trunk blood was collected and serum stored at -20°C until assayed.
Serum progesterone (Louis 32.2l3’ 1973, as modified by Convey e£_al.,
1977) and serum testosterone (Mongkonpunya s£.al., 1975) were measured
using radioimmunoassays previously validated in our laboratory.

Statistical analyses

 

One way analysis of variance and Bonferroni-t statistics (Gill,
1978) were used to test for significant changes in concentrations of
progesterone, testosterone, weights of ovaries and uteri, and amounts
of specific binding of 125I-oFSH, 125I-hCG, and 33-E17B to granulosa

cells.

55

.a

P;

Results

Cyanoketone dose response

 

FSH increased (p<0.01) levels of its own receptor compared with
control values (Figure 9). Cyanoketone at all doses tested (0.01-l mg)
reduced (p<0.05) the FSH-induced increase in levels of FSH receptor
with maximal suppression at 0.05 mg. Ovarian and uterine weights were
similar in the control and cyanoketone-treated groups (Table 4). In
all subsequent experiments, 0.05 mg of cyanoketone was used.

Effects 2£_cyanoketone pp_FSH-induced increases ip_estradiol, LH,
and FSH receptor levels

 

 

 

 

Ovine FSH increased (p<0.01) FSH and estradiol receptor levels in
granulosa cells by 12 and 24 h post injection (Figure 10). Cyanoketone
suppressed the FSH-induced increase in FSH and estrogen receptors to
values below or near controls at 12 and 24 h. Cyanoketone alone had no
effect on estradiol or FSH receptor numbers in granulosa cells.

Levels of estradiol receptors in the cytosol were similar in saline-
and cyanoketone+FSH-treated rats (Table 5). Numbers of cytosol
receptors were slightly depressed in FSH-treated rats compared to con-
trols at 12 and 24 h.

Number of LB receptors remained unchanged throughout the experiment

(Table 6).

Figure 9.

|
_ J
200 Hi I +

cpm'25 l- FSH BOUND lug DNA

46

 

 

 

 

 

+

CONTROLS FSH 1mg 0.5019 0.1 mg 0.05mg 0.01 mg
P 1

W
CYANOKETONE
+ 200 ug FSH

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

EFFECT OF VARIOUS DOSES OF CYANOKETONE ON SPECIFIC BINDING
OF 1 I-oFSH TO GRANULOSA CELLS OF RATS. Immature rats,
hypophysectomized on day 24, were injected with cyanoketone
(0.01 to 1 mg) and oFSH 24 h later. Rats were killed 24 h
after FSH administration. Granulosa cells were collected,
and saturatfpp analysis was used to determine specific
binding of I-oFSH to granulosa cells. Bars represent
means _+: SEM (n-10 rats/group).

47

TABLE 4

EFFECT OF VARIOUS DOSES OF CYANOKETONE PLUS FSH
0N OVARIAN AND UTERINE WEIGHTSa

 

Ovarian Uterine
Treatment Weight (mg) Weight (mg)
Controls 9.22 i 0.53 36.84 i 1.80
FSH 13.40 _+_- 1.06 43.34 1: 1.93*

FSH + Cyanoketone
(0.01 mg)
(0.05 mg)
(0.10 mg)
(0.50 mg)

(1.00 mg)

10.01 _+_0.63

8.24 +_- 0.52
8.18 :_0.46
7.86 :_0.44
8.96 i 0.49

32.75

I+

2.12

32.94 1.99

H-

32.36 1.97

H-

29.92 1.58

|+

31.25 1.89

H-

 

aExperiment was conducted as

described in Materials and

Methods. Data are expressed as mean :_SEM, n - 20 rats/group.
*Statistically significant at the 0.05 level compared with

controls.

Dal

48'

Cyonoketone-I- E I73 + FSH

 

  
  

 

  

 

 

<1 1200 V

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0‘
O
O
l

 

 

400 -
Control
200 I‘ o Cyanoketone
Cyanoketone + FSH
1 1
0 l2 24

Hours 'After FSH

Figure 10. EFFECT OF CYANOKETONE ON FSH INDUCTION 0F FSH AND ESTRADIOL
BINDING SITES IN GRANULOSA CELLS 0F RATS. Hypophysecto-
mized rats (20/group) were injected with 0.05 mg of
cyanoketone (s.c.). Twenty-four h later, rats were
injected s.c. with 200 ug of oFSH. Dots represent means 1
SEM (n-10 rats/group).

49'

TABLE 5

EFFECT OF CYANOKETONE ON ESTRADIOL BINDING
TO CYTOSOL OF GRANULOSA CELLS

 

Treatment cpm/mg protein
Control - 380
FSH 200
Cyanoketone 383
Cyanoketone + FSH 392

 

Data are expressed as means. N - 20 rats/group.

TABLE 6

EFFECT OF CYANOKETONE 0N FSH INDUCTION OF LH
BINDING SITES 0N GRANULOSA CELLS

 

LH Receptor
Treatment (cpm/ug DNA)
Control 63.7 i 15.3
FSH 69.8 i 6.9
Cyanoketone 68.3 :_16.7
Cyanoketone + FSH 71.6 i 17.1

 

Data are expressed as mean :LSEM. N - 20 rats/group.

50'

Effect 2£_estrogen, progestin, 23 androgen replacement 12.
cyanoketone-treated rats

 

Estradiol reversed the cyanoketone-induced blockage of FSH-
stimulated increases in FSH (Figure 11) and estradiol receptors (Figure
12).

Progesterone partially reversed the effects of cyanoketone on the
FSH-induced increase in FSH receptors, while receptors for estradiol
were increased to levels above that after FSH administration (Figures 11
and 12, panel B). R5020 alone had no effect on levels of FSH or estra-
diol receptors compared with controls (Figures 11 and 12, panel B).
Administration of R5020 with cyanoketone prior to FSH did not increase
levels of FSH or estradiol receptors above controls. Testosterone, like
progesterone, partially reversed the effects of cyanoketone on FSH-
induced increases in FSH receptors and increased numbers of estrogen
receptors to levels similar to that with FSH alone (Figures 11 and 12,
panel C). DHT either alone or in combination with cyanoketone, had
little effect on estrogen receptors; however, FSH receptors were
increased to levels intermediate to controls and FSH-treated groups
(Figures 11 and 12, panel C).

.As shown in Table 7, cyanoketone alone had no effect on ovarian or
uterine weights, or progesterone or testosterone levels in serum. FSH
caused a slight increase in progesterone production and a significant
(p<0.05) increase in ovarian weight by 24 h. Addition of cyanoketone
prior to FSH prevented these increases. Estradiol administration
concomitant with cyanoketone prior to FSH significantly increased
(p<0.01) ovarian and uterine weights by 24 h, but had no effect on serum

concentrations of progesterone or testosterone.

51'

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54'
In summary, cyanoketone can block FSH-induced increases in FSH
receptors. Although progesterone, testosterone, R5020 and DHT can
partially reverse the inhibitory effects of cyanoketone, estradiol can

completely reverse these inhibitory effects.

Discussion

 

A single injection of oFSH alone increased levels of receptors for
FSH and estradiol, and ovarian and uterine weights within 24 h.
However, if rats were pretreated with cyanoketone, a potent inhibitor of
progesterone synthesis, all of the above actions of FSH were blocked.
Estradiol replacement was able to fully reverse the effects of
cyanoketone on FSH action. Replacement of other steroids either had no
effect or partially reversed the effects of cyanoketone.

The effects of cyanoketone on blocking FSH-induced increases in the
FSH and estradiol receptor were partially reversed with progesterone or
testosterone. One reason for the increase in FSH receptor after
replacement with progesterone or testosterone is that these steroids
could be converted directly to estradiol. Also, progesterone and
testosterone in high concentrations can interact with the estrogen
receptor in the cytosol and cause translocation of the estrogen receptor
to the nucleus, thus possibly' mimicking estrogen. action. (Evans and
Leavett, 1979; Farookhi, 1980; Nilsson 55 al., 1974). Therefore, the
effects of testosterone, progesterone, and especially estradiol on FSH
induction of FSH receptors may be mediated via the estrogen receptor.
NOnmetabolizable forms of progesterone and testosterone, R5020 and DHT,
were much less effective in reversing the effects of cyanoketone than
progesterone and testosterone. Thus, the conversion of progesterone and
testosterone to estradiol could explain some of these results. However,

I cannot rule out the possible involvement of these steroids along with

55,

S6 '
estradiol in FSH action.

Although testosterone cu: progesterone could partially reverse the
effects of cyanoketone, synthesis of estradiol appears to be required
‘for FSH to maximally increase its own receptor. I have also shown that
changes in number of estrogen receptors parallel changes in number of
FSH receptors after FSH administration (Figures 11 and 12).

From my study, it appears that estradiol is the primary steroid
involved in FSH induction of the FSH receptor in the rat. Although the
biochemical action of estradiol is unknown, these data support my
hypothesis that control of estradiol production may be important for FSH
action, which, in turn, may promote follicular development.

Next, I examined more specifically the role of estradiol in FSH
action by attempting to block estradiol action through its receptor

rather than through synthesis of estradiol.

CHAPTER III

CI628 IREIBITS FSH-INDUCED INCREASES IN RECEPTORS FOR FSH

IN THE OVARY OF THE RAI

Introduction

 

Differentiation of ovarian granulosa cells is hormonally regulated
by' an interaction. of estradiol with. the gonadotropins. Estradiol
increases responsiveness of follicles to gonadotropins (Pencharz, 1940;
Williams, 1945) while FSH promotes estrogen production and formation of
antra in ovarian follicles (Moon.s£_al3, 1975; Zeleznik gt al., 1974).

Although estradiol has no effect on FSH receptors, FSH can increase
numbers of receptors for LH and FSH (Richards §£_al3, 1976) per granu-
losa cell. However, estradiol priming prior to administration of FSH
results in numbers of receptors for FSH which are significantly greater
than when FSH is given alone (Richards 5; 3i” 1976). Since FSH
promotes estrogen synthesis by granulosa cells (Moon 25.22:, 1975), a
synergistic action of estradiol with FSH on granulosa cells may be
required for FSH to induce its own receptor.

If estradiol is required for FSH to increase its own receptor,
blocking the action of estradiol should prevent the FSH-induced
increase in FSH receptor. The anti-estrogen CI628 (ct-é-pyrrolidino-

ethoxy-phenyl-é-methoxyéi-nitrostilbene) was used to block the effects

57.

58'
of estradiol. Thus, I could evaluate if blocking the action of the

estradiol receptor has an effect on FSH-induced stimulation of the FSH

receptor.

Materials and Methods

 

Materials

The following reagents were used: (2, 4, 7, 16, 17-3H)-estradiol-
173 (137 Ci/mmol; 3H-EUB), cyclic adenosine 3', 5'-monophosphate
(cAMP) 12$I-radioimmunoassay kit (lot D0227AK), ovine FSH
(SlB90-28R,100x FSH 810, 0.56 units LH-Sl9/mg, oFSH); human FSH (LER
1951, LH 100 IU/mg, FSH 6781 IU/mg, hFSH); human chorionic gonadotropin
(CR-119, 11600 IU/mg, hCG); bovine luteinizing hormone (NIAMDD-bLH-4,
bLH); sodium thiocyanate (NaSCN, analytical grade), chloramine-T,
Trizma-HCl (reagent grade), ethylenediamine tetracetic acid (EDTA,
Sigma grade), calf thymus DNA, diethylstilbestrol (DES), estradiol-17B
(£173), charcoal (Norit A), Sigma Chemical Co.; Dextran T-70,
Pharmacia; aqueous counting scintillant (ACS), Amersham; propylene
glycol, J.T. Baker Chemical Co.; sodium metabisulfite (reagent grade),
Matheson, Coleman and Bell; Bio-Gel P-60 (100-200 mesh), Bio-Rad
Laboratories; <1-4-pyrrolidino-ethoxy-phenyl-é-methoxy-dvnitrostilbene
(CI628), Park-Davis Corp. (Ann Arbor, MI).

Animals

Immature female Sprague-Dawley rats, hypophysectomized on day 24 of
age, were obtained from Hormone Assay Laboratories (Chicago, Illinois).
Rats were housed under 12L:12D and given food and water _a_d libitum.

Aninals were decapitated between 26 and 30 days of age.

59.

60

Tissue preparation

 

Ovaries and uteri were quickly dissected, trimmed of fat, and
weighed on a Mettler balance. After weighing, ovaires were immediately
placed in ice-cold 0.01 M phosphate buffered saline (PBS; pH 7.0) and
uteri were discarded. A pair of ovaries from each treatment group was
processed for histology to insure there were no gross toxic effects of
CI628.

Granulosa cells were obtained according to the method of Zeleznik
.55'31. (1974); gentle pressure was applied to follicles and expressed
cells were collected after centrifugation at 800 x g (20 min, 4°C).
Granulosa cells were washed three times in PBS buffer. Cells were then
resuspended in ice-cold PBS (2 ovaries/ml; 0.1 ml aliquots) for FSH and
LH receptor assays.

For the estradiol receptor assay, granulosa cells were washed three
times in ice-cold TED buffer (10 mM Tris, 1.5 mM EDTA, pH 7.4), homo-
genized in a glass Dounce homogenizer, and centrifuged at 4,000 x g for
20 min at 4°C. Pellets were washed three times in ice-cold TED buffer
and resuspended in 0.5 ml TED for the nuclear receptor assay.

Iodination procedures

 

Ovine FSH (10 ug/10 ul) or hCG (5 ug/10 ul) was iodinated by the
chloramine-T method as previously described (Spicer e_t_ 11:, 1981).
Specific activity for radioactive oFSH and hCG was 24 cpm/pg and 19
cpm/pg, respectively.

Measurement of specific FSH and £5 binding sites after hormone
treatments

 

 

 

 

Saturation analyses were used to measure available specific binding

sites for LH and FSH. Aliquots (0.1 ml) of resuspended cells were

61
incubated in triplicate at 27°C for 24 h with 1251-01.33 or 125

I-hCG
(200,000 cpm) in the presence or absence of an unlabeled, crude pre-
paration of oFSH (4 mg/ml, 0.02 ml aliquot) or hCG (1 mg/ml, 0.02 ml
aliquot). After incubation, samples were washed three times with PBS
(4°C), centrifuged at 3,000 x g for 10 min (4°C), and radioactivity in
the pellet counted in a gamma counter. Specific binding, expressed as
cpm specifically bound/ug DNA, was calculated as the difference in
counts bound in the presence (nonspecific) or absence (total) of an
excess of unlabeled hormone. DNA in samples was from 3 to .6 ug per

tube .

Estradiol receptor assay

 

I have previous identified the presence of cytosolic and nuclear
estradiol receptor sites for estradiol in granulosa cells of rats
(Chapter I). Assays for nuclear estrogen receptors were.performed as
described in Chapter I (see Appendix).

To insure that CI628 did not interfere with the nuclear exchange
assay, half of a pool of nuclei from granulosa cells was pre-incubated
with CI628. The estrogen receptor assay was then performed. The
number of receptors for estradiol were similar whether or not the
tissue was pre-incubated with CI628 (Table 8). I therefore concluded
that endogenous CI628 remaining after isolation of granulosa cells did
not interfere with the i_n £513 exchange of 3H-El7B in the nuclear
exchange assay.

Radioimmunoassays for progesterone and testosterone

 

Trunk blood was collected and serum stored at -20°C until assayed.

Serum progesterone (Louis e_t_ a_L, 1973, as modified by Convey 3311:,

62
1977) and serum testosterone (Mongkonpunya 35 31., 1975) were measured

using radioimmunoassays previously validated in our laboratory.

TABLE 8

ESTRADIOL BINDING T0 GRANULOSA CELLS WITH AND
WITHOUT PREINCUBATION WITH CI628

 

Treatment 'cpm/ug DNA
with CI628 preincubation 253.3 i;4.9
without CI628 preincubation 275.3 : 5.7

 

Experiment was conducted as described in Materials and Methods.
Data are expressed as mean i_SEM. N-S.

Radioimmunoassay for cAMP

 

Cyclic adenosine monophosphate levels in granulosa cells were

125I-RIA kit from New England Nuclear (1°t

determined utilizing a cAMP-
D0227A K).

Dose response for CI628

 

To determine the dose of CI628 which would block estrogen action
without major agonistic effects, 0.1, l, 5, or 10 mg CI628/0.2 ml
propylene glycol was given subcutaneously (s.c.). Six hours later,
hFSH (2 ug/0.2 ml PBS) was administered s.c. Animals were decapitated
12 h after hFSH was injected.

Effect g£_CI628 2§_FSH induction 2£_FSH receptors

 

 

Hypophysectomized rats were divided into five groups (20

rats/group): (l) saline-treated, (2) CI628-treated (1 mg/0.2 ml

63.
propylene glycol), (3) hFSH-treated (2 ug/0.2 ml PBS), (4)
CI628-treated, then hFSH 61t1 later, and (5) C1628 plus estradiol (2
mg/0.2 ml propylene glycol), then hFSH 6 h later. Animals were

decapitated 0, 6, 12, or 24 h after an injection of hFSH.

Scatchard analyses 2£_FSH and estradiol receptors after-CI628
administration

 

 

 

For the FSH Scatchard plot, pools of granulosa cells (20

3 125I-oFSH with

rats/group) were incubated with 20 to 800 x 10 cpm of
or without an excess of oFSH. For the Scatchard plots for estradiol,

granulosa cell nuclei were incubated with 1 to 20 nM of 3H-E with or

173
without excess DES. Assay procedures for FSH and estradiol were as
described above.

Histology

Tissue was prepared for histology by fixation in Bouins fluid
followed by dehydration. in ethanol. Tissue was then embedded in
paraffin, sectioned, and stained with hematoxylin and eosin, and

evaluated under a microscope for signs of atresia.

Effect 2£_CI628 23_FSH stimulation 2£_cAMP

 

 

Maximum stimulation of cAMP after hFSH (2 ug/rat) was determined.
Rats were initially injected s.c. with saline or hFSH (2 ug/rat). Then
6 or 12 h later, rats were injected a second time with 2 ug of hFSH in
the tail vein and content of cAMP in gmanulosa cells was determined.
Animals were killed 30, 60, and 120 min after the tail vein injection
of FSH. Granulosa cells were quickly expressed in 0.2 ml PBS.
Expressed cells for each rat were immediately placed in a tube with 0.8
m1 of distilled water at 100°C for 10 min. Tubes were then spun at

15,000 x g for 20 min (4°C). The supernatant was decanted and frozen

64 '
until assayed.

From the results of the time response (Table 9), the adenylate
cyclase system was more responsive after a tail vein injection of hFSH
at 12 h rather than 6 h after the initial s.c. injection of hFSH.
Sixty min after the second tail vein injection of hFSH, cAMP levels
were 'maximally increased. Therefore, these times ‘were chosen. for
future experiments.

Rats (20 rats/group) were then divided into four treatment groups:
(1) saline-treated, (2) CI628-treated, (3) hFSH-treated, and (4)
CI628-treated plus hFSH 6 h later. Twelve h after FSH, all groups
received a tail vein injection of 2 ug of hFSH. Sixty min later, rats
were decapitated, and granulosa cells expressed and processed as
described above.

Statistical analysis

 

One way analysis of variance and Bonferroni-t statistics (Gill,
1978) were used to test for significant changes in concentrations of

progesterone, testosterone, cAMP content, weights of ovaries and uteri,

125 125

and amounts of specific binding of I-oFSH, I-hCG, and 3H-E to

173

granulosa cells. Scatchard plots (Scatchard, 1949) were analyzed by

linear regression.

65'

TABLE 9

TIME COURSE OF hFSH STIMULATION OF cAMP CONTENT IN GRANULOSA
CELLS OF RATS

 

cAMP
Treatment (pmoles/ug DNA)
hFSH- 6 h
30 min 1.23 j; 0.32
hFSH- 12 h 1.01 i_0.26
30 min 3.99 i 0.13*
60 min 5.77 t 0.19*
120 min 1.17 i 0.21

 

The experiment was conducted as described in Materials and Methods.
Data are expressed as mean + SEM, n=10 rats/group.

*Statistically significant at the 0.05 level compared with controls.

Results

Dose response for CI628

 

There was a significant (p<0.01) increase in receptors for FSH
(Figure 13, panel A) and 317B (Figure 13, panel B) 12 h after FSH
administration. These increases were suppressed (p<0.05) by all doses
of CI628. Maximum suppression (p<0.01) occurred with 1 mg of C1628.
CI628 alone had no significant effect on FSH or estradiol receptor
numbers. To examine whether CI628 had any estrogenic effects, ovarian
and uterine weights were obtained. CI628 at all doses tested had no
effect on. ovarian or uterine weights (Table, 10). Histologically,
ovaries of CI628-treated rats appeared similar to ovaries of controls.
Since 1 mg of CI628 was as effective as 5 or 10 mg, the 1 mg dosage of
CI628 was used in all subsequent experiments.

Effect 2£_CI628 22_FSH induction of receptors for FSH, LH, and
estradiol

 

 

At 0, 6, 12, and 24 h, levels of receptors for FSH were similar in
saline- and CI628-treated animals (Figure 14, top panel). From 6 to 24
h after FSH alone, numbers of FSH receptors were increased
significantly (p<0.01) over controls. Although administration of CI628
prior to FSH prevented the FSH-induced increase in the FSH receptor,
administration of estradiol concomitant with CI628 was able to reverse
this effect.

Numbers of estradiol receptors in the nucleus were similar and
unchanged in CI628- and saline-treated groups (Figure l4,bottom panel).

Number of nuclear receptors for estradiol was significantly (p<0.01)

66.

Figure 13.

67

O)
1
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CONTROL FSH (0.1mg) (1mg) (O\.1rng) (1mg)

USES CIEZB
-+2ugFSH

 

EFFECT OF VARIOUS DOSES OF CI628 ON SPECIFIC BINDING OF
5 3

I-oFSH TO GRANULOSA CELLS AND H-E17 T0 NUCLEI 0F
GRANULOSA CELLS. Immature rats hypophysectomized (n=20
rats/group) on day 24 were injected with four doses of
CI628 (0.1, l, 5, or 10 mg) alone or in combination with 2
ug of hFSH. CI628 was injected 6 h prior to hFSH, and rats
(20/group) were killed before (0 h) and 12 h after hFSH was
injected. Granulosa cells were collected, and safigration
analysesJ used to determine specific binding of I-oFSH
(A) or H-E (B) to granulosa cells. Data are expressed
as cpm apeclfgcally bound/ug DNA. Bars represent means :'
standard error. Results obtained with 5 and 10 mg of CI628
(data not shown) were similar to those with 1 mg.

68'

TABLE 10

EFFECT OF VARIOUS DOSES OF CI628 AND FSH ON OVARIAN AND UTERINE

 

WEIGHTS
Ovarian Uterine
Treatment Weight(mg) Weight(mg)
Controls 11.0 :_0.6 47.4 :_3.2
FSH 9.3 _+_- 0.4 46.4 i 0.5
CI628 ( 0.1 mg) 10.4 i 0.4 45.1 i_0.8
( 1.0 mg) 9.6:0.8 47.4_+_0.6
( 5.0 mg) 10.3 i_0.6 48.4 i 0.2
(10.0 mg) 10.5 i 1.1 50.1 i 6.2
FSH+CI628 ( 0.1 mg) 10.2 i 0.7 ' 46.3 :_3.4
( 1.0 mg) 9.4 i 0.2 41.4 i 3.3
( 5.0 mg) 10.4 i 1.1 47.2 i 0.8
(10.0 mg) 10.3 i 0.9 46.1 i 0.8

 

Experiment was conducted as described in legend of Figure
13. Data are expressed as mean + SEM, n - 20 rats/group.

69

 
 
   

 

 

‘2‘ 22—

o FSH

g h-

\ |8- 0C1628+E,7B
2 +FSH'

3 _

o

a: I4—

I

(Li) P"

0! IO-

N

T... _.

N r—

‘9 6 CI628

:5 '- aza: Saline

2 2.. C1628+FSH
o.

O 1 L 1 1 l

 

   
 

 

éé 22r-
0 ’ . C1628+E
or :73
:1 18 - +F$H
\
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c F-
3
m '4 -
g. >-
LIJ :-
m' IO FSH
I L
NA 6
Io _
f: .. C1628+FSH
2 2 _- _>—a_a Saline
0. CI 628
U l l l l l

 

O 6 I2 IS 24
Hours After FSH

Figure 14. EFFECT ‘OF CI628 ON FSH INDUCTION 0F FSH AND NUCLEAR
ESTRADIOL BINDING sn‘ts 0N GRANULOSA CELLS. Hypophy-
sectomized rats (20/group) were divided into 5 groups: 1)
saline-treated, 2) CI628-treated (1 mg), 3) hFSH-treated (2
ug), 4) CI628-treated with hFSH 6 h later, and 5) CI628
plus E173 (2 mg) with hFSH 6 h later. Rats were killed at
0, 6, and 12 h. Dots represent means 1: SEM. (n =- 20
rats/group).

70'
elevated by 12 h after FSH alone but decreased (p<0.01) by 24 h. CI628
treatment prior to FSH prevented the FSH- induced increase in estradiol
receptors. However, levels of estradiol receptors increased (p<0.01)
over controls beginning 6 h after treatment with hFSH.

Levels of receptors for L1! (hCG) were unchanged unless estradiol
administration preceeded an FSH injection. In this group (CI628+E17B+
FSH), LH receptor numbers increased 3 to 4-fold at 6, 12, and 24 h
(Table 11).

To insure that increases in FSH: and E178 receptors after FSH
injection were due to an increase in numbers of receptors and not a
change in affinity, Scatchard analyses were performed. At 0 h,
affinities and binding capacity for FSH or estrogen receptors were
similar for saline-treated and CI628-treated animals (Figure 15, Table
12). At 6 h, there were no changes in numbers of receptors for
estrogen in all groups. However, number of FSH receptors were
increased after FSH administration. By 12 h, numbers of FSH and
estrogen receptors were increased further after FSH administration
while levels of FSH receptors in other treatment groups were similar to
controls. Affinity and number of estradiol or FSH receptors for CI628
or CI628+FSH groups 6 and 12 h after treatment were similar to values

at 0 h (Figure 15, Table 12).

Effect of CI628 on FSH-induced changes in ovarian and uterine
weights and plasma levels of progesterone and testosterone

 

 

 

 

Ovarian and uterine weights were similar in saline-treated, CI628-
treated, and CI628 plus FSH-treated animals (Table 13). Rats injected
with hFSH had ovarian and uterine weights similar to saline-treated

controls at 6 and 12 h, but by 24 h ovarian and uterine weights had

71'

TABLE 11

EFFECT OF CI628 ON FSH INDUCTION 0F LH BINDING SITES
0N GRANULOSA CELLS

 

LB RECEPTOR

(cpm/ug DNA)
Treatment 0 h 6 h 12 h 24 h
Control 206.8 :_19.6 189.3 :_ 9.2 190.7 : 30.3 237.5 :_ 6.4
hFSH ------- 245.1 1 29.2 188.5 1 17.3 192.8 1 11.9
CI628 247.0 i_10.2 279.4 : 16.3 258.4 :- 7.8 282.4 1 17.8
CI628+hFSH ------- 204.1 : 13.1 185.0 1 24.9 207.8 1 15.2
CI628+E173+ -------- 244.0 i_24.1 695.0 : 48.0* 770.5 i_18.4*

hFSH

 

Experiment conducted as described in legend of Figure 14.
* Statistically significant at the 0.01 level compared with controls.

72'

FSH RECEPTOR

 
 
  
   
   

0 l2h after FSH
X 6h ofter FSH
0 Oh

   

 

L111!
20 I00 I80 260 340 400 480

M (xld'2)FSH BOUND

r 8. ESTRADIOL RECEPTOR
07—-

 
 
 
   

0 l 2h after FSH
x 6h after FSH

0.5 _ 0 Oh

0.3 \\

 

 

1 1 1 1 J 1
IO 30 50 7O 90 no I20

M(xlo"2) Eng BOUND

Figure 15. EFFECTS OF CI628 ON hFSH INDUCTION 0F FSH AND NUCLEAR
ESTRADIOL RECEPTORS. Rats were hypophysectomized on day 24
and divided into five treatment groups. Pools of granulosa
cells were obtained at 0, 6, and 12 h after administration
of hFSH. Only Scatchard plots for data from the
saline-treated group at 0 h (0) and FSH-treated groups at 6
(X) and 12 h (o) are shown since binding affinities of FSH
(panel A) or estradiol (panel B) for their receptors and
number of FSH or estradiol receptors in the saline, CI628,
and CI628+FSH groups were similar at 0, 6, and 12 h.

73

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74

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75 '
increased significantly (p<0.05). Ovarian and uterine weights in rats
injected with estradiol and CI628 prior to FSH were heavier (p<0.05)
than controls at all times sampled.

Testosterone levels were similar in all groups across all times
studied and averaged 0.24 ng/ml (n-15). FSH caused a 2-fold (p<0.05)
increase in serum progesterone levels at 24 h compared to controls, but
pre-treatment with estradiol concomitant with CI628 prior to FSH signi-
ficantly increased (p<0.05) progesterone levels over controls at 6, 12,
and 24 h. Levels of progesterone were unchanged in all other treatment
groups.

Effect 2£_CI628 23 FSH stimulation of cAMP

 

Levels of cAMP were similar in salines' and CI628-treated rats
(Table 14). FSH treatment significantly increased (p<0.01) cAMP levels

over controls; whether or not rats were pre-treated with CI628.

76

TABLE 14

EFFECT OF C1628 ON hFSH STIMULATION
OF CAMP IN GRANULOSA CELLS

 

cAMP
Treatment (pmoles/ug DNA)
Saline-treated 0.96 t_0.09
CI628 0.95 i 0.13
tha 6.10 i 0.45*
CI628 + thn 6.70 i 0.54*

 

a The experiment was conducted as described in Materials and Methods
(Chapter III). Data are expressed as mean :_SEM, n - 10
rats/group. The experiment was repeated twice with similar
results.

* Statistically significant at the 0.01 level compared with
controls.

Discussion

 

Growth and differentiation of ovarian follicles are promoted by
pituitary gonadotropins and gonadal steroids (Pencharz, 1940; Williams,
1945; Zeleznik st 31., 1974; Moon ££_213, 1975). Administration of the
antiestrogen cis-clomiphene can prevent increased ovarian weight and
follicular growth induced by FSH (Nakano $3.2i'3 1982). Furthermore,
FSH plus clomiphene prevents an increase in tertiary follicle develop-
ment and suppresses mitosis of granulosa cells induced by FSH.
Tamoxifen, another antiestrogen, can inhibit FSH-stimulated follicle
growth and estrogen synthesis (Watson and Howson, 1977). This suggests
that FSH-induced follicle growth and estrogen synthesis might be
mediated by estrogens.

In hypophysectomized rats, estradiol (administered for 1-4 days)
has little effect on numbers of FSH receptors per granulosa cell,
although it enhances the increase in FSH receptors after administration
of FSH (Louvet and Vaitukaitus, 1976; Richards ‘35 .31., 1976).
Estradiol enhancement of FSH stimulation of FSH receptor is time
dependent (Ireland and Richards, 1978). Estradiol-priming for 12 to 24
h, but not 6 h, enhances the FSH-stimulated increase in its own
receptor. From the data in this chapter, hFSH can increase its own
receptor by 6 h with no increase in estradiol receptors (Figures 14 and
15) until 12 h. Thus, changes in the nuclear estradiol receptor may be
independent of changes in the FSH receptor. Alternatively, the

possible explanation for this increase in FSH receptor without a change

77.

78'

in E178 receptor is that the amount of nuclear estradiol receptor in
granulosa cells prior to FSH may have been sufficient to enable a
synergism of estradiol with FSH. In turn, this could result in FSH
induction of its own receptor. Also, an increase in nuclear estradiol
receptor could have occurred but may have been undetected in our assay
system. However, since administration of CI628 prior to hFSH blocked
the increase in FSH receptor at 6 h (Figure 14), it appears that the
estrogen receptor is linked to FSH induction of its own receptor. As
there was no increase in LH receptor or changes in testosterone levels
at this time, significant contamination of the FSH preparation with LH,
and thus synergism of FSH and LH was unlikely.

Although the. mechanism. by ‘which FSH. and. estradiol interact in
granulosa cells is unknown, the effects do not seem to be at the level
of cAMP stimulation. CI628 does not block the ability of FSH to
increase levels of cAMP. From my receptor studies, the number of
receptors for FSH in the FSH-treated animals was approximately 3 to
6-fold higher (Figures 13 and 14) than FSH receptors present in the
CI628+FSH- treated rats 12 h after an injection of hFSH, yet the amount
of cAMP stimulated after FSH administration was similar in both groups.
Therefore, the amount of cAMP produced could be independent of the
amount of receptors present. After a critical number of receptors are
bound with hormone, maximum cAMP response appears to occur. This
phenomena of "spare receptors" occurs in other tissues (Catt and Dufau,
1973; Mendelson st 31., 1975; Naor gt Elf, 1980). Also, increases in
FSH-responsive adenylate cyclase in granulosa cells of preovulatory

follicles are not directly related to increases in FSH binding sites

79'

per granulosa cell (Jonassen 35.31., 1982). However, though the cAMP
response was similar in the hFSH- and CI628+hFSH-treated groups, the
stimulation of progesterone and increases in ovarian and uterine
weights were significantly different in these groups. This indicates
that the interaction of FSH and estradiol appears to occur distal to
the receptor-adenylate cyclase-cAMP response system. And, though cAMP
may increase in the absence of changes in numbers of FSH or E17B
receptors, the actions of FSH and estradiol may not be expressed unless
concomitant increases occur in number of receptors for each of these
hormones. For example, estradiol induction of proteins which are later
phosphorylated may be required for FSH induction of its own receptor,
progesterone synthesis, and ovarian and uterine weight gains.

My study indicates that FSH induction of its own receptor requires
estradiol, although I cannot categorically rule out involvement of
other steroids. The exact mechanism by which estradiol interacts with
FSH remains to be determined. However, estradiol appears to be

required for FSH action, and estradiol appears to exert its effects at

some point after the FSH receptor-adenylate cyclase-cAMP interaction.

GENERAL DISCUSSION

The main objective of this dissertation was to determine if estra-
diol is required for FSH to increase the number of FSH receptors in
granulosa cells. As the preparations of granulosa cells used in my
studies probably had some contamination of thecal cells, it is possible
that some of my results are due to FSH. action. on thecal cells.
However, FSH binding appears to be only to granulosa cells in the rat
(Richards and Midgley, 1976). Thus, FSH action should be limited to
granulosa cells.

Based on my results, estradiol appears to be required for FSH to
maximally increase the level of FSH receptors. The ability of
progesterone and testosterone to partially reverse the inhibitory
action of cyanoketone is in part due to conversion of these steroids to
estradiol as the number of receptors for estradiol was increased after
progesterone or testosterone addition. However, as the
non-metabolizable forms of these steroids had some effect on reversing
the inhibitory effects of cyanoketone prior to FSH, progesterone and
testosterone may also have some direct effects. To determine if
progesterone and testosterone have direct effects, CI628 can be
injected into hypophysectomized rats to block estrogen receptor action.
Progesterone and testosterone could then be administered followed by

FSH and numbers of receptors for FSH and estradiol determined. If

80.

81 '
progesterone and testosterone are involved in FSH action independent of
the estradiol receptor, there would be an increase in numbers of
receptors for FSH without any’ effect on numbers of receptors for
estradiol. In other experiments, anti-progestins and anti-androgens
along with an aromatase inhibitor could be used to further determine
the role of these steroids in FSH action.

The second objective of the dissertation was to determine if the
interaction of estradiol and FSH occurred at the level of cAMP
production. As CI628 had no effect on FSH stimulation of cAMP
production, the interaction of estradiol and FSH appears to be distal
to the cAMP-adenylate cyclase system perhaps involving protein kinases
or other intracellular proteins.

The effects of estradiol alone on protein kinase activity remain
controversial (DeAngelo gtual., 1975; Richards e£_al,, 1979). However,
after concomitant administration of estradiol and FSH, there is a
10-fold increase in granulosa cell content of RII, the regulatory
subunit of type II cAMP-dependent protein kinase (Richards and Rolfes,
1980). Richards SE..El3 (1983) have also demonstrated that after
administration of FSH for 3 days, five proteins were induced in antral
follicles that are then phosphorylated by protein kinase. Since FSH
stimulates production of estradiol (Moon 5£“21., 1975), it is possible
that estradiol induces synthesis of these five follicular proteins. As
receptors for progesterone and testosterone also interact with DNA,
these steroids may play a role in protein synthesis.

Both calcium and the calciumrdependent regulatory protein,

calmodulin, appear to be required for regulation of steroidogenesis in

82 '
granulosa cells by LH and FSH (Carnegie and Tsang, 1983; Tsang and
Carnegie, 1983). This participation in gonadotropic regulation of
steroidogenesis is independent of stage of follicular maturation and
cellular differentiation (Carnegie and Tsang, 1984). Whether estradiol
has any effects on the calcium-calmodulin system is unknown, however,
estradiol could induce calmodulin or proteins that affect this system.

Although the exact mechanism of interaction of estradiol and FSH in
follicular development is unknown, a. model speculating their
interaction in granulosa cells follows.

Even though initiation of follicular growth may not require
hormones (Pederson, 1970; Pederson and Peters, 1968; 1971), basal
levels of gonadotropins are required for continuation of growth
(Richards and Midgley, 1976). Granulosa cells of all follicles appear
to posses receptors for FSH whereas receptors for LH are present on
theca cells (Eshkol and Lunenfeld, 1968; Richards and Midgley, 1976;
Richards E£.§l3’ 1976). Only granulosa cells of large, preovulatory
follicles have receptors for LH (Channing and Kammerman, 1974;
Zeleznik e_t_ £11., 1974; Amsterdam e_t. 31., 1975; Richards and Midgley,
1976; Nimrod £5 51., 1977). As synthesis of estradiol requires the
interaction of LH and FSH with their receptors in follicles, the basal
levels of gonadotropins present in early follicular development are
important for estradiol synthesis.

Thus, in early follicular development (Figure 16), LE binds to its
receptor in theca cells to stimulate testosterone synthesis. FSH,
after binding to FSH receptors on granulosa cells, stimulates cAMP

production, increases protein kinase activity and stimulates the

83.

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aromatase enzyme system. Testosterone is then converted to estradiol.
Estradiol binds to its cytoplasmic receptor and is translocated to the
nucleus where it stimulates transcription of specific mRNAs. These
mRNAs are translated into specific proteins in the cytosol. These
induced proteins (which could be protein kinases, calmodulin or other
proteins) can interact with existing proteins, protein kinases or the
calcium-calmodulin system triggered by FSH stimulation. There are then
further increases in intracellular cAMP, aromatase activity as well as
induction of receptors for FSH.

As the follicle develops from preantral through antral stages
(Figure 17), theca cells acquire more receptors for LH. Testosterone
production increases providing more substrate for estradiol synthesis.

However, most follicles on the ovary do not ovulate, but become
atretic (Baker, 1963). Alterations in the functions of gonadotropins
and estradiol are involved in this process. In atretic follicles,
there is a shift from production of estradiol to progesterone synthesis
(Uilenbroek £5 21., 1980; Hubbard _e_t_:_ 31., 1981; Braw at a_l_., 1981;
Terranova, 1981). This shift occurs at the same time as a decrease in
hCG and FSH binding (Carson it. 31., 1979; Uilenbroek e_t_ 31., 1980).
There are also decreases in aromatase as well as enzymes involved in
androgen production (Moor g£_ 21:, 1978; Uilenbroek. SE. 313, 1980;
Hubbard £3.3lf’ 1981; Braw st 11., 1981). Administration of estradiol
has prevented atresia in hypophysectomized rats, however its exact role
in preventing atresia is unknown (Ingram, 1959; Harmon 33 31., 1975).

Synthesis of estradiol appears to be an important step in control

of follicular development. Estradiol is required for FSH to maximally

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86
increase receptors for FSH as well as increase receptors for LH in
granulosa cells. Also, estradiol can enhance FSH-stimulated cAMP
production and increase aromatase activity. Therefore, control of

estradiol synthesis or action is an important tool in promoting

maturation of follicles and preventing atresia.

SUMMARY AND CONCLUSIONS

The studies presented in this dissertation examine the requirement
of estradiol for FSH action. It has been shown (Chapter 1) that
granulosa cells contain nuclear and cytosolic receptors for estradiol
which have properties similar to estrogen receptors in other tissues.

Since blockage of synthesis of estrogen with cyanoketone (Chapter
2) or inhibition of binding of estradiol to its receptor with CI628
(Chapter 3) prevented FSH induction of FSH receptors, estradiol appears
to be required for FSH action. Although progesterone and testosterone
can partially reverse .the inhibitory effects of cyanoketone, non-
metabolizable forms of these steroids had less effect. Therefore,
although I cannot rule out the importance of other steroids, estradiol
is indeed required for FSH to maximally stimulate increases in FSH
receptors.

The influence of estradiol on FSH action appears not to be mediated
through the adenylate cyclase-cAMP system as CI628 did not block FSH
stimulation of cAMP production.

In conclusion, estradiol is required for FSH to increase its own
receptor. This interaction, however, occurs distal to the
cAMP-adenylate cyclase system. Although the biochemical action of
estradiol is unknown, these data support my hypothesis that control of

estradiol production is important in the control of FSH action, which,

87 .

88'

in turn, promotes follicular development.

APPEND IX

APPENDIX

Estradiol Receptor Assay

 

Collect tissue, trim of fat, place in TED buffer on

ice

Express granulosa cells in TED ( 1 ml) in a homogen-
izer (2 ovaries/12 x 75 mm polypropylene tube)

Centrifuge at 800 x g for 20 min (4°C)

Wash cells 3 times in TED buffer (2 m1/wash)

Homogenize cells in 2 m1 TED with Dounce
homogenizer

Centrifuge at 4,000 x g for 20 min, 4°C

FOR THE NUCLEAR ASSAY

wash pellets 3 timeg in 2 m1 TED
for 10 min (4 C)

resuspend nuclei in 0.4 m1 TED

add TED buffer so final volume
is 0.25 ml

for Scatchard plots, add 3H-E

(1-20 nM) in D; for saturat on

analysis, add H-E17 (50,000
cpm) in 0.05 mI TED

add SOO-fold excess DES in 0.03
ml to NSB tube

add 0.1 ml of granulosa nuclei to
3 tubes plus 0.1 ml to a tube for
for DNA determination

incubate on ice for 1 h

89,

FOR THE CYTOSOL ASSAY

supernatant is further centri-
fuged at 106,000 x g (Type030
rotor, Beckman) for 1 h (4 C)

remove supernatant with Pasteur
pipette-exclude fat layer

for Scatchard plots, add 3H-E 73

(1-20 nM) in TED; for saturation

analysis, add H-E1 B (50,000
cpm) in 0.05 ml TED

add SOO-fold excess DES in 0.03
ml to NSB tube

add 0.5 ml of supernatant
incubate for 3 h at 4°C

add 1 ml of DCC to each tube (on
ice)

90

add 0.05 ml of TED buffer contain-
ing 3 M NaSCN to all tubes

incubate overnight at 4°C

add 0.3 m1 DCC to all tubes (on
ice)

incubate for 15 min on ice

centrifuge at 8,000 x g for 10
min

decant supernatant into scintil-
lation vials

add 10 ml AC8 to vials and count

Definitions:

incubate for 15 min on ice

centrifuge at 8,000 x g for 10
min

decant supernatant into scintil-
lation vials

add 10 ml ACS to vials and count

TED buffer: 10 mM Tris, 1.5 mM EDTA, pH 7.4
DES: diethylstilbestrol disolved in methanol

NSB: non-specific binding
NaSCN: sodium thiocyanate

DCC: 1% charcoal, 0.052 Dextran, in TED buffer

ACS: aqueous counting scintillant

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