Hormonal interactions in the control of granulosa cell differentiation

0022-473lj83 $3.00+0.00

J. sleI'oid Biochrm. Vol. 19, No. L pp. 17 32. 1983

Copyright © i 983 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

HORMONAL INTERACTIONS IN THE CONTROL OF GRANULOSA CELL DIFFERENTIATION 1. H.

DaRRINGTON,

H. L.

McKERACHER,

A.

K. CHAN

and R. E.

GORE-LANGTON

Banting and Best Department of Medical Research, University of Toronto. Toronto and Department of Physiology. University of Western Ontario. London. Canada SUMMARY

The main emphasis of this paper is on the changes in function of granulosa cells as they undergo cytodifferentiation in follicles developing from the preantral to the antral stage. and on the hormones present in the milieu of gonadotrophins and steroids which are essential for these events to proceed normally. We found that FSH alone could induce aromatase activity in cultures of immature granulosa cells and that this effect could be duplicated by dibutyryl cyclic AMP. Incubation of cell sonicates under optimal conditions indicated that FSH acted on granulosa cells to increase the cellular concentration of active aromatase. Prior treatment with androgens augmented the FSH effect. Progesterone synthesis is another differentiated function which can be induced in culture by FSH alone and augmented in the presence of androgens. In assessing the enzymes involved in progesterone synthesis we found that cholesterol side-chain cleavage activity had similar hormonal requirements whereas 3f3-hydroxysteroid dehydrogenase activity was stimulated by FSH alone. FSH also stimulates cyclic AMP binding activity in cultured granulosa cells during cytodifferentiation. These proteins represent another class of intracellular proteins, quite distinct from the steroidogenic enzymes, which increase as the granulosa cells mature. The ability of FSH to induce the appearance of LH and prolactin receptors, and stimulate the secretion of plasminogen activator and proteoglycans is reviewed. It is concluded that the appearance of steroidogenic enzymes and other intracellular proteins, cell-surface and secreted proteins as wei! as morphological maturation of granulosa cells require the presence of FSH. In the "turning-on" of some of these differentiated functions androgens playa permissive role. Having established events which occur during normal development of the follicle, we considered ways by which this overall process could be interrupted and fertility controlled. Here we describe the ways by which prolactin and LHRH interfere with the normal process of granulosa cell cytodifferentiation.

respond to the growth stimulus they develop to a certain stage but their usual fate is to undergo atresia during the preantral stages. Those which are rescued to undergo the final stages of growth from the preantral to the antral stage represent only a small percentage of those present at birth. The maintenance of fertility depends therefore upon establishing the correct environment in which this final crucial phase can be completed. The thecal and granulosa cells of the follicle are responsible for maintaining the microenvironment in which the oocyte develops and these cells differentiate in response to the cyclical pattern of gonadotropin secretion characteristic of the estrous cycle. The differentiated functions expressed by the cells in response to gonadotropins include changes in steroid secretion, intracellular and cell-surface proteins and secreted factors (Fig. I). The main emphasis of this manuscript will be on the changes in function of granulosa cells as they differentiate in the antral follicle, and on the hormones present in the milieu of gonadotropins and steroids which are essential for these events to proceed normally. Information about the control of granulosa cell differentiation has burgeoned over the last few years due to the establishment of culture conditions under which cells would remain viable and acquire differentiated functions in response to hormonal stimuli. The second part of the manuscript will

INTRODUCTION

In reproductive biology there are still many basic and fundamental questions which remain to be answered at the cellular level. In spite of the major advances in molecular biology, in the control of gene expression, in genetic engineering and gene cloning, the gametes still hang onto their secrets. It is almost 9 years since we isolated the Sertoli cell which we believed would give us the secrets of life and provide us with the critical ingredients for germ cell development and the triggers for meiosis. Since that time we have learned a good deal about the differentiation of the Sertoli cell, but how these changes influence germ cell development is still not understood. The control of gametogenesis in the ovary is equally intriguing. Shortly after birth the ovary contains a full complement of oocytes, each one arrested at the end of the prophase of meiosis and surrounded by a single layer of granulosa cells. What causes follicular growth arrest when the oocyte is at this stage of meiosis is not known. Also the factors required for reinitiation of follicular growth are not known. We are aware of the complexity of the control mechanisms involved when we see that some follicles start to grow shortly after birth whereas others do not grow for weeks or decades (depending upon the species), even though all the follicles are seemingly exposed to the same fluctuations in hormones. Once the follicles 17

J. H.

18

DoRRINGTON

et al.

preantral

antral

FSH, LH Estrogen, androgen

Granulosa cell differentiation: I. Aromatase 2. Cholesterol side-chain cleavage 3. 3/:?-hydroxysteroid dehydrogenase 4. cAMP binding sites 5. Receptors for LH, PRL 6. Plasminogen activator 7. Proteoglycans

primordial

Fig. I. Protein changes occurring in granulosa cells as they differentiate in follicles developing from the preantal to the antral stage.

deal with the actions of two naturally occurring hormones, prolactin and luteinizing hormone-releasing hormone (LHRH), which interfere with the norm, I process of granulosa cell differentiation. PROTEIN CHANGES WHICH OCCUR AS GRANULOSA CELLS DIFFERENTIATE IN FOLLICLES FROM THE PREANTRAL TO THE ANTRAL STAGE

Enzymes involved in steroidogenesis Aromatase. Aromatase is a key enzyme in the ovary and is induced in the follicle during normal development to the preovulatory stage. Estrogen secretion in the rat rises over a 24 h period to a maximum during early proestrus when FSH levels are slightly elevated and follicular growth is rapid, but before the beginning of the LH surge. The estrogens produced by the follicle act as signals from the ovary to various target organs, i.e. hypothalamus-pituitary and uterus, to synchronize the events which are characteristic of the normal cycle. In addition to these extragonadal actions, estrogens act locally within the follicle to stimulate granulosa cell proliferation and promote the rapid phase of follicular growth during normal folliculogenesis [I]. When the granulosa cells are primed with estrogen, FSH rapidly induces the formation of specific LH receptors on the surface of these cells in readiness for the LH surge which causes ovulation [2]. LH stimulates the thecal cells to produce the androgen substrates which diffuse into the granulosa cell layers where, in the rat ovary, the aromatase is localized almost exclusively. The induction of the aromatase enzyme complex and the regulation of the activity to ensure adequate levels of estrogen are of prime importance in co-ordinating the events which lead to ovulation. We have established that FSH

induces aromatase actIvIty in cultures of immature granulosa cells isolated from preantral follicles and that this effect can be duplicated by dibutyryl cAMP. The induction of aromatase activity was one of the earliest examples of the ability of granulosa cells to differentiate in culture in a chemically defined medium. This was an important finding since it showed that in a medium depleted of serum one essential function i.e. estrogen synthesis, could be "turned on" by FSH. In our initial studies, aromatase activity was assessed by measuring the production of estrogen by radioimmunoassay, and this has been reviewed in detail [3]. More recently, we have developed a radiometric assay of aromatase activity which measures the stereospecific release of 3H from [lp- 3H]-testosterone to give 3H 2 0. The use of 3H 2 0 release as a specific assay for aromatization in granulosa cells has been validated as described previously [4]. Using this assay we confirmed the earlier findings which demonstrated that FSH stimulated the aromatization of testosterone in granulosa cell cultures. The time course of the appearance of aromatase activity in granulosa cell cultures following the addition of FSH shortly after plating the cells is shown in Fig. 2. The activity in cell sonicates was assayed by measuring the release of 3H 2 0 from [lp.3H]-testos. terone under optimal conditions [4]. Aromatase activity showed little change before 16 h, then increased over the subsequent 32 h period. The incubation of cell sonicates under optimal conditions indicated that FSH increased aromatase activity by a mechanism which was independent of permeability effects on the availability of substrate and cofactors. The dosedependent increase in aromatase activity indicates that FSH acts on granulosa cells to increase the cellular concentration of active aromatase either by de novo synthesis or by facilitating post-translational

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Fig. 2. Time course of the induction of aromatase activity by 50 ng Sairam FSH S-1528 C2(ml in cultured rat granulosa cells using a cell-free aromatase assay. Aromatase activity was assessed by incubating the sonicates with [lp- 3H]-testosterone for I h under optimal conditions, and measuring the amount of 3H zO released. Values are the mean ± SE (/1 = 4). events. The placental aromatase enzyme consists of cytochrome P450 with heme prosthetic group in association with NADPH-eytochrome c (P450) reductase and phospholipid [5]. The granulosa cell enzyme closely resembles the placental aromatase in its substrate specificity, requirement for NADPH and molecular oxygen, apparent mechanism of action (i.e. cis-l,2 dehydrogenation), stabilization by dithiothreitol and relative insensitivity to carbon monoxide inhibition [4]. Therefore, the induction of FSH of a similar complex in granulosa cells could occur by synthesis, activation or membrane integration of one or more of the components. Maintaining the cells in culture for 18 h before treatment with FSH reduced the time lag of the response to 90 min, after which the aromatase activity continued to increase for several hours (Fig. 3). Prior treatment from the time of plating with dihydrotestosterone (DHT) (or testosterone) potentiated the response to FSH. The ability of androgens to augment the action of FSH on aromatase activity supports the work of Hillier and De Zwart[6] and is consistent with the synergistic action of DHT on FSH-induced progesterone synthesis [7]. This action of DHT may be related to its ability to augment FSH-stimulated cAMP production [8]. The rapid effect of FSH on aromatase activity can be inhibited by puromycin indicating that on-going protein synthesis is required. The relatively rapid effect of FSH on aromatase activity when added 18 h after plating compared with the effect when added at the time of plating (i.e. 1.5 vs 16 h) is intriguing. This may indicate that the cells require time to attach, restructure and secrete extracellular matrix before the expression of FSH actions can be manifested.

Enzymes involved in progesterone biosynthesis. In addition to synthesizing androgens and estrogens, the rat follicle produces progesterone. Progesterone levels are elevated at two periods during the estrous cycle [9, 10]. One peak of progesterone secretion from

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Fig. 3. Time course of the induction of aromatase activity by FSH after pre-incubation with DHT (5 x 10- 7 M) for 18 h after plating the cells. Aromatase activity was determined by incubating the cells with [lp- 3H]-testosterone for I h and measuring the amount of 3H zO released. the follicle occurs concomitantly with the LH and FSH surge on the afternoon of proestrus. On the morning of the next day (estrus) the levels of progesterone return to basal levels and remain low until the corpus luteum is formed and progesterone levels are again elevated. As the granulosa cells approach the preovulatory stage they acquire the capacity to synthesize progesterone. The hormonal requirements for the initiation of the synthesis of progestational steroids from endogenous precursors by cultures of granulosa cells from immature rats have been established by a number of investigators [7, II]. Treatment of cells with both FSH and androgens caused a dramatic increase in synthesis of progestational steroids, whereas androgen (testosterone and DHT) alone was ineffective and FSH alone caused only a small increase (Fig. 4). Progesterone synthesis therefore is another differentiated function which can be induced in culture. The synthesis of progesterone by granulosa cells is principally regulated by two enzymatic steps, firstly, the conversion of cholesterol to pregnenolone involves the cholesterol side-chain cleavage system and subsequently, the metabolism of pregnenolone to progesterone results from the action of the 11 5 -3{J-hydroxysteroid dehydrogenase and the 11 5-11 4 isomerase. We have investigated the requirements which cause the increase in the activity of each of the enzymes as the granulosa cells differentiate. Cholesterol side-chain cleavage actIVIty was assessed by determining the sum of the products, i.e. pregnenolone and its metabolites progesterone and 20a-hydroxypregn-4-en-3-one in the culture medium by specific radioimmunoassays. Granulosa cells from preantral follicles had little cholesterol side-chain cleavage activity (Fig. 5). Treatment with FSH stimu-

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Fig. 4. Progesterone secretion by cultured granulosa cells from 25-day-old rats pretreated with estradiol-]? P (I mg! day) for 3 days. FSH: purified Reichert FSH, 0.25 Jlg/ml; DHT: 0.5 JIM 1?P-hydroxy-5cx-androstan-3-one; T: 0.5 JIM testosterone; E 2 : 0.5 JIM estradiol-I? p. Secretion by cells cultured in medium alone or containing LH, DHT, T or E2 was undetectable. Armstrong and Dorrington[7].

Fig. 5. Interaction between FSH and DHT (5 x 10- 7 M) to stimulate cholesterol side-chain cleavage activity. Granulosa cells were isolated from 25-day-old DES-primed rats. Cholesterol side-chain cleavage activity was assessed from the sum of the total products (pregnenolone, progesterone and 20a-hydroxypregn-4-en-3-one) measured by RIA. Values are the means ± SE (n = 4).

lated the actIVIty by 24 h of culture and this was greater after 48 and 72 h. DHT alone was ineffective, but when added together with FSH, cholesterol sidechain cleavage activity was stimulated synergistically. Nimrod[ 12, 13] has considered several possible ways by which androgens may facilitate the effect of FSH on progesterone synthesis. He found that androgens did not influence the amount of 125I_FSH bound to granulosa cells or the activity of adenylate cyclase and phosphodiesterase. Androgens potentiated the effects of dibutyryl cyclic AMP and choleratoxin [14] indicating that androgens exert their effect at a step subsequent to cyclic AMP formation. The second step in the pathway involving 3P-hydroxysteroid dehydrogenase was measured by the conversion of [3H]-pregnenolone to labelled products.

The synthesis of eH]-progestational steroids was detected in cultures treated with testosterone or DHT but this effect was low compared to the effect of FSH alone. Testosterone did not act synergistically with FSH to stimulate enzyme activity. CH]-Progesterone was the major product synthesized in FSH-treated cultures, smaller amounts of eH]-20ex-hydroxypregn-4-en-3-one were found whereas eH]17ex-hydroxyprogesterone was undetectable [3]. The activity of the 3P-hydroxysteroid dehydrogenase assayed under optimal conditions was also increased in cells which had been treated with FSH [3]. The stimulatory effect of FSH was characterized by increases in both the Vmax and K m values of the enzyme [15]. As summarized in Fig. 6, the appearance of the

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Fig. 6. Major pathways for the biosynthesis of steroids in rat granulosa cells indicating the enzymes which are influenced by FSH and DHT.

Granulosa cell differentiation domain of proteins required for the synthesis of steroids by granulosa cells requires the presence of FSH.

cAMP-binding sites An increase in cAMP-binding sites is associated with the differentiation of a number of cell types, including the differentiation of cultured fibroblasts into adipocytes [16J and the differentiation of mouse neuroblastoma cells [17]. Richards and Rolfes recently showed that when immature hypophysectomized rats were treated with FSH the cAMP-binding sites in granulosa cells were increased [18]. In our studies of cultured granulosa cells we found that FSH caused a 6~1O-fold stimulation of cAMP binding activity in cells after 66 h of hormonal treatment (Fig. 7). We have not yet established the identity of the cAMP-binding components, however, Richards and Rolfes[ 18J showed in their in vivo studies that the cAMP was bound to two major proteins of molecular weight similar to that of the regulatory subunit II of the cAMP-dependent protein kinase. One can speculate that this may provide a means by which the cAMP signal is amplified within the cell as differentiation proceeds, but it is puzzling that the increase in cAMP binding was not accompanied by an increase

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Fig. 7. Specific binding of [3H]-3'S -AMP to the 20,000 g supernatant fraction from granulosa cells cultured for 66 h with 500 ng NIH-FSH-SI4/ml or 1.0 mM dbcAMP. Cells were homogenized by hand in 10 mM Tris, pH 7.4, containing 1.5 mM EDTA and the homogenate was centrifuged at 20,000 g for 20 min. Supernatants were incubated with 0.121JM [3HJ-cAMP with or without 12IJM unlabelled cAMP for 4 h. cAMP-binding components were adsorbed to millipore filters and the unbound ligand was washed with phosphate-buffered saline [18]. The [3HJ-cAMP bound in the presence of 100-fold excess of cold cAMP was regarded as the non-specific binding of eH]-cAMP to the cytosol.

21

in protein kinase activity. The physiological significance of increased cAMP binding activity in granulosa cells therefore is not clear and requires a more detailed study of the proteins involved before this can be delineated. At present, these proteins represent another class of intracellular proteins, quite distinct from the steroidogenic enzymes, which change as the granulosa cells differentiate.

Appearance of hormone receptors LH receptors. During the development of the Graafian follicle, the granulosa cells acquire LH receptors in preparation for the LH surge which causes the resumption of meiosis, ovulation and subsequent luteinization of the cells. Granulosa cells from immature rats do not bind 125I_hCG, assessed both by autoradiography and binding to freshly isolated cells [19]. Granulosa cells obtained after treatment of the animals with FSH for 2 days were able to bind significant amounts of hCG. Later, Richards et al.[2J observed that the ability of FSH to increase the number of LH receptors increased progressively following 13 days of estradiol pretreatment. FSH can also induce the appearance of hCG receptor sites in cultures of granulosa cells isolated from DES-treated hypophysectomized rats [20]. Prolactin receptors. In the rat ovary, prolactin receptors are predominantly located in the corpus luteum, however, a significant number are also present on the plasma membrane of granulosa cells from large follicles. The number of prolactin receptors on granulosa cells of immature rats is low but increases as the cells differentiate in follicles developing from the preantral to the large antral stage. Formation of prolactin receptors is under hormonal control: Richards and Williams[2lJ showed that FSH administration to hypophysectomized rats for 4 days increased the number of prolactin receptors 2-fold, whereas estrogen priming for 4 days followed by FSH for I day caused a 6-fold increase. Wang et al.[22J confirmed that FSH treatment in vivo would induce prolactin receptors in granulosa cells from DEStreated immature hypophysectomized rats and showed that FSH could also stimulate this process in cultured cells. The prolactin receptors induced by FSH were functional as shown by the stimulatory effect of prolactin on progesterone synthesis. From the experiments described above one can conclude that FSH acts directly on undifferentiated granulosa cells previously primed with DES or estrogen to induce prolactin receptors. The physiological conditions required for the induction of both LH receptors and prolactin receptors appear to be the same and this re-emphasizes the role of FSH in the regulation of cell differentiation by its ability to modulate the population of cell surface proteins.

Secreted proteins The gap junctions which exist between adjacent granulosa cells do not restrict the movement of pro-

22

1. H. DoRRINGTON et al.

teins and other large molecules in the intercellular space. Consequently, the follicular fluid contains components which are present in plasma and lymph. In addition to these plasma proteins, the follicular fluid contains other proteins which are secreted by the granulosa cells. Zachariae[23] found that 3sS04-labelled material appeared in the follicular fluid at the time of antrum formation in rabbit ovaries. Mueller et al.[24] showed that FSH treatment of hypophysectomized, DES-treated immature rats would stimulate the incorporation of 35S04 into proteoglycans. The enhanced incorporation occurred at the same time as antrum formation but whether proteoglycan synthesis is a prerequisite for antrum formation is not clear. Proteoglycans were present on the surface of granulosa cells as assessed by both light and electron microscopy. Subsequently, granulosa cells were isolated, maintained in culture and shown to secrete proteoglycans in response to exogenous FSH. Ax and Ryan[25] identified chondroitin-like material and heparin sulphate in follicular fluid, and found that the concentration of both of these components decreased as the follicle matured. Gebauer et al.[26] showed that rat ovarian slices synthesized heparin-like substances and suggested that the depolymerization of these mucopolysaccharides may play a role in ovulation related events. In this regard, the work of Salomon et al.[27] may be of physiological significance since they showed that gonadotropin-stimulated ovarian adenylate cyclase was inhibited by heparin. At the time of ovulation the follicle wall initially weakens and is subsequently degraded to allow the release of the ovum. There has been some conjecture as to the mechanism by which this occurs. The possi-

bility that enzymatic degradation of the follicle wall takes place gained support from the demonstration that plasminogen activator synthesis by the follicle was correlated with ovulation [28]. Granulosa cells in vivo secrete increasing amounts of plasminogen activator as the time of follicular rupture approaches. Granulosa cells isolated prior to ovulation and maintained in culture can be stimulated by both FSH and LH to produce plasminogen activator. FSH was more effective than LH in this regard. Plasminogen, the substrate for plasminogen activator, is present in the fluid in the follicle, and it seems likely that the plasmin produced may be involved in the weakening of the follicular wall and ovulation. The appearance of steroidogenic enzymes and other intracellular proteins, cell-surface and secreted proteins, as well as morphological maturation of granulosa cells require the presence of FSH, indicating that the differentiation of these cells is specific for FSH. In the "turning-on" of some of these differentiated functions androgens playa permissive role.

HORMONES WHICH INTERFERE WITH THE NORMAL PROCESS OF GRANULOSA CELL DIFFERENTIATION

Having established some of the events which occur during the normal development of the follicle, we are now in a better position to consider ways in which the overall process can be interrupted and fertility can be controlled. Here I shall consider the ways in which two naturally occurring hormones, prolactin and LHRH interfere with the normal process of granulosa cell differentiation.

HYPERPROLACllNEMIA

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ANOVULAliON

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Fig. 8. Possible mechanisms involved in the inhibition of ovarian function by prolactin. The heavy arrows indicate elevated and sustained levels of prolactin.

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Our work on prolactin stemmed from the clinical observations that in patients with hyperprolactinemia associated with physiological and pathological states, follicular growth and maturation did not proceed normally and ovulation did not usually occur [29]. Follicular growth, ovulation and menstruation can be restored in these patients by treating with bromocriptine, a dopaminergic agent which inhibits the release of prolactin from the pituitary causing a rapid decline in circulating prolactin [30]. The suckling stimulus causes an increase in prolactin levels, resulting in impairment of ovarian function which gives rise to an anovulatory state throughout lactation in many mammals. Since prolactin is a naturally occurring contraceptive agent, the mechanism by which it interferes with normal follicular development is clearly an important issue in understanding the control of ovarian function. The antigonadal activity of prolactin may be due to a direct effect on the ovary or an indirect effect on the hypothalamic-pituitary axis (Fig. 8). The study of McNatty[31] is of interest in resolving this issue, since he found that patients who had high levels of plasma prolactin had normal levels of FSH, suggesting that the suppressive effect of prolactin on intrafollicular activity was not due to reduced circulating gonadotropins. Since prolactin binds specifically to granulosa cells in large follicles it is possible that prolactin arrests follicular development by a direct action on granulosa cells. To test this hypothesis, we looked for possible effects of prolactin on aromatase activity in the cultured rat granulosa cell system. No effect of prolactin alone was detected during the 72 h culture period but this was predicted since undif-

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Fig. 9. Effect of 300ng NIH-FSH-SI3/ml and l/lg NIH-P-S7 ovine prolactin/ml on aromatase activity of granulosa cells in culture. At each time point four cultures from each treatment group were incubated with 0.1 /lCi [lp- 3HJ-testosterone for 2 h at 37°C. The medium was removed and the 3H 20 content was determined.

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Fig. 10. PRL dose-dependent inhibition of FSH-induced aromatase activity in granulosa cells after 48 and 72 h of treatment in culture. Granulosa cells were isolated from DES-primed rats at 25 days of age, plated in culture and treated with 300 ng NIH-FSH-SI3/ml (which maximally stimulates aromatase activity) together with graded doses of NIH-P-S7 ovine PRL. Aromatase activity was assessed after 48 and 72 h by incubating the cells for 2 h with 0.7 and 0.3/lCi [lp- 3HJ-testosterone (O.25/lM), respectively and measuring the 3H 20 released. Results are the mean ± SE of triplicate cultures from single representative experiments. Dorrington and Gore-Langton[32].

ferentiated granulosa cells from immature animals do not contain specific prolactin binding sites [21]. Prolactin did not influence the action of FSH on aromatase activity for the first 24 h of treatment but at 48 and 72 h it suppressed the apparent effect of FSH (Fig. 9). As described above, FSH stimulated the appearance of specific prolactin receptor sites on granulosa cells in vitro and this may explain the 24 h lag before the effects of prolactin on FSH-induced aromatase activity were observed. The inhibitory action of prolactin was not due to cell death since the rate of incorporation of [3HJ-leucine into TeA-insoluble material was the same in those cells treated with FSH and with FSH and prolactin [32]. The effect of prolactin on FSH-induced aromatase activity was dosedependent; the ID so value was approximately 50 ng/ml, which is within the physiological range found in human follicular fluid (Fig. 10). The effects of prolactin were reversible within 24 h after the removal of the hormone from the culture medium and the ability of FSH to elevate aromatase activity was restored [32]. Little is known of the mechanism by which prolactin exerts its effects in granulosa cells. Presumably, prolactin binds to receptors on the cell surface to trigger events that lead to changes in steroidogenesis. Prolactin inhibited (Buh cAMP plus MIX-stimulated aromatase activity in a manner similar to its effects on the FSH response suggesting that prolactin influences a step which is distal to FSH-induced cAMP production. Prolactin does not influence aromatase activity by restricting the availability of substrate and cofactors to the enzyme system, as shown by its ability to

24

J. H.

DoRRINGTON

et al.

Table 1. Effects of FSH and prolactin on the aromatase activity of sonicates of ovarian granulosa cells using an optimal cell free assay Treatment in culture

3H 2 0 (d.p.m./mg protein) x 10- 3 0.4 ± 0.1 12.2 ± 0.4

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Granulosa cells isolated from 25-day-old DES-primed rats were cultured for 48 h with FSH (300 ng NIH-FSH-SI3/ml) and/or prolactin (I JIg NIH-P-S7/ml). Sonicates were prepared and incubated with 0.31lCi [lp- 3H]-testosterone (0.25 JIM) under optimal conditions for I h. Aromatase activity was assessed from the amount of 3H 2 0 released. Values are the mean ± SE (n = 3).

reduce the amount of active aromatase induced by FSH when the enzyme was assayed in cell sonicates under optimal conditions (Table 1). From this data, we conclude that prolactin acts directly on rat granulosa cells to inhibit the induction of aromatase activity by FSH, resulting in a decrease in the amount of estrogen synthesized. Since estrogen is a granulosa cell mitogen, a consequence of this inhibitory action of prolactin would be impaired granulosa cell proliferation and abnormal follicular development. The antigonadal effects of high levels of prolactin in women are consistent with the notion that they may be caused, at least in part, by an inhibition of estrogen production. This was supported by McNatty[31] in his studies on the relationship between plasma prolactin and the concomitant hormonal and morphological changes taking place within the follicles. Those women who had elevated plasma levels of prolactin also had high levels in their follicular fluid. The high levels of prolactin in the follicular fluid were associated with low levels of FSH and estrogen, even though the plasma levels were normal. When these ovaries were examined, the size and number of antral follicles isolated during the follicular phase of the cycle were normal, but each follicle contained less than 50% of the full complement of granulosa cells, and were devoid of aromatase activity. These follicles were not competent to develop further and in most cases the oocytes were degenerating. McNatty et al.[33] also showed that high concentrations of prolactin suppressed progesterone synthesis by human granulosa cells in vitro. The inhibitory effect of prolactin on both progesterone and estradiol synthesis has been confirmed recently by Demura et al.[34] using an ovarian perfusion technique. HCG stimulated the synthesis of both steroids but this effect was abolished when prolactin was infused simultaneously. In women, the inhibitory effects of prolactin on steroidogenesis may establish a microenvironment in which follicles can no longer develop normally and ovulate. Lactational amenorrhoea is characterized by hyperprolactinemia, normal levels of FSH and LH and reduced levels of progesterone and estrogen [35]. In

women, breast-feeding provides an adequate means of contraception provided the nursing sessions are frequent. This is exemplified by the Kung-San huntergatherers of northwestern Botswana who feed their babies every few minutes, resulting in a birth-spacing of up to 44 months. In this regard, McNeilly[35] has documented an impressive relationship between the number and duration of suckling episodes, the serum levels of prolactin and the resumption of follicular activity. The levels of prolactin were related to the duration and frequency of suckling sessions, and declined in parallel with the decrease in suckling time as supplementary food was introduced into the diet. Resumption of ovarian activity, increased estrogen secretion and ovulation were associated with the decline in serum levels. Other factors may also contribute to the contraceptive action of breast-feeding, for example, changes in the pulsatile release of LH and impairment of the positive-feedback response of gonadotropins to estrogens.

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Fig. II. [3H]-3',5'-AMP specifically bound to the 20,000 9 supernatant fraction from granulosa cells cultured in the presence of FSH (500 ng NIH-FSH-SI4/rnl) and/or prolactin (l/lg NIH-P-S7/ml). cAMP binding activity was measured as previously described.

25

Granulosa cell differentiation Prolactin also inhibited the FSH stimulation of 3',5'-AMP binding to the 20,000 g supernatant fraction of cultured rat granulosa cells as shown in Fig. II. This effect of prolactin was not observed after 21 h but at 40 and 64 h the suppressive action on the FSHinduced response was apparent. FSH promotes granulosa cell development by sequentially inducing the synthesis of "domains" of proteins involved in the differentiated functions of the cell. The process involved in the synthesis of at least two of these proteins, aromatase and 3',5'-AMP binding protein, by FSH is impaired in the presence of prolactin and the cells can no longer differentiate normally.

Luteinizing hormone-releasing hormone (LH RH) LHRH and the potent LHRH agonists, once considered to have potential in enhancing fertility due to their ability to release gonadotropic hormones. were more recently found to inhibit reproductive processes [36]. These inhibitory properties of LHRH have been attributed to both direct and indirect effects on the hypothalamic-pituitary-gonadal axis as shown in Fig. 12. The acute effects of LHRH agonists administered to rats are to enhance the numbers of LHRH receptors [37, 38J and stimulate the secretion of gonadotropins [39--42]. However, chronic treatment with large doses results in pituitary desensitization to LHRH with the subsequent diminution of gonadotropin secretion [43-45]. The initial action of LHRH is to increase the secretion of gonadotropins and has led to the suggestion that gonadal impairment might also result from the desensitization of gonadal cells as a result of excessive hormonal stimulation [46]. Desensitization of Leydig cells is well

documented and involves a decrease in LH/hCG-stimulated adenylate cyclase activity [47] and a downregulation of LH receptors [48]. Desensitization and consequent steroidogenic lesions [49, 50J result in a refractory period in which gonadotropins fail to stimulate maximal steroid synthesis. Similar effects occur in ovarian luteal cells where reduced gonadotropin responsiveness in vivo results from treatment with high doses of hCG; this process involving loss of LH receptors [51J and reduced adenylate cyclase activity [52]. Heterologous densensitization of LH and FSH-stimulated adenylate cyclase has been demonstrated in granulosa cells of antral follicles in the rat [53, 54]. A direct extrapituitary mechanism for the gonadal inhibition by LHRH has also been demonstrated [55, 56J, this effect being mediated via specific LHRH receptors, which have been identified on granulosa cells [57, 58] and luteal cells [58. 59] of rat ovaries. LHRH binding sites have now been demonstrated on various other tissues, including placenta [60J and adrenals [58]. The physiological basis for LHRH receptors on gonadal cells is not yet understood. LHRH-like substances have been reported in the gonads, placenta, pancreas, various CNS structures and several tumour types [61 J, suggesting a much wider role for LHRH or LHRH-Iike molecule(s) than was originally suspected. It has also been suggested that LHRH receptors may playa role in normal regulatory responses of the gonads to a locally produced peptide resembling the brain LHRH [62]. The effects of LHRH on rat granulosa cells have been most widely studied. LHRH and the potent agonist analogues inhibit FSH -stimulated estrogen and progesterone secretion in vivo [55].

(!)PilUilar y gonodot roph de ..n.llizalion to GnRH Indirecl actions of exogenous GnRH

LH • FSH

® desensitization Oyarion

(-)

-------- ... -._-

10 gonodolropllin.

® Olrecl action. 01 Ixogenoul GnRH

FSH or LH- Oependenl rupon se.

Fig. 12. Possible mechanisms by which LHRH may impair ovarian function.

26

J. H.

DoRRINGTON

These effects might be contributory factors in the observed inhibition of follicular maturation and ovulation [63-65], delay of implantation [66] and termination of pregnancy in its early stages [67]. The impairment of rat granulosa cell estrogen secretion in vitro by the highly active LHRH agonist, [o-Ser-(Bu t)6,des-Gly-NHz'°J-LHRH ethylamide, is apparently due to inhibition of the action of FSH to induce the aromatase enzyme (Fig. 13). A similar inhibition of the effect of cholera toxin on aromatase activity [68] indicates that the effects of LHRH or the LHRH agonist (LHRH-A) are not specific to FSH action. The stimulation of aromatase activity by dibutyryl cyclic AMP in the presence of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (MIX), was also previously shown to be partially inhibited by LHRH-A suggesting that one site of action may be distal to cyclic AMP synthesis [68]. The stimulation of estrogen and progesterone production in granulosa cell cultures by cyclic AMP analogs was also reported to be inhibited by LHRH and an agonist [69]. We do not know whether these inhibitory effects of LHRH on the actions of cyclic AMP derivatives are due entirely to increased cyclic AMP catabolism as discussed later, or whether other steps in the action of cyclic AMP are directly influenced. In addition to showing the anti-gonadotropic actions of LHRH-A, we had previously observed that basal levels of aromatase activity in rat granulosa cells were stimulated by treatment with LHRH-A, this effect being much greater in the presence of MIX [68]. These results indicate that the inhibitory actions of LHRH are not merely due to non-specific suppressive effects on granulosa cells. The inhibitory and stimula-

12 ¢

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D····

·.·.:b ··············b 24

LHRH

48

Time in culture.

72

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Fig. 13. Modulation of aromatase activity in granulosa cells by LHRH and/or FSH. Granulosa cells were cultured for periods of 24, 48 or 72 h in the presence or absence of maximally stimulating doses of FSH and/or LHRH agonist (10- 7 M). Aromatase activity was assessed at the end of each time period; by incubating the cells with [lp- 3H]-testosterone for 2 h and measuring the amount of 3H 2 0 released. Each value is a mean ± SE (n = 4).

et al. 7

•'0

y

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)( 5 l:

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Fig. 14. Dose-response curve for the action of LHRH alone on aromalase activity. The cells were cultured in the presence of graded doses of LHRH-A (10- 1 °-10- 6 M) for 48 h at which time the aromatase activity was assessed, by measuring the amount of 3H 2 0 released from [IP_ 3 H]testosterone during a 2 h incubation. Each value is a mean ± SE (n = 4).

tory actions of LHRH-A on aromatization occur at similar relatively high concentrations of LHRH-A, suggesting that both effects represent pharmacological actions. This view might need to be revised if a natural LHRH-like peptide is later demonstrated to be present in similar concentrations in the follicular environment, or if such a peptide has an even greater affinity for the same LHRH receptors. The levels of LHRH in human plasma, presumed to be of hypothalamic origin, have recently been re-evaluated with improved methodology and the highest levels appear well below the concentrations having inhibitory effects in vitro; 10 pg/ml (8 x 10- 12 M) in plasma collected during the periovulatory period [70]. The dose-response curve for stimulation of aromatase activity in rat granulosa cells in culture by LHRH-A is shown in Fig. 14; the ED so is 3.8 x 10- 9 M and maximal stimulation was observed at about 10- B M. The inhibitory effect of LHRH-A on aromatization is apparently by an irreversible blockade of the stimulatory action of FSH. This was shown by treating granulosa cell cultures for 24 h with FSH in the presence of 10- 7 M LHRH-A, at which time the medium containing the LHRH-A was removed and fresh medium added containing FSH. The inhibition which occurred during the first 24 h continued for a further 48 h despite the removal of the LHRH-A (Fig. 15). The direct inhibitory and stimulatory effects of LHRH-A demonstrated on estrogen synthesis and aromatase activity in granulosa cell cultures have also been observed for the enzymes involved in progesterone synthesis. Treatment of cell cultures with LHRH-A and FSH prevented much of the FSH-stimulated increase in cholesterol side-chain cleavage (CS-CC) activity, assessed by the measurement of total secreted products (Fig. 16). The majority of the steroids produced were secreted into the culture medium, and consisted mainly of progesterone and

27

Granulosa cell differentiation 7

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Fig. 15. Irreversible inhibition of FSH-induced aromatase activity by LHRH agonist. Granulosa cells were isolated from 25-day-old DES-primed rats. Cells were cultured with FSH alone or with FSH plus LHRH agonist (10. 7 M). In one group, cells were treated with FSH + LHRH for 24 h after which the cells were washed and subsequently treated with FSH alone. Aromatase activity was assessed from the release of 3H 20 from [Ip- 3H}testosterone during a 2h incubation of the cells. Values are means ± SE (n = 4).

0

Fig. 16. Cholesterol side-chain cleavage activity of rat granulosa cells cultured in the presence of LHRH agonist (10- 7 M) and/or FSH. The total secreted products were measured as pregnenolone, progesterone and 20ex-hydroxypregn-4-en-3-one individually determined by RIA. Each value is a mean ± SE (n = 4). -t I

~

20ct-hydroxypregn-4-en-3-one. LHRH-A alone was also capable of independently stimulating the total products from the CS-CC step. By examining the conversion of eH}pregnenolone to CH}progesterone in a cell-free incubation with added cofactor, we have demonstrated that the 3p-hydroxysteroid dehydrogenase (3P-HSD) is regulated by FSH and LHRH-A in a fashion similar to that previously shown for CS-CC and aromatase. Whereas FSH treatment caused an increase in the 3P-HSD activity in the cell extracts and LHRH-A inhibited this effect, LHRH-A alone significantly stimulated the activity (Fig. 17). Clark and Marsh[7 J) previously repor,ted stimulation of progesterone accumulation in rat granulosa cells by a different LHRH analogue. Inhibitory and stimulatory effects of a LHRH agonist on 3P-HSD activity have also been reported by Jones and Hsueh[15]. Their results indicate that while FSH increases both the Vmn and K", of the 3P-HSD, the stimulation by LHRH increased only the Vmax in comparison to basal enzyme activity. A quantitative comparison of the effects of FSH and LHRH-A on the three enzymatic steps which we have examined are summarized in Table 2. Each enzyme step was stimulated by LHRH-A alone, but to a lesser extent than with FSH. LHRH-A partially

72

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FSH + LHRH-A

Fig. 17. ,15-3p-Hydroxysteroid dehydrogenase activity in rat granulosa cells cultured with LHRH agonist (10- 1 M) and/or FSH for 72 h. The activity was measured in a cellfree system under optimal conditions using eH]-pregnenolone as the substrate. [3H]-Progesterone was the only product obtained after a 20 min incubation and this was purified by TLC.

inhibited the response obtained with FSH stimulation. In addition to the above observations, Hsueh and Jones[72) have shown that LHRH also stimulated the 200:-hydroxysteroid dehydrogenase, indicating

Table 2. Summary of effects of LHRH agonist and FSH on steroidogenic enzymes in granulosa cells after 72 h in culture Enzyme

FSH

Fold-stimulation of control activity LHRH-A FSH LHRH-A + FSH 3.6

4.0 3.3

31.7 6.0 29.8

17.4 3.0 7.3

1. H. DORRINGTON et al.

28

that the inhibition of FSH-stimulated progesterone production might in part be due to increased progesterone metabolism to 201X-hydroxypregn-4-en-3one. Our measurements of progesterone and 201X-hydroxypregn-4-en-3-one also suggest enhanced metabolism of progesterone in cells stimulated with LHRH-A alone or in combination with FSH. In addition to its effects on steroidogenic enzymes, LHRH-A also suppressed the FSH-stimulated increase in eH]-3',5'-AMP binding activity. This parameter was slightly stimulated by the addition of LHRH-A alone. The mechanism(s) by which LHRH-A inhibits FSH-stimulated end responses while being capable of independently stimulating the same processes is at least partially understood. Knecht and Catt[73J have suggested that the inhibitory effects of LHRH are attributable to a reduction in cyclic AMP accumulation, resulting from inhibition of the FSH-stimulated decrease in activity of cyclic nucleotide phosphodiesterase (PDE) and to inhibition of the FSHstimulated increase in adenylate cyclase activity. The effect of LHRH-A in vitro to prevent the decrease in PDE activity was apparent only after 24 and 48 h of treatment, as determined from measurements of cyclic AMP accumulation [73]. It was not reported whether LHRH-A caused an elevation in PDE levels above basal levels, in the absence of FSH action. It is therefore possible that LHRH-A acts in this regard to inhibit FSH action rather than to increase PDE activity. A LHRH facilitated decrease in FSH-stimulated adenylate cyclase activity was also observed after 24 h [73J, and has since been attributed to a decrease in the FSH receptor content and to an inhibition of the FSH-regulated increase in its own receptor [74]. In order to further investigate the mechanisms by which LHRH-A causes its effects, we have looked at the interaction of FSH and LHRH-A on cyclic AMP accumulation over 4 h (Table 3). The in vitro model in

which cells were cultured for 18 h before treatment was found to be most suitable for relating cyclic AMP production to subsequent steroidogenic responses since these occurred after only a brief delay. Using this model, FSH caused a rapid accumulation of cyclic AMP with maximal levels at 30-60 min and declining production over 2-4 h. LHRH-A (10- 7 M) partially inhibited the relatively large cyclic AMP responses to FSH over 0-120 min. Stimulation with LHRH-A alone caused no detectable increase in cyclic AMP over 0-60 min, but very low levels were detectable for the periods 60-120 min and 120-240 min. These levels were significantly greater than the levels in untreated cells over the same periods. The phosphodiesterase inhibitor (MIX) did not influence the FSH-stimulated cyclic AMP accumulation over 0-120 min, but did enhance the levels of cyclic AMP accumulation over 120-240 min. In the presence of MIX, LHRH-A caused low levels of cyclic AMP accumulation to be detected at each incubation period from 0 to 4 h. These cyclic AMP levels were similar to those levels in untreated cultures at 0-30 min, but were significantly greater than control levels at later incubation periods when cyclic AMP accumulation in control cultures had declined. MIX also partially reversed the inhibitory effect of LHRH-A on FSH-stimulated cyclic AMP accumulation for each incubation period. In other experiments, in which cells were treated at the time of cell plating, we have observed that LHRH-A continued to inhibit FSH-stimulated cyclic AMP accumulation at 24, 48 and 72 h of culture. Also, LHRH-A alone caused higher levels of cyclic AMP accumulation than in control cells over the same periods. Our findings during the first 4 h following stimulation by FSH or FSH plus LHRH-A are consistent with observations by Knecht et al.[75J using a different culture regime and a different LHRH analog. Their observations also showed that the LHRH analog caused a marked decline in FSH-stimulated cyclic AMP accumulation

Table 3. Effects of FSH, LHRH-A and MIX on the rate of cyclic AMP accumulation by rat granulosa cells in culture

Treatments None FSH LHRH-A FSH + LHRH-A MIX FSH + MIX LHRH-A + MIX FSH + LHRH-A + MIX

Period from time of treatment (min) 0-30 30-60 6b-120 120-240 (pmol cyclic AMP accumulated min - 1 mg protein - 1) 0.201 ± 0.053 16.3 ± 3.1 ND 5.36 ± 0.20 0.160 ± 0.028 16.7 ± 0.7 0.233 ± 0.028 7.99 ± 0.32

ND 16.9 ± l.l ND 10.8 ± 0.4 0.033 ± 0.033 16.2 ± l.l 0.089 ± 0.011 13.0 ± 0.06

ND 9.11 ± 0.09 0.0196 ± 0.0[03 6.75 ± 0.11 0.016 ± 0.007 9.53 ± 0.51 0.120 ± 0.042 8.77 ± 0.35

0.040 1.81 0.156 2.82 0.101 3.09 0.235 3.76

± 0.013 ± 0.13 ± 0.013 ± 0.36 ± 0.013 ± 0.07 ± 0.Dl8

± 0.36

Cells were cultured for 18 h without treatment, at which time the medium was replaced with fresh medium and the treatments made: NlH-FSH-14 (500 ng/mI); LHRH-A (10- 7 M); MIX (0.4 mM). Medium was removed at the end of each incubation period and assayed for cyclic AMP. Fresh medium was added for subsequent incubation periods. ND = not detectable.

Granulosa cell differentiation between 30-48 h, and that this inhibitory action was blocked by MIX. These results add further support to their suggestion that LHRH acts primarily to inhibit the late effects of FSH, by a mechanism involving increased cyclic AMP catabolism. In addition, our results indicate a small stimulatory effect of LHRH-A alone on cyclic AMP accumulation in rat granulosa cells. The same LHRH agonist was reported to cause a transient inhibition of basal cyclic AMP accumulation at I h in porcine granulosa cells in culture [76]. LHRH-A alone has been shown to mimic FSH action by stimulating steroidogenic enzyme activities, although the responses were considerably less than with FSH. This action of LHRH-A would be consistent with an increase in cyclic AMP production, since cyclic AMP appears to mediate the actions of LH and FSH on steroid synthesis [77]. However, the effect of LHRH-A on cyclic AMP accumulation was very small in comparison with the stimulation obtained with FSH, and the increased cyclic AMP levels obtained with LHRH-A were detectable only later than I h after treatment, or earlier if MIX was present. It is not known whether the mechanisms for stimulation of cyclic AMP production differ for LHRH-A and FSH, although quantitatively the effects were very different. The very low levels of cyclic AMP accumulating after LHRH-A treatment could be sufficient to obtain minimal steroidogenic responses, since it has been previously demonstrated in Leydig cells that stimulation of testosterone production by hCG occurs at concentrations well below that required to obtain even detectable increases in cyclic AMP [78, 79]. Similar observations with Sertoli cells indicate that FSH or cholera toxin could stimulate several late responses at concentrations which did not elicit detectable cyclic AMP production [80]. It cannot be ruled out that other cyclic AMP-independent mechanisms may also be involved in the action of LHRH. The stimulatory effect of LHRH on 201X-hydroxysteroid dehydrogenase activity in rat granulosa cells [72] is probably not mediated by cyclic AMP since the response to FSH is less than that to LHRH, while FSH is able to stimulate much greater cyclic AMP production. It is also not known whether the inhibitory effects of LHRH-A on FSH action are mediated via similar mechanisms to that involved in the stimulatory properties. In view of the stimulatory action of LHRH-A, it is conceivable that LHRH-A is antagonistic to FSH action by a mechanism in which LHRHreceptor and FSH-receptor complexes compete for the activation of adenylate cyclase. The less effective activation obtained with LHRH could thus be inhibitory in the presence of FSH. This mechanism would be consistent with the effect of LHRH agonist to reduce the sensitivity of rat luteal cells to stimulation by hCG or epinephrine, without reducing the maximal responses [81]. Such a mechanism might also account for inhibition of the stimulatory effect of cholera toxin which involves activation of adenylate cycS.B. 19-I(A)'-'"

29

lase by a mechanism different from that of FSH. In contrast with effects on other steroidogenic enzymes, the stimulation of the 20a-hydroxysteroid dehydrogenase by FSH was not inhibited by concomitant treatment with LHRH, but rather was enhanced further [72]. However, regulation of this enzyme is unusual in that it is only stimulated to a small extent by FSH action [72]. The inhibition of steroid synthesis by LHRH-A appears to show some specificity, since PGE-stimulated synthesis of progesterone, 20a-hydroxypregn-4en-3-one and l7p-estradiol in rat granulosa cells cultured for 48 h was not inhibited (Gore-Langton and Reddoch, unpublished observations). This lack of effect of LHRH-A may be due to maintained high levels of POE activity in PGE-stimulated cultures, since MIX greatly enhanced the responses to PGE 2 alone or in combination with LHRH-A. There was some inhibition by LHRH-A of cyclic AMP accumulation in the culture medium assayed after 48 h of culture. Ranta el al.[74] have reported that LHRH-A fails to inhibit the stimulation of adenylate cyclase by PGE or isoproterenol. In summary, various actions of LHRH have been demonstrated on rat granulosa cells in culture, these effects being either inhibitory in the presence of FSH or stimulatory when LHRH is added alone. The direct inhibitory actions on FSH-induced steroid synthesis may contribute to the observed inhibition of reproductive processes in vivo by exogenously administered LHRH or LHRH agonist, although other indirect mechanisms may also be acting. REFERENCES

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