Intraovarian mechanisms in the hormonal control of granulosa cell differentiation in rats A. J. W. Hsueh, P. B. C. Jones, E. Y. Adashi, Christina Wang, L.-Z. Zhuang and T. H. Welsh, Jr Department of Reproductive Medicine, M-025, University of California, San Diego, La Jolla, California 92093, U.S.A.

Summary. The present review summarizes our studies on the hormonal control of the differentiation of granulosa cells in vitro. Studies on the hormonal regulation of granulosa cell oestrogen and progestagen biosynthesis by various regulatory agents facilitate our understanding of the control processes involved in follicular maturation and luteinization in vivo. This multiple hormonal control mechanism also provides an interesting model for future studies on the diverse mode of hormone action.

Introduction Ovarian follicles have been shown to be the basic functional unit of the and consist of an outer layer of theca cells which encircle inner layers of granulosa cells. The granulosa cells, in turn, surround the innermost -cumulus cell complex. In response to cyclic pituitary gonadotrophin secretion, the various follicular compartments interact in a highly integrated manner to secrete sex steroids (oestrogens and progestagens) and to produce a fertilizable ovum. occurs in one of the two during a given cycle in many species. Although pituitary gonadotrophins (LH, FSH and prolactin) are the major regulators of follicular development (Richards & Midgley, 1976) and the blood concentrations of these hormones perfusing the two ovaries are identical, not all follicles respond to pituitary gonadotrophins during a given cycle. Only a limited number of the 'selected' follicles ovulate during the life span of the females, while most of the follicles become atretic. After ovulation, the granulosa cells undergo profound changes in their hormonal responsiveness and their capacity to produce steroids. These luteinized granulosa cells constitute the major component of the corpora lutea and are the main source of ovarian progestagens. In rats, the follicular granulosa cells transform from an FSH-dominated cell type to a cell type mainly controlled by LH and prolactin. To understand the basis for the disparate maturation of ovarian follicles and the luteinization of maturing granulosa cells, functional and morphological correlates of intraovarian changes in different hormonal environments are required. In the present review, our attempts to elucidate intraovarian control mechanisms during the differentiation of granulosa cells to luteal cells in vitro will be discussed. Since several extensive reviews on the hormonal regulation of granulosa cell functions are available (Channing, 1970; Richards & Midgley, 1976; Bjersing, 1978; Richards, 1979 ; Dorrington & Armstrong, 1979 ; Hillier, Zeleznik, Knazek & Ross, 1980), we do not intend to provide a comprehensive survey of the literature on in-vivo studies concerning the hormonal control of follicle maturation. Instead, we will summarize the results from our laboratory concerning the hormonal regulation of cultured granulosa cells in vitro.

0022-4251 /83/050325-18S02-00/0 © 1983 Journals of Reproduction & Fertility Ltd

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access Granulosa cell differentiation: induction of hormonal responsiveness in vitro

To study the hormonal control of the functions of granulosa cells during a given stage of development in a defined experimental condition, it is necessary to synchronize the maturation of a large group of follicles to obtain a homogeneous population of granulosa cells as well as to establish a functional culture system for in-vitro studies. An experimental model was set up to yield large numbers of relatively undifferentiated granulosa cells from preantral follicles of immature hypophysectomized rats implanted with oestrogen (diethylstilboestrol or oestradiol) capsules (Richards & Midgley, 1976; Hillier et al., 1980). Hypophysectomy eliminates possible influences of endogenous gonadotrophins on ovarian granulosa cells while treatment with oestrogens enhances the proliferation of granulosa cells. The follicular granulosa cells are completely separated from blood vessels and other cell types by the basement membrane lining the follicle; hence, it is relatively easy to isolate pure granulosa cells. Bjersing & Carstensen (1964) performed short-term incubation of granulosa cells and demonstrated the steroidogenic capacity of these cells in vitro. Several groups further modified this procedure and cultured the 'pure' granulosa cells in serum-containing medium to study progestagen biosynthesis (Channing, 1970). Rat granulosa cells cultured in serum-containing media, however, do not respond to gonadotrophins by the induction of aromatases or gonadotrophin receptors (Nimrod, Tsafriri & Lindner, 1977). This is presumably due to the presence of uncharacterized serum inhibitory factors, e.g. epidermal growth factor (Hsueh, Welsh & Jones, 1981), which interfere with FSH action. Further improvement of the culture conditions was achieved by use of serum-free culture conditions. Primary cultures of granulosa cells maintained in serum-free medium retain hormonal responsiveness in vitro (Dorrington, Moon & Armstrong, 1975; Erickson & Hsueh, 1978a) and undergo hormonal differentiation in a way similar to that in vivo. The preantral follicles obtained from immature hypophysectomized, oestrogen-treated rats contain multiple layers of granulosa cells and a fully grown oocyte which is arrested in the dictyate stage of meiotic prophase. These granulosa cells possess cell membrane receptors for FSH and intracellular receptors for oestrogen, and other steroids (Richards, 1975, 1979 ; Schreiber & Ross, 1976; Schreiber & Hsueh, 1979). In response to FSH treatment in vitro, the granulosa cells luteinize to become cells resembling those obtained from mature antral follicles and the . The maturation and differentiation of the cultured granulosa cells to 'granulosa-luteal' cells is accompanied by sequential acquisition of hormone receptors and profound changes in the hormonal responsiveness of these cells. We have studied the in-vitro differentiation of granulosa cells to 'granulosa-luteal' cells by investigating changes in hormone receptor content using radioligand receptor assay. We also studied the hormonal regulation of the steroidogenic capacity of these cells by measuring oestrogen and progestagen biosynthesis. In the primary cultures of granulosa cells, treatment with FSH and A4- increases the formation of oestrogen, and 20a-dihydroprogesterone (Wang, Hsueh & Erickson, 1979). These cultured cells contain all the necessary enzymes for de-novo biosynthesis of progesterone by the cholesterol—pregnenolone biosynthetic pathway (Text-fig. 1). FSH treatment presumably increases cholesterol through de-novo synthesis or enhanced utilization of serum lipoproteins. This is accompanied by increases in the activities of cholesterol side-chain cleavage enzymes (which convert cholesterol to pregnenolone) in the mitochondria and 3ß-hydroxysteroid dehydrogenase (3ß-HSD)/A5-A4-isomerase (which converts pregnenolone to progesterone) in the microsomal fraction (Jones & Hsueh, 1982a, b). Since isomerase activity appears to be in excess, production of progesterone from pregnenolone is mainly regulated by 3ß-HSD. The bioactive progesterone may be further metabolized by 20a-hydroxysteroid dehydrogenase (20a-HSD) to a much less active progestagen, 20a-dihydroprogesterone. In contrast to theca cells, granulosa cells possess negligible concentrations of 17a-hydroxylase or 17-20 desmolase to convert progestagens into . Treatment of granulosa cells with FSH,

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access CELL MEMBRANE

CHOLESTEROL MITOCHONDRIA ESTER SMOOTH ENDOPLASMIC RETICULUM

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ESTROGENS

Text-fig. 1. The steroidogenic pathway involved in progestagen and oestrogen biosynthesis in the granulosa cell. 20a-OH-P = 20a-dihydroprogesterone. however, induces high concentrations of aromatase enzymes which convert androgens to oestrogens (Text-fig. 1). Furthermore, granulosa cells also contain 17ß-hydroxysteroid dehydrogen¬ ase which converts androstenedione to testosterone or oestrone to oestradiol. In most studies described below, androstenedione was added to the culture medium to serve as the substrate for aromatases and the production of total oestrogens was measured. In addition to its role as aromatase substrate, androgens have also been shown to augment FSH-stimulated progestagen production through an androgen receptor-mediated mechanism (Daniel & Armstrong, 1980; Hillier & deZwart, 1981). In contrast to the stimulatory actions of FSH, treatment of granulosa cells from preantral follicles with LH/hCG, prolactin or adrenergic agents does not enhance oestrogen or progestagen biosynthesis (Wang et al., 1979). In addition, radioligand receptor studies also demonstrate a low concentration of receptors for LH/hCG (Erickson, Wang & Hsueh, 1979) or prolactin (Navickis, Jones & Hsueh, 1982) in these cells. However, FSH treatment of granulosa cells for 2 days in vitro increases the concentration of LH and prolactin receptors and the induction of receptors for these peptide hormones is accompanied by an enhancement in the ability of LH and prolactin to stimulate steroidogenesis (Text-fig. 2). Subsequent treatment of these FSH-primed cells with LH increases oestrogen and progestagen production (Wang et al., 1981). In contrast, treatment with prolactin stimulates progesterone production without affecting aromatase activity (Wang et al., 1979). This stimulatory action of prolactin is shared by human growth hormone, indicating that the actions of these protein hormones are mediated through lactogenic receptors. In FSH-primed granulosa cells, treatment with prolactin also enhances the formation of LH receptors (Jones & Hsueh, 1981b). Conversely, treatment with LH maintains prolactin receptors in FSH-primed granulosa-luteal cells (Navickis et al., 1982). FSH treatment in vitro also increases the responsiveness of granulosa cells to adrenergic agents. In FSH-primed cells, treatment with ( —)epinephrine, ( —)norepinephrine, ( )isoproterenol (a ß- — adrenergic agonist) or terbutaline (a selective ß2-adrenergic agonist) stimulates progesterone production without affecting aromatase activity or LH receptor content (Adashi & Hsueh, 1981). In contrast, treatment with carbamylcholine (a cholinergic agonist), methoxamine (an a- adrenergic agonist) or dobutamine (a selective ß !-adrenergic agonist) is without effect on progesterone biosynthesis. Furthermore, progesterone production induced by epinephrine is blocked by a selective ß2-antagonist (IPS 339) but a ß,-antagonist (practolol) has only about 014%

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access activity. These results suggest that FSH treatment induces an increase in the responsiveness of granulosa cells to ß2-adrenergic agents. The cultured granulosa cells therefore undergo differentiation in response to FSH treatment in vitro and acquire multiple hormone receptors (Text-fig. 2). Although FSH-primed granulosa cells closely resemble luteal cells, we have termed these FSH-primed cells as 'granulosa-luteal' cells because theca cells probably also contribute to formation of the rat corpus luteum.

FSH m PREANTRAL FOLLICLES

FSH FSH

ANTRAL ti LH Adrenergic FOLLICLES agents Î200-OH-P R PRL Adrenergic PRL agents

tP t f PRL® t LH® Text-fig. 2. Hormone-induced differentiation of cultured granulosa cells into granulosa-luteal cells. R = receptors; PRL = prolactin; E = oestrogens; = progesterone; 20 - - = 20a- dihydroprogesterone.

Hormonal regulation of oestrogen biosynthesis Oestrogen production by ovarian follicles plays an important role in the co-ordination of reproductive cycles of the female. Not only do ovarian oestrogens regulate the release of pituitary gonadotrophins, these steroid hormones are also important for the growth and differentiation of female accessory sex organs. In addition, oestrogens are essential for the development of ovarian follicles through local actions. In granulosa cells, oestrogens increase intercellular gap junction formation, stimulate oestrogen receptor content and enhance cellular proliferation. Oestrogens also synergize with FSH in the formation of the follicular antrum and granulosa cell LH receptors. Therefore, studies on the hormonal regulation of granulosa cell aromatase activity are essential for the elucidation of follicular maturation.

FSH and LH As discussed earlier, FSH is the prime inducer of aromatases and treatment with FSH increases the ability of granulosa cells to convert androgens to oestrogens. Since granulosa cells lack the

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access enzymes required to convert progestagens into androgens, these cells depend upon theca cells to provide the substrate for aromatases. Ovarian FSH receptors have been shown to be present exclusively in the granulosa cells (Nimrod, Erickson & Ryan, 1976) while LH treatment significantly enhances theca cell androgen production (Leung & Armstrong, 1980). Since optimal oestrogen biosynthesis in hypophysectomized animals requires treatment with both FSH and LH (Simpson, Li & Evans, 1951), as well as the participation of the two ovarian cell types (theca and granulosa; Falck, 1959), a 2-cell-2-gonadotrophin hypothesis has been formulated to explain the mechanism of oestrogen production (Short, 1962; Baird, 1977; Moor, 1977; Dorrington & Armstrong, 1979). Although all our in-vitro studies were performed with saturating concentrations of androgens, the extrapolation of these in-vitro findings to the in-vivo state should consider the distinct possibility that availability of the androgen substrates may be subject to additional hormonal regulation. FSH induces aromatase activity with a lag time of 24 h and in a dose-dependent manner (Erickson & Hsueh, 1978a). Furthermore, the stimulatory effect of FSH is blocked by actinomycin D or cycloheximide, suggesting the involvement of RNA and protein synthesis in the induction process (Wang et al., 1982). Cultures of human granulosa cells from 4-6 mm follicles of normal and polycystic ovaries also produce oestrogen in response to FSH treatment (Erickson, Hsueh, Quigley, Rebar & Yen, 1979). Treatment with FSH derived from various species (sheep, rat, human, pig) also stimulates aromatase activity in rat granulosa cells. The responsiveness of rat granulosa cells to FSH is further enhanced by the concomitant addition of a phosphodiesterase inhibitor, resulting in an assay sensitivity which is approximately 2000-fold greater than that of the classical Steelman- Pohley assay for FSH (Hsueh & Erickson, 1978a). Further refinement of this system should contribute to the development of a sensitive in-vitro bioassay for FSH. Since FSH also stimulates cAMP production by granulosa cells, we tested the influence of cAMP analogues and cAMP-inducing agents (cholera toxin and prostaglandin E-2) on aromatase activity. Like FSH, these agents also stimulate aromatase activity, indicating the possible mediatory role of cAMP in the action of FSH (Wang et ai, 1982). As mentioned earlier, treatment with FSH increases LH receptor content in cultured granulosa cells. In FSH-primed cells, LH treatment maintains oestrogen production at the level previously induced by FSH, but does not increase it further (Wang et al., 1981). Since highly purified LH was used, the LH action cannot be explained by the degree of FSH contamination of the LH preparation. It therefore appears possible that FSH induction of aromatase activity is important for oestrogen production by granulosa cells of the preantral follicles. Once LH receptors have been induced in granulosa cells of the mature antral follicles, LH may assume a dominant role in controlling granulosa cell oestrogen biosynthesis. During the preovulatory period, LH alone may be sufficient to stimulate theca androgen biosynthesis as well as to maintain granulosa cell aromatase activity, resulting in the preovulatory oestrogen surge. These studies further modify the 2-cell-2- gonadotrophin theory for oestrogen biosynthesis and emphasize the important role (and possibly the sole action) of LH during the preovulatory stage of follicular maturation.

Prolactin Although LH treatment of FSH-primed granulosa cells stimulates both oestrogen and progestagen production, prolactin treatment increases progestagen production but not aromatase activity. This result indicates the existence of divergent biosynthetic pathways for oestrogens and progestagens in the granulosa cell. Also, it is possible that FSH induces heterogeneous subpopulations of granulosa cells with different hormonal responsiveness. In FSH-primed granulosa cells, treatment with prolactin inhibits basal aromatase activity in a dose-dependent manner (Wang et al., 1980). Under a high dose (1 µg/ml) of prolactin, oestrogen formation is suppressed by > 90%. Treatment of FSH-primed cells with FSH or LH augments aromatase activity while concomitant prolactin treatment suppresses the aromatase activity

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access induced by the gonadotrophins. This finding suggests a direct inhibitory action of prolactin upon the granulosa cells. Thus, in addition to its well-recognized luteotrophic action in the rat corpus luteum, prolactin may also exert important regulatory actions upon oestrogen production by the developing follicles. Since oestrogens increase prolactin production by the pituitary, these studies further suggest the existence of a negative feedback mechanism by which the production of ovarian oestrogen and pituitary prolactin is regulated. The observed prolactin suppression of aromatase activity may also explain the reduction of ovarian oestrogen production during hyperprolactinemic states. These in-vitro studies were confirmed in hypophysectomized rats and pro-oestrous rats (Dorrington & Gore-Langton, 1981 ; Wang & Chan, 1982) and the effect may be mediated through a prolactin suppression of cAMP production by the granulosa cells.

Oestrogens Although treatment with oestrogens alone does not affect granulosa cell aromatase activity, our recent studies indicate an important role of oestrogens in enhancing the gonadotrophin stimulation of granulosa cell oestrogen production (Adashi & Hsueh, 1982; Zhuang, Adashi & Hsueh, 1982). Treatment with 10 ng ovine FSH/ml for 3 days stimulates aromatase activity while concomitant addition of synthetic oestrogens (hexoestrol > moxoestrol > ethinyl oestradiol » chlorotrianisene and mestranol), or natural oestrogens (oestradiol-17ß = oestrone > oestradiol-17a » oestriol) enhances FSH-stimulated aromatase activity in a dose-dependent manner. Oestradiol-17ß stimulates FSH action with an ED50 value of 9 -9 M (Adashi & Hsueh, 1982), which is within the physiological levels of follicular oestradiol (Baird & Fraser, 1975). In FSH-primed granulosa cells, treatment with oestrogen also enhances LH-stimulated aromatase activity (Zhuang et al., 1982). These in-vitro findings suggest that oestrogens within the microenvironment of ovarian follicles may exert a local autoregulatory effect on their own production via an ultra-short loop positive feedback mechanism. This self-amplification of oestrogen production may be important in maintenance of the dominant follicle(s). It has been suggested that the selected follicle(s), by way of its oestrogen production, reduces FSH levels, thereby impeding the maturation of other follicles (Zeleznik, 1981). However, it is not known how the selected follicle itself continues to mature in the face of declining FSH levels. Our present findings suggest that intrafollicular oestrogens enhance the actions of FSH to stimulate aromatases. Once a 'chosen' follicle(s) is producing a critical amount of oestrogens, it then has the capacity to produce more oestrogens than other maturing follicles and the selection of the dominant follicle(s) is secured. In contrast, follicles destined to undergo atresia may not be able to produce enough follicular oestrogens to counteract the adverse effects of FSH deprivation. Since oestrogen treatment enhances aromatase activity induced by both FSH and LH, this autoregulatory ovarian positive feedback mechanism is essential for the initiation of the preovulatory surge of oestrogens. Thus, oestrogens secreted by the dominant follicles not only exert positive feedback action at the hypothalamic-pituitary unit to increase LH and FSH release, but may also act through intraovarian positive feedback mechanisms. This resultant 'cascading' feedback forms a closed-loop positive-control mechanism which is responsible for the preovulatory surges of pituitary gonadotrophins and ovarian oestrogens as well as the ultimate ovulation process. Administration of clomiphene citrate to women with anovulatory infertility increases the circulating levels of both FSH and LH, leading to follicular maturation and ovulation. It has been postulated that the release of pituitary gonadotrophins by clomiphene citrate represents an anti- oestrogenic action of this agent through competition with oestrogen for pituitary and/or hypothalamic oestrogen receptors. Treatment with clomiphene citrate also enhances aromatase activity in granulosa cells stimulated by FSH and LH in vitro (Zhuang et al, 1982). Thus, clomiphene citrate acts as an oestrogen rather than an antioestrogen at the granulosa cells. This compound may act independently of the hypothalamic-pituitary unit to enhance the induction and

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access maintenance of ovarian oestrogen production by LH and FSH. Although the clinical efficacy of clomiphene citrate may derive partially from its interaction with the pituitary and in initiating gonadotrophin release, our findings indicate that clomiphene citrate also directly augments the gonadotrophin-stimulated production of oestrogens at the ovarian level.

Gn-RH

Treatment with Gn-RH and its agonists in vivo causes paradoxical inhibitions of both female and male reproductive functions (Hsueh & Jones, 1981, 1982). We have demonstrated that Gn-RH may exert an extrapituitary, direct inhibitory action on ovarian granulosa cells (Hsueh & Erickson, 1979; Hsueh & Ling, 1979). The stimulatory effect of FSH and LH on aromatase activity of the cultured granulosa cells is blocked by concomitant treatment with Gn-RH or its agonists in a dose- dependent manner. Furthermore, the inhibitory effect of Gn-RH is prevented by concomitant addition of a potent Gn-RH antagonist, suggesting that the action of these peptides is mediated through stringent stereospecific ovarian recognition sites. Furthermore, Gn-RH treatment inhibits aromatase activity induced by cAMP analogues and cAMP-inducing agents (cholera toxin and prostaglandin E-2) in the cultured granulosa cells (Jones & Hsueh, 1981b; Gore-Langton, Lacroix & Dorrington, 1981). The inhibitory action of Gn-RH in vitro was confirmed in vivo in hypophysectomized rats treated with FSH and Gn-RH (Hsueh, Wang & Erickson, 1980). These findings serve as the basis to explain the observed paradoxical inhibitory effect of Gn-RH and its agonists on ovarian oestrogen production and various female reproductive functions in vivo (for review, see Hsueh & Jones, 1981).

Epidermal growth factor This factor, EGF, has long been known for its mitogenic action in a variety of epidermal and non-epidermal cells (Carpenter & Cohen, 1979). Since EGF is present in the serum and serum- containing media have been shown to attenuate the FSH stimulation of granulosa cell functions, we studied the effect of EGF under serum-free culture conditions. EGF, but not fibroblast growth factor, inhibits the FSH stimulation of aromatase activity in granulosa cells in a dose-dependent manner with an EDS0 value of ~0-2 ng EGF/ml. Higher concentrations of EGF inhibit FSH action by 70-90% (Hsueh et ai, 1981 ; Jones, Welsh & Hsueh, 1982). Similarly, other investigators have demonstrated the ability of EGF to inhibit the FSH stimulation of LH receptor content in cultured granulosa cells (Mondschein & Schomberg, 1981a). These findings, along with the reported stimulatory effect of androgens upon EGF synthesis by the mouse submaxillary gland (Barthe, Bullock, Mowszowicz, Bardin & Orth, 1974), raise intriguing questions regarding a possible endocrine role of EGF in the regulation of reproductive functions.

Glucocorticoids Hyperactivity of the causes disturbances in female reproductive functions. Administration of pharmacological doses of glucocorticoids blocks ovulation in women and laboratory animals as well as delays implantation in rodents (Hagino, 1972). Although glucocorticoids may act at the hypothalamic-pituitary level to suppress gonadotrophin release, one cannot rule out possible direct effects of the adrenal steroids on the ovary. In granulosa cells, treatment with cortisol or dexamethasone inhibits the FSH-induced increases in aromatase activity (Hsueh & Erickson, 1978b). Cortisol at "6 M inhibits oestrogen production by 50% whereas dexamethasone is effective at 10~8 m. Thus, glucocorticoids may directly affect ovarian oestrogen production by acting at the granulosa cell level through specific glucocorticoid receptors (Louvet, Baislic, Bayard & Boulard, 1977). The inhibitory effect of glucocorticoids upon oestrogen pro¬ duction does not represent a general suppression of granulosa cell functions because progesterone

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access production by these cells was increased by glucocorticoid treatment (Adashi, Jones & Hsueh, 1981). Since the effective doses of glucocorticoids were higher than circulating levels of these steroids, these findings indicate that glucocorticoids may not interfere with normal ovarian oestrogen production under physiological conditions. However, prolonged hyperactivity of the adrenal gland during stressed states and the use of pharmacological doses of dexamethasone for the therapy of hypoadrenalism may result in a direct suppression of ovarian oestrogen biosynthesis.

Hormonal regulation of progestagen biosynthesis—modulation of steroidogenic enzymes FSH and LH As discussed earlier, FSH treatment stimulates progesterone production by cultured granulosa cells. The FSH modulation of various steroidogenic enzymes involved in progestagen biosynthesis has been studied (Text-fig. 3a). FSH appears to stimulate progestagen production (i.e. progesterone plus 20a-dihydroprogesterone production) by stimulating pregnenolone biosynthesis (probably at the side-chain cleavage (SCC) enzyme step; Jones & Hsueh, 1982b), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activity (Jones & Hsueh, 1982a). FSH also causes a small but significant increase in the activity of 20a-hydroxysteroid dehydrogenase (20a-HSD). In the presence of cyanoketone, an inhibitor of 3ß-HSD, FSH stimulates pregnenolone production in a dose-dependent manner (ED50 ~007 NIH-FSH-S1 mi.u./ml) compared with un¬ treated controls. Additionally, 25-hydroxycholesterol, a soluble substrate for the SCC enzymes, enhances FSH-stimulated, but not basal, pregnenolone production, suggesting that FSH treatment increases the activity of side-chain cleavage enzymes. FSH treatment for 2 days also increases 3ß- HSD activity by ~ 6-fold in a dose-dependent manner with an ED50 value of ~0-06 NIH-FSH-S1 mi.u./ml (Jones & Hsueh, 1982a). Since serum FSH concentrations have been shown to vary from 001 to 1 0 NIH-FSH-S1 mi.u./ml during the rat oestrous cycle (Smith, Freeman & Neill, 1975), the FSH stimulation of pregnenolone biosynthesis and 3ß-HSD activity observed in the cultured granulosa cells probably reflects physiological phenomena. Treatment with LH/hCG stimulates progestagen production by FSH-primed granulosa cells. The mechanism by which LH/hCG exerts its action in the granulosa cell has also been studied (P. B. C. Jones & A. J. W. Hsueh, unpublished observation). LH/hCG increases progestagen production by stimulating pregnenolone biosynthesis and 3ß-HSD activity. In the presence of cyanoketone, hCG stimulates pregnenolone production by 3-4-fold during a 2-day incubation. Treatment with hCG also stimulates 3ß-HSD activity by ~ 2-fold, while hCG does not affect 20a- HSD activity.

Cholesterol ...t..) ß u c »· |SCC 3ß_HSD Pregnenolone—£ ~ Progesterone Pregnenolone-* Progesterone 20a-HSD 20a-HSD

20a -hydroxypregn-4-en-3-one 20a -hydroxypregn-4-en-3-one Text-fig. 3. Mechanisms by which (a) FSH and Gn-RH and (b) prolactin and ß2-adrenergic agents regulate progestagen biosynthesis in cultured granulosa cells. In (b) the granulosa cells have been primed with FSH and the inhibitory action of Gn-RH is also shown. SCC = side- chain cleavage enzyme; 3ß-HSD = 3ß-hydroxysteroid dehydrogenase; 20a-HSD = 20a- hydroxysteroid dehydrogenase; 20a-hydroxypregn-4-en-3-one (20a-OH-P) = 20a-dihydro- progesterone ; R = receptors.

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access Prolactin As discussed earlier, in-vitro treatment with FSH increases the steroidogenic responsiveness of granulosa cells to prolactin. Prolactin-stimulated progesterone production appears to be the result of stimulation of pregnenolone biosynthesis and 3ß-HSD activity, as well as an inhibition of 20a- HSD activity (Text-fig. 3b). Prolactin stimulates pregnenolone production in a dose-dependent manner with a minimal effective dose of ~9 mi.u./ml (P. B. C. Jones & A. J. W. Hsueh, unpublished data). It is unlikely that prolactin-stimulated pregnenolone production contributes significantly to progesterone production during the rat oestrous cycle since serum prolactin levels increase to a maximal concentration of ~4 mi.u./ml (Smith et al., 1975). However, it is possible that prolactin-stimulated pregnenolone production may be important during early pregnancy when serum prolactin levels increase to ~11 mi.u./ml (Beattie et al., 1977). Prolactin treatment for 2 days also stimulates 3ß-HSD activity by 2-fold. In contrast, prolactin treatment inhibits 20a-HSD activity (maximal inhibition; ~90% decrease) with an ED50 value of 0-99 mi.u./ml (Jones & Hsueh, 1981a, d), suggesting that the major mechanism by which prolactin increases apparent progesterone production is through the inhibition of progesterone breakdown. In-vivo studies also suggest that the major effect of prolactin upon luteal steroidogenesis is due to the inhibition of progesterone metabolism (Wiest, Kidwell & Balogh, 1968; Lamprecht, Lindner & Strauss, 1969).

Adrenergic agents As discussed earlier, treatment with FSH increases the responsiveness of granulosa cells to catecholamines (Adashi & Hsueh, 1981). Adrenergic agents, specifically ß2-adrenergic agonists, appear to stimulate progesterone production by increasing pregnenolone biosynthesis and 3ß-HSD activity, as well as by inhibiting 20a-HSD activity (Text-fig. 3b). Isoproterenol treatment inhibits 20a-HSD activity in a dose-dependent manner (ED50, 3-4 10~10 m). Similar inhibition of 20 - HSD activity was observed after treatment with epinephrine, norepinephrine, and a ß2-adrenergic agonist (terbutaline sulphate), whereas a ß ¡-adrenergic agonist (dobutamine) is less effective. Furthermore, the inhibitory effect of epinephrine is blocked by concomitant treatment with a ß2- adrenergic antagonist (IPS 339) but not by a ßi-antagonist (practolol) (Jones & Hsueh, 1981d). Although there are no extensive studies on the regulation of progestagen biosynthesis by catecholamines in vivo, ovarian denervation or chemical sympathectomy decreases 3ß-HSD activity of rat CL, suggesting that catecholamines may influence 3ß-HSD activity in vivo (Burden, 1978).

Gn-RH

As discussed previously, treatment with Gn-RH or Gn-RH agonists results in the inhibition of granulosa cell oestrogen production. Similarly, these peptides suppress progesterone production stimulated by FSH, LH/hCG, prolactin, or ß2-adrenergic agonists (Jones & Hsueh, 1980, 1981a, b; Hsueh & Jones, 1981). Gn-RH inhibits FSH-stimulated progesterone production by decreasing pregnenolone production (probably at the side-chain cleavage enzyme step) and 3ß-HSD activity, as well as by increasing 20a-HSD activity (Text-fig. 3a). The major effects of Gn-RH are probably due to the decreased pregnenolone production coupled with the increased conversion of progesterone to 20a-dihydroprogesterone. Similarly, Gn-RH appears to inhibit LH/hCG- stimulated progesterone production by inhibiting pregnenolone production and by increasing 20a- HSD activity. In contrast, Gn-RH inhibits prolactin- or ß2-adrenergic agonist-stimulated progesterone production by increasing 20a-HSD activity without inhibiting pregnenolone production (Text-fig. 3b). Treatment with Gn-RH alone also modulates progestagen biosynthesis by cultured granulosa cells (Jones & Hsueh, 1981 a, c). In cultured granulosa cells, Gn-RH stimulates small but significant increases in pregnenolone production in a dose-dependent manner (ED50 ~ 30 nM) to 3-9-fold the

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access control level (Jones & Hsueh, 1982b). Gn-RH treatment for 2 days in vitro also stimulates the activities of 3ß-HSD (10 nM-Gn-RH : -40% increase) and 20a-HSD (ED50 9 µ; maximal effect at 1 µ : 9-fold increase). These stimulatory effects of Gn-RH on enzyme activities are associated with increases in the apparent maximal velocity of the enzyme and a small but significant increase in the production of progesterone and 20a-dihydroprogesterone (Jones & Hsueh, 1982a, b). Furthermore, the stimulatory effect of Gn-RH on 20a-HSD activity is prevented by concomitant treatment with a Gn-RH antagonist or cycloheximide, suggesting the involvement of specific Gn-RH receptors and the requirement of protein synthesis in the induction process (Jones & Hsueh, 1981c). The physiological significance of the stimulatory action of Gn-RH is presently unknown.

Androgens and progestagens Treatment with androgens stimulates progestagen production by cultured granulosa cells (Schomberg, Stouffer & Tyrey, 1976; Armstrong & Dorrington, 1976; Lucky, Schreiber, Hillier, Schulman & Ross, 1977). The action of androgens can be prevented by concomitant addition of antiandrogens and this process is believed to be mediated by androgen receptors found in the granulosa cells (Schreiber & Ross, 1976). Treatment with androgens also enhances FSH-stimulated progesterone production in a synergistic manner and the actions of these agents are associated with increases in the activities of side-chain cleavage enzymes (Nimrod, 1981). These studies suggest that theca androgens may play an important role in the regulation of granulosa cell progestagen biosynthesis. Fanjul, Ruiz de Galarreta & Hsueh (1983) have shown that a synthetic progestagen, R5020, has a facilitatory action on gonadotrophin-stimulated progesterone synthesis in cultured granulosa cells.

Glucocorticoids Treatment with increasing concentrations of various natural and synthetic corticoids increases FSH-stimulated progesterone production (Adashi et al., 1981). The stimulatory actions of the corticoids correlated with their glucocorticoid, rather than their mineralocorticoid, potencies. This augmentation of FSH-stimulated progesterone production may be accounted for, at least in part, by the stimulation of 3ß-HSD activity and the inhibition of 20a-HSD activity. The physiological significance of the synergistic effect of glucocorticoids on the FSH-stimulated accumulation of progesterone remains uncertain. Nevertheless, the presence of glucocorticoid receptors in rat granulosa cells (Louvet et al., 1977) and the isolation of a specific cortisol-binding globulin from porcine (Mahajan & Little, 1978) would suggest a possible role for adrenal corticoids in ovarian functions.

Epidermal growth factor Epidermal growth factor (EGF) stimulates progestagen production by cultured granulosa cells through specific membrane receptors (Hsueh, Welsh & Jones, 1981 ; Jones, Welsh & Hsueh, 1982). In contrast to the inhibition of granulosa cell aromatase activity described earlier, EGF treatment enhances FSH-stimulated 20a-dihydroprogesterone production without affecting progesterone production by granulosa cells. This is accompanied by an enhancement of FSH-stimulated pregnenolone biosynthesis and the stimulation of 3ß-HSD activity. Thus, EGF enhancement of FSH-stimulated progestagen production appears to result from synergistic increases in pregnenolone biosynthesis and 20a-hydroxysteroid dehydrogenase activity. This yields a net increase in 20a-dihydroprogesterone but not progesterone production. Furthermore, treatment with EGF alone dose-dependently increases the production of pregnenolone, progesterone and 20a-dihydroprogesterone. Although these studies provide additional evidence for possible endocrine functions of EGF, the physiological significance of these findings remains to be elucidated.

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access Phorbol ester

Phorbol esters are tetracyclic diterpenes originally identified in the seed oil of the Croton plant and are used experimentally as co-carcinogens. We have utilized 12-0-tetradecanoyl-phorbol-13- acetate (TPA), a phorbol ester with potent tumour-promoting activity, to investigate the influence of these co-carcinogens upon gonadotrophin-stimulated steroidogenesis in primary cultures of ovarian granulosa cells (Welsh & Hsueh, 1982). FSH-stimulated production of progesterone and 20a-dihydroprogesterone is suppressed in a dose-dependent fashion by TPA, whereas the phorbol ester analogue 4a-phorbol-12,13-didecanoate (4a-PDD) is inactive. The physiological relevance of these findings remains to be established ; however, the effects observed in these studies occur at dosages consistent with the reported dissociation equilibrium constant (Kd) for phorbol ester binding to rat pituitary cells (Jaken, Tashijan & Blumberg, 1981). Existence of endogenous phorbol ester-like growth promoting factors is suggested by the demon¬ stration of a phorbol ester binding protein in sera (Horowitz, Greenbaum & Weinstein, 1981; Shoyab & Todaro, 1982). Further investigation with TPA and the granulosa cell model may assist elucidation of cellular mechanisms associated with carcinogenesis or steroidogenesis.

The sensory world of granulosa cells: multiple hormone receptor systems studies reveal the regulation of the diverse functions of granulosa cells by multiple hormones and the acquisition of various hormonal receptors during the maturation of granulosa cells to luteal cells. As summarized in Text-fig. 4, receptors for many physiological and non- physiological regulators have been found in the granulosa-luteal cells. Physiological regulator receptors

.

Peptide hormone receptors (FSH, LH. PRL, GH, GnRH, EGF)

Neurotransmitter receptor 1 ( GABA) - Steroid hormone ß2 -adrenergic, receptors Granulosa-luteal cell Prostaglandin receptor — (E.P.G.T.) / 1 \ »Lipoprotein receptor (Cholera toxin) Phorbolbol A) Bacterial (Concanavalin ester;r lectin receptor toxin receptor \ / non-physiological regulator receptors Text-fig. 4. The sensory world of granulosa-luteal cells. EGF = epidermal growth factor; GH = growth hormone; E = oestrogen; = progestagen; G = glucocorticoid; = testosterone; GABA = gamma-aminobutyric acid.

Using charcoal adsorption and column Chromatographie assays, intracellular receptors for oestrogen (Richards, 1975), progestagen (Schreiber & Hsueh, 1979), androgen (Schreiber & Ross, 1976) and glucocorticoids (Louvet et al., 1977) have been demonstrated in the cytoplasmic fractions

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access of granulosa cells. These receptors are of high affinity and stereospecificity as indicated by the binding of the receptors to specific synthetic ligands (R5020 for progestagen receptors, R1881 for androgen receptors and dexamethasone for glucocorticoid receptors). After binding to respective specific ligands, these intracellular cytoplasmic receptors presumably undergo transformation and translocation to the nuclei to initiate genomic events, which ultimately alter the functions of the granulosa cell. The presence of specific receptors for various protein hormones (FSH, LH and lactogens) has been demonstrated in the plasma membrane of granulosa cells by radioligand receptor assays. Similarly, cell membrane receptors for several regulatory peptides (Gn-RH and EGF) have also been demonstrated (Jones, Conn, Marian & Hsueh, 1980 ; Jones et al., 1982) while the receptors for insulin and platelet-derived growth factor are also presumably present in these cells (May, McCarty, Reichert & Schomberg, 1980; May & Schomberg, 1981; Mondschein & Schomberg, 1981b). Receptors for two neurotransmitters have been demonstrated in the granulosa cells. Adrenergic receptors of the ß2 subtype were found in the membrane of granulosa cells using specific ligand (125I-hydroxybenzylpindolol) (Aguado, Petrovic & Ojeda, 1982) while our recent results identified the presence of binding sites for gamma-aminobutyric acid (GABA) in the rat granulosa cell by using [3H]muscimol, a specific GABA agonist, as the ligand (Schaeffer & Hsueh, 1982). The adrenergic receptors are believed to mediate the stimulation of progestagen biosynthesis by ß2- agonists (Adashi & Hsueh, 1981) while the physiological role of the ovarian GABA 'receptors' is presently unknown. It is, however, of interest to note that the majority of the ovarian GABA binding sites are associated with the granulosa cells while GABA and a GABA-synthesizing enzyme (glutamate decarboxylase) are present in high concentration in whole ovarian homogenates but not in enriched granulosa cells (Schaeffer & Hsueh, 1982). These studies suggest a potential modulation of granulosa cell functions by GABA. The possible existence of muscarinic cholinergic receptors was studied using [3H]quinuclidinyl benzilate (a potent cholinergic antagonist) with negligible binding detected in the ovarian tissue (Schaeffer & Hsueh, 1980). However, one cannot rule out the presence of nicotinic cholinergic receptors in the granulosa cells. In addition, specific receptors for prostaglandin E-2 and F-2a are presumably present in the cell membrane of granulosa cells. PGE-2 stimulates both oestrogen and progestagen biosynthesis (Wang et al., 1981) while PGF-2a is luteolytic and was shown to stimulate the activity of 20a- hydroxysteroid dehydrogenase in FSH-treated granulosa cells (Jones & Hsueh, 198Id). Activation of membrane receptors by peptide hormones, neurotransmitters and prostaglandins is believed to be important for the induction of various steroidogenic enzymes and the differentiation of granulosa cells. The membrane receptors of several of these hormones (FSH, LH, adrenergic agent and prostaglandins) are clearly coupled to adenyl cyclases in the cell membrane and the actions of these agents are believed to be mediated through the activation of protein kinase and the subsequent phosphorylation of cellular proteins. In contrast, several other hormones (prolactin and EGF) do not stimulate cAMP production and the possible existence of specific second messenger molecules remains to be elucidated. Furthermore, serum lipoproteins stimulate progestagen production by cultured rat granulosa cells (Schreiber, Hsueh, Weinstein & Erickson, 1980) and cell membrane binding sites for serum high-density lipoproteins have been demonstrated (Christie, Gwynne & Strauss, 1982). In human ovarian follicular fluid, high-density lipoproteins have been identified (Simpson, Rochelle, Carr & MacDonald, 1980). At binding of these receptors with the lipoproteins, the lipoprotein-receptor complexes cluster into specialized "coated pits" in the cell membrane and presumably enter the cells by receptor-mediated endocytosis (Goldstein, Anderson & Brown, 1979). The receptors may be recycled back to the cell surface while the lipoproteins are degraded to liberate cholesterol, which is incorporated into the intracellular cholesterol pool for progestagen biosynthesis. Receptors for two non-physiological regulators (concanavalin A and cholera toxin) may also be present in the granulosa cell (Lee & Ryan, 1979; Wang et al., 1981). Cholera toxin is a bacterial

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access toxin known to interact with the gangliosides in the cell membrane to activate adenyl cyclase and consequently steroidogenesis, while concanavalin A binds to specific carbohydrate residues of the granulosa cell membrane. Concanavalin A induces capping of its binding sites in the surface of rabbit granulosa cells and the 'capped' membrane has been suggested to serve as an organizing site for microtubule polymerization (Albertini & Clark, 1975). The multiple hormonal receptors are involved in the mediation of the actions of their respective ligands in an integrated fashion. This, in turn, results in successful ovulation and optimal ovarian steroidogenesis for the maintenance of accessory sex organ growth and pregnancy. The in-vitro culture of ovarian granulosa cells not only yields important clues for understanding the hormonal regulation of follicular maturation, but also provides an interesting model for cellular and molecular studies on the regulation of multiple hormone receptors and the mechanism of action of these hormones.

Different modes of cell-to-cell communication: the granulosa cell as a model hormonal target cell

The ovarian granulosa cells not only provide an interesting system for elucidating the mechanism of action of peptide hormones, steroid hormones, neuromodulators, prostaglandins, lipoproteins and other regulatory agents, but also serve as an excellent model for understanding the diverse mode of cell-to-cell communication.

Endocrine control

Most of the classical hormones are acting on the granulosa cells through an endocrine control mechanism. These hormones are secreted from their gland of synthesis, traverse the circulatory system and reach the ovarian follicles to exert their action. These hormones include the pituitary gonadotrophins (FSH, LH and prolactin) as well as pancreatic insulin and possibly epidermal growth factor from the submaxillary gland. The adrenal steroids, glucocorticoids, are also included in this category. Although the lipoproteins from the liver are not 'hormones' in a strict sense, their regulation of granulosa cell progestagen biosynthesis is mediated through a similar mechanism. The production of oestrogens and progestagens in response to pituitary FSH and LH constitutes the basis for a closed-loop feedback mechanism. The gonadal steroids exert both negative and positive influence upon the hypothalamic-pituitary axis to modulate gonadotrophin production, while the gonadotrophins stimulate gonadal steroidogenesis. In addition, cultured granulosa cells secrete a protein factor 'inhibin' which has been shown to act on cultured cells to suppress FSH, but not LH, release (Erickson & Hsueh, 1978b). Since FSH may increase 'inhibin' production by granulosa cells, there may exist an additional closed-loop feedback mechanism between pituitary FSH release and granulosa cell 'inhibin' production. Although the regulatory roles of the above-mentioned hormones are well-documented, it is important to note that changes in the blood levels of these circulating hormones are not necessarily related to the functions of individual follicles due to possible changes in local ovarian vasculature and hence the delivery of the hormones to the granulosa cells.

Neuronal or neuromodulatory control The ovaries are innervated by various peripheral nerves (Burden, 1978; Bahr, Kao & Nalbandov, 1974). This neuronal input may directly affect ovarian steroidogenesis, modulate ovarian responsiveness to other hormones, or affect the intraovarian blood flow. The possible vascular action may, in turn, affect the delivery of circulating hormones to various follicles.

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access Although the stimulatory role of adrenergic agents on progestagen biosynthesis has been conclusively demonstrated (Adashi & Hsueh, 1981), the physiological significance of these findings remains to be clarified. Adrenergic nerves have been identified in the ovarian cortex adjacent to the follicles in cat and guinea pig ovaries (Burden, 1972) and the concentrations of epinephrine and norepinephrine have been shown to be 10-fold higher in porcine follicular fluid as compared to the systemic circulation (Veldhuis, Harrison & Hammond, 1980). Thus, catecholamines may modulate follicular steroidogenesis. A second possible neuromodulatory agent in the follicles in GABA. As discussed earlier, a high concentration of GABA and a GABA-forming enzyme have been found in the non-granulosa cell compartment of the ovary while GABA binding sites are present in the granulosa cells (Schaeffer & Hsueh, 1982). The possible modulation of granulosa cell functions by GABA, however, remains to be elucidated. Recently, a possible role of regulatory peptides as neurotransmitters has been demonstrated. A Gn-RH-like peptide has been identified in the sympathetic ganglia of the frog and is released in response to the stimulation of presynaptic fibres (Jan, Jan & Kuffler, 1979). This peptide interacts with specific post-synaptic receptors to elicit changes in membrane potentials. Since Gn-RH has been shown to bind to granulosa cell receptors and exert profound influences on granulosa cell functions, it is conceivable that Gn-RH or Gn-RH-like substances may be released by ovarian nerves to modulate granulosa cell functions (Hsueh & Jones, 1981).

Paracrine control

Studies in many laboratories have suggested that the secretory product of a given cell type may regulate the functions of its neighbouring cells by a paracrine control mechanism, i.e. without traversing the systemic circulation. For example, androgens secreted by theca cells not only serve as substrate for granulosa cell aromatase enzymes but also play important paracrine roles in the regulation of granulosa cell functions. Specifically, androgens augment the gonadotrophin stimulation of oestrogen and progestagen biosynthesis as well as the FSH induction of LH receptors (Richards, 1979; Armstrong & Dorrington, 1976; Hillier & deZwart, 1981). Although the demonstration of a Gn-RH-like substance in the ovary is not conclusive, a Gn- RH-like substance has been demonstrated in the rat Sertoli cell. This substance is believed to be involved in the regulation of functions through a paracrine control mechanism (Sharpe, Fraser, Cooper & Rommerts, 1981). Further studies are needed to demonstrate a possible paracrine role of Gn-RH-like peptides in the control of granulosa cell functions, Similarly, one cannot rule out a paracrine role of EGF, since EGF may be produced by neighbouring ovarian cells to affect granulosa cell functions. The prostaglandins are known to be important local hormones in the regulation of multiple reproductive functions. Prostaglandin biosynthesis by granulosa cells has been demonstrated (Triebwasser, Clark, LeMaire & Marsh, 1978) and prostaglandins may also play an important role in the intraovarian paracrine control of granulosa cell functions.

Exocrine control

A pheromone is a hormone that is secreted from one animal to affect cellular functions of a different individual. A Gn-RH-like substance has been detected in human, cow and rat milk (Baram, Koch, Hazum & Fridkin, 1977), and this substance may affect the pubertal changes in granulosa cell Gn-RH receptor content and ovarian steroidogenic capacity through an exocrine control mechanism (White & Ojeda, 1981). Similarly, since an EGF-like substance has been identified in the milk (Carpenter, 1980) one can also propose an exocrine role for EGF in the regulation of granulosa cell functions.

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access Autocrine control One of the unique aspects of the hormonal control of granulosa cell functions is the ability of the steroidogenic products of the granulosa cells to exert an autocrine, ultra-short loop regulation of granulosa cell functions. The demonstration of the ability of oestrogens to augment gonadotrophin- stimulated aromatase activity (Adashi & Hsueh, 1982; Zhuang et al., 1982) serves as an interesting model for further studies on the autocrine control mechanism. Since steroids are believed to be freely diffusible amongst the various cellular organelles and the oestrogens are synthesized in the smooth endoplasmic reticulum, the pathway by which granulosa cell oestrogens interact with the intracellular oestrogen receptors are not known and remain to be elucidated.

Research in the authors' laboratory is supported by NIH grants HD-14084 and HD-12303. A.J.W.H. is the recipient of NIT Research Career Development Award HD-00375. T.H. W. is the recipient of a Giannini Medical Research Foundation postdoctoral fellowship. We thank Ms Kayle Watts for excellent secretarial help, and Ms E. Tucker, C. Fabics and C. Talamantez for technical assistance.

References

Adashi, E.Y. & Hsueh, A.J.W. (1981) Stimulation of ß2- epidermal growth factor: a sensitive index of adrenergic responsiveness by follicle-stimulating hor¬ biological androgen activity. 95, 1019— mone in rat granulosa cells in vitro and in vivo. Endo¬ 1025. crinology 108, 2170-2178. Beattie, C. W., C orbiti, ., Cole, G., Corry, S., Jones, R.C., Adashi, E.Y. & Hsueh, A.J.W. (1982) augment Koch, K. & Tracy, J. (1977) Mechanism of the the stimulation of ovarian aromatase activity by fol¬ postcoital contraceptive effect of LH-RH in the rat. I. licle-stimulating hormone in cultured granulosa cells. Serum hormone levels during chronic LH-RH ad¬ J. biol. Chem. 257, 6077-6083. ministration. Biol. Reprod. 16, 322-332. Adashi, E.Y., Jones, P.B.C. & Hsueh, A.J.W. (1981) Bjersing, L. (1978) Maturation, morphology and endo¬ Synergistic effect of glucocorticoids on the stimula¬ crine function of the follicular wall in mammals. In tion of progesterone production by follicle-stimu¬ The Vertebrate Ovary, pp. 181-214. Ed. R. E. Jones. lating hormone in cultured rat granulosa cells. Endo¬ Plenum Press, New York. crinology 109, 1888-1894. Bjersing, L. & Carstensen, H. (1964) The role of the Aguado, L.Í., Petrovic,S.L. & Ojeda, S.R. (1982) Ovarian granulosa cell in the biosynthesis of ovarian steroid ß-adrenergic receptors during the onset of : hormones. Biochim. Biophys. Acta 86, 639-640. characterization, distribution and coupling to stero¬ Burden, H.W. (1972) Adrenergic innervation in ovaries idogenic responses. Endocrinology 110, 1124-1132. of the rat and guinea pig. Am. J. Anat. 133, 455-461. Albertini, D.F. & Clark, J.I. (1975) Membrane-micro- Burden, H.W. (1978) Ovarian innervation. In The tubule interactions: concanavalin A capping induced Vertebrate Ovary, pp. 615-638. Ed. R. E. Jones. redistribution of cytoplasmic microtubules and col- Plenum Press, New York. chicine binding proteins. Proc. natn. Acad. Sci. Carpenter, G. (1980) Epidermal growth factor is a major U.S.A. 72, 4976-4980. growth promoting agent in human milk. Science, Armstrong, D.T. & Dorrington, J.H. (1976) Androgens N.Y. 210, 198-199. augment FSH-induced progesterone secretion by Carpenter, G. & Cohen, S. (1979) Epidermal growth cultured rat granulosa cells. Endocrinology 99, 1411- factor. Ann. Rev. Biochem. 48, 193-216. 1414. Channing, C.P. ( 1970) Influences of the in vivo and in vitro Bahr, J., Kao, L. & Nalbandov, A.V. (1974) The role of hormonal environment upon luteinization of granu¬ catecholamines and nerves in ovulation. Biol. Reprod. losa cells in tissue culture. Recent Prog. Harm. Res. 26, 10, 273-290. 589-622. Baird, D.T. (1977) Evidence in vivo for the two-cell Christie, M.H., Gwynne, J.T. & Strauss, J.F., HI (1982) hypothesis of oestrogen synthesis by the sheep Binding of human high density lipoproteins to mem¬ Graafian follicle. J. Reprod. Fert. 50, 183-185. branes of luteinized rat ovaries. J. Steroid Biochem. Baird, D.T. & Fraser, I.J. (1975) Concentrations of 14, 671-678. oestrone and oestradiol in follicular fluid and ovarian Daniel, S.A.J. & Armstrong, D.T. (1980) Enhancement of venous blood of women. Clin. Endocr. 4, 259-266. follicle-stimulating hormone-induced aromatase ac¬ Baram, T., Koch, Y., Hazum, E. & Fridkin, M. (1977) tivity by androgens in cultured rat granulosa cells. -releasing hormone in milk. Science, Endocrinology 107, 1027-1033. N. Y. 198, 300-302. Dorrington, J.H. & Armstrong, D.T. (1979) Effects of Barthe, P.L., Bullock, L.P., Mowszowicz, I., Bardin, FSH on gonadal functions. Recent Prog. Horm. Res. C.W. & Orth, D.N. (1974) Submaxillary gland 35, 301-342.

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access Dorrington, J.H. & Gore-Langton, R.E. (1981) Prolactin actions of gonadotropin-releasing hormone. Endo¬ inhibits oestrogen synthesis in the ovary. Nature, crine Reviews 2, 437-461. Lond. 290, 600-602. Hsueh, A.J.W. & Jones, P.B.C. (1982) Direct hormonal Dorrington, J.H., Moon, Y.S. & Armstrong, D.T. (1975) modulation of ovarian granulosa cell maturation: -17ß biosynthesis in cultured granulosa cells effect of gonadotropin-releasing hormone. In Follicu¬ from hypophysectomized immature rats; stimulation lar Maturation and Ovulation, pp. 19-33. Eds R. by follicle-stimulating hormone. Endocrinology 97, Rolland, E. V. Van Hall, K. P. McNatty & J. 1328-1331. Schoemaker. Excerpta Medica, Amsterdam. Erickson, G.F. & Hsueh, A.J.W. (1978a) Stimulation of Hsueh, A.J.W. & Ling, N.C. (1979) Effect of an aromatase activity by follicle stimulating hormone in antagonistic analog of gonadotropin-releasing hor¬ rat granulosa cells in vivo and in vitro. Endocrinology mone upon ovarian granulosa cell function. Life Sci. 102, 1275-1282. 25, 1223-1230. Erickson, G.F. & Hsueh, A.J.W. (1978b) Secretion of Hsueh, A.J.W., Wang, C. & Erickson, G.F. (1980) Direct "inhibin" by rat granulosa cells in vitro. Endocrinology inhibitory effect of gonadotropin-releasing hormone 103, 1960-1963. upon follicle-stimulating hormone induction of Erickson, G.F., Hsueh, A.J.W., Quigley, M.E., Rebar, receptor and aromatase activity R.W. & Yen, S.S.C. (1979) Functional studies of in rat granulosa cells. Endocrinology 106, 1697-1705. aromatase activity in human granulosa cells from Hsueh, A.J.W., Welsh, T.H., Jr & Jones, P.B.C. (1981) normal polycystic ovaries. J. clin. Endocr. Metab. 49, Inhibition of ovarian and testicular steroidogenesis 514-519. by epidermal growth factor. Endocrinology 108, 2002- Erickson, G.F., Wang, C. & Hsueh, A.J.W. (1979) FSH 2004. induction of functional LH receptors in granulosa Jaken, S., Tashjian, A.H., Jr & Blumberg, P.M. (1981) cells cultured in a chemically-defined medium. Relationship between biological responsiveness to Nature, Lond. 279, 336-337. phorbol esters and receptor levels in GH4C, rat Falck, B. (1959) Site of production of oestrogen in rat pituitary cells. Cancer Res. 41, 4956-4960. ovary as studied in micro-transplants. Acta physiol. Jan, Y.N., Jan, L.Y. & Kuffler, S.W. (1979) A peptide as scand. 47, Suppl. 163, 1-101. a possible transmitter in sympathetic ganglia of the Fanjul, L.F., Ruiz de Galarreta, CM. & Hseuh, A.J.W. frog. Proc. natn. Acad. Sci. U.S.A. 76, 1501-1505. (1983) Progestin augmentation of gonadotropin stim¬ Jones, P.B.C. & Hsueh, A.J.W. (1980) Direct inhibitory ulated progesterone production by cultured rat effect of gonadotropin-releasing hormone upon luteal granulosa cells. Endocrinology 112, 405-407. luteinizing hormone receptor and steroidogensis in Goldstein, J.L., Anderson, R.G.W. & Brown, M.S. (1979) hypophysectomized rats. Endocrinology 107, 1930- Coated pits, coated vesicles and receptor-mediated 1936. endocytosis. Nature, Lond. 279, 679-685. Jones, P.B.C. & Hsueh, A.J.W. (1981a) Direct stimula¬ Gore-Langton, R.E., Lacroix, M. & Dorrington, J.H. tion of ovarian progesterone metabolizing enzyme by (1981) Differential effects of luteinizing hormone- gonadotropin-releasing hormone in cultured granu¬ releasing hormone on follicle-stimulating hormone- losa cells. J. biol. Chem. 256, 1248-1254. dependent responses in rat granulosa cells and Sertoli Jones, P.B.C. & Hsueh, A.J.W. (1981b) Direct effects of cells in vitro. Endocrinology 108, 812-819. gonadotropin-releasing hormone and its antagonist H agino, . (1972) The effect of synthetic corticosteroids upon ovarian functions stimulated by FSH, prolactin on ovarian function in the baboon. J. clin. Endocr. and LH. Biol. Reprod. 24, 747-759. Metab. 35, 716-721. Jones, P.B.C. & Hsueh, A.J.W. (1981c) Regulation of Hillier, S.G. & deZwart, F.A. (1981) Evidence that ovarian 20a-hydroxysteroid dehydrogenase by granulosa cell aromatase induction/activation by gonadotropin-releasing hormone and its antagonist in Biochem. FSH is an androgen receptor-regulated process in vitro and in vivo. J. Steroid 14, 1169-1175. vitro. Endocrinology 109, 1303-1305. Jones, P.B.C. & Hsueh, A.J.W. (198Id) Regulation of Hillier, S.G., Zeleznik, A.J., Knazek, R.A. & Ross, G.T. progesterone metabolizing enzyme by adrenergic (1980) Hormonal regulation of preovulatory follicle agents, prolactin and prostaglandins in cultured rat maturation in the rat. J. Reprod. Fert. 60, 219-229. ovarian granulosa cells. Endocrinology 109, 1347- Horowitz, A.D., Greenbaum, E. & Weinstein, LB. (1981) 1354. Identification of receptors for phorbol ester tumor Jones, P.B.C. & Hsueh, A.J.W. (1982a) Regulation of promoters in intact mammalian cells and of an ovarian 3ß-hydroxysteroid dehydrogenase by gona¬ inhibitor of receptor binding in biologic fluids. Proc. dotropin-releasing hormone and follicle-stimulating natn. Acad. Sci. U.S.A. 78, 2315-2319. hormone in cultured rat granulosa cells. Endocrin¬ Hsueh, A.J.W. & Erickson, G.F. (1978a) A sensitive in ology 110, 1663-1671. vitro bioassay for FSH. Endocrinology 102, Suppl. 102, Jones, P.B.C. & Hsueh, A.J.W. (1982b) Pregnenolone Abstr. 55. biosynthesis by cultured rat granulosa cells : modula¬ Hsueh, A.J.W. & Erickson, G.F. (1978b) Glucocorticoid- tion by follicle-stimulating hormone and gonado¬ inhibition of FSH-induced production in tropin-releasing hormone. Endocrinology 111, 713- cultured rat granulosa cells. Steroids 32, 639-648. 721. Hsueh, A.J.W. & Erickson, G.F. (1979) Extrapituitary Jones, P.B.C., Conn, P.M., Marian, J. & Hsueh, A.J.W. action of gonadotropin releasing hormone: direct in¬ (1980) Binding of gonadotropin-releasing hormone hibition of ovarian steroidogenesis. Science, N. Y. agonist to rat ovarian granulosa cells. Life Sci. 27, 204, 854-855. 2125-2132. Hsueh, A.J.W. & Jones, P.B.C. (1981) Extrapituitary Jones, P.B.C., Welsh, T.H., Jr & Hsueh, A.J.W. (1982) Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access Regulation of ovarian progestin production by follicular development: a 1978 perspective. Recent epidermal growth factor in cultured rat granulosa Prog. Harm. Res. 35, 343-373. cells. J. biol. Chem. 257, 11268-11273. Richards, J.S. & Midgley, A.R., Jr (1976) Protein Lamprecht, S.A., Lindner, H.R. & Strauss, J.R. (1969) hormone action: a key to understanding ovarian Induction of 20a-hydroxysteroid dehydrogenase in follicular and luteal cell development. Biol. Reprod. rat corpora lutea by pharmacological blockade of 14, 82-94. pituitary prolactin secretion. Biochim. biophys. Acta SchaefTer, J.M. & Hsueh, A.J.W. (1980) Acetylcholine 187, 133-143. receptors in the rat anterior . Endocrin¬ Lee, C.Y. & Ryan, R.J. (1979) The porcine ovarian ology 106, 1377-1381. follicle. V. Binding of concanavalin A to granulosa SchaefTer, J.M. & Hsueh, A.J.W. (1982) Identification of cells during follicle maturation. Biol. Reprod. 21,973- gamma-aminobutyric acid (GABA) and its binding 977. sites in the ovary. Life Sci. 30, 1599-1604. Leung, P.C.K. & Armstrong, D.T. (1980) Interactions of Schomberg, D.W., Stouffer, R.L. & Tyrey, L. (1976) steroids and in the control of steroido¬ Modulation of progestin secretion in ovarian cells genesis in the . Ann. Rev. Physiol. 42, by 17ß-hydroxy-5cc-androstan-3-one (dihydrotesto- 71-82. sterone): a direct demonstration in monolayer cul¬ Louvet, J.P., Baislic, M., Bayard, F. & Boulard, C. (1977) ture. Biochem. Biophys. Res. Commun. 68, 77-85. Glucocorticoid receptors in rat ovarian granulosa cell Schreiber, J.R. & Hsueh, A.J.W. (1979) Progesterone cytosol. Endocrinology 100, Suppl., Abstr. 601. "receptor" in rat ovary. Endocrinology 105, 915-919. Lucky, A.W., Schreiber, J.R., Hillier, S.G., Schulman, Schreiber, J.R. & Ross, G.T. (1976) Further characteriza¬ J.D. & Ross, G.T. (1977) Progesterone production by tion of a rat ovarian testosterone receptor with cultured preantral rat granulosa cells : stimulation by evidence for nuclear translocation. Endocrinology 99, androgens. Endocrinology 100, 128-133. 590-596. Mahajan, D.K. & Little, A.B. (1978) Specific cortisol Schreiber, J.R., Hsueh, A.J.W., Weinstein, D.B. & binding protein in porcine follicular fluid. Biol. Erickson, G.F. (1980) Plasma lipoproteins stimulate Reprod. 17, 834-842. progestin production by rat ovarian granulosa cells May, J.V. & Schomberg, D.W. (1981) Granulosa cell cultured in serum. J. Steroid Biochem. 13, 1009-1014. I. & differentiation in vitro; effect of insulin on growth Sharpe, R.M., Fraser, H.M., Cooper, Rommerts, and functional integrity. Biol. Reprod. 25, 421-431. F.F.G. (1981) Sertoli-Leydig cell communication via May, J.V., McCarty, K., Jr, Reichert, L.E., Jr & an LHRH-like factor. Nature, Lond. 290, 785-787. Schomberg, D.W. (1980) Follicle-stimulating hor¬ Short, R.V. (1962) Steroids in the follicular fluid and the mone-mediated induction of functional luteinizing corpus luteum of the mare. A "two-cell type" theory hormone/human chorionic gonadotropin receptors of ovarian steroid synthesis. J. Endocr. 24, 59-63. during monolayer culture of porcine granulosa cells. Shoyab, M. & Todaro, G.J. (1982) Partial purification Endocrinology 107, 1041-1049. and characterization of a binding protein for biologi¬ Mondschein, J.S. & Schomberg, D.W. (1981a) Growth cally active phorbol and ingenol esters from murine factors modulate gonadotropin receptor induction in sera. J. biol. Chem. 257, 439^*45. granulosa cell cultures. Science, N.Y. 211, 1179— Simpson, E.R., Rochelle, D.B., Carr, B.R. & MacDonald, 1180. P.C. (1980) Plasma lipoproteins in follicular fluid of Mondschein, J.S. & Schomberg, D.W. (1981b) Platelet- human ovaries. J. clin. Endocr. Metab. 51, 1469-1471. derived growth factor enhances granulosa cell lutein¬ Simpson, M.E., Li, C.H. & Evans, H.M. (1951) Syner- izing hormone receptor induction by follicle-stimu¬ gism between pituitary follicle stimulating hormone lating hormone and serum. Endocrinology 109, 325- and human chorionic gonadotropin. Endocrinology 327. 48, 370-383. Moor, R.M. (1977) Site of steroid production in ovine Smith, M.S., Freeman, M.E. & NeUI, J.D. (1975) The Graafian follicles in culture. J. Endocr. 73, 143-150. control of progesterone secretion during the estrous Navickis, R.J., Jones, P.B.C. & Hsueh, A.J.W. (1982) cycle and early pseudopregnancy in the rat: prolac¬ Modulation of prolactin receptors in cultured rat tin, gonadotropin and steroid levels associated with granulosa cells by FSH, LH and GnRH. Molec. cell. rescue of the corpus luteum of pseudopregnancy. Endocr. 27, 77-88. Endocrinology 96, 219-226. Nimrod, A. (1981) On the synergistic action of androgen Triebwasser, W.F., Clark, M.R., LeMaire, W.J. & and in vitro and FSH on progestin secretion by cultured rat Marsh, J.M. (1978) Localization synthesis granulosa cells. Cellular and mitochondrial meta¬ of prostaglandins in components of rabbit pre¬ bolism. Molec. cell. Endocr. 21, 51-62. ovulatory Graafian follicles. Prostaglandins 16, 621- Nimrod, ., Erickson, G.F. & Ryan, K.J. (1976) A 632. T.S. & J.M. specific FSH receptor in rat granulosa cells : proper¬ Veldhuis, J.D., Harrison, Hammond, (1980) ties of binding in vitro. Endocrinology 98, 56-64. ß2-adrenergic stimulation of ornithine decarboxylase Nimrod, ., Tsafriri, A. & Lindner, H.R. (1977) In vitro activity in porcine granulosa cells in vitro. Biochim. induction of binding sites for hCG in rat granulosa Biophys. Ada 627, 123-130. cells by FSH. Nature, Lond. 267, 632-633. Wang, C. & Chan, V. (1982) Divergent effects of pro¬ Richards, J.S. (1975) Estradiol receptor content in rat lactin on estrogen and progesterone production by cells rat follicles. Endo¬ granulosa cells during follicular development : modi¬ granulosa of Graafian fication by estradiol and gonadotropins. Endo¬ crinology 110, 1085-1093. crinology 97, 1174-1184. Wang, C, Hsueh, A.J.W. & Erickson, G.F. (1979) Richards, J.S. (1979) Hormonal control of ovarian Induction of functional prolactin receptors by fol-

Downloaded from Bioscientifica.com at 09/30/2021 10:42:11PM via free access licle-stimulating hormone in rat granulosa cells in vivo White, S.S. & S.R. (1981) in ovarian and Ojeda, Changes in vitro. J. biol. Chem. 254, 11330-11336. LHRH receptor content during the onset of puberty Wang, C, Hsueh, A.J.W. & Erickson, G.F. (1980) in the female rat. Endocrinology 108, 347-349. Prolactin inhibition of estrogen production by cul¬ Wiest, W.G., Kidwell, W.R. & Balogh, K., Jr (1968) Pro¬ tured rat granulosa cells. Molec. Cell. Endocr. 20, 135- gesterone catabolism in the mechanism for 144. regulatory progestational potency during pregnancy. Endo¬ Wang, C, Hsueh, A.J.W. & Erickson, G.F. (1981) LH crinology 82, 844-859. stimulation of estrogen secretion in cultured granu¬ Zeleznik, A.J. (1981) Premature elevation of losa cells. Molec. cell. systemic Endocr. 24, 17-28. estradiol reduces serum levels of follicle-stimulating Wang, C, Hsueh, A.J.W. & Erickson, G.F. (1982) The hormone and lengthens the of the role of cyclic AMP in the induction of estrogen and in rhesus monkeys. Endocrinology progestin synthesis in cultured granulosa cells. Molec. 109, 352-355. cell. Endocr. 25, 73-83. Zhuang, L.Z., Adashi, E.Y. & Hsueh, A.J.W. (1982) Welsh, T.H., Jr & Hsueh, A.J.W. (1982) Phorbol ester Direct enhancement of gonadotropin-stimulated inhibition of ovarian and testicular steroidogenesis in ovarian estrogen biosynthesis by estrogen and clomi¬ vitro. Endocrinology 110, Suppl., 179, Abstr. 397. phene citrate. Endocrinology 110, 2219-2221.

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