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Acute liver failure has extremely high mortality without liver transplantation. We attempted to determine the value of abdominal CT scanning and liver biopsy in its management. A retrospective analysis of patients with acute liver failure was performed; demographic, clinical, radiologic and histopathologic features were noted. Over a period of 13 years, 177 patients were evaluated. The mean age was 39 years and 63% were females. The patients were divided into three groups. Fourteen percent survived with medical management (group I), 37% died (group II), and 49% had liver transplantation (group III). Most patients showed diffuse low density of the liver on CT scanning and the proportions were similar in the three groups. Moderate to large ascites was not present in group I but occurred in 31% of patients in group II and in 15% in group III. Mean hepatic volumes were similar in the three groups; however, 97% of the patients with a liver volume of less than 1000 ml either died or required liver transplantation. Liver biopsies among patients with spontaneous recovery (group I) were distinguished by the presence of regenerative changes and a hepatic parenchymal necrosis of less than 50%. These results suggest that in patients with acute liver failure a liver volume of less than 1000 ml and/or hepatic parenchymal necrosis of greater than 50% is indicative of a poor prognosis. This information may assist decision making in such patients, in particular, regarding the need for liver transplantation.

Extensive remodeling of the follicular extracellular matrix occurs during the process of ovulation. This remodeling involves the breakdown of collagen, which is regulated, in part, by the action of the metalloproteinase collagenase and its associated inhibitors. In the present study, follicular metalloproteinase inhibitors were characterized to determine whether they were serum-borne or of ovarian origin, possibly a tissue-derived inhibitor known as tissue inhibitor of metalloproteinase (TIMP). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in an in vitro fertilization program. Chromatographic separation of follicular fluid on Sepharose 6B resulted in two peaks of inhibitory activity. The large molecular radius (Mr) inhibitor was similar in size to the serum-borne metalloproteinase inhibitor alpha 2-macroglobulin (i.e. Mr 700,000) whereas the small Mr inhibitor approximated the size of TIMP (i.e. Mr 29,000). Incubation of aliquots from either of the two peaks of inhibitor activity or an alpha 2-macroglobulin standard with an antibody to alpha 2-macroglobulin decreased the inhibitory activity in both the large Mr peak and the alpha 2-macroglobulin standard by 86.6 +/- 1.7% and 71.5 +/- 7.7% (n = 4, P less than 0.005), respectively, implying cross-reactivity with the alpha 2-macroglobulin antibody. The inhibitory activity in the small Mr peak, however, was unchanged. Northern analysis of total granulosa cell RNA demonstrated TIMP messenger RNA (mRNA) in all eight granulosa cell samples examined whereas alpha 2-macroglobulin mRNA was virtually undetectable. A positive correlation (r = 0.85, P less than 0.01) was observed between the levels of TIMP mRNA and the ratio of the follicular estradiol-progesterone concentration. However, inhibitor activity in the follicular fluid was not correlated with the levels of TIMP mRNA (r = 0.05). These findings confirm the presence of alpha 2-macroglobulin in follicular fluid and demonstrate that human preovulatory granulosa cells contain mRNA for TIMP, an inhibitor that regulates metalloproteinases such as collagenase, gelatinase, and proteoglycanase. Additionally, the expression of TIMP mRNA is steroid related and may be hormonally regulated. It is proposed that TIMP produced in the granulosa cell compartment in conjunction with alpha 2-macroglobulin from the serum may act to control the site and extent of ovarian connective tissue remodeling.

The modulation of ovarian steroidogenesis by epidermal growth factor (EGF) was investigated in cultured rat granulosa cells. Granulosa cells, obtained from ovaries of immature, hypophysectomized, estrogen-treated rats, were incubated for 2 days with EGF, follicle-stimulating hormone (FSH), or EGF plus FSH. Treatment with EGF did not affect estrogen production, but stimulated progestin (i.e. progesterone and 20 alpha-hydroxy-pregn-4-en-3-one) production in a dose-dependent manner. Stimulation of progestin production by EGF appears to be the result of an increase in pregnenolone biosynthesis as well as increases in the activities of 20 alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase/isomerase. Treatment with FSH increased both estrogen and progestin production by cultured granulosa cells. When cells were treated concomitantly with EGF, FSH-stimulated estrogen production was inhibited, while progestin production was further enhanced. The EGF enhancement of FSH-stimulated progestin production appears to be the result of synergistic increases in pregnenolone biosynthesis and 20 alpha-hydroxysteroid dehydrogenase activity, resulting in substantial increases in 20 alpha-hydroxypregn-4-en-3-one but not progesterone production. The effects of EGF were shown to be time-dependent. The concept of a direct action of EGF on rat granulosa cells is reinforced by the demonstration of high affinity (Kd approximately 3 X 10(-10) M), low capacity (approximately 5,000 sites/cell) EGF binding sites in these cells. Thus, EGF interacts with specific granulosa cell receptors to stimulate progestin but to inhibit estrogen biosynthesis.

Prolactin (Prl), beta 2-adrenergic agents and human chorionic gonadotropin (hCG) are luteotropic in rats, whereas gonadotropin releasing hormone (GnRH) exerts direct inhibitory effects on ovarian steroidogenesis. The present study examined the modulation of the progestin biosynthetic pathway by the luteotropic agents, as well as the actions of GnRH. Rat granulosa cells were primed with follicle-stimulating hormone (FSH) to increase their responsiveness to the luteotropic agents. Subsequent treatment for 2 days with Prl, terbutaline (a beta 2-adrenergic agonist) or hCG stimulated the production of progesterone, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P), pregnenolone and the activity of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). In contrast, treatment with Prl or terbutaline, but not hCG, inhibited 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity by decreasing the apparent maximal velocity of the enzyme with no change in its Km value. Concomitant treatment with GnRH inhibited progesterone, but increased 20 alpha-OH-P production stimulated by Prl or terbutaline. These effects were associated with a stimulation of 20 alpha-HSD activity, while neither 3 beta-HSD activity nor pregnenolone biosynthesis was decreased. In contrast, GnRH inhibited progesterone production in hCG-treated cells without affecting 20 alpha-OH-P production. This was associated with an inhibitory effect of GnRH on pregnenolone biosynthesis with no effect upon 3 beta-HSD activity. Thus, Prl and the beta 2-agonist stimulate progesterone production in granulosa cells by increasing pregnenolone production and 3 beta-HSD activity as well as by decreasing 20 alpha-HSD activity, while hCG stimulates progesterone production by increasing pregnenolone production and 3 beta-HSD activity. The inhibitory effect of GnRH on Prl- or terbutaline-stimulated progesterone production appears to result from a preferential increase in 20 alpha-HSD activity, while the GnRH inhibition of hCG-stimulated progesterone production appears to result from a preferential inhibition of pregnenolone production.

Harris (1) first proposed that the anterior pituitary gland is controlled by hypothalamic releasing factors. After the subsequent purification and sequencing of gonadotropin-releasing hormone (GnRH) (2, 3) and the synthesis of potent GnRH agonists and antagonists (4, 5), many laboratories have investigated the effects of these peptides on various reproductive functions. In both males and females, administration of GnRH or its potent agonists results in sustained increases in serum gonadotropins. In women, administration of GnRH causes preovulatory-like surges of gonadotropins (6, 7). In several animal species, a single injection of GnRH induces preovulatory- like surges of LH that trigger ovulation (8–11). Since pituitary gonadotropins are essential for normal gonadal functions and hypothalamic GnRH was believed to act solely on the pituitary gland, treatment with high doses of GnRH and its agonists had been predicted to be a potential means for enhancing fertility. Paradoxically, long term administration...

The direct effects of adrenocortical steroids on basal and FSH-stimulated production of progesterone by ovarian granulosa cells were investigated in vitro. Granulosa cells from immature, hypophysectomized, estrogen-treated rats were cultured for 2 days in the presence or absence of FSH and/or corticoids. Treatment with FSH resulted in substantial increases in the accumulation of progesterone, whereas dexamethasone by itself was only minimally effective. However, concomitant treatment with increasing concentrations of various native and synthetic corticoids led to dose-dependent increases (200-500%) in the FSH-stimulated accumulation of progesterone. The stimulatory potencies of the corticoids tested correlated with their glucocorticoid rather than their mineralocorticoid potencies (dexamethasone greater than cortisol much greater than aldosterone). Treatment with dexamethasone also led to significant increases in the accumulation of progesterone stimulated by prostaglandin E2, cholera toxin, or dibutyryl cAMP. In the presence or absence of FSH, treatment with dexamethasone, cortisol, or corticosterone led to significant decreases in the activity of 20 alpha-hydroxysteroid dehydrogenase. In addition, treatment with dexamethasone led to significant increases in basal and FSH-stimulated activities of 3 beta-hydroxysteroid dehydrogenase. Our findings indicate that glucocorticoids synergize with FSH and other trophic agents in the accumulation of progesterone in accordance with their glucocorticoid rather than mineralocorticoid potencies and that this direct effect may be accounted for, at least in part, by the stimulation of 3 beta-hydroxysteroid dehydrogenase and the inhibition of 20 alpha-hydroxysteroid dehydrogenase.

The mechanism by which GnRH inhibits ovarian progesterone production was investigated by studying the GnRH modulation of pregnenolone biosynthesis in cultured rat granulosa cells. Granulosa cells from hypophysectomized, estrogen-treated rats were incubated for 2 days with various hormones in vitro. Pregnenolone production was measured in the presence of cyanoketone, which inhibits the conversion of pregnenolone to progesterone. FSH stimulated pregnenolone production in a dose-dependent manner (ED50, 4.47 ng/ml). Concomitant treatment with GnRH resulted in a dose-dependent decrease in FSH-stimulated pregnenolone production (ID50, 6.3 x 10(-9) M; maximal decrease, approximately 50%). In contrast, treatment with high doses of GnRH alone stimulated pregnenolone production (ED50, 2.95 x 10(-8) M) reaching a maximal level of about 10% that induced by FSH. Treatment with a GnRH antagonist, [Ac-D-Phe1, D-pCl-Phe2, D-Trp3,6]GnRH, did not affect either basal or FSH-stimulated pregnenolone production, but blocked both inhibitory and stimulatory effects of GnRH. The addition of 25-hydroxycholesterol, a soluble substrate for side-chain cleavage enzymes, enhanced FSH-stimulated pregnenolone production, but failed to overcome the inhibitory action of GnRH. GnRH also inhibited progesterone production stimulated by 8-bromo-cAMP and cholera toxin. This action of GnRH, however, was not associated with an inhibition of pregnenolone biosynthesis, but appeared to be due to a preferential increase in the metabolism of progesterone to 20 alpha-hydroxypregn-4-en-3-one. Thus, in addition to the reported GnRH stimulation of progesterone metabolism to 20 alpha-hydroxypregn-4-en-3-one and the GnRH inhibition of FSH-stimulated 3 beta-hydroxysteroid dehydrogenase activity, the present results demonstrate that GnRH also inhibits FSH-stimulated pregnenolone biosynthesis, probably at the side-chain cleavage enzyme step.

We have previously shown that gonadotropin releasing hormone (GnRH) and its agonists inhibit ovarian functions by a direct action on ovarian granulosa cells in vitro. A labeled GnRH agonist, [des-Gly10, D-Ser (TBu)6, Pro9-NHEt]GnRH, was used here to examine the possibility that these inhibitory actions of GnRH were mediated through specific receptors which recognize GnRH. Ovarian membrane fractions obtained from immature, hypophysectomized diethylstilbesterol-treated rats were incubated with the 125I-GnRH agonist and specific binding was determined by a filtration assay. Stereospecific, high affinity binding was detected in the ovarian membranes; the dissociation constant for the labeled GnRH agonist was determined to be 0.84 ± 0.33 × 10−10 M and the binding capacity was calculated to be 12.9 fmol/mg protein, or 0.142 fmol/μg DNA. The binding affinity for the GnRH decapeptide was 3.3 times lower than that of the GnRH agonist whereas two GnRH partial peptides did not compete for the 125I-agonist binding. After sequential treatment with FSH, LH and prolactin to the hypophysectomized female rats, the ovarian GnRH binding capacity increased per ovary, but decreased per mg ovarian protein. Furthermore, ovarian granulosa cells were isolated and their binding capacity was determined to be 25.2 fmol/mg protein, or 0.133 fmol/μg DNA, suggesting that the granulosa cells contain GnRH binding sites. Thus, this report demonstrates the presence of stereospecific, high affinity GnRH binding sites in the rat ovarian granulosa cells.

The mechanism by which gonadotropin-releasing hormone (GnRH) inhibits ovarian progesterone production was investigated by studying the GnRH modulation of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)/delta 5-delta 4-isomerase activity in cultured rat granulosa cells. Ovarian granulosa cells, obtained from immature hypophysectomized estrogen-treated rats, were incubated with various hormones in vitro, and 3 beta-HSD activity was determined by measuring the conversion of radiolabeled pregnenolone to progesterone. Treatment with FSH increased the apparent maximal velocity of the enzyme by about 6-fold in a dose-dependent manner, with an ED50 value of 3.58 ng/ml. FSH treatment also resulted in an approximately 10-fold increase in the apparent Km of this enzyme (from 0.46 to 4.98 microM). In contrast, concomitant treatment with GnRH (10(-8) M) inhibited the FSH-stimulated increase in enzyme activity by about 27%. This inhibitory effect of GnRH was associated with a decrease in the apparent maximal velocity, while the apparent Km remained unchanged. Furthermore, the inhibitory effect of GnRH was observed whether the enzyme activity was expressed per mg protein or per mg DNA. Concomitant treatment with 10(-6) M of a GnRH antagonist, [D-pGlu1,D-Phe2,D-Trp3,6]GnRH, completely blocked the inhibitory effect of GnRH. In contrast to the inhibitory effect of GnRH on FSH-stimulated 3 beta-HSD activity, treatment with GnRH alone increased enzyme activity by about 40%. This was accompanied by a slight but significant stimulation of basal progestin production by GnRH in granulosa cells. The present results coupled with the observed GnRH stimulation of 20 alpha-HSD activity reported earlier suggest that GnRH inhibits the FSH stimulation of progesterone production by decreasing the conversion of pregnenolone to progesterone as well as by increasing the metabolism of progesterone.

To evaluate the roles of plasminogen activator (PA) and PA inhibitor (PAI) in human ovulation, we obtained follicular fluid and granulosa cells from individual preovulatory follicles of patients undergoing gamete intrafallopian tube transfer. The follicular fluid samples (n = 25) were analyzed for total tissue-type PA antigen, PA enzyme activity by fibrin autography, PAI activity, PAI type 1 (PAI-1) antigen, and PAI-1 mRNA. The follicular fluid of preovulatory follicles contained low levels of total tissue-type PA antigen (less than 1 ng/mL). Fibrin autography experiments indicated little or no detectable PA activity associated with free or unbound PA. The results of the PAI activity assay and PAI-1 antigen determination support the concept of a relative abundance of PAI compared with PA. Hybridization analysis was used to measure the relative amounts of granulosa cell PAI-1 mRNA. The levels of PAI-1 mRNA correlate with follicular fluid PAI concentrations in individual follicles. Taken together, these results support the idea that there is very little free, or active, PA in follicular fluid of human preovulatory follicles, but there is an abundance of PAI. Furthermore, PAI-1 produced by the granulosa cells may represent a major form of PAI in follicular fluid.

Epidermal growth factor (EGF), but not fibroblast growth factor, inhibits the gonadotropin stimulation of estrogen and testosterone production by primary cultures of ovarian granulosa and testicular Leydig cells, respectively. EGF also inhibits gonadal steroidogenesis induced by cholera toxin and (Bu)2cAMP, and the inhibitory effect of EGF is not associated with cell proliferation. EGF may play an important endocrine role in the regulation of steroidogenic functions of gonadal cells.

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.

Hypothalamic gonadotropin releasing hormone (GnRH) and its analogs have been shown to regulate progestin production by ovarian granulosa cells treated with FSH and prolactin. The present study examines the direct modulation of an ovarian progesterone metabolizing enzyme by GnRH and its analogs in vitro and in vivo . Granulosa cells from hypophyseclomized, diethylstilbestrol-treated rats were cultured with GnRH or its analogs. After 2 days, cells were homogenized and 20α-hydroxysteroid dehydrogenase (20α-OH-SDH) activity was measured using a direct enzyme assay. GnRH stimulated 20α-OH-SDH activity in a dose-dependent manner with an ED 50 of 8.95 × 10 −9 M and a GnRH aganist ([des-Gly 10 , d -Lcu 6 , (N 2 Me)Leu 7 ,Pro 9 NHEt]GnRH) was shown to be 5.6 fold more potent than GnRH. The stimulatory effect of GnRH was inhibited by concomitant treatment with a GnRH antagonist ([ d -pGlu 1 , d -Phe 2 d -Trp 3,6 ]GnRH) with a half maximal inhibitory dose ratio of 3 1 ([antagonist]/[GnRH]). Time course studies indicated that 12 h of GnRH treatment were required to stimulate a measurable increase in enzyme activity. Cycloheximide treatment inhibited the GnRH action, suggesting that GnRH induces the synthesis of 20α-OH-SDH. Furthermore, treatment with GnRH and its analogs in hypophysectomizcd rats in vivo stimulated enzyme activity without affecting the K m of the enzyme, whereas the stimulatory effect of GnRH was blocked by concomitant treatment with a potent GnRH antagonist. These studies demonstrated that GnRH and its analogs may directly regulate the synthesis of ovarian 20α-OH-SDH in vitro and in vivo .

1. Cell Culture Approach to the Study of Granulosa Cells In 1672, Regnier de Graaf first described the differentiation of ovarian follicles into the corpus luteum. He stated that “age and coitus cause very great changes in eggs (follicles). In young animals, they are very small and in more developed ones they are larger. After coitus, they (the follicles) so alter as to resemble the globules (the corpora lutea) …, being one or more according as the animal will produce one or more fetuses.” (1) Follicles as the functional unit: Ovarian follicles have since been shown to be the basic functional unit of the ovary and consist of an outer layer of theca interna cells which encircle inner layers of granulosa cells. Granulosa cells, in turn, surround the innermost oocyte-cumulus cell complex. The maturation of ovarian follicles and their transformation into corpora lutea is regulated by a specific set of hormones and has been a major research area for ovarian physiologists for several hundred years.

To assess the role of inhibitors of proteolytic enzymes, such as plasminogen activator (PA) and collagenase in the ovulatory process, inhibitor activity and mRNA levels were examined in periovulatory rat and human ovaries. In the rat, immature animals received 20 IU of pregnant mare serum gonadotropin (PMSG) followed 52 h later by 10 IU of hCG. Ovaries were removed at intervals from 0 to 20 h after human chorionic gonadotropin (hCG) administration. Inhibitor activity for metalloproteinases, such as collagenase, increased from 60.5 +/- 4.1 inhibitor units/ovary at 0 h (i.e., time of hCG treatment) to a maximum of 218.2 +/- 11.4 units/ovary at 8 h after hCG before decreasing at 12 h (time of ovulation) and 20 h (122.2 +/- 7.9 and 71.6 +/- 8.1 units/ovary, respectively). Human follicular fluid and granulosa cells were obtained from preovulatory follicles of patients in our in vitro fertilization program. Metalloproteinase inhibitor activity was evaluated in follicular fluid as well as the levels of PA and PA inhibitor (PAI) mRNA by Northern analysis. Increasing metalloproteinase inhibitor activity was positively correlated with follicular levels of estradiol (p less than 0.001) and progesterone (p less than 0.02, N = 26). Chromatographic separation of follicular fluid resulted in two peaks of metalloproteinase inhibitor activity. The large molecular weight (MW) inhibitor had an approximate size of 700 kilodaltons (kDa) and may represent alpha 2-macroglobulin, a serum-derived inhibitor. The small MW inhibitor shared many of the characteristics of tissue-derived inhibitors of metalloproteinases. Partial purification of the small MW inhibitor by Concanavalin A-Sepharose and Heparin-Sepharose chromatography demonstrated the inhibitor to be a glycoprotein with an approximate MW = 28-29 K. Northern analysis of human granulosa cell total RNA from preovulatory follicles showed little or no detectable tissue-type PA or urokinase-type PA mRNA. In contrast, two species of PA inhibitor type-1 mRNA were detected in relative abundance. The present findings demonstrate the presence of proteolytic inhibitors in periovulatory ovaries of the rat and human. These ovarian inhibitors may play a role in regulating connective tissue remodeling during follicular rupture.

The direct modulation of 20α-hydroxysteroid dehydrogenase (20α-OH-SDH) by regulatory agents of luteal functions was examined using in vitro cultures of ovarian granulose cells from immature, hypophysectomized, estrogen-treated rats. Without FSH priming, treatment with isoproterenol, PRL, or prostaglandin F2α (PGF2α) resulted in minimal modulation of the 20α-OH-SDH activity of granulosa cells. In contrast, after in vitro FSH priming, these hormones or agents caused marked changes in 20α-OH-SDH activity. Isoproterenol treatment inhibited 20α-OH-SDH activity in a dose-dependent manner (ED50, 3.4 × 10-10 M; maximal inhibition, 62% decrease). Similar inhibition of 20α-OH-SDH activity was observed after treatment with epinephrine, norepinephrine, and a β2-adrenergic agonist (terbutalino sulfate), whereas a β1 -adrenergic agonist (dobutamine) was less effective. Furthermore, the inhibitory effect of epinephrine was blocked by concomitant treatment with a β2- antagonist (IPS 339) but not by a β1-antagonist (pract...

The effects of gonadotropin-releasing hormone (GnRH) and its potent agonist [des-Gly10, D-Leu6-N alpha Me) Leu7, Pro9,NHEt-GnRH (GnRH-A)] on ovarian luteal functions maintained by PRL were studied in vivo and in vitro. Hypophysectomized, diethylstilbestrol-treated female rats were primed with FSH for 2 days, followed by an ovulating dose of LH or hCG. Two days later, ovarian luteal functions were maintained by daily injections of 250 microgram PRL for 3 days. PRL treatment increased the serum progesterone level from 13.0 +/- 0.5 to 298 +/- 24 ng/ml and increased the ovarian hCG-binding capacity from 5.8 +/- 1.3 to 584 +/- 86 ng bound hCG/ovary. In contrast, concomitant treatment with GnRH or GnRH-A resulted in dose-dependent decreases in the PRL-induced increase of serum progesterone and ovarian LH/hCG receptor content. GnRH at 100 microgram/day caused a 60% decrease in serum progesterone and an 80% decrease in ovarian LH receptor content, whereas GnRH-A was effective at a 1-microgram dose level. Neither GnRH nor GnRH-A affected the binding affinity (Kd) of ovarian LH receptor. The direct inhibitory effects of GnRH and GnRH-A upon granulosa-luteal cell function were also tested in vitro. FSH treatment for 2 days induced functional LH and PRL receptors in cultured PRL, increased (by approximately 3-fold) progesterone production by these granulosa-luteal cells, whereas concomitant treatment with GnRH-A inhibited progesterone production in a dose-dependent manner. Thus, these studes demonstrated that GnRH and GnRH-A exert direct inhibition on ovarian luteal functions by decreasing LH receptor and progesterone production in vivo as well as inhibiting progesterone production by cultured granulosa-luteal cells in vitro.

Studies with rat ovarian cells indicate that proteolytic enzymes, such as plasminogen activator (PA), play a role in the tissue remodeling that occurs before ovulation. In the rat, gonadotropins appear to increase granulosa cell tissue-type plasminogen activator (t-PA) content by increasing the cellular concentration of t-PA mRNA as well as by modulating the activity of a specific PA inhibitor (PAI). We obtained granulosa cells from preovulatory follicles of women undergoing either in vitro fertilization or gamete intra-fallopian tube transfer in order to evaluate the roles of PA and PAI in human ovulation. Samples of granulosa cell total RNA were hybridized with probes for t-PA, urokinase-type PA, PAI type 1 (PAI-1), or inhibin A-chain (as a control). Northern analyses revealed that the RNA of granulosa cells from preovulatory follicles contained little or no detectable PA mRNA. In contrast, two species of PAI mRNA were detected in relative abundance. The signal intensity produced by the PAI-1 probe varied by about 8-fold among patient samples, suggesting that PAI-1 may be useful as a marker of follicular maturation and differentiation. These results demonstrate that human granulosa cells collected immediately before ovulation contain PA inhibitor mRNA, yet have little or no PA mRNA.

Since the purification and sequencing of gonadotropin releasing hormone (GnRH) and the subsequent synthesis of potent GnRH agonists and antagonists, many laboratories have been involved in investigating the effects of these peptides on reproductive functions. In women, administration of GnRH at a restricted time in the cycle results in preovulatory-like surges of gonadotropin (Yen et al., 1975; Grimes et al., 1975). Paradoxically, long-term administration of potent GnRH agonists is always associated with the inhibition of a variety of female reproductive functions such as ovarian steroidogenesis, ovulation, ovum transport, ovum implantation, pregnancy, uterine growth and ovarian-dependent mammary tumorigenesis.

The influence of triphenylethylene antiestrogens on FSH-stimulated production of estrogen, progesterone, and 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) was examined in cultured rat granulosa cells. The cells were cultured with increasing concentrations of FSH, with or without diethylstilbestrol (DES) or various antiestrogens. After 3 days, medium progesterone and 20 alpha-OH-P contents were determined. Cells were reincubated for an additional 8 h with delta 4-androstene-3,17-dione, and estrogen formation was measured. Steroid production was negligible by cultures treated with DES or antiestrogens alone. FSH treatment increased estrogen and progestin production, while the addition of DES (10(-7) M) further enhanced FSH-stimulated steroidogenesis. Likewise, treatment with 10(-6) M of the antiestrogens tamoxifen (Tam), nafoxidine (Naf), or clomiphene citrate (CC) also enhanced FSH-stimulated aromatase activity. In contrast, the antiestrogens each inhibited FSH stimulation of progesterone and 20 alpha-OH-P production (greater than 73% inhibition at 300 ng/ml FSH). Increasing concentrations (3 X 10(-8)-10(-6) M) of the antiestrogens augmented the stimulatory effect of FSH (10 ng/ml) on estrogen production in a dose-related manner (CC greater than Tam greater than Naf). Similar doses of these antiestrogens inhibited the stimulatory effect of FSH (300 ng/ml) on progesterone and 20 alpha-OH-P production (Tam greater than CC greater than Naf). The observed inhibition of progestin production is associated with decreases in FSH-stimulated pregnenolone biosynthesis in antiestrogen-treated cells incubated with 10(-6) M cyanoketone. Furthermore, the antiestrogens inhibited the binding of [3H]estradiol to ovarian estrogen receptors with binding affinity constants of 364, 437, and 2144 nM for CC, Tam, and Naf, respectively. Thus, antiestrogens exert disparate actions on granulosa cell estrogen and progestin biosyntheses. Like estrogens, CC, Tam, and Naf enhance FSH-stimulated aromatase activity with potencies comparable with their abilities to interact with ovarian estrogen receptors. However, unlike estrogens, the antiestrogens inhibit FSH-stimulated progestin biosynthesis, partially via suppression of pregnenolone biosynthesis. The present granulosa cell culture system provides a valuable model for elucidating the disparate actions of estrogens and antiestrogens on ovarian steroidogenesis.

Since pituitary gonadotropins are essential for normal gonadal functions and since hypothalamic LHRH was believed to act solely on the pituitary gland, treatment with high doses of LHRH or its agonists had been predicted to be a potential means for enhancing fertility. However, early studies using LHRH as an ovulation-inducing agent in anovulatory women have been only partially successful. Similarly, the early use of LHRH and LHRH agonists in the therapy of hypogonadotropic hypogonadism in men resulted in only limited success, and actual decreases in testicular steroidogenesis were reported.

The hormonal modulation of prolactin (PRL)-binding capacity of rat granulosa cells was studied. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 2 days in a serum-free medium in the presence of various hormones. FSH treatment in vitro stimulated granulosa cell PRL-binding capacity by approximately 4-6-fold in a dose-dependent manner. Concomitant treatment with 10-8 M GnRH inhibited the FSH-induced increase in PRL-binding capacity by 64%. In contrast, the inhibitory effect of GnRH was blocked by concomitant treatment with 10-6 M of a GNRH antagonists, [D-pGlu1, D-Phe2, D-Trp3,6]GnRH. PRL-binding capacity was also increased (approximately 2-fold) by in vitro treatment with cholera toxin (10 microgram/ml). In granulosa cells pre-treated with FSH in vitro for 2 days, hCG treatment for 2 additional days stimulated PRL-binding capacity in a dose-dependent manner (approximately 2-fold). Likewise, treatment with LH (100 ng/ml) also stimulated PRL-binding capacity by approximately 2-fold. These in vitro studies demonstrated that gonadotropins (FSH, LH and hCG) directly enhanced PRL binding by granulosa cells, whereas GnRH inhibited FSH action.

Possible influences of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) upon gonadal steroidogenesis were investigated in vitro. Granulosa cells from hypophysectomized, estrogen-treated rats were cultured for 2 days in medium containing 0.1 microM androstenedione. Follicle-stimulating hormone (FSH) treatment increased estrogen, progesterone, and 20 alpha-hydroxypregn-4-en-3-one production. Concomitant TPA treatment inhibited FSH-stimulated estrogen production by up to 95% [concentration that induced 50% inhibition of steroid production (ED50), 1.1 ng/ml]. TPA also inhibited FSH-stimulated progesterone (ED50, approximately 0.6 ng/ml) and 20 alpha-hydroxypregn-4-en-3-one (ED50, approximately 1.1 ng/ml) production. N6O2'-dibutyryl cyclic adenosine 3':5'-monophosphate increased steroidogenesis; however, cotreatment with TPA blocked progestin but not estrogen production. The TPA inhibition of progestin biosynthesis was accompanied by decreases in FSH-stimulated pregnenolone biosynthesis and 3 beta-hydroxysteroid dehydrogenase activity without decreasing the activity of 20 alpha-hydroxysteroid dehydrogenase. In primary cultures of rat testicular cells, human chorionic gonadotropin treatment increased testosterone production 44-fold, whereas concomitant treatment with TPA inhibited testosterone production by up to 86% (ED50, 10 ng/ml). Cholera toxin and N6O2'-dibutyryl cyclic adenosine 3':5'-monophosphate also increased testosterone production, while the actions of these agents were decreased by TPA. The TPA suppression of testosterone production was associated with a decrease in accumulation of 17 alpha-hydroxyprogesterone and androstenedione and an increase in progesterone production, suggesting a specific inhibition of 17 alpha-hydroxylase and 17,20-lyase activities. These results demonstrate the inhibitory effects of a tumor promoter upon gonadotropin-stimulated steroidogenesis by cultured rat granulosa and Leydig cells through specific regulation of steroidogenic enzymes. Additional studies may assist in further elucidation of cellular mechanisms associated with carcinogenesis and steroidogenesis.