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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 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.

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.

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.

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.

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.