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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.

Inhibin is a gonadal protein hormone that suppresses the secretion of FSH from pituitary gonadotrophs. It has previously been characterized as a heterodimer of two dissimilar subunits (alpha, 18 kilodaltons and beta, 14 kilodaltons) the smaller of which exists in two forms (beta A and beta B) and can form dimers that stimulate the secretion of FSH. In the present work, cDNA clones encoding the inhibin alpha- and beta A-subunits have been isolated from rat ovary and characterized. The alpha-inhibin cDNA predicts a precursor protein of 366 amino acids containing the 133 amino acid mature alpha-subunit at its COOH-terminus. The beta A-inhibin cDNA predicts a precursor protein of 424 amino acids containing the 116 amino acid beta A-subunit at its COOH-terminus. Analysis of rat ovarian RNA indicates that alpha-inhibin mRNA levels are stimulated by PMSG treatment in vivo. In cultured granulosa cells, FSH also stimulates alpha-inhibin mRNA, and the FSH effect is suppressed by cotreatment with GnRH. Hybridization in situ to rat ovarian tissue demonstrates that both the alpha-inhibin and beta A-inhibin mRNAs are specifically expressed in granulosa cells of the developing follicles.

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.