Gonadotropin-releasing Hormone Analogs Inhibit Primate Granulosa Cell Steroidogenesis Via a Mechanism Distinct from that in the Rat1

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Abstract
Gonadotropin-releasing hormone (GnRH) and related peptides are implicated in the local control of rat ovarian function, but evidence to date for direct effects of such peptides on primate ovarian cells is equivocal. In contrast to rat ovaries, where GnRH action is mediated through specific, high-affinity GnRH receptors, no such binding sites have been identified in primate tissue. Using undifferentiated granulosa cells from immature follicles in cyclic (luteal phase) marmoset ovaries, we have observed direct suppression of human (h) FSH-induced steroidogenesis by GnRH analogs in vitro. Granulosa cells from immature (< 1 mm diameter) follicles were incubated for 4 days in the presence of hFSH and testosterone (aromatase substrate) to stimulate cyclic AMP (cAMP) production and steroidogenesis. The additional presence of GnRH alone (up to 10 .mu.M) had no effect on FSH action. However, the GnRH agonist, [D-Ser(But)6[GnRH 1-9)-ethylamide (Buserelin, 0.1 .mu.M-10 .mu.M), caused time- and dose-dependent inhibition of estradiol (maximum inhibition = 79%; ED50 = 0.55 .mu.M) and progesterone production (maximum inhibition = 93%; ED50 = 0.1 .mu.M). Accumulation of cAMP was also inhibited by up to 54%. Paradoxically, a GnRH antagonist ([N-Ac-D-Nal(2)1, D-pCl-Phe2, D-Trp3, D-hArg(Et2)6, D-Ala10]-GnRH; 10 .mu.M) alone also inhibited hFSH-stimulated cAMP and steroid production by 40% and 70%, respectively. Moreover, the suppressive effects of the GnRH agonist on granulosa cell functions were augmented by the presence of the GnRH antagonist (10 .mu.M). In contrast, GnRH (0.01 .mu.M) completely inhibited FSH-stimulated cAMP and steroid production by immature rat granulosa cells, and this inhibition was totally reversed by an equimolar concentration of the GnRH antagonist. To investigate GnRH binding to granulosa cell-rich marmoset and rat ovarian tissue, homogenates were incubated with 125I-GnRH agonist with and without excess unlabeled GnRH agonist. No specific high affinity of the agonist to marmoset ovarian tissue was found, whereas 125I-GnRH agonist was specifically bound to rat ovarian tissue. These results for marmoset granulosa cells were quantitatively (low potency inhibition by GnRH agonist) and qualitatively (paradoxical suppression by GnRH antagonist and absence of high-affinity binding sites) distinct from those for rat granulosa cells. It is concluded that, in marmoset granulosa cells, GnRH analogs in high concentration can inhibit hFSH-stimulated steroidogenesis in vitro via a mechanism that does not involve a rat-type specific ovarian GnRH receptor.

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