Direct actions of a gonadotropin-releasing hormone agonist on steroidogenesis and aromatase activity in cultured porcine granulosa cells

The mechanism by which a gonadotropin-releasing hormone agonist (D-Ala⁶) des-Gly¹⁰-GnRH (GnRHa) modulates ovarian steroid secretion was investigated in cultured porcine granulosa cells. Ovarian granulosa cells, obtained from medium (3-5 mm) sized follicles, were incubated with various hormones in vitro and the steroid content of the spent medium determined by radioimmunoassay. The morphology of the granulosa cells were initially epithelioid, but tended to assume a fibroblast-like morphology during the course of the culture. In comparison with control cultures, it was determined that the hormone had no systemic effect on morphology or cell growth in culture. Graded doses of hFSH or 8-Br-cAMP caused significant increases in progesterone secretion, but had little effect on estradiol secre-tion. Granulosa cell response to GnRHa was generally dose-dependent, with GnRHa causing increases in both progesterone and estrogen secretion. The GnRHa-induced actions were blocked in a dose-dependent manner by a GnRH antagonist Ac-D-Ala¹, D-Phe², D-Trp^3,6 GnRH. GnRHa (1 µg) inhibited FSH- (100 ng/ml) and 8-Br-cAMP- (0.1 mM) stimulated progesterone secretion. Moreover, GnRHa stimulated basal progesterone secretion in untreated cells or in cells treated with only hCG (1 µg/ml). However, GnRHa stimulated estradiol secretion in untreated, FSH-treated, as well as hCG-treated granulosa cells. This secretion, however did not occur in cells treated with 8-Br-cAMP. GnRHa decreased progesterone secretion at the same time it increased estradiol secretion in granulosa cells isolated from large (6-12 mm) follicles. To further examine the GnRHa-induced increase in estrogen secretion, an aroma-tase assay was performed on both untreated and GnRHa treated cells. GnRHa stimulated aromatase activity over 4 d of continuous incuba-tion. Removal of GnRHa from the medium resulted in aromatase activ-ity returning to control levels.

This study demonstrated that GnRHa has both stimulatory and inhibitory actions on steroidogenesis in the cultured porcine granu-losa cell. GnRHa decreases of FSH- and 8-Br-cAMP stimulated pro-gesterone secretion suggests that some of the GnRHa actions lie distal to cAMP formation. One of these actions demonstrated by GnRHa stimulation of estrogen secretion, with or without gonado-tropins, and subsequent aromatase assay is GnRHa-induced increase in aromatase activity. Furthermore, continuous exposure to GnRHa is necessary to elicit the increased aromatase activity.