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In rat ovarian granulosa cells the effects of GnRH are determined by the state of granulosa cell development with mainly inhibitory actions in immature cells and stimulatory actions in differentiated mature cells. These developmentally related effects of GnRH may arise from changes in either one or more of the signal transduction pathways activated by GnRH. The present study therefore measured downstream signalling events associated with the activation of the phospholipase C (PLC) signal transduction pathway in both mature and immature rat ovarian granulosa cells. Results showed that GnRH produced similar total inositol phosphate and intracellular calcium ([Ca2+ ]i) responses in both immature and mature granulosa cells. In contrast to the biphasic GnRH-induced [Ca2+]i response in pituitary gonadotropes, stimulation of the endogenously expressed GnRH receptor in both immature and mature granulosa cells produced a prompt monophasic rise in [Ca2+]i. This calcium transient was abolished by pretreating either cell type with a potent GnRH receptor antagonist or the PLC inhibitor U73122 [GenBank] , demonstrating a GnRH receptor-specific activation of PLC. Similarly, pretreatment of cells with the [Ca2+]i antagonists thapsigargin or cyclopiazonic acid abolished the GnRH-induced calcium transient, whereas EGTA and nifedipine, a voltage-operated calcium channel (VOCC) antagonist, had no effect. These results suggest that in either immature or mature granulosa cells GnRH mobilises calcium from thapsigargin/cyclopiazonic acid-sensitive [Ca2+]i stores but does not involve the influx of extracellular calcium through VOCCs. We conclude that GnRH-induced stimulation of the PLC signal transduction pathway is independent of the stage of granulosa cell maturity and that alternative mechanisms account for the opposite effects of GnRH on gonadotrophin-induced steroidogenesis in mature and immature rat granulosa cells in vitro.
Journal of Endocrinology (1996) 149, 449–456
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