GPR30 Signaling in Neonatal Hamster Ovary Involves PI3 Kinase (PI3K) and ERK-1/2 Pathways.

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We have shown that estradiol (E2) stimulates the formation and development of hamster primordial follicles in vivo and in vitro. A part of the E2 action is mediated by GPR30, but the mechanisms are not known. The objective of this study was to investigate whether GPR30 signaling during primordial follicle formation involved PI3 kinase or ERK-1/2 pathway. Wortmannin or U0126 suppressed the stimulatory effect E2-BSA on primordial follicle formation. PI3 kinase protein was detected in the oocytes of 13-day old fetus (E13), but the enzyme appeared in somatic cells on postnatal day 6 (P6). The intensity of PI3K immunosignal increased in the granulosa cells of newly formed primordial follicles on P8 and primary follicles on P9. PI3K expression correlated well with the formation of primordial follicles, with a higher expression on P8, when the first cohort of primordial follicles appear. The levels of phosphoAkt (pAkt) also increased from P7 and reached a peak on P9, suggesting that PI3K action via Akt might be involved in primordial follicle formation. pAkt immunosignal was located mainly in the oocytes and in adjacent somatic cells before the formation of primordial follicles. After follicle formation, pAkt immunosignal shifted to the granulosa cells of primordial and primary follicles. Similarly, higher ERK-1/2 immunosignal was present in the oocytes and adjacent somatic cells, and increased ERK1/2 phosphorylation correlated with the appearance of the first cohort of primordial follicles. Treatment of cultured P6 ovarian cells with 10 nM E2 significantly increased the levels of pAkt and pERK-1/2 within 10 min, while 1 µM E2 had no effect. 10 nM E2 also stimulated the phosphorylation of cRaf and MEK. Whereas Wortmannin blocked E2-induced Akt phosphorylation without affecting ERK-1/2 phosphorylation, U0126 blocked E2-induced ERK1/2 phosphorylation without affecting Akt phosphorylation. Similar phosphorylation patterns were observed when cells were exposed to G1, a GPR30 specific ligand. siRNA knockdown of GPR30 significantly reduced E2- or G1-induced Akt and ERK1/2 phosphorylation, thus providing additional evidence for a GPR30-mediated action. These results together with our previous findings suggest that the mechanisms of E2-regulated primordial follicle formation may involve two parallel signaling modalities, and GPR30 mediates a part of the E2 action during primordial follicle formation. (platform)