EARLY ACTIVATION OF THE EGFR SIGNALLING PATHWAY BY LH IN PREOVULATORY FOLLICLES

The surge of the gonadotropin LH activates a cascade of events that are propagated throughout the ovarian preovulatory follicle to promote ovulation of a fertilizable egg. Critical to LH-induced ovulation is the induction of EGF-like growth factors and transactivation of EGF receptor (EGFR) signalling. Because the timing of this transactivation in relationship to oocyte re-entry into the cell cycle is not established, we sought to investigate the dynamics of LH regulation of the EGF network in cultured follicles. Preovulatory follicles from PMSG-primed mice were cultured without or with recombinant LH (rLH) and/or specific inhibitors. EGF receptor (EGFR) and MAPK phosphorylation were examined by immunoprecipitation and Western blot analyses. In cultured follicles, an increase in amphiregulin and epiregulin mRNAs was already detectable by semi-quantitative RT-PCR 30 min after rLH stimulation, and was maximal after 2 h. Similarly, EGFR phosphorylation was increased in follicles 30 min after rLH stimulation and was maximal after 2 h, thus preceding oocyte maturation by more than 1 h. EGFR activation is likely cAMP dependent, as treatment with Forskolin produced a similar effect. Moreover, rLH-induced EGFR phosphorylation is sensitive to AG1478, an EGFR kinase inhibitor, and to inhibitors of matrix metalloproteases (GM6001 and TAPI-1), suggesting the involvement of EGF-like growth factor shedding. LH-dependent but not amphiregulin-induced oocyte maturation and EGFR phosphorylation is sensitive to protein synthesis inhibition, suggesting that the proteins whose synthesis is required for LH action are the EGF-like growth factors. When granulosa cells were cultured with a combination of neutralizing antibodies against the EGFlike factors amphiregulin, epiregulin and betacellulin, EGFR phosphorylation and MAPK activation were significantly inhibited. In cultured follicles, LH-induced MAPK activation is partially inhibited by AG1478 and GM6001, indicating that this pathway is regulated in part by the EGF network, but that additional pathways are also involved. By immunocytochemistry, signal for phosphorylated MAPK was observed in mural granulosa cells of preovulatory follicles within 15–30 minutes of hCG stimulation, and propagated to cumulus cells after 1 h. Taken together, we demonstrate that the complex mechanisms involved in the rapid amplification and propagation of the LH signal within preovulatory follicles include the early activation of the EGFR-MAPK network marked by rapid shedding of EGF-like ligands and EGFR transactivation. (poster)