Endocrine Abstracts

Recent studies demonstrate an essential role of the EGF network in propagating the LH signal within ovarian preovulatory follicles. However, the molecular bases for the integration are poorly characterized. Here, we propose that the early LH signal leading to ovulation is amplified through activation of the EGF network.

For this study, preovulatory follicles from euthanized gonadotropin-primed mice were isolated and cultured with or without recombinant LH (rLH) and/or specific inhibitors. Primary granulosa cells were used in additional experiments. Analysis of EGF receptor (EGFR) and MAPK activation was performed by immunoprecipitation, western blot and immunohistochemistry (IHC). An increase in EGFR phosphorylation was detected as early as 30 minutes after LH stimulation. This activation is most likely cAMP dependent and sensitive to AG1478, an EGFR kinase inhibitor, as well as to inhibitors of matrix-metalloproteases (GM6001 and TAPI-1), suggesting the involvement of shedding of EGF-like factors in LH-induced EGFR transactivation. A target of EGFR signaling is the MAPK pathway. In IHC assays, signal for phosphorylated MAPK was observed in mural granulosa cells of preovulatory follicles within 15–30 minutes of hCG stimulation, and in both granulosa and cumulus cells after 1 h. 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. Furthermore, treatment of granulosa cells with a combination of neutralizing antibodies against amphiregulin, epiregulin and betacellulin (EGF-like factors described as regulators of ovulation) significantly inhibits EGFR phosphorylation and MAPK activation, supporting a role for these ligands in the LH-induced EGFR signaling in mural granulosa cells.

In conclusion, we provide evidence of early activation of EGF network following LH stimulation, involving rapid shedding of EGF-like ligands and EGFR transactivation. This mechanism partecipates in the rapid amplification and propagation of the LH signal within preovulatory follicles.