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General and comparative endocrinology

Human chorionic gonadotropin-dependent up-regulation of epiregulin and amphiregulin in equine and bovine follicles during the ovulatory process.


PMID 23178756

Abstract

Little is known about the expression and regulation of epiregulin (EREG) and amphiregulin (AREG) in ovarian follicles of large monoovulatory animal species. To characterize the gonadotropin-dependent regulation of EREG and AREG mRNAs in equine follicles prior to ovulation, extracts were prepared from equine follicles collected during estrus between 0 and 39h post-hCG and corpora lutea obtained on day 8 of the estrous cycle (day 0=day of ovulation). Results from RT-PCR/Southern blot analyses showed that levels of EREG and AREG mRNAs were very low in follicles obtained at 0h but increased thereafter (P<0.05), with maximal levels observed 33-39h post-hCG. This significant increase was observed in both granulosa and theca cells. Immunohistochemistry and immunoblot analyses confirmed the hCG-dependent induction of EREG protein in both cell types. RT-PCR/Southern blot analyses of ADAM17, which encodes an enzyme that cleaves and releases soluble bioactive EREG and AREG, showed that levels of its transcript were high and remained constant throughout the period studied. Studies on the hCG-dependent regulation of EREG and AREG in bovine preovulatory follicles in vivo showed that the induction of both transcripts was transient, observed predominantly at 6h post-hCG and localized only in granulosa cells. To characterize the effect of epidermal growth factor receptor (EGFR) activation on the expression of ovulation-related genes in granulosa cells of a large monoovulatory animal species, primary cultures of bovine granulosa cells were established. Results from RT-PCR analyses revealed that EREG and AREG mRNAs were induced by forskolin treatment in vitro; but the EGFR inhibitor PD153035 suppressed the forskolin-dependent induction of several ovulation-related transcripts, including PTGS2, PTGER2, TNFAIP6, PGR, MMP1, VEGFA, and CTSL2 mRNAs. Moreover, these transcripts were induced in granulosa cell cultures by EGF, an analog of EREG and AREG. Collectively, this study identifies differences in the temporal and cellular localization of EREG and AREG expression in equine and bovine preovulatory follicles, and underscores the potential role of follicular EGFR activation in the regulation of ovulation-regulated genes in large monoovulatory species.