Signální dráhy a geny regulující u prasete zrání oocytů a expanzi kumulu indukované gonadotropiny

Abstract

V podmínkách in vitro jsou meotické zrání prasečích oocytů a expanze kumulu indukovány FSH a peptidy rodiny EGF (AREG a EREG). FSH a tyto peptidy způsobují expresi genů spojených s expanzí kumulu (HAS2, PTGS2 a TNFAIP6). Za účelem zjistit signální dráhy, které kontrolují expanzi kumulu indukovanou FSH nebo AREG, prasečí komplexy oocyt-kumulus (COC) byly ošetřeny specifickými inhibitory proteinkináz. Inhibitory MAPK3/1, MAPK14 a ERBB1 signifikantně redukovaly expresi HAS2, PTGS2 a TNFAIP6. Tyto inhibitory rovněž snížily expresi AREG a EREG indukovanou FSH a LH v buňkách stěnové granulózy. Inhibitor PKA překvapivě neměl efekt na expresi AREG v COC, ale snížil expresi TNFAIP6 indukovanou AREG. Inhibitor PI3K zvýšil expresi AREG a PTGS2, ale snížil expresi EREG, HAS2 a TNFAIP6. Anlog cGMP (8-CPT-cGMP) neovlivnil expresi genů spojených s expanzí kumulu. Nicméně blokoval spontánní meiotickou maturaci prasečích oocytů a jeho efekt byl eliminován FSH. Klíčová slova: expanze kumulu, geny spojené s expanzí kumulu, meiotická maturace, FSH, amfiregulin, cGMP

In vitro, meotic maturation of porcine oocytes and cumulus expansion are induced by FSH and EGF-like peptides AREG and EREG. FSH and EGF-like peptides induce expression of cumulus expansion-related genes (HAS2, PTGS2 and TNFAIP6). To define signaling pathways that control FSH- and AREG-induced cumulus expansion, porcine cumulus-oocyte complexes were treated with specific protein kinase inhibitors. Inhibitors of MAPK3/1, MAPK14 and ERBB1 significantly reduced both FSH- and AREG-induced expression of HAS2, PTGS2 and TNFAIP6. These inhibitors decreased FSH/LH-induced expression of AREG and EREG in mural granulosa cells. Surprisingly, inhibitor of PKA had no effect on AREG expression in cumulus-oocyte complexes but the inhibitor decreased expression of TNFAIP6 induced by AREG. Inhibitor of PI3K increased expression levels of AREG and PTGS2 but EREG, HAS2 and TNFAIP6 were reduced. Expression levels of the cumulus expansion-related genes were not affected by an analog of cGMP (8-CPT-cGMP). However, 8-CPT-cGMP blocked spontaneous in vitro meiotic maturation of porcine oocytes and its effect was abolished by FSH. Key words: cumulus expansion, cumulus expansion-related genes, meotic maturation, FSH, amphiregulin, cGMP