RESUMO
The aim of this work was to assess the FSH-stimulated expression of epidermal growth factor (EGF)-like peptides in cultured cumulus-oocyte complexes (COCs) and to find out the effect of the peptides on cumulus expansion, oocyte maturation, and acquisition of developmental competence in vitro. FSH promptly stimulated expression of amphiregulin (AREG) and epiregulin (EREG), but not betacellulin (BTC) in the cultured COCs. Expression of AREG and EREG reached maximum at 2 or 4 h after FSH addition respectively. FSH also significantly stimulated expression of expansion-related genes (PTGS2, TNFAIP6, and HAS2) in the COCs at 4 and 8 h of culture, with a significant decrease at 20 h of culture. Both AREG and EREG also increased expression of the expansion-related genes; however, the relative abundance of mRNA for each gene was much lower than in the FSH-stimulated COCs. In contrast to FSH, AREG and EREG neither stimulated expression of CYP11A1 in the COCs nor an increase in progesterone production by cumulus cells. AREG and EREG stimulated maturation of oocytes and expansion of cumulus cells, although the percentage of oocytes that had reached metaphase II was significantly lower when compared to FSH-induced maturation. Nevertheless, significantly more oocytes stimulated with AREG and/or EREG developed to blastocyst stage after parthenogenetic activation when compared to oocytes stimulated with FSH alone or combinations of FSH/LH or pregnant mares serum gonadotrophin/human chorionic gonadotrophin. We conclude that EGF-like peptides do not mimic all effects of FSH on the cultured COCs; nevertheless, they yield oocytes with superior developmental competence.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células do Cúmulo/efeitos dos fármacos , Gonadotropinas/farmacologia , Oócitos/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Suínos , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Células do Cúmulo/metabolismo , Células do Cúmulo/fisiologia , Técnicas de Cultura Embrionária , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/farmacologia , Feminino , Hormônio Foliculoestimulante/farmacologia , Perfilação da Expressão Gênica , Oócitos/metabolismo , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Oogênese/genética , Partenogênese/efeitos dos fármacos , Partenogênese/genética , Partenogênese/fisiologia , Fragmentos de Peptídeos/química , Suínos/genética , Suínos/metabolismo , Suínos/fisiologiaRESUMO
The purpose of the present study was to elucidate signaling pathways by which insulin like-growth factor 1 (IGF1) promotes FSH-stimulated synthesis and retention of hyaluronic acid (HA) in pig oocyte-cumulus complexes (OCCs) cultured in serum-free medium. We found that IGF1 had no effects on FSH-stimulated production of cAMP and activation of protein kinase A in the OCCs. Immunoblotting with phospho-specific antibodies showed that FSH moderately phosphorylated v-akt murine thymoma viral oncogene homolog (AKT) and mitogen-activated kinase 3 and 1 (MAPK3/1) in cumulus cells. The exposure of OCCs to both FSH and IGF1 resulted in a significant (P < 0.05) increase in AKT and MAPK3/1 phosphorylation. An inhibitor of phosphoinositide-3-kinase (PIK3), LY 294002, significantly (P < 0.05) reduced the IGF1-enhanced phosphorylation of AKT, and inhibitors of AKT (SH6) and MAPK3/1 (U0126) significantly (P < 0.05) decreased the synthesis and retention of HA stimulated by concomitant exposure of OCCs to both FSH and IGF1. The IGF1-promoted synthesis of HA was not accompanied by an increase in the relative abundance of hyaluronan synthase 2 (HAS2) mRNA in the cumulus cells. We conclude that IGF1 promotes the FSH-stimulated synthesis and retention of HA in pig OCCs by PIK3/AKT- and MAPK3/1-dependent mechanisms.
