Melatonin attenuates nitric oxide induced oxidative stress on viability and gene expression in bovine oviduct epithelial cells, and subsequently increases development of bovine IVM/IVF embryos

Abstract

The objective of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co-culture on developmental capacity of bovine IVM/IVF embryos and to determine whether or not melatonin acts as an antioxidant in BOEC culture and subsequent embryo development. These studies examined the effects of melatonin against NO-induced oxidative stress on cell viability, lipid peroxidation (LPO) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl-2, Caspase-3 and Bax) during BOECs culture. We also evaluated the developmental rates of bovine IVM/IVF embryos with BOEC co-culture, which were pre-treated with melatonin (1,000 μM) in the presence or absence of sodium nitroprusside (SNP, 1,000 μM) for 24 h. Cell viability in BOECs treated with SNP (50-2,000 μM) decreased while melatonin addition (1-1,000 μM) increased viability in a dose-dependent manner. Cell viability in melatonin plus SNP (1,000 μM) gradually recovered according to increasing melatonin addition (1-1,000 μM). The LPO products were measured by thiobarbituric acid (TBA) reaction for malondialdehyde (MDA). Addition of melatonin in BOEC culture indicated a dose-dependent decrease of MDA, and in the SNP group among BOECs treated with SNP or melatonin plus SNP groups MDA was significantly increased compared with SNP plus melatonin groups (p<0.05). In expression of apoptosis or antioxidant genes detected by RT-PCR, Bcl-2 and antioxidant genes were detected in melatonin or melatonin plus SNP groups, while Caspase-3 and Bax genes were only found in the SNP group. When bovine IVM/IVF embryos were cultured for 6-7 days under the BOEC co-culture system pre-treated with melatonin in the presence or absence of SNP, the highest developmental ability to blastocysts was obtained in the 1,000 μM melatonin group. These results suggest that melatonin has an anti-oxidative effect against NO-induced oxidative stress on cell viability of BOECs and on the developmental competence of bovine IVM/IVF embryo co-culture with BOEC.