Oxygen tension and Antioxidants on development of porcine in vitro produced embryos

Abstract

Because of availability of pigs in biomedical research, in vitro production of porcine embryos is very crucial step. The oxygen toxicity resulted from ROS on in vitro environment could be one among numerous causes of low in vitro productivity. The purpose of this study is to investigate effects on embryo culture as change of culture condition resulted from different oxygen tension and to increase in vitro production rates of porcine embryos by supplementing some antioxidants to the media for defending of oxygen toxicity. So this study was conducted to investigate the effect of different oxygen concentration (5 and 20%) during in vitro maturation (IVM) and in vitro culture (IVC) and gene expression pattern, and to investigate effects of some antioxidants such as melatonin, flavonoids (quercetin and taxifolin) on porcine oocyte maturation and embryo further development.

First, as a result of the effect of two oxygen concentrations during IVM and IVC on porcine embryo development, there were no significant differences in oocytes nuclear maturation rate. However, on further culture in 20% IVM, the 5% IVC group showed significantly increased blastocyst formation rate compared to the 20% IVC group. According to mRNA abundance data of multiple genes, each treatment altered the expression of genes in different patterns. As a result, in low oxygen, it occurred with a higher glucose uptake and an increase in anaerobic glycolysis in the cumulus cells, whereas in high oxygen, it happened to a higher activity of mitosis-promoting factor and antioxidant response in cumulus cells. Therefore, it can be concluded that high oxygen concentration during IVM and low oxygen during IVC may alter the expression of multiple genes related to oocyte competence and significantly improves embryo development.

Second, as one of means to supplement of antioxidants, melatonin was added to maturation media. Melatonin at the 10 ng/ml concentration during maturation showed the beneficial effect on the maturation rate and further developmental competence and lower levels of ROS. Also, as results of the local expression of the endogenous melatonin, melatonin receptor-1 gene expressed in cumulus and granulosa cells surrounding on oocytes. I concluded the exogenous melatonin has beneficial effects on nuclear and cytoplasmic maturation during porcine IVM. But it is not clear whether the observed effects may be mediated by receptor binding or receptor independent, as a direct free radical scavenging.

The result of some flavonoids (quercetin and taxifolin) treatment as other antioxidants showed all quercetin and taxifolin treatment did not improve nuclear maturation of oocytes, but a significantly greater proportion of parthenogenetically activated oocytes developed into blastocysts when the IVM medium was supplemented with adequate quercetin (1ug/ml). As measurement result of levels of ROS and GSH in oocytes and embryos produced in maturation medium supplemented with quercetin or taxifolin, both treatment groups had significantly lower levels of ROS than controls, however GSH levels were different only in quercetin treated oocytes. I concluded that exogenous flavonoids such as quercein reduce ROS levels in oocytes and may work effectively on embryonic development.

In conclusion, I suggested that low oxygen tension during culture effectively contribute to in vitro embryo development of porcine oocytes and that media supplemented with exogenous antioxidant such as melatonin and quercetin on the maturation process of porcine oocyte could promote maturation rate in vitro by reducing ROS level arisen during in vitro culture.