Gene expression and epigenetic changes during the meiotic inhibition of sheep oocytes

Abstract

Meiosis inhibitors such as roscovitine have been used in different animal species with the aim of increasing the in vitro development potential of oocytes and embryos. Studies have shown, however, the culture conditions during the in vitro embryo production predispose the abnormal DNA methylation. So, this research aims to analyze the possible epigenetic changes in terms of citosine methylation and hydroxymethylation in sheep oocytes undergoing treatment with roscovitine and to evaluate the heritability of these changes during the in vitro embryo development. For this, oocytes from sheep ovaries, obtained from slaughterhouse, will be cultivated at 38.5ºC and 5% CO2 in air, for 13 hours, in a specific maturation medium (Control) supplemented with 75µM of roscovitine (Treatment). Then, certain proportion of oocytes will be cultivated for further 17 hours in inhibitor-free medium, under the same conditions described above. At the end of 13 and 30 hours of in vitro culture, oocytes from both experimental groups will be assessed for global level of DNA methylation and hydroxymethylation, by immunocytochemistry and for expression of genes DNMT1, DNMT3a, DNMT3b, TET1, TET2, TET3 by RT-qPCR. Some of the oocytes from both groups, after 30 hours of culture, will be assigned to the in vitro embryo production. The zygote, morula and blastocyst obtained will be submitted to the same analysis described for oocytes. The results will be evaluated by ANOVA and Tukey test. The cleavage and blastocyst rate also will be evaluated and analyzed by Chi-Square test. (AU)