| Obtaining high quality mature oocytes and its subsequent embryos is the key technology for animal production and assisted reproduction. When compared to the natural growing conditions, oocytes and embryos cultured in vitro may occur abnormal epigenetic modifications and thereby decreasing the quality or even causing the death of oocytes and embryos. L-Ascorbic acid is the essential nutrients for human, recent studies have unveiled that it can widely influence epigenetic modifications, and can improve the developmental competence of embryos. However, the functional revelance of L-Ascorbic acid during oocyte meiotic maturation and its underlying mechanism by which affecting embryonic development remain largely unknown. For example, whether L-Ascorbic acid can affect the meiotic program of mouse oocytes; improve the quality of mouse oocytes and embryos; alleviate the abnormal epigenetic modifications caused by in vitro culture conditions etc.Herein, we employed the techniques of oocytes and embryos in vitro culture, Immunofluorescence, Western Blot and RT-PCR to investigated the effects of L-Ascorbic acid on in vitro oocyte meiotic maturation and embryos developmented, and determine the optimal usage concentration of L-Ascorbic acid in oocyte and embryo culture. The main results areas follows:1. After different concentrations of L-Ascorbic acid treatment on mouse oocytes, the GVBD rates of all the treatment groups were decreased; The 50 ug/ml treatment can significantly improve the first polar body emissions rate while the100 ug/ml treatment decreased significantly(P< 0.05), however, the 200 ug/ml and 1000 ug/ml treatment both could significantly decreased the GVBD rate (P< 0.05);2. Immunofluorescence results showed that:after 100 ug/ml L-Ascorbic acid treatment, the chromosome arranged abnormally; 200ug/ml and 1000 ug/ml treatment led the defectives in chromosome misalignment and spindle assembly;3.After different concentrations of L-Ascorbic acid treatment on mouse oocytes, the expression of DNMT1 protein in each treatment group improved while 100 ug/ml treatment improved it significantly; The immunofluorescent analysis of 5-me showed that 200 ug/ml and 1000 ug/mltreatment significantly improve the 5-mc level (P<0.05);4.The spindle checkpoint BubRl proteins expression of the oocytes matured in vivo were lower than that of oocytes cultured in vitro after different concentration of L-Ascorbic acid (P<0.01);5. After different concentrations of L-Ascorbic acid treatment on mouse oocytes,50 ug/ml L-Ascorbic acid treatment can reduce the expression level of p-ERK while 100 ug/ml could significantly increased the expression level of p-ERK (P< 0.05);6.RT-PCR results showed that the main methylation regulation gene in mouse oocytes is TET3, After different concentrations of L-Ascorbic acid treatment, the mRNA level of methylation gene DNMT1 and demethylation gene TET3 were both increased while the mRNA level of totipotency gene Kif4 was decreased; 100 ug/ml treatment improved the mRNA level of OCT4 which was decreased in other treatment groups;7.1n 2 cell stage, the 5-mc level of the mouse embryos in vivo were higher than that of cultured in vitro, the 5-hmc level of mouse embryos in vivo were significant higher than that of cultured in vitro (p<0.05); In 8cell、Morula、Blastula stage, the 5-mc level of mouse embryosin vitro are very significant higher than in vivo (p<0.01); In 8cell、 Blastulastage, the 5-hmc level of mouse embryos in vitro are very significant higher than in vivo (p<0.01), in Morula stage, the 5-hmc level of mouse embryos in vitro are higher than in vivo;8. In morula stage, the mRNA levels of demethylation gene TET2 in vivo is significant higher than that of in vitro(P<0.05); TET3 play a major role in 2 cell stage, the mRNA level of TET3 in vivo was significantly higher than in vitro (P<0.05);9.50 ug/ml L-Ascorbic acid significantly improved the embryos development ratein all stages (2 cell and 8 cell, P<0.05; Morula and Blastula, P<0.01). |
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