Application Of Ascorbic Acid In The Regulation Of DNA Methylation In Yak Oocytes And IVF Embryos

Yak is a unique species that dwells on the Qinghai-Tibet Plateau,and has important economic value to the herdsmen.Due to the Yak’s low natural reproduction rate,in vitro fertilization（IVF）has undoubtedly become the main method to solve this problem.However,the quality of Yak oocytes is a key factor for successful fertilization and embryonic development.Aflatoxin B₁ is the most dangerous mycotoxin in feed,and contamination of feed cannot be completely avoided,and it is highly stable after contamination.Consumption of AFB₁ contaminated feeds can lead to reproductive toxicity of mammal,destroy the epigenetic modification and quality of oocytes,and affect fertilization.Therefore,the prevention and mitigation of aflatoxin induced toxicity is the most effective way to supplement drugs or antioxidants.Ascorbic acid（AA）is widely used in cell culture as an antioxidant and has proven to be an irreplaceable cofactor of TET dioxygenase in DNA methylation regulation.However,the impact of AA in the regulation of DNA methylation in yak IVF embryos and whether it can protect yak oocytes from the toxic effects of AFB₁ are still unknown.In this study,DNA methylation was used to evaluate the effects of AA on the development of yak preimplantation embryos and the protective effect of AA on oocytes exposed to AFB₁.It aims to elucidate the specific mechanism behind the possible occurrence.1.IVF embryos were cultured with different concentration of AA,evaluating the rate of blastocyst apoptosis and number of blastocyst cells by the statistics of cleavage rate,blastocyst rate and apoptosis（TUNEL）staining.The results showed that,compared with the control group,adding 50μg/mL AA significantly increased the number of blastocyst cells,decreased the expression of pro-apoptotic gene BAX while increased the expression of apoptosis inhibiting gene BCL-2,and inhibited the rate of blastocyst apoptosis.Other concentrations（100,200μg/mL）had negative effects on these indicators.Therefore,the 50μg/mL AA was selected for subsequent experiments.2.The toxic effect of AFB₁ on oocytes and the protective effect of AA were evaluated using seven indicators:the first polar body rate,sperm-egg binding ability,parthenogenetic activated embryonic development rate,ROS level,early apoptosis,mitochondrial distribution and integrity of actin filaments.The results indicated that,first,the exposure of AFB₁ was not conducive to the meiotic maturation of oocytes,hinders the first polar body rate of oocytes,reduces the ability of sperm-egg binding and parthenogenetic activation of embryo development,resulting in the quality of mature oocytes decline.Secondly,AFB₁ toxicity has a destructive effect on the integrity of actin filaments.Finally,AFB₁ caused oxidative stress damage to oocytes,interfered with the even distribution of mitochondria,promoted early oocyte apoptosis.After adding 50μg/mL AA,these indicators can be restored to levels close to the control group,indicating that AA can protect oocytes from the toxic effects of AFB₁.In addition,the detection of DNA methylation immunofluorescence（5mC）and methyltransferase（DNMT1,DNMT3b,DNMT3b）found that compared with the control group,the toxic effect of AFB₁ on mature oocytes changed the DNA methylation levels.Adding 50μg/mL of AA can significant improvement the DNA methylation level of mature oocytes.3.Immunofluorescence staining and fluorescent quantitative PCR were used to detect the dynamic expression of DNA methylation（5mC,5hmC,TET3）and related methylation gene（DNMT1,DNMT3b,DNMT3b,TET3）at all stages of IVF embryonic development.Fluorescence staining results showed that pre-implantation yak embryos followed the classic pattern of DNA demethylation and remethylation,and remethylation occurs at the blastocyst stage.Secondly,the unique expression pattern of TET3 in cytoplasm plays a key role in the demethylation mechanism.Compared with the control group,adding 50μg/mL AA concentration enhanced the fluorescence signal intensity of 5mC and 5hmC at all stages of the embryo,and changed the expression level of methyltransferase DNMT1 and DNMT3a,but did not change the expression level of methyltransferase DNMT3b.Compared with the control group,adding 50μg/mL AA concentration significantly enhanced the fluorescence signal intensity of TET3 at the 2-cell,4-cell,8-cell and mulberry embryo stage,while the signal intensity at the blastocyst stage was significantly reduced.The fluorescence signal intensity trend was similar to its mRNA transcription abundance.4.The pluripotent genes（CDX2,POU5F1,SOX2,NANOG）at blastocyst stage were detected by methylation sequencing and fluorescence quantitative PCR.According to the results,CDX2 pluripotency gene promoter region showed a low level of methylation while SOX2,POU5F1 pluripotency genes and NANOG promoter region presented medium level of methylation.Compared with the control group,adding 50μg/mL AA concentration reduced the methylation level of the NANOG promoter region,but did not change the methylation level of the promoter region of the other pluripotency genes.Fluorescence quantitative results showed that compared with the control group,adding 50μg/mL AA concentration increased the expression of pluripotency genes CDX2,POU5F1 and NANOG,but had no significant effect on the expression of pluripotency gene SOX2.The above research results show that ascorbic acid can protects yak oocytes from quality reduction and aberrant DNA methylation caused by AFB₁ exposure,thereby helps regulate DNA methylation of yak pre-implantation embryos,increases the number of blastocyst cells.