Ascorbic acid increases demethylation in somatic cell nuclear transfer embryos of the pig (Sus scrofa)
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Objective: Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT). Methods: Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR. Results: Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid. Conclusion: Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.
Abstract
Objective: Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT). Methods: Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR. Results: Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid. Conclusion: Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.
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