Abstract

ObjectiveInvestigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT).MethodsPorcine 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.ResultsAscorbic 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.ConclusionAscorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.

Highlights

  • We investigated the influence of Ten-eleven translocation 3 (TET3) knockdown on the effect of ascorbic acid on porcine somatic cell nuclear transfer (SCNT) em­ bryo development

  • Effect of treatment with ascorbic acid at various concentrations on the in vitro development of SCNT embryos Our results showed that treatment of porcine SCNT embryos with 500 ng/mL ascorbic acid for 20 h resulted in greater cleavage (52.82%±6.43% vs 43.05%±3.03%, p

  • The results showed that siRNA3 markedly reduced the mRNA level of TET3 in porcine oocytes (Figure 2, p

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Summary

Objective

Investigated the effect and mechanism of ascorbic acid on the development of por­ cine 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. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Results: Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. 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 in­ creased TET3 mediated demethylation of somatic nucleus

MATERIAL AND METHODS
F: GTTCAGCCAAACGACCATCT R: TCTCGATACTTGTCCGCTTTC F: CCCGTGGTTACCTCTTCTTCC R
RESULTS
DISCUSSION
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