Analysis of apoptosis and methyltransferase mRNA expression in porcine cloned embryos cultured in vitro.
OBJECTIVE
The purpose of this study was to investigate the relationship of porcine somatic cell nuclear transfer (SCNT) embryo developmental competence with embryonic cell apoptosis and DNA methylation.


METHODS
The apoptotic incidence was examined via comet assay, and the mRNA expression of genes implicated in apoptosis (Bcl-2) and DNA methylation (Dnmt1, Dnmt3a) was determined using real-time RT-PCR.


RESULTS
Comet assay showed that the SCNT embryos exhibited significantly higher apoptotic rate at 2-cell stage (8.3% versus 2.1%, P<0.05), 16-cell stage (27.3% versus 19.2%, P<0.05) and morula (37.5% versus 26.9, P<0.05) compared with IVF embryos. Compared with IVF embryos, a higher Bcl-2 mRNA expression pattern was observed in SCNT embryos before the 8-cell stage and differed significantly at 2- and 4-cell stages (P<0.05). After the 16-stage, Bcl-2 mRNA expression pattern became significantly lower in SCNT group (P<0.05). The relative expression level of Dnmt1 mRNA showed a higher expression level in oocytes, then sharply decreased and started to increase slightly after the 8-cell (IVF embryos) or 16-cell stage (SCNT embryos). Dnmt1 mRNA expression in IVF embryos appeared to have been lower than that of SCNT group before 16-cell stage embryos, especially at 4- and 8-cell stages (P<0.05). Although a trend for a similar increase of Dnmt3a expression was observed in IVF and SCNT embryos after 8-cell embryos, SCNT group resulted in much higher Dnmt3a mRNA abundance compared with the IVF group, particularly after 16-cell embryos (P<0.05).


CONCLUSIONS
The results showed that low efficiency of porcine SCNT technology may be associated with either embryonic apoptosis or incomplete reprogramming of donor nuclear caused by abnormal Dnmts mRNA expression.
Ju
Shiqiang
S
College of Veterinary Medicine, Nanjing Agricultural University, Jiangsu, 210095, China.
Rui
Rong
R
Lu
Qing
Q
Lin
Pengfei
P
Guo
Huili
H
D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D019976:Cloning, Organism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism; D019175:DNA Methylation / Q000235:genetics; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000378:metabolism*; D047108:Embryonic Development / Q000235:genetics; D005307:Fertilization in Vitro; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D053652:Nuclear Transfer Techniques; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine
D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D004248:DNA (Cytosine-5-)-Methyltransferases