Effects of cumulus cells on in vitro maturation of oocytes and development of cloned embryos in the pig.
The purpose of this study was to investigate the role of porcine cumulus cells (CC) in oocyte maturation and somatic cell nuclear transfer (SCNT) embryo development in vitro. Denuded pig oocytes were co-cultured with CC or routinely cultured in maturation medium without a feeder layer. Porcine CC inactivated with mitomycin C or non-inactivated were used for the feeder layer in co-culture with porcine SCNT embryos to investigate comparatively the developmental competence of cloned embryos. The DNA damage aspects of apoptosis and expression pattern of genes implicated in apoptosis (Fas/FasL) as well as the mRNA expression of DNA methyltransferase (Dnmt1, Dnmt3a) of porcine SCNT embryos were also evaluated by comet assay or real-time RT-PCR, respectively. The results showed that co-culture with CC improved the extrusion rate of pbI (49.3% vs 31.5%, p<0.05) and survival rate (75.7% vs 53.3%, p<0.05) of denuded oocytes, but had no effects on blastocyst developmental rate or 2-cell-stage survival rate of in vitro fertilization embryos. Co-culture with CC inactivated by mitomycin C improved the blastocyst developmental rate (26.6% vs 13.0%, p<0.05) and decreased the apoptotic incidence (27.6% vs 46.2%, p<0.05) of porcine cloned embryos. Co-culture with inactivated CC reduced Fas and FasL mRNA expression of cloned embryos at the blastocyst stage compared with NT controls (p<0.05), but there were no differences in Dnmt1 and Dnmt3a mRNA expression among groups. Co-culture with inactivated cumulus cell monolayer significantly increased blastocyst formation and decreased the apoptotic incidence in porcine cloned embryos during in vitro development.
College of Veterinary Medicine, Nanjing Agricultural University, Jiangsu, China.
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D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001755:Blastocyst / Q000502:physiology; D019976:Cloning, Organism; D018920:Coculture Techniques / Q000662:veterinary; D054885:Cumulus Cells / Q000502:physiology*; D004249:DNA Damage / Q000235:genetics; D046149:Embryo Culture Techniques / Q000662:veterinary; D047108:Embryonic Development / Q000502:physiology*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D015870:Gene Expression; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary; D008297:Male; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000254:growth & development*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D013552:Swine