Effect of oocyte mitochondrial DNA haplotype on bovine somatic cell nuclear transfer efficiency

Abstract

The development capability of reconstructed bovine embryos via ovum pick-up (OPU)-somatic cell nuclear transfer (SCNT) technique has been influenced by the maternal lineage of oocyte cytoplasm, but the underlying mechanism remains unclear. Since mitochondria are the richest maternal-inherited organelle, in this study, we intended to clarify the effect of mtDNA haplotypes on cloning efficiency. By PCR-RFLP method, we identified mtDNA haplotypes A and B, differing in six restriction sites. Reconstructed embryos with haplotype A cytoplast achieved better fusion and blastocyst formation rate (64.6% and 39.4%), as compared with haplotype B (53.6% and 26.3%; P < 0.05). To further evaluate the role of mitochondria, the quantity of mtDNA, ATP content, and mRNA level of mtDNA-encoded COXI, COXIII in both oocytes were measured. Our data indicated that mtDNA copy number in haplotype A oocyte was significantly higher than that in haplotype B oocyte, both at the GV (10(5.03 +/- 0.69) vs. 10(4.81 +/- 0.86) copies/oocyte) and MII stages (10(5.31 +/- 0.71) vs. 10(5.13 +/- 0.63) copies/oocyte; logarithmically transformed values; P < 0.05). ATP content in type A oocyte was also greater at the GV (1.67 +/- 0.09 vs. 1.27 +/- 0.1 pmol) and MII stages (5.18 +/- 0.07 vs. 2.68 +/- 0.03 pmol; P < 0.05). Similarly, the mRNA expression level of mtDNA-encoded COXI and COXIII in haplotype A oocyte was significantly higher comparing to haplotype B oocyte (3.3 +/- 2.0 x 10(3) vs. 0.68 +/- 0.45 x 10(3); 24.9 +/- 10.5 x 10(3) vs. 9.4 +/- 3.3 x 10(3), respectively; P < 0.05). The data suggest that mitochondrial structure, quantity, and function may significantly affect the developmental competence of reconstructed embryos.