Effects of the removal of cytoplasm on the development of early cloned bovine embryos.

Oocyte cytoplasm plays a prominent role in cloned embryonic development. To investigate the influence of oocyte cytoplasmic amount on cloned embryo development, we generated bovine somatic cell nuclear transfer (SCNT) embryos containing high (30-40% of the cytoplasm was removed), medium (15-25% of the cytoplasm was removed) and low (<10% of the cytoplasm was removed) nucleocytoplasmic volume ratios (N/C) using enucleated metaphase II oocyte as recipient, and fibroblast as donor nucleus, and analyzed the expression levels of ND1, Cytb and ATPase6, as well as the embryonic quality. The results indicated: (1) the process of embryonic development was not influenced by <40% of cytoplasm removal; (2) the rate of blastocyst formation, the total number of blastomere and the ratio of ICM to TE were inversely proportional to the N/C; (3) SCNT embryos with reduced volume equal to 75-85% or >90% of an intact oocyte volume showed similar karyotype structure of the donor cells; (4) the number of mtDNA copy was larger in low N/C embryos than that in medium or high N/C embryos, and the expression levels of each gene hardly varied from the 2-cell to 8-cell stage, while the expression levels increased dramatically at the blastocyst stage; (5) from 16-cell to the blastocyst stage, the change of the expression level of each gene was not significant between low N/C embryos and IVF embryos, but it was more significant than those of high or medium N/C embryos. The results suggest that the decrease of mtDNA copy number and mitochondrial gene expression may be related to the impairment in early embryonic development, and removal of <10% adjacent cytoplasm volume may be optimal for bovine SCNT embryo development.