255 GENE EXPRESSION OF Cox 5a, 5b, AND 6b1 AND THEIR ROLES IN PRE-IMPLANTATION OF MOUSE EMBRYOS

Abstract

Despite clear evidence of regulation of mitochondrial respiration by nuclear encoded genes, cytochrome oxidase (Cox), little information is available on their expression and functional roles during early embryonic development. To examine the role of Cox in oocyte maturation and embryogenesis, we first characterized mRNA and protein levels of nuclear encoded genes, Cox 5a, 5b, and 6b1, in mouse oocytes and during early embryogenesis, using real-time RT-PCR and immunocytochemistry. We then examined the possible role of these genes in oocyte maturation and pre-implantation development using RNA interference analysis. The relative abundances of Cox 5a, 5b, and 6b1 transcripts was measured by real time RT-PCR. After normalization by comparison to histone H2a mRNA levels, the mRNA expression of Cox 5a, 5b, and 6b1 were found to be considerable in mature oocytes and zygotes, but reduced slightly in 2-cell embryos. From the 2-cell to the blastocyst stage, mRNA expression is dependent on the number of blastomeres, as expression increases only gradually with development. Immunocytochemical studies revealed that Cox 5a, 5b, and 6b1 proteins were expressed in all blastomeres of the blastocyst. Injection of Cox 5a, 5b, or 6b1 siRNA into GV stage oocytes decreased expression of the target mRNA specifically, while not affecting the expression of mRNAs for the other subunits in mature oocytes. Similarly, each siRNA injection into zygotes specifically reduced target mRNA expression at the 2-cell, morula and blastocyst stages (P < 0.05). Silencing of mRNA expression by RNA interference (siRNA) did not inhibit oocyte maturation or developmental events up to the morula and blastocyst stages. The expression level of mtDNA9, as well as overall levels of mitochondrial mRNAs, was not different following injection of siRNA for Cox 5a, 5b, or 6b1. However, it is evident that the number of mitochondria in siRNA treated blastocysts was greatly reduced, and they appeared to be morphologically abnormal. Significantly higher apoptosis and lower cell numbers were observed in siRNA treated blastocysts. Real time RT PCR revealed that silencing of Cox 5a, 5b, and 6b1 decreased mRNA and protein levels of E-cadherin. These results suggest that the Cox subunits, Cox 5a, 5b, and 6b1, play an important role in mitochondrial function during pre-implantation development.

This work was funded by a grant from the National Research Laboratory Program in Korea.