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Gene Expression of Cox5a, 5b, or 6b1 and Their Roles in Preimplantation Mouse Embryos¹

National Research Laboratory,³ Department of Animal Sciences, Chungbuk National University, Chungbuk 361–763, South Korea Laboratory of Reproductive Biology & Infertility,⁴ Samsung Cheil Hospital & Women's Healthcare Center, Sungkyunkwan University School of Medicine, Seoul 100–380, Korea

ABSTRACT

To investigate the role of nuclear encoded genes in mitochondrial function during oocyte maturation and early embryogenesis we examined the expression pattern and function of the cytochrome oxidase (Cox) subunits, Cox5a, 5b, and 6b1 during oocyte maturation and early embryo development. Transcription of Cox5a, 5b, or 6b1 was observed in oocytes and during early development; their expression levels were abundant in mature oocytes (MII) and zygotes (1C), and lowest at the 2-cell stage (2C), gradually increasing from 4-cell to blastocyst stage. Immunocytochemical studies revealed that COX5A, 5B, or 6B1 proteins were expressed in all blastomeres of the blastocyst. Silencing of mRNA expression by RNA interference (siRNA) did not inhibit oocyte maturation or developmental events up to the morula and blastocyst stages, but disrupted mitochondrial distribution. Significantly higher apoptosis and lower cell numbers were observed in siRNA-treated blastocysts. Real time RT-PCR revealed that silencing of Cox5a, 5b, or 6b1 did not alter mRNA levels of Bcl-xL (Bcl2l1), but increased transcription levels of proapoptotic genes, Bax and caspase 3 (Casp3). Furthermore, mRNA and protein levels of E-cadherin (CDH1) were decreased in siRNA microinjected blastocysts. These results suggest that gene expression of the Cox subunits, Cox5a, 5b, and 6b1 is not required for embryo developmental events up to the blastocyst stage. The loss of these genes leads to mitochondrial dysfunction that results in apoptosis of the blastocyst stage embryos.

apoptosis, embryo, oocyte development

² Correspondence: Nam-Hyung Kim, Department of Animal Sciences, Chungbuk National University, Gaesin-dong, Cheongju, Chungbuk, 361–763, South Korea. FAX: 82 43 272 8853; nhkim{at}chungbuk.ac.kr

¹ Supported by the Ministry of Science and Technology (NRL), the Ministry of Agriculture and Forestry (Bio-Organ Production Project), and Research Center for Bioresource and Health in Chungbuk National University.