246 FUNCTIONAL ANALYSIS OF Sebox DURING OOCYTE MATURATION AND EARLY EMBRYOGENESIS BY RNAi

Abstract

Previously, we found that the skin-embryo-brain-oocyte homeobox (Sebox) gene was highly expressed in germinal vesicle (GV)-stage oocytes. The function of Sebox is unknown as yet; thus the objective of this study was to determine the role(s) of Sebox during oocyte maturation and early embryogenesis using RNA interference (RNAi). Cumulus-free GV oocytes were collected from 4-week-old ICR mice 48 h after pregnant mare serum gonadotropin (PMSG) injection. The expression pattern of Sebox mRNA was evaluated in various tissues, including gonads, oocytes, and embryos at different developmental stages. To determine the role of Sebox in oocyte maturation and preimplantation embryonic development, Sebox dsRNA was microinjected into the cytoplasm of GV oocytes and pronuclear (PN)-stage embryos, respectively. After Sebox RNAi, phenotype and maturation rates were recorded prior to measuring changes in Sebox mRNA and protein concentration. RNA content was determined by RT-PCR and protein content was determined by oocyte dot blot. Anti-Sebox antibody was produced against a 14 amino acid synthetic peptide corresponding to residues 67–80. Additionally, chromosomal status of the oocytes was confirmed by orcein staining, while spindle shape was confirmed by immunofluorescence staining. Sebox mRNA was ubiquitous in adult mice tissues, with relatively high expression in the brain and ovary. Expression of Sebox was detected in oocytes, granulosa cells, and theca cells. Sebox mRNA was highly expressed from GV up to 2-cell-stage embryos, but dramatically decreased afterward in later developmental stages. Sebox dsRNA microinjection into GV oocytes resulted in a markedly decreased Sebox mRNA (80%) and protein (70%) expression. However, Sebox RNAi resulted in rates in MII oocytes (82%) similar to that of control (87%) and the buffer injection (80%) group during in vitro maturation. Sebox RNAi did not affect the spindle and chromosomal organizations of the MII oocytes. But microinjection of Sebox dsRNA into PN embryos inhibited preimplantational embryo development and blocked at the 2-cell stage (79%). Results suggest that the Sebox gene is not related to the regulation of oocyte nuclear maturation in mice. However, we concluded that Sebox is a new entry of maternally expressed homeobox gene that is involved in the regulation of early embryogenesis, especially at the 2-cell block.

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-311-E00067).