Changes in gene expression associated with oocyte meiosis after Obox4 RNAi

OBJECTIVE Previously, we found that oocyte specific homeobox (Obox) 4 plays significant role in completion of meiosis specifically at meiosis I-meiosis II (MI-MII) transition. The purpose of this study was to determine the mechanism of action of Obox4 in oocyte maturation by evaluating downstream signal networking. METHODS The Obox4 dsRNA was prepared by in vitro transcription and microinjected into the cytoplasm of germinal vesicle oocytes followed by in vitro maturation in the presence or absence of 0.2 mM 3-isobutyl-1-metyl-xanthine. Total RNA was extracted from 200 oocytes of each group using a PicoPure RNA isolation kit then amplified two-rounds. The probe hybridization and data analysis were used by Affymetrix GeneChip® Mouse Genome 430 2.0 array and GenPlex 3.0 (ISTECH, Korea) software, respectively. RESULTS Total 424 genes were up (n=80) and down (n=344) regulated after Obox4 RNA interference (RNAi). Genes mainly related to metabolic pathways and mitogen-activated protein kinase (MAPK) signaling pathway was changed. Among the protein kinase C (PKC) isoforms, PKC-alpha, beta, gamma were down-regulated and especially the MAPK signaling pathway PKC-gamma was dramatically decreased by Obox4 RNAi. In the cell cycle pathway, we evaluated the expression of genes involved in regulation of chromosome separation, and found that these genes were down-regulated. It may cause the aberrant chromosome segregation during MI-MII transition. CONCLUSION From the results of this study, it is concluded that Obox4 is important upstream regulator of the PKC and anaphase-promoting complex action for maintaining intact germinal vesicle.