Characterization and Functional Analysis of Obox4 during Oocyte Maturation by RNA Interference

Lee, Hyun Seo; Lee, Kyung Ah

Abstract

OBJECTIVE
Previously, we identified differentially expressed genes between GV and MII stage mouse oocytes using ACP technology.1 When we study one of GV selective genes, Obox family, we found Obox4 mRNA expression in ovaries that has been reported as expressed exclusively in testis. Therefore, this study was conducted for characterization and functional analysis for Obox4.
METHODS
Expression of Obox4 mRNA was examined in gonads and oocytes by RT-PCR. To determine the role of Obox4 in oocyte maturation, Obox4 dsRNA was microinjected into the cytoplasm of GV oocytes followed by 16 h of incubation in the plain medium or by 24 h of incubation in the medium containing IBMX. After RNAi, phenotypes and maturation rates were observed, change in mRNA expression was evaluated, and chromosomal status was confirmed by orcein staining.
RESULTS
Obox4 has minimal expression in the ovary compared to that of the other family members. When oocytes were cultured for 16 h in M16 medium after RNAi, maturation rate was not changed significantly, compared with that of non-injected or bufferinjected control oocytes. Surprisingly, however, when oocytes were cultured for 24 h in M16 containing IBMX, in which oocytes were supposed to arrest at GV stage, Obox4 RNAi oocytes were advanced to MI and MII. Spindle structure was disappeared and the chromosomes were condensed in the oocytes after Obox4 RNAi.
CONCLUSIONS
This is the first report on the expression of Obox4 in the ovary and oocytes. Results of the study suggest that Obox4 plays a crucial role in spindle formation and chromosome segregation during meiosis in oocytes. In addition, Obox4 may play an important role in cAMP-dependent signal cascades of GV-arrest in mouse oocytes.