Gas6 downregulation impaired cytoplasmic maturation and pronuclear formation independent to the MPF activity.

Abstract

Previously, we found that the growth arrest-specific gene 6 (Gas6) is more highly expressed in germinal vesicle (GV) oocytes than in metaphase II (MII) oocytes using annealing control primer (ACP)-PCR technology. The current study was undertaken to investigate the role of Gas6 in oocyte maturation and fertilization using RNA interference (RNAi). Interestingly, despite the specific and marked decrease in Gas6 mRNA and protein expression in GVs after Gas6 RNAi, nuclear maturation including spindle structures and chromosome segregation was not affected. The only discernible effect induced by Gas6 RNAi was a change in maturation promoting factor (MPF) activity. After parthenogenetic activation, Gas6 RNAi-treated oocytes at the MII stage had not developed further and arrested at MII (90.0%). After stimulation with Sr(2+), Gas6-silenced MII oocytes had markedly reduced Ca(2+) oscillation and exhibited no exocytosis of cortical granules. In these oocytes, sperm penetration occurred during fertilization but not pronucleus (PN) formation. By roscovitine and colcemid treatment, we found that the Gas6 knockdown affected cytoplasmic maturation directly, independent to the changed MPF activity. These results strongly suggest that 1) the Gas6 signaling itself is important to the cytoplasmic maturation, but not nuclear maturation, and 2) the decreased Gas6 expression and decreased MPF activity separately or mutually influence sperm head decondensation and PN formation.