Rbx1 Protein Function In Mouse Oocytes

In mammals, oocytes are arrested at the diplotene stage of the first meiotic prophase. Meiosis maturation is controled by a variety of complex regulatory mechanisms. Our lab successfully constructed the protein expression lineage of mouse MII oocytes through proteomics technology. The purpose of our research was to find and study proteins important for oocyte or embryo development .In the base of the lineage, we further study on the interesting proteins hinted by bioinformatics and previous papers .Here, we selected the RING-H2 finger protein Rbx1 for further investigation.RBX1 (RING box protein-1) or ROC1 (regulator of cullins-1) is the RING component of SCF (Skp1, Cullins, F-box proteins) E3 ubiquitin ligases, which regulate diverse cellular processes by targeting various substrates for degradation. However, the function of RBX1 in the meiosis of mammals remains uncharacterized.In the present study, we investigate the expression, location and physiological functions of Rbx1 in mouse oocyte. First of all, We confirmed its existence in mouse ovary and GV oocytes by western blot. The result of immunofluorescence showed that Rbx1 protein localized in the cytoplasm and nucleus of GV oocyte. During the spontaneous maturation process of in vitro oocyte , the localization of Rbx1 changed associated with chromosome condensation and movement, co-localizated with alpha-Tubulin in metaphase and in anaphase.Rbx1 protein seperated with the seperation of homologous chromsomes, One half remains in oocyte, and the other discharges into the first polar body. When oocyte progress into the second meiotic metaphase, chromosomes re-condensed to form the board order, Rbx1 protein was localized to the spindle again.Nocodazole was used to depolymerization of microtubules, The result showed that along with microtubule depolymerization, spindle disappearing, Rbx1 protein d diffused into the cytoplasm. The dynamic localization of Rbx1 proteins during the oocyte maturation process and its correlation with Tubulin, suggesting that Rbx1 may play an important role in the process of oocyte maturation. We futher studied its biological fuction using microinjection and living cells workstation.The knockdown effect of Rbx1 siRNA microinjection was confirmed byReal Time PCR , the expression level of Rbx1 was decreased by 83.33%. Continuous observation using living cells workstation showed that, compared with control group, the first polar body dilivery rate decreased when Rbx1 knockdown. the first polar body dilivery rate in control group was 70.07% , while in the experimental group, this rate was 53.82%. Meanwhile, down regulation of Rbx1 significantly slowdowned the first polar body dilivery., The control group exclude 50% of the first polar body within 10h,while the time of the knockdown group was around 12h.These results suggest that, Rbx1 protein knockdown affected the mouse oocytes first polar body exclusion.According to the above phenotype, we then further studied the mechanism of Rbx1 protein during the mouse oocyte maturation. Previous studies have shown that Rbx1 proteins affect the growth and development of the mouse oocyte, and have found that the growth inhibition is due to the aggregation of proliferation-related protein, which can not be efficiently degraded, thus affecting the normal growth and development. According to the literature, we find that Emi1 (early mitosis inhibitor 1) or early mitosis of the original protein,play an important role in mouse oocyte maturation. In vitro studies have suggested that, Emi1 protein is the substrate of SCFβ-trcp E3 ubiquitin ligase .So we detect the Emi1 protein expression after Rbx1 knockdown using Western Blot method . The results showed that, Emi1 protein, normally was undetectable in MI oocyte ,but accumulated at high levels in Rbx1 knockdown oocytes. This result suggests that the impact of Rbx1 protein in the process of the first polar body extrusion may be caused by Emi1 protein accumulation.In summary, we studied the location of Rbx1 protein in mouse oocytes .In mouse oocyte maturation, Rbx1 protein location changes with the dynamic movement of chromosome .And co-localization with alpha-Tubulin in metaphase. Decreased Rbx1 Expression inhibited and delayed the first polar body extrsion.Further studies revealed that these effects is caused by Emi1 protein accumulation.