The Role Of ASB7 During Mouse Oocytes Meiosis

The orderly progress of oocyte meiosis is the key to produce high-quality mature eggs.Any error in the process may lead to the aneuploid eggs.The aneuploid eggs increase the risk of female infertility,miscarriage and defective fetuses.Numerous studies have shown that aging is often accompanied by the decline of female fertility.Aneuploid eggs is one of the important reasons for decline of fertility.But the underlying mechanism remains to be explored.ASB7,as a member of the ASB protein family,belongs to the CRL(Cullin-ring ligases)subclass of E3 ubiquitin ligases containing the RING domain.Studies have shown that ASB7 can regulate DDA3(Differential display and activated by p53)through ubiquitination to regulate spindle dynamics and genome integrity,thereby promoting the process of mitosis.However,the role of ASB7 in meiosis has not been reported.In this study,by immunofluorescence labeling and laser confocal microscopy scanning,this research shown that the ASB7 protein co-localizes with the chromosome during recovery of meiosis in mouse oocytes,suggesting that it may play an important role in this process.To test this hypothesis,specific Asb7-si RNA was microinjected into oocytes to explore the effect of ASB7 knockdown(ASB7-KD)during oocyte meiosis.The results showed that when the ASB7 protein was knocked down,the maturation ratio of oocytes was significantly lower than that of the control groups,and the ratio of symmetric division was significantly increased.It is worth noting that the proportion of ASB7-KD oocytes blocked at MI(Metaphase I)was significantly increased compared to control groups,suggesting that ASB7 is involved in oocyte meiosis regulation.The migration of spindle and the formation of actin caps are essential for the extrusion of Pb1 during oocyte maturation.By immunofluorescence staining,this research shown that in ASB7-KD oocytes,the spindle migration the to the cortex during the Meta-Anaphase I was affected,and the proportion of actin caps formed during the MII(Metaphase II)was significantly lower than that in control groups.Based on the special subcellular location of the ASB7 protein and its influence during the maturation process,we speculated that the ASB7 protein may be related to the assembly of the meiotic apparatus of mouse oocytes.Confocal microscopy confirmed that ASB7-KD resulted in a high proportion of spindle assembly defects and chromosomal alignment errors.By chromosome spreading and kinetochore labeling,this research further revealed that the proportion of aneuploidy in ASB7-KD oocytes was approximately twice that of the control groups.The arrangement and pulling of chromosomes depend on the interaction between the kinetochore and the microtubules from the spindle poles.By analyzing the kinetochore-microtubules(K-MT)interaction in MI oocytes,this research found that normal oocytes exhibit typical two-way K-MT attachment,while the K-MT in ASB7-KD oocytes,the proportion of K-MT misconnections has increased significantly.K-MT misconnections can activate the SAC(Spindle assembly checkpoint),and SAC is essential for the accurate separation of chromosomes and completion of meiosis.By immunolabeling Bub R1,an important component of SAC,the SAC system in ASB7-KD oocytes was greatly activated,which may be one of the reasons why ASB7-KD oocytes arrested at MI.The abnormal meiosis of ASB7-KD oocytes is very similar to the defective phenotypes in aging mouse oocytes,suggesting that the lack of ASB7 protein may be a potential way for aging to affect the quality of oocytes.Western blot results showed the expression level of ASB7 in aging mouse oocytes was only 51% of that in the young groups.Importantly,this research found ASB7 overexpression in aging mouse oocytes can partially improve the related phenotypes of meiotic defects and reduce the frequency of aneuploidy.In summary,this study confirmed that ASB7 is a factor that regulates oocyte meiosis and maturation,and participates in the control of oocyte aging.The related findings will provide potential molecular target for improving oocyte maturation system in vitro and evaluating oocyte quality.