【作者】 刘源；

【导师】 顾玲；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2021， 硕士

【摘要】 卵母细胞减数分裂的有序进行,是产生优质成熟卵子的关键。减数分裂的异常会导致非整倍体卵子的发生率升高,从而增加雌性不孕、流产和胎儿出生缺陷的风险。大量研究表明,雌性动物的年龄增长往往伴随着其生育能力的下降,非整倍体卵子的产生是生育力降低的重要原因之一,但其潜在的机制仍有待阐明。ASB7(Ankyrin repeat and SOCS box 7)属于ASB蛋白家族中含有RING结构域的E3泛素连接酶的CRL(Cullin-ring ligases)亚类。已有研究表明,ASB7可以通过泛素化调节DDA3(Differential display and activated by p53)来调控纺锤体动力学和基因组完整性,从而促进有丝分裂进程。然而ASB7在减数分裂中的作用未见报道。在本研究中,通过免疫荧光标记结合激光共聚焦扫描,发现ASB7蛋白在小鼠卵母细胞减数分裂恢复时与染色体共定位,提示其可能在此过程中发挥着重要的作用。为了验证这一假设,本研究通过向卵母细胞内注射特异性的Asb7-si RNA,探究ASB7敲减(ASB7 knockdown,ASB7-KD)对卵母细胞减数分裂的影响。结果显示,当ASB7蛋白被敲减后,卵母细胞发育成熟的比例明显降低,而对称分裂的比例显著增加。其中值得注意的是,ASB7-KD卵母细胞阻滞在减数第一次分裂中期(Metaphase I,MI)的比例相较于对照组显著增加,提示ASB7参与调控了卵母细胞减数分裂进程。纺锤体迁移和微丝帽形成对于卵母细胞成熟过程中极体的排出至关重要。通过免疫荧光染色,本研究发现ASB7-KD卵母细胞中纺锤体在减数第一次分裂中后期向皮质区的迁移受到了影响,且在MII(Metaphase II)期微丝帽形成的比例显著低于对照组。基于ASB7蛋白特殊的亚细胞定位及其对成熟过程的影响,推测ASB7蛋白可能与小鼠卵母细胞的减数分裂装置装配有关。共聚焦显微扫描证实ASB7敲减导致卵母细胞减数分裂过程中纺锤体组装缺陷和染色体排列错误的比率显著升高。通过染色体铺片和动粒标记的方法,进一步研究发现,ASB7-KD卵母细胞的非整倍体发生率约为对照组的两倍。染色体的排列和牵引取决于动粒和来自纺锤体两极微管之间的互作。通过分析MI期卵母细胞内动粒-微管(Kinetochore-Microtubules,K-MT)相互作用,本研究发现正常卵母细胞呈现典型的双向K-MT附着,而ASB7-KD卵母细胞中K-MT错误连接的比例明显增加。K-MT的错误连接可激活纺锤体组装检验点(Spindle assembly checkpoint,SAC),而后者对染色体的准确分离和减数分裂的完成至关重要。通过免疫标记SAC的重要组分Bub R1,注意到ASB7-KD卵母细胞内的SAC系统被大幅激活,这可能是其发育停滞在MI期的原因之一。ASB7-KD卵母细胞的减数分裂异常与老化小鼠卵母细胞的缺陷表型非常相似,提示ASB7蛋白可能是衰老影响卵母细胞质量的潜在途径。Western blot试验结果表明老化鼠卵母细胞中ASB7的蛋白表达水平仅为年轻组的51%。重要的是,在老化鼠卵母细胞中过表达ASB7可以部分改善其减数分裂缺陷的相关表型,并降低非整倍体的发生频率。综上所述,本研究证实ASB7是调节卵母细胞减数分裂成熟的因子,并参与了卵母细胞的老化控制,相关发现将为改进卵母细胞体外成熟体系和卵子质量评估提供潜在的分子靶标。更多还原

【Abstract】 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.更多还原