Generation And Phenotype Analyses Of Oocyte Specific Cul4b Gene Knockout Mouse Model

Mammalian ovaries contain follicles and corpus luteum at different developmental stages,and have the function of producing egg cells and releasing mature egg cells.Hormones synthesized and secreted by the ovary are necessary for follicular growth,maintenance of normal reproductive cycle and reproductive capacity,development of female second sex characteristics and metabolic function.The number of primordial follicles will not increase after the mammal is born.After the formation of primordial follicles,dormant primordial follicles will be activated in an orderly manner,also known as initial recruitment.After puberty,the antral follicles continue to develop to mature follicles under the action of follicle stimulating hormone periodically.This process is called cyclic recruitment,and the antral follicles that fail to enter the cyclic recruitment will go atresia.The follicles that have grown to maturity will ovulate under the action of luteinizing hormone,and the remaining follicular membrane cells and granular cells will form the corpus luteum after ovulation.Because the process of follicle activation and development is irreversible,if the process of follicle activation is out of control,it will cause the follicle to be exhausted early in life and premature ovarian failure.Premature ovarian failure can produce symptoms such as loss of fertility,which is one of the causes of female infertility and seriously affects the physical and mental health of women.Previous studies in our laboratory found that mutation in the CUL4B gene led to X-linked mental retardation syndrome.In addition to mental retardation,patients also showed symptoms of language and motor developmental delay,obesity,short stature,and hypogonadism.CUL4B is a member of the Cullin protein family.Cullin proteins are important scaffold proteins,which are involved in the formation of the largest class of ubiquitin ligase complex in eukaryotes(Cullin-RING E3 ligase complex).CUL4B protein participates in the formation of Cullin 4B-RING E3 ubiquitin ligase complex(abbreviated as CRL4B complex).Its C-terminus binds to Ring protein Rbx/ROC,thereby recruiting E2 ubiquitin binding enzyme,and its N-terminus binds to adaptor protein DDB1,and DDB1 further binds to the substrate receptor protein(DCAF),and specifically binds to the substrate protein through DCAF.The CRL4B complex catalyzes either polyubiquitination for proteosomal degradation or regulate gene expression by epigenetic way,and plays important roles in various tissues and various pathophysiological processes.In order to clarify the physiological function of the CUL4B gene and its mechanism underlines X-linked mental retardation syndrome,previously our laboratory established the Cul4b gene knockout mouse model by the Cre/loxp system.The study found that the global Cul4b gene knockout mice showed severe developmental arrest in the early embryonic development and died before 9.5 days of embryonic development.Subsequently,mice models were used to clarify the roles of the Cul4b gene in neural development,differentiation of mouse myeloid cells,adipocyte differentiation,and islet delta cell development.Mutations in the CUL4B gene can cause symptoms of hypogonadism in patients,suggesting that CUL4B may play an important role in the reproductive system.Studies have shown that global(Sox2-Cre)or germ cell-specific(Vasa-Cre,or Ddx4-Cre)knockout of the Cul4b gene can cause infertility in male mice.Other members of the CRL4A/B complex play important roles in ovarian development.Knocking out Ddb1,Dcaf1 or Dcaf 13 at different stages of oocyte development can lead to premature ovarian failure or abnormal embryonic development.The above results suggest that CUL4B may also play an important role in ovarian development.In this study,we will investigate the role of CUL4B in ovarian development by knocking out the Cul4b gene in activated oocytes(Zp3-Cre).In order to obtain mice that specifically knocked out the Cul4b gene in oocytes of activated follicles,we mated the Cul4bflox/flox gene targeting mice with Zp3-Cre+/-transgenic mice to generate Zp3-Cr;Cul4bflox/flox gene knockout female mouse model.CUL4B immunohistochemistry showed that CUL4B was expressed in the nuclei of oocytes of wild-type female mice of different ages.In the ovary of Zp3-Cre;Cul4bflox/flox female mice,CUL4B staining was still present in the primordial follicles,however,there was no CUL4B staining in the primary follicles and oocytes in the later period,demonstrating that CUL4B was knocked out in the oocytes of activated follicles in Zp3-Cre;Cul4bflox/flox female mice.The fertility test showed that Zp3-Cre;Cul4bflox/flox female mice lost their fertility,suggesting that CUL4B plays an important role in the female reproduction of mice.In order to find the reason for the loss of fertility of Zp3-Cre;Cu14bflox/flox female mice,we first conducted morphological analysis of mice ovarian tissues.H&E staining,MVH and FOXO1 immunohistochemistry staining showed that the ovary size and morphology of Zp3-Cre;Cu/4bflox/flox female mice at different ages did not change significantly,and the number,size and morphology of various follicles were comparable to the littermate control female mice.The structure and number of corpus luteum in the ovaries of the knockout mice after sexual maturity are not significantly different from the female littermates,suggesting that the ovulation function of Zp3-Cre;Cul4bflox/flox female mice are normal.The TUNEL staining also showed that the oocytes of Zp3-Cre;Cul4bflox/flox female mice did not show more apoptosis.The above results suggest that knocking out the Cul4b gene in oocytes of activated follicles has no significant effect on the development of the primary follicle to ovulation.Zp3-Cre;Cul4bflox/flox female mice may lose their fertility due to abnormal embryonic development,which need further characterization.In order to analyze the molecular mechanism of the infertility of Zp3-Cre;Cul4bflox/flox gene knockout female mice,we analyzed the changes of protein and mRNA expression in ovarian tissues of gene knockout female mice.Western blot showed that the expression levels of phosphatase PP2AA subunit(PP2A-A)and C subunit(PP2A-C)were decreased in ovarian tissue of Zp3-Cre;Cul4bflox/flox gene knockout female mice.The real-time quantitative PCR experiments found that knocking down the Cul4b gene in ovarian tissue cultured in vitro resulted in increased expression of Figla and Nobox.In addition after knocking down the Cul4b gene in granulocytes cultured in vitro,the morphology of knockdown granulocytes did not change significantly compared with the control cells,however,the results of EdU incorporation experiments showed that the proliferation capacity of Cul4b knockdown granulocytes was significantly reduced.In summary,this study generated a mouse model in which Cul4b was deleted from oocytes of activated follicles,and found that lack of CUL4B in oocytes of activated follicles resulted in loss of fertility.Further study will need to characterize the underlying mechanisms.