Effects Of Knocking Out Cul4b Gene In Oocytes On Follicular Development And Early Embryonic Development

Background:The ovary is one of the most important reproductive organs in female mammals,which produces and releases eggs.Follicle is the smallest structural and functional unit in the ovary,which can be divided into primordial follicles,primary follicles,secondary follicles,antral follicles and preovulatory follicles.Primary follicles and subsequent follicles are collectively referred to as activated follicles.After an individual is born,the number of primordial follicles directly determines the reproductive potential of a female individual.The egg combines with the sperm in the fallopian tube to form a fertilized egg,initiating the early embryonic development process.The process of early embryonic development can be summarized as the process of a continuous division of the fertilized egg and cell differentiation.CUL4B belongs to the Cullin family,whose members,as scaffold proteins,form the largest class of ubiquitin ligase complex in eukaryotes-Cullin Ring E3 ubiquitin ligase complexes(CRL complexes).The CRL4B complexes can catalyze polyubiquitination of substrate proteins and proteasome-mediated degradation,or catalyze histone monoubiquitination to regulate gene transcription through epigenetic modifications,thereby playing important roles in a variety of tissues and pathophysiological processes.Mutations in the CUL4B gene cause an X-linked mental retardation syndrome.The patients have the symptoms of mental retardation,short stature,abnormal behavior and hypogonadism.In addition,multiple members of the CRL4 complexes play important roles in follicular development and early embryonic development.The specific knockout of Ddb1,Dcaf1 or Dcaf13 genes in follicles at different stages can lead to premature ovarian failure or early embryonic development abnormalities,respectively,suggesting that CUL4B may play important roles in follicular development and early embryonic development.Objective:To investigate the effects of knocking out the Cul4b gene in oocytes from different stages of follicles on mouse follicular development and early embryonic development.Methods and Results:1.Zp3-Cre+/-transgenic mice were crossed with Cul4bflox/flox mice to generate a female mouse model of specifically knocking out the Cul4b gene in oocytes of activated follicular.Immunofluorescence staining and immunohistochemical staining confirmed that CUL4B was knocked out in oocytes of activated follicles of Zp3-Cre+/-;Cul4bflox/flox mice,and that CUL4A did not compensate for CUL4B.The in vivo fertilization and in vitro fertilization experiments showed that the development of blastocysts from Zp3-Cre+/-;Cul4bflox/flox mice was impaired.Immunofluorescence and qPCR results showed that the expression of NANOG in early embryos from Zp3-Cre+/-;Cul4bflox/flox mice was reduced.2.Gdf9-Cre+/-transgenic mice were crossed with Cul4bflox/flox mice to generate a female mouse model of specifically knocking out the Cul4b gene in oocytes of primordial follicles.Immunofluorescence staining and immunohistochemical staining confirmed that CUL4B was knocked out in oocytes of primordial follicles of Gdf9-Cre+/-;Cul4bflox/flox mice.Immunohistochemical staining of ovarian tissues and follicles counting showed that the morphology of ovaries and follicles of Gdf9-Cre+/-;Cul4bflox/flox mice was normal,but the number of primordial follicles was decreased.Conclusions:In this study,it was found that the specific knockout of the Cul4b gene in oocytes of activated follicles leads to impaired and poorly developed blastocysts and decreased NANOG expression in early embryos.The specific knockout of the Cul4b gene in oocytes of primordial follicles results in a decreased number of primordial follicles.