The Study Of Centrosome Protein CEP72 In Mouse Spermatogenesis

Objective: Male infertility is an important public health problem,and abnormal spermatogenesis is one of the main reasons for male infertility.The centrosome,as the main microtubule organizing center（MTOC）,is composed of centrioles and pericentriolar material（PCM）.It is essential for spindle formation,chromosome separation,cytokinesis and other cellular activities.Abnormalities of the centrosome can lead to diseases such as oligospermia,asthenospermia,microcephaly and dwarfism.The CEP family constitutes the PCM around the centrosome.CEP72,as a member of CEP family,regulates spindle assembly,microtubule formation and chromosome arrangement during mitosis,while its function in meiosis has not been reported.Therefore,this study took mice as the model to explore the function and mechanism of centrosomal protein CEP72 in the process of mouse spermatogenesis,hoping to provide some theoretical reference for the candidate genes of male infertility,the clinical diagnosis,treatment and research of oligospermia and asthenospermia.Methods:（1）RT-PCR detects the expression of Cep72 in various tissues at the transcription level.（2）The polyclonal rabbit anti-CEP72 antibody was produced by prokaryotic expression system,and further detect the expression of CEP72 in various tissues by Western blot at the protein level.（3）Prepare Cep72 knockout mice by CRISPR-Cas9 gene editing technology,and verify at the genome level and protein level.（4）Exploring whether the spermatogenic ability of Cep72 knockout mice is affected by histomorphological analysis.（5）Exploring whether the sperm morphology of Cep72 knockout mice is affected by costaining with PNA and DAPI.（6）Exploring whether the flagella structure of Cep72 knockout mice is affected by electron microscopy.（7）Exploring whether the meiosis process of Cep72 knockout mice is affected by spermatocyte chromosome spreading and karyotype analysis experiments.（8）The fertility of Cep72 knockout mice was measured by counting the number of offspring and reproductive interval.Result:（1）At the transcriptional level,Cep72 was highly expressed in testis,there was also a.small amount of expression in other tissues（2）We successfully prepared CEP72 polyclonal rabbit antibody,the high expression of CEP72 in the reproductive organs testis was further confirmed at the protein level.（3）Through the verification at genome level and protein level,we successfully prepared Cep72 knockout mice.（4）After Cep72 was knocked out,there was no significant difference in the morphology and weight of the mouse testis.The HE staining results showed that the morphology of the mouse seminiferous tubules was normal,there were various levels of spermatocytes,and no significant difference in the number of sperm in epididymis compared with WT mice（5）The abnormal rate of sperm head in Cep72 knockout mice increased significantly.（6）The abnormal rate of sperm flagella in Cep72 knockout mice increased significantly.（7）DNA double-strand break（DSB）formation,repair,chromosome synapsis,and homologous recombination were normal during the process of meiosis in spermatocytes of Cep72 knockout mice.（8）The fertility of Cep72 knockout mice was not affected.Conclusion:Cep72 plays a regulatory role during the assembly of the head and flagella of mice sperm,but it will not damage the fertility of mice,and has no decisive influence on spermatogenesis.