Functional Study Of WHSC1 In Mouse Spermatogenesis

PartⅠAcquisition and phenotype exploration of WHSC1 conditional knockout miceObjective: To study the effect of WHSC1 gene deletion on mouse reproduction,we conditionally knocked out WHSC1 gene in male mouse germ cells,and provide a potential target for the diagnosis and treatment of male infertility.Methods: Cre recombinase is a site-specific recombinase and mediates recombination between two Loxp sites.With gene editing technology CRISPR/Cas9,together with Cre-Lox P recombinase system(Stra8-cre transgenic mice)to acquire mice with conditionally knockout of WHSC1 in male germ cells,and the experimental group and the control group mice were obtained by genetic identification.Immunohistochemistry of frozen sections confirmed success of knockout in mice.Fertility tests were further used to evaluate impact on male reproduction.HE/PAS staining of the testicular and epididymis paraffin section was also used for studying how spermatogenesis process was impacted.Results: 1.After germ cells of male mice were specifically knocked out of WHSC1,the testis and epididymis of adult mice became significantly smaller,the average diameter of seminiferous tubules became smaller,and even local vacuolation appeared.2.The number of sperm in the epididymis is greatly reduced,the average litter size is reduced,and fertility is also dampened.Conclusions: Conditionally knock out of WHSC1 gene in male mice was successful.Depletion of this gene dampened male fertility,testis and epididymal development,sperm morphology and structure,sperm motility and sperm acrosome formation.PartⅡ Exploration of the effect of WHSC1 on spermatogenesis in miceObjective: To explore the mechanism of the effect of WHSC1 gene deletion on spermatogenesis,and to provide new diagnosis and treatment ideas for males with oligoasthenospermia.Methods: On the basis of the first part of the research,phenol-chloroform extraction of mouse testis RNA sequencing was performed to explore the possible targets of WHSC1.Immunofluorescence of frozen sections was used to study potential changes of histone modifications such as H3K36me2 and H4K16 ac.Sperm smear was further performed to examine abnormal morphology and localization of PNA and other markers.Results: 1.After WHSC1 was knocked out,RNA-seq showed that most of the genes related to spermatogenesis did not change significantly.2.Compared with the control group,the level of H3K36me2 in spermatogenic cells within seminiferous tubules of WHSC1 conditional knockout mice was greatly decreased.3.H4K16 ac in round spermatids and elongating spermatids of WHSC1 conditional knockout mice was significantly increased.4.Sperm head of WHSC1 conditional knockout mice was abnormal.Conclusions: WHSC1 regulates spermatogenesis-related epigenetic processes rather than gene expression networks by catalyzing H3K36me2 modification,to ensure successful spermatogenesis.WHSC1 inhibits H4K16 hyperacetylation,thereby inhibiting the integration of excessive protamine into the sperm genome.