Effect And Mechanism Study Of GKAP1 Gene Knockout On Spermatogenesis In Mice

Objective: The regulation and molecular mechanisms of mammalian spermatogenesis have always been the focus of research.In previous studies,many genes and their protein products in the testis have been shown to play an important role in spermatogenesis.However,there are still many genes that are highly expressed in the testis,but their function is still unclear.In this study,we constructed a GKAP1 knockout mouse model to study its effects on testis and explore its function in spermatogenesis.Methods and Results:1.Phylogenetic analysis was used to show the conservation of GKAP1 protein,and its high expression in testis was detected by Western-blot and immunofluorescence techniques revealed that it was mainly expressed in spermatocytes and round sperm cells,the knockout mice model were constructed using CRISPR-Cas9 technology;2.GKAP1 knockout mice did not find fertility abnormalities.CASA(computerassisted semen analysis)showed normal sperm motility,but testicular sperm and epididymal sperm count increased,and knockout mouse epididymis swollen;3.Flow cytometry and Western-blot detected the apoptosis in the epididymal sperm the result showed that there was no significant difference between GKAP1 knockout and WT mouse epididymal sperm,so the increase of sperm was not related to epididymis;4.The proliferation and apoptosis signals in the testis were detected by immunohistochemistry.It was found that the proliferation of cells in the testis of GKAP1 knockout mice was not different from that of WT,but the number of apoptosis of germ cells was significantly reduced;5.Knockout of GKAP1 causes a slight increase in c GK-Iα expression in the testis,resulting in increased activation of CREB phosphorylation,which in turn promotes increased expression of certain inhibitory proteins,ultimately resulting in decreased caspase-3 activation;6.Detection of GKAP1 m RNA expression levels in infertile male testis.Conclusions:1.Knockout of GKAP1 causes a decrease in germ cell apoptosis and increased sperm production in the testis;2.The knockdown of GKAP1 mainly reduces the apoptosis level of spermatogenic cells through the cGK-Iα pathway.