The Mechanism Of Akap4 Deletion For Fibrous Sheath Dysplasia And Mice Infertility

Introduction:In the 21st century,the quality of male sperm continues to decline due to the change of human life style,the aggravation of environmental pollution and the increase of reproductive age.Sperm has to go a long distance to meet the oocyte and fertilize,sperm movement is mainly based on the tail of the flagella swing,so sperm tail movement is a necessary condition for normal fertilization.Asthenospermia is a cormon cause of male infertility.Male infertility accounts for about 50%of all infertility.The genetic background of male infertility is highly complex,because the morphology of sperm and histological phenotype of testis are extremely complex.According to statistics,there are at least 2000 genes involved in spermatogenesis.About 90%of male infertility is due to decreased sperm count and/or asthenospermia.A-kinase anchoring protein 4(AKAP4),an x-linked member of the AKAPs scaffold protein family,anchors cAMP-dependent protein kinaseA(PKA),which plays an important role in the assembly of fibrous sheath of the principal of the sperm tail during the process of sperm formation in late meiosis and the motor function of the mature sperm tail.The fibrous sheath,consisting of two longitudinal and transverse costal columns,accounts for most of the whole length of the sperm tail and is involved in signal transduction and glycolysis.Proteomic analyses have shown that the fibrous sheath is composed of at least 26 proteins,among which AKAP4 is the most abundant protein in the fibrous sheath.The results of immunoelectron microscopy have shown that AKAP4 is expressed in the longitudinal column(CL)and transverse rib(TR)of the fibrous sheath,and the gene encoding AKAP4 is only transcribed in the late stage of sperm meiosis.Previous studies have shown that the abnormality of AKAP4 gene can lead to the loss of fibrous sheath of human sperm,decreased activity and shortened tail,but the influence of AKAP4 gene on the formation of related proteins in the tail of human sperm has not been systematically studied.Akap4 gene knockout mouse model was constructed to study the role of Akap4 gene in spermatogenesis,providing a new direction for the treatment of male infertility.Purposes:To investigate the role of Akap4 gene in mice spermatozoa formation;To study the effect of Akap4 gene deficiency on other components of FS proteins in mice and the role of Akap4 gene regulation network in the process of spermatogenesis in the testis;To explore the possible mechanism of male infertility caused by deficiency of FS related proteins;To find a new way to diagnose and treat male infertility.Methods:Akap4 gene knockout mouse model was constructed to study spermatozoa motility morphology.The ultrastructure of spermatozoa was observed by electron microscopy.Immunofluorescence technique was used to localize and analyze the FSproteins.PAS staining,immunofluorescence and ultrastructure of the seminiferous tubules of testis were observed.Results:The Akap4 gene knockout(KO)mice were successfully constructed by CRISPR/Cas9 technology,and the sperm motility and forward movement activity of Akap4-KO mice were significantly decreased(P<0.01).Akap4-KO mice had no normal sperm,and the short ratio of the sperm tail was about 91.9%(P<0.01).Through scanning and transmission electron microscopy,it was found that the longitudinal column of the fibrous sheath of the principal piece of Akap4-KO mice spermatozoa was missing,resulting in the disorderly distribution of the 3 and 8 outer dense fiber(ODF)in the abnormally enlarged tail.The normal 9+2 microtubule structure was destroyed and the central microtubule was displaced.The high electron density can be seen near the membrane,which was the residual structure of traverse column.According to testicular transmission electron microscopy,no normal fibrous sheath structire was found in the spermatozoa of Akap4-KO mice.Immunofluorescence showed that the ODF 3 and 8 and 9+2 microtubules in the spermatozoa of Akap4-KO mice presented a disorderly distribution in the tail after the middle piece.The expression of AKAP3,GAPDHS5CABYR was not affected and the expression was aecreased.AKAP4 protein was not detected in spermatozoa of Akap4-KO mice.Single-cell RNA sequencing results showed that the deletion of Akap4 gene blocks the stepl5-16 of mice spermatozoa at the elongation stage,confirmed that Akap4 gene played an important role in the Step 15 and Step 16 of spermatogenesis.Conclusion:The deletion of Akap4 gene resulted in abnormal fibrous sheath of mice spermatozoa and lower sperm motility,and finally leads to male infertility in mice.Akap4 gene deletion had no significant effect on spermatogenesis in mice.It was first confirmed that the deletion of Akap4 gene caused the deletion of the longitudinal coltunn of the sperm fibous sheath,and caused the outer dense fibers 3 and 8 to detach from the 9+2 microtubule structure,and it was first confirmed that the transverse column was still partially residual.Akap4 gene deletion had no effect on CABYR and GAPDHS expression of fibrous sheath components,but the expression levels of CABYR and GAPDHS were down-regulated Akap4gene deletion affected the assembly of fibrous sheath structure in the tail of mice spermatozoa,which confirmed that it had no significant effect on the initiation of the formation process of the tail of mice spermatozoa.The deletion of Akap4 gene blocked the step15-16 of mice spermatozoa at the elongating stage,which confirmed that Akap4 gene plays an important role in the Step 15 and Step 16 of spermatogenesis.lt provides a new direction for the treatment of male infertility.