Pathogenic Mechanisms Of Multiple Morphological Abnormalities Of Sperm Flagella Caused By Lrrc46 And Cfap61 Knockout

As the third category diseases affecting human health worldwide,infertility affects millions of couples of child-bearing age,bringing significantly negative impacts on families and society.Male factors account for about 45%-50%of infertility cases,and genetic factors control about 15%of male infertility.Any abnormalities in the testis,epididymis,vas deferens and endocrine system will lead to abnormal sperm function and quality,and cause male infertility.Clinically,male infertility can be divided into azoospermia,oligozoospermia,asthenozoospermia,teratozoospermia,and multiple combinations of these diseases.The genetic basis of male infertility is very complex,and at least 2,000 genes have been found to be involved in various stages of spermatogenesis.Asthenospermia includes multiple morphological abnormalities of the sperm flagella(MMAF)and primary sperm motility disorder(PCD).Multiple morphological abnormalities of the sperm flagella,abbreviated as MMAF,is a severe malformation of sperm flagellum caused by teratospermia,which proposed by French scientist Professor Ray in 2014.According to the 5th edition of Human Semen Evaluation Criteria of the World Health Organization,MMAF is mainly characterized by several types,such as short,coiled,and irregular flagella.Resulting in decreased sperm motility and fertilization,MMAF eventually caused male infertility.The ultrastructural abnormalities were observed by transmission electron microscopy,including sperm flagellar fibrous sheath,mitochondrial sheath,central microtubule,peripheral microtubule,and inner and outer dynein arms.The etiology of MMAF is complex and heterogeneous.Currently,the known pathogenic genes of MMAF can only explain about 60%of MMAF cases.Therefore,the discovery of new genetic factors remains a major challenge in the field of male infertility genetics.With the rapid development of high-throughput sequencing technology,researchers used whole exon sequencing(WES)and bioinformatics analysis techniques to conduct genetic studies on inbreeding families and sporadic male infertility patient cohorts.Researchers have successively identified a series of pathogenic genes of MMAF,including AKAP,DNAH,CCDC,CFAP and TTC family genes.In this paper,focused on the susceptibility gene families of CFAP and LRRC family,we attempt to discover some new pathogenic genes that cause male infertility.The cilia and flagella related proteins(CFAPs)family is a group of proteins related to the development and function of cilia and flagella,which play important roles in the biological occurrence,normal assembly,and maintenance of motor function in cilia and flagella.In the process of biological evolution,the function of leucine rich repeat sequence proteins(LRRCs)family proteins are highly conserved in many ciliated and flagellated organisms.Currently,multiple CFAP and LRRC family genes have been reported to be associated with MMAF and PCD.The evolution of CFAP61 protein is highly conserved.In unicellular organisms such as chlamydomonas and tetrahymena,its homologous protein is FAP61.In chlamydomonas,FAP61 is involved in the formation of flagella and cilia.The structure of flagellar calmodulin and spoke-associated complex(CSC)is composed of flagellar associated protein 61(FAP61),flagellar associated protein 91(FAP91),and flagellar associated protein 251(FAP251),connecting to the base of radiospoke protein 2(RS2)and radiospoke protein 3(RS3).In tetrahymena,mutations in FAP61 or FAP251 lead to the deletion of RS3 structures,affecting the wave shape and frequency of the cilia or flagella on the cell surface,leading to a decrease in the swimming ability of the cell.LRRC46 is a candidate susceptibility gene for ciliary related diseases such as familial prostate cancer(PCa),advanced serous cancer(HGSOC),glioblastoma,nasopharyngeal carcinoma(NPC),and female ectopic pregnancy(ECT).Focused on LRRC and CFAP family proteins in databases such as BioGPS,NCBI,HPA,and MGI,we found that there were no relevant report on the phenotypes and functions of LRRC46 and CFAP61 in the reproductive system.Via CRISPR/Cas9 technology,we constructed Lrrc46 and Cfap61 gene knockout mice,and observed important biological events including the growth and development,spermatogenesis,sperm morphology,and sperm function,with an attempt to explore the potential functions and mechanisms of Lrrc46 and Cfap61 in the development of the male reproductive system.In this study,we identified two pathogenic genes caused MMAF,namely Lrrc46 and Cfap61.We demonstrate that the Lrrc46 and Cfap61 knockout mouse exhibit typical MMAF phenotypes including sperm with short,coiled,and irregular flagella,and lead to infertility in male mice.Lrrc46 and Cfap61 are located at the tail of mouse and human sperm flagella,which suggests that Lrrc46 and Cfap61 may affect the development and stability of sperm flagella.CFAP61 and LRRC46 may be involved in MMAF or other ciliary diseases leading to male infertility.These results provided potential theoretical guidance for molecular diagnosis,genetic counseling,and clinical intervention of male infertility.