The Role And Functional Mechanism Of SYMPK In The Meiosis Of Male Germ Cells

Background Infertility has become a global social problem,affecting numerous childbearing couples,of which male infertility accounts for approximately 50%.For male infertility,non-obstructive azoospermia(NOA)resulting from meiotic abnormalities by hereditary factors is one of the most important causes.At present,gene mutations that lead to NOA have not been sufficiently elucidated,especially genetic pathogenic factors contributing to the disability of key regulators during meiosis.Objective In this study,the conditional gene knockout(c KO)mouse model of SYMPK was constructed to explore its role and mechanism in male meiosis.Methods Western blot(WB)and immunofluorescence(IF)experiments were implemented to uncover tissue-related expression levels of SYMPK and its cytological locations in spermatogenic cells.Transgenic mice with lox P sequences knocked into the SYMPK genome,were generated,and then mated with mice carrying germline-specific Cre recombinase to achieve SYMPK deficiency in spermatocytes.In c KO mice,paraffin-embedded tissue sectioning and IF technologies were applied to analyze meiotic and spermatogenetic phenotypes,while the apoptosis of spermatocytes were detected by TUNEL staining.Immunoprecipitation with high-resolution mass spectrometry,WB,and IF experiments were combined to identify SYMPK’s interactors.Transcriptomes in spermatogenesis were mapped by RNA-seq and RT-q PCR.Besides,alternative splicing events and corresponding protein expression levels were verified with transcriptomics cluster analysis,RT-PCR,as well as WB.Results SYMPK protein was highly expressed in mouse testis and enriched in spermatogenesis,mainly located in the nucleus of spermatogonia,spermatocytes,round spermatozoa,and elongated spermatids at partial developmental stages.Germline-conditional knockout of SYMPK led to male sterility,which exhibited primarily spermatogenetic and meiotic abnormalities.The spermatogenetic disorders were characterized by meiotic diplotene arrest with increased spermatocyte apoptosis,while defects in chromosome synapsis,DNA damage repair and homologous recombination accounted for the abnormal meiosis.In male germ cells,SYMPK interacted with DDX5 and PRPF8,of which the deficiency resulted in disordered alternative splicing events and transcriptional expression levels of genes related to spermatogenesis and meiosis.Conclusions SYMPK mutation leads to male infertility by NOA resulting from meiotic disorders.Moreover,SYMPK controls the transcriptomics and alternative splicing events through interacting with DDX5 and PRPF8 during spermatogenesis.