Function Of RAD51AP2 In Meiotic Recombination Of Sex Chromosome During Spermatogenesis

Meiosis is an inevitable process of producing haploid gametes during spermatogenesis,which not only ensures stable transmission of genetic materials among species,but also is an important source of genetic diversity.In meiosis,a series of complex and ordered events occur between homologous chromosomes,including pairing,synapsis,recombination and separation,of which,recombination is the core event,yet pairing and synapsis play an accessary role for recombination.Meiotic recombination involves three processes:programmed DNA double-strand breaks(DSBs)formation on homologous chromosomes,strand exchange of homologous chromosomes and eventually formation of the physical linkage in order to facilitate accurate separation of chromosomes later.The sex chromosome differs from autosomes in chromosome morphology and belongs to a heteromorphic chromosome so that recombination is spatiotemporally limited in a very small homologous region,namely the pseudoautosomal region(PAR).Meiotic recombination abnormality causes infertility,birth defects and multiple inherited diseases,such as Down syndrome and Klinefelter syndrome(47,XXY).It is of great scientific significance to explore the key issues of meiotic reorganization,for example which proteins are involved and how they function.However,so far,little is known about the meiotic recombination,especially in sex chromosome.As a "natural model",patients with male reproductive disorders provide us with an opportunity to further study spermatogenesis.To discover new molecules that regulate meiosis in spermatocytes,on the basis of testicular histological analysis,we selected patients with abnormal spermatocyte development from the database of human reproductive diseases to performed whole-exome sequencing,bioinformatics analysis,focused on a potential candidate mutant,RAD51-associated protein 2,RAD51AP2.The T-vector monoclonalizaton of target gene and Sanger sequencing confirmed that the patient was a RAD51AP2 cmpound heterozygous mutation(Exonl:c.943-946delTTTT,p.Lys315Leufs*2;Exon3:c.3391-3395insTTG GT,p.Arg1131Leufs*19).We generated two kinds of mouse RAD51AP2 antibodies,using Western blot and fluorescence immunostaining we found that RAD51AP2 was specifically expressed in mouse testis,and localized on spermatocyte chromosomal axis depended on SPO11-mediated DSBs;co-immunoprecipitation(co-IP)results showed that RAD51AP2 interacts with the recombinase RAD51,these results suggest that RAD51AP2 is likely to participate in the meiotic recombination.To confirm whether the compound heterozygous mutation in this patient is a causative mutation,we used the CRISPR/Cas9 gene editing technology to prepare Rad51ap2 homozygous deletion mouse(Rad51ap2-/-),Rad51ap2 mutant mice(Rad51ap2MUT/MUT)and compound heterozygous mutation mice similar to the patient(Rad51ap2*),and found that all these male mice showed the identical defects in spermatogenesis,including the metaphase I of meiosis(MMI)spermatocytes can be found,most of the MMI cells occurred lagging chromosome or appeared apoptotic.In order to find out the reasons,we analyzed the meiotic prophase progression by combining meiotic chromosomal spreading with fluorescence immunostaining and found that Rad51ap2 knockout mice was the similar with the control in the process of autosome pairing,synapsis and recombination;XY synapsed normally in the early pachynema of Rad51ap2 knockout mice(H1t as a marker),but a high percentage of XY separation began in the mid-pachynema(51.09%)and diplonema(51.36%),suggesting that the recombination repair likely had some problems.Further analysis revealed that the recombination related proteins such as RAD51 in knockout mouse were persistently located in the isolated XY PAR,indicating that the DSB repair in the PAR region was impaired.Co-IP experiments further confirmed that RAD51AP2 interacts with RAD51 through the C-terminus,whereas RAD51AP2 loses its interaction with RAD51 in Rad51ap2MUT/MUT mice lacking the C-terminal 4 amino acids(SRPI),indicating that the interaction between RAD51AP2 and RAD51 is crucial to recruit itself to the chromosomal axis and function.In summary,our study confirmed that Rad51ap2 is a key gene that guarantees the completion of meiotic recombination of sex chromosome.The loss of RAD51AP2 function does not affect the meiotic behavior of the autosome,but only results in failure of DSB repair in the XY PAR area,physical connection between XY cannot be formed,and meiosis cannot be completed,eventually leading to spermatogenesis disorders.These findings reveal that DSBs repair mechanism of sex chromosome is different from that of autosomes,providing new insights into the understanding of meiotic recombination,especially the meiotic recombination of sex chromosome.It also provides a theoretical basis for genetic counseling and diagnosis and treatment of male infertility.