C14ORF39/SIX6OS1 Is Essential For The Initiation And Extension Of Chromosome Synapsis During Meiosis

Synaptonemal complex(SC),formed between paired homologous chromosomes during the prophase of meiosis I,is a tripartite proteinaceous zipper-like structure that facilitates interhomolog crossover formation and successful production of gametes.C14orf39/Six6osl encodes a novel central element of SC,and its deletion results in chromosomal asynapsis,which ultimately leads to infertility in mice.In general,deficiency in any one of the genes encoding SC components causes meiotic arrest and infertility.However,only mutations in SYCP3 and SYCE1 have been associated with human male infertility so far.Thus,whether and how other mutations in SC encoding genes affect human spermatogenesis is still unclear.In this study,using whole-exome sequencing analysis of the DNA from infertile patients with spermatocyte development arrest(SDA),we identified a homozygous nonsense mutation(c.958G>T)in C14orf39.Subsequent Sanger sequencing confirmed the existence of this mutation at the genome and mRN A levels,and immunostaining on the testicular sections revealed that the nonsense mutation would produce a truncated protein p.Glu320*.To verify whether this mutation causes infertility,we generated the mouse model,named Six6os1?C/?C which carries a similar mutation to the patient by CRISPR/Cas9 technology.We found that the testicular size of Six6os1?C/?C mice reduced to about one-third of the control.Histological analysis showed that no post-meiotic cells in the seminiferous tubules of mutant mice.By analyzing the progression of meiotic prophase I,we found that the advanced spermatocytes in Six6os1?C/?C mice were arrested at the pachytene-like stage.Furthermore,programmed DNA double-strand breaks(DSBs)were formed in Six6os1?C/?C spermatocytes,but these DSBs cannot be efficiently repaired as crossovers through homologous recombination(HR).More importantly,the pachytene-like spermatocytes of Six6os1?C/?C mice displayed discontinuous SYCP1 and SYCE1 signals,which is different from the reported phenotypes of knockout mice.To confirm the above findings,we generated the Six6os1 knockout mice,whose advanced spermatocytes showed the absence of SYCP1 and SYCE1 signals.These reSults suggested that chromosome synapsis has been initiated but cannot be completed in Six6os1?C/?C spermatocytes.Besides,yeast two-hybrid results showed that the N-terminal of SIX60S1 containing two predicted coiled-coil motifs interacts with SYCE1,and the C-terminal of SIX6OS1 mediates itself polymerization.Therefore,we argue that SIX6OS1 truncated protein generated by the nonsense mutation initiates synapsis through the interaction with SYCE1 but cannot get complete synapsis due to lacking C-terminal.Taken together,infertile patients with SDA provide an opportunity for us to study the regulation of meiosis.Combined with the functional study of the mouse model,on the one hand,we identified and validated that the mutation in C14orf39/SIX6OS1 indeed causes human infertility;on the other hand,we found that C14ORF39/SIX6OS1 is essential for the initiation and extension of chromosome synapsis during meiosis.Our research has deepened our understanding of the SC assembly.