| The 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.To date,eight SC components have been identified in mammals.Among them,C14orf39/Six6os1 encodes a novel central element of SC.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 thesis,using whole-exome sequencing and bioinformatics 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 mRNA levels,and immunostaining on the testicular sections revealed that the nonsense mutation resulted in a truncated protein(p.Glu320*).The results of immunoelectron microscopy and super-resolution microscopy imaging showed co-localization of SIX60S1 and SYCE1.Interestingly,the C14ORF39/SIX60S1 truncated protein caused by the nonsense mutation can still be located in the synapsed regions of chromosomes.To clarify whether this mutation causes infertility,we generated a mouse model named Six6os1ΔC/ΔC that carries a mutation similar to that in the patient by CRISPR/Cas9 technology.We found that the testis weight of Six6os1ΔC/ΔC mice was reduced to about one-third of the control.Histological analysis showed that there were no post-meiotic cells in the seminiferous tubules of mutant mice.By analyzing the progression of meiotic prophase I,we found that the spermatocyte development of Six6os1ΔC/ΔC mice was arrested at the pachytene-like stage.Moreover,programmed DNA double-strand breaks(DSBs)are formed normally in Six6os1ΔC/ΔC spermatocytes,but these DSBs cannot be efficiently repaired as crossovers through homologous recombination(HR).More importantly,the transverse filament SYCP1 and central element SYCE1 signals in pachytene-like spermatocytes of Six6os1ΔC/ΔC mice were discontinuous,which is different from the phenotypes of the reported knockout mice.To confirm our findings,we generated new Six6os1 knockout mice and found that the advanced spermatocytes in these mice had no SYCP1 and SYCE1 signals.These results suggest that homologous chromosomes in Six6os1ΔC/ΔC spermatocytes indeed undergo incomplete synapsis.Moreover,yeast two-hybrid results showed that the N-terminal of SIX60S1,containing two predicted coiled-coil motifs,binds to SYCE1,and the C-terminal of SIX60S1 mediates its self-polymerization,thereby explaining the phenotype.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 confirmed that the homozygous mutation in C14orf39/SIX60S1 indeed causes human infertility;on the other hand,we found that C14ORF39/SIX6OS1 promotes the initial assembly of the SCs via its N-terminal interaction with SYCE1,and facilitates the extension of synapsis through its C-terminal self-polymerization.Our research has helped us better understand the SC assembly. |
