Temperature Sensitivity Analysis Of Meiotic Defects In Caenorhabditis Elegans

Meiosis is a special way of cell division that produces sperms or eggs for fertilization.The precise separation of chromosomes during meiosis is very important.If the chromosomes are incorrectly separated,it will have a serious impact on human health,leading to infertility,abortion and defects of newborns.There are a series of regulatory mechanisms in the process of meiosis to ensure the normal separation of chromosomes,such as homologous chromosome recognition,pairing,synapsis,DNA double-strand break(DSB),the formation of crossover(CO),chromosome remodeling,etc.Therefore,studying meiosis is of great significance to human reproductive health.It is of great significance to human reproductive health to clarify the regulation mechanism of the precise separation of meiotic chromosomes and how these regulation links are affected by external environmental factors.The synaptonemal complex(SC)is a structurally conserved protein complex consisting of a central region and axial elements on both sides.The proper assembly and disaggregation of SC is essential for the correct separation of chromosomes during meiosis.We discovered a new central region protein of SC in Caenorhabditis elegans and named it SYP-5.The adult survival rate of the offspring of the syp-5 mutant under high temperature(25℃)is significantly lower than that under normal culture temperature(20℃),and the proportion of male increases significantly at high temperatures.While the rate of adult survival and the proportion of male of wild-type did not change significantly at different culture temperatures,indicating that the reproductive phenotype of the syp-5 mutant is temperature sensitive but not the wild-type.Since the loss of SYP-5 causes a variety of meiotic events to be disordered,the temperature-sensitive phenotype of the syp-5 mutant is caused by the loss of the SYP-5 protein.Therefore,we explored the temperature sensitivity of syp-5 mutants based on the defective phenotype of syp-5mutants.This paper mainly studies the temperature-sensitive mechanism of syp-5 mutants in four aspects: assembly and depolymerization of the synaptonemal complex,synapsis,homologous recombination and chromosome remodeling.First,the defects of assembly and disaggregation of the synaptonemal complex in the syp-5 mutant not change with the culture temperature of C.elegans,proving that the temperature sensitivity of syp-5 mutants was not related to defects in assembly and depolymerization of synaptic complex.Second,we performed plate phenotype analysis on mutants,with partial or complete synapsis defect,cultured at 20℃ and 25℃.The adult survival rate of zim-3,syp-4(22K),and lab-1 with partial chromosomal synapsis defects decreased significantly with the increase of the culture temperature,showing similar reproductive phenotype to syp-5 mutant.The reproductive phenotypes of other mutants with partial or complete synapsis defects were similar to those of the wild type,and they did not show sensitivity to temperature.This result indicates that the temperature sensitivity of syp-5 mutant has nothing to do with chromosome synapsis defect.Third,by comparing and analyzing the formation of DNA double-strand breaks and crossover between wild-type and syp-5 mutants,we found that syp-5 mutants have defects in homologous recombination.Although the number of DSBs in the syp-5 mutant is not consistent with the wild type and increases significantly at multiple stages,this increase is the same at different temperatures,so the DNA double-stranded breaks defect of the syp-5 mutant is not temperature sensitive.However,the defects of crossover formation of syp-5 mutants was more serious with the increase of culture temperature of C.elegans.Therefore,defect in crossover formation is one of the reasons for the temperature sensitivity of syp-5 mutants.Observe the Phosphorylation of Histone H3 and localization of AIR-2 on the chromosomes of wild-type and syp-5 mutants at the diakinesis.Unlike wild-type,the abnormal localization of AIR-2 in syp-5 mutants at 25℃ is more serious than at 20℃.It proves that abnormal chromosome remodeling in the syp-5 mutant is affected by temperature.This conclusion was subsequently confirmed by temperature shift experiments.Finally,the spindle assembly and chromosome separation of meiosis were further observed at different temperatures.In the syp-5 mutants cultured at 25℃,aberrant chromosome separation in the late stage of meiosis I and were accompanied by abnormal spindle morphology.In summary,the temperature-sensitive phenotype exhibited by the syp-5 mutant is not only related to the formation of crossovers but also to chromosomal remodeling.These findings reveal the synergistic effect of meiotic defects and temperature on reproduction,which will provide a useful reference for the prevention and treatment of human reproductive diseases.