Problem:Condensed sperm chromatin is relatively resistant to chemical and physical treatments.The purpose ofthis study is to explore the highest temperature that sperm can tolerate to produce live offspring and possibledamages caused by heat treatment.Material and methods:Sperm heated in a water bath for 30 minutes were microinjectedinto mouse oocytes.Fertilization,embryo development and 1-cell embryo karyotypes were evaluated.Epigeneticchanges including DNA methylation and histone modifications were evaluated by immunofluorescent staining.Results:Mouse sperm treated at 800C for 30min were able to support full-term development after intracytoplasmicinjection into the oocytes.Heat treatment destroyed integrity of sperm chromatin in a temperature-dependentmanner.However, once became a pronucleus, heat-treated sperm chromatin could undergo normal active DNAdemethyaltion and histone demethylation.Sperm treated at 950C for 30 min underwent incomplete spermchromatin decondensation, and thus failed to support embryo development.Combined with artificial oocyte activation, mouse sperm could maintain relatively high fertilization rate after 650C heating for 30min. But the blastocyst rate and implantation rateof heated group weremuch lower compared to those of fresh sperm group.We further compared the patterns of DNAmethylation and histone modification including H3K9,H3K27 tri-methylation and H4K12 acetylation betweenembryos derived from heated and fresh sperm.No significant differences in AcH4K12,H3K9-Trill,and DNAmethylation were observed between fresh- and heated-sperm derived embryos from zygote to blastocyst stage.However,we detected defective reprogramming of H3K27-Trill in heated-sperm derived blastocysts,which couldbe one of the reasons for low implantation rate of embryos derived from heated-sperm.Conclusion:The highest temperature for mammalian sperm treatment that can produce living offspring is 800C.The developmental competence decrease of heated sperm is caused by destroyed integrity of sperm chromatin anddefective epigenetic reprogramming in embryos.
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