| Background:Sperm chromatin is highly condensed and relatively resistant to chemical and physical treatments. The purpose of this study is to explore the highest temperature that sperm can tolerate and still produce live offspring. We detected the epigenetics and karyotypes of embryo derived from heated sperm to explore the reason underlied the compromised fertilization ability,embryonic development and blastocyst fomation rate and low implantation rate.Methods:Mouse sperm were heated in a water bath at(50,65,80or95℃for30minutes before they were microinjected into mouse oocytes. Fertilization, embryo development and1-cell embryo karyotypes were evaluated. Epigenetic reprogramming including DNA methylation and histone H3K4-Tri-methylation were evaluated by immunofluorescent staining. Preimplantation embryo epigentics reprogramming including DNA methyaltion,H4K12(ACH4K12) acetylation, H3K9trimethylation (H3K9-TriM), H3K27-TriM was also examined by IF methods.Results:The ability of mouse sperm to activate the egg after ICSI was heat sensitive; only20%of eggs were activated by sperm that had been heated to50℃and none by sperm heated to80℃. However, if eggs were activated artificially, mouse sperm subjected to80℃for30min were able to produce live offspring, while95℃treatment affected sperm decondensation after intracytoplasmic sperm injection (ICSI). Once the heat-treated sperm nucleus had developed into a pronucleus, sperm chromatin was able to undergo normal active DNA demethylation and histone methylation. Aberrant chromosome rates increased from16.3%to100%when the temperature was raised from50℃to95℃.The patterns of DNA methylation, histone H4K12(ACH4K12) acetylation, H3K9trimethylation (H3K9-TriM), and H3K27trimethylation (H3K27-TriM) in preimplantation embryos derived from65℃-heated sperm were investigated. Although no evident changes in global DNA methyaltion, histone H4K12(ACH4K12) acetylation, and H3K9trimethylation (H3K9-TriM) was found, significantly low level of H3K27-TriM, which was thought to be one of the reasons for low efficiency of mouse cloning, was found in the inner cell mass (ICM) of heated-sperm derived blastocysts. Thus, defective modification of H3K27-TriM might contribute to compromised development of embryos derived from heated sperm.Conclusion:Heat treatment destroys integrity of sperm chromatin in a temperature-dependent manner.80℃was the highest temperature that mouse sperm could withstand and still produce live offspring. Without oocyte activation, sperm substained little fertilization ability after65℃treatment. However, heated sperm restored the oocyte activation after oocyte activation. Heat destroyed chromosome integrity and the effection was temperature dependent. Defective H3K27-TriM reprogramming of blastocyst derived from heated sperm was detected, which were the reason of compromised blastocyst fomation rate and low implantation rate. |
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