| In recent years,the number of infertility has increased significantly in China.Cryopreservation of human sperm is an important technology for male conservation.However,freeze-thaw results in structural and functional damage of sperm and low qualified rate of semen in using.It is an urgent scientific and technological problem to study the mechanism of freeze-thaw injury and explore a new method to protect human sperm from freeze-thaw injury so as to improve the clinical pregnancy rate.Aim:To study the effects of freeze-thaw on six sperm function-related proteins?HTL-1b?HEL-2?HTL-4?HTL-5,HEL-1a and HEL-3?and freeze tolerance of sperm to provide molecular basis for the cryopreservation of sperm.Method:The sperm was from Beijing Human Sperm Bank,through the Ethical Review of Institute of Science and Technology,National Health and Family Planning Commission.According to the standard procedure of the fifth edition of WHO,semen was routinely tested.The standard of sperm bank storage was C?60×106/ml and PR?%??50 before freezing;the standard was PR%?40 and CPR?60×106/ml after thawing.Semen samples that met the standard of sperm bank before and after freeze-thaw were used as qualified group?100semens in total?.Semen samples that met the standard of sperm bank before freezing and not meet the standard after thawing were used as unqualified group?40 semens in total?.Six kinds of sperm surface proteins were localized and quantitatively detected by immunofluorescence staining and laser confocal microscopy imaging.ZEN2.0 and sperm quality analysis software V1.1.1.63 were used to collect and analyze the data.SPSS.17 and excel 2010 were used to analyze the fluorescence intensity and fluorescence expression rate.Result:?1?The effect of freeze-thaw on total samples:Compared with pre-freezing,in the freeze-thaw PR?%?decreased by 28%,and PR+NP?%?decreased by 27%;The fluorescence intensity and expression rate of HTL-1b protein in the head decreased by 39%and 21%respectively,while the intensity in the tail decreased by 26%.The fluorescence intensity and expression rate of the other five proteins decreased by 32%and 15%in HEL-2protein,52%and 42%in HTL-4 protein,31%and 17%in HTL-5 protein,3.71 and 2.04 times in HEL-1a protein,51%and 53%in HEL-3 protein.?2?The effect of freeze-thaw on qualified group:Compared with pre-freezing,in the freeze-thaw PR?%?decreased by 24%,PR+NP?%?decreased by 23%after freeze-thaw;The fluorescence intensity and expression rate of HTL-1b protein decreased by 38%and 22%in the head,the intensity 22%decreased by 26%in the tail,31%and 14%in the HEL-2 protein,65%and 65%in the HTL-4 protein,30%and18%in the HTL-5 protein,3.60 and 1.94 times in the HEL-1a protein,51%and 54%in the HEL-3 protein.?3?The effect of freeze-thaw on the unqualified group:Compared with the pre-freezing group,in the freeze-thaw PR?%?decreased by 38%,PR+NP?%?decreased by36%after freeze-thaw.The fluorescence intensity and expression rate of HTL-1b protein decreased by 41%and 19%in the head,36%in the tail,35%and 17%in the HEL-2 protein,27%and 5%in the HTL-4 protein,36%and 16%in the HTL-5 protein,4.11 and 2.35 times in the HEL-1a protein,48%and 50%in the HEL-3 protein.?4?Compared with the results of qualified group,the vitality of unqualified group decreased by 6.6%?PR%?and 6.3%?PR+NP%?before freezing,23%?PR%?and 22%?PR+NP%?after freezing.The fluorescence intensity and expression rate of HTL-1b protein in the tail of unqualified group decreased by 36%and 26%respectively after freezing.The fluorescence expression rate of HTL-1b protein in the head of unqualified group decreased by 13%before freezing.The fluorescence intensity and expression rate of HTL-4 protein increased by 50%before freezing.The fluorescence intensity and expression rate of HEL-3 protein decreased by 18%and 16%respectively before freezing.There were statistical differences in the above results.Conclusion:In the freeze-thaw,four proteins?HTL-1b?HEL-2?HTL-5 and HEL-3?were significantly down-regulated,and two proteins?HTL-4 and HEL-1a?were significantly up-regulated in 140 semen samples collected from sperm bank.The freeze tolerance of unqualified sperm was worse than that of qualified sperm.The differential expression of three proteins?HTL-1b,HTL-4,and HEL-3?between qualified and unqualified sperm was inferred to be related to the freeze tolerance of sperm. |
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