冷冻复苏过程对人精子印记基因SNRPN和GRB10DNA甲基化及表达的影响  

DNA methylation and expression of SNRPN and GRB10imprinted genes in human semen freezing-thawing process

作　　者：周雪[1] 王燕蓉[1] 田龙[1] 马良宏[2] 颜贝 田稼[2] 张帆[2] 周岳[2] 王红燕[1,2] ZHOU Xue WANG Yanrong TIAN Long MA Lianghong YAN Bei TIAN Jia ZHANG Fan ZHOU Yue WANG Hongyan(Key Laboratory of Fertility Maintenance, Ministry of Education, Ningxia Medical University ＆ Key Laboratory of Reproduction and Genetics General Hospital of Ningxia Medical University, Ningxia Human Sperm Bank, Ningxia 750004, Yinchuan, China)

机构地区：[1]宁夏医科大学生育力保持教育部重点实验室宁夏生殖与遗传重点实验室 [2]宁夏医科大学总医院宁夏人类精子库,宁夏银川750004

出　　处：《山东大学学报（医学版）》2017年第1期54-59,共6页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(81460243;81560249);宁夏回族自治区科技攻关项目(2012ZYS239);宁夏医科大学生殖与遗传基础及临床创新团队人才项目(FGCT201505)

摘　　要：目的探讨精液冷冻复苏过程对人精子印记基因SNRPN、GRB10甲基化状态和mRNA表达的影响。方法将20份人精液标本平行分为2组:新鲜组(n=20)和冷冻组(n=20)。采用计算机辅助精子分析(CASA)进行两组精液的常规参数检测;Percoll密度梯度离心法纯化精子,SNRPN、GRB10基因的甲基化率采用Sequenom DNA测序分析;SNRPN、GRB10基因的mRNA表达采用qRT-PCR定量分析。结果冷冻组精子浓度、精子活力、前向运动精子百分比均低于新鲜组(P<0.05),冷冻组不活动精子百分比显著高于新鲜组(P<0.05);冷冻组精子SNRPN mRNA表达显著低于新鲜组(P<0.05);冷冻组精子GRB10启动子区亚片段检测的-902Cp G位点(即转录起始点上游989 bp处)的甲基化率显著下降(P<0.05);GRB10 mRNA表达较新鲜组显著升高(P<0.05)。结论冷冻复苏过程影响人精子GRB10、SNRPN基因的DNA甲基化和mRNA表达,提示精子库常规使用的精液冻储技术可能改变配子的表观遗传信息,进而调控基因的表达。Objective To assess the effect of human semen frozen-thawing process on sperm DNA methylation status and mRNA expressions of SNRPN and GRB10 imprinted genes. Methods The semen samples were collected from 20 healthy men,and each sample was divided into two equalaliquots： fresh group（ n = 20） and cryopreserved group（ n =20）. Routine semen parameters was tested by computer-aided sperm analysis（ CASA）. Each semen sample was purificated by Percoll density gradient centrifugation. The DNA methylation status of SNRPN promoter region neighbouring fragment and GRB10 promoter region fragment were determined by Sequenom DNA sequencing. The mRNA expressions of SNRPN and GRB10 were evaluated by qRT-PCR. Results Compared with fresh group,the semen parameters including sperm concentration,percentage of sperm motility and forward-moving sperms all decreased in cryopreservedgroup（ P〈0. 05）,and percentage of unforward-moving sperm increased significantly（ P〈0. 05）. Compared with fresh group,the mRNA expression of SNRPN decreased significantly in cryopreserved group（ P〈0. 05）. DNA methylation status on the forth methylation site（- 902 Cp G point） of GRB10 promoter region fragment remarkably decreased（ P〈0. 05） and the mRNA expression of GRB10 increased significantly in cryopreserved group（ P〈0. 05）. Conclusion The freezing-thawing process affects DNA methylation status and mRNA expressions of human sperm SNRPN and GRB10 imprinted genes. It suggests that the semen freezing technique routinely used in the human sperm bank may alter the epigenetic information of the gametes and consequently regulate the expression of related genes.

关 键 词：精液冷冻 DNA甲基化 小分子核糖体蛋白多肽N 生长因子受体结合蛋白10 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学] Q786[医药卫生—基础医学]