p,p’-DDE致大鼠跨代雄性生殖毒性效应及Igf2 DMR2区域甲基化的可能机制  

Transgenerational reproductive toxicity in male rats induced by p,p'-DDE and possible mechanism of Igf2 DMR2 methylation

作　　者：宋杨[1,2] 高明[2] 王禹[2] 吴南翔[2] 

机构地区：[1]杭州师范大学医学院预防医学系,浙江杭州311121 [2]浙江省医学科学院卫生学研究所环境医学研究室,浙江杭州310013

出　　处：《环境与职业医学》2017年第2期106-111,共6页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金(编号:81102161;81573196);浙江省自然科学基金(编号:LY14H260004);浙江省医药卫生科技计划(编号:201475777)

摘　　要：[目的]建立宫内p,p’-DDE暴露跨代大鼠模型,观察雄性子代生殖毒性是否具有亲源性遗传特征及可能的表观遗传机制。[方法]SPF级的SD大鼠于孕8~15天给予每天每千克体重100 mg p,p’-DDE灌胃,另设溶剂对照组采用玉米油灌胃,孕鼠自由分娩,保留所有雄性仔鼠(F1代),同组别不同窝别的F1代仔鼠于出生后90天按雌雄比1∶1交配,产生F2代仔鼠。染毒组和对照组雌雄F2代按以下分组设计两两交配后,产生F3代仔鼠,分组为:(1)雌雄均为对照组(C♂-C♀),(2)雌雄均为染毒组(DDE♂-DDE♀),(3)染毒组雄性与对照组雌性(DDE♂-C♀),(4)对照组雄性与染毒组雌性(C♂-DDE♀)。用计算机辅助精子分析系统(CASA)分析3代雄性仔鼠成年后的精子质量,亚硫酸氢钠基因组测序法检测Igf2 DMR2区域甲基化水平,实时荧光定量PCR分析印记基因表达。[结果]p,p’-DDE可诱导F1、F2及F3代DDE♂-DDE♀、DDE♂-C♀精子数量和活力下降,F1代精子数量和活力分别从(70.63±11.79)×106/m L、(76.75±7.57)%下降至(50.00±13.08)×106/m L、(62.42±9.40)%;F2代精子数量和活力分别从(82.86±38.60)×106/m L、(82.14±6.44)%下降至(43.75±25.04)×106/m L、(60.88±25.03)%;F3代DDE♂-DDE♀精子数量及活力分别从(68.89±41.37)×106/m L、(68.56±10.67)%下降至(21.66±4.83)×106/m L和(29.33±32.70)%,F3代DDE♂-C♀精子数量和活力下降为(28.00±16.60)×106/m L和(34.60±31.60)%,差异均有统计学意义(均P<0.05)。F1~F3代雄性仔鼠精子细胞Igf2 DMR2区域(1、16、18位点)低甲基化,Igf2转录下调,H19转录上调,Igf2基因的转录水平和Igf2 DMR2甲基化成正相关(r=0.806 5)。[结论]Igf2 DMR2区域甲基化改变可能在p,p’-DDE诱导的跨代雄性大鼠生殖毒性中起重要作用。[Objective] To establish a transgenerational rat model with in utero p, p＇-DDE exposure and observe inherited characteristics and possible epigenetic mechanism. [ Methods ] Only the pregnant rats （FO） were administered with p, p＇-DDE （100 mg/kg per day according to body weight） or corn oil at the critical time of testis development （from gestation day 8 to 15）. Male rats （90 days old） of F1 generation were 1：1 mated with female of the same maternal treatment to produce F2 progeny. To reveal whether the male or female germ line was responsible for the transgenerational phenotype, F3 progeny was generated by intercrossing the control and treated male and female of F2 generation and divided as following groups： 1） control male plus control female （C ♂ -C♀）, 2） exposed male plus exposed female （DDE ♂ -DDE♀）, 3） exposed male plus control female （DDE ♂-C ♀）, and 4） control male plus exposed female （C ♂ -DDE ♀）. Sperm count and motility of male offspring of F1-F3 were analyzed with the Computer-Aided Sperm Analysis （CASA）. Igf2 DMR2 methylation was analyzed with bisulfite genomic sequencing. Gene expression was analyzed with real-time quantitative PCR. [ Results ] p, p＇-DDE decreased the sperm numbers and motility in F1 and F2 generations and DDE ♂-C♀ and DDE ♂-DDE♀ in F3 generation. In F1 generation, sperm numbers and motility decreased from （70.63 ± 11.79）× 10^6/mL and （75.75 ± 7.57）% to （70.63 ± 11.79） × 10^6/mL and （62.42 ± 9.40）% after p, p＇-DDE treatment, respectively. In F2 generation, sperm numbers and motility decreased from （82.86 ± 38.60） × 10^6/mL and （82.14 ± 6.44%）% to （43.75 ± 25.04） × 10^6/mL and （60.88 ± 25.03）% afterp, p＇-DDE treatment, respectively. As for DDE ♂ -C♀ in F3 generation, p, p＇-DDE decreased the sperm numbers and motility from （68.89 ± 41.37） × 10^6/mL and （68.56 ± 10.67）% to （21.66± 4.83） × 10^6/mL and （29.33 ± 32.70）%, respectively. As

关 键 词：IGF2 甲基化 p p'-DDE 雄性生殖 

分 类 号：R114[医药卫生—卫生毒理学]