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[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 intrauterine p, p’-DDE exposure inter-generational rat model and observe whether progeny reproductive toxicity of male progenies has genetic characteristics and possible epigenetic mechanisms. [Methods] SD rats of SPF grade were given daily 100 mg p / d of body weight per day for 8-15 days and p’-DDE was given intragastrically. Another solvent control group was fed with corn oil and pregnant rats were given free delivery. All the male Offspring (F1 generation), the same group of different brood F1 generation offspring 90 days after birth by male and female ratio of 1: 1 mating, generation F2 offspring. The male and female F2 generation of the infected group and the control group were designed by the following grouping to produce F3 generation offspring, which were grouped as follows: (1) Both male and female were control group (C♂-C♀), (2) both male and female were dyed (DDE ♂-DDE ♀), (3) DDE ♂-C♀ in male and control groups, and (4) male and female C♂-DDE♀ in control group. The adult sperm motility of 3 generations of male offspring was analyzed by computer assisted sperm analysis system (CASA), methylation level of Igf2 DMR2 region was detected by sodium bisulfite sequencing, and imprinted gene expression was analyzed by real-time fluorescence quantitative PCR. [Results] The numbers and viability of DDE♂-DDE♀ and DDE♂-C♀ in F1, F2 and F3 were decreased with p and p’-DDE. The number and viability of sperm in F1 were decreased from (70.63 ± 11.79) × 106 / (82.86 ± 38.60) × 106 / m L, (82.14 ± 8.66) × 106 / m L and (62.42 ± 9.40)% respectively when compared with the control group (68.89 ± 41.37) × 106 / m L, (68.56 ± 10.66) × 106 / m L and (60.88 ± 25.03)% respectively; (28.00 ± 16.60) × 106 / m L and (34.60) × 106 / m L and (29.33 ± 32.70)%, respectively. The results showed that the number and viability of DDE♂-C♀ in the F3 generation decreased to (21.66 ± 4.83) ± 31.60)%, the differences were statistically significant (all P <0.05). The hypomethylation, Igf2 transcription and H19 transcription were upregulated in Igf2 DMR2 region (1, 16 and 18 sites) in F1 ~ F3 male offspring mice. The Igf2 gene transcription level was positively correlated with Igf2 DMR2 methylation (r = 0.806 5). [Conclusion] The methylation of Igf2 DMR2 may play an important role in reproductive toxicity of transplanted male rats induced by p, p’-DDE.