下丘脑Polycomb蛋白关键基因甲基化在孕期双酚A暴露致子代雌鼠青春期发育提前中的作用

The role of hypothalamus polycomb gene methylation in bisphenol A exposure during pregnancy and premature puberty in female offspring

目的探讨下丘脑Polycomb蛋白组(PcG)关键基因Eed和Ezh甲基化在孕期双酚A(BPA)暴露致子代雌鼠青春期发育提前的中作用。方法采用随机数字表法将40只受孕CD-1小鼠平均分为对照组(玉米油)和低、中、高剂量暴露组(BPA染毒剂量分别为8、40、200mg/kg),于受孕1~18d每天进行BPA灌胃染毒。子代雌鼠出生后21~33d观察阴道开口情况;出生后34d处死,收集下丘脑组织,检测下丘脑Eed和Ezh基因的甲基化水平。采用阴道开口提前率、阴道开口时间、第一次动情时间和阴道开口提前天数评价仔鼠青春期发育提前情况。以阴道开口提前天数为因变量,BPA暴露为自变量,通过路径模型分析Eed和Ezh基因甲基化在BPA暴露致子代雌鼠青春期提前中的作用。结果孕期低、中、高剂量BPA暴露组子代雌鼠出生28d的阴道开口率[分别为40.00%(29/72)、47.62%(25/53)、37.84%(20/53)]均高于对照组[14.06%(9/64)];孕期低、中、高剂量BPA暴露组子代雌鼠阴道开口时间P50(P25,P75)分别为28(26,30)、28(26,29)和28(26,30)d,第一次动情时间分别为31(27,32)、30(27,31)和31(28,33)d,均早于对照组的阴道开口时间[30(28,31)d]和第一次动情时间[32(30,33)d](P值均<0.05);孕期低、中、高剂量BPA暴露组子代雌鼠Eed1(1.61%~1.82%)、Eed2(1.36%~1.43%)和Ezh2(2.87%~3.05%)甲基化水平均高于对照组(分别为1.47%、1.26%、2.56%)(P值均<0.05)。路径模型分析结果显示,BPA暴露对子代雌鼠青春期发育提前无直接作用,经Eed2和Ezh2甲基化的间接作用路径系数分别为0.045和0.142。结论孕期母体BPA暴露会通过下丘脑PcG蛋白关键基因Eed和Ezh甲基化导致子代雌鼠青春期发育提前。

Objective To explore the role of hypothalamus Polycomb Group (PcG) gene (Eed, Ezh) methylation in the relationship between bisphenol A (BPA) exposure during pregnancy and premature puberty in female offspring.Methods A total of 40 pregnant CD-1 mice were randomly and averagely assigned into four groups: control group (corn oil) and low, middle and high BPA-exposed groups (the poisonous doses were 8 mg/kg, 40 mg/kg, and 200 mg/kg, respectively) by random number table method. Each group was administered by gavage from gestational day (GD) 1 to 18. The vaginal opening of female offspring was observed from postnatal day (PND) 21 to 33. All female offsprings were sacrificed, and hypothalamus was remained on the PND 34. The methylation levels of Eed and Ezh in the hypothalamus were measured. The early puberty of CD-1 mice was evaluated by the rate of vaginal opening in advance, initial time of vaginal opening, the first estrus occurrence and vaginal opening days in advance. The path model was used to explore the role of Eed and Ezh gene methylation in the early puberty of female offspring with maternal BPA exposed including the number of days of vaginal opening in advance as a dependent variable and BPA exposure as an independent variable.Results The rate of vaginal opening on the 28 day in each maternal BPA-exposure group [low, middle and high BPA-exposed groups were 40.00%(29/72), 47.62%(25/53) and 37.84%(20/53), respectively] was higher than that rate in the control group [14.06%(9/64)]. Similarly, the P50(P25, P75) values of initial time of vaginal opening in low, middle and high BPA-exposed group were 28 (26, 30), 28 (26, 29), 28 (26, 30) days, respectively and the P50(P25, P75) values of the first estrus occurrence in low, middle and high BPA-exposed group were 31 (27, 32), 30 (27, 31), 31 (28, 33) days, respectively, which were earlier than those in the control group [initial time of vaginal opening was 30(28, 31) days, and the first estrus occurrence was 32(30, 33) days](all P values<0.05). Compared with the control group (the methylation levels of Eed1, Eed2, Ezh2 were 1.47%, 1.26%, 2.56%, respectively), the methylation levels of Eed1 (1.61%-1.82%), Eed2 (1.36%-1.43%) and Ezh2 (2.87%-3.05%) in female offspring were significantly higher in BPA-exposed groups (all P values<0.05). The results of path model analysis showed that BPA had no direct influence on puberty in advance, but had an indirect effect on puberty in advance (indirect effect path coefficient was 0.045 and 0.142, respectively) by mediating methylation of Eed2, and Ezh2.Conclusion Early puberty in female offspring induced by maternal exposure to BPA during pregnancy through the increased methylation levels of hypothalamus PcG gene (Eed, Ezh) in female offspring.