长期低剂量氯氰菊酯对小鼠卵巢功能的影响

Effect of long-term and low-dose cypermethrin exposure on ovarian function in mice

目的 探讨长期低剂量氯氰菊酯(CYP)暴露对小鼠卵巢功能的影响。方法 研究分为体内实验和体外实验。体内实验,将15只体重为17~19 g的3周龄ICR雌性小鼠随机分为3组,每组5只,予以0.02 mg·kg^(-1)·d^(-1)CYP的为极低剂量组,予以0.2 mg·kg^(-1)·d^(-1)CYP的为低剂量组,予以0.01%无水乙醇的为对照组,连续处理6个月。于第6个月观察小鼠动情周期,检测血清卵泡刺激素(FSH)和抗苗勒管激素(AMH)水平,分离单侧卵巢组织进行卵泡计数。体外实验,3周龄ICR雌鼠腹腔注射10 U孕马血清促性腺素(PMSG)46~48 h后获取未成熟卵丘-卵母细胞复合体(COCs),分别置于0μmol/L CYP+体外成熟(IVM)培养液(对照组)、100μmol/L CYP+IVM培养液(极低剂量组)和200μmol/L CYP+IVM培养液(低剂量组)中处理16~18 h后,计数MⅡ卵母细胞,并分析各组卵母细胞第一极体(PB1)排出率、活性氧(ROS)及线粒体膜电位情况。结果 体内实验结果显示,与对照组比较,极低剂量组和低剂量组小鼠体重、子宫系数未见明显改变,卵巢系数稍有减少,但差异无统计学意义(P>0.05);与对照组比较,低剂量组小鼠动情周期紊乱,血清FSH水平显著升高[(17.53±4.04)U/L vs.(5.67±0.52)U/L,P<0.05],AMH水平显著下降[(0.47±0.15)ng/ml vs.(1.41±0.24)ng/ml,P<0.05],窦卵泡数显著减少[(22.80±4.49)个vs.(84.00±14.98)个,P<0.05],其余各级卵泡数比较无显著性差异(P>0.05)。体外实验结果显示,与对照组比较,低剂量组卵母细胞PB1排出率和卵母细胞线粒体膜电位显著下降(P<0.05),卵母细胞内ROS水平显著升高(P<0.05)。结论 长期低剂量(0.2 mg·kg^(-1)·d^(-1))CYP暴露可通过影响卵母细胞的发育导致卵巢储备功能下降。

Objective:To explore the effects of long-term and low-dose cypermethrin(CYP)exposure on ovarian function in mice.Methods:Fifteen 17-19 g,3-week old ICR female mice were randomly assigned into three groups with 5 mice for each group:the very low-dose group,the low-dose group and the control group,and the mice were exposed to 0.02 mg·kg^(-1)·d^(-1) CYP,0.2 mg·kg^(-1)·d^(-1) CYP and 0.01%absolute ethanol respectively for six months.At the 6 th month,the estrous cycle,serum levels of FSH and anti-Mullerian hormone(AMH)in mice were observed,and unilateral ovaries were isolated for follicles counting.Moreover,the immature cumulus oocyte complexes(COCs)were extracted after intraperitoneal injection of 10 U of pregnant horse serum gonadotropin(PMSG)into 3-week old ICR female mice for 46 to 48 hours.Then,the COCs were randomly divided into three groups,the COCs in the control group,very low-dose group and low-dose group were exposed to CYP at doses of 0μmol/L,100μmol/L and 200μmol/L.After 16-18 hours,the number of MⅡoocytes was counted,and the expulsion rate of the first polar body(PB1),reactive oxygen species(ROS)and mitochondrial membrane potential of each group were analyzed.Results:The results of in vivo experiments showed that the body weight and uterine coefficient of mice in the very low dose group and the low dose group did not significantly change,while the ovarian coefficient slightly decreased,but the difference was not significant(P>0.05).Compared with the control group,the estrous cycle of mice in the low dose group was disordered,serum FSH levels[(17.53±4.04)U/L vs.(5.67±0.52)U/L,P<0.05]were significantly increased,AMH levels[(0.47±0.15)ng/ml vs.(1.41±0.24)ng/ml,P<0.05]were significantly decreased,and the number of antral follicles[(22.80±4.49)vs.(84.00±14.98),P<0.05]were significantly decreased,but there was no significant difference in number of follicles at other stages(P>0.05).The results of in vitro experiments showed that compared with the control group,the PB1 expulsion rate and mitochondrial membrane potential were significantly decreased,and the level of ROS in oocytes was significantly increased in the low-dose group(P<0.05).Conclusions:Long-term and low-dose cypermethrin(0.2 mg·kg^(-1)·d^(-1))exposure may leads to diminished ovarian reserve by affecting the development of oocyte.