邻苯二甲酸二(2-乙基己基)酯及其代谢产物MEHP对体外大鼠卵泡发育的影响

目的利用大鼠腔前卵泡体外培养方法研究邻苯二甲酸二(2-乙基己基)酯(DEHP)及其代谢产物邻苯二甲酸单(2-乙基己基)酯(MEHP)对卵泡体外发育的影响。方法机械性分离大鼠腔前卵泡,单个卵泡在96孔板中连续培养。MEHP设2.5、5、10、20、40和80μg/ml6个浓度,DEHP设33.7、67.5、125、250、500、1000nmol/L6个浓度,同时设二甲基亚砜(DMSO)溶剂对照和阴性对照。每组16～20个卵泡,3次重复。隔天换1/2液,在培养第10天诱导排卵,观察卵泡体外发育情况。结果DEHP对第11天卵泡存活率、有腔形成率、异常卵泡形成率和卵丘-卵母细胞复合体(COCs)排出率无明显的影响,与溶剂对照组差异无显著性(P0.05)。第2天暴露10μg/ml以上剂量的MEHP48h后,第11天卵泡存活率明显下降(54.76%±3.37%),与对照组(91.57%±1.32%)比较差异有显著性(P0.05),剂量与卵泡存活率之间存在一定相关性(R2=0.92);COCs排出(24.99%±3.95%)明显减少,与对照组(52.78%±8.45%)比较差异有显著性(P0.05);异常卵泡出现率(45.24%±3.37%)明显升高,与对照组比较差异有显著性(P0.05);暴露20μg/mlMEHP有腔形成率(46.18%±1.67%)减少,与对照组(85.91%±5.03%)比较差异有显著性(P0.05),且有明显剂量-反应关系。结论MEHP可以抑制体外大鼠腔前卵泡的生长发育。

Effect of DEHP and its metabolite MEHP on in vitro rat follicular development

Objective To use rat preantral follicle culture as a novel bioassay in order to study the effects of di2-ethylhexyl phttmlate(DEHP) and its metabolite mono-2-ethylhexyl phthalate(MEHP) on folliculogenesis in vitro.Methods The preantral follicles were mechanically dissected from ovaries and equally randomized to to 96well plates.Six dose groups were setup to be acute exposed in 2.5、5,10,20,40 and 80μg /ml MEHP concentration,33.7,67.5,125,250,500 and 1000nmol /L DEHP concentration on day 2,while another control group used solvent only,each group had 16 to 20 follicles.After subsequently cultured individually for 10 days,the follicles were induced to ovulate for 20h,follicle development and oocyte maturation were observed.Results Day11 follicle survival rate(54.76% ± 3.37%) had significantly dropped after exposure to > 10μg /ml MEHP concentration,comparing to the control group(91.57% ± 1.32%),the difference is statistical significant(P < 0.05).There is correlation between dosage and follicle survival rate,with correlation coefficient R2 = 0.92.Abnormal follicle rate(45.24% ± 3.37%) is remarkably higher,and the difference is statistically significant(P < 0.05) comparing to the control group.At the concentrations of > 20μg /ml MEHP,follicle growth and differentiation were dependently impaired:antral follicles(46.18% ± 1.67%) had significantly reduced(P < 0.05) comparing to the control group(85.91% ± 5.03%).DEHP showed no adverse effects upon follicle development,there were no significant defference between DMSO and control.Conclusion At the dose of > 10μg /ml,MEHP could inhibit the in-vitro rat follicle development,DEHP seemed no adverse effects upon follicle development.