环境雌激素双酚A暴露干扰仔鼠卵巢发育的研究  

Research of Bisphenol A Exposure Interfering Ovarian Development in Offspring Mice(Mus musculus)

作　　者：张石磊 包佳鹭 史万玉[2] 钟秀会[1,2] ZHANG Shi-Lei;BAO Jia-Lu;SHI Wan-Yu;ZHONG Xiu-Hui(College of Animal Science and Technology,Hebei Agricultural University,Baoding 071001,China;College of Traditional Chinese Veterinary Medicine,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]河北农业大学动物科技学院,保定071001 [2]河北农业大学中兽医学院,保定071001

出　　处：《农业生物技术学报》2020年第1期108-118,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31872506)

摘　　要：双酚A(bisphenol A,BPA)是一种无处不在的环境雌激素。为探究小鼠(Mus musculus)孕期和哺乳期连续暴露BPA对子代卵巢发育的影响,。本研究将8周龄孕0 d母鼠随机分为7组,每组20只,分别通过饮水给予蒸馏水和0.05、0.5、5、10、20和50 mg/kg BPA。从母鼠孕0 d开始直到仔鼠21 d断奶,仔鼠并未直接接触BPA,而是通过孕期胎盘传递和哺乳期乳汁传递间接接触BPA,小鼠均于孕20 d分娩,故仔鼠间接接触BPA时间为41 d。结果显示,母鼠染毒BPA剂量≥20 mg/kg时,仔鼠血清和卵巢中BPA含量显著增加(P<0.05)。器官指数测定结果显示,染毒BPA剂量≥10 mg/kg可使仔鼠卵巢指数显著增大(P<0.05)。苏木精-伊红染色(hematoxylin-eosin staining,HE)病理组织切片的观察结果则证明,极低剂量BPA(≥0.05 mg/kg)即可使仔鼠卵巢产生损伤,卵泡颗粒细胞层出现破损。细胞凋亡原位末端转移酶标记技术(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay,TUNEL)实验结果显示,母鼠染毒BPA≥20 mg/kg时卵巢颗粒细胞发生细胞凋亡(P<0.05)。免疫组化结果显示,母鼠染毒BPA剂量≥20 mg/kg时仔鼠卵巢雌激素受体α(estrogen receptorα,ERα)表达量显著增多(P<0.05);而母鼠染毒BPA剂量为50 mg/kg时其子代雌鼠卵巢ERβ表达量显著升高。转录组测序结果显示,母鼠染毒BPA后仔鼠卵巢相较于空白对照组核糖体通路发生变化,其中核糖体蛋白L3(ribosomal protein L3,Rpl3)、Rpl21、Rpsa基因表达显著下调、Rps2、Rps28、Rpl26、Rpl32和Rpl10a基因表达显著上调。这些基因分别与胚胎生殖系统发育受阻、细胞凋亡和卵巢早熟等相关。荧光定量PCR验证了转录组结果。本研究证实了BPA的生殖毒性,在基因层面揭示了核糖体功能蛋白合成异常与BPA的生殖毒性有关,且低剂量BPA抑制核糖体蛋白表达而高剂量BPA促进核糖体蛋白表达,证明了BPA在扰乱卵巢发育的两面性,即低剂量抑制�Bisphenol A(BPA)is a ubiquitous environmental endocrine disruptor.This study was conducted to investigate the effects of BPA exposure to the dams during gestation and lactation on ovarian development in offspring.In this experiment,8-week-old female mice(Mus musculus)at pregnant 0 d were randomly divided into 7 groups with 20 each group,and distilled water and 0.05,0.5,5,10,20,50 mg/kg BPA were given through drinking water from 0 d pregnant to 21 d weaned.In this test,the offspring mice did not directly contact BPA,but indirectly contacted BPA through placental transmission and lactation during breastfeeding.And the mice gave birth at 20 d of pregnancy,so the indirect contact time of BPA was 41 d.The period of exposure was from gestation day 0 until the weaning of the F1 mice for 41 d.The results showed that the BPA content in the serum and ovary of the offspring mice was significantly increased when the dose of BPA≥20 mg/kg(P<0.05).Organ index showed that the dose of BPA≥10 mg/kg resulted in a significant increase in the ovarian index of the pups(P<0.05).The observation of hematoxylin-eosin staining(HE)pathological tissue sections showed that low doses of BPA(≥0.05 mg/kg)could damage the ovaries of the offspring and cause irregular architecture in follicular granule cell layers.And the pathological changes of the ovary also increase as the dose increases.The results of terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling assay(TUNEL)showed that the ovarian granulosa cells were apoptotic when the BPA exposure was at or over 20 mg/kg(P<0.05).The expression of estrogen receptor α(ERα)in the ovary significantly increased when BPA exposed at no less than 20 mg/kg(P<0.05).And the dose of BPA of 50 mg/kg had significant affect on ER β expression in ovarian progeny 21-day-old mice.The results of transcriptome sequencing showed that there were 81 significant differentially expressed genes in the ovary of the offspring compared with the control group,38 of which were up-regulated and 43 gen

关 键 词：双酚A 卵巢发育 原位末端转移酶标记技术(TUNEL) 转录组测序 核糖体 

分 类 号：S814[农业科学—畜牧学]