【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,即低剂量抑制卵巢发育,高剂量促进卵巢发育。使用剂量的不同或许是目前关于BPA研究结果产生矛盾的原因。本研究中,子代小鼠并未直接接触BPA,但仔鼠卵巢的发育依然受到了BPA的影响,并进一步证实了BPA通过影响卵巢核糖体功能扰乱卵巢正常发育。本研究结果意味着家畜使用含BPA的塑料制品可能会影响后代卵巢发育,因此在家畜养殖中避免使用含BPA塑料制品将有助于保证后代生殖健康,提高畜家畜殖效益。更多还原

【Abstract】 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 ERβ 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 genes were down-regulated. Differentially expressed genes were enriched in transcriptional functions and ribosomal metabolic pathways, in which the ribosomal genes ribosomal protein L3(Rpl3), Rpl21 and Rpsa were significantly down-regulated, and the Rps2、Rps28、Rpl26、Rpl32 and Rpl10 a were significantly upregulated. These genes are associated with developmental arrest of the reproductive system of the embryo,apoptosis and ovarian precocity. The expression levels of Rps2, Rpl21 and Rpsa in all 7 groups of ovarian were detected by real-time PCR, which was consistent with the results of transcriptome, but the effect of BPA on Rps2 varied with doses. When BPA dose was 0.05 mg/kg to 20 mg/kg, the expression of Rps2 was lower than that of the control group, but the expression level was higher than that of the control group at the dose of 50 mg/kg. These results indicated that BPA could be indirectly transmitted to progeny mice through the placenta and milk of maternal mice. Ingestion of BPA could cause lesions in the ovaries of offspring mice, affected the development and function of the ovaries. Further studies at the genetic level revealed that abnormalities in ribosomal functional protein synthesis were associated with reproductive toxicity of BPA,and that low-dose BPA inhibited ribosomal protein expression while high-dose BPA promoted ribosomal protein expression, demonstrating the duality of BPA in ovarian development. The difference in doses used may be the reason for the current contradiction in the results of the BPA study. In this study, the offspring mice did not directly contact BPA, but the ovarian development of the pups was still affected by BPA, and it was further confirmed that BPA disrupted the normal ovarian development by affecting ovarian ribosomal function. The results of this study imply that the use of BPA-containing plastic products in pregnant livestock may affect offspring ovarian development, Which means that avoiding the use of BPA-containing plastic products in livestock breeding can help ensure the reproductive health of offspring, improve the breeding efficiency of livestock.更多还原