【作者】 刘玉；

【导师】 齐忠权；

【作者基本信息】 厦门大学 ， 生理学， 2017， 博士

【摘要】 生殖和发育功能障碍是当前严重影响人体健康的主要公共卫生问题之一。外源因素如疾病、应激、化学物质作用于雌性生殖系统可以导致下丘脑-垂体-性腺轴功能紊乱,卵巢周期改变,致使人群中自发性流产率、子代发育异常、生育力下降等显著增加。近年来,环境雌激素(Environmental estrogens,EEs)的暴露被认为是除了衰老、糖尿病和吸烟之外,影响女性生殖功能的最大风险因素。大量研究报道了 EEs进入体内后模拟雌激素作用,干扰内分泌系统,打破激素平衡,破坏卵巢与子宫正常的生理活动,最终造成不良的生殖结果。然而,作为雌性生殖过程中最基本的环节,EEs是否会干扰到卵母细胞的减数分裂过程以及相关机制还没有报道。本文以小鼠为模型,选择了两个具有代表性的EEs:甲氧滴滴涕和代锌锰森,探讨它们对卵母细胞的毒理学作用,并试图寻找相应的应对策略。甲氧滴滴涕是一种有机氯杀虫剂,在20世纪70年代滴滴涕被禁止使用后,作为其替代品被广泛地应用于世界各地的病虫害防治领域。甲氧滴滴涕具有高雌激素效应,通过抑制甾体激素合成途径中酶的活性使雄性激素合成受阻。在小鼠实验中,已经有报道甲氧滴滴涕暴露会引起不规则的动情周期,降低孕酮水平并影响后代发育。在本研究中,我们通过卵母细胞体外成熟(In vitro maturation,IVM)模型,将小鼠卵母细胞置于含有甲氧滴滴涕的培养基中,考察其对小鼠卵母细胞的毒性作用,我们发现甲氧滴滴涕能够降低小鼠卵母细胞的成熟能力,抑制第一极体的排放。免疫荧光的结果显示,在甲氧滴滴涕条件下培养的小鼠卵母细胞,纺锤体组装和染色体的中板排列出现了严重的异常,微管组织中心蛋白从两极处掉落,纺锤体组装检验点蛋白BubR1和Mad2在中后期转换中保持激活状态。此外,甲氧滴滴涕会影响小鼠卵母细胞内活性氧(Reactive oxygen species,ROS)的水平,作为ROS标记的探针二氯荧光素-乙酰乙酸酯在甲氧滴滴涕条件下荧光强度明显上升;此外,我们观察到以ROS为中介的细胞毒性作用在甲氧滴滴涕处理的小鼠卵母细胞中均有发生,表现为DNA双链断裂和脂质过氧化的发生。最后,我们还观察到在甲氧滴滴涕条件下培养的小鼠卵母细胞内,线粒体的分布会发生改变。总之,这些数据表明甲氧滴滴涕会降低小鼠卵母细胞的发育潜能,这种细胞毒性作用是通过其打破小鼠卵母细胞中的氧化应激平衡而实现的。代森锰锌是一种锰/锌络合的亚乙基二硫代氨基甲酸盐,是农业中应用最广泛使用的真菌抑制剂之一。代森锰锌在真菌内作用的靶标是线粒体,其能够干扰ATP的合成,和呼吸链中氨基酸中的巯基交联,引起代谢紊乱和凋亡发生,但是代森锰锌的这种毒性作用不具有物种选择性,其富集于农作物进入其他生物体后常常会诱发生殖毒性。以前的研究表明,代森锰锌暴露于雌性个体将导致不规律的发情周期,孕酮水平降低,同窝幼崽数量减少并出现死胎的现象。在此次研究中,我们试图用具有雌激素和抗凋亡效应的天然产物白藜芦醇缓解代森锰锌所引起的雌性小鼠生殖毒性。我们将小鼠分成四组,分别为:阴性对照组、阳性对照(代森锰锌)组,低浓度白藜芦醇组(100 mg/L)和高浓度白藜芦醇组(200 mg/L)。四周后,我们通过后代仔鼠、母鼠卵巢和卵母细胞三个水平来评价各组雌鼠的生殖情况。我们发现白藜芦醇可以减轻代森锰锌的不利影响,如所生小鼠数量和质量下降,卵巢重量降低和成熟卵泡数量减少。此外,代森锰锌组小鼠的卵母细胞在体外培养时,发育到原核和二细胞的比例明显下降,并且微丝系统出现异常,这也在白藜芦醇缓解组中得到改善。我们进一步确认了代锌锰森可以从细胞凋亡和表观遗传修饰两个方面影响小鼠卵母细胞,表现为Annexin-V阳性细胞比例增加,ROS水平上升,线粒体分布异常并发生去极化,H3K9和H3K27相应位点甲基化程度降低,而白藜芦醇在这些方面对代森锰锌所造成的细胞毒性均具有缓解作用。更多还原

【Abstract】 Reproductive and developmental dysfunction is one of the major public health problems that seriously affect human health.Exogenous factors such as disease,stress and chemical substances interfere hypothalamus-pituitary-gonadal axis,leading altered ovarian cycle,decreased fertility,offspring dysplasia and spontaneous abortion.In recent years,exposure to environmental estrogens(EEs)is considered as a great risk factor affecting women’s reproductive function,in addition to aging,diabetes and smoking.A large number of studies have reported that EEs interfere with the endocrine system,break the hormone balance,destroy ovarian and uterine normal physiological activities,and ultimately cause adverse reproductive results.However,as a basic part of the female reproductive process,whether EEs would disturb oocyte meiotic maturation and the related mechanisms have not been reported.In this paper,we selected methoxychlor and mancozeb as representative to study their effects on oocytes,also with an attempt to find the corresponding protective methods.Methoxychlor(MXC)is an organochlorine pesticide that has been widely usedas a substitute for pesticide control in the world since the ban of DDT occurred in the 1970s.Previous studies linked MXC exposure with hyperestrogenism and other reproductive disorders,which reflect its ability to suppress the activity of steroidogenic enzymes.In female mice,in utero developmental exposure to MXC through their mother led to irregular estrous cycles,decreased progesterone levels,reduced litter sizes,and more frequent delivery of dead fetuses.In this study,we used in vitro maturation(IVM)model to investigate the effect of MXC on oocyte meiosis.Our results showed that maturation rates of MXC-treated oocytes were lower than those of controls,which was due to abnormal spindle morphologies,dysfunctioned microtubule organized centers(MTOCs)and activated spindle assembly checkpoints BubRl and Mad2.In addition,MXC increased the level of reactive oxygen species(ROS)in mouse oocytes,with the evidence of increased fluorescence intensity of ROS-labeled probe 2’,7’-dichlorofluorescin diacetate.We also observed the ROS-mediated cytotoxicity occurs in MXC-treated mouse oocytes,such as DNA double-strand breaks and lipid peroxidation.Finally,we found the distribution of mitochondria was changed in mouse oocytes under MXC conditions.In conclusion,these data suggested that MXC reduces the developmental potential of mouse oocytes,and this was achieved by impairing cellular ROS metabolism.Mancozeb,a mixture of ethylene-bis-dithiocarbamate manganese and zinc salts,is one of the most widely used fungicides in agriculture.Mancozeb could lead to mitochondria dysfunction,cellular anti-oxidation enzymes depletion and apoptotic pathways activation.Previous studies indicated the exposure of mancozeb through mother would lead to irregular estrous cycles,decreased progesterone levels,reduced litter sizes,and more frequent delivery of dead fetuses.In this study,we attempt to relieve mancozeb-induced female reproductive toxicity with resveratrol,a natural product with estrogen and anti-apoptotic effects.We divided the mice into four groups:negative control group,positive control(mancozeb)group,low concentration of resveratrol(100 mg/L)group and high concentration of resveratrol(200 mg/L)group.After 4 weeks,we evaluated the reproductive behivors of each group with the status of offspring,ovaries and oocytes.Results showed that resveratrol could alleviate mancozeb’s adverse effects,such as declined fertility,decreased ovary weight and primary follicles.Besides,mancozeb treated oocytes displayed suboptimal developmental competence and this can also be improved by treatment of resveratrol.More detailed investigation of these processes revealed that mancozeb increased reactive oxygen species,causing cell apoptosis and abnormal epigenetic modifications,and resveratrol can block these cytotoxic changes.Collectively,our results showed that resveratrol can alleviate mancozeb induced infertility and this was mainly through the correction of apoptotic tendency and the abnormity of cellular epigenetic modification.更多还原