代表性内分泌干扰物致小鼠性别发育和性别稳定异常的作用机制

发布时间：2018-06-15 23:17

本文选题：内分泌干扰物 + 性别发育　；参考：《大连理工大学》2015年博士论文

【摘要】：内分泌干扰物(Endocrine disrupting chemicals, EDCs)广泛存在于空气、土壤、水体等环境介质中,能够干扰动物性别发育过程及性别稳定维持从而造成生物种群的性别比例失衡、生物个体性早熟、性反转等性别发育异常现象。最近研究发现在哺乳动物的性别发育过程中,除性染色体上的关键基因,位于常染色体上一系列转录因子和信号分子的调控对双潜能性腺的分化具有关键作用,性成熟后诸多性别决定基因也参与维系性腺的正常功能和性别稳定过程。因此,基于常染色体上的性别决定基因研究EDCs对哺乳动物性别发育过程和性别稳定维持的基因调控通路的影响,对阐明其生殖和发育毒性机理具有重要意义。本论文以抗雌激素效应化合物他莫昔芬(Tamoxifen, TAM)为模型化学物,研究其对小鼠性别发育和性别稳定维持过程常染色体上关键调控基因的影响,并基于这一研究结果进一步选择两种典型环境内分泌干扰物—双酚A(Bisphenol A, BPA)和三丁基锡(Tributyltin, TBT),探讨其干扰小鼠性别发育过程的分子调控机理。本论文的主要研究内容和结果如下：1.胚胎期暴露TAM对小鼠性别决定基因的影响。ICR孕鼠在妊娠第10.5天(days post-coitum, dpc)腹腔注射1 mg/kg-bw TAM,13.5 dpc取胎鼠生殖嵴,采用实时反转录多聚酶链式反应(Realtime reverse transcription polymerase chain reaction, Realtime RT-PCR)、整胚原位杂交对性别决定基因表达进行检测。结果发现性别决定关键期暴露TAM能够诱导雌性生殖嵴中血小板源生长因子受体-a(Platelet-derived growth factor receptor-a, Pdgfra)过表达且表达模式与雄性生殖嵴类似,表明TAM能够诱导睾丸特异性体腔血管生成。同时TAM显著上调小鼠雌性生殖嵴中睾丸特异性转录因子Sry相关基因9(SRY-related HMG box gene 9, Sox9)表达,但下调卵巢决定关键因子—叉头状转录因子基因2(Forkhead/winged helix transcription factor gene 2, Foxl2)表达。SOX9和FOXL2蛋白表达水平的变化和基因表达变化一致。小鼠胚胎生殖嵴体外培养实验进一步证实TAM能够显著上调雌性生殖嵴中Pdgfra和成纤维细胞生长因子9(Fibroblast growth factor-9, Fgf9)表达,并降低雄性生殖嵴中Fgf9的表达。雌二醇和睾酮的比值(E2/T)在TAM暴露组中也显著降低。结果提示TAM抑制小鼠雌性生殖嵴常染色体上雌性特异性关键基因表达,激活雄性决定基因的表达,干扰性别发育过程,从而异常起始雌性生殖嵴中睾丸发育调控通路。2.性成熟期TAM暴露对雌性小鼠性别稳定调控通路关键基因的影响。向性成熟的ICR雌性小鼠,连续7d分别腹腔注射75或225 mg/kg-bw TAM。暴露结束5d后,收集小鼠血清和卵巢,采用Realtime RT-PCR、免疫组织化学、蛋白免疫印迹(Western blot)对性别决定基因和蛋白的表达和分布进行检测。结果发现TAM上调了卵巢中睾丸索成分血小板内皮细胞粘附分子-1 (Platelet/endothelial cell adhesion molecule 1, PECAM-1)表达,表明TAM诱导卵巢产生睾丸特异性血管内皮细胞。此外,TAM还能够上调Sox9和Fgf9基因表达；抑制Foxl2.无翼乳房肿瘤病毒相关整合位点4(Wingless-related MMTV integration site 4, Wnt4)和其下游基因卵泡抑制素(Follistatin, Fst)的表达。TAM暴露导致激素水平紊乱,雌二醇、孕酮、促卵泡生成素和黄体生成素水平降低,而睾酮含量增加。结果表明性成熟期TAM暴露可以抑制卵巢维持因子Foxtt和Wnt4及其下游基因的表达,而激活睾丸决定因子Sox9和Fgf9,启动Sox9和Fgf9之间的反馈调节通路,从而干扰性成熟雌性小鼠卵巢功能维持和性别稳定。3.胚胎期和哺乳期暴露BPA对雄性仔鼠性别发育调控通路关键基因的影响。ICR小鼠孕鼠从0.5 dpc直至出生后21d(Postnatal day, PND21)连续暴露0.1 mg/kg-bw BPA。取PND0、7、28、49雄性仔鼠,收集血清及睾丸组织。采用Realtime RT-PCR、Western blot和免疫组织化学等方法对参与性别发育过程的关键基因和蛋白进行检测。结果发现,BPA未显著影响雄性仔鼠性成熟期FGF9及SOX9蛋白的表达,但抑制出生早期和性成熟期抗缪勒氏管激素(Anti-Mullerian hormone, Amh)、Sox9和Fgf9表达。同时BPA暴露下调了雄性仔鼠各时期锌指转录因子3ATA结合蛋白4(Zinc finger transcription factor GATA-binding protein 4, Gata4)的表达,使其在青春期未正常表达上调。此外,BPA诱导Foxl2、Wnt4、Fst和X染色体DSS-AHC决定区基因1(DSS-AHC critical region on the X chromosome gene 1, Dea1)表达上调。结果提示发育早期BPA暴露可抑制雄性仔鼠睾丸调控通路,激活卵巢调控通路,揭示了BPA诱导雄性动物雌性化的潜在机制。4.胚胎期和哺乳期暴露TBT对雄性仔鼠性别发育调控通路关键基因的影响。ICR小鼠孕鼠从0.5 dpc直至PND21连续暴露20μg/kg-bw TBT。仔鼠出生后,在PND0、7、28、49取雄性小鼠血清及睾丸组织。采用Realtime RT-PCR、Western blot和免疫组织化学等方法对参与性别发育的关键基因和蛋白进行检测。结果发现胚胎期暴露TBT能够增加成年雄性仔鼠血清中E2/T,而对雌性仔鼠血清中E2/T无影响。TBT显著抑制PND0时Sox9、Fgf9和Amh基因表达水平,随时间延长无显著影响。TBT改变了生精细胞与精子形成相关基因Gata4的表达趋势,导致其表达异常升高并诱导其过早表达。TBT暴露未显著改变雄性仔鼠睾丸中卵巢发育特异性基因Foxl2等表达水平和时间规律。结果表明胚胎期和哺乳期暴露TBT干扰子代雄性性别发育过程,至成年期性激素分泌仍呈紊乱状态。本论文研究了EDCs中三种代表性化学物对小鼠常染色体上性别决定基因的影响,发现基因调控通路的紊乱和其造成的性别发育和性别稳定异常一致,揭示了EDCs新的生殖发育毒性作用的潜在机制。该结果有助于阐明环境内分泌干扰物暴露与哺乳动物及人类的性别比例异常和生殖障碍之间的关联,同时有助于揭示发育早期接触EDCs对机体功能发育及后期健康的长期影响机制,为EDCs的健康风险评价提供科学依据。
[Abstract]:Endocrine disrupting interferons (Endocrine disrupting chemicals, EDCs) are widely existed in air, soil, water and other environmental mediums, which can interfere with the sex development of animals and maintain sex stability, resulting in the gender imbalance of biological population, individual precocious precocity, sex reversal and other sex dysplasia. Recent studies have found that breastfeeding In the process of sex development in animals, the key genes on the sex chromosome, the regulation of a series of transcription factors and signal molecules on the autosomes play a key role in the differentiation of the dual potential gonads. After sexual maturation, many sex determination genes are also involved in the normal function and gender stability of the gonads. Therefore, the autosomal based autosomes are on the autosomes. The effect of EDCs on the sex development process and the gene regulation pathway of gender stability in mammals is of great significance to elucidate the mechanism of reproductive and developmental toxicity. This paper uses the anti estrogen effect compound tamoxifen (Tamoxifen, TAM) as a model chemical, and studies its sex development and sex in mice. The key regulatory genes on the autosomes in the stable maintenance process, and based on the results of this study, we further selected two typical environmental endocrine disruptors - bisphenol A (Bisphenol A, BPA) and three butyl tin (Tributyltin, TBT) to discuss the molecular regulation mechanism that interfered with the sex development of mice. The results are as follows: 1. the effects of exposure to TAM on the sex determination genes of mice in the embryo period.ICR mice were intraperitoneally injected with 1 mg/kg-bw TAM (days post-coitum, DPC) on day 10.5 of pregnancy, and the reproductive crista of fetal mice was taken by 13.5 dpc, and the real time reverse transcription polymerase chain reaction (Realtime reverse transcription polymerase) was used as a whole embryo. The expression of sex determined gene was detected by in situ hybridization. The results showed that exposure to TAM in the critical stage of sex determination could induce the overexpression of -a (Platelet-derived growth factor receptor-A, Pdgfra) in female reproductive crista, and the expression pattern was similar to that of male reproductive crista, indicating that TAM can induce testicular specific body cavity. At the same time, TAM significantly up-regulated the expression of Sry related gene 9 (SRY-related HMG box gene 9, Sox9) in the female reproductive crista of mice, but down regulated the key factor of the ovarian decision - the fork head transcription factor gene 2 (Forkhead/winged helix transcription factor gene 2) and the expression of water In vitro culture experiment of mouse embryo reproductive crista further confirmed that TAM could significantly increase the expression of Pdgfra and fibroblast growth factor 9 (Fibroblast growth factor-9, Fgf9) in female reproductive crista, and reduce the expression of Fgf9 in the male reproductive crista. The ratio of estradiol and testosterone (E2/T) in TAM exposure The results suggest that TAM inhibits the expression of key female specific key genes on the autosomes of female reproductive crista, activates the expression of the male determining genes and interferes with the process of sex development, so that the abnormal initiation of the testicular development regulation pathway in the female reproductive crista in the female reproductive crista is to regulate the sexual stability of female mice by TAM exposure in the.2. sexual maturity stage. The effect of bond gene. The expression and distribution of sex determined genes and proteins were detected by Realtime RT-PCR, immunohistochemical staining and protein immunoblotting (Western blot) in ICR female mice of mature ICR, after continuous 7d intraperitoneal injection of 75 or 225 mg/kg-bw TAM. exposures to 5D, and the expression and distribution of sex determined genes and proteins were detected. The expression of the platelet endothelial cell adhesion molecule -1 (Platelet/endothelial cell adhesion molecule 1, PECAM-1) in the ovary of the ovary showed that TAM induced the ovary to produce testicular specific vascular endothelial cells. In addition, TAM could also up regulate the expression of Sox9 and Fgf9 genes, and inhibit the Foxl2. pterygoid tumor virus related integration site 4 (Wingl). Ess-related MMTV integration site 4, Wnt4) and its downstream gene follicle inhibin (Follistatin, Fst) expression.TAM exposure leads to hormone levels disorder, estradiol, progesterone, follicle stimulating hormone and luteinizing hormone levels decrease, and testosterone levels are increased. The results show that TAM exposure at sexual maturity can inhibit ovarian maintenance factor Foxtt and Wnt. The expression of 4 and its downstream genes, while activating the testicular determinants Sox9 and Fgf9, and starting the feedback regulation pathway between Sox9 and Fgf9, thus interfering with the ovarian function maintenance of the mature female mice and the gender stable.3. embryo and the lactating stage, the effect of BPA on the key genes of sex development of male offspring in male mice.ICR mice from 0.5 DPC. 21d (Postnatal day, PND21) was continuously exposed to 0.1 mg/kg-bw BPA. to collect PND0,7,28,49 male offspring and collect serum and testicular tissues. The key genes and proteins involved in sex development process were detected by Realtime RT-PCR, Western blot and immunohistochemistry. The results showed that BPA did not significantly affect male offspring sex. The expression of FGF9 and SOX9 protein at mature stage, but inhibit the expression of Anti-Mullerian hormone, Amh, Sox9 and Fgf9 in the early stage of birth and sexual maturity, while BPA exposure down regulated the expression of 3ATA binding protein 4 of the zinc finger transcription factor of the male offspring (Zinc finger transcription). In addition, BPA induced Foxl2, Wnt4, Fst and X chromosome DSS-AHC determinant gene 1 (DSS-AHC critical region on the X) expression up-regulated. The results suggest that early developmental exposure inhibits the regulation of testicular regulation in male offspring, activates the ovarian regulation pathway, and reveals the induced male movement. The potential mechanism of physical feminization.4. embryo and lactation stage exposure TBT effect on the key genes of sex development control pathway in male offspring mouse.ICR mice pregnant mice from 0.5 DPC until PND21 continuous exposure to 20 g/kg-bw TBT. offspring were born in PND0,7,28,49 male mice serum and testicular tissue. Realtime RT-PCR, Western blot and immune group were used. The detection of key genes and proteins involved in sex development was detected by weave chemistry. The results showed that exposure to TBT in embryo stage could increase the E2/T in the serum of adult male offspring, but there was no effect of.TBT on the expression level of Sox9, Fgf9 and Amh in the serum of female offspring, and no significant influence of.TBT on the E2/T. The expression trend of cell and sperm formation related gene Gata4, resulting in abnormal expression of its expression and induction of premature expression of.TBT exposure did not significantly change the expression level and time regularity of the ovarian developmental specific gene Foxl2 in the testicles of male offspring. The results showed that the embryo and lactation period exposed TBT interfering the male sex development process to adulthood. The effect of three representative chemicals on the sex determination genes on the autosomes of EDCs was studied in this paper. It was found that the disorder of the gene regulation pathway was unusually consistent with the sex development and gender stability, which revealed the potential mechanism of EDCs's new reproductive toxicity. It helps to elucidate the relationship between environmental endocrine disruptors exposure and the sexual abnormality of sex and reproductive disorders in mammals and humans, and helps to reveal the long-term mechanism of the impact of early exposure to EDCs on the functional development and later health of the body, and provides a scientific basis for the assessment of the health risk of EDCs.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R114

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