典型环境污染物对性别决定和性别稳定性的干扰作用机制研究

According to recent research, besides Sry gene on the Y chromosome and Dax1 gene on the X chromosome, some autosomal genes, such as Sox9, Fox12, and Wnt4 etc., could also play important roles in the gonadal differentiation and development as well as the stability of gender. Abnormal expression or mutation of any of these genes could be able to induce gonadal dysgenesis and gender alteration. This was the first study to hypothesize that environmental contaminants might induce gonadal dysgenesis and gender alteration, through the potential interruption in sex determination- and sex stability-related gene expression. This experimental animal-based study aims to 1) reveal the impact of environmental contaminants on the expression of sex determination-related gene(s) in the embryonic gonadal primordial germ cells and alteration in the gonadal histology; 2) find out the impact of environmental contaminants on the expression of sex stability-related gene(s) in the adult mouse testis or ovary and variation in the histology and sex hormone levels; and 3) demonstrate the environmental contaminants interference on sex determination and sex stability and their mechanisms. The expected results from this study would not only reveal the new toxicity of environmental contaminants that is helpful to further explore the action mechanism of environmental hormones and human reproductive disorders, but also provide scientific supports for protecting and improving the human reproductive health and drawing up the prevention strategies.

近年的研究发现，除了Y染色体上的Sry和X染色体上的Dax1基因之外，常染色体上的Sox9、Foxl2和Wnt4等基因，在性腺分化和发育以及性别稳定中起着非常重要的作用，并且其中某一个基因的表达异常或突变，均可以引起性腺发育异常和性别转化。本项目首次提出，环境污染物通过干扰性别决定和性别稳定性相关基因表达，引起性腺分化异常和性别转化的假说。欲通过动物实验，弄清典型环境污染物对胚胎性腺原基细胞中性别决定基因表达以及性腺组织学影响；弄清对成年小鼠睾丸或卵巢中性别稳定性相关基因的表达、组织学改变和性激素水平的影响。阐明环境污染物对性别决定和性别稳定性的干扰作用及其机理。研究结果，不仅首次发现环境污染物的新毒性，有助于进一步深入阐明环境激素的作用机制和人类生殖障碍原因，将为保障和提高人类生殖健康，建立预防对策提供科学依据。

本项目首次提出，环境污染物通过干扰性别决定和性别稳定性相关基因表达，引起性腺分化异常和性别转化的假说。研究目的是通过动物实验，弄清典型环境污染物对胚胎性腺原基细胞中性别决定基因表达以及性腺组织学影响；弄清对成年小鼠睾丸或卵巢中性别稳定性相关基因的表达、组织学改变和性激素水平的影响；阐明环境污染物对性别决定和性别稳定性的干扰作用及其机理。.. 研究选取他莫昔芬、双酚A、三丁基锡和邻苯二甲酸二(2-乙基己基)酯(DEHP)几种不同类型的代表性内分泌干扰物。采用实时定量PCR和整胚原位杂交(WISH)方法检测了性别决定调控通路关键基因表达量的变化，采用蛋白质印记(Western blot)法检测细胞标志因子和分化调控关键蛋白。结果发现性别决定关键期暴露于几种典型环境内分泌干扰物，干扰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)的精确表达时序，且影响Sry上游基因生长阻滞及DNA损伤诱导45 γ基因(Gadd45g)和锌指转录因子基因4(Gata4)的表达涉及的通路为：Gadd45g → Gata4/Fog2 → Sry → Sox9 → Fgf9。此外，胚胎期环境内分泌干扰物暴露影响卵巢决定关键因子－叉头状转录因子基因2(Forkhead/winged helix transcription factor gene 2, Foxl2) 、无翼乳房肿瘤病毒相关整合位点4 (Wnt4)、β链蛋白(β-catenin)和卵泡抑制素(Fst)的表达水平。结果提示环境内分泌干扰物通过扰乱生殖嵴中雄性决定基因和雌性特异性关键基因的表达干扰睾丸/卵巢发育调控通路，从而干扰性别发育过程。胚胎期DEHP暴露导致仔鼠出生后支持细胞分化和功能损伤。性成熟期他莫昔芬暴露可以抑制卵巢维持因子Foxl2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。.. 研究结果揭示了环境内分泌干扰物新的雄性和雌性生殖发育毒性作用的潜在机制，为环境内分泌干扰物的健康风险评价提供科学依据，结果有助于阐明环境内分泌干扰物暴露与野生哺乳动物及人类生殖障碍之间的关联。

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