Impacts of Genetic and Environmental Factors on Reproductive Organ Development

1.确定胎儿性腺中体细胞谱系的来源，并研究它们如何获得器官特异性身份 我们确定了胎儿支持细胞中维持其身份的机制。支持细胞来源的AMH和激活素B通过表达AMH和激活素B受体作为自身的自分泌和旁分泌因子。这两个因素维持支持细胞的睾丸程序，当两者都不存在时，颗粒细胞程序出现，导致部分性逆转。Amh/Inhbb双敲除睾丸的不完全性逆转（卵睾丸结构和雌性化转录组和细胞群体）进一步表明其它因子，例如其它TGF-β蛋白14、26、38 - 40，可以补偿Amh和Inhbb的损失。dKO雄性的卵睾丸结构与其他卵睾丸病例相似29，33;然而，dKO雄性中卵睾丸的形成明显不同。在大多数情况下，卵睾丸在XY小鼠胚胎中睾丸确定开始时建立，性腺的中心部分遵循睾丸程序，并且两侧极同时发育成卵巢结构32，41。 然而，在缺乏AMH和Inhbb的情况下，胎儿睾丸首先正常形成，只有当支持细胞转分化成雌性对应细胞时，卵巢结构域才出现。最有趣的是，与出生前消失的大多数卵睾丸病例相反，dKO卵睾丸不仅维持到成年，而且能够维持精子发生和卵泡发生。最后，移植实验的结果支持AMH和激活素B的双重性质：（1）AMH和激活素B对胎儿支持细胞具有自分泌/旁分泌性质，（2）AMH和激活素B具有能够使胎儿卵巢雄性化的freemartin因子的内分泌性质。 这些结果不仅推进了我们对性腺性别分化的理解，而且还提供了对人类卵睾丸和性发育障碍病例背后机制的深入了解。 2.调查子宫内暴露于内分泌干扰物对胎儿生殖器官发育的影响及其对成年生育能力的持续影响 胎儿生殖器官的形成依赖于类固醇激素和信号分子之间复杂的相互作用，因此使这一过程成为内分泌干扰物的主要目标。已知模拟或干扰类固醇激素和信号分子作用的化学品或化合物对胎儿生殖器官形成具有有害影响，并在受影响动物成年后对生育力产生长期影响。砷是一种在地下水和食品中发现的人类致癌物质，已知会影响生殖系统。小鼠胚胎暴露于砷会导致卵巢和生殖道的癌症发展。为探讨妊娠期砷暴露对个体成年后的长期影响，本研究用人类相关剂量的饮用水对孕鼠进行砷暴露。当一些暴露的男性胎儿发育到成年时，他们变得肥胖并出现代谢问题，如葡萄糖耐受不良。我们的研究结果表明，在子宫内砷暴露对代谢的潜在影响。 3.破译外生殖器形成的过程 我们已经确定了一个新的细胞群，从后肠迁移到雄性小鼠胚胎的外生殖器。该细胞群对孤儿核受体Nr5a1呈阳性，其用作该细胞群的谱系标志物。Nr5a1+细胞群通过EGF/Neuregulin途径调节尿道周围间充质细胞的功能和分化来促进尿道闭合。如果没有Nr 5a 1+细胞群，尿道闭合就无法正常发生，从而导致尿道下裂，这是最常见的出生缺陷之一。我们将利用这些知识和我们建立的模型来探讨母体热应激对男性后代尿道下裂发病率的潜在影响。由于气候变化，孕妇在怀孕期间面临暴露于高温的风险。怀孕期间的这种热应激可能导致男性后代尿道下裂的发病率增加。出生时患有尿道下裂的男性在以后的生活中容易患上其他疾病（不育症、肾脏疾病等），影响生殖结果和生活质量。我们的工作将建立一个急需的动物模型，以更好地了解产妇热应激对尿道下裂的影响。研究成果将为未来的研究奠定基础，并为该领域提供关键知识。我们的最终目标是根据该项目的结果，制定减轻高温对母亲及其后代健康的系统性影响的方法。

1. Identify the sources of somatic cell lineages in the fetal gonads and investigate how they acquire their organ-specific identities we identified a mechanism in fetal Sertoli cells that maintains their identity. Sertoli cell-derived AMH and activin B act as autocrine and paracrine factors on themselves through their expression of AMH and activin B receptors. These two factors maintain the testis program in Sertoli cells and when both are absent, the granulosa cell program emerges leading to partial sex reversal. The incomplete sex reversal of the Amh/Inhbb double knockout testis (ovotestis structure and feminized transcriptomes and cell populations) further indicates that other factors, such as other TGF-beta proteins 14,26,38-40, could compensate for the loss of Amh and Inhbb. The ovotestis structure of the dKO male resembles that of other ovotestis cases29,33; however, the formation of the ovotestis in dKO males is distinctly different. The ovotestis, in most cases, is established at the onset of testis determination in XY mouse embryos with the central part of the gonad following the testicular program and the flanking poles simultaneously developing into an ovarian structure32,41. In the absence of Amh and Inhbb, however, the fetal testes formed properly at first, with the ovarian domains only arising later when Sertoli cells transdifferentiate into their female counterparts. Most intriguingly, in contrast to most ovotestis cases that disappear before birth, the dKO ovotestes are not only maintained into adulthood, but also capable of sustaining both spermatogenesis and folliculogenesis. Finally, the results of the transplantation experiments support the dual properties of AMH and activin B: (1) AMH and activin B have autocrine/paracrine properties on fetal Sertoli cells and (2) AMH and activin B possess the endocrine property of the freemartin factors that can masculinize the fetal ovary. These results not only advance our understanding of gonadal sex differentiation, but also provide insight into the mechanisms behind cases of ovotestis and disorders of sex development in humans. 2. Investigate the effects of in utero exposure to endocrine disruptors on the development of fetal reproductive organs and its lingering impacts on fertility in adulthood Formation of fetal reproductive organs relies on an intricate interaction between steroid hormones and signaling molecules, therefore making this process a prime target of endocrine disruptors. Chemicals or compounds that mimic or interfere with the action of steroid hormone and signaling molecules are known to have detrimental impacts on fetal reproductive organ formation and long-term impacts on fertility when the affected animals reach adulthood. Arsenic, a human carcinogen found in underground water and food products, is known to impact reproductive systems. Exposure of mouse embryos to arsenic led to cancer development in the ovary and reproductive tracts. To investigate whether arsenic exposure during gestation has a long term impact on the individuals when they reach adulthood, we exposed pregnant mice with human relevant dose of arsenic in the drinking water. When some of the exposed male fetuses are allowed to develop to adulthood, they became obese and developed metabolic problems such as glucose intolerance. Our findings demonstrate a potential impact of in utero arsenic exposure on metabolism. 3. Decipher the process of external genitalia formation We have identified a novel cell population that migrates from the hindgut into the external genitalia in the male mouse embryo. This cell population is positive for the orphan nuclear receptor Nr5a1, which serve as a lineage marker for this population. The Nr5a1+ cell population facilitates urethra closure by modulating the function and differentiation of peri-urethra mesenchymal cells via the EGF/Neuregulin pathway. Without the Nr5a1+ cell population, urethral closure fails to occur normally, consequently leading to hypospadias, one of the most common birth defects. We will use this knowledge and the models that we established to interrogate the potential impacts of maternal heat stress on the incidence of hypospadias in male offspring. As a result of climate change, pregnant women are at risk of being exposed to high temperatures during gestation. Such heat stress during pregnancy could lead to an increase in the incidence of hypospadias in male offspring. Males born with hypospadias are susceptible to other disorders later in life (infertility, kidney diseases, etc.), which affects reproductive outcomes and quality of life. Our work will establish a much needed animal model to better understand the impacts of maternal heat stress on hypospadias. The research outcomes will lay the foundation for future research and provide critical knowledge to the field. Our ultimate goal is to develop ways, based on the results of this project, to mitigate the systemic impacts of high heat on the health of mothers and their offspring.