Impacts of Genetic and Environmental Factors on Reproductive Organ Development

遗传和环境因素对生殖器官发育的影响

• 批准号: 8553809
• 负责人: Hung Chang Yao
• 金额: $ 140.7万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553809
• 关键词: Activins; Adrenal Cortex; Adrenal Glands; Adult; Affect; Androgens; Arsenic; Birth; Bisexual; Cell Lineage; Cells; Decision Making; Defect; Development; Developmental Biology; Diethylstilbestrol; Dose; Ductal Epithelium; Embryo; Embryology; Embryonic Development; Endocrine Disruptors; Environment; Environmental Risk Factor; Epididymis; Epithelium; Erinaceidae; Estrogen Receptor alpha; Estrogen Receptors; Exhibits; Exposure to; Female; Fertility; Gene Expression; Gene Mutation; Genetic; Genital system; Goals; Gonadal structure; Growth; Histology; Human; In Vitro; Infertility; Knock-out; Lead; Maintenance; Malignant neoplasm of testis; Mesenchymal; Mesenchyme; Mesonephric structure; Microarray Analysis; Modeling; Molecular; Morphogenesis; Mus; Mutant Strains Mice; Oocytes; Organ; Ovarian; Ovarian Follicle; Ovary; Pathway interactions; Pattern; Perinatal Exposure; Phenotype; Play; Postdoctoral Fellow; Primordium; Process; Production; Proliferating; Prostate; Recruitment Activity; Regulation; Research; Resistance; Rodent; Role; Somatic Cell; Sonic Hedgehog Pathway; Source; Sperm Count Procedure; Stem cells; Structure of mesonephric duct; Testicular Dysgenesis Syndrome; Testing; Testis; Time; Tissues; Vas deferens structure; Visit; Yasmin; activin A; bisphenol A; capsule; cell type; design; estrogenic endocrine disruptor; fetal; gene environment interaction; granulosa cell; granulose; in utero; in vivo; insight; interest; male; mature animal; novel; paracrine; pre-doctoral; progenitor; receptor; regenerative; reproductive; research study; sex; sperm cell; stem cell population; theca cell

1. Understand how different somatic cell lineages are established in the mouse ovary Successful production of oocytes depends upon a somatic cell environment capable of supporting oocyte development. Granulosa and theca cells, the somatic cell types in the ovary, surrounds the oocytes and initiate oocyte growth by forming the ovarian follicles. Granulosa cells are derived from somatic cell precursors of the gonadal primordium and are the first cell type to appear in the fetal ovary. Theca cells, on the other hand, appear only after the ovarian follicles are formed and are thought to be recruited from the mesenchymal cells in the ovarian Interstitium by granulosa cells. In an effort to identify the origin of theca cells, Chang Liu, a visiting predoctoral fellow, discovered that the progenitor cells of theca cells actually come from an extra-ovarian source: the fetal mesonephros adjacent to the ovary. By using genetic lineage tracing approach, Chang found that the mesenchymal cells in the fetal mesonephros migrate into the ovary and become theca cells. This finding provides a paradigm shift on the origin of this cell type as well as the regulation of follicle formation. Experiments using FACS and conditional genetic approaches are conducted to isolate the theca progenitors and to study what control their differentiation. It is interesting to note that mesenchymal cells in the mesonephros are positive for estrogen receptor alpha (ERα), suggesting that the theca progenitor cells could be a target of estrogenic endocrine disruptors. 2. Define the cellular and molecular processes that lead to formation of the Wolffian duct, the precursor of the male reproductive tract Wolffian duct is the embryonic precursor of adult male internal reproductive tract including epididymis, vas deferens, and prostate. Wolffian duct forms in embryos of both sexes but remain only in the male embryo due to the presence of testis-derived androgens. In the female embryos, the lack of androgens leads to degeneration of the Wolffian duct. Although the maintenance of Wolffian duct by androgen is well characterized, it is not known what trigger the initial establishment of the Wolffian duct. Heather Franco, an IRTA postdoc fellow, has identified that the epithelium of the Wolffian duct produces Sonic hedgehog (Shh), a paracrine regulator that plays critical role in formation of various organs. The receptor and intracellular regulator of Shh are present in the mesenchyme surrounding the Shh-positive Wolffian duct epithelium, suggesting a potential epithelium/mesenchyme interaction via Shh. Indeed in the Shh knockout embryo, the mesenchyme surrounding the Wolffian duct undergoes degeneration early, leading to a complete disappearance of Wolffian duct before birth. The ongoing experiments are designed to identify novel regulators downstream of Shh in the Wolffian duct and study whether this pathway is susceptible to endocrine disruptors. Arsenic has been shown recently to affect the Shh pathway in vitro, opening a new direction that could provide mechanistic insight into arsenic action in vivo. We will be examining whether in utero exposure to arsenic has an impact on Shh-regulated organs such as the Wolffian duct. 3. Investigate effects of in utero exposure to endocrine disruptors on fetal testis development Testicular dysgenesis syndrome (TDS) manifests as genital abnormalities, testicular cancer, reduced sperm count, or infertility. TDS in humans has increased dramatically over the past 50 years. It is postulated that TDS originates from defects in embryonic development of the testis that are induced by genetic mutations, or environmental insults, or a combination of both (gene-environment interaction). Endocrine disruptors, such as diethylstilbestrol (DES), have been shown to induce TDS in rodent embryos. We discovered that loss of activin A in the mouse fetal testis leads to focal dysgenesis of testis cords, a phenotype similar to mouse fetal testes exposed to DES and bisphenol A (BPA) in utero. When the activin A mutant mice reach adulthood, their testes are significantly smaller and produce less sperm, phenotypes resembling human TDS. We therefore hypothesize that altered activin function and exposure to DES or BPA interact as part of a common mechanism for TDS. Karina Rodriguez and Yasmin Crespo-Mejias are conducting a small scale experiment testing whether low dose of BPA exposure (0.5 and 50 ug/kg/day) in utero could affect testis cord morphogenesis (histology) and gene expression (microarray) in CD1 embryos. We expect that DES and/or BPA treatment will affect the activin A pathway and cause testis cord dysgenesis. The long-term goal of this study is to identify the mechanism of BPA action by testing whether mouse embryos lacking estrogen receptors became resistant to BPA exposure. 4. Characterize the stem cells in the adrenal gland and investigate their developmental potential Adrenal gland is one of the few organs that have regenerative capability in adult animals. In an effort to understand how adrenal glands, an organ that derives from the same primordium as the gonads, develop in the mouse embryos, we discovered that the capsule surrounding the adrenal cortex is a source of stem cells. The expansion of the stem cell population in the capsule requires Sonic hedgehog (Shh) derived from the adrenal cortex. Mouse embryos lacking Shh had a significant decrease in adrenal cortex expansion, resulting in hypoplastic adrenal glands at the time of birth. Shh is expressed in the cortex of developing adrenal while the receptor Ptch1 and its intracellular regulators Gli1 and Gli2 are present in the adrenal capsule. Using genetic lineage tracing approaches, we found that the Gli1-positive cells in the capsule migrate into the adrenal cortex. In the absence of Shh, the Gli1-positive cells fail to proliferate, leading to a significant reduction in the growth and size of adrenal cortex. Erica Ungewitter, an IRTA postdoctoral fellow, has embarked a set of new experiments with the goal to isolate the stem cells in the adrenal capsule and characterize these cells in vitro. She has successfully cultured the adrenal stem cells and will inject these cells into various steroidogenic organs to investigate whether these cells are capable of differentiating into other steroidogenic cell lineages. Erica will also perform microarray analysis to identify downstream regulators of Shh that define the stem cell lineage in the adrenal gland.

1. 了解不同体细胞谱系如何在小鼠卵巢中建立 卵母细胞的成功产生取决于能够支持卵母细胞发育的体细胞环境。颗粒细胞和卵泡膜细胞是卵巢中的体细胞类型，围绕卵母细胞并通过形成卵泡来启动卵母细胞生长。颗粒细胞源自性腺原基的体细胞前体，是出现在胎儿卵巢中的第一种细胞类型。另一方面，卵泡膜细胞仅在卵泡形成后出现，并且被认为是由颗粒细胞从卵巢间质中的间充质细胞招募的。为了确定卵泡膜细胞的起源，访问博士前研究员刘畅发现卵泡膜细胞的祖细胞实际上来自卵巢外：与卵巢相邻的胎儿中肾。通过使用遗传谱系追踪方法，张发现胎儿中肾的间充质细胞迁移到卵巢并成为卵泡膜细胞。这一发现为这种细胞类型的起源以及卵泡形成的调节提供了范式转变。使用 FACS 和条件遗传方法进行实验来分离卵泡膜祖细胞并研究控制其分化的因素。有趣的是，中肾间充质细胞对雌激素受体α（ERα）呈阳性，这表明卵泡膜祖细胞可能是雌激素内分泌干扰物的目标。 2. 定义导致男性生殖道前身沃尔夫管形成的细胞和分子过程 沃尔夫管是成年男性内生殖道的胚胎前体，包括附睾、输精管和前列腺。沃尔夫管在两性胚胎中形成，但由于睾丸来源的雄激素的存在，仅保留在雄性胚胎中。在雌性胚胎中，雄激素的缺乏会导致沃尔夫管退化。尽管雄激素对沃尔夫管的维持已得到很好的表征，但尚不清楚是什么触发了沃尔夫管的初始建立。 IRTA 博士后研究员希瑟·佛朗哥 (Heather Franco) 发现，沃尔夫管上皮会产生声波刺猬 (Shh)，这是一种旁分泌调节剂，在各种器官的形成中发挥着关键作用。 Shh 的受体和细胞内调节因子存在于 Shh 阳性沃尔夫管上皮周围的间质中，表明上皮/间质可能通过 Shh 相互作用。事实上，在Shh敲除胚胎中，沃尔夫管周围的间充质很早就发生了退化，导致沃尔夫管在出生前完全消失。正在进行的实验旨在识别 Wolffian 管中 Shh 下游的新型调节因子，并研究该通路是否容易受到内分泌干扰物的影响。最近，砷在体外被证明可以影响 Shh 通路，开辟了一个新的方向，可以为砷在体内的作用机制提供深入的了解。我们将研究在子宫内暴露于砷是否会对诸如沃尔夫管等受嘘调节的器官产生影响。 3. 研究子宫内暴露于内分泌干扰物对胎儿睾丸发育的影响 睾丸发育不全综合征 (TDS) 表现为生殖器异常、睾丸癌、精子数量减少或不育。过去 50 年来，人类的 TDS 急剧增加。据推测，TDS 源于睾丸胚胎发育缺陷，这些缺陷是由基因突变、环境损伤或两者的组合（基因-环境相互作用）引起的。内分泌干​​扰物，例如己烯雌酚 (DES)，已被证明会在啮齿动物胚胎中诱导 TDS。我们发现，小鼠胎儿睾丸中激活素 A 的缺失会导致睾丸索局灶性发育不全，这种表型类似于在子宫内暴露于 DES 和双酚 A (BPA) 的小鼠胎儿睾丸。当激活素 A 突变小鼠成年后，它们的睾丸明显变小，产生的精子也更少，其表型类似于人类 TDS。因此，我们假设激活素功能的改变和暴露于 DES 或 BPA 相互作用是 TDS 常见机制的一部分。 Karina Rodriguez 和 Yasmin Crespo-Mejias 正在进行一项小规模实验，测试子宫内低剂量 BPA 暴露（0.5 和 50 ug/kg/天）是否会影响 CD1 胚胎中睾丸索形态发生（组织学）和基因表达（微阵列）。我们预计 DES 和/或 BPA 治疗会影响激活素 A 通路并导致睾丸发育不全。这项研究的长期目标是通过测试缺乏雌激素受体的小鼠胚胎是否对 BPA 暴露产生抵抗力来确定 BPA 的作用机制。 4. 表征肾上腺干细胞并研究其发育潜力 肾上腺是成年动物中少数具有再生能力的器官之一。为了了解肾上腺（一种与性腺源自同一原基的器官）如何在小鼠胚胎中发育，我们发现肾上腺皮质周围的囊是干细胞的来源。胶囊内干细胞群的扩张需要来自肾上腺皮质的音猬因子（Shh）。缺乏Shh的小鼠胚胎肾上腺皮质扩张显着减少，导致出生时肾上腺发育不全。 Shh 在发育中的肾上腺皮质中表达，而受体 Ptch1 及其细胞内调节因子 Gli1 和 Gli2 则存在于肾上腺囊中。使用遗传谱系追踪方法，我们发现胶囊中的 Gli1 阳性细胞迁移到肾上腺皮质中。在缺乏Shh的情况下，Gli1阳性细胞无法增殖，导致肾上腺皮质的生长和大小显着减少。 IRTA 博士后研究员 Erica Ungewitter 开展了一系列新实验，目的是分离肾上腺囊中的干细胞并在体外表征这些细胞。她已经成功培养了肾上腺干细胞，并将这些细胞注射到各种类固醇生成器官中，以研究这些细胞是否能够分化为其他类固醇生成细胞谱系。 Erica 还将进行微阵列分析，以确定 Shh 的下游调节因子，这些调节因子定义了肾上腺中的干细胞谱系。