1. Identify the sources of somatic cell lineages in the fetal gonads and investigate how they acquire their organ-specific identities Organs are composed of common cell types, such as fibroblasts and vasculature endothelial cells, and specialized cell types that define the unique functions of the organs. These specialized cells are thought to originate from organ-specific progenitor cells and acquire their identity during embryogenesis. My lab is using the gonads as a model organ to study how progenitor cells make decision to differentiate into various tissue-specific cell types. Testis and ovary derive from a common primordium during embryogenesis. The primordium, through cell automatous fate determination and intercellular signaling, gives rise to cell types unique to testis (Sertoli, Leydig, and peritubular myoid cells) and ovary (granulosa, theca, and unknown somatic stem cells). Using genetic lineage tracing mouse models, we first characterize the progenitor cells in the primordium and study their sex specification process. For example in the fetal ovary, we discover for the first time the lineage transition of granulosa cells from progenitor state (Steroidogenic factor 1 or SF1 positive) to initial fate specification (FOXL2 positive). This lineage transition process is actively suppressed in the fetal testis by the TGFβpathway, therefore allowing the progenitor cells to differentiate into FOXL2-negative Sertoli cells. We have also discovered that theca cells, a mesenchymal cell type supporting development of the ovarian follicles, consist of at least two populations that come from complete different origins. These findings provide a paradigm shift on the origin of the cell types in the gonads as well as the regulation of gonadal formation. It is worth noting that the progenitor cells in the gonads are positive for various steroid receptors such as estrogen and androgen receptors, suggesting that these progenitor cells could be targets of endocrine disruptors. 2. Define the cellular and molecular processes that lead to sexually dimorphic establishment of the reproductive tracts Before sexual differentiation occurs, embryos are anatomically bisexual as they possess both male and female reproductive tracts. These two tracts derive from two separate progenitor systems in the fetal mesonephros: Wolffian duct for the male tract and Mllerian duct for the female tract. In the male embryos, Wolffian ducts are maintained by testis-derived androgens while Mllerian duct undergo regression induced by anti-Mllerian hormone, also a product of testes. Female embryos, which do not produce androgens or anti-Mllerian hormone (AMH), experience the opposite where Mllerian ducts are maintained and Wolffian ducts undergo regression. In search of novel regulators in this process, we discovered the presence of orphan nuclear receptor COUPTFII in the mesenchyme of the mesonephros. Inactivation of COUPTFII specifically in the mesonephric mesenchyme leads to maintenance of both Wolffian and Mllerian ducts in the male and female mouse embryos, a typical case of pseudohermaphroditism. The affected embryos still have sex-specific production of hormones (androgen and AMH), indicating that hormonal abnormality is not responsible for the pseudohermaphroditic phenotypes of the reproductive tracts. Instead, COUPTFII in the mesenchyme of the mesonephros appears to be a molecular switch that controls the decision-making process of the identity of reproductive tract progenitors. This novel finding provides new mechanistic insights into the dimorphic establishment of reproductive tract. Ongoing experiments are to identify the upstream and downstream regulators of COUPTFII in this process. 3. 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, has been shown to affect the Hedgehog pathway, one of the signaling pathways important for the formation of reproductive organs. Exposure of moues embryos to arsenic leads to cancer development in the ovary and reproductive tracts, organs where the Hedgehog pathway is functional. To investigate whether arsenic exposure targets the Hedgehog pathway in the fetal gonads and reproductive tract, we expose pregnant mice with various doses of inorganic arsenic in the drinking water. We found a dose-dependent and organ-specific effect of arsenic exposure on the Hh pathway: arsenic activates the Hedgehog pathway in the ovary whereas decrease its activity in the fetal testis. When some of the exposed female fetuses are allowed to develop to adulthood, they exhibit precocious onset of puberty (vaginal opening) compared to the control littermate. The Hedgehog pathway is known to control steroidogenesis in the ovary and testis. We therefore hypothesize that altered Hedgehog activity by in utero arsenic exposure could contribute to premature steroid (i.e. estrogen) production and consequent early onset of puberty in the affected female. Our findings are the first to demonstrate a potential impact of arsenic exposure on female reproductive systems. We are collaborating with Jean Harry and Mike Walkkes at NTP to study the neurological impacts of arsenic on these animals.
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