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.

Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2012
Total Cost
$1,406,984
Indirect Cost
City
State
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Zip Code
Zhao, Fei; Franco, Heather L; Rodriguez, Karina F et al. (2017) Elimination of the male reproductive tract in the female embryo is promoted by COUP-TFII in mice. Science 357:717-720
Ungewitter, Erica; Rotgers, Emmi; Bantukul, Tanika et al. (2017) From the Cover: Teratogenic Effects of in Utero Exposure to Di-(2-Ethylhexyl)-Phthalate (DEHP) in B6:129S4 Mice. Toxicol Sci 157:8-19
Liu, Chang; Rodriguez, Karina; Yao, Humphrey H-C (2016) Mapping lineage progression of somatic progenitor cells in the mouse fetal testis. Development 143:3700-3710
Kang, Hong Soon; Chen, Liang-Yu; Lichti-Kaiser, Kristin et al. (2016) Transcription Factor GLIS3: A New and Critical Regulator of Postnatal Stages of Mouse Spermatogenesis. Stem Cells 34:2772-2783
Rodriguez, Karina F; Ungewitter, Erica K; Crespo-Mejias, Yasmin et al. (2016) Effects of in Utero Exposure to Arsenic during the Second Half of Gestation on Reproductive End Points and Metabolic Parameters in Female CD-1 Mice. Environ Health Perspect 124:336-43
Nicol, Barbara; Yao, Humphrey H-C (2015) Gonadal Identity in the Absence of Pro-Testis Factor SOX9 and Pro-Ovary Factor Beta-Catenin in Mice. Biol Reprod 93:35
Liu, Chang; Peng, Jia; Matzuk, Martin M et al. (2015) Lineage specification of ovarian theca cells requires multicellular interactions via oocyte and granulosa cells. Nat Commun 6:6934
Archambeault, Denise R; Yao, Humphrey Hung-Chang (2014) Loss of smad4 in Sertoli and Leydig cells leads to testicular dysgenesis and hemorrhagic tumor formation in mice. Biol Reprod 90:62
Barsoum, Ivraym B; Kaur, Jaspreet; Ge, Renshan S et al. (2013) Dynamic changes in fetal Leydig cell populations influence adult Leydig cell populations in mice. FASEB J 27:2657-66
Ungewitter, E K; Yao, H H-C (2013) How to make a gonad: cellular mechanisms governing formation of the testes and ovaries. Sex Dev 7:7-20

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