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Differential Estrogen Sensitivity Among Populations of African Clawed Frogs

Abstract

Estrogens are a class of steroid hormones ubiquitous and crucial to organismal reproductive success throughout the vertebrate phylum. It is physiologically important to both males and females, playing a crucial role in gonad development, spermatogenesis, oogenesis, bone development, and sex behavior. Localized overproduction of the estrogen is significant in the development and progression of hormone-dependent cancers (e.g. cancers of the breasts, endometrium, etc.). Many chemicals that increase the production of estrogen or mimic its action have contributed to the global decline of aquatic life due to adverse effects on species’ development and reproductive health. Comparable responses to environmental contaminants have been observed in humans. It is evident that not all populations, individuals, or tissues are equally affected by hormone-driven ailments. I aim to elucidate the basis of population differences in estrogen susceptibility using the African clawed frog (Xenopus laevis). This knowledge will not only contribute to the field of developmental endocrinology, but may also hold great significance in environmental toxicology and animal conservation.

Chapter One reviews the role of estrogen in amphibian development, the impacts of endocrine disruptors on animal health, and the significance of acknowledging and characterizing response variation in animal models used in scientific studies.

Chapter Two describes a study where I examined how two source populations of X. laevis display different susceptibilities to the feminizing effects of 17-beta estradiol (E2). All-male, ZZ-tadpoles from a store-bought population, Xenopus Express (XE), and from a wild-caught population, San Diego (SD), were treated with 3.00 ng/ml E2 starting at 10 days post-hatch until metamorphosis. Animals were then sacrificed, gonad sex was determined via dissection, and sex ratios were calculated. Experiments were conducted with multiple parents within each population as well as with familial replicates in order to verify sex reversal trends. The data indicated that, within a species, there are inherent differences in gonad susceptibility to the feminizing effects of E2 and implied varying susceptibility to the feminizing effects of endocrine disruptors.

In Chapter Three, I examined whether differences in E2 susceptibility span tissue types and developmental stages. Adult, SD and XE males were treated with increasing concentrations of E2 for 12 hours. Expression of E2-responsive genes, vtga2 and esr1, in the liver was quantified via qRT-PCR and compared between the two populations. XE and SD animals showed different fold-changes in vtga2 expression in response to E2 treatment, and there were disparities between vtga2 and esr1 expression. These genes may be used as indicators of E2 exposure, but may not necessarily serve as effective tools to measure the degree of in vivo exposure.

Chapter Four discusses the results of an RNA-Seq experiment examining differential gene transcription between control and E2-treated SD and XE groups. All male, ZZ tadpoles were treated with 3.00 ng/ml E2 starting at 10 days post hatch (dph) and sacrificed when they reached NF stages 52-53 (during which sex differentiation occurs). The gonads were then dissected out, mRNA was extracted, and a cDNA library was constructed and sequenced. ahr.S, cyp2c8.2.L, cyp19a1.L, dmrt1.S, and hsd17b7.S were differentially expressed genes between the two populations that we recognized to have a role in sex differentiation and steroidogenesis. Based on the expression profiles, we proposed a mechanism underlying susceptibility to feminization, which involves both the suppression of masculinizing genes and the upregulation in feminizing genes.

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