Date of Award

12-2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Forestry and Natural Resources

First Advisor

Maria S. Sepulveda

Committee Chair

Maria S. Sepulveda

Committee Member 1

Jennifer L. Bay

Committee Member 2

Cecon T. Mahapatra

Committee Member 3

GuangJun Zhang

Abstract

Natural and synthetic estrogenic and androgenic compounds are continuously released into aquatic ecosystems. Exposure of teleost fishes to these contaminants can negatively impact sex differentiation and reproductive output. Specifically, development of gonadal intersex in gonochoristic (fixed sex) fish species has been studied extensively in relation to exposure to this class of compounds. The main objectives of this dissertation were to: 1) conduct field and laboratory studies to investigate the molecular signaling pathways behind the development of gonadal intersex; and 2) establish molecular biomarkers and assays for testing the ability of environmental pollutants to develop this condition using a battery of molecular, cellular and organ-level tools.

First, we conducted a literature review to summarize all available articles reporting gonadal intersex in wild populations of gonochoristic fish (Chapter 1). We also included the limited information available on this topic in amphibians. We analyzed studies from across the globe, identified families and species with reported cases of gonadal intersex, and highlighted the contaminants often linked with this condition. In addition, we discussed the current knowledge of molecular signaling pathways behind the development of gonadal intersex and summarized molecular biomarkers tested and others that require further investigation. We then conducted a field study investigating the prevalence of testicular oocytes (TOs), the most prevalent form of gonadal intersex, in a sentinel freshwater fish species, the smallmouth bass Micropterus dolomieu, inhabiting the St. Joseph River and its tributaries in northern Indiana (Chapter 2). This constitutes the first study of this nature. Sites on this river were previously identified as having medium to high intersex induction potential based on contaminant quantification and estrogen equivalence estimations. We reported prevalence and severity of gonadal intersex reaching 100% in some sites, and significant decreases in prevalence and increases in severity of TOs occurrence after the spawning season. We evaluated changes in the transcription levels of several genes involved in sex differentiation and gonadal development. Significantly higher levels of vitellogenin ( vtg) transcripts were found in livers of males with TOs, but only when sampled in the spawning season. Further, we quantified contaminant levels in surface water to identify possible correlations between contamination levels and the observed prevalence of gonadal intersex. Multiple sites had detectable levels of endocrine disruptors, but no correlations with the prevalence or severity of TOs was recorded.

In order to develop molecular biomarkers and assays that test contaminants’ ability to develop gonadal intersex, short-term laboratory exposures were performed using Japanese medaka Oryzias latipes, a fish model with a well-understood sex determination mechanism and high sensitivity to exogenous hormone exposure (Chapter 3). First, we identified a gene, ovary structure protein 1 (osp1), with strong female-specific expression during gonadal differentiation and observed a significant downregulation in its expression in females following short-term (10 d) exposure to a potent synthetic androgen, 17β-trenbolone. Importantly, this decrease in osp1expression was correlated with changes in ovarian phenotype, namely ovarian intersex, later in life. We decided to further utilize this promising molecular biomarker by incorporating it in a visual in vivo reporter assay for rapid detection of contaminants with estrogenic/androgenic potential (Chapter 4). For this purpose, we built a pOSP1-AcGFP (promoterOSP1-Aequorea coerulescens green florescence protein) Japanese medaka transgenic line with osp1 promoter region driving the expression of a reporter protein, AcGFP. After establishing this line, we tested its use in an in vivo visual reporter system for identifying estrogenic contaminants. Significant upregulation in fluorescence intensity was recorded in 30 d post hatch females following a 24 h exposure to 500 ng/L of a synthetic potent estrogen, ethinyl estradiol.

Overall, our results support earlier findings suggesting that gonadal intersex is highly prevalent in impacted rivers across the US and that smallmouth bass are highly sensitive to developing this condition. We also conclude that hepatic and plasma VTG are promising biomarkers for diagnosing gonadal intersex, but only in males sampled during the spawning season. Our findings also support the hypothesis that molecular biomarkers, such as osp1 , are sensitive tools that can be used for early detection of the effects of contaminants with estrogenic and androgenic activity on fish and are ideal endpoints in wide-scale contaminant screening assays.

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