Abstract

Persistent, anthropogenic contaminants collectively known as endocrine-disrupting chemicals (EDCs), are considered to be potential threats to wildlife reproductive fitness.¹⁰ One possible mechanism by which EDCs may affect reproductive fitness is competitive binding to key steroid receptors in gonadal tissue. To determine the relative binding affinities of steroids versus putative EDCs to steroid receptors in dolphin reproductive tissues, reproductive tissues, were obtained by the Georgia Aquarium's Dolphin Conservation Field Station (GADCFS) from stranded bottlenose dolphins (four male, three female) during 2010 and 2011. We detected steroid receptors immunohistochemically (IHC) with steroid reporters conjugated^4,5 to fluorescein isothiocyanate (FITC),⁶ and subsequently calculated relative binding affinities. The steroids were estradiol (E2), testosterone (T2), and progesterone (P4). EDCs were selected from the Dolphin Health and Environmental Risk Assessment (HERA) results^7,8 The final EDC candidate pool included: polychlorinated biphenyls (PCB)-118, 137, 153; dichlorodiphenyltrichloroethane (DDT); dichlorodiphenyldichloroethylene (DDE); pentabromodiphenylether (BDE47); perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA). EDCs were conjugated to BSA in the same manner as the steroids, with part of each resulting conjugate solution being bound to FITC as before, and part set aside for competitive binding assays. FITC-conjugated EDCs were ligated to each of the tissue samples to determine individual binding affinities, while the BSA-only conjugates were used in competitive binding assays on gonad and uterus samples. Competition between each EDC was conducted with E2 on ovary samples, T2 on testis samples and P4 on uterine samples. All slides were imaged with a fluorescent microscope (Provis AX70^TM with MagnaFire software), followed by imaging of one slide of each tissue type from each assay on a confocal microscope (Zeiss LSM^TM). The Allred method1 was used to visually score all slides prior to calculating binding affinities, yielding the following results. Native steroids most strongly targeted receptors along the boundaries of glands, follicles and vessels in reproductive tissues. E2 and T2 showed some binding success in all tissues, but was strongest in female and male gonadal and tract tissue respectively. P4 binding was primarily successful in uterine and ovarian tissues, and showed limited binding in any male tissues. All EDCs showed at least minimal binding to all tissues when not placed in competition with native steroids. In competitive assays with E2, PCBs 118, 137 and 153 and PFOS showed minimal disruption of E2; PCB-180, DDE, DDT, BDE47, PFOA displayed extensive disruption. In competitive assays with T2, PCBs 118 and 137 showed minimal disruption, while PCBs 153 and 180 showed moderate disruption; DDE, DDT, BDE47, PFOA and PFOS showed extensive disruption. In competitive assays with P4, none of the PCBs disrupted P4 binding; DDE, DDT and BDE-47 showed moderate disruption; PFOA and PFOS showed extensive disruption. Fluorescent monochrome images were merged and analyzed in both ImageJ (Fiji release)^2,3 and ImmunoRatio,⁹ both of which are validated for IHC detection of steroid receptors. We conclude that endocrine-disruptors are tissue and receptor specific, and that both legacy and emerging contaminants are of concern to dolphin stocks, particularly those facing other significant environmental stressors.

Acknowledgements

We gratefully thank Dr. Gregory Bossart and Dr. Patricia Fair for support and access to HERA project results and samples, and Mr. George Biedenbach and Mr. Mike Denney (GADCFS) for tissue samples. Financial support was provided by the FAU National Alumni Association, the FAU Graduate Student Association, FAU Student Financial Aid scholarships, the Department of Biological Sciences, and the Captain Vincent Saurino Award.

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