Embryonic estrogen exposure recapitulates persistent ovarian transcriptional programs in a model of environmental endocrine disruption†

Abstract

Estrogens regulate key aspects of sexual determination and differentiation, and exposure to exogenous estrogens can alter ovarian development. Alligators inhabiting Lake Apopka, FL are historically exposed to estrogenic endocrine disrupting contaminants and are characterized by a suite of reproductive abnormalities, including altered ovarian gene expression and abated transcriptional responses to follicle stimulating hormone. Here, we test the hypothesis that disrupting estrogen signaling during gonadal differentiation results in persistent alterations to ovarian gene expression that mirror alterations observed in alligators from Lake Apopka. Alligator embryos collected from a reference site lacking environmental contamination were exposed to estradiol-17 beta or a non-aromatizable androgen in ovo and raised to the juvenile stage. Changes in basal and gonadotropin-challenged ovarian gene expression were then compared to Apopka juveniles raised under identical conditions. Assessing basal transcription in untreated reference and Apopka animals revealed a consistent pattern of differential expression of key ovarian genes. For each gene where basal expression differed across sites, in ovo estradiol treatment in reference individuals recapitulated patterns observed in Apopka alligators. Among those genes affected by site and estradiol treatment were three aryl hydrocarbon receptor (AHR) isoforms, suggesting that developmental estrogen signaling might program sensitivity to AHR ligands later in life. Treatment with gonadotropins stimulated strong ovarian transcriptional responses, however, the magnitude of responses was not strongly affected by steroid hormone treatment. Collectively, these findings demonstrate that precocious estrogen signaling in the developing ovary likely underlies altered transcriptional profiles observed in a natural population exposed to endocrine disrupting contaminants.

Assessing the Ability of Developmentally Precocious Estrogen Signaling to Recapitulate Ovarian Transcriptomes and Follicle Dynamics in Alligators from a Contaminated Lake

“…Animal Husbandry, Follicle-Stimulating Hormone Administration, and Sample Collection All experiments described herein were reviewed and approved by the institutional animal care and use committee at the Medical University of South Carolina (Charleston, SC), and alligator egg collections were approved and permitted by the Florida Fish and Wildlife Conservation Commission. The husbandry for animals used in this study has been previously described in detail (Galligan et al 2019;Hale et al 2019). Briefly, alligator eggs were collected shortly after oviposition from two sites in Florida, AP (Orange county, FL) and Lake Woodruff National Wildlife Refuge (WO; Volusia county, FL).…”

“…To ensure that neonates were housed with similarly sized individuals, all animals were weighed and sorted by body mass every 2 wk. At 2 months of age, the animals were switched to a feeding schedule based on body mass to ensure relative homogeneity of animal size (Hale et al 2019). Briefly, animals weighing <92 g were fed daily, animals weighing 92-132 g were fed three times per week, and animals weighing >132 g were fed twice weekly.…”

“…Patterns in RNAseq read count were independently compared via quantitative real-time polymerase chain reaction (qPCR) using matched samples for 12 genes described as part of a preceding study (Hale et al 2019). Genes assessed via qPCR were aromatase (CYP19A1, LOC102566432), follistatin (FST), ERa (ESR1), ERb (ESR2), FSH receptor (FSHR), androgen receptor (AR), aryl hydrocarbon receptor 1A (AHR1A), aryl hydrocarbon receptor 1B (AHR1B, LOC102568094), aryl hydrocarbon receptor 2 (AHR2, LOC102576203), progesterone receptor (PGR), glucocorticoid receptor (GR or NR3C1), and anti-Mullerian hormone (AMH).…”

“…Among wildlife models, studies on a population of American alligators inhabiting a highly contaminated freshwater system, Lake Apopka (AP), in central Florida (Orange County, FL) helped to pioneer our early understanding of how contaminant exposure during embryonic stages determines future reproductive health. There, developmental exposures to a complex mixture of organochlorine pesticides (OCPs) (Heinz et al 1991;Rauschenberger et al 2007Rauschenberger et al , 2009 have been associated with a suite of reproductive abnormalities, including ovarian and testicular morphological abnormalities (Guillette et al 1994) and shifts in gonadal transcription that persist into juvenile stages (Hale et al 2019;Kohno et al 2008;Moore et al 2012). Reproductive pathologies at AP have been repeatedly observed in juvenile animals collected as eggs and raised under controlled laboratory settings, demonstrating a persistent site-of-origin effect that appears to have developmental origins (Guillette et al 1994;Hale et al 2019;Moore et al 2012).…”

Incubation Temperature and Maternal Resource Provisioning, but Not Contaminant Exposure, Shape Hatchling Phenotypes in a Species with Temperature-Dependent Sex Determination

“…Dichlorodiphenyltrichloroethane (DDT) is a widespread and persistent organochlorine pesticide, and its primary metabolite, dichlorodiphenyldichloroethylene (DDE), is capable of disrupting androgen signaling (Kelce et al, 1995). Impaired reproductive function is associated with EDC exposure (including elevated levels of DDE) in a wild alligator population in Florida (Guillette et al, 1994(Guillette et al, , 1999, and experimental work has demonstrated that these alterations to reproductive function originate during development (Moore et al, 2012;Hale et al, 2019;Hale and Parrott, 2020). In laboratory studies, in ovo exposure to DDE induces female-biased sex ratios in turtles and alligators (Willingham and Crews, 1999;Milnes et al, 2005b).…”