Altered sex hormone concentrations and gonadal mRNA expression levels of activin signaling factors in hatchling alligators from a contaminated Florida lake

Abstract

Activins and estrogens participate in regulating the breakdown of ovarian germ cell nests and follicle assembly in mammals. In 1994, our group reported elevated frequencies of abnormal, multioocytic ovarian follicles in 6 month old, environmental contaminant-exposed female alligators after gonadotropin challenge. Here, we investigated if maternal contribution of endocrine disrupting contaminants to the egg subsequently alters estrogen/inhibin/activin signaling in hatchling female offspring, putatively predisposing an increased frequency of multioocytic follicle formation. We quantified basal and exogenous gonadotropin-stimulated concentrations of circulating plasma steroid hormones and ovarian activin signaling factor mRNA abundance in hatchling alligators from the same contaminated (Lake Apopka) and reference (Lake Woodruff) Florida lakes, as examined in 1994. Basal circulating plasma estradiol and testosterone concentrations were greater in alligators from the contaminated environment, whereas activin/inhibin betaA subunit and follistatin mRNA abundances were lower than values measured in ovaries from reference lake animals. Challenged, contaminant-exposed animals showed a more robust increase in plasma estradiol concentration following an acute follicle stimulating hormone (FSH) challenge compared with reference site alligators. Aromatase and follistatin mRNA levels increased in response to an extended FSH challenge in the reference site animals, but not in the contaminant-exposed animals. In hatchling alligators, ovarian follicles have not yet formed; therefore, these endocrine differences are likely to affect subsequent ovarian development, including ovarian follicle assembly.

Figure 1

Basal circulating steroids concentrations and ovarian mRNA expression levels, bars report means±SEM, in hatchling alligators: estradiol and sample sizes (A), testosterone (B), Inha (C), Inhba (D), Fst (E), and Cyp19a1 (F) in Lake Woodruff and Lake Apopka alligators. Days after hatching: white bars = one, gray bars = two, and black bars = five. All mRNA expression sample means are normalized using ribosomal protein L8 (Rpl8) expression and standardized for each endpoint to Lake Woodruff female, day 1 expression = one. Horizontal line above bars indicates statistical significance by lake of origin (P<0.05). Different letters above the bars indicate age by lake of origin statistical significances.

Figure 2

Circulating steroid concentrations and ovarian mRNA expression levels in 2 day old FSH-challenged alligators. Bars report means±SEM: estradiol and sample sizes (A), testosterone (B), Inha (C), Inhba (D), Fst (E), and Cyp19a1 (F) in Lake Woodruff (black bars) and Lake Apopka (white bars) alligators. All mRNA expression sample means are normalized using ribosomal protein L8 (Rpl8) expression and standardized for each endpoint to Lake Woodruff female, vehicle treated expression = one. Horizontal lines above bars indicate statistical significance by treatment (P<0.05). Asterisks above bars indicate significant difference by lake of origin. Different letters above the bars indicate age by lake of origin statistical significances.

Figure 3

Circulating steroid concentrations and ovarian mRNA expression levels in 5 day old FSH-challenged alligators. Bars report means±SEM: estradiol and sample sizes (A), testosterone (B), Inha (C), Inhba (D), Fst (E), and Cyp19a1 (F) in Lake Woodruff (black bars) and Lake Apopka (white bars) alligators. All mRNA expression sample means are normalized using ribosomal protein L8 (Rpl8) expression and standardized for each endpoint to Lake Woodruff female, vehicle treated expression = one. Horizontal lines above bars indicate statistical significance by treatment (P<0.05). Asterisks above bars indicate significant difference by lake of origin. Different letters above the bars indicate age by lake of origin statistical significances.