Heather J. Hamlin

Agriculture Alters Gonadal Form and Function in the Toad Bufo marinus

Background Many agricultural contaminants disrupt endocrine systems of wildlife. However, evidence of endocrine disruption in wild amphibians living in agricultural areas has been controversial. Typically, studies on the effects of pollutants on wildlife attempt to compare polluted with unpolluted sites. Objectives We took a novel approach to address this question by explicitly quantifying the relationship between gonadal abnormalities and habitats characterized by differing degrees of agricultural activity. Methods We quantified the occurrence of gonadal abnormalities and measures of gonadal function in at least 20 giant toads (Bufo marinus) from each of five sites that occur along a gradient of increasing agricultural land use from 0 to 97%. Results The number of abnormalities and frequency of intersex gonads increased with agriculture in a dose-dependent fashion. These gonadal abnormalities were associated with altered gonadal function. Testosterone, but not 17β-estradiol, concentrations were altered and secondary sexual traits were either feminized (increased skin mottling) or demasculinized (reduced forearm width and nuptial pad number) in intersex toads. Based on the end points we examined, female morphology and physiology did not differ across sites. However, males from agricultural areas had hormone concentrations and secondary sexual traits that were intermediate between intersex toads and non-agricultural male toads. Skin coloration at the most agricultural site was not sexually dimorphic; males had female coloration. Conclusions Steroid hormone concentrations and secondary sexual traits correlate with reproductive activity and success, so affected toads likely have reduced reproductive success. These reproductive abnormalities could certainly contribute to amphibian population declines occurring in areas exposed to agricultural contaminants.

Birth defects in wildlife: the role of environmental contaminants as inducers of reproductive and developmental dysfunction.

The etiology of adverse pregnancy outcomes is not well understood. Wildlife observations provide considerable evidence that environmental contaminants can play a critical role in reproductive and developmental dysfunction. Early evidence leading to a widespread awareness of the impact of environmental chemicals on surrounding wildlife was observed in the Laurentian Great Lakes. A suite of reproductive and congenital defects was identified in birds, reptiles, and mammals alike that were attributed to high concentrations of organochlorine pesticides and industrial chemicals. Due to the ubiquitous and persistent nature of many anthropogenic chemicals, these defects, including thyroid dysfunction, hatching success, egg shell thinning, and gross birth deformities, have since been identified in numerous wildlife populations across the world. Certain wildlife taxa such as amphibians are especially vulnerable to chemical perturbation and are suffering alarming population declines. Amphibian field studies have found severe hindlimb and other developmental abnormalities and it has been demonstrated that the greater the agricultural intensity, the greater the number and severity of defects in toad populations. Alligators living in contaminated lakes have shown a significant reduction in penis size and fish exposed to tributyltin have shown tail deformities and abnormal eye development. Physiological and molecular responses to chemical insult are often conserved across vertebrates, alerting scientists and medical professionals alike that greater attention needs to be paid to the roles environmental contaminants play in the etiology of congenital disorders in both humans and wildlife.

Embryos as targets of endocrine disrupting contaminants in wildlife.

Environmental contaminants are now a ubiquitous part of the ecological landscape, and a growing literature describes the ability of many of these chemicals to alter the developmental trajectory of the embryo. Because many environmental pollutants readily bioaccumulate in lipid rich tissues, wildlife can attain considerable body burdens. Embryos are often exposed to these pollutants through maternal transfer, and a growing number of studies report long-term or permanent developmental consequences. Many biological mechanisms are reportedly affected by environmental contaminants in the developing embryo and fetus, including neurodevelopment, steroidogenesis, gonadal differentiation, and liver function. Embryos are not exposed to one chemical at a time, but are chronically exposed to many chemicals simultaneously. Mixture studies show that for some developmental disorders, mixtures of chemicals cause a more deleterious response than would be predicted from their individual toxicities. Synergistic responses to low dose mixtures make it difficult to estimate developmental outcomes, and as such, traditional toxicity testing often results in an underestimate of exposure risks. In addition, the knowledge that biological systems do not necessarily respond in a dose-dependent fashion, and that very low doses of a chemical can prove more harmful than higher doses, has created a paradigm shift in studies of environmental contaminant-induced dysfunction. Although laboratory studies are critical for providing dose-response relationships and determining specific mechanisms involved in disease etiology, wildlife sentinels more accurately reflect the genetic diversity of real world exposure conditions, and continue to alert scientists and health professionals alike of the consequences of developmental exposures to environmental pollutants.

Androgens modulate song effort and aggression in Neotropical singing mice

Androgens are an important class of steroid hormones involved in modulating the expression and evolution of male secondary sex characters. Vocalizations used in the context of aggression and mate attraction are among the most elaborate and diverse androgen-dependent animal displays as reflected in a rich tradition of studies on bird song and anuran calls. Male Alstons singing mice (Scotinomys teguina) commonly emit trilled songs that appear to function in male-male aggression. In this study, we experimentally manipulated androgens in singing mice to assess their role in modulating aggression and song effort. Testosterone- and DHT-treated animals retained aggressive and song attributes similarly. However, castrated mice administered empty implants showed more subordinate behavior and sang fewer songs that were shorter, lower in power, higher in frequency, and less stereotyped. The extensive effects of androgens on a suite of phenotypes highlight their role in linking gonadal status with decisions about investment in reproductive behaviors.

The culture and early weaning of larval haddock (Melanogrammus aeglefinus) using a microparticulate diet

Abstract In order to effectuate haddock, Melanogrammus aeglefinus , as a practical aquaculture candidate, they must be able to successfully wean onto a formulated diet. This weaning should be accomplished soon after hatching in order to limit financial bottlenecks associated with the high cost of live feed production. Two experiments were designed to determine the earliest point at which haddock could be successfully weaned onto a formulated food. The first examined start weaning at 14, 21, 28, and 35 days post-hatch (dph) at 8.5°C. Control animals were given only live feeds throughout the experiment. Samples of larvae were taken weekly for length and dry weight analysis. Haddock did not wean as effectively as cod onto the commercial diet (Biokyowa™), and survival was compromised for all treatments examined. There were no differences in survival for any of the weaning periods investigated with averages ranging from 2.5% to 6.3%. However, the survival of the control group was significantly higher at 37.9%. The second experiment examined weaning at later stages and at an increased temperature. This experiment examined start weaning periods of 30, 35, and 42 days post-hatch. The 42-day start weaning period yielded results similar to those obtained with the live feed control, however survival and growth was compromised for both the 30- and 35-day weaning periods.

Endocrine Activity of Extraembryonic Membranes Extends beyond Placental Amniotes

Background During development, all amniotes (mammals, reptiles, and birds) form extraembryonic membranes, which regulate gas and water exchange, remove metabolic wastes, provide shock absorption, and transfer maternally derived nutrients. In viviparous (live-bearing) amniotes, both extraembryonic membranes and maternal uterine tissues contribute to the placenta, an endocrine organ that synthesizes, transports, and metabolizes hormones essential for development. Historically, endocrine properties of the placenta have been viewed as an innovation of placental amniotes. However, an endocrine role of extraembryonic membranes has not been investigated in oviparous (egg-laying) amniotes despite similarities in their basic structure, function, and shared evolutionary ancestry. In this study, we ask whether the oviparous chorioallantoic membrane (CAM) of chicken (Gallus gallus) has the capability to synthesize and receive signaling of progesterone, a major placental steroid hormone. Methodology/Principal Findings We quantified mRNA expression of key steroidogenic enzymes involved in progesterone synthesis and found that 3β-hydroxysteroid dehydrogenase, which converts pregnenolone to progesterone exhibited a 464 fold increase in the CAM from day 8 to day 18 of embryonic development (F5, 68 = 89.282, p<0.0001). To further investigate progesterone synthesis, we performed explant culture and found that the CAM synthesizes progesterone in vitro in the presence of a steroid precursor. Finally, we quantified mRNA expression and performed protein immunolocalization of the progesterone receptor in the CAM. Conclusions/Significance Collectively, our data indicate that the chick CAM is steroidogenic and has the capability to both synthesize progesterone and receive progesterone signaling. These findings represent a paradigm shift in evolutionary reproductive biology by suggesting that endocrine activity of extraembryonic membranes is not a novel characteristic of placental amniotes. Rather, we hypothesize that these membranes may share an additional unifying characteristic, steroidogenesis, across amniotes at large.

Nitrate‐Induced Goiter in Captive Whitespotted Bamboo Sharks Chiloscyllium plagiosum

Elasmobranch susceptibility to goiter formation in captive environments has been well documented. Until recently, most public aquariums operated under the belief that the etiology of goiter in elasmobranchs was nutritional and specifically caused by insufficient dietary iodine. Recent studies have demonstrated that high environmental nitrate (NO3-N) inhibits the ability of the thyroid gland to utilize available iodide, resulting in thyroid gland overstimulation by thyroid stimulating hormone and ultimately leading to the development of goiter. The objective of this study was to evaluate the effects of high environmental nitrate concentrations on thyroid function in juvenile whitespotted bamboo sharks Chiloscyllium plagiosum. In July 2008, five juveniles (80-150 g) were exposed to a low-nitrate environment (NO3-N concentration < 1 mg/L of water) and five were exposed to an elevated-nitrate environment (NO3-N = 70 mg/L) for 29 d in a flow-through natural seawater system. Nitrate exposure did not affect growth rates (e.g., weight, length, and condition factor) and did not alter free plasma thyroxine concentrations during the 29-d experimental period. However, histological examination of thyroid glands from sharks exposed to elevated nitrate revealed the development of diffuse hyperplastic goiter. With increasing restrictions on water use, most modern aquaria operate as recirculating systems, which results in higher and more chronic nitrate exposure for captive animals. Goiter is one of the most common health problems in captive elasmobranchs, and this study suggests that nitrate exposure is an important factor in the etiology of this disease.

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

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.

Environmental Influence on Yolk Steroids in American Alligators (Alligator mississippiensis)

The egg yolk serves as a significant source of maternally derived steroids that are available to the embryo during early development. Altered deposition of yolk steroids can change the developmental trajectory of the embryo and have long lasting or permanent consequences. Alligators from contaminated environments have shown significant reproductive and developmental dysfunction, and it is unclear if altered deposition of yolk steroids could be a contributing factor. Alligator eggs were collected from Lake Woodruff (a reference lake), Lake Apopka (a site of known agricultural contamination), and the Merritt Island National Wildlife Refuge (MINWR) (home of the Kennedy Space Center and a site of heavy metal contamination). The yolks of eggs at embryonic stages 12 (prior to sex determination) and 24 (post-sex determination) were evaluated for concentrations of progesterone, 17-beta estradiol, and testosterone. Yolk concentrations of progesterone were significantly lower at embryonic stage 12 in eggs from Lake Apopka and MINWR when compared to eggs from Lake Woodruff. Yolk concentrations of 17-beta estradiol were significantly lower at embryonic stage 12 in eggs from MINWR when compared to the other two sites. Reductions in yolk 17-beta estradiol concentrations from embryonic stage 12 to 24 were significantly attenuated in eggs from MINWR versus that of Lakes Woodruff and Apopka. This study suggests that altered deposition of yolk steroids, and possibly differential utilization by the embryo, could be a contributory mechanism in the reproductive and developmental abnormalities seen in alligators from contaminated locales.

Posthatching development of Alligator mississippiensis ovary and testis

We investigated ovary and testis development of Alligator mississippiensis during the first 5 months posthatch. To better describe follicle assembly and seminiferous cord development, we used histochemical techniques to detect carbohydrate‐rich extracellular matrix components in 1‐week, 1‐month, 3‐month, and 5‐month‐old gonads. We found profound morphological changes in both ovary and testis. During this time, oogenesis progressed up to diplotene arrest and meiotic germ cells increasingly interacted with follicular cells. Concomitant with follicles becoming invested with full complements of granulosa cells, a periodic acid Schiffs (PAS)‐positive basement membrane formed. As follicles enlarged and thecal layers were observed, basement membranes and thecal compartments gained periodic acid‐methionine silver (PAMS)‐reactive fibers. The ovarian medulla increased first PAS‐ and then PAMS reactivity as it fragmented into wide lacunae lined with low cuboidal to squamous epithelia. During this same period, testicular germ cells found along the tubule margins were observed progressing from spermatogonia to round spermatids located within the center of tubules. Accompanying this meiotic development, interstitial Leydig cell clusters become more visible and testicular capsules thickened. During the observed testis development, the thickening tunica albuginea and widening interstitial tissues showed increasing PAS‐ and PAMS reactivity. We observed putative intersex structures in both ovary and testis. On the coelomic aspect of testes were cell clusters with germ cell morphology and at the posterior end of ovaries, we observed “medullary rests” resembling immature testis cords. We hypothesize laboratory conditions accelerated gonad maturation due to optimum conditions, including nutrients and temperature. Laboratory alligators grew more rapidly and with increased body conditions compared with previous measured, field‐caught animals. Additionally, we predict the morphological maturation observed in these gonads is concomitant with increased endocrine activities. J. Morphol. 2010.