Optimizing hormone extraction protocols for whale baleen: Tackling questions of solvent:sample ratio and variation.

Obtaining endocrine data from alternative sample types such as baleen and other keratinized tissues has proven a valuable tool to investigate reproductive and stress physiology via steroid hormone quantification, and metabolic stress via thyroid hormone quantification in whales and other vertebrates. These alternative sample types provide an integrated measure of plasma levels over the period that the structure was growing, thus capturing months or even years of an individual's endocrine history. Additionally, their robust and stable keratin matrix allows such samples to be stored for years to decades, enabling the analysis and comparison of endocrine patterns from past and modern populations. However, the extraction and analysis of hormones from baleen and other keratinized tissues remains novel and requires both biological and analytical validations to ensure the method fulfills the requirements for its intended use. We utilized baleen recovered at necropsy from southern right whales (Eubalaena australis) that died at Península Valdés, Argentina, using a commercially available progesterone enzyme immunoassay (EIA) to address two methodological questions: 1) what is the minimum sample mass required to reliably quantify hormone content of baleen samples analyzed with commercially available EIAs, and 2) what is the optimal ratio of solvent volume to sample mass, i.e., the ratio that yields the maximum amount of hormone with high accuracy and low variability between replicates. We concluded that masses of at least 20 mg should be used whenever possible, and extraction is best performed using an 80:1 ratio of solvent to sample (volume of solvent to sample mass; µl:mg). These results can help researchers to make informed methodological decisions when using a destructive extraction method with rare or unique specimens.

Perchlorate disrupts embryonic androgen synthesis and reproductive development in threespine stickleback without changing whole-body levels of thyroid hormone.

Perchlorate, an environmental contaminant, disrupts normal functioning of the thyroid. We previously showed that perchlorate disrupts behavior and gonad development, and induces external morphological changes in a vertebrate model organism, the threespine stickleback. Whether perchlorate alters these phenotypes via a thyroid-mediated mechanism, and the extent to which the effects depend on dose, are unknown. To address these questions, we chronically exposed stickleback to control conditions and to three concentrations of perchlorate (10, 30 and 100ppm) at various developmental stages from fertilization to reproductive maturity. Adults chronically exposed to perchlorate had increased numbers of thyroid follicles and decreased numbers of thyrocytes. Surprisingly, T4 and T3 levels in larval, juvenile, and adult whole fish chronically exposed to perchlorate did not differ from controls, except at the lowest perchlorate dose, suggesting a non-monotonic dose response curve. We found no detectable abnormalities in external phenotype at any dose of perchlorate, indicating that the increased number of thyroid follicles compensated for the disruptive effects of these doses. In contrast to external morphology, gonadal development was altered substantially, with the highest dose of perchlorate causing the largest effects. Perchlorate increased the number both of early stage ovarian follicles in females and of advanced spermatogenic stages in males. Perchlorate also disrupted embryonic androgen levels. We conclude that chronic perchlorate exposure may not result in lasting adult gross morphological changes but can produce lasting modifications to gonads when compensation of T3 and T4 levels occurs by thyroid follicle hyperplasia. Perchlorate may therefore affect vertebrate development via both thyroidal and non-thyroidal mechanisms.

Hypothalamic remodeling of thyroid hormone signaling during hibernation in the arctic ground squirrel.

Hibernation involves prolonged intervals of profound metabolic suppression periodically interrupted by brief arousals to euthermy, the function of which is unknown. Annual cycles in mammals are timed by a photoperiodically-regulated thyroid-hormone-dependent mechanism in hypothalamic tanycytes, driven by thyrotropin (TSH) in the pars tuberalis (PT), which regulates local TH-converting deiodinases and triggers remodeling of neuroendocrine pathways. We demonstrate that over the course of hibernation in continuous darkness, arctic ground squirrels (Urocitellus parryii) up-regulate the retrograde TSH/Deiodinase/TH pathway, remodel hypothalamic tanycytes, and activate the reproductive axis. Forcing the premature termination of hibernation by warming animals induced hypothalamic deiodinase expression and the accumulation of secretory granules in PT thyrotrophs and pituitary gonadotrophs, but did not further activate the reproductive axis. We suggest that periodic arousals may allow for the transient activation of hypothalamic thyroid hormone signaling, cellular remodeling, and re-programming of brain circuits in preparation for the short Arctic summer.

Estimating lean mass over a wide range of body composition: a calibration of deuterium dilution in the arctic ground squirrel.

Calculating body water through isotope dilution has become a useful way to nondestructively estimate body composition in many species. The most accurate estimates using this method require calibration against proximate chemical analysis of body composition for individual species, but no studies to our knowledge have calibrated this method on a hibernating mammal that seasonally undergoes dramatic changes in body composition. We use deuterium oxide to estimate total body water in captive arctic ground squirrels, Urocitellus parryii, and compare two approaches of calculating lean mass from total body water, both calibrated against lean mass based on proximate analysis. The first method uses a single tissue hydration constant to calculate lean mass from total body water; the second method uses a predictive equation to calculate lean mass from total body water and body mass. We found that the predictive equation performs better over the large range of body composition common to this species. Distillation of blood samples did not affect lean mass estimates from either calculation method. These findings indicate that isotope dilution using a predictive equation should work well as an alternative to destructive methods in other small mammals that undergo radical changes in body composition across their annual cycle.

Interannual variation in the adrenal responsiveness of black-legged kittiwake chicks (Rissa tridactyla).

Over the last decade, field endocrinologists have explored the efficacy of using plasma corticosterone concentrations of breeding seabirds as an indicator of food availability and predictor of breeding success. However, studies have been predominately confined to adults and the results have been inconsistent. We examined the relationship between the productivity of black-legged kittiwakes (Rissa tridactyla) and the baseline and stress-induced corticosterone levels of 12-15 day-old kittiwake chicks in a multi-year study in Chiniak Bay, Kodiak, Alaska. We predicted that corticosterone levels would negatively correlate with productivity. Productivity decreased across years from relatively high levels in 2002, to very low levels in 2004 and 2005. Baseline corticosterone levels of the chicks did not increase consistently across years. Stress-induced corticosterone levels were statistically indistinguishable in high productivity years but increased significantly in low productivity years. The decline in kittiwake productivity coincided with warming ocean conditions, which, historically, are linked to declines in forage abundance for kittiwakes. Inconsistent changes in baseline corticosterone levels suggest some adult kittiwakes were able to buffer their chicks from poor foraging conditions. However, large increases in stress-induced corticosterone levels during the low productivity years indicate chicks were physiologically stressed for some period prior to sampling. Our results suggest that stress-induced corticosterone levels of kittiwake chicks are effective indicators of productivity in poor years, whereas the changes in baseline corticosterone levels across years are not as pronounced and therefore may not be as reliable.