Stress and reproductive events detected in North Atlantic right whale blubber using a simplified hormone extraction protocol.

As studies quantifying steroid hormones in marine mammal blubber progress, methodological refinements may improve the utility and consistency of blubber hormone measurements. This study advances blubber extraction methodologies by testing a simplified extraction protocol that reduces time and complexity compared to a protocol widely used in cetacean blubber studies. Using blubber samples archived from remote biopsy (n = 21 live whales) and necropsy collection (n = 7 dead whales) of North Atlantic right whales (NARW; Eubalaena glacialis) of known life history states, we performed analytical and biological validations to assess the feasibility of measuring reproductive (testosterone, progesterone) and glucocorticoid (cortisol) hormones in blubber via enzyme immunoassay following the simplified extraction. Analytical validations (parallelism, accuracy, extraction efficiency, repeatability) showed the simplified extraction produced similar results to the extended protocol, offering a more efficient and consistent technique. In live, apparently healthy whales, blubber testosterone concentrations (mean ± SE) were significantly higher in males (2.02 ± 0.36 ng/g) compared to females (0.81 ± 0.15 ng/g). Blubber progesterone was highest in a confirmed pregnant female (60.3 ng/g), which was 12-fold greater than the mean concentration of non-pregnant females (4.56 ± 0.88 ng/g). Blubber cortisol concentrations in whales that died from anthropogenic causes averaged 5.31 ± 2.28 ng/g, whereas most live, healthy whales had cortisol values below 1 ng/g. Among living whales, a whale actively entangled in fishing gear had the highest blubber cortisol measurement (3.51 ng/g), exhibiting levels similar to whales that died from acute entanglement (2.88 ± 0.42 ng/g). Overall, the highest blubber cortisol concentration (18.0 ng/g) was measured in a dead whale with a severe chronic entanglement, approximately 30-fold greater than mean blubber cortisol of apparently healthy whales (0.58 ± 0.11 ng/g). The methodological approach presented here provides a reference for researchers interested in an alternative, streamlined technique for hormone extraction of cetacean blubber and contributes to the diverse tool set for stress and reproductive assessments of endangered NARWs.

Ultrasound imaging improves hormone therapy strategies for induction of ovulation and in vitro fertilization in the endangered dusky gopher frog (Lithobates sevosa).

Establishing captive breeding populations of amphibians is an important conservation strategy to safeguard against ongoing declines of wild populations and provide broodstock for reintroduction programs. The endangered dusky gopher frog (DGF) has never naturally reproduced in captivity and requires breeding intervention to sustain the population. Methods for inducing ovulation in female DGFs using hormone therapies have not been evaluated. To address this need, we tested four exogenous hormone treatments to induce ovulation in DGFs (n = 11/treatment), including: treatment (A) gonadotropin-releasing hormone agonist (GnRHa); (B) GnRHa with dopamine antagonist metoclopramide hydrochloride; (C) GnRHa and human chorionic gonadotropin (hCG) and (D) GnRHa with hCG following two low hCG priming doses. Treatments B, C and D resulted in a significantly greater (P < 0.0125) number of ovulating females compared to the control (no hormone); Treatment A was not different from control. For ovulating females, the number of eggs, relative fecundity and cleavage rates of eggs were compared between the four hormone treatments and initial ultrasound grade. Between treatments, there was no difference in number of eggs or relative fecundity; however, Treatments A and D resulted in higher (P < 0.05) cleavage rates than Treatment C, but were not different from Treatment B. Ultrasound imaging was used to assess the ovarian state of DGF females prior to and following hormone therapy. A grading scale (Grades 1-5) was developed to characterize ovarian states. Ultrasound grade was found to be a significant (P = 0.002) predictor for ovulation following hormone treatment, with only high-grade females (Grades 3-4) ovulating in response to hormones. Ultrasound grade did not influence egg numbers or cleavage rate (P > 0.05). Results demonstrate multiple hormone therapies are available for stimulating ovulation in female DGFs and ultrasonography is a valuable tool to inform hormone therapy. Ultimately, these reproductive technologies are critical to enhance breeding and reintroduction efforts for the DGF.

Biological sex identification in the endangered dusky gopher frog (Lithobates sevosa): a comparison of body size measurements, secondary sex characteristics, ultrasound imaging, and urinary hormone analysis methods.

BACKGROUND: Accurate sex identification techniques are important for wildlife demographic studies and for genetic management of captive breeding colonies. Various non-invasive methods for identification of biological sex in the weakly dimorphic endangered dusky gopher frog (DGF; Lithobates sevosa) were explored to support planned recovery efforts for this species including breeding and augmentation of wild populations. METHODS: Body size (snout-vent length and body weight) measurements, observation of nuptial pads, ultrasound imaging, and urinary hormone analysis for testosterone and estrone were performed on 27 male and 19 female DGFs. For each method, the mean and range of measurement values were determined for male and female DGFs housed in a captive breeding population. The ability of these methods to accurately predict the true biological sex of the individuals was assessed retrospectively. RESULTS: Body size measurements were of limited use for sex identification purposes, as males and females demonstrated overlapping body lengths and weights. Observation of the presence/absence of nuptial pads in males and females, respectively, proved to be accurate and easy to perform in most cases. Ultrasound imaging was useful for predicting the sex of female frogs, particularly when females were gravid. Commercial enzyme immunoassay kits were validated to measure urinary hormones in the DGF. Mean urinary testosterone (males: 2.22 ± 0.38 ng/ml; females: 0.92 ± 0.11 ng/ml) and estrone (males: 0.08 ± 0.01 ng/ml; females: 1.50 ± 0.39 ng/ml) concentrations were significantly (p < 0.05) different between the sexes. However, there was some overlap in hormone concentrations between the sexes. When a ratio of testosterone (T) to estrone (E) concentrations was calculated for each individual, males demonstrated significantly greater T/E ratios compared to females (p < 0.05). Use of this ratio showed greater accuracy in predicting the sex of the animal compared to using testosterone or estrone concentrations alone. CONCLUSIONS: Monitoring for presence/absence of nuptial pads and using urinary testosterone to estrone hormone ratios were the most accurate methods for identifying the biological sex of adult DGFs. Urinary hormone measurements for sex identification may be useful in other weakly dimorphic and monomorphic amphibian species in both ex situ and in situ settings.

Examining factors that may influence accurate measurement of testosterone in sea turtles.

Differences in reported testosterone concentrations in male sea turtle blood samples are common in the veterinary literature, but may be accounted for by differences in sample handling and processing prior to assay. Therefore, our study was performed to determine best practices for testosterone analysis in male sea turtles (Caretta caretta and Chelonia mydas). Blood samples were collected into 5 collection tube types, and assay validation and measured testosterone concentrations were compared across different sample storage (fresh, refrigerated 1 week, or frozen), extraction (unextracted or ether-extracted), and processing treatment (untreated, homogenized, or dissociation reagent) conditions. Ether-extracted and dissociation reagent-treated samples validated in all conditions tested and are recommended for use, as unextracted samples validated only if assayed fresh. Dissociation reagent treatment was simpler to perform than ether extraction and resulted in total testosterone concentrations ~2.7-3.5 times greater than free testosterone measured in ether-extracted samples. Sample homogenization did not affect measured testosterone concentrations, and could be used to increase volume in gelled samples. An annual seasonal testosterone increase was observed in both species when ether extraction or dissociation reagent treatment was used. Annual deslorelin implant treatments in a Chelonia mydas male resulted in suppression of seasonal testosterone following the fourth treatment. Seasonal testosterone patterns resumed following discontinuation of deslorelin. Comparison of in-house and commercially available enzyme immunoassay kits revealed similar patterns of seasonal testosterone increases and deslorelin-induced suppression. Our study highlights the importance of methodological validation and provides laboratorians with best practices for testosterone enzyme immunoassay in sea turtles.

Regulation of survivin by ErbB2 signaling: therapeutic implications for ErbB2-overexpressing breast cancers.

In breast cancer, overexpression of ErbB2 or aberrant regulation of survivin, a member of the inhibitor of apoptosis family, is associated with resistance to chemo/hormone therapy and predicts for a poor clinical outcome. A functional link between the two predictive factors has not been previously shown. Here, using genetic and pharmacologic approaches to block ErbB2 signaling, we show that ErbB2 regulates survivin protein expression in ErbB2-overexpressing breast cancer cells. Selective knockdown of ErbB2 using small interfering RNA markedly reduced survivin protein, resulting in apoptosis of ErbB2-overexpressing breast cancer cell lines such as BT474. Alternatively, inhibition of ErbB2 signaling using lapatinib (GW572016), a reversible small-molecule inhibitor of ErbB1/ErbB2 tyrosine kinases, at pharmacologically relevant concentrations, leads to marked inhibition of survivin protein with subsequent apoptosis. The effect of lapatinib on survivin seems to be predominantly posttranslational, mediated by ubiquitin-proteosome degradation as lactacystin, a proteosome inhibitor, reverses these effects. Furthermore, lapatinib down-regulated the expression of His-tagged survivin, which was under the transcriptional control of a heterologous promoter, providing additional evidence supporting a posttranslational mechanism of regulation. In contrast, trastuzumab and gefitinib failed to down-regulate survivin in ErbB2-overexpressing breast cancer cells. Importantly, the clinical relevance of these findings was illustrated in patients with ErbB2-overexpressing breast cancer whose clinical response to lapatinib was associated with marked inhibition of survivin in their tumors. These findings shed new light on the mechanism by which ErbB2 overexpression protects against apoptotic stimuli in breast cancer and identifies therapeutic interventions to improve clinical outcomes in these aggressive tumors.