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Journal: General and comparative endocrinology

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The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0-4°C did not strongly affect fGCM concentrations within 24h, simplifying sample collection when immediate storage at -20°C is not feasible. The two cheetahs in Germany also received an injection of [(3)H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs.

Concepts: Cortisol, Chromatography, High performance liquid chromatography, Cushing's syndrome, Adrenocorticotropic hormone, Adrenal cortex, Addison's disease, Cheetah

28

Susceptibility during fasting has been reported for the common vampire bat (Desmodus rotundus), to the point of untimely deaths after only 2 to 3 nights of fasting. To investigate the underlying physiology of this critical metabolic condition, we analyzed serum insulin levels, pancreatic islets morphometry and immunocytochemistry (ICC), static insulin secretion in pancreas fragments, and insulin signaling mechanism in male vampire bats. A glucose tolerance test (ipGTT) was also performed. Serum insulin was found to be lower in fed vampires compared to other mammals, and was significantly reduced after 24 hours fasting. Morphometrical analyses revealed small irregular pancreatic islets with reduced percentage of ß-cell mass compared to other bats. Static insulin secretion analysis showed that glucose-stimulated insulin secretion was impaired, as insulin levels did not reach significance under high glucose concentrations, whereas the response to the amino acid leucin was preserved. Results from ipGTT showed a failure on glucose clearance, indicating glucose intolerance due to diminished pancreatic insulin secretion and/or decreased β-cell response to glucose. In conclusion, data presented here indicate lower insulinemia and impaired insulin secretion in D. rotundus, which is consistent with the limited ability to store body energy reserves, previously reported in these animals. Whether these metabolic and hormonal features are associated with their blood diet remains to be determined. The peculiar food sharing through blood regurgitation, reported to this species, might be an adaptive mechanism overcoming this metabolic susceptibility.

Concepts: Insulin, Islets of Langerhans, Pancreas, Glucose tolerance test, Insulin resistance, Vampire bat, Common Vampire Bat, Vampire

28

Sexual maturation in the stickleback is controlled by photoperiod. The aim of this study was to find out whether changes in feedback effects exerted by sex steroids could mediate the photoperiodic effect, which is regarded to be of an all-or-nothing character. To that end, males were castrated and treated with different doses of testosterone (T) and in one experiment also with the aromatase inhibitor fadrozole (AI) and kept under different photoperiods. In control fish, long day (LD 16:8) stimulated maturation, associated with more hypertrophied kidneys (a secondary sexual character) and higher levels of pituitary lhb and fshb mRNA than under short day conditions (LD 8:16). Under LD 8:16, low doses of T suppressed both lhb and fshb mRNA levels. However, with the use of high doses of T and/or longer photoperiods the inhibitory effects on lhb and fshb mRNA levels became less clear or instead positive effects were observed. Under intermediate photoperiod conditions, the negative feedback effect of a low dose of T on fshb was more prominent with shorter photoperiods, whereas no such shift was observed for lhb mRNA. The inhibitory effect of the low dose of T on lhb mRNA levels under LD 8:16 was abolished by AI, whereas the stimulatory effect of the high dose of T was not. The negative feedback effects were more marked under short days than under long days, whereas positive feedback effects were more marked under long days. The suppression of both fshb and lhb mRNA levels by low androgen levels, especially under short days, may inhibit maturation completely unless a rise of androgens above threshold levels would allow complete maturation.

Concepts: Testosterone, Androgen, Control theory, Feedback, Negative feedback, Three-spined stickleback, Photoperiodism, Gasterosteiformes

28

Studies on the role of thyroid hormones (THs) in teleost fish physiology have deployed the synthetic goitrogens, methimazol (MMI), propilthiouracil (PTU) and thiourea (TU) that are used to treat human hyperthyroidism. However, the action of the goitrogens, MMI, PTU and TU at different levels of the hypothalamic-pituitary-thyroid (HPT) axis in teleosts is largely unknown. The central importance of the hypothalamus and pituitary in a number of endocrine regulated systems and the cross-talk that occurs between different endocrine axes makes it pertinent to characterize the effects of MMI, PTU and TU, on several endpoints of the thyroid system. The marine teleost, sea bream (Sparus auratus) was exposed to MMI, PTU and TU (1mg/kg wet weight per day), via the diet for 21days. Radioimmunoassays (RIA) of plasma THs and ELISA of the TH carrier transthyretin (TTR) revealed that MMI was the only chemical that significantly reduced plasma TH levels (p<0.05), although both MMI and PTU significantly (p<0.05) reduced plasma levels of circulating TTR (p<0.05). Histological analysis of the thyroid tissue revealed modifications in thyrocyte activity that explain the reduced circulating levels of THs. MMI also significantly (p<0.05) up-regulated transcript abundance of liver deiodinase 1 and 2 while significantly (p<0.05) decreasing TRβ expression in the pituitary, all hallmarks of HPT axis action of goitrogens in vertebrates. The results indicate that in the sea bream MMI is the most effective goitrogen followed by PTU and that TU (1mg/kg wet weight for 21days) failed to have a goitrogenic effect. The study highlights the non-uniform effect of goitrogens on the thyroid axis of sea bream and provides the basis for future studies of thyroid disrupting pollutants.

Concepts: Thyroid disease, Hyperthyroidism, Thyroid, Endocrine system, Actinopterygii, Goitrogen, Teleostei, Goitrin

28

The neurohypophyseal hormone arginine vasotocin (AVT) mediates behavioral and reproductive plasticity in vertebrates, and has been linked to the behavioral changes associated with protogyny in the bluehead wrasse (Thalassoma bifasciatum). In this study, we sequenced full-length cDNAs encoding two distinct V1a-type AVT receptors (v1a1 and v1a2) from the bluehead wrasse, and examined variation in brain and gonadal abundance of these receptor transcripts among sexual phases. End point RT-PCR revealed that v1a1 and v1a2 transcripts varied in tissue distribution, with v1a1 receptor mRNAs at greatest levels in the telencephalon, hypothalamus, optic tectum, cerebellum and testis, and v1a2 receptor transcripts most abundant in the hypothalamus, cerebellum and gills. In the brain, v1a1 and v1a2 mRNAs both localized by in situ hybridization to the dorsal and ventral telencephalon, the preoptic area of the hypothalamus, the ventral hypothalamus and lateral recess of the third ventricle. Quantitative real-time RT-PCR revealed that relative abundance of these two receptor mRNAs varied significantly in brain and gonad with sexual phase. Relative levels of v1a2 mRNAs were greater in whole brain and isolated hypothalamus of terminal phase (TP) male wrasse compared to initial phase (IP) males or females. In the gonad, v1a1 mRNAs were at levels 2.5-fold greater in the testes of IP males - and 4-5-fold greater in the testes of TP males - compared to the ovaries of females. These results provide evidence that V1a-type AVT receptor transcript abundance in the hypothalamus and gonads of bluehead wrasse varies in patterns linked to sexual phase, and bestow a foundation for future studies investigating how differential expression of v1a1 and v1a2 teleost AVT receptors links to behavioral status and gonadal function in fish more broadly.

Concepts: Neuroanatomy, Hypothalamus, Brain, Testicle, Reproductive system, Gamete, Ovary, Gonad

28

Non-invasive measurement of urinary corticosterone and testosterone metabolites in amphibians provides opportunities for endocrine studies of responses to physiological and psychological stressors. Typically, corticosterone metabolite concentrations increase in frog urine within 1-2h of a mild capture and handling stress protocol. However, no study has investigated the effect of duration of manual restraint on the changes in corticosterone and reproductive hormones in amphibians. We quantified urinary corticosterone and testosterone metabolite responses for 8h following various durations of manual restraint (control, 5, 15 or 30min) in adult male cane toads (Rhinella marina) under controlled laboratory conditions. All toads had a corticosterone stress response over 8h to our standard capture and handling stressor. The mean corticosterone stress response was significantly higher after 15 or 30min restraint in comparison to the control (no restraint) or to 5min restraint. Manual restraint for 5, 15 or 30min caused a significant reduction in urinary testosterone concentrations over 8h. We also provide a novel method of quantifying plasticity in corticosterone stress responses in amphibians with respect to restraint duration using the concept of a “reaction norm”. The reaction norm, which was calculated as slope of the regression line of integrated corticosterone response against restraint duration, was 9.69 (pg corticosterone/μg creatinineh)/min for male toads. In summary, corticosterone and testosterone responses to restraint are affected by restraint duration in male toads. Glucocorticoid reaction norms can be applied to study the change in physiological stress hormonal response with respect to restraint duration in other amphibian species.

Concepts: Reproduction, Hormone, Adrenal cortex, Amphibian, Frog, Toad, Toads, True toad

28

Nitric oxide (NO), a short-lived freely diffusible radical gas that acts as an important biological signal, regulates an impressive spectrum of physiological functions in vertebrates including fishes. The action of NO, however, on thyroid hormone status and its role in the integration of acid-base, osmotic and metabolic balances during stress are not yet delineated in fish. Sodium nitroprusside (SNP), a NO donor, was employed in the present study to investigate the role of NO in the stressed air-breathing fish Anabas testudineus. Short-term SNP treatment (1mM; 30min) interacted negatively with thyroid axis, as evident in the fall of plasma thyroxine in both stressed and non-stressed fish. In contrast, the cortisol responsiveness to NO was negligible. SNP challenge produced systemic alkalosis, hypocapnia and hyperglycemia in non-stressed fish. Remarkable acid-base compensation was found in fish kept for 60 min net confinement where a rise in blood pH and HCO(3) content occurred with a reduction in PCO(2) content. SNP challenge in these fish, on the contrary, produced a rise in oxygen load together with hypocapnia but without an effect on HCO(3) content, indicating a modulator role of NO in respiratory gas transport during stress response. SNP treatment reduced Na(+), K(+) ATPase activity in the gill, intestine and liver of both stressed and non-stressed fish, and this suggests that stress state has little effect on the NO-driven osmotic competence of these organs. On the other hand, a modulatory effect of NO was found in the kidney which showed a differential response to SNP, emphasizing a key role of NO in kidney ion transport and its sensitivity to stressful condition. H(+)-ATPase activity, an index of H(+) secretion, downregulated in all the organs of both non-stressed and stressed fish except in the gill of non-stressed fish and this supports a role for NO in promoting alkalosis. The data indicate that, (1) NO interacts antagonistically with T(4) (2) modifies respiratory gas transport and (3) integrates acid-base and osmotic actions during stress response in air-breathing fish. Collectively, this first evidence in fish indicate that NO promotes compensatory physiologic modification and that can reduce the magnitude of stress-induced acid-base and osmotic disturbance.

Concepts: Kidney, Physiology, Fish, Hormone, Thyroid, Thyroid hormone, Anabas testudineus, Thyroglobulin

27

We validate fecal glucocorticoid (GC) and thyroid (T3) hormone metabolite measures in the Critically Endangered Hawaiian monk seal for the first time, and examine variation in the concentrations of these hormones in individuals across the species' range. We test hypotheses that monk seals from declining subpopulations have relatively high GCs and low T3 on average suggesting impacts of food limitation, and that this hormone pattern is more apparent in immature animals compared to adults, as food limitation is specifically indicated as a principal cause of poor body condition and survival of juvenile monk seals. We opportunistically sampled scat from 84 individually identifiable monk seals during the 2010 breeding season from two geographic regions, the main Hawaiian Islands (MHI) and the Northwestern Hawaiian Islands (NWHI). The MHI subpopulation of monk seals is growing, whereas subpopulations at many sites in the NWHI are in decline. Best fit general linear models predicting variation in GCs and T3 (examined separately) were similar (after accounting for significantly elevated hormone concentrations associated with molt and possibly lactation); both included sample date, region, and monk seal age as predictors. GC concentrations were significantly lower in MHI versus NWHI monk seals and decreased as the breeding season progressed. T3 concentrations were significantly lower in immature monk seals compared to adults. GC and T3 concentrations were positively correlated at 4 NWHI sites; prey may be adequate for physiological growth or maintenance at these sites but relatively stressful to acquire. GCs were highest at French Frigate Shoals, (a NWHI site) while T3 was relatively low here, indicating a possible signal of food limitation. GCs were lowest in the MHI. Disturbance associated with living near a high human population in the MHI appears to impact monk seal physiology less than other stressors encountered in the remote and highly protected NWHI where human presence is extremely low.

Concepts: Hawaiian Islands, Earless seal, Laysan, Caribbean Monk Seal, Mediterranean Monk Seal, Hawaiian Monk Seal, Northwestern Hawaiian Islands, French Frigate Shoals

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The capa peptide family, originally identified in the tobacco hawk moth, Manduca sexta, is now known to be present in many insect families, with increasing publications on capa neuropeptides each year. The physiological actions of capa peptides vary depending on the insect species but capa peptides have key myomodulatory and osmoregulatory functions, depending on insect lifestyle, and life stage. Capa peptide signalling is thus critical for fluid homeostasis and survival, making study of this neuropeptide family attractive for novel routes for insect control. In Dipteran species, including the genetically tractable Drosophila melanogaster, capa peptide action is diuretic; via elevation of nitric oxide, cGMP and calcium in the principal cells of the Malpighian tubules. The identification of the capa receptor (capaR) in several insect species has shown this to be a canonical GPCR. In D. melanogaster, ligand-activated capaR activity occurs in a dose-dependent manner between 10(-6) and 10(-12) M. Lower concentrations of capa peptide do not activate capaR, either in adult or larval Malpighian tubules. Use of transgenic flies in which capaR is knocked-down in only Malpighian tubule principal cells demonstrates that capaR modulates tubule fluid secretion rates and in doing so, sets the organismal response to desiccation. Thus, capa regulates a desiccation-responsive pathway in D. melanogaster, linking its role in osmoregulation and fluid homeostasis to environmental response and survival. The conservation of capa action between some Dipteran species suggests that capa’s role in desiccation tolerance may not be confined to D. melanogaster.

Concepts: Genetics, Biology, Organism, Genome, Insect, Chromosome, Drosophila melanogaster, Drosophila

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Salivary glands, although widely considered as typically exocrine, may also release specific proteins in an endocrine manner. However, endocrine release of salivary gland proteins is not generally acknowledged since the evidences are not easily demonstrable. Submandibular salivary glands (SMG) of male Syrian hamsters express male-specific secretory proteins (MSP) visible in SDS-PAGE of SMG extracts, as major bands and also detectable in immunoblots of whole-saliva and urine as low MSP crossreactions. We report here that MSP is localized in acinar cells of SMG and acute treatment with isoproterenol (IPR; non-specific β1/β2-adrenergic agonist) results in considerable release of MSP in SMG-saliva. Moreover, acute IPR treatment markedly depletes SMG-MSP in a dose- and time-dependent manner. However, MSP depleted from SMG, far exceeds that recovered in SMG-saliva. Blood, submandibular lymph nodes and kidney of IPR-treated males showed MSP crossreactions and SDS-PAGE of their urine revealed profuse MSP excretion; this was undetectable in IPR-treated-SMG-ablated males, confirming that a substantial amount of MSP depleted from SMG after IPR treatment enters circulation and is excreted in urine. Treatments with specific β1- or β2-adrenergic agonists also reduced SMG-MSP levels and resulted in copious urinary excretion of MSP. Co-treatments with specific β1/β2-blockers indicated that above effects of IPR, β1- and even β2-agonists are very likely mediated by β1-adrenoceptors. MSP’s detection by SDS-PAGE in urine after β-agonist treatment is a compelling and easily demonstrable evidence of release into circulation of a salivary gland protein. The possible means (endocrine-like or otherwise) of MSP’s release into circulation and significance of its presence in saliva, blood and urine of male hamsters are discussed.

Concepts: Blood, Secretion, Pancreas, Glands, Hamster, Salivary gland, Exocrine system, Submandibular gland