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Iodine, an essential component of thyroid hormones, can only be obtained through the diet. The sodium iodide symporter (NIS) transports iodide across mammalian intestinal and thyroid epithelia to deliver iodide for thyroid hormone production. Using reverse transcription-polymerase chain reaction (RT-PCR) we confirmed that mRNA for a homolog of mammalian NIS is expressed in comparable locations, both sub-pharyngeal thyroid tissue and intestine, in multiple teleost fish species, supporting a conserved mechanism for intestinal-thyroid iodine transport across vertebrates. To determine when in embryogenesis NIS expression is initiated we utilized in situ hybridization (ISH) during development of zebrafish (Danio rerio) embryos. This revealed expression of nis as early as 2 days post fertilization (dpf) along the dorsal surface of the yolk sac, suggesting a function to import iodine from yolk. To evaluate the potential for maternal deposition of iodine in yolk, RT-PCR and further in situ staining of ovarian tissue in gravid female zebrafish confirmed NIS mRNA presence in the ooplasm and granulosa layer of early stage follicles. This further suggests that maternally-deposited NIS mRNA may be available for early embryogenesis. Unexpectedly, ISH in embryos revealed robust nis expression in the central nervous system throughout days 2-5 days post fertilization, with adult whole brain ISH localizing expression in the hypothalamus, cerebellum, and optic tectum. RT-PCR on whole brain tissue from five species of adult fish representing three taxonomic orders likewise revealed robust CNS expression. These unexpected locations of nis expression suggest novel, as yet undescribed reproductive and neural functions of NIS in teleost species., (Published by Elsevier Inc.)

Neutrophil myeloperoxidase (MPO) catalyzes the H 2 O 2 -dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N -retinylidene- N -retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N -retinylidene- N -retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Thyroxine (T 4 ) undergoes dynamic daily cycles in the perciform fish the red drum, Sciaenops ocellatus, that are inversely timed to cycles of thyrotropin (TSH) subunit mRNA expression in the pituitary gland. We have proposed that these daily cycles are regulated by negative feedback of circulating T 4 on expression of pituitary thyroid hormone deiodinase type 3 (Dio3), such that elevated circulating T 4 results in diminished pituitary thyroid hormone catabolism and consequent increased negative feedback on expression of TSH subunits during the day. To determine whether thyroid hormones function to modulate expression of pituitary deiodinase enzymes we developed an immersion technique to administer physiological doses of T 3 and T 4 in vivo. Immersion in T 4 or T 3 significantly inhibited the mRNA expression of the TSH α and β subunits from 4 to 66h of immersion. Pituitary Dio3 expression was significantly diminished by T 3 and T 4 at 22h. These results indicate that both T 4 and T 3 are capable of negative feedback regulation of TSH subunit expression in red drum at physiological concentrations and on a time scale consistent with the T 4 daily cycle. Furthermore, thyroid hormones negatively regulate Dio3 expression in the pituitary in a manner suggesting that negative thyroxine feedback on Dio3 promotes the release of TSH subunits from TH inhibition and may be an important mechanism for generating daily thyroid hormone cycles. These results highlight a potentially important role for D3 in mediating thyroid hormone feedback on TSH expression, not previously described in other species., (Published by Elsevier Inc.)

Prolonged food deprivation in mammals typically reduces glucose, insulin, and thyroid hormone (TH) concentrations, as well as tissue deiodinase (DI) content and activity, which, collectively, suppress metabolism. However, in elephant seal pups, prolonged fasting does not suppress TH levels; it is associated with upregulation of adipose TH-mediated cellular mechanisms and adipose-specific insulin resistance. The functional relevance of this apparent paradox and the effects of glucose and insulin on TH-mediated signaling in an insulin-resistant tissue are not well defined. To address our hypothesis that insulin increases adipose TH signaling in pups during extended fasting, we assessed the changes in TH-associated genes in response to an insulin infusion in early- and late-fasted pups. In late fasting, insulin increased DI1, DI2, and THrβ-1 mRNA expression by 566%, 44%, and 267% at 60 min postinfusion, respectively, with levels decreasing by 120 min. Additionally, we performed a glucose challenge in late-fasted pups to differentiate between insulin- and glucose-mediated effects on TH signaling. In contrast to the insulin-induced effects, glucose infusion did not increase the expressions of DI1, DI2, and THrβ-1 until 120 min, suggesting that glucose delays the onset of the insulin-induced effects. The data also suggest that fasting duration increases the sensitivity of adipose TH-mediated mechanisms to insulin, some of which may be mediated by increased glucose. These responses appear to be unique among mammals and to have evolved in elephant seals to facilitate their adaptation to tolerate an extreme physiological condition., (Copyright © 2016 the American Physiological Society.)

Norepinephrine (NE), the primary neurotransmitter of the sympathetic nervous system, has been reported to be a chemoattractant for enterohemorrhagic Escherichia coli (EHEC). Here we show that nonpathogenic E. coli K-12 grown in the presence of 2 μM NE is also attracted to NE. Growth with NE induces transcription of genes encoding the tyramine oxidase, TynA, and the aromatic aldehyde dehydrogenase, FeaB, whose respective activities can, in principle, convert NE to 3,4-dihydroxymandelic acid (DHMA). Our results indicate that the apparent attractant response to NE is in fact chemotaxis to DHMA, which was found to be a strong attractant for E. coli. Only strains of E. coli K-12 that produce TynA and FeaB exhibited an attractant response to NE. We demonstrate that DHMA is sensed by the serine chemoreceptor Tsr and that the chemotaxis response requires an intact serine-binding site. The threshold concentration for detection is ≤5 nM DHMA, and the response is inhibited at DHMA concentrations above 50 μM. Cells producing a heterodimeric Tsr receptor containing only one functional serine-binding site still respond like the wild type to low concentrations of DHMA, but their response persists at higher concentrations. We propose that chemotaxis to DHMA generated from NE by bacteria that have already colonized the intestinal epithelium may recruit E. coli and other enteric bacteria that possess a Tsr-like receptor to preferred sites of infection., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Food deprivation in mammals is typically associated with reduced thyroid hormone (TH) concentrations and deiodinase content and activity to suppress metabolism. However, in prolonged-fasted, metabolically active elephant seal pups, TH levels are maintained, if not elevated. The functional relevance of this apparent paradox is unknown and demonstrates variability in the regulation of TH levels, metabolism and function in food-deprived mammals. To address our hypothesis that cellular TH-mediated activity is upregulated with fasting duration, we quantified the mRNA expression and protein content of adipose and muscle deiodinase type I (DI1) and type II (DI2), and TH receptor beta-1 (THrβ-1) after 1, 3 and 7 weeks of fasting in northern elephant seal pups (N=5-7 per week). Fasting did not decrease the concentrations of plasma thyroid stimulating hormone, total triiodothyronine (tT3), free T3, total thyroxine (tT4) or free T4, suggesting that the hypothalamic-pituitary-thyroid axis is not suppressed, but rather maintained during fasting. Mean mRNA expression of adipose DI1 and DI2 increased threefold and fourfold, respectively, and 20- and 30-fold, respectively, in muscle. With the exception of adipose DI1, protein expression of adipose DI2 and muscle DI1 and DI2 increased twofold to fourfold. Fasting also increased adipose (fivefold) and muscle (fourfold) THrβ-1 mRNA expression, suggesting that the mechanisms mediating cellular TH activity are upregulated with prolonged fasting. The data demonstrate a unique, atypical mechanism of TH activity and regulation in mammals adapted to prolonged food deprivation in which the potential responsiveness of peripheral tissues and cellular TH activity are increased, which may contribute to their lipid-based metabolism.

The role of thyrotropin (thyroid-stimulating hormone, TSH) in driving peripheral thyroid function in non-mammalian species is still poorly understood. Thyroxine (T₄), the principal hormone released from the thyroid gland in response to TSH stimulation, circulates with a robust daily rhythm in the teleost fish the red drum. Previous research suggests that the red drum T₄ cycle is circadian in nature, driven by TSH secretion in the early photophase and inhibited by T₄ feedback in the early scotophase. To determine whether TSH is produced in a pattern consistent with feedback inhibition by this T₄ cycle, we used quantitative real time PCR (qPCR) to quantify the daily cycle of expression of the pituitary TSH subunits GSUα, and TSHβ. We found that TSH expression cycled inversely to, and 6-12 h out of phase with, the T₄ cycle, consistent with the hypothesis that TSH secretion drives the T₄ cycle. To examine the potential role of deiodinases in negative feedback regulation of this TSH cycle, we also utilized qPCR to assess the pituitary expression patterns of the TH activating enzyme outer-ring deiodinase (Dio2) and the TH deactivating enzyme inner-ring deiodinase (Dio3). Dio2 was not expressed with an obvious daily cycle, whereas Dio3 expression mirrored the expression of TSH. These results are consistent with circulating T₄ providing the negative feedback signal controlling both TSH production and Dio3 expression in the pituitary, and suggest that TH inactivation by inner ring deiodination is an important component of TSH negative feedback control., (Published by Elsevier Inc.)

Fish are becoming an increasingly important research species as investigators seek alternatives to mammalian models. Combined positron emission tomography/computed tomography with ¹⁸F-fluorodeoxyglucose (FDG-PET/CT) is a powerful new technology that has been extensively applied for high-resolution imaging in mammals but not fish. CT scanning provides detailed anatomical three-dimensional imaging. PET scanning detects areas of cellular activity using radio-labelled molecular probes with specific uptake rates appropriate to the tissue involved. FDG-PET is used in oncology because tissues with high glucose uptake, such as neoplasms, are intensely radio-labelled. PET/CT combines the two technologies, so that images acquired from both devices are merged into one superimposed image, thus more precisely correlating metabolic activity with anatomical three-dimensional imaging. Our objective was to determine if fish can be viable replacement animals in cancer studies using this technique by analysing the similarities between fish and humans in glucose uptake in select organs across multiple fish species. Rapid, quantifiable glucose uptake was demonstrated, particularly in brain, kidneys and liver in all imaged fish species. Standard uptake values for glucose uptake in the major organ systems of fish were more similar to those of humans than mice or dogs, indicating that fish may serve as effective alternative animal models using this technology. Applications for this technique in fish may include oncogenesis and metabolism studies as well as screening for environmental carcinogenesis., (© 2013 John Wiley & Sons Ltd.)

Thyroid (TH) and growth (GH) hormones, and insulin-like growth factor I (IGF-I) are anabolic regulators in fish and responsive to nutrient intake. A study was conducted to determine if previously reported growth effects of dietary arginine (ARG) in channel catfish were related to the activation of endocrine axes. In a first experiment, catfish were fed incremental levels of ARG (0.5 - 4% of diet) for 6 weeks and sampled at 2-week intervals. In a second experiment, fasted (48h) fish were fed a single ration of ARG (0.5 or 4% of diet) and sampled at various intervals (0 to 72h postprandial, PP). Experiment 1 did not reveal any influence of ARG on circulating TH, GH, or IGF-I despite the significantly increased growth of fish fed ARG-enriched diets. In experiment 2, feeding the 4% ARG diet significantly increased the amplitude of pulsatile plasma GH levels and also significantly increased IGF-I mRNA in liver and muscle, (at 2h PP) and plasma IGF-I levels (at 6h PP). Although relatively infrequent sampling failed to reveal alterations in TH or GH levels in response to ARG-induced growth activation, PP high frequency sampling unveiled high amplitude pulsatile GH secretions and may be important in activating IGF production in target tissues. Additionally, expressed and secreted IGF-I exhibited discernible patterns which closely correlate with ARG-induced growth effects in catfish., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Thyrotropin (TSH) is a pituitary glycoprotein hormone heterodimer that binds to its G-protein coupled receptor (TSH-R) at the thyroid to promote the synthesis and secretion of thyroid hormone. Very little is known about TSH-TSH-R interactions in teleost fish. Mammalian gonadotropins have been reported to have an intrinsic ability to activate teleost fish TSH-Rs, suggesting the TSH-R in teleost fish is more promiscuous than in other vertebrates. In this study we utilized the goldfish T(4)-release response and recombinant human TSH analogs as in vivo tools to evaluate the structural constraints on hormone-receptor interactions. We found that four positively charged lysines substituted for neutral or negatively charged amino acids within positions 11-20 of the glycoprotein hormone subunit α (GSUα) significantly increased biological activity of hTSH in fish, as it does in mammals. We further found that bovine follicle stimulating hormone but not luteinizing hormone, whose GSUα subunits also contain four lysine or arginine amino acid residues in the N-terminal portion of GSUα, was thyrotropic in goldfish, suggesting gonadotropin β subunit contributes to the heterothyrotropic activity. Though recombinant human FSH did not produce a dose-dependent increase in T(4), thyrotropic activity could be acquired with the addition of positively charged amino acids at the N-terminal portion of its GSUα, confirming the importance of the charge on those amino acids for activation of the goldfish TSH-R. These studies demonstrate that mammalian glycoprotein hormone analogs can be utilized to evaluate the conservation of receptor binding and activation mechanisms between fish and mammals., (Published by Elsevier Inc.)

Darier's disease (DD), caused by mutations in the endoplasmic reticulum (ER) Ca(2+) ATPase ATP2A2 (SERCA2b), is a skin disease that exhibits impaired epidermal cell-to-cell adhesion and altered differentiation. Although previous studies have shown that keratinocyte Ca(2+) sequestration and fluxes are controlled by sphingolipid signaling, the role of this signaling pathway in DD previously has not been investigated. We show here that sphingosine levels increase and sphingosine kinase (SPHK1) expression decreases after inactivating SERCA2b with the specific SERCA2 inhibitors thapsigargin (TG) or small interfering RNA to SERCA2b. Conversely, inhibiting sphingosine lyase rescues the defects in keratinocyte differentiation, E-cadherin localization, desmoplakin (DP) translocation, and ER Ca(2+) sequestration seen in TG-treated keratinocytes. Here, we report early evidence that the keratinocyte sphingolipid and Ca(2+) signaling pathways intersect in ATP2A2-controlled ER Ca(2+) sequestration, E-cadherin and DP localization, and Ca(2+)-controlled differentiation, and thus may be important mediators in DD.

Two critical defensive functions of the outer epidermis, the permeability barrier and antimicrobial defense, share certain structural and biochemical features. Moreover, three antimicrobial peptides (AMPs), i.e., mouse β-defensin 3 (mBD3), mouse cathelicidin antimicrobial peptide (mCAMP), and the neuroendocrine peptide, catestatin (Cst), all localize to the outer epidermis, and both mBD3 and mCAMP are secreted from the epidermal lamellar bodies with other organelle contents that subserve the permeability barrier. These three AMPs are upregulated in response to acute permeability barrier disruption, whereas conversely, mCAMP-/- mice (unable to combat Gram-positive pathogens) also display abnormal barrier homeostasis. To determine further whether these two functions are co-regulated, we investigated changes in immunostaining for these three AMPs in skin samples in which the permeability barrier function in mice had been either compromised or enhanced. Compromised or enhanced barrier function correlated with reduced or enhanced immunohistochemical expression of mCAMP, respectively, but conversely with Cst expression, likely due to the role of this AMP as an endogenous inhibitor of cathelicidin expression. mBD3 expression correlated with experimental barrier perturbations, but poorly with developmental changes in barrier function. These studies show that changes in cathelicidin and Cst expression parallel changes in permeability barrier status, with a less clear relationship with mBD3 expression.

Few studies have examined the underlying hormonal mechanisms that mediate reproductive cyclicity, male pregnancy and reproductive behaviour in syngnathids. Progress in these areas has been hampered by the small size of most species in the family and a lack of validated techniques for assessing endocrine function. Research on a relatively small number of species has suggested that androgens are likely regulators of spermatogenesis and the development of the male brood pouch prior to pregnancy whereas prolactin and corticosteroids synergistically promote brood pouch function during pregnancy. No evidence supports a reversal of reproductive steroid hormone function in sex-role reversed behaviour, but neuropeptides such as arginine vasotocin or isotocin should be examined for their role in regulating parturition and mating behaviour. The diversity of reproductive patterns exhibited by syngnathids suggests that they will provide a unique opportunity to assess how hormonal regulation of integumentary function, gametogenesis and reproductive behaviour have evolved within a teleost lineage. Additionally, their coastal distribution and embryo retention make them potentially important subjects for studies on the effect of endocrine disruption on fitness., (© 2011 The Authors. Journal of Fish Biology © 2011 The Fisheries Society of the British Isles.)

Psychological stress (PS) exerts well-known negative consequences for permeability barrier function in humans and mice, and deterioration of barrier function appears to be attributable largely to excess production of endogenous glucocorticoids (GC). More recently, PS has been shown to compromise antimicrobial defense, also by GC-dependent mechanisms. We assessed here changes in a third antimicrobial peptide (AMP); i.e., the neuropeptide, catestatin (Cst), which also is expressed in the outer epidermis, and previously shown to be regulated by changes in permeability barrier status. In these studies, PS again provoked a decline in both mouse cathelicidin (CAMP) and mouse β-defensin 3 (mBD3) expression, in a GC-dependent fashion. In contrast, Cst immunostaining instead increased after short-term PS, but then began to decline with more sustained PS. In cultured keratinocytes, we showed further that GC downregulate Cst expression, but β-adrenergic blockade increased immunostaining for Cst in the face of long-term PS. Furthermore, β-adrenergic blockade also upregulated CAMP and mBD3 expression. Together, these results suggest that both endogenous GC and β-adrenergic signaling regulate AMP expression.

Background: Endoplasmic reticulum (ER) Ca(2+) depletion, previously shown to signal pathological stress responses, has more recently been found also to trigger homeostatic physiological processes such as differentiation. In keratinocytes and epidermis, terminal differentiation and barrier repair require physiological apoptosis and differentiation, as evidenced by protein synthesis, caspase 14 expression, lipid secretion and stratum corneum (SC) formation., Objectives: To investigate the role of Ca(2+) depletion-induced ER stress in keratinocyte differentiation and barrier repair in vivo and in cell culture., Methods: The SERCA2 Ca(2+) pump inhibitor thapsigargin (TG) was used to deplete ER calcium both in cultured keratinocytes and in mice. Levels of the ER stress factor XBP1, loricrin, caspase 14, lipid synthesis and intracellular Ca(2+) were compared after both TG treatment and barrier abrogation., Results: We showed that these components of terminal differentiation and barrier repair were signalled by physiological ER stress, via release of stratum granulosum (SG) ER Ca(2+) stores. We first found that keratinocyte and epidermal ER Ca(2+) depletion activated the ER-stress-induced transcription factor XBP1. Next, we demonstrated that external barrier perturbation resulted in both intracellular Ca(2+) emptying and XBP1 activation. Finally, we showed that TG treatment of intact skin did not perturb the permeability barrier, yet stimulated and mimicked the physiological processes of barrier recovery., Conclusions: This report is the first to quantify and localize ER Ca(2+) loss after barrier perturbation and show that homeostatic processes that restore barrier function in vivo can be reproduced solely by releasing ER Ca(2+), via induction of physiological ER stress., (© 2010 No claim to original US government works. BJD © 2010 British Association of Dermatologists 2010.)

Full-length cDNAs for thyrotropin β (TSHβ) and glycoprotein hormone α (GSUα) subunits were cloned and sequenced from the red drum (Sciaenops ocellatus). The cDNAs for TSHβ (877 bp) and GSUα (661 bp) yielded predicted coding regions of 126 and 94 amino acid proteins, respectively. Both sequences contain all invariant cysteine and putative glycosylated asparagines characteristic of each as deduced by comparison with other GSUα and TSHβ sequences from representative vertebrate species. Multiple protein sequence alignments show that each subunit shares highest identity (79% for the TSHβ and 86% for the GSUα) with perciform fish. Furthermore, in a single joint phylogenetic analysis, each subunit segregates most closely with corresponding GSUα and TSHβ subunit sequences from closely related fish. Tissue-specific expression assays using RT-PCR showed expression of the TSHβ subunit limited to the pituitary. GSUα mRNA was predominantly expressed in the pituitary but was also detected in the testis and ovary of adult animals. Northern hybridization revealed the presence of a single transcript for both TSHβ and GSUα, each close in size to mRNA transcripts from other species. Dot blot assays from total RNA isolated from S. ocellatus pituitaries showed that in vivo T3 administration significantly diminished mRNA expression of both the TSHβ and GSUα subunits and that goitrogen treatment caused a significant induction of TSHβ mRNA only. Both TSHβ and GSUα mRNA expression in the pituitary varied significantly in vivo over a 24-h period. Maximal expression for both TSHβ and GSUα occurred during the early scotophase in relation to a peak in T4 blood levels previously documented. These results suggest the production of TSH in this species which may serve to drive daily cycles of thyroid activity. Readily quantifiable, variable, and thyroid hormone-responsive pituitary TSH expression, coupled with previously described dynamic daily cycles of circulating T4 and extensive background on the growth, nutrition, and laboratory culture of red drum, suggests that this species will serve as a useful model for experimental studies of the physiological regulation of TSH production.

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia. The disease is caused by inactivating mutations in fibroblast growth factor 23 (FGF23), Klotho (KL), and uridine diphosphate-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). In vitro studies indicate that GALNT3 O-glycosylates a phosphaturic hormone, FGF23, and prevents its proteolytic processing, thereby allowing secretion of intact FGF23. In this study we generated mice lacking the Galnt3 gene, which developed hyperphosphatemia without apparent calcifications. In response to hyperphosphatemia, Galnt3-deficient mice had markedly increased Fgf23 expression in bone. However, compared with wild-type and heterozygous littermates, homozygous mice had only about half of circulating intact Fgf23 levels and higher levels of C-terminal Fgf23 fragments in bone. Galnt3-deficient mice also exhibited an inappropriately normal 1,25-dihydroxyvitamin D level and decreased alkaline phosphatase activity. Furthermore, renal expression of sodium-phosphate cotransporters and Kl were elevated in Galnt3-deficient mice. Interestingly, there were sex-specific phenotypes; only Galnt3-deficient males showed growth retardation, infertility, and significantly increased bone mineral density. In summary, ablation of Galnt3 impaired secretion of intact Fgf23, leading to decreased circulating Fgf23 and hyperphosphatemia, despite increased Fgf23 expression. Our findings indicate that Galnt3-deficient mice have a biochemical phenotype of tumoral calcinosis and provide in vivo evidence that Galnt3 plays an essential role in proper secretion of Fgf23 in mice.

The underlying molecular mechanisms that control milk yield and milk protein yield in domestic animals are not completely understood. In this study, the galactopoietic response to exogenous growth hormone (GH) was used as an experimental model to investigate the role of translation initiation and elongation in the regulation of milk protein synthesis in the mammary gland. A slow-release formula of commercially available GH was administered via a single subcutaneous injection to 4 lactating cows (GH group). A further 4 cows were given a single subcutaneous injection of saline (control group). Changes in mRNA transcript level and protein phosphorylation status of key members of the mammalian target of rapamycin (mTOR) pathway were assessed in mammary gland tissues of these animals using quantitative real-time PCR and Western blotting. The GH treatment enhanced the phosphorylation of ribosomal protein S6 and increased the protein abundance of eukaryotic initiation factor 4E (eIF4E) and eukaryotic elongation factor 2 (eEF2) proteins in the mammary gland of GH-treated animals. These results indicate a link between milk protein synthesis and the regulation of mRNA translation. The GH treatment did not change mRNA abundance of ribosomal protein S6, eIF4E, and eEF2, nor did it change the mRNA (mTOR, eEF2 kinase) or protein abundance of eEF2 kinase. These results demonstrate that GH administration changes mRNA translation initiation and elongation possibly via the mTOR pathway (suggested by the increased levels of ribosomal protein S6 phosphorylation), indicating that the mTOR pathway might be a potential control point in the regulation of milk protein synthesis in the mammary gland.

Thyrotropin (TSH), a pituitary glycoprotein hormone that stimulates the thyroid gland, has been cloned and sequenced from over a dozen teleost fish species. Although TSH is established as a primary driver of systemic thyroid status in mammals, its importance in the regulation of fish thyroid function is still uncertain. We review recent studies indicating that TSH structure is highly conserved across species representing six teleost families. These studies have found TSH messenger RNA consistently expressed in teleost pituitary tissue, although ectopic expression, particularly in gonads, has also been observed. They have also provided evidence for negative feedback inhibition of TSH expression by thyroid hormones, as well as stimulation by hypothalamic peptides. Descriptive studies have found increased TSHbeta expression associated with life history events thought to be promoted by thyroid hormones. These results, coupled with the discovery of a G-protein coupled TSH receptor in several teleost species, supports an active and conserved role for TSH in the regulation of teleost thyroid function. The relative importance of central pathways in regulating thyroid hormone provision to targets and the identity of a proposed thyrotropin-inhibiting factor in teleost fish are still unanswered questions whose resolution will be facilitated by development of methods to measure circulating TSH and its secretion from the pituitary gland.

We collected adult cave swallows (Petrochelidon fulva) and cliff swallows (P. pyrrhonota) during the breeding seasons in 1999 and 2000 from eight locations along the Rio Grande from Brownsville to El Paso (unless otherwise specified, all locations are Texas, USA) and an out-of-basin reference location. Body mass, spleen mass, hepatosomatic index (HSI), gonadosomatic index (GSI), thyroxine (T4) in plasma, DNA damage measured as the half-peak coefficient of variation of DNA content (HPCV) in blood cells, as well as acetylcholinesterase and butyrylcholinesterase in brain were compared with concentrations of organochlorines, metals, and metalloids in carcasses to determine potential effects of contaminants on swallows during the breeding season. Concentrations of 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE) were significantly greater in swallows from El Paso than in those from most locations, except for Pharr and Llano Grande. All swallows from these three locations had p,p'-DDE concentrations of 3 microg/g wet weight or greater. Swallows from El Paso either had or shared the highest concentrations of p,p'-DDE, polychlorinated biphenyls, and 13 inorganic elements. Swallows from El Paso exhibited greater spleen mass and HPCV values as well as lower T4 values compared with those from other locations. Thyroxine was a potential biomarker of contaminant exposure in swallows of the Rio Grande, because it was negatively correlated with p,p'-DDE and Se. Spleen mass was positively correlated with selenium and HSI and negatively correlated with body mass, GSI, Mn, and Ni. Overall, the present study suggests that insectivorous birds living in areas of high agricultural and industrial activity along the Rio Grande bioaccumulate environmental contaminants. These contaminants, particularly p,p'-DDE, may be among multiple factors that impact endocrine and hematopoietic function in Rio Grande swallows.

The accumulation of mRNA encoding for hepatic and intestinal T3-receptor (TR) and body and liver masses were measured in fed and 3-week fasted juvenile and swim up stage rainbow trout embryos. Plasma and total body thyroid hormone (TH) levels were measured for juvenile and swim up stages, respectively. Fasted juveniles exhibited a lower hepatosomatic index (HSI), liver mass and plasma T4 and T3 concentrations than fed animals, but there were no changes in body mass or the accumulation of mRNA encoding for either of the TR(alpha) or TR(beta) isoforms in liver or intestine. TR(beta) mRNA accumulation was greater than TR(alpha) mRNA accumulation in both tissues. Fasted embryos had lower whole body TH levels and body, liver and intestinal tract masses, in addition to a lower intestinosomatic index. However, there was no change in HSI. Fasting did not affect whole body or hepatic TR(alpha) and TR(beta) mRNA accumulation, although intestinal tract TR(alpha) and TR(beta) mRNA accumulation was lower in the fasted embryos. The HSI and body mass changes in fasted juvenile and embryo stages, respectively, indicated that both developmental stages were impacted by fasting. Both stages also showed evidence of decreased TH production. The lower TR gene expression in the intestinal tract of fasted embryos may suggest a role for THs in the transitional stage of intestinal development during this period of development.

Several experiments were conducted to investigate the dynamics of central regulation of thyroid function in the red drum, Sciaenops ocellatus, by manipulating a well-characterized circadian rhythm of T(4) secretion. In the first experiment, red drum were reared under either a long (16L:8D) or short (8L:16D) photoperiod and fed at the same time relative to dawn. The same feeding time under different photoperiods maintained the same phase relationship between T(4) cycles under each photoperiod. This suggests that the circadian clock that determines when the hypothalamus-pituitary-thyroid (HPT) axis is activated is comprised of a feeding-entrained oscillator and a light-entrained oscillator that interact to determine the phase of the T(4) rhythm. Additionally, the amplitude of the main T(4) peak of the cycle was inversely related to the frequency of feeding, while the duration of the main T(4) peak was directly related to feeding frequency under a long photoperiod. Feeding time appears to modify the diurnal profile of circulating T(4) by stimulating post-prandial T(4) secretion that subsequently results in negative feedback on the HPT axis to regulate thyroidal status. In following experiments, red drum immersed in T(3), in lieu of a meal at a specific time that would diminish the main T(4) peak, exhibited a dose-dependent decline in amplitude of the T(4) cycle. This demonstrates that T(3) can exert negative feedback on the HPT axis of red drum to maintain appropriate thyroid hormone concentrations. These data are consistent with a dynamic and physiologically important central component of the regulation of thyroid function in fish., (Copyright 2003 Wiley-Liss, Inc.)

Previous studies with red drum and other species have indicated that diurnal rhythms of circulating thyroid hormones (thyroxine, T(4), and 3-5-3'-triiodo-L-thyronine, T(3)) are synchronized to the light cycle, and not to time of feeding. In this study we set out to address the effects of various lighting regimes on thyroid hormone levels in the red drum. The first experiment was undertaken to determine the effects of long and short photoperiods on diurnal thyroid hormone rhythms, growth rate, feed efficiency and food consumption. Red drum raised under a long photoperiod (16L:8D) grew significantly larger and exhibited greater feed efficiency than their short photoperiod (8L:16D) counterparts. There were no changes in food consumption or the diurnal profile of plasma thyroid hormones, e.g. increased peak amplitude or duration, that would explain this increase in growth rate and feed efficiency. The second experiment was undertaken to determine if diurnal thyroid hormone rhythms in the red drum originate from an endogenous circadian clock. To address this question, red drum were housed under a 12L:12D photoperiod and fed once daily at variable times before the lighting was switched to constant dim illumination for up to 3 days. The rhythm of circulating T(4) levels persisted for two complete cycles with constant amplitude in fish that continued to be fed during constant dim illumination, and did not appear to entrain to feeding. The T(4) rhythm also persisted for three complete cycles under constant conditions in feed-restricted fish, although with a diminished amplitude over time. To our knowledge, these data provide the first evidence for a free-running circadian rhythm of plasma T(4) levels in a fish. These findings implicate the involvement of an endogenous circadian clock that determines when the hypothalamo-pituitary-thyroid axis is activated.

The more biologically active thyroid hormone 3,5,3'-triiodothyronine (T(3)), is primarily derived from peripheral deiodination of thyroxine (T(4)). We characterized hepatic deiodination for a commercially important, warm water teleost fish, the red drum (Sciaenops ocellatus). Low K(m) outer-ring deiodination (ORD) activity was determined by production of free iodide ((125)I) upon incubation of hepatic microsomes with radiolabeled T(4). HPLC analysis demonstrated that (125)I, and T(3) were produced in equal amounts, thereby validating 125I as a measure of T(3) production. A small amount of 3,3',5'-triiodothyronine (reverse T(3)) was also produced by inner-ring deiodination. Production of (125)I was linear over a range of 0--100 microg protein/ml and for incubations of 30 min--4 h. Maximal ORD activity was measured at pH 6.6, 50 mM dithiothreitol (DTT) and an incubation temperature of 20 degrees C. Double reciprocal plots demonstrated that the average apparent K(m) was 5.1 nM and the average V(max) was 3.7 pmol T(4) converted/h per mg protein. ORD was not inhibited by propylthiouracil but was 50% inhibited by 90 microM of iodoacetic acid and 7 microM of gold thioglucose. The substrate analog preference was T(4) = tetraiodoacetic acid = reverse T(3) > triiodoacetic acid >> T(3). In relation to other tissues, ORD for liver>gill>intestine>kidney. Similar hepatic deiodination activity was present in adult wild, aquacultured and laboratory-reared red drum, but in adult wild red drum the optimum temperature was higher. Red drum hepatic low-K(m) deiodination activity appears to most closely resemble rainbow trout hepatic and mammalian Type II deiodination. Evidence of inner-ring T(4) deiodination suggests a more active hepatic iodothyronine catabolic pathway than in other teleost species.

To characterize seasonal changes in thyroid function in a terrestrial reptile, thyroid hormones were measured over a period of 2 years in desert tortoises, Gopherus agassizii, maintained at the Desert Tortoise Conservation Center in Las Vegas, Nevada. In all samples, triiodothyronine was nondetectable (less than 0.1 ng/ml). Thyroxine (T(4)) exhibited distinct cycles in both sexes, being lowest during hibernation and rising toward the time of emergence. Females exhibited only one peak in T(4), during the early spring. In males, T(4) levels peaked in early spring and again in late summer. The desert tortoise has distinct activity patterns that include increased feeding, mating, and locomotor activity in the early spring and increased mating and combat in the late summer. In an experiment to determine whether food intake influences T(4), food was withheld for 2 weeks. Compared to continuously fed controls, T(4) declined significantly in unfed tortoises, but increased significantly within 36 h of refeeding, indicating that thyroid activity is responsive to nutrient intake. The second seasonal peak of T(4) only in males suggests that male reproductive activity in late summer is associated with thyroid activation. To evaluate this possibility, adult, subadult, and juvenile males were sampled during the months of the second seasonal peak in T(4). Although all three age groups showed similar foraging and thermoregulatory behaviors, T(4) peaked in July only in the reproductively active adults, which also exhibited significantly higher testosterone levels. Elevated T(4) in desert tortoises is thus associated with periods of increased feeding and reproductive activity, supporting a role for thyroid hormones in these energy-demanding activities., (Copyright 2001 Academic Press.)

Because thyroid hormones play a critical role in the regulation of metabolism, the low metabolic rates reported for manatees suggest that thyroid hormone concentrations in these animals may also be reduced. However, thyroid hormone concentrations have yet to be examined in manatees. The effects of captivity, diet and water salinity on plasma total triiodothyronine (tT(3)), total thyroxine (tT(4)) and free thyroxine (fT(4)) concentrations were assessed in adult West Indian manatees (Trichechus manatus). Free-ranging manatees exhibited significantly greater tT(4) and fT(4) concentrations than captive adults, regardless of diet, indicating that some aspect of a captive existence results in reduced T(4) concentrations. To determine whether this reduction might be related to feeding, captive adults fed on a mixed vegetable diet were switched to a strictly sea grass diet, resulting in decreased food consumption and a decrease in body mass. However, tT(4) and fT(4) concentrations were significantly elevated over initial values for 19 days. This may indicate that during periods of reduced food consumption manatees activate thyroid-hormone-promoted lipolysis to meet water and energetic requirements. Alterations in water salinity for captive animals did not induce significant changes in thyroid hormone concentrations. In spite of lower metabolic rates, thyroid hormone concentrations in captive manatees were comparable with those for other terrestrial and marine mammals, suggesting that the low metabolic rate in manatees is not attributable to reduced circulating thyroid hormone concentrations.

Available data in cyprinid and salmonid species indicate that nutrient intake sustains thyroidal rhythmicity and that time of feeding may influence the amplitude, but not the phase, of diurnal thyroid hormone cycles. Several experiments were conducted to characterize the nature of thyroidal rhythmicity in a more derived perciform teleost, the red drum. These studies were designed to test the following hypotheses: (1) that feeding time will alter the amplitude of the thyroid hormone rhythm without altering its phase and (2) that food deprivation will diminish the amplitude of the thyroid hormone rhythm. Circulating T(4) levels in this species exhibit high-amplitude diurnal rhythms, whereas circulating T(3) levels fluctuate within a more narrow range. Fish were reared under a 12L:12D photoperiod and fed 5% body weight once daily either at dawn or at dusk. Feeding time had no discernible effect on the phase of the T(4) cycle, but altered the amplitude of the cycle. Dawn-fed fish had significantly greater mean peak levels of T(4) than dusk-fed fish, although there was no difference in daily mean levels in both groups of fish. When red drum were deprived of food, significant declines in plasma glucose, HSI, and liver glycogen content occurred within 3 days. When red drum were sampled once per day after 3, 7, or 11 days of food deprivation there were no consistent changes in circulating T(4) and T(3) levels compared to those of fed controls. However, significant declines in circulating T(4) and T(3) levels in response to food deprivation were detected with a diurnal sampling protocol. Within 3 days of food deprivation, T(4) levels were significantly reduced compared to those in fed controls and not significantly different from T(4) levels after 10 days of food deprivation. T(3) levels exhibited a stepwise decline in circulating levels during food deprivation. These data indicate that both feeding time and nutrient status exert their effects on thyroid hormone rhythms by modifying the amplitude of these cycles. These data also underscore the importance of incorporating a consideration of endocrine rhythmicity into sampling protocols., (Copyright 2000 Academic Press.)

Adrenocortical responsiveness to turning stress was examined in wild, reproductively-active olive ridley sea turtles (Lepidochelys olivacea) in relation to their mass nesting (arribada) behavior. We hypothesized that the high sensitivity threshold (HST) observed in ovipositing sea turtles is associated with a diminished sensitivity of the hypothalamo-pituitary-adrenal (HPA) axis to stressful stimuli in arribada females. We tested this hypothesis by determining whether arribada females exhibited an increased activation threshold of the HPA axis to an imposed stressor (turning stress). Mean basal corticosterone (B) and glucose levels were below 1.0 ng/ml and 60 mg/dl, respectively. Basal B remained unchanged throughout a 24-hr period in basking females. Most animals responded to turning stress with elevated mean B levels (up to 6.5 ng/ml after 6 hr) and no increase in circulating glucose. Nearly 50% of females (and none of the males) were refractory to the stimulation. Males exhibited the most rapid response, with B levels significantly elevated by 20 min over basal levels. Among females, arribada and solitary nesters exhibited a slower rate of response than basking, non-nesting animals. These results demonstrate that olive ridleys exhibit stress-induced changes in circulating B which are slower than those observed in most reptilian and in mammalian, avian, and piscine species. Furthermore, the presence of refractory females and the relatively slower increase in B in arribada and solitary nesters indicate a hyporesponsiveness of the HPA axis to turning stress in nesting olive ridleys. The hyporesponsiveness may be part of a mechanism to facilitate arribada nesting. J. Exp. Zool. 284:652-662, 1999., (Copyright 1999 Wiley-Liss, Inc.)

The ability of West Indian manatees (Trichechus manatus latirostris and Trichechus manatus manatus) to inhabit both freshwater and marine habitats presents an interesting model to study osmoregulation in sirenians. Blood samples were analyzed from manatees held in fresh- and saltwater and from wild animals captured in fresh-, brackish, and saltwater for concentrations of aldosterone, arginine vasopressin, plasma renin activity, Na+, K+, Cl-, and osmolality. Two separate experiments were also conducted on captive animals to evaluate osmoregulatory responses to acute saltwater exposure and freshwater deprivation. Spurious differences were observed in plasma electrolyte and osmolality among the captive and wild groups. Wild brackish water animals exhibited the highest vasopressin concentrations, while wild freshwater manatees had the highest aldosterone levels. A significant correlation between mean vasopressin and osmolality was demonstrated for captive and wild animals. When freshwater animals were acutely exposed to saltwater, osmolality, Na+, and Cl- increased 5.5%, 8.0%, and 14%, respectively, while aldosterone decreased 82.6%. Saltwater animals deprived of freshwater exhibited an almost twofold increase in aldosterone during the deprivation period and a fourfold decrease when freshwater was again provided. Within this group, osmolality increased significantly by 3.4% over the course of the study; however, electrolytes did not change. The lack of consistent differences in electrolyte and osmolality among wild and captive groups suggests that manatees are good osmoregulators regardless of the environment. The high aldosterone levels in wild freshwater animals may indicate a need to conserve Na+, while the high vasopressin levels in wild brackish-water manatees suggest an antidiuretic state to conserve water. Vasopressin levels appear to be osmotically mediated in manatees as in other mammals.

The seasonal reproductive cycle of the Kemp's ridley sea turtle (Lepidochelys kempi) was studied under seminatural conditions at the Cayman Turtle Farm, Grand Cayman, British West Indies, from June 1987 to July 1988. Male L. kempi displayed a prenuptial rise in serum testosterone 4 to 5 months prior to the mating period (March). Male testosterone then declined sharply during the mating period. Female L. kempi also displayed a prenuptial rise in serum testosterone, estradiol, and total calcium 4 to 6 months prior to the mating period (March). Female testosterone and estradiol declined during the nesting period (April to July) immediately following the mating period (March). Elevated levels in female estradiol and total calcium corresponded with the period of vitellogenesis as determined from gel electrophoresis and ultrasonography. Serum thyroxine also fluctuated seasonally with elevated levels observed in females associated with the period of vitellogenesis. L. kempi displayed a distinct seasonal reproductive cycle in captivity. Nesting in the captive study group corresponded with nesting in the wild population at Rancho Nuevo, Mexico (April to July). Female endocrine cycles during the nesting period were similar to those observed in the wild population.

Captive immature green (Chelonia mydas) and Kemp's ridley (Lepidochelys kempi) sea turtles were examined to determine if a hibernation-like state could be induced under controlled conditions. Both species demonstrated that they are able to acclimate to cold temperatures behaviorally. However, the two species appeared to respond differently to decreasing temperature. Whereas the green turtles tolerated the onset of cold water temperatures by reducing swimming activity, the ridleys became very agitated and active as they were exposed to temperatures below 20 degrees C. Nevertheless, both species displayed semi-dormant behavior at temperatures below 15 degrees C, coming to the surface to breathe periodically at intervals of up to three hours. At low temperatures, venous blood pO2 and pCO2 decreased, whereas venous blood pH increased. Feeding also decreased as either species was exposed to cold temperature: greens (at 15 degrees C) and ridleys (at 20 degrees C) decreased food consumption to 50% of control levels, and ceased feeding below 15 degrees C. Thus, these species tolerated temperature drops and the associated hypophagia. They did not exhibit cold-stunning behavior, as has been observed in wild sea turtles exposed to rapid temperature drops, or prolonged periods of hibernation-like dormancy, as has been proposed for wild sea turtles during cold winter months.

Estrogen stimulation of the production of teh yolk protein precursor vitellogenin was demonstrated in immature Kemp's ridley sea turtles. 17beta-Estradiol injection elicited an increase in serum estrogen, protein, protein phosphorus, and total calcium within 7 days. Associated with these changes was the appearance of a single, dimethylformamide-precipitable, 205-kDa estradiol-induced serum protein, which became the predominant serum protein identified by polyacrylamide gel electrophoresis. The effects of estradiol injection were maintained for 3 months following termination of estradiol administration, despite a decline in serum estrogen levels. Although studies in other species have indicated that changes in circulating vitellogenin levels can influence thyroid hormone transport, no changes were observed throughout the study in total blood levels of thyroxine or triiodothyronine or protein binding of these hormones. We conclude that in the immature Kemp's ridley estrogen induces the synthesis and secretion of a vitellogenin which resembles that of other reptiles, but which does not influence thyroid hormone transport. The prolonged presence of vitellogenin in the blood may be due to a lack of an active ovarian uptake mechanism in these immature animals.

Four separate 8-week feeding trials were conducted to assess the effects of supplementing semipurified diets with either triiodothyronine (T3) or thyroxine (T4) at 0, 2, 10, and 50 mg/kg on growth and body composition of juvenile red drum (Sciaenops ocellatus) held in artificial brackish water (6‰) and artificial seawater (32‰). At both levels of salinity, increasing doses of T3 resulted in fish with reduced weight gain, feed efficiency, condition factor (weight × 100/length(3)), and muscle ratio (muscle weight × 100/body weight), as well as a lighter body color. Significant (p < 0.05) effects of T3 on the proximate composition of whole body, liver, and muscle were variable, generally reflecting decreased lipid and protein storage in liver and muscle, respectively. The two highest doses of T3 given to seawater adapted fish increased survival. Dietary T4 supplementation had no distinctive effects on appearance, growth or proximate body composition. These results indicate that whereas T3 may function to regulate protein and lipid metabolism in red drum, dietary supplementation with T3 leads to a hyperthyroidism-induced catabolic state. The elevated endogenous thyroid hormone levels found in fish fed optimal diets may thus adequately supply tissue needs during juvenile growth.

Juvenile red drum (Sciaenops ocellatus) were cultured at 25°C on a variety of diets and blood sampled over eight weeks to examine the relationship between growth and plasma thyroid hormone levels. Maximum growth rates were achieved on formulated experimental diets and a simulated natural shrimp diet. Associated with these maximal rates was a significant increase in triidothyronine (T3), but no consistent change in thyroxine (T4). Reduced rations of diets resulted in low growth rates associated with significantly lowered levels of T3 but not T4. To determine whether weight gain could be increased by application of exogeneous hormone, diets were supplemented with T3 or T4 at 2, 10, and 50 mg hormone/kg diet. Significantly elevated T3 was induced by supplementation with 10 and 50 mg T3/kg diet, although there were no indications of an anabolic effect of T3 incorporation, and 50 mg T3/kg diet was in fact associated with decreased weight gain. Incorporation of T4 into diets had no effect on growth or T3, and had effects on T4 which were small and inconsistent, indicating that T4 may not be effectively absorbed from the gut. No difference was found in response to hormone feeding between low (6 ppt) or high (35 ppt) water salinity. T3 levels thus appear to closely parallel growth in fish on unsupplemented diets, whereas T4 which were small and manipulation. Supplementation with T3 is not an effective means of stimulating growth in red drum fed optimum diets. Whereas thyroid hormones may function to regulate intermediary metabolism in red drum, elevated endogenous thyroid hormone levels appear adequate to supply tissue needs during juvenile growth in culture.

Thyroxine (T4) level in the blood of newly hatched cockerels were measured at different times after the injection of bovine thyrotropin. A linear response to increasing doses of mammalian thyrotropin was seen when a two-injection protocol was used with a fifteen-hour interval between injections. Blood T4 levels peaked 5 hr after the second injection and declined thereafter. This response was shown to be highly specific for thyrotropin from both mammalian and avian sources; gonadotropins, prolactin, and growth hormone had negligible activity, although the last two hormones were able to synergise with thyrotropin under certain circumstances to augment the response. Experiments conducted at different times of day indicated that diurnal fluctuations in the response of the thyroid to exogenous thyrotropins may exist. As a result, animals were injected and bled at the same time of day in all subsequent experiments. Under these circumstances, in vivo thyroxine release in cockerels appears to be precise, simple, and sensitive bioassay for thyrotropin. This bioassay can be used to demonstrate that thyrotropin purified from ostrich pituitaries is distinct from gonadotropin and active in an avian species.

Thyrotropins (TSH) and gonadotropins (luteinizing hormone, LH, and follicle-stimulating hormone, FSH) purified from ovine, bovine, ostrich, sea turtle, and bullfrog pituitary glands were tested for their ability to simulate the thyroid glands of four species of amphibians (three anurans and a urodele), a reptile (the slider turtle), and a bird (the cockerel). All animals tested responded specifically to mammalian, sea turtle, and ostrich thyrotropins; any thyroid-stimulating activity of the gonadotropins from these species could be accounted for by their contamination with thyrotropin. In addition, the three anuran species showed a high degree of specificity for bullfrog TSH; bullfrog LH exhibited low thyrotropic activity in amphibians. In contrast, bullfrog LH was more potent than bullfrog TSH in stimulating the thyroid gland of the cockerel. This bird therefore resembles reptiles in showing a "heterothyrotropic" response to bullfrog LH. The hormonal specificity of thyroid response to these pituitary glycoprotein hormones therefore varies distinctly among representatives of different vertebrate classes. Although the tetrapods tested were generally specific for thyrotropin in their thyroid responses to homologous and heterologous pituitary hormones, occasionally a lack of specificity for heterologous hormones existed which could be shared by species from closely-related classes. The varied patterns seen indicate that independent evolution of the functional characteristics of the thryotropin receptor has occurred to different degrees within each vertebrate class.

To determine whether increases in gonadotropin levels are capable of altering thyroid function in goldfish, plasma thyroid hormone levels were measured following induced changes in endogenous gonadotropin secretion and injection of carp gonadotropin. Radio-frequency lesions placed in the nucleus preopticus periventricularis or monosodium-L-glutamate-induced lesions of the posterior nucleus lateralis tuberis (NLT) of the hypothalamus were capable of stimulating significant increases in plasma gonadotropin levels, but were without effect on plasma triiodothyronine (T3) or thyroxine (T4) at time intervals ranging from 5 hr to 10 days. Likewise, injections of a superactive analog of gonadotropin-releasing hormone resulted in profound increases in gonadotropin levels without associated changes in thyroid hormones. No changes in the circulating levels of T4 or T3 were observed in response to injection of purified carp gonadotropins whereas injection of bovine thyrotropin or carp pituitary extracts stimulated significant increases in T4. Radiofrequency lesions of the pituitary stalk or of the anterior NLT also resulted in significant increases in circulating levels of T4, but not of T3, at 10 and 30 hr postlesion. These results demonstrate that direct acute stimulation of circulating thyroid hormone levels is not an intrinsic action of endogenous goldfish gonadotropin and that activation of the reproductive system, leading to ovulation in some cases, is without effect on blood total thyroid hormone levels. Additionally, these results confirm that hypothalamic inhibition of the pituitary-thyroid axis exists in this teleost fish and demonstrate that interruption of this inhibition results in a time-dependent, high-magnitude increase in circulating thyroxine levels.

Blood samples were taken from white suckers (Catostomus commersoni) during their annual spring spawning migration and analyzed by radioimmunoassay for gonadotropin (GtH), estradiol-17 beta (E2), testosterone (T), 11-ketotestosterone (11-KT), 17 alpha-hydroxyprogesterone (17-P), 17 alpha-hydroxy-20 beta-dihydroprogesterone (17, 20-P), androstenedione (A), and cortisol. GtH, 17-P, and 17,20-P levels were low in prespawning fish of both sexes, rose to their highest levels in ovulated females and spawning males, and then fell to low levels in spent fish. In females, E2, T, and A levels were high in prespawning fish and declined significantly at ovulation, dropping to lowest levels in spent fish. In males, 11-KT, T, and A levels were highest in prespawning fish, and lowest in spent fish. Cortisol levels were highest in spermiating males and ovulated females. Plasma profiles of the sex steroids in the white sucker are very similar to those observed in the rainbow trout (Salmo gairdneri).

Enzymatically dispersed goldfish pituitary cells or freshly prepared goldfish pituitary fragments continue to secrete gonadotropin spontaneously in a column perifusion system. After the establishment of basal secretion rates, treatment of dispersed pituitary cells with 5 and 500 nM dopamine, or pituitary fragments with 50 and 100 nM dopamine, decreased the amount of gonadotropin released into the perifusate. Perifusion with 500 nM dopamine also abolished the gonadotropin-release response to a 10 nM solution of a luteinizing hormone-releasing hormone analogue in both perifusion systems. Perifusion of pituitary dispersed cells or fragment preparations obtained from sexually regressed goldfish with 50 nM norepinephrine consistently increased the amount of gonadotropin released into the perifusate. These results provide in vitro evidence for direct dopamine inhibition of spontaneous gonadotropin release, blockade by dopamine of gonadotropin-releasing hormone actions, and norepinephrine stimulation of gonadotropin secretion in goldfish.

Salmon and mammalian gonadotropin-releasing hormones (sGnRH, mGnRH) were tested for their ability to stimulate in vitro gonadotropin (GtH) release from superfused goldfish pituitary fragments. A two minute exposure to either peptide was sufficient to stimulate a dose-dependent increase in GtH release which reached maximum levels in 15 minutes and returned to baseline within one hour. Both peptides were approximately equipotent in stimulating GtH release, as was a superactive analog of mGnRH. These results demonstrate that sGnRH is capable of directly stimulating GtH release from teleost pituitary tissue, and that structural differences between the three peptides tested do not result in significant differences in in vitro bioactivity.

White suckers (Catostomus commersoni; Cypriniformes, Teleosteii) spawning in a small stream in central Alberta were captured during different stages of their spawning migrations in 1981 and 1982, blood was sampled, and the fish were examined to determine their reproductive condition. Blood samples were analyzed for gonadotropin (GtH), growth hormone (GH), triiodothyronine (T3), and thyroxine (T4) by radioimmunoassay. GtH levels in both sexes were lowest prior to the onset of spawning, increased significantly in spawning males, females in which germinal vesicle migration had begun, and ovulated females and then dropped significantly in spent fish of both sexes. GH was lowest in prespawning females, increased significantly at ovulation, and remained high in spent females. In contrast, GH levels in males were relatively constant throughout spawning. In both sexes, highest T4 levels were found in prespawning fish, and T4 decreased significantly in spent fish. Although a similar decline was seen in T3 in 1981, in 1982 there were no T3 changes associated with changes in reproductive condition. No significant diurnal variations were detected in the levels of GtH or T3; T4 levels appeared to vary on a diurnal basis in prespawning males only. Spawning activity in both sexes therefore appears to be associated with increases in GtH occurring at ovulation in females and at the initiation of spawning in males. GH levels may also be related to reproductive condition in females, but not in males. The relationship of thyroid hormone levels to reproductive condition is less clear, however, and these levels may reflect both endocrine and environmental influences on thyroid function.