Negative feedback regulation of thyrotropin subunits and pituitary deiodinases in red drum, Sciaenops ocellatus.

Thyroxine (T4) 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 T4 on expression of pituitary thyroid hormone deiodinase type 3 (Dio3), such that elevated circulating T4 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 T3 and T4in vivo. Immersion in T4 or T3 significantly inhibited the mRNA expression of the TSH α and ß subunits from 4 to 66h of immersion. Pituitary Dio3 expression was significantly diminished by T3 and T4 at 22h. These results indicate that both T4 and T3 are capable of negative feedback regulation of TSH subunit expression in red drum at physiological concentrations and on a time scale consistent with the T4 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.

Cyclic mRNA expression of thyrotropin subunits and deiodinases in red drum, Sciaenops ocellatus.

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 TSHß. 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.

Using PET/CT imaging to characterize 18 F-fluorodeoxyglucose utilization in fish.

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.

Thyrotropic activity of recombinant human glycoprotein hormone analogs and pituitary mammalian gonadotropins in goldfish (Carassius auratus): insights into the evolution of thyrotropin receptor specificity.

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 ß 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.

Reproductive endocrinology of Syngnathidae.

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.

Endoplasmic reticulum Ca2+ depletion activates XBP1 and controls terminal differentiation in keratinocytes and epidermis.

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.

Initiation and elongation steps of mRNA translation are involved in the increase in milk protein yield caused by growth hormone administration during lactation.

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.

Effect of fasting on thyroid hormone levels, and TR(alpha) and TR(beta) mRNA accumulation in late-stage embryo and juvenile rainbow trout, Oncorhynchus mykiss.

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.

The effects of photoperiod on growth rate and circulating thyroid hormone levels in the red drum, Sciaenops ocellatus: evidence for a free-running circadian rhythm of T(4) secretion.

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.

Characterization of hepatic low-K(m) outer-ring deiodination in red drum (Sciaenops ocellatus).

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.

Seasonality in plasma thyroxine in the desert tortoise, Gopherus agassizii.

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.

Thyroid hormone concentrations in captive and free-ranging West Indian manatees (Trichechus manatus).

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.

The effects of photoperiod and feeding on the diurnal rhythm of circulating thyroid hormones in the red drum, Sciaenops ocellatus.

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.

Basal and stress-induced corticosterone levels in olive ridley sea turtles (Lepidochelys olivacea) in relation to their mass nesting behavior.

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.

Osmoregulation in wild and captive West Indian manatees (Trichechus manatus).

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.

Seasonal reproductive cycle of the Kemp's ridley sea turtle (Lepidochelys kempi).

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.

Estrogen induction of plasma vitellogenin in the Kemp's ridley sea turtle (Lepidochelys kempi).

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.

Simulated hibernation of sea turtles in the laboratory: I. Feeding, breathing frequency, blood pH, and blood gases.

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.

Effects of dietary thyroid hormones on the red drum (Sciaenops ocellatus).

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.