Variable effects of goitrogens in inducing precocious metamorphosis in sea lampreys (Petromyzon marinus)

The ability of different goitrogens (anti-thyroid agents) to induce precocious metamorphosis in larval sea lampreys (Petromyzon marinus) was assessed in four separate experiments. Two of these goitrogens (propylthiouracil [PTU] and methimazole [MMI]) are inhibitors of thyroid peroxidase-catalyzed iodination, and three (potassium perchlorate [KClO(4)], potassium thiocyanate [KSCN], and sodium perchlorate [NaClO(4)]) are anionic competitors of iodide uptake. Because, theoretically, all of these goitrogens prevent thyroid hormone (TH) synthesis, we also measured their influence on serum concentrations of thyroxine and triiodothyronine. All goitrogens except PTU significantly lowered serum TH concentrations and induced metamorphosis in some larvae. The incidence of metamorphosis appeared to be correlated with these lowered TH concentrations in that KClO(4), NaClO(4), and MMI treatments resulted in the lowest serum TH concentrations and the highest incidence of metamorphosis in sea lampreys. Moreover, fewer larvae metamorphosed in the KSCN and low-KClO(4) treatment groups and their serum TH concentrations tended to be greater than the values in the aforementioned groups. MMI treatment at the concentrations used (0.087 and 0.87 mM) was toxic to 55% of the exposed sea lampreys within 6 weeks. The potassium ion administered as KCl did not alter serum TH concentrations or induce metamorphosis. On the basis of the results of these experiments, we have made the following conclusions: (i) In general, most goitrogens other than PTU can induce metamorphosis in larval sea lampreys, and this induction is coincident with a decline in serum TH concentrations. (ii) The method by which a goitrogen prevents TH synthesis is not directly relevant to the induction of metamorphosis. (iii) PTU has variable effects on TH synthesis and metamorphosis among lamprey species. (iv) Unlike in protochordates, potassium ions do not induce metamorphosis in sea lampreys and are not a factor in the stimulation of this event.     

Harderian glands of golden hamsters: morphological and biochemical responses to thyroid hormones

Summary Manipulation of circulating levels of thyroid hormones modifies Harderian gland structure and porphyrin concentrations in male and female golden hamsters. Specifically, thyroxine (T₄) and triiodothyronine (T₃) induce the morphological conversion of the Harderian glands of females to approximate those of the male. Further, porphyrin concentrations are markedly decreased by this treatment. This effect occurs in ovariectomized animals as well, indicating that the gonads are not involved. Suppression of thyroid function by potassium perchlorate (KClO₄) drastically reduces Harderian gland weight in both males and females. However, KClO₄ decreases porphyrin levels in the Harderian glands of females and increases it in the male. Concurrently, KClO₄ also induces a morphological conversion of the Harderian glands of males to the female type. This effect is evident in photoperiods of either 14:10 (h) or 8:16 (h).     

Changes in whole body concentrations of thyroid hormones and cortisol in metamorphosing conger eel

Summary To clarify the hormonal regulation of metamorphosis of the conger eel (Conger myriaster), changes in whole body concentrations of thyroid hormones, thyroxine (T₄) and triiodothyronine (T₃), and cortisol during metamorphosis were examined, as well as the changes in the histological activity of the thyroid gland. In larvae before metamorphosis, T₄ and T₃ levels were less than 5 and 0.15 ng·g^-1 respectively. Levels of T₄ increased to about 30 ng·g^-1 during early metamorphosis, and decreased subsequently. Levels of T₃ increased gradually in early metamorphosis, and then increased abruptly to about 2.0 ng·g^-1 in late metamorphosis. Before metamorphosis, cortisol levels of the leptocephali less than 11 cm in total length were greater than 200 ng·g^-1. Cortisol levels decreased rapidly in larger premetamorphic leptocephali, and low levels were maintained throughout the metamorphic period. Histological observation revealed an activation of the thyroid gland in early metamorphosis; thyroid follicle epithelial cells became columnar and their nuclei larger. Active uptake of colloid by these cells and intensive vascularization of the gland were also observed. By the end of metamorphosis, follicle epithelial cells became squamous, indicating a low level of glandular activity. These results suggest that thyroid hormone plays an important role in regulation of conger eel metamorphosis.Abbreviations AL anal length - TL total length - T ₃ triiodothyronine - T ₄ thyroxine     

Endogenous thyroid hormone synthesis in facultative planktotrophic larvae of the sand dollar Clypeaster rosaceus: implications for the evolutionary loss of larval feeding

Critical roles of hormones in metamorphic life history transitions are well documented in amphibians, lampreys, insects, and many plant species. Recent evidence suggests that thyroid hormones (TH) or TH-like compounds can regulate development to metamorphosis in echinoids (sea urchins, sand dollars, and their relatives). Moreover, previous research has provided evidence for endogenous hormone synthesis in both feeding and nonfeeding echinoderm larvae. However, the mechanisms for endogenous synthesis remain largely unknown. Here, we show that facultatively planktotrophic larvae (larvae that reach metamorphosis in the absence of food but have the ability to feed) from the subtropical sea biscuit Clypeaster rosaceus can synthesize thyroxine endogenously from incorporated iodine (I(125)). When treated with the goitrogen thiourea (a peroxidase inhibitor), iodine incorporation, thyroxine synthesis, and metamorphosis are all blocked in a dose-dependent manner. The inhibitory effect on metamorphosis can be rescued by administration of exogenous thyroxine. Finally, we demonstrate that thiourea induces morphological changes in feeding structures comparable to the phenotypic plastic response of larval structures to low food conditions, further supporting a signaling role of thyroxine in regulating larval morphogenesis and phenotypic plasticity. We conclude that upregulation of endogenous hormone synthesis might have been associated with the evolution of nonfeeding development, subsequently leading to morphological changes characteristic of nonfeeding development.     

The effects of corticosteroid hormones and thyroid hormones on lymphocyte viability and proliferation during development and metamorphosis of Xenopus laevis

Abstract. Metamorphosis in the South African clawed frog, Xenopus laevis , is characterized by a striking loss of lymphocytes in the thymus, liver, and spleen. Changes in the proliferative responses of splenocytes and thymocytes to T cell mitogens and semi-allogeneic cells are also observed at metamorphosis. Because the levels of circulating thyroid hormones (TH) and corticosteroid hormones (CH) increase dramatically during the climax of metamorphosis, we have investigated the possible role of TH and CH as mediators of the changes in lymphocyte numbers or lymphocyte function. Here we report on the in vitro effects of CH and TH on lymphocyte viability and on phytohemagglutinin-P (PHA)-stimulated lymphocyte proliferation at prometamorphosis and climax of metamorphosis. We have observed consistently significant inhibition of proliferation by corticosterone. In contrast, we have observed inconsistent inhibition of proliferation by both thyroxine (T₄) and triiodothyronine (T₃). In short-term studies, the viability of thymocytes and splenocytes was reduced in the presence of CH but not TH.
These observations are consistent with a hypothesis that loss of larval lymphocytes and changes of lymphocyte function at metamorphosis may be due to elevated concentrations of CH rather than TH.
Because CH have been shown to enhance TH-induced effects during metamorphosis, we looked at the combined effects of these agents on PHA-stimulated lymphocyte proliferation. While each agent was inhibitory in several experiments, there was no significantly greater inhibition when splenic lymphocytes were cultured with both.     

Deiodination activity in extrathyroidal tissues of the atlantic hagfish, Myxine glutinosa

We measured low substrate (<1 nM) thyroid hormone (TH) deiodination activities in liver, muscle, intestine, and brain microsomes of Atlantic hagfish fasted for 2 weeks and found extremely low thyroxine (T(4)) outer-ring deiodination (T(4)ORD) and inner-ring deiodination (T(4)IRD) as well as 3,5,3'-triiodothyronine (T(3)) IRD activities. T(3)ORD, 3',5'-triiodothyronine (rT(3)) ORD and rT(3)IRD activities were undetectable. Hagfish deiodinating pathways resembled those of teleosts in requiring a thiol cofactor (dithiothreitol, DTT) and in their inhibition by established deiodinase inhibitors and by TH analogues. However, under optimal pH and DTT conditions intestinal T(4)ORD activity exceeded that of liver about 10-fold. This contrasts with the situation in teleosts but resembles that reported recently in larval and adult lampreys, suggesting the intestine as a primary site of TH deiodination in lower craniates.     

Effect of alloxan and insulin immunoneutralization on circulating thyroid hormone levels in larval landlocked sea lampreys,petromyzon marinus

The effects of alloxan, an insulin (INS)-secreting cell toxin, and INS immunoneutralization on circulating levels of thyroid hormones (thyroxine, T(4); triiodothyronine, T(3)) were examined in larval landlocked sea lampreys, Petromyzon marinus. Animals were injected intraperitoneally with either (Experiment 1) saline (0.6%) or alloxan (20 or 200 microg/g body weight), or with (Experiment 2) normal rabbit serum or anti-lamprey INS. Alloxan (200 microg/g) decreased plasma T(3), but not T(4), in larvae by about 45-80%. Three, six, or nine hr after acute immunoneutralization of lamprey INS with anti-lamprey INS, plasma T(3) levels decreased by 13-30%, relative to controls. These data indicate that INS deficiency can regulate the thyroid system of larval lampreys. There is some suggestion that INS may mediate the metamorphic processes by modulating thyroid hormone concentrations.     

Thyroid hormones are necessary for the metamorphosis of tarpon Megalops cyprinoides leptocephali

This study investigates the effects of thyroxine (T₄), triiodothyronine (T₃) and thiourea (TU) on the metamorphosis of tarpon Megalops cryprinoides leptocephali. TU is an anti-thyroid hormone drug that inhibits the production of T₄ and T₃ in the thyroid tissue. Fully grown tarpons leptocephali were collected at the river mouth and, in the laboratory, were immediately treated with 100 ppb T₄, 10 ppb T_3, or 300 ppm TU. The appropriate concentrations were validated in a preliminary dose response experiment. Morphological and physiological characteristics that indicate metamorphic processes were measured every 2 days. T₄ and T₃ slightly speeded up the metamorphosis of tarpons compared with the control group. The experimental treatments produced accelerated reductions in length, increases in head/body ratio, swimbladder development, and loss of body water and sodium. In contrast, TU treatment caused metamorphic stasis with complete inhibition of metamorphosis between days 6 and 8. Thyroid hormone treatment stimulated fast otolith growth while TU treatment stopped otolith growth between days 6 and 9. Leptocephali in T₄, T₃ and control groups completed metamorphosis in 10-14 days, but TU-treated tarpons remained in the metamorphic leptocephalus stage more than 22 days. In addition, the inhibition of leptocephalus metamorphosis by 300 ppm TU can be reversed in the presence of 10 ppb T₃. These results indicate that thyroid hormones are involved in regulating the metamorphosis of leptocephali.     

Molecular and cellular changes in skin and muscle during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus) are accompanied by changes in deiodinases expression

Flatfish metamorphosis is the most dramatic post-natal developmental event in teleosts. Thyroid hormones (TH), thyroxine (T4) and 3,3??-5??-triiodothyronine (T3) are the necessary and sufficient factors that induce and regulate flatfish metamorphosis. Most of the cellular and molecular action of TH is directed through the binding of T3 to thyroid nuclear receptors bound to promoters with consequent changes in the expression of target genes. The conversion of T4 to T3 and nuclear availability of T3 depends on the expression and activity of a family of 3 selenocysteine deiodinases that activate T4 into T3 or degrade T4 and T3. We have investigated the role of deiodinases in skin and muscle metamorphic changes in halibut. We show that, both at the whole body level and at the cellular level in muscle and skin of the Atlantic halibut (Hippoglossus hippoglossus) during metamorphosis, the coordination between activating (D2) and deactivating (D3) deiodinases expression is strongly correlated with the developmental TH-driven changes. The expression pattern of D2 and D3 in cells of both skin and muscle indicate that TH are necessary for the maintenance of larval metamorphic development and juvenile cell types in these tissues. No break in symmetry occurs in the expression of deiodinases and in metamorphic developmental changes occurring both in trunk skin and muscle. The findings that two of the major tissues in both larvae and juveniles maintain their symmetry throughout metamorphosis suggest that the asymmetric changes occurring during flatfish metamorphosis are restricted to the eye and head region.     

High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity

Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in triglyceride (TG) synthesis using diacylglycerol (DAG) and fatty acyl-CoA as substrates. In the liver, the production of VLDL permits the delivery of hydrophobic TG from the liver to peripheral tissues for energy metabolism. We describe here a novel high-content, high-throughput LC/MS/MS-based cellular assay for determining DGAT activity. We treated endogenous DGAT-expressing cells with stable isotope-labeled [¹³C₁₈]oleic acid. The [¹³C₁₈]oleoyl-incorporated TG and DAG lipid species were profiled. The TG synthesis pathway assay was optimized to a one-step extraction, followed by LC/MS/MS quantification. Further, we report a novel LC/MS/MS method for tracing hepatic TG synthesis and VLDL-TG secretion in vivo by administering [¹³C₁₈]oleic acid to rats. The [¹³C₁₈]oleic acid-incorporated VLDL-TG was detected after one-step extraction without conventional separation of TG and recovery by derivatizing [¹³C₁₈]oleic acid for detection. Using potent and selective DGAT1 inhibitors as pharmacological tools, we measured changes in [¹³C₁₈]oleoyl-incorporated TG and DAG and demonstrated that DGAT1 inhibition significantly reduced [¹³C₁₈]oleoyl-incorporated VLDL-TG. This DGAT1-selective assay will enable researchers to discern differences between the roles of DGAT1 and DGAT2 in TG synthesis in vitro and in vivo.     

Variations in larval growth and metabolism of an estuarine shrimp Palaemonetes pugio during toxicosis by an insect growth regulator

1. Exposure of the estuarine shrimp, Palaemonetes pugio, to a juvenile hormone analogue (⩾8 μg methoprene 1⁻¹) throughout larval development inhibited successful completion of metamorphosis.2. Methoprene exposure retarded growth in early larval stages and postlarvae, but enhanced growth in premetamorphic larvae.3. Respiration rates of early larvae were elevated by methoprene exposure, but not so older larvae or post larvae.4. Lower net growth efficiency (K₂ values) in methoprene-exposed early larvae suggests that increased metabolic demands reduced assimilated energy available for growth.5. Modifications in O:N ratios of premetamorphic larvae and postlarvae suggest that methoprene altered substrate utilization patterns during metamorphosis.     

Induction of metamorphosis by thyroid hormone in anuran small intestine cultured organotypically in vitro

Summary We have developed an organ culture system of the anuran small intestine to reproduce in vitro the transition from larval to adult epithelial form which occurs during spontaneous metamorphosis. Tubular fragments isolated from the small intestine ofXenopus laevis tadpoles were slit open and placed on membrane filters in culture dishes. In 60% Leibovitz 15 medium supplemented with 10% charcoal-treated serum, the explants were maintained in good condition for at least 10 days without any morphologic changes. Addition of triiodothyronine (T₃) at a concentration higher than 10⁻⁹ M to the medium could induce cell death of larval epithelial cells, but T₃ alone was not sufficient for proliferation and differentiation of adult epithelial cells. When insulin (5 μg/ml) and cortisol (0.5 μg/ml) besides T₃ were added, the adult cells proliferated and differentiated just as during spontaneous metamorphosis. On Day 5 of cultivation, the adult cells rapidly proliferated to form typical islets, whereas the larval ones rapidly degenerated. At the same time, the connective tissue beneath the epithelium suddenly increased in cell density. These changes correspond to those occurring at the onset of metamorphic climax. By Day 10, the adult cells differentiated into a simple columnar epithelium which possessed the brush border and showed the adult-type lectin-binding pattern. Therefore, the larval epithelium of the small intestine responded to the hormones and transformed into the adult one. This organ culture system may be useful for clarifying the mechanism of the epithelial transition from larval to adult type during metamorphosis.     

Proximate body composition of the larval,metamorphosing and downstream migrant stages in the life cycle of the Southern Hemisphere lamprey,Geotria australis

Synopsis The water, total lipid, protein and ash content have been measured in larval, metamorphosing (stages 1–7) and downstream migrantGeotria australis caught in Western Australia between October 1977 and August 1979. The total lipid content of ammocoetes changed markedly with season and increased with body size. Although, unlike other species, the ammocoetes ofG. australis continue to increase in length during the latter part of larval life, the relative amount of total lipid still rose during this period, eventually reaching levels equivalent to approximately 14% of the wet body weight at the commencement of metamorphosis. During the six months between the onset of metamorphosis and the downstream migration, total lipid declined to approximately 8%. Assays for phospholipid of larval and metamorphosingG. australis indicated that changes in total lipid were almost entirely due to variations in neutral lipid. Changes in the percentage amount of total lipid were accompanied by an inverse but slightly greater amount of change in percentage water. During metamorphosis, the absolute amount of total lipid in a standard animal declined from 122 mg at stage 1 to 53 mg at stage 7, whereas water rose initially from 597 mg at stage 1 to 638 mg at stage 3, before declining to 442 mg by stage 7. Although the percentage amount of protein and ash tended to increase slightly during larval life, neither showed conspicuous seasonal changes. In both relative and absolute terms, protein declined during metamorphosis. A comparison of the data on the proximate body composition inG. australis and holarctic lampreys shows that different strategies have been employed to accumulate large amounts of fat by the end of larval life and to utilize protein during metamorphosis.     

Daily endocrine profiles in parr and smolt Atlantic salmon

To elucidate possible mechanisms behind the endocrine control of parr–smolt transformation, the daily plasma profiles in thyroid hormones (TH; free thyroxine (FT₄), total thyroxine (TT₄), and total 3,5,3′-triiodothyronine (TT₃)), growth hormone (GH) and cortisol were studied in Atlantic salmon parr and smolts under simulated-natural winter (8 L:16D) and spring (16.5 L:7.5D) photoperiods, respectively. Overall, TT₄, TT₃ and GH levels were higher in smolts than in parr, whereas FT₄ levels fluctuated within the same range in parr and smolts. Significant diurnal changes in plasma TH were present in parr. Both FT₄ and TT₄ levels increased during the photophase and decreased during the scotophase, while TT₃ levels followed an inverse pattern. Growth hormone showed no significant changes in parr. Changes in FT₄, TT₄, GH, and cortisol, but not TT₃, levels, were observed in smolts with peak levels during both the photophase and scotophase for FT₄, TT₄ and GH. Plasma cortisol was not assayed in parr but in smolts the peaks were associated with dusk and dawn. In addition to the general increases in TH, GH and cortisol, the distinct endocrine differences in nighttime levels between parr in the winter and smolts in the spring suggest different interactions between TH, GH, cortisol and melatonin at these different time points. These spring scotophase endocrine profiles may represent synergistic hormone interactions that promote smolt development, similar to the synergistic endocrine interactions shown to accelerate anuran metamorphosis. The variations in these diurnal rhythms between parr and smolts may represent part of the endocrine mechanism for the translation of seasonal information during salmon smoltification.     

Inhibition of thyrotropin binding to human thyroid plasma membranes by thyroid hormones and "reverse triiodothyronine".

Triiodothyronine, reverse triiodothyronine and thyroxine were found to inhibit ¹²⁵I labelled thyrotropin binding to human thyroid plasma membranes in vitro. Both the thyrotropin binding and the effect of the above iodoamino-acids on this binding were pH, temperature and time dependent, 50% inhibition of thyrotropin binding was observed at 2×10^?7M concentration of reverse triiodothyronine or thyroxine and at 1.1 × 10^?6M concentration of triiodothyronine. The kinetic studies of thyrotropin binding revealed that the maximal capacity of receptor sites for the pituitary hormone is unaffected by the presence of thyroid hormones. On the other hand the association and dissociation constants for thyrotropin binding changed when iodoaminoacids were present in the incubation medium /Ka 8.13 × 10⁷M^?1 vs 1.6 × 10⁸M^?1 and Kd 1.14 × 10^?8M vs 4.55 × 10^?9M respectively, depending on the pH/. The double reciprocal plots showed competitive mechanism of inhibition. The present study suggest that triiodothyronine, reverse triiodothyronine and thyroxine are able to modify the thyrotropin binding to membrane receptors.     

Expression and regulation of miR-1, -133a, -206a, and MRFs by thyroid hormone during larval development in Paralichthys olivaceus

MiR-1, miR-133a, and miR-206a have been identified as muscle-specific miRNAs. They play multiple crucial roles in the regulation of muscle development. Here, we show that these miRNAs were differentially expressed during the larval development of flounder, and specifically expressed in skeletal muscle and heart in adult tissues/organs. The expression levels of these miRNAs were significantly changed by thyroid hormone (TH) or thiourea (TU) treatment during metamorphosis from 17 dph (days post hatching) to 42 dph. In addition, the expression levels of MyoD and Myf5 mRNAs markedly increased at 14 dph (pre-metamorphosis) compared to metamorphic stages, and their expression levels are far above the myogenin during larval development. Moreover, these MRFs (myogenic regulatory factors) expression were directly or indirectly regulated by thyroid hormone or thiourea during metamorphosis. All the results suggest that miRNAs and MRFs might be involved in signaling pathway of TH or TU-mediated flounder metamorphosis.     

The effect of thyroid hormones on the aerobic development of locomotor and cardiac muscles in the barnacle goose

The role of thyroid hormones in the development of the locomotor and cardiac muscles of the barnacle goose (Branta leucopsis) was investigated. From 2 weeks of age, goslings were treated with thyroxine, triiodothyronine, or methimazole (a thyroid inhibitor). Birds were killed at 6 weeks (n = 5) or 9 weeks (n = 4) and various locomotor and cardiac muscle masses recorded and tissue samples taken for analysis of citrate synthase activity. The effects of thyroxine and triiodothyronine were not significantly different from each other, except in the case of the iliofibularis at 9 weeks. The mass-specific citrate synthase activity of the iliofibularis, semimembranosus muscles and liver were significantly increased by thyroid hormone treatment. Cardiac muscle showed a significant hypertrophy at 9 weeks of age following treatment with thyroxine. Hypothyroidism induced by treatment with methimazole exhibited the greatest effect on the pectoralis muscle, reducing citrate synthase activity by 33%. Mass-specific citrate synthase activity of the pectoralis and pectoralis mass (% body wt.) were found to be highly correlated (r ² = 0.74) at 6 weeks of age. It is suggested that thyroid hormones may be involved in controlling the tissue-specific timing of the maturation of locomotor and cardiac muscles. Accepted: 16 September 1996     

Larval metamorphosis of the mussel Mytilus galloprovincialis Lamarck, 1819 in response to neurotransmitter blockers and tetraethylammonium

The metamorphic response of pediveliger larvae of Mytilus galloprovincialis to the neurotransmitter blockers chlorpromazine, amitriptyline, rauwolscine, idazoxan, atenolol and butoxamine, and to tetraethylammonium chloride (TEA) was investigated through a series of bioassays. Chlorpromazine, amitriptyline and idazoxin inhibited larval metamorphosis induced by 10^?4 M epinephrine. The concentration that inhibited metamorphosis by 50% (IC₅₀) for chlorpromazine and amitriptyline was 1.6 × 10^?6 M and 6.6 × 10^?5 M, respectively. Idazoxan was less effective with an IC₅₀ of 4.4 × 10¹³ M. Moreover, these three inhibitors showed no toxicity at any of the concentrations tested. The larval metamorphic response to K⁺ was not inhibited by 10^?3 M tetraethylammonium chloride after 96 h. Thus, the neurotransmitter blockers chlorpromazine and amitriptyline are inhibitors of larval metamorphosis, and will be useful tools for antifouling studies.     

Specific thyroxine receptors in mammary cytosol from lactating cattle

Specific thyroxine (T₄) binding was identified in bovine mammary cytosol preparations. Binding specificity of 3,5,3′ triiodothyronine (T₃) with respect to T₄ was less than 1%. The halftime of T₄ binding and displacement at 4°C was 1 and 20 minutes, respectively. Scatchard analyses demonstrated the presence of two T₄ binding components with dissociation constants of 3.61 × 10^?10 and 7.73 × 10^?8M. The high affinity binding component had a molecular weight of ～ 100,000, a T₄ binding capacity of 5.05 × 10^?12moles/mg protein, and was destroyed by boiling or protease treatment. High-affinity, low capacity T₄ binding was not found in bovine serum and was unique to mammary tissue.