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Amphibian metamorphosis has long been used as model to study postembryonic development in vertebrates, a period around birth in mammals when many organs/tissues mature into their adult forms and is characterized by peak levels of plasma thyroid hormone (T3). Of particular interest is the remodeling of the intestine during metamorphosis. In the highly-related anurans Xenopus laevis and Xenopus tropicalis, this remodeling process involves larval epithelial cell death and de novo formation of adult stem cells via dedifferentiation of some larval cells under the induction of T3, making it a valuable system to investigate how adult organ-specific stem cells are formed during vertebrate development. Here, we will review some studies by us and others on how T3 regulates the formation of the intestinal stem cells during metamorphosis. We will highlight the involvement of nucleosome removal and a positive feedback mechanism involving the histone methyltransferases in gene regulation by T3 receptor (TR) during this process., (Published by Elsevier Inc.)

In amphibians, thyrotropin (TSH), corticotropin (ACTH) and prolactin (PRL) are regarded as the major pituitary hormones involved in metamorphosis, their releasing factors being corticotropin-releasing factor (CRF), arginine vasotocin (AVT), and thyrotropin-releasing hormone (TRH), respectively. It is also known that thyrotropes and corticotropes are equipped with CRF type-2 receptor and AVT V1b receptor, respectively. As for PRL cells, information about the type of receptor for TRH (TRHR) through which the action of TRH is mediated to induce the release of PRL is lacking. In order to fill this gap, an attempt was made to characterize the TRHR subtype existing in the PRL cells of the anterior pituitary gland of the bullfrog, Rana catesbeiana. We cloned cDNAs for three types of bullfrog TRHRs, namely TRHR1, TRHR2 and TRHR3, and confirmed that all of them are functional receptors for TRH by means of reporter gene assay. Analyses with semi-quantitative reverse transcription-PCR and in situ hybridization revealed that TRHR3 mRNA is expressed in the anterior lobe and that the signals reside mostly in the PRL cells. It was also noted that the expression levels of TRHR3 mRNA in the anterior pituitary as well as in the PRL cells of metamorphosing tadpoles elevate as metamorphosis progresses. Since the pattern of changes in TRHR3 mRNA levels in the larval pituitary is almost similar to that previously observed in the pituitary PRL mRNA and plasma PRL levels, we provide a view that TRHR3 mediates the action of TRH on the PRL cells to induce the release of PRL that is prerequisite for growth and metamorphosis in amphibians., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The Mexican axolotl (Ambystoma mexicanum) is a salamander species that does not undergo metamorphosis, resulting in the retention of juvenile characteristics in the mature breeding stage (paedomorphosis). Here we review the endocrinological studies investigating the proximate cause of axolotl paedomorphosis with a focus on the hypothalamo-pituitary-thyroid (HPT) axis. It is well established that axolotl paedomorphosis is a consequence of low activity of the HPT axis. The pituitary hormone thyrotropin (TSH) is capable of inducing metamorphosis in the axolotl, which indicates that all processes and interactions in the HPT axis below the pituitary level are functional, but that TSH release is impaired. In metamorphosing species, TSH secretion is largely controlled by the hypothalamic neuropeptide corticotropin-releasing hormone (CRH), which seems to have lost its thyrotropic activity in the axolotl. However, preliminary experiments have not yet confirmed a role for faulty CRH signalling in axolotl paedomorphosis. Other hypothalamic factors and potential pituitary inhibitors need to be investigated to identify their roles in amphibian metamorphosis and axolotl paedomorphosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Anuran metamorphosis involves the transformation of an aquatic tadpole into a juvenile frog. This process is completely dependent upon thyroid hormones (THs). Although much research has been focused on changes in gene expression programs during this postembryonic developmental period, transitions in the metabolic profiles are relatively poorly understood. Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) is a technique that generates highly multiplexed mass spectra while retaining spatial location information on a thin tissue section. Reconstructed ion heat maps are correlated with morphology of the tissue section for biological interpretation. The present study is the first to use whole-body MALDI-MSI on tadpoles to gain insights into anuran metamorphosis. Approximately 1000 features were detected in each of five tissues examined (brain, eye, liver, notochord, and tail muscle) from premetamorphic North American bullfrog (Rana [Lithobates] catesbeiana) tadpoles. Of these detected metabolites, 1700 were unique and 136 were significantly affected by exposure to 50 nM thyroxine for 48 h. Of the significantly-affected metabolites, 64 features were tentatively identified using the MassTRIX annotation tool. All tissues revealed changes in lipophilic compounds including phosphatidylcholines, phosphatidylinositols, phosphatidylglycerols, phosphatidylethanolamines, and phosphatidylserines. These lipophilic compounds made up the largest portion of significantly-affected metabolites indicating that lipid signaling is a major target of TH action in frog tadpoles., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Early life-stages of amphibians rely on the innate immune system for defense against pathogens. While thyroid hormones (TH) are critical for metamorphosis and later development of the adaptive immune system, the role of TH in innate immune system development is less clear. An integral part of the innate immune response are pro-inflammatory cytokines - effector molecules that allow communication between components of the immune system. The objective of this study was to characterize the expression of key pro-inflammatory cytokines, tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β) and interferon-γ (IFN-γ), throughout amphibian development and determine the impacts of thyroidal modulation on their expression. Xenopus laevis were sampled at various stages of development encompassing early embryogenesis to late prometamorphosis and cytokine expression was measured by real-time PCR. Expression of TNFα and IL-1β were transient over development, increasing with developmental stage, while IFN-γ remained relatively stable. Functionally athyroid, premetamorphic tadpoles were exposed to thyroxine (0.5 and 2 μg/L) or sodium perchlorate (125 and 500 μg/L) for seven days. Tadpoles demonstrated characteristic responses of advanced development with thyroxine exposure and delayed development (although to a lesser extent) and increased thyroid gland area and follicular cell height with sodium perchlorate exposure. Exposure to thyroxine for two days resulted in decreased expression of IL-1β in tadpole trunks. Sodium perchlorate had negligible effects on cytokine expression. Overall, these results demonstrate that cytokine transcript levels vary with stage of tadpole development but that their ontogenic regulation is not likely exclusively influenced by thyroid status. Understanding the direct and indirect effects of altered hormone status may provide insight into potential mechanisms of altered immune function during amphibian development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Corticosterone (CORT), a principal glucocorticoid in amphibians, is known to regulate diverse physiological processes including growth and metamorphosis of anuran tadpoles. Environmental stressors activate the neuroendocrine stress axis (hypothalamus-pituitary-interrenal axis, HPI) leading to an acute increase in CORT, which in turn, helps in coping with particular stress. However, chronic increase in CORT can negatively affect other physiological processes such as growth and metamorphosis. Herein, we studied the effect of exogenous CORT on larval growth, antipredator behaviour and metamorphic traits of Hylarana indica. Embryonic exposure to 5 or 20μg/L CORT did not affect their development, hatching duration as well as larval growth and metamorphosis. Exposure of tadpoles to 10 or 20μg/L CORT throughout larval development caused slower growth and development leading to increased body mass at stage 37. However, body and tail morphology of tadpoles was not affected. Interestingly, larval exposure to 5, 10 or 20μg/L CORT enhanced their antipredator response against kairomones in a concentration-dependent manner. Further, larval exposure to increasing concentrations of CORT resulted in the emergence of heavier froglets at 10 and 20μg/L while, delaying metamorphosis at all concentrations. Interestingly, the heavier froglets had shorter hindlimbs and consequently shorter jump distances. Tadpoles exposed to 20μg/L CORT during early, mid or late larval stages grew and developed slowly but tadpole morphology was not altered. Interestingly, exposure during early or mid-larval stages resulted in an enhanced antipredator response. These individuals metamorphosed later but at higher body mass while SVL was unaffected., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Insulin and ecdysone signaling play a critical role on the growth and development of insects including Bombyx mori. Our previous study showed that Bombyx larvae reached critical weight for metamorphosis between day 3.5 and 4 of the fifth larval instar. The present study showed that the effect of insulin on the accumulation of glycogen in fat body of Bombyx larvae depends on the critical growth period. When larvae are in active growth period (before reaching critical weight), insulin caused increased accumulation of glycogen, while its treatment in larvae at terminal growth period (after critical period) resulted in an increased mobilization of glycogen. During terminal growth period, insulin and 20-hydroxyecdysone (20E) showed an antagonistic effect on the accumulation of fat body glycogen in fed, food deprived and decapitated larvae as well as in isolated abdomens. Insulin treatment decreased the glycogen content, whereas, 20E increased it. Food deprivation and decapitation caused an increase in the transcript levels of insulin receptor (InR) and this increase in InR expression might be attributed to a decrease in synthesis/secretion of insulin-like peptides, as insulin treatment in these larvae showed a down-regulation in InR expression. However, insulin showed an up-regulation in InR in isolated abdomens and it suggests that in food deprived and decapitated larvae, the exogenous insulin may interact with some head and/or thoracic factors in modulating the expression of InR. Moreover, in fed larvae, insulin-mediated increase in InR expression indicates that its regulation by insulin-like peptides also depends on the nutritional status of the larvae. The treatment of 20E in fed larvae showed an antagonistic effect on the transcript levels since a down-regulation in InR expression was observed. 20E treatment also led to a decreased expression of InR in food deprived and decapitated larvae as well as in isolated abdomens. Insulin and 20E also modulated the expression level of ecdysone receptors (EcRB1 and USP1). 20E treatment showed an up-regulation in expression of ecdysone receptors, but only in fed larvae, whereas insulin treatment showed a down-regulation in the expression of EcRB1 and USP1 in all the experimental larvae studied. Further, the data indicates that an up-regulation of ecdysone receptors is associated with an increase in fat body glycogen content, whereas an up-regulation of insulin receptor expression causes glycogen mobilization. The study, therefore, suggests that the insulin and ecdysone signaling are linked to each other and that both insulin and ecdysone are involved in regulating the carbohydrate reserves in B. mori., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F 1 F 0 ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Global climate change leading to increased temperatures may affect shifts in physiological processes especially in ectothermic organisms. Temperature-dependent shifts in developmental rate in particular, may lead to life-long changes in adult morphology and physiology. Combined with anthropogenic changes in the chemical environment, changes in developmental outcomes may affect adult functionality. The purpose of this study is to determine 1) if small increases in diel water temperature affect the development of Arizona tiger salamander (Ambystoma tigrinum nebulosum) larvae, and 2) if this change interacts with exposure to the common environmental thyroid disrupting compound, perchlorate. Larvae between Watson and Russell developmental stages 8-13 were exposed to ammonium perchlorate (AP) at doses of 0, 20 or 200ppb and then raised at either ambient or a 0.9°C elevated above ambient temperature for 81days in outdoor enclosures. During the first 5 treatment weeks, AP treatment induced slower development and smaller snout-vent length (SVL) of exposed larvae, but only in the elevated temperature group. During the later stages of development, the small increase in temperature, regardless of AP treatment, tended to decrease the time to metamorphosis and resulted in a significantly smaller body mass and worse body condition. Our results suggest that even small diel water temperature increases can affect the developmental process of salamanders and this shift in the water temperature may interact with a common environmental contaminant., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Knowledge about sensitivities and responses of amphibian larvae to sex steroids and the chemicals alike is the first step towards understanding and assessing the effect of diverse chemicals that interfere with gonadal development and other endocrine functions. Herein, we used Microhyla ornata to determine the role of sex steroids on its gonad differentiation and sex ratio. Our results show that the exposure to increasing concentrations of estradiol-17β throughout larval development did not affect gonad differentiation resulting in 1:1 sex ratio at metamorphosis. But, females emerging from estradiol-17β treatment had larger ovaries with larger sized follicles. Further, testes of some males contained lumens, the number of which was dose dependent. Similarly, exposure to testosterone propionate had negligible effects on gonad differentiation. However, the mean diameter of the largest follicles was smaller in treated ovaries. Treatment of tadpoles with tamoxifen had no effect on gonad differentiation and ovary development while testicular development was accelerated at the highest concentration. Similarly, treatment of tadpoles with cyproterone acetate had little effect on gonad differentiation as well as development, hence the sex ratios at the end of metamorphosis. Further, in tadpoles exposed to increasing concentrations of formestane, gonad differentiation was normal resulting in 1:1 sex ratio. Thus, in M. ornata, both estradiol and testosterone are essential for the development of ovaries and testes respectively but, they are not critical to gonadal differentiation. Hence, the effects of sex steroids and other endocrine disrupting chemicals could be species-specific; different species may have differential sensitivities to such chemicals., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In amphibians, although genetic factors are involved in sex determination, gonadal sex differentiation can be modified by exogenous steroid hormones suggesting a possible role of sex steroids in regulating the process. We studied the effect of testosterone propionate (TP) and estradiol-17β (E2) on gonadal differentiation and sex ratio at metamorphosis in the Indian skipper frog, Euphlyctis cyanophlyctis with undifferentiated type of gonadal differentiation. A series of experiments were carried out to determine the optimum dose and sensitive stages for gonadal sex reversal. Our results clearly indicate the importance of sex hormones in controlling gonadal differentiation of E. cyanophlyctis. Treatment of tadpoles with 10, 20, 40, and 80μg/L TP throughout larval period resulted in the development of 100% males at metamorphosis at all concentrations. Similarly, treatment of tadpoles with 40μg/L TP during ovarian and testicular differentiation resulted in the development of 90% males, 10% intersexes and 100% males respectively. Treatment of tadpoles with 10, 20, 40, and 80μg/L E2 throughout larval period likewise produced 100% females at all concentrations. Furthermore, exposure to 40μg/L E2 during ovarian and testicular differentiation produced 95% females, 5% intersexes and 91% females, 9% intersexes respectively. Both TP and E2 were also effective in advancing the stages of gonadal development. Present study shows the effectiveness of both T and E2 in inducing complete sex reversal in E. cyanophlyctis. Generally, exposure to E2 increased the larval period resulting in significantly larger females than control group while the larval period of control and TP treated groups was comparable., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Metamorphosis, which depends upon a fine balance between two groups of lipid-soluble hormones such as juvenile hormones (JHs) and ecdysteroids, is an important feature in insect evolution. While it is clear that the onset of metamorphosis depends on the decrease of JH levels, the way in which these hormones exert their activities is not fully understood in Triatominae species. The discovery of a Drosophila melanogaster mutant resistant to the treatment with the JH analog methoprene, led finally to the description of the methoprene-tolerant gene in Tribolium castaneum (TcMet) as a putative JH receptor. Here we present the genomic and functional characterization of an ortholog of the methoprene-tolerant gene in the hemimetabolous insect Rhodnius prolixus (RpMet). The analysis of the R. prolixus gene showed that the exonic structure is different from that described for holometabolous species, although all the critical protein motifs are well conserved. Expression analysis showed the presence of RpMet mRNA in all the tested tissues: ovary, testis, rectum, Malpighian tubules and salivary glands. When juvenile individuals were treated with RpMet specific double strand RNA (dsRNA), we observed abnormal molting events that resulted in individuals with morphological alterations (adultoids). Similarly, treatment of newly emerged fed females with dsRNA resulted in an abnormal development of the ovaries, with eggs revealing anomalies in size and accumulation of yolk, as well as a decrease in the amount of heme-binding protein. Altogether, our results validate that RpMet is involved in the transduction of JH signaling, controlling metamorphosis and reproduction in R. prolixus., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In holometabolous insects the fall to zero of the titer of Juvenile Hormone ends its still poorly understood "status quo" mode of action in larvae. Concurrently it initiates metamorphosis of which the programmed cell death of all internal tissues that actively secrete proteins, such as the fat body, midgut, salivary glands, prothoracic glands, etc. is the most drastic aspect. These tissues have a very well developed rough endoplasmic reticulum, a known storage site of intracellular Ca(2+). A persistent high [Ca(2+)]i is toxic, lethal and causal to apoptosis. Metamorphosis becomes a logical phenomenon if analyzed from: (1) the causal link between calcium toxicity and apoptosis; (2) the largely overlooked fact that at least some isoforms of Ca(2+)-ATPases have a binding site for farnesol-like endogenous sesquiterpenoids (FRS). The Ca(2+)-ATPase blocker thapsigargin, like JH a sesquiterpenoid derivative, illustrates how absence of JH might work. The Ca(2+)-homeostasis system is concurrently extremely well conserved in evolution and highly variable, enabling tissue-, developmental-, and species specificity. As long as JH succeeds in keeping [Ca(2+)]i low by keeping the Ca(2+)-ATPases pumping, it acts as "the status quo" hormone. When it disappears, its various inhibitory effects are lifted. The electrical wiring system of cells, in particular in the regenerating tissues, is subject to change during metamorphosis. The possibility is discussed that in vertebrates an endogenous farnesol-like sesquiterpenoid, probably farnesol itself, acts as a functional, but hitherto completely overlooked Juvenile anti-aging "Inbrome", a novel concept in signaling., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Barfin flounder larvae exhibit unique black coloration, as well as left-right asymmetry in juvenile stage as in other flatfish. In this study, we first assessed the changes in melanophores with development and then investigated their responsiveness to melanin-concentrating hormone (MCH) during metamorphosis. Larval-type melanophores appeared on both sides of the body before metamorphosis, whereas adult-type melanophores appeared only on the ocular side after metamorphosis. Even in the individuals of this species displaying black coloration, the density of larval-type melanophores was similar to that in transparent larvae of other species. However, unlike in transparent larvae, larval-type melanophores completely dispersed in the black larvae of this species. Therefore, the black coloration during larval stages was mainly due to dispersion, and not the density, of larval-type melanophores. In vitro MCH treatment revealed, for the first time, the responsiveness of melanophores in larval stages. On the ocular side, larval-type melanophores aggregated against MCH during larval stages, while, in the larvae at later metamorphic stages and in juveniles, larval-type melanophores did not aggregate, although aggregation of adult-type melanophores was noted. In contrast, on the blind side, the responsiveness of larval-type melanophores to MCH was consistently present from larval to juvenile stages. The metamorphic transition of MCH responsiveness from larval- to adult-type melanophores only on the ocular side suggests the larval (therefore, immature) nature of the blind side skin. We propose that the inhibited development, and thus the retention of the larval-type skin leads to the formation of the blind side characteristics and is the central mechanism for the flatfish asymmetry., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Because thyroid hormones (THs) are conserved modulators of development and physiology, identification of compounds adversely affecting TH signaling is critical to human and wildlife health. Anurans are an established model for studying disruption of TH signaling because metamorphosis is dependent upon the thyroid system. In order to strengthen this model and identify new gene transcript biomarkers for TH disruption, we performed DNA microarray analysis of Xenopus laevis tadpole tail transcriptomes following treatment with triiodothyronine (T(3)). Comparison of these results with previous studies in frogs and mammals identified 36 gene transcripts that were TH-sensitive across clades. We then tested molecular biomarkers for sensitivity to disruption by exposure to wastewater effluent (WWE). X. laevis tadpoles, exposed to WWE from embryo through metamorphosis, exhibited an increased developmental rate compared to controls. Cultured tadpole tails showed dramatic increases in levels of four TH-sensitive gene transcripts (thyroid hormone receptor β (TRβ), deiodinase type II (DIO2), and corticotropin releasing hormone binding protein (CRHBP), fibroblast activation protein α (FAPα)) when exposed to T(3) and WWE extracts. TRβ, DIO2, and CRHBP were identified as TH sensitive in other studies, while FAPα mRNA transcripts were highly TH sensitive in our array. The results validate the array and demonstrate TH-disrupting activity by WWE. Our findings demonstrate the usefulness of cross-clade analysis for identification of gene transcripts that provide sensitivity to endocrine disruption. Further, the results suggest that development is disrupted by exposure to complex mixes of compounds found in WWE possibly through interference with TH signaling., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Flatfish begin life as bilaterally symmetrical larvae that swim up-right, then abruptly metamorphose into asymmetrically shaped juveniles with lateralized swimming postures. Flatfish metamorphosis is mediated entirely by thyroid hormone (TH). Changes in flatfish swim posture are thought to be regulated via vestibular remodeling, although the influence of TH on teleost inner ear development remains unclear. This study addresses the role of TH on the development of the three otolith end-organs (sacculus, utricle, and lagena) during southern flounder (Paralichthys lethostigma) metamorphosis. Compared with pre-metamorphosis, growth rates of the sacculus and utricle otoliths increase dramatically during metamorphosis in a manner that is uncoupled from general somatic growth. Treatment of P. lethostigma larvae with methimazol (a pharmacological inhibitor of endogenous TH production) inhibits growth of the sacculus and utricle, whereas treatment with TH dramatically accelerates their growth. In contrast with the sacculus and utricle otoliths that begin to form and mineralize during embryogenesis, a non-mineralized lagena otolith is first visible 10-12 days after hatching. The lagena grows during pre- and pro-metamorphosis, then abruptly mineralizes during metamorphic climax. Mineralization of the lagena, but not growth, can be induced with TH treatment, whereas treatment with methimazol completely inhibits lagena mineralization without inhibiting its growth. These findings suggest that during southern flounder metamorphosis TH exerts differential effects on growth and development among the three types of otolith., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In submammalian vertebrates, corticotropin-releasing factor (CRF) acts as an anorexigenic neuropeptide as well as a potent stimulator of corticotropin and thyrotropin release from the pituitary. As a step for demonstrating the involvement of CRF in the feeding regulation of anuran larvae, which are known to stop feeding toward the metamorphic climax, we studied firstly the changes in the distribution of CRF-like immunoreactivity (CRF-LI) in the brain of metamorphosing bullfrog larvae. Neuronal cell bodies showing CRF-LI were invariably present in the thalamic regions throughout larval development. Cells with CRF-LI were also found in the hypothalamus. The number of cells with CRF-LI in the hypothalamus, but not in the thalamus, showed a significant increase as metamorphosis progressed. Immunoreactive nerve fibers were observed mainly in the median eminence, and became abundant as metamorphosis proceeded. The number of cells showing CRF-LI in the hypothalamus as well as the density of immunoreactive fibers in the median eminence decreased at the end of metamorphosis. Secondly, we examined the effect of intracerebroventricular (ICV) injection of CRF on the food intake in the premetamorphic larvae. ICV injection of CRF at 10 pmol/g body weight (BW) induced a significant decrease of food intake during 15 min. The CRF-induced anorexigenic action was blocked by the treatment with a CRF receptor antagonist [alpha-helical CRF(9-41)] at 100 pmol/g BW. The results suggest the involvement of CRF in the accomplishment of metamorphosis through the pituitary and in the feeding restriction that occurs during the later stages of metamorphosis through the central nervous system., (Copyright 2010 Elsevier Inc. All rights reserved.)

Thyroid hormone (TH) is essential for proper development in vertebrates. TH deficiency during gestation and early postnatal development produces severe neurological, skeletal, metabolism and growth abnormalities. It is therefore important to consider environmental chemicals that may interfere with TH signaling. Exposure to environmental contaminants that disrupt TH action may underlie the increasing incidence of human developmental disorders worldwide. One contaminant of concern is the xenoestrogen bisphenol A (BPA), a chemical widely used to manufacture polycarbonate plastics and epoxy resins. The difficulty in studying uterus-enclosed mammalian embryos has hampered the analysis on the direct effects of BPA during vertebrate development. As TH action at the cellular level is highly conserved across vertebrate species, amphibian metamorphosis serves as an important TH-dependent in vivo vertebrate model for studying potential contributions of BPA toward human developmental disorders. Using Xenopus laevis as a model, we and others have demonstrated the inhibitory effects of BPA exposure on metamorphosis. Genome-wide gene expression analysis revealed that surprisingly, BPA primarily targets the TH-signaling pathway essential for metamorphosis in Xenopus laevis. Given the importance of the genomic effects of TH during metamorphosis and the conservation in its regulation in higher vertebrates, these observations suggest that the effect of BPA in human embryogenesis is through the inhibition of the TH pathway and warrants further investigation. Our findings further argue for the critical need to use in vivo animal models coupled with systematic molecular analysis to determine the developmental effects of endocrine disrupting compounds., (Published by Elsevier Inc.)

Corticosteroids (CS) act synergistically with thyroid hormone (TH) to accelerate amphibian metamorphosis. Earlier studies showed that CS increase nuclear 3,5,3'-triiodothyronine (T(3)) binding capacity in tadpole tail, and 5' deiodinase activity in tadpole tissues, increasing the generation of T(3) from thyroxine (T(4)). In the present study we investigated CS synergy with TH by analyzing expression of key genes involved in TH and CS signaling using tadpole tail explant cultures, prometamorphic tadpoles, and frog tissue culture cells (XTC-2 and XLT-15). Treatment of tail explants with T(3) at 100 nM, but not at 10 nM caused tail regression. Corticosterone (CORT) at three doses (100, 500 and 3400 nM) had no effect or increased tail size. T(3) at 10 nM plus CORT caused tails to regress similar to 100 nM T(3). Thyroid hormone receptor beta (TRbeta) mRNA was synergistically upregulated by T(3) plus CORT in tail explants, tail and brain in vivo, and tissue culture cells. The activating 5' deiodinase type 2 (D2) mRNA was induced by T(3) and CORT in tail explants and tail in vivo. Thyroid hormone increased expression of glucocorticoid (GR) and mineralocorticoid receptor (MR) mRNAs. Our findings support that the synergistic actions of TH and CS in metamorphosis occur at the level of expression of genes for TRbeta and D2, enhancing tissue sensitivity to TH. Concurrently, TH enhances tissue sensitivity to CS by upregulating GR and MR. Environmental stressors can modulate the timing of tadpole metamorphosis in part by CS enhancing the response of tadpole tissues to the actions of TH., (Copyright 2010 Elsevier Inc. All rights reserved.)

Anuran (frog) metamorphosis has long-served as a model of how thyroid hormones regulate post-embryonic development in vertebrates. However, comparatively little is known about urodele (salamander) metamorphosis. We conducted a detailed time-course study of induced metamorphosis in the Mexican axolotl (Ambystoma mexicanum) that probed metamorphic changes in morphology and gene expression in the skin. Using morphometrics, quantitative PCR, histology, and in situ hybridization we demonstrate that the development of transcriptional markers is fundamental to the resolution of early metamorphic events in axolotls. We then use linear and piecewise linear models to identify a sequence of morphological and transcriptional changes that define larval to adult remodeling events throughout metamorphosis. In addition, we show that transcriptional biomarkers are expressed in specific larval and adult cell populations of the skin and that temporal changes in these biomarkers correlate with tissue remodeling. We compare our results with other studies of natural and induced metamorphosis in urodeles and highlight what appear to be conserved features between urodele and anuran metamorphosis.

During the transformation from larval tadpole to juvenile frog, there are critical periods of metamorphic development and sex differentiation that may be particularly sensitive to endocrine disruption. The aim of the present study was to identify sensitive developmental periods for estrogenic endocrine disruption in the northern leopard frog (Rana pipiens) using short, targeted exposures to the synthetic estrogen, ethinylestradiol (EE2). Post-hatch tadpoles (Gosner stage 27) were exposed over five distinct periods of metamorphosis: early (stage 27-30), mid (stage 30-36), early and mid (stage 27-36), late (stage 36-42), and the entire metamorphic period (chronic; stage 27-42). For each period, animals were sampled immediately following the EE2 exposure and at metamorphic climax (stage 42). The effects of EE2 on metamorphic development and sex differentiation were assessed through measures of length, weight, developmental stage, days to metamorphosis, sex ratios and incidence of gonadal intersex. Our results show that tadpoles exposed to EE2 during mid-metamorphosis were developmentally delayed immediately following exposure and took 2 weeks longer to reach metamorphic climax. In the unexposed groups, there was low proportion (0.15) of intersex tadpoles at stage 30 and gonads appeared to be morphologically distinct (male and female) in all individuals by stage 36. Tadpoles exposed early in development displayed a strong female-biased sex ratio compared to the controls. Moreover, these effects were also seen at metamorphic climax, approximately 2-3 months after the exposure period, demonstrating that transient early life-stage exposure to estrogen can induce effects on the reproductive organs that persist into the beginning of adult life-stages.

Although amphibian sex determination is genetic, it can be manipulated by exogenous hormone exposure during sexual differentiation. The timing of sexual differentiation varies among anuran amphibians such that species may or may not be a tadpole during this period, and therefore, may or may not be exposed to aquatic contaminants. Estrogenic contamination is present in amphibian habitats worldwide. We examined three species with varying somatic and ovarian developmental rates to assess their susceptibility to estrogenic contaminants. American toads (Bufo americanus), gray treefrogs (Hyla versicolor), and Southern leopard frogs (Ranasphenocephala) were exposed as larvae to 17-beta-estradiol (10(-7)M), three concentrations of a widespread herbicide (1, 3, 30 ppb atrazine), or a solvent control (ethanol). Somatic and ovarian developmental stages as well as time to metamorphosis were recorded. Toads and treefrogs were examined at three weeks and metamorphosis, while leopard frogs were examined at three, six, and nine weeks as well as at metamorphosis. Our results demonstrate that each species displays heterochronic somatic and ovarian development. Further, the more rapid of the two rates determines the susceptibility to estrogenic contaminants. These results suggests that amphibians with shorter larval periods, and therefore quicker somatic developmental rates (i.e. American toads, gray treefrogs), are more susceptible to somatic treatment effects (i.e. prolonged time to metamorphosis) due to estrogenic contaminants. Moreover, the results suggest that amphibians with relatively rapid ovarian development (i.e. Southern leopard frogs) are more susceptible to gonadal treatment effects caused by estrogenic contaminants.

Estrogen or eco-estrogenic chemicals can disrupt normal gonadal sex differentiation, causing intersex formation and feminization in amphibians. The cellular basis for estrogen-induced sex reversal is not well understood. In the present study, we investigated the concentration- and stage-dependent effects of estradiol (E(2)) exposure during the larval period on histological characteristics of gonadal sex differentiation and gonadal sex steroid secretion in vitro in the African clawed frog, Xenopus laevis. Embryos were exposed to E(2) (1, 10, or 100 microg/L) or vehicle control through metamorphosis and then allowed to develop in untreated medium for 2-mo post-metamorphosis. To investigate gonadal sex differentiation and development during and after exposure, gonadal samples were collected at different developmental stages. Gonadal sex differentiation did not occur before NF stage 52 in any group. At NF stage 54-55 primordial germ cells (PGCs) were observed in both cortical and medullary regions of developing tadpoles gonads in the control, 1 and 10 microg/L E(2) treatments, but were observed only in the cortical region of tadpoles exposed to 100 microg/L E(2). E(2) increased the percent of spermatocytes, spermatids, and spermatozoa compared to controls. Larval E(2) exposure did not alter hCG-induced gonadal testosterone secretion in vitro but significantly increased E(2) secretion from ovaries of juvenile frogs. Our results indicate that E(2) exposure during larval development appears to prevent PGC migration to the medulla of developing gonads in a concentration-dependent manner. The degree of PGC migration to the medulla may be related to the degree of E(2)-induced intersex formation and feminization in X. laevis. E(2) exposure during the larval period accelerates spermatogenesis and can increase ovarian E(2) secretion in juvenile frogs.

Larval-stage summer flounder (Paralichthys dentatus) were immersed in the corticosteroid-receptor blocker RU486 to test the effects of cortisol deficiency on salinity tolerance. Premetamorphic larvae held at 10 (near isosmotic) or 30 (hyperosmotic) parts per thousand ( per thousand) seawater survived well over 5d in 0, 0.012, or 0.12 microM RU486. However, at concentrations of 1.2 or 3.6 microM RU486, mortality was significantly greater for larvae in 30 per thousand compared to larvae in 10 per thousand. In a separate experiment, the ability of RU486 to inhibit tolerance to hyperosmotic medium (30 per thousand) was confirmed; immersion at 1.2 microM RU486 induced mortality of larvae in the metamorphic climax stage held at 30 per thousand, but not 0 or 10 per thousand. Mortality due to RU486 in pre- or prometamorphic stage larvae was prevented by concurrent immersion in cortisol at concentrations approximately 10-200 times greater than RU486, indicating that the action of RU486 was specific to antagonism of cortisol. The efficacy of 1.2 microM RU486 in reducing survival in 30 per thousand was found to be stage-dependent and exhibited the following hierarchy for fastest time to 50% mortality: prometamorphosis>metamorphic climax>premetamorphosis. In a 5-d pretreatment of pre- or prometamorphic larvae by immersion in 20 microM cortisol and/or 0.12 microM RU486 at 30 per thousand, only RU486 had a limited effect on decreasing survival when larvae were challenged with abrupt exposure to 50 per thousand. In total, the results evidence for the first time a necessary role for cortisol in seawater tolerance of a larval marine teleost.

Using RT-PCR, two cDNAs encoding preprotemporins were cloned from a total RNA preparation of the skin of Tago's brown frog Rana tagoi. Preprotemporin-1TGa cDNA directs the synthesis of temporin-1TGa (FLPILGKLLSGIL.NH2) previously isolated from R. tagoi skin. Preprotemporin-1TGb cDNA directs the synthesis of a novel 16-amino-acid-residue peptide (AVDLAKIANKVLSSLF.NH2) that, atypically for members of the temporin family, inhibits the growth of Gram-negative bacteria more effectively than Gram-positive bacteria. Preprotemporin-1TGa mRNA and preprotemporin-1TGb mRNA were not detected in skin prior to the onset of metamorphosis (stage 35) but the levels of the transcripts increased markedly during metamorphosis reaching a maximum at stage 38. Exposure of adult animals to 10(-8) M triiodothyronine (T3) for 72 h enhanced expression of the preprotemporin-1TGb gene (approximately threefold) but did not significantly change the level of expression of the preprotemporin-1TGa gene. Exposure of the animals to 10(-8) M T3 and 10(-6) M bisphenol A, an endocrine disrupting chemical that potently inhibits the action of thyroid hormones (THs), reduced expression of the preprotemporin-1TGb gene by 10-fold and the preprotemporin-1TGa gene by threefold. We propose that T3-stimulated synthesis of antimicrobial peptides is important in protecting the animal against microorganisms, particularly at metamorphosis and during skin moulting, but environmental pollutants can inhibit peptide synthesis and render the animal susceptible to invasion by pathogens.

The thyroid hormone plays an essential role in the metamorphosis in flatfish, during which external asymmetry (for example, eye relocation and pigmentation) is established. However, no information is available on the expression mechanisms of metamorphic asymmetry. Since the presence of malformed juveniles (having either ocular or blind side characteristics on both sides) was known in spotted halibut (Verasper variegatus), the effect of the timing of thyroid hormone treatment was investigated. When thyroxine (T4, 10 or 30 ng/ml) was administered to the hypothyroid larvae (continuously receiving 30 microg/ml thiourea) from 25 days after hatching (DAH), the occurrence of one type of symmetrical juvenile (symmetrical pseudoalbino having blind side characteristics on both sides) increased to more than three times, and the occurrence was significantly greater than that observed in the control group. In the fish in which T4 treatment was initiated prior to 15 DAH or after 60 DAH, the occurrence of another type of symmetrical juvenile (symmetrical ambicolorate having ocular side characteristics on both sides) became more than two times, and its occurrence was significantly greater than that observed in the control group. These results suggest that both sides of the larval body independently possess the potential to become either the ocular or the blind side of the juvenile body and that the timing of thyroid hormone increase is the determining factor of "which side to become." Based on previous studies, since thyroid hormone is expected to induce blind side characteristics, the differential responsiveness to thyroid hormone-longer in the left side (blind side in normal juvenile) than the right-is strongly suggested as the central mechanism for metamorphic asymmetry in spotted halibut.

Entanglement of functions between the adrenal (or interrenal) and thyroid axis has been well described for all vertebrates and can be tracked down up to the level of gene expression. Both thyroid hormones and corticosteroids may induce morphological changes leading to metamorphosis climax in the neotenic Mexican axolotl (Ambystoma mexicanum). In a first series of experiments, metamorphosis was induced with an injection of 25 microg T(4) on three alternate days as judged by a decrease in body weight and tail height together with complete gill resorption. This injection also resulted in elevated plasma concentrations of T(3) and corticosterone. Previous results have indicated that the same dose of dexamethasone (DEX) is ineffective in this regard (Gen. Comp. Endocrinol. 127 (2002) 157). In a second series of experiments low doses of T(4) (0.5 microg) or DEX (5 microg) were ineffective to induce morphological changes. However, when these submetamorphic doses were injected together, morphological changes were observed within one week leading to complete metamorphosis. It is concluded that thyroid hormones combined with corticosteroids are essential for metamorphosis in the axolotl and that only high doses of either thyroid hormone or corticosteroid can induce morphological changes when injected separately.

Effects of bisphenol A (BPA) on embryonic and larval development were investigated. In Xenopus laevis blastulae treated with 2.5-3.0 x 10(-5) M BPA or with 10(-5) M 17beta estradiol (E2), malformation of the head region, scoliosis (curved vertebrate), and suppression of organogenesis were observed. In addition, 10(-5)-10(-4) M BPA blocked tri-iodothyronine (T3)-inducible resorption of the tail segments from premetamorphic (stage 52-54) larvae in vitro. When stage 52 tadpoles were immersed in 1.0-2.5 x 10(-5) M BPA, deceleration of both spontaneous and thyroxin (T4)-induced metamorphic changes occurred. Furthermore, BPA suppressed thyroid hormone receptor (TR) beta gene expression both in vivo and in vitro. Thus, we concluded that BPA at the concentrations examined affects both embryonic development and larval metamorphosis.

An in vitro experimental system was devised to assess the direct effects of the goitrogen, potassium perchlorate (KClO(4)), on radioiodide uptake and organification by the larval lamprey endostyle. Organification refers to the incorporation of iodide into lamprey thyroglobulin (Tg). Histological and biochemical evidence indicated that endostyles were viable at the termination of a 4h in vitro incubation. A single iodoprotein, designated as lamprey Tg, was identified in the endostylar homogenates by polyacrylamide gel electrophoresis and Western blotting. Lamprey Tg was immunoreactive with rabbit anti-human Tg serum and had an electrophoretic mobility similar to that of reduced porcine Tg. When KClO(4) was added to the incubation medium, both iodide uptake and organification by the endostyle were significantly reduced relative to controls as determined by gamma counting, and gel-autoradiography and densitometry, respectively. Western blotting showed that KClO(4) significantly lowered the total amount of lamprey Tg in the endostyle. Based on the results of this in vitro investigation, we conclude that KClO(4) acts directly on the larval lamprey endostyle to inhibit thyroidal activity. These data support a previous supposition from in vivo experimentation that KClO(4) acts directly on the endostyle to suppress the synthesis of thyroxine and triiodothyronine, resulting in a decrease in the serum levels of these two hormones., (Copyright 2002 Elsevier Science (USA))

When the juvenile hormone analog fenoxycarb was topically applied to the silkworm Bombyx mori at the beginning of the 3rd or 4th (penultimate) instar, an extra larval molt was induced. The 5th instar period was shortened to about 5 days and the extra 6th instar ranged from 8 to more than 20 days, depending on the dose applied. Starvation before fenoxycarb treatment strongly enhanced the incidence of extra molting up to 100%. When 1 ng was applied in the 4th instar after a 2-day starvation, most larvae underwent an extra molt, metamorphosed to pupae, then to fertile adults. Combining starvation and fenoxycarb application thus induces a perfect extra molt efficiently. In perfect extra molting larvae, profiles of total ecdysteroid titer during the 4th and 5th instars were similar to that during the 4th instar in the control, and the ecdysteroid profile during the extra 6th instar was similar to that during the control 5th (last) instar. At ecdysteroid peaks, 20-hydroxyecdysone (20E) and ecdysone (E), generally regarded as the active molting hormone and its precursor, had similar titers in the 6th instar, whereas E was much less than 20E in the 4th and 5th instars in the extra molting larvae. E was also abundant only in the last larval instar in the control. These results suggest that both 20E and E contents are important for regulation of larval molt and metamorphosis in silkworms and that fenoxycarb triggers the extra molt by inducing an additional larval molt type of ecdysteroid surge before the last larval instar., (Copyright 2002 Elsevier Science (USA))

In amphibians, there is a close interaction between the interrenal and the thyroidal axes. Hypothalamic corticotropin-releasing hormone or related peptides stimulate thyroidal activity by increasing thyrotropin synthesis and release, while corticosterone accelerates both spontaneous and thyroid hormone-induced metamorphosis. One of the mechanisms that is thought to contribute to this acceleration is a corticosterone-induced change in peripheral deiodinating activity. The present experiments were designed to investigate further the effects of glucocorticoid treatment on amphibian deiodinase activities and to explore the possible role of these effects in metamorphosis. Neotenic axolotls (Ambystoma mexicanum) were treated either acutely or chronically with dexamethasone (DEX) and changes in type II and type III iodothyronine deiodinase (D2 and D3) activities were studied in liver, kidney, and brain. In addition, gill length, tail height, and body weight were measured at regular intervals in the chronically treated animals in search of metamorphosis-related changes. A single injection of 50 microg DEX decreased hepatic D3 activity (6-48 h) while it increased D2 activity in brain (6-48 h) and to a lesser extent in kidney (24 h). These changes were accompanied by an increase in plasma T(3) levels (48 h). Samples taken during chronic treatment with 20 or 100 microg DEX showed that both hepatic D2 and D3 activities were decreased on day 26, while renal D3 activity was decreased but only in the 20 microg dose group. All other deiodinase activities were not different from those in control animals. At 25 days, all DEX-treated axolotls showed a clear reduction in gill length, tail height, and body weight, changes typical of metamorphosis. Prolongation of the treatment up to 48 days resulted in complete gill resorption by days 44-60. Although probably several mechanisms contribute to these DEX-induced metamorphic changes, the interaction with thyroid function via a sustained downregulation of hepatic D3 may be one of them., (Copyright 2002 Elsevier Science (USA))

The bicolored frog, Rana curtipes, is endemic to Western Ghats of Southern India, having a prolonged larval life. In this species, gonadal differentiation is of the semidifferentiated type. The gonads initially differentiate into ovaries in all the individuals at Gosner stage 25. Later, in genetic males, the oocytes degenerate and testicular differentiation occurs at stages 30-31. Exposure of R. curtipes tadpoles to 50 microg/L of testosterone (T) or estradiol-17beta (E(2)) during stages 24-26 or 29-32 did not affect gonadal sex differentiation and proportion of males and females at metamorphosis. In all the groups, the sex ratio was almost 1:1 as in the controls. Likewise, exposure of tadpoles to low concentration of steroids (12.5 microg/L T or E(2)) throughout larval development was ineffective in altering the sex ratio or the gonadal sex differentiation. On the other hand, exposure to higher concentrations of steroids (25 and 50 microg/L) throughout the larval development (stages 25-45) significantly skewed the sex ratio toward the male or female direction, depending upon the sex steroid used. Thus, exposure to T or E(2) throughout the larval period could produce 93% males or 79% females, respectively, indicating the ability of these steroids to cause sex reversal. This study shows a possible absence of a "critical stage" that is sensitive to sex steroids for gonadal sex reversal in this frog., (Copyright 2001 Academic Press.)

This study focuses on the effect of octopamine (OA) on metamorphosis of the silkworm Bombyx mori and the red flour beetle Tribolium freemani Hinton. Titers of OA and juvenile-hormone esterase (JHE) were measured at various larval and pupal stadia of both insects. Effects of OA, OA agonists, and antagonists on metamorphosis and JHE activity were also examined. At day 2, peaks of OA and JHE activity were observed in third instars, and at day 3, a sharp peak of OA was observed, followed by a large peak of JHE activity at day 4 in last instars of B. mori. However, no peaks of OA and JHE activity were observed in fourth instars. A high titer of OA appeared at days 2-4, followed by a peak of JHE activity at day 7 and the second OA peak at day 9 after the start of assay of T. freemani. At pupation, a small peak of OA and the highest activity of JHE were observed. The effects of OA on JHE activity were examined in vitro, because the relationship could be responsible for triggering pupation in B. mori and T. freemani larvae. Exogeneous OA (0.1-10 mM) stimulated the JHE activity of final instars (day 2) of B. mori in vitro. Similarly, the presence of OA (10 mM) activated the JHE activity of newly ecdysed T. freemani pupae in vitro. OA antagonists chlorpromazine and gramine delayed the start of spinning and reduced the JHE activity of B. mori, when applied in diet at 10-100 ppm. Some OA agonists stimulated the pupation and JHE activity of T. freemani larvae reared under crowded conditions, when topically applied. Thus, OA may contribute to activation of the events preparatory to a pupal molt, i.e., the secretion of OA increases JHE activity followed by stimulation of pupation., (Copyright 1999 Academic Press.)

The current study examined the role of steroids in primary and secondary sex differentiation in the African reedfrog (Hyperolius argus: Hyperolidae). This species is sexually dimorphic: males have a solid green dorsum and females are reddish-brown with large white spots. This study is the first to report the effects of sex steroids on the development of a sexually dichromatic species and the first to examine the role of sex steroids on development of the vocal sac. Both males and females metamorphosed solid green without spots. Approximately 2 months after metamorphosis, control females transformed to the female-typical color pattern. Control males never developed this color pattern (remained green), but developed vocal sacs. To examine the role of sex steroid hormones on primary (gonadal differentiation) and secondary (vocal sac development and dorsal coloration) sex differentiation, testosterone (T) or estradiol-17beta (E(2)) were administered throughout larval development. At metamorphosis, 53% of the controls were males, based on gross gonadal morphology and histology of a subsample. Both doses of T produced 100% males. All E(2)-treated animals had ovarian cavities and/or follicles when examined histologically (at both doses) but 50% had testicular tissue in addition to these ovarian characteristics. Both doses of T induced vocal sac development and both doses of E(2) induced female coloration. Thus, both T and E(2) induced secondary sex characteristics (vocal sac development and dorsal color change, respectively) but E(2) produced hermaphroditic gonads, whereas T induced complete sex reversal., (Copyright 1999 Academic Press.)

Immediately premetamorphic larval sea lampreys (Petromyzon marinus) (>/=120 mm in length) were treated for 4, 8, or 16 weeks with one of two concentrations of either exogenous thyroxine (T4; 1 or 0.5 mg/L) or 3,5,3'-triiodothyronine (T3; 1 or 0.25 mg/L) in the presence or absence of the goitrogen potassium perchlorate (KC1O4; 0.05%) as well as with KC1O4 alone. Larvae from all treatments were examined for signs of metamorphosis, changes in serum T4 and T3 concentrations (serum T4 and serum T3), and changes in intestinal T4 outer-ring (5') deiodination to T3 (T4ORD). KC1O4 depressed both serum T4 and T3 and induced metamorphosis in 80% of larvae treated for 8 weeks or longer. However, neither effect was observed in larvae exposed to KC1O4 combined with either thyroid hormone (TH). These data confirm previous suggestions that exogenous TH blocks KC1O4-induced metamorphosis by elevating serum TH concentrations, and provide evidence that declines in serum TH concentrations are mandatory for precocious metamorphosis. Serum T4, but not serum T3, was elevated following exogenous T4 treatment in the presence or absence of KC1O4. This maintenance of serum T3 at control levels, in the presence of a T4 challenge, was not due to decreases in intestinal T4ORD activity, since T4ORD activity was not affected by any treatments in the study. Exogenous T3 elevated both serum T4 and T3. However, serum T3 in T3-treated larvae decreased with time, suggesting a stringent T3 regulation. Elevation of serum T4 following T3 treatment may have been a result of either inhibition of T4 metabolism, or stimulation of T4 secretion by the endostyle. Based on these results, we conclude that (i) exogenous TH blocks KClO4-induced metamorphosis in sea lampreys and (ii) serum T3 is maintained at control levels despite elevations in serum T4 (its immediate precursor), but this does not involve any changes in intestinal T4ORD activity., (Copyright 1998 Academic Press.)

The response of grouper larvae to the thyroid hormones, thyroxine (T4) and triiodothyronine (T3), was examined. Two-, 3-, and 4-week-old grouper larvae were reared in seawater containing either T4 or T3 at 0.01, 0.1, and 1 ppm. T4 or T3 induced metamorphosis in all age groups in a dose-dependent manner. Regardless of the size of the larvae, metamorphosis was completed in 2 days in larvae treated with 1 ppm of either T4 or T3; 3-4 days in larvae exposed to 0.1 ppm; and 5-6 days in larvae immersed in 0.01 ppm. None of the fish in the control group completed metamorphosis during this period. Compared with the control fish, survival rates were higher in groups exposed to 0.01 ppm and lower in those exposed to 1 ppm of T3. In 4-week-old larvae, T4 treatment (0.01 to 1.0 ppm) resulted in higher survival compared to the control. These results suggest that a dose of 0.01 ppm is appropriate for acceleration of metamorphosis and improvement of survival in 3- and 4-week-old grouper larvae. A lower dose may be apropriate for earlier stages., (Copyright 1998 Academic Press.)

This study was designed to examine the role of somatostatin in regulating changes in lipid metabolism of larvae and metamorphosing landlocked sea lamprey, Petromyzon marinus. Larvae and animals in late metamorphosis (stage 6 on a 7-stage scale) were injected intraperitoneally once per day for 2 days with either saline (0.6%) or somatostatin-14 (SS-14; 500 ng/g body wt). Injection of SS-14 into larval and stage 6 metamorphosing animals resulted in elevated plasma fatty acids levels. In larvae, SS-14-induced hyperlipidemia was supported by enhanced lipolysis, as indicated by increased triacylglycerol lipase (TGL) activity in the liver and kidney. Mobilization of larval renal lipid was accompanied by reduced TG synthesis, as indicated by decreased diacylglycerol acyltransferase (DGAT) activity. In stage 6 metamorphosing lamprey, SS-14 did not significantly affect TGL activity; however, SS-14 significantly reduced fatty acid synthesis, as measured by acetyl-CoA carboxylase activity, in kidney, liver, and muscle, as well as muscular TG synthesis. SS-14-stimulated lipid depletion is reminiscent of the pattern of lipid metabolism displayed by P. marinus during their spontaneous metamorphosis-an observation which suggests that somatostatin may play a role in metamorphosis-associated changes in lipid metabolism in this species., (Copyright 1998 Academic Press.)

During normal pupal-adult development, programmed cell death of the prothoracic gland of Manduca sexta proceeds via apoptosis. By employing the DNA laddering technique, the earliest sign of DNA fragmentation in the cells comprising the gland occurred on Day 5 of pupal-adult development and DNA fragmentation peaked 1 day later. Since juvenile hormone (JH) is known to prevent adult development and since prothoracic gland degeneration occurs during the initial stages of adult development, we wished to determine the possible role of JH in prothoracic gland maintenance. JH was injected into pupae and the glands analyzed by DNA laddering and in situ TUNEL labeling. The administration of JH prevented apoptosis of the prothoracic gland even 11 days after JH injection into young pupae. The prothoracic glands remained intact and their ability to synthesize ecdysteroids was maintained at a fairly active level as ascertained by radioimmunoassay after in vitro incubation. The control glands had degenerated by this time and were almost devoid of ecdysteroidogenesis capability. Ultrastructural analysis confirmed that JH-treated prothoracic gland cells were rescued from the apoptotic sequence, i.e., nuclear condensation, cytoplasmic budding, and cell fragmentation. They exhibited a preserved smooth endoplasmic reticulum and intercellular channel system typical of active prothoracic gland cells. The composite data suggest that JH can both maintain and stimulate the prothoracic gland. Therefore, during normal pupal-adult metamorphosis the absence of JH is prerequisite for both the initiation and completion of prothoracic gland degeneration.

Larval sea lampreys (Petromyzon marinus) measuring 100-119 mm in length were exposed to thyroxine (T4; 10 mg liter-1) or 3,5,3'-triiodothyronine (T3; 1 mg liter-1) in the presence and absence of the goitrogen potassium perchlorate (KClO4; 0.01%), for 4-24 weeks. Every 4 weeks, treated and untreated (control) groups of sea lampreys were examined for external signs of metamorphosis and serum was assayed for T4 and T3 concentrations. Precocious metamorphosis was observed following 8, 12, and 24 weeks of KClO4 treatment; however, metamorphosis was not observed in any control, or T4-, T3-, T4+KClO4-, and T3+KClO4-treated larvae. In addition, serum T4 and T3 concentrations were 62 and 72% lower in KClO4-treated individuals than in control animals, respectively. Treatment with exogenous thyroid hormones (TH), in the presence or absence of KClO4, resulted in serum T4 concentrations which were significantly greater (1.2- to 58-fold) than those of the controls in all sampling periods except one, but serum T3 concentrations were not significantly elevated in more than 50% of the cases. TH+KClO4 treatments produced serum T3 concentrations which were significantly greater than those of KClO4-treated animals and never less than those of controls. These data indicate that larval sea lampreys have a tremendous capacity to take up and store exogenous T4 in their serum, but the uptake and/or serum storage of T3 appears to be stringently regulated. Also, the absence of both metamorphosis and a decline in serum TH concentrations in TH+KClO4-treated animals suggests that a decline in serum TH concentrations may be an essential factor contributing to the induction of metamorphosis by KClO4.

Changes in the pituitary cells involved in amphibian metamorphosis were studied in Bufo arenarum tadpoles with inhibited thyroid function. After hatching, larvae were treated for 5 months with potassium perchlorate (KClO4), a goitrogenic substance known to prevent absorption of iodine from water or food, resulting in impaired thyroid hormone synthesis. Treated larvae continued to grow but halted their development in premetamorphosis, showing hyperplastic thyroid glands with disorganized follicles lacking colloid. Thyrotrop (TSH), lactotrop (PRL), somatotrop (GH), and corticotrop (ACTH) cells were stained immunocytochemically and the changes observed were evaluated morphometrically using an automatic image analyzer. Pars distalis volume increased in treated larvae. Morphometric results showed that, in treated tadpoles, TSH and PRL cell populations and cell volumes increased compared to normal larvae at the same stage. Changes in the GH and ACTH cell morphometry were slight. These results indicate that in Bufo tadpoles, after chronic thyroid hormone withdrawal, TSH, PRL, GH, and ACTH cells are able to develop and that thyroid hormones exert a strong feedback control on the synthesis and storage of TSH and PRL.

Metamorphosis was induced in neotenic axolotls by immersion of the animals in a solution of thyroid hormone. Hematology of the axolotls was examined before, during, and after metamorphosis. There was a transient decrease in numbers of certain white blood cells during metamorphic climax and a permanent shift in the pattern of circulating cells. The eosinophilic granulocyte was the dominating leukocyte type in neotenes and in metamorphosing animals up to midclimax. Lymphocytes and neutrophilic granulocytes (polymorphs) significantly decreased during midclimax. In postmetamorphic axolotls, lymphocytes and polymorphs predominated. The observed decrease of some leukocytes in metamorphosing animals accords with a transient immunosuppression at metamorphic climax. Metamorphosed axolotls showed a humoral immune response (increase in circulating plasma cells) after repeated antigen challenge, whereas neotenic axolotls did not. Alterations in both cellular and humoral immunity are suggested to occur in both young and adult axolotls following experimental induction of metamorphosis.

Thyroidal control of the transition of myosin isoforms during flounder metamorphosis was examined by the administration of either only thiourea (TU), a potent inhibitor of thyroid hormone synthesis, or thyroxine (T4) together with TU into premetamorphic larvae. Immersion of premetamorphic larvae in 400 microM of TU inhibited the appearance of the adult-type DTNB (5,5'-dithio-bis-nitrobenzoic acid) light chain, LC2, whereas administration of T4 with TU induced a precocious appearance of LC2 and decreased the relative amount of the larval-type DTNB light chain, LC2*. TU was administered into juveniles just after completion of metamorphosis. The treatment did not affect the composition of the myosin light chains. These results suggest that thyroid hormone irreversibly turns on the switch for transition of the DTNB light chains from larval to adult type during metamorphosis of the flounder.

Prolactin has an antimetamorphic action in amphibian tadpoles. In the Japanese flounder, Paralichthys olivaceus, ovine prolactin (oPRL, 100 ng/ml) antagonized the stimulatory effect of triiodothyronine (T3, 1 ng/ml) on the resorption of the dorsal fin rays of prometamorphic larvae in vitro. Ovine growth hormone (oGH) at the same dose was without effect. Injections with oPRL (50 ng/fish, six times) into prometamorphic larvae also delayed the resorption of the dorsal fin rays without affecting the rates of eye migration and settling, while oGH was again without effect. The changes in the expression of both PRL and GH mRNAs in the pituitary during metamorphosis were monitored by in situ hybridization using cDNA probes. Both PRL and GH genes were increasingly expressed during successive metamorphic stages. These results are discussed in light of the possible interactions of PRL and GH with thyroid hormones in the control of development in the flounder.

Despite substantial information on the role of the pituitary-thyroid and pituitary-interrenal axes in controlling amphibian metamorphosis, the hypothalamic hormones responsible for controlling the activity of these axes have not been identified. The mammalian thyrotropin-releasing hormone (TRH) does not regulate the thyroid axis of tadpoles; however, corticotropin releasing hormone (CRH) stimulates the release of thyrotropin from bullfrog tadpole pituitary glands in vitro and may thus function as a central regulator of the thyroid axis during metamorphosis. I tested the possibility that a CRH-like peptide is involved in controlling amphibian development by treating tadpoles of two anuran species, the western spadefoot toad Scaphiopus hammondii, and the North American bullfrog, Rana catesbeiana, with neuropeptides and monitoring their effects on metamorphosis. Injection of spadefoot toad tadpoles with ovine (o) CRH (2 micrograms/animal every other day for 3 weeks) or the amphibian CRH-like peptide sauvagine (SV) significantly decreased their time from hatching to metamorphic climax (Gosner stage 42; frontlimb emergence) and their body weight and body length at climax compared with vehicle-injected controls; whereas, TRH had no effect and arginine vasotocin produced a small but significant lengthening of the larval period but did not alter body size at climax. In an acute response experiment, S. hammondii tadpoles (in Gosner stages 36-38--late prometamorphosis) treated with oCRH or SV (2 micrograms/animal) exhibited significantly elevated whole-body thyroxine (T4) content at 2 and 6 hr after injection; whereas, treatment with TRH (2 micrograms/animal) did not significantly alter whole-body T4. R. catesbeiana tadpoles treated with oCRH or SV (surgical implantation of ELVAX pellets impregnated with 100 micrograms peptide and injections of peptides at 5 micrograms/animal once every 3 days) exhibited accelerated spontaneous and triiodothyronine (T3)-induced metamorphosis as assessed by changes in tail height, hind limb development, and body weight; TRH had no effect. Injections of a pool of antisera generated against CRH-like peptides (rat/human CRH, oCRH, SV) slowed T3-induced metamorphosis when compared with normal serum-injected controls. These results support the hypothesis that a CRH-like peptide(s) is involved in the central control of metamorphosis of anuran amphibians, and may act, at least in part, through stimulation of the thyroid axis.

Molting in pycnogonid larvae, Pycnogonum litorale, is accelerated by exposing the animals to 20-hydroxyecdysone at concentrations of 10 to 1000 ppm during Days 5 to 9 after the first ecdysis. Hormone application at the beginning or at the end of the molt cycle, as well as treatment all through the molt cycle, delays or even inhibits the molt by upsetting the sequence of molting processes.

Tail fin regression can be induced in anuran amphibians with L-thyroxine (T4). This regression can be antagonized with prolactin (PRL). Previous work had suggested that protein kinase C (PKC) was involved in PRL action. To address this issue further, the effect of a potent and selective inhibitor of protein kinase C on in vitro tail fin regression was investigated. T4-induced regression of tail fin pieces from Rana pipiens tadpoles could be antagonized by adding PRL or the PKC inhibitor H-7 to the medium. H-7 inhibited fin regression in a dose-dependent manner, with a half-maximal effective concentration of about 10(-5) M. The H-7 analogue, HA-1004 (which is not a selective inhibitor of PKC), was without effect. These results suggest a possible role for PKC in tail fin regression and may be useful in elucidating the antimetamorphic action of PRL.

Attempts to identify a hypothalamic molecule that stimulates thyrotropin (TSH) secretion from amphibian pituitary have been unsuccessful to date. The effects of mammalian (ovine and human) corticotropin-releasing factor (CRF) on the thyroid function of prometamorphic (Taylor & Kollros stages XI-XVII) (Taylor and Kollros, 1946) Rana perezi larvae were studied. Chronic treatments with both ovine and human CRF (oCRF, hCRF) stimulated metamorphosis while delaying larval growth. Chronic hCRF (1 microgram) administration induced 3.2- and 5.3-fold increases in whole body concentration of thyroxine (T4) and triiodothyronine (T3), respectively. In contrast, the 0.5-microgram dose of hCRF stimulated a significant (3.4-fold) increase in whole body concentration of T4 but not of T3. Histological studies of the thyroid gland revealed a 22% increase in the number of follicles per section as a result of the chronic treatment with oCRF (1 microgram). Acute oCRF (2 micrograms) treatment induced a significant increase in T4 concentration at 4 hr (1.3-fold) and 8 hr (2.3-fold) postinjection. T3 concentration was not altered. These results support previous reports and lead us to conclude that a CRF-like peptide, and not TRH, is involved in the regulation of thyroid activity in anuran amphibians during metamorphosis.

Tadpole erythrocyte nuclei contain specific T3 binding sites which increase in number during spontaneous or T3-induced metamorphosis. In the present studies the increase in the number of T3 binding sites after a T3 injections appeared to be completely prevented by cycloheximide and actinomycin D, inhibitors of protein synthesis and RNA synthesis, respectively. However, in some experiments the effect was not statistically significant. The increase in sites was prevented only if the inhibitors were administered at 0 or 24 hr after T3 injection, but not at 48 or 72 hr after T3. When tadpole erythrocytes were incubated with T3 in vitro in M199 culture medium, the number of nuclear T3 binding sites increased within 48 hr. This increase was highly sensitive to inhibition by cycloheximide (maximal at 1 x 10(-6) M) or actinomycin D (maximal at 0.02 micrograms/ml). These inhibitor concentrations only slightly reduced the incorporation of labeled precursors. The T3 concentration required to induce a half-maximal increase in binding sites in vitro was about the same as the T3 concentration at which half the binding sites were occupied. The T3 binding sites had a high affinity for thyroid hormone analogs which stimulate metamorphosis. These results support the designation of the T3 binding sites as T3 receptors. They show that the tadpole erythrocytes respond to T3 with an increase in the number of T3 binding sites without the involvement of other tissues. It is proposed that this is a receptor induction involving synthesis of RNA and protein.

To determine if prolonged larval exposure to juvenile hormone (JH) could influence the decision to metamorphose, Drosophila melanogaster larvae were reared from hatching on medium containing either of the JH mimics, methoprene or 2-[1-methyl-2-(4-phenoxyphenoxy)-ethoxy]-pyridine (S31183). The latter was 23 times more active as a JH mimic in the white puparial assay (ED50 = 0.22 pmole). Larval development and pupariation were unaffected except at 1000 ppm methoprene and 10 ppm or higher S31183 where larval life was prolonged with increased mortality in the second instar. Adult eclosion was prevented by concentrations greater than 1 ppm methoprene and 0.1 ppm S31183. At low concentrations only adult abdominal development was affected, but at the higher concentrations an increasing percentage was blocked at the pupal stage. This latter effect was considerably diminished when the treatment was begun in the mid second instar. The methoprene-resistant mutations, Met1 and Met2, were 10 and 6 times more resistant to S31183 in the white puparial assay and about 20 times more resistant in the larval feeding experiments than the wild-type, indicating that the effects seen are typical of JH. These studies suggest that excess JH may affect adult development of imaginal structures if present at the onset of postembryonic cell proliferation of the imaginal discs or histoblasts. Thus, commitment for adult differentiation must occur early during this proliferative phase.

This study examined the effects of photoperiod and three different dosages of melatonin on the rate of metamorphosis and weight gain in Xenopus laevis. Exposure of larvae to 23L:1D resulted in lower mean body weight and a retarded metamorphic rate in comparison to larvae exposed to 1L:23D. Larvae reared in either photoperiod and treated with exogenous melatonin demonstrated a dose-dependent suppression of weight attained, with short photoperiod larvae showing a more dramatic effect. Analysis of growth patterns indicate that photoperiod and exogenous melatonin have a greater effect on weights prior to Nieuwkoop and Faber stage 56 in the development of Xenopus. Larvae exposed to 1L:23D and exogenous melatonin metamorphosed at an accelerated rate when compared to larvae exposed to 23L:1D and the same dosages of melatonin. In both photoperiod regimens an exogenous melatonin concentration of 45 micrograms/100 ml resulted in an accelerated metamorphic rate, whereas 225 and 450 micrograms/100 ml retarded metamorphic rate of larvae in comparison to controls.