Ontogeny of thyroid tissue and tissue thyroid hormone clearance in rainbow trout embryos reared at two temperatures

The development of the thyroid tissue and clearance of thyroid hormones was studied in rainbow trout embryos reared at 8.5 and 5.5 °C, in order to determine if these events are affected by temperature in the same manner as growth rate. Although the change in growth rate was found to be predictable using the degree-day model, the pattern of whole body thyroid hormone clearance was not similarly related to rearing temperature. Further, although the thyroid follicles appeared at different times in embryos reared at the two temperatures, the differences could not be predicted using the degree-day model. These results suggest that the development of the thyroid tissue and the clearance of yolk hormones are not directly related to rearing temperature. Immunohistochemical staining of the thyroid tissue indicated two phases of thyroid activity, one associated with hormonogenesis, and the other a later phase of hormone release. However, there was no increase in 'whole body' thyroid hormone level associated with the time of appearance of colloid in the follicles. Further, evidence was presented to suggest that the extraction method used did not remove thyroid hormone associated with the colloid in the follicles. There was also evidence of sporadic increases in 'whole body' thyroid hormone content, after the development of phase 2 thyroid follicles which might indicate pulsatile release of T4 and T3.     

The thyroid gland and thyroid hormones in sheepshead minnow (<Emphasis Type="Italic">Cyprinodon variegatus</Emphasis>) during early development and metamorphosis

The sheepshead minnow is widely used in ecotoxicological studies that only recently have begun to focus on disruption of the thyroid axis by xenobiotics and endocrine disrupting compounds. However, reference levels of the thyroid prohormone thyroxine (T4) and biologically active hormone 3,5,3′-triiodothyronine (T3) and their developmental patterns are unknown. This study set out to describe the ontogeny and morphology of the thyroid gland in sheepshead minnow, and to correlate these with whole-body concentrations of thyroid hormones during early development and metamorphosis. Eggs were collected by natural spawning in our laboratory. T4 and T3 were extracted from embryos, larvae and juveniles and an enzyme-linked immunoassay was used to measure whole-body hormone levels. Length and body mass, hatching success, gross morphology, thyroid hormone levels and histology were measured. The onset of metamorphosis at 12-day post-hatching coincided with surges in whole-body T4 and T3 concentrations. Thyroid follicles were first observed in pre-metamorphic larvae at hatching and were detected exclusively in the subpharyngeal region, surrounding the ventral aorta. Follicle size and thyrocyte epithelial cell heights varied during development, indicating fluctuations in thyroid hormone synthesis activity. The increase in the whole-body T3/T4 ratio was indicative of an increase in outer ring deiodination activity. This study establishes a baseline for thyroid hormones in sheepshead minnows, which will be useful for the understanding of thyroid hormone functions and in future studies of thyroid toxicants in this species.     

鳙、团头鲂和短盖巨脂鲤早期发育阶段甲状腺激素含量变化研究

鳙、团头鲂和短盖巨脂鲤胚胎与仔鱼体内三碘甲腺原氨酸和四状腺素含量的变化，采用放射免疫测定法进行测定，研究发现胚胎阶段的Ｔ３和Ｔ４含较稳定，出膜后仔鱼阶段的Ｔ３和Ｔ４含量逐渐下降。     

Development of thyroid gland and changes in thyroid hormone levels in Leptocephali of Japanese Eel (Anguilla japonica)

Development of the thyroid gland of the Japanese eel (Anguilla japonica) was studied with the use of tank-reared fish. A single thyroid follicle was first found in larvae at 29 days post hatching (dph), total length (TL) 12 mm. Until reaching 25 mm in TL (100 dph), leptocephali had one or two follicles per individual. The inner colloid of the follicles was weakly immuno-positive against the anti-thyroxine (T₄) antibody. The number of thyroid follicles and the immunoreactivity later increased as the larvae grew. Thyroid hormones (TH) T₄ and triiodothyronine (T₃) were not detected in premetamorphic larvae by radioimmunoassays, but became detectable during metamorphosis. The maximum level of T₄ was seen in fish at the end of metamorphosis and in just-metamorphosed juveniles, whereas T₃ reached the highest level during metamorphosis and declined toward the end of metamorphosis. The results indicated that the thyroid gland first became active during metamorphosis in the development of eel.     

A reference growth curve for nutritional experiments in zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) and changes in whole body proteome during development

Zebrafish is one of the most used vertebrate model organisms in molecular and developmental biology, recently gaining popularity also in medical research. However, very little work has been done to assess zebrafish as a model species in nutritional studies in aquaculture in order to utilize the methodological toolbox that this species represents. As a starting point to acquire some baseline data for further nutritional studies, growth of a population of zebrafish was followed for 15 weeks. Furthermore, whole body proteome was screened during development by means of bi-dimensional gel electrophoresis and mass spectrometry. Fish were reared under best practice laboratory conditions from hatching until 103 days post-fertilization (dpf) and regularly fed ad libitum with Artemia nauplii from 12 dpf. A growth burst occurred within 9–51 dpf, reaching a plateau after 65 dpf. Fork length and body weight were significantly lower in males than in females from 58 dpf onwards. Proteomics analysis showed 28 spot proteins differently expressed through development and according to sex. Of these proteins, 20 were successfully identified revealing proteins involved in energy production, muscle development, eye lens differentiation, and sexual maturation. In summary, zebrafish exhibited a rapid growth until approximately 50 dpf, when most individuals started to allocate part of the dietary energy intake for sexual maturation. However, proteomic analysis revealed that some individuals reached sexual maturity earlier and already from 30 dpf onwards. Thus, in order to design nutritional studies with zebrafish fed Artemia nauplii, it is recommended to select a period between 20 and 40 dpf, when fish allocate most of the ingested energy for non-reproductive growth purposes.     

Effects of thyroid hormone supplementation of canola meal-based diets on growth,and interrenal and thyroid gland physiology of rainbow trout (Salmo gairdneri)

Rainbow trout fed a 26% canola meal-based (CM) diet for 12 weeks at 15°C exhibited reduced growth, lower feed conversion, enlarged thyroid glands and lower plasma thyroid hormone (TH) levels than comparable fish fed equinitrogenous, equicaloric soybean meal-based (SB) diets. Supplementation of the SB diets with either T₄ (20 mg/kg) or T₃ (10 or 20 mg/kg) had no effect on the growth rate, feed conversion and thyroid histology of the trout. However, plasma T₄ levels weredepressed in trout fed the T₄- and high T₃-supplemented SB diets. In trout fed T₄- and T3-supplemented CM diets the growth rates and feed conversion were not significantly different from those of the SB-fed groups. Moreover, in the T₄-supplemented group, plasma T₄ levels were in the normal range. However, thyroid enlargement was evident in all the CM-fed fish, and plasma T₃ levels were markedly elevated in groups fed the T₃-supplemented CM diets. The data suggest that antithyroid components in the CM diets inhibited TH synthesis (but not their release), and impaired TH clearance from the circulation. There were no significant differences in plasma cortisol levels in the 8 treatment groups, nor were there differences in the histological appearance of the interrenal gland. However, when the data from SB- and CM-fed fish were pooled, plasma cortisol levels in the SB-fed fish were significantly lower than in the CM-fed animals. Glucosinolates at a level of 164 mg/kg diet were toxic to young trout, but the effect was ameliorated by dietary TH supplementation.     

Dynamic expression pattern of corticotropin-releasing hormone,urotensin I and II genes under acute salinity and temperature challenge during early development of zebrafish

Corticotropin-releasing hormone (CRH), urotensin I (UI) and urotensin II (UII) are found throughout vertebrate species from fish to human. To further understand the role of crh, uI and uII in teleosts during development, we investigated the expression pattern of crh, uI, uIIα and uIIβ genes, and their response to acute salinity and temperature challenge during early development of zebrafish, Danio rerio. The results reveal that crh, uI, uIIα and uIIβ mRNA are detected from 0hpf, and the expression levels increase to a maximum at 6 days post fertilization (dpf), with the exception of uIIα that peak at 5dpf. Exposure of zebrafish embryos and larvae to acute osmotic (30ppt) stress for 15 min failed to modify expression levels of crh, uI, uIIα and uIIβ mRNA from levels in control fish except at 6dpf when uIIα and uIIβ were significantly (P < 0.05) modified. Exposure of embryos and larvae to a cold (18 °C) or hot stress (38 °C) generally down-regulated mRNA levels of crh, uI, uIIα and uIIβ apart from at 3dpf. The results indicate that the contribution of crh, uI, uIIα and uIIβ genes to the stress response in zebrafish may be stressor-specific during early development. Overall, the results from this study provide a basis for further research into the developmental and stressor-specific function of crh, uI, uIIα and uIIβ in zebrafish.     

Stimulatory effect of thyroid hormones improves larval development and reproductive performance in alligator gar (Atractosteus spatula) and spotted gar (Lepisosteus oculatus)

Development of alligator gar (Atractosteus spatula) and spotted gar (Lepisosteus oculatus) larvae was evaluated after exposure of embryos to thyroid hormones (TH) by two different methods of administration. For the first method, alligator gar embryos were placed in a water bath with triiodothyronine (T₃; 1 ppm for 2 h), and control treatment embryos were placed in a water bath without T₃. For the second method, spotted gar adult males and females were injected with different TH during induced spawning: a group was injected with T₃ [20 mg kg^?1 body weight (BW)], a second group with thyroxine (T₄; 20 mg kg^?1 BW), a third group with thyroid‐stimulating hormone (TSH; 4 IU kg^?1BW) and a control group with dimethyl sulfoxide. Both methods resulted in increases in the concentrations of T₃ and T₄ in embryos of both species. An increase in the alkaline enzymatic activity of TH‐treated larvae was observed as well as an increase in the RNA:DNA, RNA:Dry weight and DNA:Dry weight ratios at hatching. Some positive effects of TH application were: a significant increase in the hatching rate of spotted gar larvae from TH‐injected parents and a higher percentage of successful spawns. Evenness of larval sizes and a higher survival rate were observed in alligator gar larvae. Snout development was accelerated by TH treatment in both species.     

牙鲆碱性磷酸酶基因调控序列的功能分析及甲状腺激素对其调节作用

为分析牙鲆碱性磷酸酶基因5'调控区和第一内含子调控序列的功能,本研究运用DNA重组技术将PCR扩增得到的牙鲆碱性磷酸酶基因的5′调控区序列、第一内含子序列及5′调控区和第一内含子串联序列分别插入启动子缺失的增强型绿色荧光蛋白表达载体pAcGFP1-1中,得到pAcGFP1-5′ALP、pAcGFP1-Intron1和pAcGFP1-AI报告基因表达载体。通过脂质体介导重组质粒转染中国仓鼠卵巢细胞(CHO)并检测荧光信号。转染后细胞状态良好,24 h后发现,在倒置荧光显微镜下均可看到不同强度的绿色荧光信号,且荧光信号随时间的延长而增强,其中转染pAcGFP1-AI后的荧光信号强度最强。以上结果表明,牙鲆碱性磷酸酶基因5′调控区和第一个内含子序列均具有启动子活性,且第一内含子与5′调控区存在协同效应,两者协同能增强基因的表达。为了解甲状腺激素T3对调控序列启动子活性的调节作用,用不同浓度的T3处理转染的CHO细胞,24 h后发现加入50、75和100 nM的T3都增强了绿色荧光蛋白的表达,而且信号强度随T3浓度的增加而增强。这表明甲状腺激素T3对牙鲆碱性磷酸酶基因调控序列的启动子活性有一定的增强作用。本研究为深入阐明牙鲆碱性磷酸酶基因转录表达的分子机制提供了实验依据。     

Developmental changes in pituitary–thyroid axis, and formation of gonads in leptocephali and glass eels of Anguilla spp.

SUMMARY: Pituitary, thyroid gland and gonads in leptocephali of Japanese eel Anguilla japonica (19.8–32.6 mm in total length), A. obscura (45.0 mm), and A. bicolor pacifica (49.5 mm) and those in glass eels of the Japanese eel were histologically and immunohistochemically examined in order to observe the developmental changes of these endocrine organs in the Anguillidae. The pituitary, consisting of adenohypophysis and neurohypophysis in Japanese eel leptocephali over 22.5 mm, did not contain thyroid stimulating hormone (TSH) immunoreactive cells. Such cells were, however, detectable in the more developed pituitaries of leptocephali of A. obscura and A. bicolor pacifica and in those of glass eels. Conversely, thyroxine (T₄)-immunoreactive thyroid follicles could be detected in all specimens, both leptocephalic and glass eel. Only in glass eels, gonads were found in the body cavity, and these gonads harbored one or two primordial germ cells (PGC) per cross-section. Our results indicate that thyroid hormones (TH) production started prior to TSH production, and that TSH and TH are both secreted during the metamorphosis from leptocephalus to glass eel. Therefore, it is plausible that the TSH–TH axis is involved in the metamorphosis from leptocephalus to glass eel, but not in the early growth from preleptocephalus to leptocephalus.     

Thyroid hormone‐induced swim bladder and eye maturation are transduced by IGF‐1 in zebrafish embryos

Maternally derived thyroid hormones (THs) deposited in yolk promote fish embryogenesis and survival, and understanding early regulatory mechanisms could lead to improved seedstock production. We have tested the hypothesis that some thyroid actions may be mediated by insulin‐like growth factor I (IGF‐1), another promoter of embryo development. Differentiation and performance were assessed in embryos treated with THs in the presence or absence of an IGF‐1 receptor blocking peptide. Treatment with the TH triiodothyronine (T₃) promoted IGF‐1 gene expression at days 1 and 5, and advanced swim bladder and eye development, but blocking the IGF‐1 receptor eliminated the swim bladder and eye effects. Growth and survival at 1 week of age were impaired by the IGF‐1 receptor blocking peptide alone, but concurrent treatment with T₃ partially restored these indices. Our results confirm interaction of T₃ and IGF‐1 regulatory signalling in zebrafish embryogenesis and transduction by IGF‐1 of thyroid‐driven swim bladder and eye maturation.     

Determination of periods of sensitivity to low-salinity and low-temperature conditions during the early development of cultured Japanese eel Anguilla japonica larvae with respect to the rate of morphological deformity at completion of yolk resorption

The present study analyzed the rate of occurrence of deformities at completion of yolk resorption after Japanese eel larvae were exposed to low-salinity (29 psu) or low-water-temperature (20 °C) conditions (after they had been reared under suitable conditions: 34 psu, 25 °C) at various points between 0 and 8 days post-fertilization (dpf) in order to determine the period of sensitivity of the larvae to low-salinity or low-temperature conditions. No significant differences were observed between the rate of deformed larvae obtained in the groups reared under suitable conditions and the corresponding rates in groups that were exposed to low-salinity or low-temperature conditions at 4 dpf or later, but the frequency of normal larvae showed a downward trend in experimental groups that were subjected to low-salinity or low-temperature conditions at 3 dpf or earlier. Occurrence rates of pericardial edema were significantly higher in experimental groups that were exposed to low-salinity or low-temperature conditions at 2 dpf or earlier. Therefore, it was concluded that the rearing conditions for Japanese eel larvae should be maintained at 34 psu salinity and 25 °C until at least 4 dpf in order to reduce the occurrence of larval deformity at completion of yolk resorption.     

Selenium supplementation changes glutathione peroxidase activity and thyroid hormone production in Senegalese sole (Solea senegalensis) larvae

Dietary selenium (Se) influences the glutathione peroxidase (GPx) activity and the thyroid hormone system in vertebrates, but these relations are poorly described in fish. The present study aimed to evaluate the effect of Se supplementation on Senegalese sole larvae, focusing on GPx activity and thyroid status. Two groups of larvae were fed on either control (C) or Se‐enriched live feed, from 2 days after hatch (DAH) until 34 DAH. Senegalese sole larvae fed Se‐enriched live feed significantly increased their whole‐body Se, compared to larvae from C group. The activity of GPx was higher in larvae from Se group, suggesting that the levels of this mineral were suboptimal in the control diet. TH levels were also higher in larvae fed Se‐enriched live feed, suggesting that Se may have a positive influence on the TH production in early life stages of marine fish larvae.     

Profiles of thyrotropin,thyroid hormones,follicular cells and type I deiodinase gene expression during ontogenetic development of tilapia larvae and juveniles

The aims of the present study are to determine whether triiodothyronine (T3) and/or thyroxine (T4) in tilapia larvae is gifted through the mother, and to investigate the change profiles of thyrotropin (TSH), thyroid follicular cells and type I deiodinase (D1) gene expression following larval development. T3 and T4 contents were measured using radioimmunoassay, thyrotropin was observed using immunocytochemistry, and the D1 gene was cloned and measured using real-time PCR. Results indicated that the β-TSH-immunoreactive cells (thyrotropin ICC) signals were detected at 9 dph (i.e., 9 days of post-hatching). Thyroid follicular cells were observed first at 3 dph, while the T3 contents of the whole body gradually decreased before 11 dph. T4 contents were detected until 13 dph, with higher secretion during 19–21 dph. In addition, the T3 synthesis was not inhibited by thiourea (TU) before 13 dph, but the TU response in the larvae appeared after 13 dph. Type I deiodinase (D1: GenBank accession number KC591724) was found to contain 2444 bases and encoded 248 amino acids. The D1 mRNA expression began to increase at 13 dph, with a higher expression during 15–19 dph. These results suggested that the T3 contents were maternally derived before 13 dph. Both thyroid hormonal changes and some parameters related to thyroid hormone synthesis in ontogenetic tilapia are discussed.     

Improved protocol for rapid zebrafish growth without reducing reproductive performance

Despite widespread use of zebrafish Danio rerio (Hamilton) as a model organism for finfish aquaculture, genetics and molecular biology, husbandry methods and nutrition have not been standardised. We characterise a protocol for rearing zebrafish using a recently developed combination of environmental parameters that yield rapid growth rates: high densities of live food organisms (marine rotifers and brine shrimp nauplii), low salinity, continuous light and algal turbidity during larval and early juvenile stages (5–21 days post fertilisation, dpf) followed by a transition to conventional rearing methods (dark/light photoperiod, freshwater). We assessed a range of larval fish densities in a stagnant water system with continuous ad libitum feeding until 21 dpf and periodic water changes. Fish stocking densities of 12–22 fish/L gave maximum growth rate and growth rate declined as stocking density increased past this threshold. Survival rates during 5–21 and 21–42 dpf were 87 ± 9 and 98 ± 3% respectively. Mean ± SD weight and length were 64 ± 9 mg and 19.4 ± 1.0 mm at 21 dpf and 245 ± 28 mg and 30.4 ± 1.5 mm at 42 dpf. Fertility was assessed at 66 ± 3 dpf. Mean clutch size was 232 ± 124 oocytes/female and fertilisation rate was 72 ± 16%. This protocol will significantly increase productivity in zebrafish facilities.