Thyroid and pituitary gland development from hatching through metamorphosis of a teleost flatfish, the Atlantic halibut

Fish larval development, not least the spectacular process of flatfish metamorphosis, appears to be under complex endocrine control, many aspects of which are still not fully elucidated. In order to obtain data on the functional development of two major endocrine glands, the pituitary and the thyroid, during flatfish metamorphosis, histology, immunohistochemistry and in situ hybridization techniques were applied on larvae of the Atlantic halibut (Hippoglossus hippoglossus), a large, marine flatfish species, from hatching through metamorphosis. The material was obtained from a commercial hatchery. Larval age is defined as day-degrees (D =accumulated daily temperature from hatching). Sporadic thyroid follicles are first detected in larvae at 142 D (27 days post-hatch), prior to the completion of yolk sack absorption. Both the number and activity of the follicles increase markedly after yolk sack absorption and continue to do so during subsequent development. The larval triiodothyronine (T3) and thyroxine (T4) content increases, subsequent to yolk absorption, and coincides with the proliferation of thyroid follicles. A second increase of both T3 and T4 occurs around the start of metamorphosis and the T3 content further increases at the metamorphic climax. Overall, the T3 content is lower than T4. The pituitary gland can first be distinguished as a separate organ at the yolk sack stage. During subsequent development, the gland becomes more elongated and differentiates into neurohypophysis (NH), pars distalis (PD) and pars intermedia (PI). The first sporadic endocrine pituitary cells are observed at the yolk sack stage, somatotrophs (growth hormone producing cells) and somatolactotrophs (somatolactin producing cells) are first observed at 121 D (23 days post-hatch), and lactotrophs (prolactin producing cells) at 134 D (25 days post-hatch). Scarce thyrotrophs are evident after detection of the first thyroid follicles (142 D ), but coincident with a phase in which follicle number and activity increase (260 D ). The somatotrophs are clustered in the medium ventral region of the PD, lactotrophs in the anterior part of the PD and somatolactotrophs are scattered in the mid and posterior region of the pituitary. At around 600 D , coinciding with the start of metamorphosis, somatolactotrophs are restricted to the interdigitating tissue of the NH. During larval development, the pituitary endocrine cells become more numerous. The present data on thyroid development support the notion that thyroid hormones may play a significant role in Atlantic halibut metamorphosis. The time of appearance and the subsequent proliferation of pituitary somatotrophs, lactotrophs, somatolactotrophs and thyrotrophs indicate at which stages of larval development and metamorphosis these endocrine cells may start to play active regulatory roles.This work has been carried out within the projects ‘‘Endocrine Control as a Determinant of Larval Quality in Fish Aquaculture’’ (CT-96-1422) and ‘‘Arrested development: The Molecular and Endocrine Basis of Flatfish Metamorphosis’’ (Q5RS-2002-01192), with financial support from the Commission of the European Communities. However, it does not necessarily reflect the Commission’s views and in no way anticipates its future policy in this area. This project was further supported by the Swedish Council for Agricultural and Forestry Research and Pluriannual funding to CCMAR by the Portuguese Science and Technology Council

The Impact of Initial Energy Reserves on Growth Hormone Resistance and Plasma Growth Hormone-Binding Protein Levels in Rainbow Trout Under Feeding and Fasting Conditions

The growth hormone (GH)–insulin-like growth factor I (IGF-I) system regulates important physiological functions in salmonid fish, including hydromineral balance, growth, and metabolism. While major research efforts have been directed toward this complex endocrine system, understanding of some key aspects is lacking. The aim was to provide new insights into GH resistance and growth hormone-binding proteins (GHBPs). Fish frequently respond to catabolic conditions with elevated GH and depressed IGF-I plasma levels, a condition of acquired GH resistance. The underlying mechanisms or the functional significance of GH resistance are, however, not well understood. Although data suggest that a significant proportion of plasma GH is bound to specific GHBPs, the regulation of plasma GHBP levels as well as their role in modulating the GH–IGF-I system in fish is virtually unknown. Two in vivo studies were conducted on rainbow trout. In experiment I, fish were fasted for 4 weeks and then refed and sampled over 72 h. In experiment II, two lines of fish with different muscle adiposity were sampled after 1, 2, and 4 weeks of fasting. In both studies, plasma GH, IGF-I, and GHBP levels were assessed as well as the hepatic gene expression of the growth hormone receptor 2a (ghr2a) isoform. While most rainbow trout acquired GH resistance within 4 weeks of fasting, fish selected for high muscle adiposity did not. This suggests that GH resistance does not set in while fat reserves as still available for energy metabolism, and that GH resistance is permissive for protein catabolism. Plasma GHBP levels varied between 5 and 25 ng ml−1, with large fluctuations during both long-term (4 weeks) fasting and short-term (72 h) refeeding, indicating differentiated responses depending on prior energy status of the fish. The two opposing functions of GHBPs of prolonging the biological half-life of GH while decreasing GH availability to target tissues makes the data interpretation difficult, but nutritional regulatory mechanisms are suggested. The lack of correlation between hepatic ghr2a expression and plasma GHBP levels indicate that ghr2a assessment cannot be used as a proxy measure for GHBP levels, even if circulating GHBPs are derived from the GH receptor molecule

Identification of two isoforms of Atlantic halibut insulin-like growth factor-I receptor genes and quantitative gene expression during metamorphosis

Insulin-like growth factor-I (IGF-I) is an important regulator of growth and development in vertebrates. Both the endocrine and paracrine actions of IGF-I are mediated through ligand-binding to a membrane-bound IGF-I receptor (IGF-IR). The characterization of this receptor and subsequent expression studies thus help elucidate the endocrine regulation of developmental processes. As other flatfish species, the Atlantic halibut (Hippoglossus hippoglossus) undergoes a dramatic larval metamorphosis. This process is largely under endocrine control, and data indicate that IGF-I could be a key regulator. IGF-I content increases up to late pre-metamorphosis and decreases during metamorphosis. The IGF-IR has, however, not been studied during flatfish metamorphosis. To examine IGF-IR gene expression, two IGF-IR mRNA were cloned and sequenced. These partial sequences share high identity (≥ 95%) and similarity (≥ 97%) with other fish IGF-IR and lower identity (≥ 77%) and similarity (≥ 83.5%) with Japanese flounder insulin receptors. The expression of mRNA for both IGF-IR was analyzed by quantitative real-time RT-PCR during six larval developmental stages from pre- to post-metamorphosis. IGF-IR1 and IGF-IR2 mRNA are differentially expressed during metamorphosis, but if this indicates an isoform-specific regulation of developmental processes by circulating and/or locally-secreted IGF-I is unclear. Both IGF-IR genes are down-regulated in halibut larvae experiencing arrested metamorphosis, suggesting the IGF-I system is critical for metamorphic success in halibut

Cloning of Atlantic halibut growth hormone receptor genes and quantitative gene expression during metamorphosis

To gain insight into the possible regulatory role of the growth hormone (GH)-insulin-like growth factor I (IGF-I) system in flatfish metamorphosis, body GHR gene expression as well as IGF-I protein content was quantified in larval Atlantic halibut throughout metamorphosis (developmental stages 5–10). The cDNA of the full-length GH receptor (hhGHR) was cloned from adult liver and characterized. The hhGHR shows common features of a GHR, including a (Y/F)GEFS motif in the extracellular domain, a single transmembrane region, and an intracellular domain containing a Box 1 and Box 2. Additionally, a truncated GHR (hhGHRtr), similar to turbot and Japanese flounder GHRtr, was cloned and sequenced. These sequences are highly similar to the full-length and truncated GHRs in turbot (89%/86%) and Japanese flounder (93%/91%) with lower identity with other fish type I GHR (⩽81%) and type II GHRs (⩽58%). A quantitative real-time RT-PCR assay was used to measure hhGHR and hhGHRtr mRNA content in normally and abnormally metamorphosed individuals at six developmental stages, from early pre-metamorphosis to post-metamorphosis, when the fish is considered a juvenile. The level of hhGHR gene expression was highest at pre-metamorphic stage 6 and at stage 8 at the onset of metamorphosis, and then decreased during metamorphic climax and post-metamorphosis. Expression of hhGHRtr reached highest levels at stage 6 and then decreased to post-metamorphosis. The ratio of expression between the full-length and the truncated GHR (hhGHR:hhGHRtr) varied among stages and was highest at the onset of metamorphosis and at metamorphic climax. A radioimmunoassay was used to measure halibut IGF-I body content throughout metamorphosis. IGF-I increases from early metamorphosis to the onset of metamorphosis and then decreases towards post-metamorphosis. In comparison between normally and abnormally metamorphosing larvae, IGF-I content, hhGHR and hhGHRtr mRNA levels were reduced in the abnormal fish. These data indicate that the GH-IGF-I system either has a regulatory role in metamorphosis, or is being affected as a consequence of the abnormal metamorphosis

Involvement of growth hormone-insulin-like growth factor I

The role of growth hormone (GH) and insulinlike growth factor-I (IGF-I) in the tissue remodeling associated with the transition of a symmetrical larva to an asymmetrical juvenile during flatfish metamorphosis is unknown. In order to investigate the potential role of these hormones in the remodeling of cranial bone and soft tissue that accompanies eye migration during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus) larvae, tissuespecific gene expression was monitored by in situ hybridization for Atlantic halibut type I growth hormone receptor (hhGHR), type II hhGHR, and insulin-like growth factor-I receptor (hhIGF-IR). Polyclonal antibody generated against the extracellular domain of type I hhGHR was used for the immunohistochemical localization of type I GHR protein. Type I hhGHR, type II hhGHR, and hhIGF-IR mRNA were expressed in fibroblasts, frontal bone osteocytes, and dorsal chondrocytes at the onset of metamorphosis (stage 8),during metamorphic climax (stage 9), and in fully metamorphosed juveniles (stage 10). Type I GHR protein showed similar expression patterns to those of type I hhGHR mRNA, except in chondrocytes in which little GHR protein was detected. The localization of GHR and IGF-IR transcripts and GHR protein in cranial structures that undergo remodeling is intriguing and suggests that, in addition to thyroid hormones, the GH-IGF-I system is involved in morphological transformations during metamorphosis in Atlantic halibut.We thank Heiddis Smáradóttir, Arnar Jónsson, and Øystein Saele for larval sampling, and Nádia Silva for methodological assistance