Masatomo Tagawa

Presence of thyroxine in eggs and changes in its content during early development of chum salmon, Oncorhynchus keta.

In order to examine the role of thyroid hormones during salmonid development, techniques were developed for quantitative extraction of thyroxine from eggs, whole embryos, and alevins of chum salmon (Oncorhynchus keta) at various stages of development. Frozen eggs, embryos, alevins, or fry were homogenized in ice-cold methanol. The homogenate was centrifuged, and the supernatant was washed with a mixture of chloroform and 0.05% CaCl2. The aqueous layer was lyophilized, and the residue was redissolved in barbital buffer for thyroxine radioimmunoassay (RIA). Serial dilutions of the egg or tissue extracts gave inhibition slopes that were parallel to that of the thyroxine standard in the RIA. Immunoreactivity of the extracts coeluted with thyroxine standard in reverse-phase HPLC on an ODS column. Recovery of thyroxine from egg and tissue extracts was estimated from the recovery of 125I-labeled thyroxine added to the initial homogenates. Thyroxine content of eggs just after fertilization was 4-5 ng/egg, and this level was maintained until hatching. A decrease in thyroxine content was seen during yolk absorption. Total thyroxine increased to about 10 ng/fish, a level higher than that in the unfertilized egg, at the time of complete yolk absorption, and decreased within 10 days to a low level of 1 ng/fish. These findings are discussed in relation to the role of maternal thyroid hormones during early development and also to the onset of larval thyroid function.

Thyroxine surge in metamorphosing flounder larvae

The tissue concentration of thyroxine (T4) in larval flounder (Paralichthys olivaceus) was studied at various stages of their metamorphosis using a specific radioimmunoassay developed for samples obtained by quantitative extraction of the hormone from fish eggs and larvae. T4 concentrations were below the limit of detection in extracts of fertilized eggs and larvae until the end of prometamorphosis. Several dorsal fin rays continued growing from premetamorphosis until the end of prometamorphosis. The hormone became detectable at the onset of metamorphic climax when the elongated dorsal fin rays stopped growing. A sharp increase in tissue T4 concentration was observed in midclimax of metamorphosis, and the high level of T4 (11-13 ng/g body weight) was maintained until the end of the climax. The period of elevated T4 levels coincided with resorption of the elongated dorsal fin rays. The right eye completed translocation from the right to the left side of the body during metamorphic climax. The tissue T4 level declined to about a half of the peak value during postclimax, and then increased moderately in later adult-type juveniles. The results of an additional experiment, in which fertilized eggs and newly hatched larvae were treated with T4 and/or thiourea, suggested the presence of negative feedback regulation of pituitary-thyroid axis, implying an involvement of thyroid hormones even in early premetamorphic larvae. These results suggest that metamorphic climax is induced by a surge of thyroid hormone, and that thyroid hormone may also regulate development before and after the metamorphosis.

DEVELOPMENT OF THE PITUITARY, THYROID AND INTERRENAL GLANDS AND APPLICATIONS OF ENDOCRINOLOGY TO THE IMPROVED REARING OF MARINE FISH LARVAE

Abstract A key to success in the culture of marine fish is the mass production of high quality fry, a process largely dependent on successful first feeding and normal development and growth of fish larvae. In this regard it is important to examine the structural and functional development of the endocrine system during early ontogeny of marine fish. To characterize early endocrine development a comparative study was conducted with various marine fish. The age when the pituitary gland differentiated, assessed by immunohistochemical detection of GH and PRL, varied among species, but generally coincided with the time of eye pigmentation, when yolk absorption is very rapid in most marine fish larvae. Thyroid follicles and pancreatic islets first appeared at about the same time, while interrenal cells appeared later. Tissue concentrations of thyroid hormones decreased during embryonic development to nearly undetectable levels at the completion of yolk absorption. During early larval life thyroid hormone levels increased gradually and were significantly elevated during the transformation to the juvenile stage, especially in flatfish. Tissue concentrations of cortisol showed nearly the same developmental pattern, but increased prior to thyroid hormone levels. GH and PRL exhibited different developmental patterns. GH increased continuously during larval growth while PRL exhibited nearly the same pattern as cortisol in Japanese flounder. Based on these findings it is concluded that the endocrine system becomes functional before the completion of yolk absorption in most marine fish, when the switch from a maternal (yolk) to an endogenous larval hormone source occurs. A preliminary experiment on T 4 supplementation indicated that intact protein ingestion by pinocytosis in the rectal epithelium and fat absorption in the intestinal epithelium were enhanced. Potential benefits of exogenous hormone treatment are discussed particularly in relation to digestive functions.

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

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

Changes in tissue and blood concentrations of thyroid hormones in developing chum salmon

The changes in tissue and blood concentrations of thyroxine (T4) and triiodothyronine (T3) were examined during development of the chum salmon (Oncorhynchus keta). Extraction methods previously established for tissue T4 were also validated for tissue T3, by parallel displacement curves to T3 standard in the radioimmunoassay and by the same elution patterns of immunoreactivity in a HPLC system. The T3 concentration of the eggs just after fertilization (4-9 ng/g) was lower than the T4 concentration (5-15 ng/g). Both T4 and T3 concentrations in the whole body decreased steadily during yolk absorption, primarily due to the decline of the hormone content in the yolk. Both T4 and T3 were detected in blood plasma at later stages of yolk absorption, and the plasma levels increased toward the end of yolk absorption. At the end of yolk absorption, when the larvae emerge from the gravel bed, a transient increase in whole body concentrations of T4 and T3 was observed. Plasma levels of T4 were always greater than the T3 levels. Thyroid follicles began to develop during the early stages of yolk absorption. These findings suggest important roles of maternal thyroid hormones for developing salmon embryos during yolk absorption.

Thyroid hormones in eggs of various freshwater, marine and diadromous teleosts and their changes during egg development

Thyroid hormone concentrations in unfertilized eggs of 26 species of various freshwater, marine and diadromous teleosts were examined, together with changes in their concentrations during egg development in some species. Significant quantities of both thyroxine (T4) and triiodothyronine (T3) were found in eggs of all species examined. Mean T4 and T3 concentrations in eggs varied from 0.04 (marbled sole) to 15.00 ng/g (chum salmon), and from 0.07 (goldfish) to 9.95 ng/g (Pacific herring), respectively. T4 concentrations were significantly greater than T3 concentrations in eggs of most freshwater fishes, whereas T3 concentrations were greater in seawater fishes. During the course of development, thyroid hormones in eggs decreased markedly before hatching. These findings suggest that thyroid hormones are consistently present in teleost eggs, and thus may play an important role in the egg development.

DISTRIBUTIONAL CHANGES IN BRANCHIAL CHLORIDE CELLS DURING FRESHWATER ADAPTATION IN JAPANESE SEA BASS LATEOLABRAX JAPONICUS

Abstract Distributional changes in branchial chloride cells were examined in Japanese sea bass (Lateolabrax japonicus) juveniles transferred from seawater (SW) to fresh water (FW) during their migration season toward low salinity habitat in nature. Chloride cells were identified by immunocytochemistry with a specific antiserum for Na+,K+-ATPase. In fish reared in SW as controls, branchial chloride cells were localized exclusively in the filaments and absent in the lamellae. When sea bass were transferred from SW to FW, chloride cells emerged in gill lamellae, starting at the proximal part of the lamellae and thereafter spread over the lamellar epithelium. On 7th and 15th days after FW transfer, chloride cells were mostly found on the lamellae, whereas the number of filament chloride cells was decreased. These results suggest that, in Japanese sea bass juveniles, chloride cells in the gill lamellae are important in FW adaptation, and that lamellar chloride cells originated from the filaments and migrated to the lamellae during FW adaptation.

Asymmetrical development of bones and soft tissues during eye migration of metamorphosing Japanese flounder, Paralichthys olivaceus.

Abstract. The symmetrical body of flatfish larvae dramatically changes into an asymmetrical form after metamorphosis. Eye migration results in the most significant asymmetrical development seen in any vertebrate. To understand the mechanisms involved in eye migration, bone and cartilage formation was observed during metamorphosis in laboratory-reared Japanese flounder, Paralichthys olivaceus, by using whole-body samples and histological sections. Most of the hard tissues of the cranium (parasphenoid, trabecular cartilage, supraorbital canal, and supraorbital bar) exist symmetrically in the larval period before metamorphosis and develop by twisting in the same direction as that in which the eye migrates. An increase in skin thickness beneath the eye was observed only on the blind side at the beginning of eye migration; this was the first definitive difference between the right and left sides of the body. The pseudomesial bar, a peculiar bone present only in flatfishes, developed from this thick skin and grew dorsad. Novel sac-like structures were found and named retrorbital vesicles. The retrorbital vesicle of the blind side grew larger and faster than that of the ocular side when the right eye moved most dramatically, whereas no difference was observed between the volume of right and left connective tissue in the head. The asymmetrical presence and growth of the pseudomesial bar together with inflation of the retrorbital vesicle on the blind side may be responsible for right eye migration during metamorphosis in the Japanese flounder.

Hypoosmoregulatory ability of eyed-stage embryos of chum salmon

To evaluate the osmoregulatory ability of eyedstage embryos of laboratory-reared chum salmon, Oncorhynchus keta, we examined changes in osmolality of the perivitelline fluid and blood following transfer to 50 and 100% seawater (SW), together with morphological changes in chloride cells present in the yolk sac membrane. Transfer to SW did not cause any significant change in the whole egg weight. However, the embryos shrank when the eggs were transferred to SW, whereas the perivitelline space increased at the expense of the embryo. Osmolality of the perivitelline fluid increased rapidly to reach environmental levels 3 h after transfer, indicating that the egg shell is permeable to ions and water. Blood osmolality increased after transfer to SW, reached a peak level at 3 h, and then decreased gradually. The chloride cells in the yolk sac membrane became activated following transfer, as shown by increased cell size and frequent appearance of apical openings. These results indicate that the eyed-stage embryos of chum salmon possess hypoosmoregulatory ability and that chloride cells in the yolk sac membrane may be involved in salt extrusion, in place of gill chloride cells, during the late embryonic stage.

Effects of bovine TSH on the tissue thyroxine level and metamorphosis in prometamorphic flounder larvae

Bovine thyroid-stimulating hormone (TSH) was microinjected (0.2 microliter/fish) into prometamorphic flounder larvae and the effects on metamorphosis as well as the tissue thyroxine (T4) and triiodothyronine (T3) levels were studied. After a single injection of TSH (5 mIU/g), the tissue T4 concentration increased markedly after 5 hr, reached a peak after 10 hr, and decreased subsequently. T4 concentration after 24 hr was still higher than in saline-injected fish but returned to the control level 48 hr after the injection. On the other hand, tissue T3 concentration was kept lower than the detectable level (0.2 ng/g) throughout the experimental period of 72 hr after a single injection of TSH (5 mIU/g). TSH treatment also accelerated the process of metamorphic climax, such as shortening of the second fin ray and eye migration. These results suggest that an increased secretion of TSH from the pituitary stimulates the thyroid, resulting in a surge of the tissue T4 concentration which induces the climax of the flounder larvae.