Yasuaki Takagi

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.

Stimulation of non-specific immune functions in seawater-acclimated rainbow trout, Oncorhynchus mykiss, with reference to the role of growth hormone.

The influence of acclimation to seawater (SW) and growth hormone (GH) administration on immune functions was examined in the rainbow trout (Oncorhynchus mykiss). After 3 days acclimation to dilute SW (12 parts per thousand, ppt), an increase in plasma lysozyme activity was observed compared to the fish kept in fresh water (FW). No change was seen in plasma immunoglobulin M (IgM) levels. When they were transferred from dilute SW to full-strength SW (29 ppt) after a single intra-peritoneal injection of ovine or salmon GH, plasma sodium levels of GH-treated fish were significantly lower than those of the control fish injected with Ringers solution 24 h after the transfer. The plasma level of IgM was not influenced by GH injection in the fish kept in FW nor in those transferred to SW. The administration of GH increased plasma lysozyme activity in the fish in FW, but no further increase was seen after SW transfer. The production of superoxide anions in peripheral blood leucocytes was stimulated by GH in both FW and SW. These results suggest that GH is involved in the stimulation of the non-specific immune functions in SW-acclimated salmonids.

Molecular cloning and expression of an otolith matrix protein cDNA from the rainbow trout, Oncorhynchus mykiss

The fish otolith is a hard tissue consisting of calcium carbonate and organic matrices. The matrix proteins play important roles in otolith formation, but little is known about the nature of these proteins. In this study, matrix proteins were extracted from the otoliths of rainbow trout, Oncorhynchus mykiss, and chum salmon, Oncorhynchus keta. EDTA-soluble matrix proteins were separated by SDS-PAGE, revealing two major components in the otoliths of both species with apparent molecular masses of 55 and 43 kDa. N-terminal and some internal amino acid sequences of the 55-kDa otolith matrix protein were determined. A cDNA fragment encoding this protein of O. mykiss was amplified by reverse transcription PCR using two degenerate primers designed from the amino acid sequences. A cDNA encoding this protein was obtained by screening a saccular cDNA library using the amplified cDNA fragment as a probe. Nucleotide sequence analysis revealed that the cDNA clone has a sequence of 2.5 kb and the open reading frame encoding 344 amino acid residues. Northern blot analysis showed that mRNA of this protein is expressed specifically in the sacculus, and consistently during the day.

Cloning and Expression of a cDNA Encoding an Insoluble Matrix Protein in the Gastroliths of a Crayfish, Procambarus clarkii

Abstract In the crayfish Procambarus clarkii, the gastroliths are formed as a paired structure in the stomach during the premolt period, and contain calcium carbonate and a small amount of an organic matrix. In this investigation, a cDNA encoding an insoluble matrix protein was isolated from P. clarkii. The open reading frame encoded 505 amino acid residues including two unique repeated sequences. The N-terminal half of the amino acid sequence, which included 10-amino-acid repeats, exhibited a high degree of similarity to that of involucrin, a protein synthesized in human keratinocytes. Northern blot analysis revealed that mRNA encoding the matrix protein is specifically expressed in the gastrolith discs during the premolt period in which the gastroliths formed. In the gastrolith discs, levels of expression of this mRNA correlated increases in weights of the gastroliths concomitant with their formation. Organ culture of the gastrolith discs suggested that expression of mRNA in the discs is induced by molting hormone, 20-hydroxyecdysone. These results reinforced the relationship between the matrix protein and formation of the gastroliths. Functional analysis showed that the protein inhibits calcium carbonate crystallization in a solution system, suggesting that the protein plays a role in the calcification of the gastroliths.

Estradiol-17β and nutritional status affect calcium balance, scale and bone resorption, and bone formation in rainbow trout, Oncorhynchus mykiss

Abstract The effects of estradiol-17β (E2) on bone resorption and formation as well as its effects on scale resorption were investigated in rainbow trout in order to elucidate the role of the hormone in calcium mobilization from calcified tissues, and to clarify the importance of scale and bone as calcium reserves during sexual maturation. Furthermore, the effects of nutritional status on calcified tissues and E2-induced calcium mobilization were studied. In fed as well as fasted rainbow trout, E2 treatment increased scale osteoclastic activity measured as tartrate-resistant acid phosphatase activity, and reduced scale calcium content, suggesting that E2 increases scale resorption in both the fed and fasted fish. Using histomorphometry, E2 treatment was found to decrease pharyngeal bone resorption in fed, but not in fasted rainbow trout. The E2 effect on rainbow trout bone is consistent with its physiological role in mammals and birds where E2 has been reported to decrease bone resorption. It appears therefore that rainbow trout protect their skeleton and instead use scales as a source of calcium during E2-induced calcium mobilization. The formation of pharyngeal bone was decreased by fasting, and the importance of the nutritional status for the activity of the bone cells in rainbow trout is therefore emphasized.

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.

Characterization of ootolith soluble‐matrix producing cells in the saccular epithelium of rainbow trout (Oncorhynchus mykiss) inner ear

Although the organic matrix may play an important role in the growth of teleost otoliths, cellular contributions to the production of the organic matrix have been studied in only a small number of teleost species with limited methods, and are still poorly understood. In order to characterize saccular epithelial cells which produce otolith matrix, antiserum was raised against an EDTA‐soluble fraction of otolith matrix (otolith soluble‐matrix, OSM) of the rainbow trout. The components in the OSM and in the endolymph were characterized by immunoblotting. The saccular epithelium was immunohistochemically stained with the antiserum and the ultrastructure of OSM immunoreactive cells was studied. By immunoblotting, multiple components (> 94.0 kDa [smeared] and 43.0 kDa) in the OSM reacted with the antiserum, whereas only one band (> 94.0 kDa) was detected in the endolymph. Under immunohistochemical staining, reactions to the antiserum were observed in columnar cells lined at the most peripheral region of the sensory epithelium, transitional epithelial cells, and squamous epithelial cells. Electron microscopic observations revealed that all three types of cells were equipped with extended rough endoplasmic reticulum and prominent Golgi apparatus, suggesting the active production of organic material(s). Dilations of translucent vesicles, apocrine‐like extrusions of cytoplasm, and vesicles containing many minute globules were frequently associated with the apical surface of these cells. Some ruptured vesicles were observed, releasing their contents into the endolymphatic space. The present study identified columnar cells lining the most peripheral region of the sensory epithelium, transitional epithelial cells, and squamous epithelial cells as the OSM‐producing cells. We suggest that the OSM components are secreted and dissolved into the endolymph and subsequently deposited onto the otolith. Anat Rec 254:322–329, 1999.

Meshwork arrangement of mitochondria‐rich, Na+,K+‐ATPase‐rich cells in the saccular epithelium of rainbow trout (Oncorhynchus mykiss) inner ear

Electrolyte composition of the teleost fish inner ear endolymph is characterized by a high potassium concentration. From the ultrastructural characteristics, the mitochondria‐rich cells (MRCs) in the inner ear epithelium are suggested to regulate the ionic composition of the endolymph.

Developmental sequence of bone-resorbing cells induced by intramuscular implantation of mineral-containing bone particles into rainbow trout, Oncorhynchus mykiss

Mineral-containing bone particles (BPs) were implanted intramuscularly into rainbow trout (Oncorhynchus mykiss) to investigate the sequence of appearance of bone-resorbing cells. A fibrous substance first surrounded the implanted BPs and was gradually replaced by connective tissue containing capillaries. Two weeks after BP implantation, relatively small multinucleated cells (type-1 cells), whose cytoplasm stained deeply with hematoxylin, appeared along the surfaces of the BPs. At later stages (after 4–8 weeks), the majority of cells which appeared to be resorbing the BPs were multinucleated cells whose cytoplasm stained deeply with eosin (type-2 cells). Type-2 cells contained more nuclei than type-1 cells. Electron-microscopical observations revealed that type-2 cells had the characteristic features of osteoclasts: the presence of numerous mitochondria, vacuoles and granules, and a differentiation of the cell membrane and cytoplasm into a ruffled border and clear zone, respectively. A tartrate-resistant acid phosphatase activity, which is an established characteristic of osteoclasts in terrestrial vertebrates, but which had not previously been examined in teleosts, was demonstrated histochemically in the type-2 cells. Development of type-2 cells was closely correlated with the development of connective tissue. These findings suggest that the development of a capillary network around the implanted BPs enables circulating osteoclast-progenitors to reach the surface of the BPs.