Nobuko Naito

Molecular biology of major components of chloride cells

Current understanding of chloride cells (CCs) is briefly reviewed with emphasis on molecular aspects of their channels, transporters and regulators. Seawater-type and freshwater-type CCs have been identified based on their shape, location and response to different ionic conditions. Among the freshwater-type CCs, subpopulations are emerging that are implicated in the uptake of Na(+), Cl(-) and Ca(2+), respectively, and can be distinguished by their shape of apical crypt and affinity for lectins. The major function of the seawater CC is transcellular secretion of Cl(-), which is accomplished by four major channels and transporters: (1). CFTR Cl(-) channel, (2). Na(+),K(+)-ATPase, (3). Na(+)/K(+)/2Cl(-) cotransporter and (4). a K(+) channel. The first three components have been cloned and characterized, but concerning the K(+) channel that is essential for the continued generation of the driving force by Na(+),K(+)-ATPase, only one candidate is identified. Although controversial, freshwater CCs seem to perform the uptake of Na(+), Cl(-) and Ca(2+) in a manner analogous to but slightly different from that seen in the absorptive epithelia of mammalian kidney and intestine since freshwater CCs face larger concentration gradients than ordinary epithelial cells. The components involved in these processes are beginning to be cloned, but their CC localization remains to be established definitively. The most important yet controversial issue is the mechanism of Na(+) uptake. Two models have been postulated: (i). the original one involves amiloride-sensitive electroneutral Na(+)/H(+) exchanger (NHE) with the driving force generated by Na(+),K(+)-ATPase and carbonic anhydrase (CA) and (ii). the current model suggests that Na(+) uptake occurs through an amiloride-sensitive epithelial sodium channel (ENaC) electrogenically coupled to H(+)-ATPase. While fish ENaC remains to be identified by molecular cloning and database mining, fish NHE has been cloned and shown to be highly expressed on the apical membrane of CCs, reviving the original model. The CC is also involved in acid-base regulation. Analysis using Osorezan dace (Tribolodon hakonensis) living in a pH 3.5 lake demonstrated marked inductions of Na(+),K(+)-ATPase, CA-II, NHE3, Na(+)/HCO(3)(-) cotransporter-1 and aquaporin-3 in the CCs on acidification, leading to a working hypothesis for the mechanism of Na(+) retention and acid-base regulation.

The duality of teleost gonadotropins.

The duality of salmon gonadotropins has been proved by biochemical, biological, and immunological characterization of two chemically distinc gonadotropins. GTH I and GTH II were equipotent in stimulating estradiol production, whereas GTH II appears to be more potent in stimulating maturational steroid synthesis. The ratio of plasma levels and pituitary contents of GTHs and the secretory control by a GnRH suggest that GTH I is the predominant GTH during vitellogenesis and early stages of spermatogenesis in salmonids, whereas GTH II is predominant at the time of spermiation and ovulation. GTH I and GTH II are found in distinctly separate cells. In trout, GTH I is expressed first in ontogeny, whereas GTH II cells appear coincident with the onset of spermatogenesis and vitellogenesis, and increase dramatically at the time of final reproductive maturation. Comparison of the amino acid sequences of polypeptides and the base sequences of cDNA revealed that salmon GTH I β is more similar to bovine FSHβ than bovine LHβ and salmon GTH II β shows higher homology to bovine LHβ than to bovine FSHβ. The existence of two pituitary gonadotropins in teleosts as well as tetrapods suggests that the divergence of the GTH gene took place earlier than the time of divergence of teleosts from the main line of evolution leading to tetrapods.

Salmonid pituitary gonadotrophs. I, Distinct cellular distributions of two gonadotropins, GTH I and GTH II

Using antisera specific for the beta subunits of two distinct coho salmon gonadotropins, GTH I and GTH II, an immunocytochemical study of rainbow trout and Atlantic salmon pituitaries was done. Cells which immunostained with anti-GTH I beta were distributed in the periphery of the glandular cords of the proximal pars distalis (PPD), in close association with somatotrophs. On the other hand, cells immunostained with anti-GTH II beta were located in the central parts of the glandular cords of the PPD. Neither the GTH I-producing nor the GTH II-producing cells stained with antisera against chum salmon growth hormone or the beta subunit of human thyroid-stimulating hormone. Moreover, GTH I and GTH II were localized in distinctly different cells. In no case was colocalization of these GTHs in the same cell observed. Finally, it was concluded that classification of GTH cells as globular and vesicular forms does not reflect the type of hormone produced by the cell, but may reflect differences in the physiological conditions of the cells.

Differential production and regulation of gonadotropins (GTH I and GTH II) in the pituitary gland of rainbow trout, Oncorhynchus mykiss, during ovarian development

SummaryBiosynthesis of salmon gonadotropins, GTH I and GTH II, during ovarian development, were examined by means of in situ hybridization histochemistry and indirect immunocytochemistry. In rainbow trout pituitary glands, expression of GTH Iβ- and IIβ-subunit genes appeared separately in distinct cells (GTH I- and GTH II-cells), whereas the GTH α-subunit gene was expressed in both cell-types. In the GTH I-cells, coordinated increases in GTh, α and Iβ messenger ribonucleic acids (mRNAs) occurred coincident with the onset of vitellogenesis, indicating active synthesis of GTH I during vitellogenesis. In contrast, in the GTH II-cells, both GTH α-and IIβ-mRNA signals markedly increased from a later stage of vitellogenesis and persisted throughout oocyte maturation and ovulation, supporting the idea that GTH II is actively synthesized as a maturational GTH. GTH α-mRNA levels in the GTH I-cells selectively decreased prior to final oocyte maturation, although Iβ-mRNA levels remained elevated, thus suggesting a decline of biosynthesis of GTH I after vitellogenesis. These findings clarify how the synthesis of GTH I and GTH II are coordinated in the piscine pituitary, and indicate that the expression of GTH subunit genes during gametogenesis is regulated differentially in a cell-specific manner, both temporally and spatially.

Salmonid pituitary gonadotrophs. II. Ontogeny of GTH I and GTH II cells in the rainbow trout (Salmo gairdneri irideus).

Immunocytochemistry of rainbow trout pituitary gonadotrophs (GTH I- and GTH II-producing cells) during gametogenesis was investigated. GTH I and GTH II were found in distinctly different cells in all stages of reproductive development that were examined. Only GTH I cells were present in trout prior to puberty. GTH II appeared in addition to GTH I coincident with the onset of vitellogenesis and spermatogenesis. Both GTH I and GTH II cells were found in trout at the time of final reproductive maturation, although the number of GTH II cells was greater than that of GTH I cells. These data indicate that GTH I and GTH II are localized in separate cells in the trout pituitary throughout gametogenesis, and that synthesis of GTH I and GTH II varies during reproductive development.

Immunocytochemical identification of melanin-concentrating hormone in the brain and pituitary gland of the teleost fishes Oncorhynchus keta and salmo gairdneri

SummaryMelanin-concentrating hormone (MCH) has been purified from the chum salmon pituitary. Its complete amino acid sequence has recently been established. To identify the precise site of origin of MCH, immunostaining was performed in the brain and pituitary gland of the chum salmon and the rainbow trout using a highly sensitive and specific antiserum raised against synthetic MCH. In these two salmonid species immunoreactivity for MCH was detected in neurons and neuronal processes in the pars lateralis of the nucleus lateralis tuberis (NLT) in the basal hypothalamus. Numerous positive-staining processes of these MCH-neurons project to the pituitary gland, extending into neurohypophysial tissues within the pars intermedia and, to a lesser extent, into the pars distalis. No pituitary cells showed cross-reactivity. These results suggest that MCH is biosynthesized in the neurons of the NLT/pars lateralis and released in the neurohypophysis. On the other hand, prominent but less numerous MCH-positive processes could be traced to the pretectal area in which projection of both optic and pineal fibers has been detected using tracers. This observation suggests that the synthesis and/or release of MCH might be under the influence of either of these photosensory neurons. Moreover, the existence of an extrahypothalamic projection from MCH-positive neurons suggests that, in addition to melanin-concentration, MCH might be involved in other neuronal functions, perhaps serving as neuromodulator in the brain.

Isolation and properties of chum salmon prolactin.

A highly purified prolactin (PRL) was isolated from the chum salmon pituitary by extraction with acid acetone, gel filtration on Sephadex G-25 and ion-exchange chromatography on CM-Sephadex C-25 with a yield of 1 mg/g of wet tissue. It was 10-15 times more potent than ovine PRL in sodium-retaining activity for juvenile rainbow trout adapted to 50% seawater. The salmon PRL emerged as a single and symmetrical peak on Sephadex G-100 with Ve/Vo = 2.0. Polyacrylamide gel electrophoresis revealed only one band at pH 4.3, whereas no band was seen at pH 7.5. The isoelectric point was estimated to be 10.3 by gel electric focusing. The circular dichroism spectrum of the salmon PRL was similar to that of tilapia PRL, showing an alpha-helix content of 50%. The salmon PRL had a molecular weight of 23,400 daltons by gel filtration and 22,300 daltons by sodium dodecyl sulfate gel electrophoresis, with a single NH2-terminal residue, isoleucine, and a single COOH-terminal residue, half-cystine. In the sequence comparison with those of mammalian PRLs and growth hormones, the clusters of invariant residues were found in both terminal regions, although the disulfide at NH2-terminal of mammalian PRLs was missing. Specific salmon PRL antisera were prepared in rabbits giving a precipitin reaction against the salmon PRL and a pituitary extract of tilapia in agar diffusion but no cross reaction with purified mammalian PRLs. The antibody was localized specifically in PRL cells of the chum salmon pituitary.

Ontogeny of pituitary cell-types and the hypothalamo-hypophysial relationship during early development of chum salmon, Oncorhynchus keta

The ontogeny of pituitary cell-types, hypothalamic neurons and their fibers, was studied immunocytochemically during development of chum salmon. Five weeks after fertilization (eyed stage embryos), 5 cell-types were detected in the adenohypophysial (AHP) anlage; prolactin (PRL)-, growth hormone (GH)-, adrenocorticotropin (ACTH)-, melanotropin (α-MSH)-, and thyrotropin (TSH)-producing cells. The PRL-, GH- and ACTH-cells were relatively well developed as compared with MSH- and TSH-cells. Gonadotropes, however, were not detected even 3 weeks after hatching (10 weeks after fertilization). The neurohypophysis (NHP), on the other hand, began to grow around the eyed stage. Neuroendocrine fibers, not only from the tuberal hypothalmus (melanin-concentrating hormone) but also from the preoptic regions (vasotocin and somatostatin), reached the NHP during the last week of embryonic life. In the developing pituitary, the ratio between the length of the boundary between the AHP and the NHP, and the area of the AHP, was stable, being approximately 1–4%. The coordinated development of the AHP and NHP in chum salmon seems to result in the development of the characteristic hypothalamo-hypophysial relationship by the time of hatching.

Immunocytochemical identification of the prolactin-secreting cells in the teleost pituitary with an antiserum to chum salmon prolactin

An antiserum raised to highly purified chum salmon (Oncorhynchus keta) prolactin (sPRL) was used to identify prolactin-producing cells in the adenohypophysis of 15 species of teleosts by the immunocytochemical peroxidase-antiperoxidase method. In the chum salmon, the only pituitary cells that reacted with sPRL antibody were the PRL cells organized as follicular structures in the rostral pars distalis. When the antiserum was absorbed with sPRL, on the other hand, no immunoreactive cell was observed in the pituitary, indicating the specificity of the antiserum. Furthermore, the antibody to sPRL reacted only with PRL cells in the pituitaries of three species of salmonids, a plecoglossid, eel, carp, goldfish, killifish, tilapia, and five species of marine fishes, thus showing no species specificity of the antibody among the teleosts tested. The PRL cells of the eel decreased in number and also in immunoreactivity after adaptation to seawater for 1 month. On the other hand, highly immunoreactive PRL cells were observed in the pituitaries of marine fishes, although the cells were much fewer in number than in eels and in other fishes in fresh water.

Coexistence of immunoreactivity for melanin-concentrating hormone and α-melanocyte-stimulating hormone in the hypothalamus of the rat

Coexistence of immunoreactivity for melanin-concentrating hormone (MCH) and alpha-melanocyte-stimulating hormone (alpha-MSH) within rat hypothalamic neurons has been examined by the unlabeled antibody enzyme method. Neurons exhibiting both MCH- and alpha-MSH-like immunoreactivities were found in the dorsolateral hypothalamus, whereas no MCH-like immunoreactive perikarya were seen in the arcuate nucleus, where some neurons were stained with alpha-MSH antiserum. There seem to be two distinct alpha-MSH-like immunoreactive neurons in the rat hypothalamus, one exhibiting coexistence with MCH-like immunoreactivity and the other not showing any cross-reaction with MCH antiserum.