Purification and characterization of luteinizing hormone from pituitary glands of rockfish, Sebastes schlegeli.

To acquire greater knowledge of the reproductive function of luteinizing hormone (LH) in the viviparous rockfish Sebastes schlegeli, LH from the pituitary glands of mature rockfish was isolated, purified, and localized and its biological activity was characterized. The molecular mass of purified LH was estimated to be approximately 33 kDa, similar to that of known LH. When rockfish LH was purified by reverse-phase high-performance liquid chromatography, its N-terminal amino acid sequences were found to coincide with those of predicted cDNA sequences of rockfish gonadotropin α (ssGTHα) and ssLHß mature peptides. Immunocytochemical analysis using antisera against ssGTHα (molecular weight [MW], ~14.5 kDa) and ssLHß (MW, ~18.5 kDa) indicated that the LH-producing cells are mainly distributed throughout the proximal pars distalis and along the periphery of the pars intermedia. Further, in vitro ovarian follicle analysis demonstrated that purified intact rockfish LH significantly enhances E(2) secretion in a dose-dependent manner. This is the first report on the purification and characterization of LH from a viviparous teleost, and these results will enable future research and increase our understanding of the mechanisms underlying the maturation of such fish.

Follicle-stimulating hormone induces spermatogenesis mediated by androgen production in Japanese eel, Anguilla japonica.

Follicle-stimulating hormone (FSH) plays important roles in spermatogenesis. However, the biologic activity of FSH can vary in different vertebrate classes, and the definitive function of FSH has not been established. In this study, we investigated the functions of FSH on spermatogenesis using an in vitro culture system for Japanese eel testis. The eel Fsh receptor was expressed in testis tissue during the whole process of spermatogenesis, mainly by Leydig cells that produce steroid hormones and by Sertoli cells surrounding type A spermatogonia and early type B spermatogonia. In an in vitro organ culture, recombinant eel Fsh (r-eFsh) induced complete spermatogenesis from the proliferation of spermatogonia to spermiogenesis during 36 days of culture; also, spermatozoa were observed in the testicular fragments. Spermatogenesis induced by r-eFsh was inhibited by trilostane, a specific inhibitor of 3beta-hydroxysteroid dehydrogenase. However, trilostane did not inhibit spermatogenesis induced by 11-ketotestosterone. These results clearly show that the main function of FSH in eel is to induce spermatogenesis via stimulating androgen production.

Lack of circadian regulation of in vitro melatonin release from the pineal organ of salmonid teleosts.

In many teleost species, the photoreceptive pineal organ harbors the circadian clock that regulates melatonin release in the pineal organ itself. However, the pineal organ of three salmonids (rainbow trout Oncorhynchus mykiss, masu salmon Oncorhynchus masou, and sockeye salmon Oncorhynchus nerka) did not exhibit circadian rhythms in melatonin release when maintained under constant darkness (DD) in vitro, suggesting that the pineal organs of all salmonids lack the circadian regulation of melatonin production. To test this hypothesis, the pineal organ of seven salmonids (common whitefish Coregonus lavaretus, grayling Thymallus thymallus, Japanese huchen Hucho perryi, Japanese charr Salvelius leucomaenis pluvius, brook trout Salvelius fontinalis, brown trout Salmo trutta and chum salmon Oncorhynchus keta) and closely related osmerids (ayu Plecoglossus altivelis altivelis and Japanese smelt Hypomesus nipponensis) were individually maintained in flow-through culture at 15 degrees C under several light conditions. Under light-dark cycles, the pineal organ of all species showed a rhythmic melatonin release with high rates during the dark phase. Under DD, the osmerid pineal organs exhibited circadian rhythms in melatonin release with high rates only during the subjective-night but the salmonid pineal organs constantly released melatonin at high rates. Under constant light, melatonin release was suppressed in all species. The pineal organ of rainbow trout maintained at different temperature (15, 20 or 25 degrees C) under DD released melatonin with high rates but the amount of melatonin released was temperature-sensitive (highest at 20 degrees C). Thus, melatonin release from the pineal organ of osmerids is regulated by both light and circadian clock but the circadian regulation is lacking in salmonids. These results indicate that ancestral salmonids lost the circadian regulation of melatonin production after the divergence from osmerid teleosts.

Molecular cloning and characterization of cortical rod protein in the giant freshwater prawn Macrobrachium rosenbergii, a species not forming cortical rod structures in the oocytes.

The formation of cortical rod structures is a characteristic of fully mature oocytes in penaeid prawns, but such structures are absent from oocytes of giant freshwater prawn Macrobrachium rosenbergii. In the present study, we first demonstrated the presence of a 30-kDa protein, which was immunologically related to kuruma prawn cortical rod protein (CRP), in the ovary of giant freshwater prawn, and subsequently purified this protein. Furthermore, a cDNA encoding the CRP-like protein was isolated. Based on the high homology (98%) in the amino acid sequence with kuruma prawn CRP, the 30-kDa protein has been identified as a CRP homologue of giant freshwater prawn, designated mrCRP. The RT-PCR analysis revealed that mrCRP mRNA was present in the ovary from a prawn with a gonadosomatic index (GSI) of 0.2. Western blot analysis revealed the presence of a CRP-immunoreactive band of 30kDa in the ovary with GSI of 1.6. By immunocytochemistry, CRP-immunopositive signals were detected in the ovary with GSI of 0.9, that had started to accumulate considerable amounts of vitellins and lipids in the peripheral cytoplasm. With progress of vitellogenesis, mrCRP was apparently accumulated in the mature oocytes, although it was not detectable, presumably because a relatively small amount of mrCRP was masked with large amounts of vitellin and lipids. In giant freshwater prawn without forming cortical rod structures, our findings indicate that the oocytes produce mrCRP, a homologue of CRP found in penaeid prawns.

Ocular melatonin rhythms in teleost fish.

Melatonin (N-acetyl-5-methoxytryptamine) is synthesized in the pineal organ and the retina of vertebrates. In some teleost species, ocular melatonin levels can exhibit a circadian periodicity with elevated levels during the dark phase under light-dark (LD) cycles and this periodicity can persist even under constant dark (DD) cycles. However, reversed melatonin profiles and an absence of circadian ocular melatonin rhythms have also been reported. In this study, we investigated the daily rhythms of ocular melatonin in 32 teleost species under LD cycles. The melatonin profiles could be classified into three types: (1) normal profiles, with higher melatonin levels during the dark phase than the light phase; (2) reversed profiles, with higher levels during the light phase than the dark phase; (3) no significant differences in melatonin levels. We also studied whether ocular melatonin exhibits circadian rhythms under DD in selected species. Our results showed that ocular melatonin exhibited circadian rhythms in some but not all of the species examined. These results indicate that ocular melatonin rhythms in teleost fish exhibit species-specific variations as a result of the changes in the regulatory mechanisms during the course of evolution.

Effects of growth hormone and cortisol on the downstream migratory behavior in masu salmon, Oncorhynchus masou.

The effects of ovine growth hormone (oGH) and cortisol on downstream migratory behavior in yearling (1(+)) smolts and underyearling (0(+)) parr of masu salmon, Oncorhynchus masou, were examined during the downstream migratory period in spring using artificial raceways. In May, each of 22 1(+) smolts and 0(+) parr were implanted with cholesterol pellets containing 250 microg of oGH and/or 2 mg of cortisol. Their downstream migratory behavior was subsequently observed in artificial raceways, along with control groups 4-23 days after implantation. In 1(+) smolts, the frequency of downstream migratory behavior was 23%, 18%, 72%, and 82% in the control, oGH, cortisol, and oGH+cortisol-treated groups, respectively. The frequency was significantly higher in the cortisol and oGH+cortisol-treated groups than in the control and oGH-treated groups. In 0(+) parr, the frequency of downstream migratory behavior in the cortisol (82%) and cortisol+oGH-treated (90%) groups was significantly higher than in the control (18%) and oGH-treated (0%) groups. These results indicate that cortisol is an important endocrine factor inducing downstream migratory behavior in both 1(+) smolt and 0(+) parr of masu salmon.

Prolactin gene expression and gill chloride cell activity in fugu Takifugu rubripes exposed to a hypoosmotic environment.

To evaluate a possible involvement of prolactin (PRL) in low-salinity tolerance of a marine pufferfish Takifugu rubripes, or fugu, gene-expression profiles of PRL in the pituitary and PRL receptor (PRLR) in the osmoregulatory organs were investigated in fish exposed to 25%-dilute seawater (SW). Following transfer from full-strength (100%) SW to 25% SW, plasma osmolality and Na(+) and Cl(-) levels were slightly decreased on day 1, which were restored on days 3 and 7. Expression levels of PRL mRNA in the pituitary was significantly increased in response to 25% SW transfer, which was in sharp contrast with a remarkable decrease in growth hormone (GH) mRNA levels. These profiles suggest that PRL and GH are involved in hyper- and hypoosmoregulation, respectively, as is the case with euryhaline teleosts. Expression levels of PRLR mRNA in the gill and intestine were not significantly different from the initial levels, whereas, PRLR mRNA expression in the kidney was significantly higher on day 7 than the initial levels. Although transfer to 25% SW did not affect the average size of Na(+)/K(+)-ATPase-immunoreactive chloride cells in the gills, both size and density of apical openings of chloride cells became significantly smaller after transfer to 25% SW. These findings suggest that the possible hypoosmotic action of PRL is mediated by PRLR expressed in the osmoregulatory organs, and that low-salinity tolerance of fugu may involve reduction of an ion-secreting function of gill chloride cells. To further evaluate long-term effects of the low-salinity environment on growth and osmoregulation, fugu were raised in 25% and 100% SW for a prolonged period of 8 weeks. They grew similarly in 25% and 100% SW, and there was no significant difference in body weight and standard length at any weekly sampling point. The plasma osmolality was maintained at about 345mOsm/kg.H(2)O in both media, whereas the gill Na(+)/K(+)-ATPase activity was significantly lower in 25% SW than 100% SW. Gene expression of PRL in the pituitary was higher in 25% SW than in 100% SW; conversely, expression levels of GH were lower in 25% SW than in 100% SW. These findings support a hyperosmotic action of PRL and a hypoosmotic, rather than growth-promoting, action of GH in this marine teleost.

Dynamics of vitellogenin synthesis in juvenile giant freshwater prawn Macrobrachium rosenbergii.

The dynamics of vitellogenin (Vg) mRNA expression and patterns of Vg and vitellin distribution in the hepatopancreas and ovary of juvenile Macrobrachium rosenbergii were examined using real-time RT-PCR and immunohistochemical methods. Eyestalk ablation was seen to induce rapid development of the gonads and Vg synthesis in females. In the female hepatopancreas, Vg mRNA expression was observed several days following ablation, after which levels increased gradually with increasing gonadosomatic index (GSI). Vitellin accumulation in the oocytes also increased with increasing Vg mRNA synthesis; expression was however negligible in the ovary. Hemolymph Vg levels in females ranged from 0.04 to 2.2 mg/ml. SDS PAGE/Western blotting analysis of hemolymph samples revealed that juvenile Vg was composed of 199 and 90 kDa subunits; the 102 kDa subunit present in adult female Vg (Okuno et al., 2002. J Exp Zool 292:417-429) could not be detected at any stage of vitellogenesis in juveniles. Vg was not detectable in non-ablated juveniles. The results of this study confirmed that the mode of involvement of eyestalk factors in regulating vitellogenesis is intrinsic to both juveniles and adults, and that a basic pattern of Vg synthesis and processing is conserved. However, the fact that juveniles are not able to produce the same Vg levels observed in adult females, and do not reach high GSI levels culminating in spawning suggests that other factors and physiological conditions specific to adult females are necessary to demonstrate full reproductive ability.

Prolactin and prolactin receptor expressions in a marine teleost, pufferfish Takifugu rubripes.

To investigate the physiological significance of prolactin (PRL) in a marine teleost, pufferfish (or fugu), Takifugu rubripes, we cloned and characterized cDNAs encoding its PRL and PRL receptor (PRLR) from the pituitary and gills, respectively. The fugu PRL cDNA consisted of 995 bp and encoded a protein of 213 amino acids. The PRLR, consisting of 510 amino acids, contained a putative signal peptide, an extracellular domain with two pairs of cysteines, a WSXWS motif, a single transmembrane domain, and a cytoplasmic (intracellular) domain with box 1 and box 2 regions, all of which are characteristic of the cytokine receptor superfamily. Reverse transcription-PCR showed the expression of PRLR mRNA in osmoregulatory organs, such as gills, kidney, and intestine, whereas pufferfish PRL mRNA was detected only in the pituitary. Furthermore, in situ hybridization revealed the expression of pufferfish PRLR in branchial chloride cells, kidney tubule cells, and intestinal epithelia. The PRL-gene expression levels in the pituitary were about five times higher in 25%-diluted seawater than in full-strength seawater. These results suggest that fugu PRL regulates water and electrolyte balances through PRLR expressed in the osmoregulatory organs, as is the case with freshwater-adapted euryhaline species.

Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus.

In penaeid shrimp species, ovarian development is characterized by the accumulation of a major yolk protein (vitellin) and the formation of cortical rods in the oocytes. The process is considered to be under the control of a neuroendocrine organ in the eyestalk (the X-organ sinus gland complex). In the present study, the synthesis of vitellogenin (VTG, precursor of vitellin) and two kinds of cortical rod proteins (cortical rod protein, CRP; thrombospondin, MjTSP) was induced by bilateral eyestalk ablation (removal of the X-organ sinus gland complex) in immature female kuruma prawn, Marsupenaeus japonicus, and the synthesis process was monitored over a 7-day period after the ablation. The ovarian weight and hemolymph VTG levels increased in the ablated females. The VTG mRNA levels in the ovary increased concomitantly with vitellin accumulation in the ovary after eyestalk ablation. On the other hand, the CRP and MjTSP protein levels in the ovary increased after eyestalk ablation, whereas the CRP and MjTSP mRNA levels in the ovary did not change concomitantly. The results suggest that the regulatory mechanism of gene expression by eyestalk hormones is different between VTG (transcriptional control) and CRP-MjTSP (translational control).

Understanding hypoxia-induced gene expression in early development: in vitro and in vivo analysis of hypoxia-inducible factor 1-regulated zebra fish insulin-like growth factor binding protein 1 gene expression.

Insulin-like growth factor binding protein 1 (IGFBP-1) is a hypoxia-inducible gene that plays an important role in regulating embryonic growth and development under hypoxic stress. The molecular mechanisms underlying hypoxia-induced IGFBP-1 gene expression in the embryonic tissues are not well understood. Here we report that the hypoxia-inducible factor 1 (HIF-1) pathway is established in early embryogenesis and mediates hypoxia-induced IGFBP-1 expression. Hypoxia increased the HIF-1 activity, and HIF-1alpha overexpression or CoCl2 treatment resulted in elevated IGFBP-1 expression in zebra fish embryos. Although the zebra fish IGFBP-1 promoter contains 13 consensus hypoxia response elements (HREs), deletion and mutational analysis revealed that only the HRE positioned at -1090/-1086 is required for the hypoxia and HIF-1 induction. Further experiments revealed that there is an HIF-1 ancillary sequence (HAS) adjacent only to the functional HRE. Mutation of this HAS greatly reduced the responsiveness of the IGFBP-1 promoter to hypoxia and HIF-1. The HAS does not directly bind to HIF-1 or affect the binding of the HRE to HIF-1. The HAS is bound to a nuclear protein(s), and this HAS binding activity is reduced by hypoxia. These results suggest that HIF-1 mediates hypoxia-induced IGFBP-1 gene expression in early development by selectively interacting with the -1090/-1086 HRE and its adjacent HAS.

Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome.

Neurons that synthesize and release GnRH are essential for the central regulation of reproduction. Evidence suggests that forebrain GnRH neurons originate in the olfactory placode and migrate to their final destinations, although this is still a matter of controversy. X-linked Kallmann syndrome (X-KS), characterized by failed gonadal function secondary to deficient gonadotropin secretion, is caused by a mutation in KAL1, which is suggested to regulate the migration of forebrain GnRH neurons. Because rodents lack Kal1 in their genome and have GnRH neurons scattered throughout their forebrain, the development of forebrain GnRH neurons and the pathogenesis of X-KS have been difficult to study. In the present study, we generated transgenic medaka that expressed green fluorescent protein under the control of the gnrh1 and gnrh3 promoters for analyzing forebrain GnRH neuronal development. Our data revealed the presence of the following four gnrh1 neuronal populations: an olfactory region-derived ventral preoptic population, a dorsal preoptic population that migrates from the dorsal telencephalon, a medial ventral telencephalic population that migrates from the anterior telencephalon, and a nonmigratory ventral hypothalamic population. We found that all forebrain gnrh3 neurons, extending from the terminal nerve ganglion to the anterior mesencephalon, arise from the olfactory region and that trigeminal ganglion neurons express gnrh3. Maternal gnrh3 expression was also observed in oocytes and early embryos. We subsequently identified a KAL1 ortholog and its paralogous form in the medaka. Consistent with the X-KS phenotype, antisense knockdown of the medaka KAL1 ortholog resulted in the disruption of forebrain GnRH neuronal migration. Thus, these transgenic medaka provide a useful model system for studying GnRH neuronal development and disorders of GnRH deficiency.

Steroidogenic activities of follicle-stimulating hormone in the ovary of Japanese eel, Anguilla japonica.

To clarify the physiological functions of follicle-stimulating hormone (FSH) during oogenesis in Japanese eel, Anguilla japonica, the steroidogenic activities of recombinant Japanese eel FSH (rjeFSH) were assessed in the eel ovary. Female eel were injected with salmon pituitary homogenate to enhance the ovarian development, and the ovaries at different developmental stages were subjected to steroidogenic bioassay. These ovaries could be classified into three types according to oocyte growth and development of ovarian follicular cells. The type-A ovary possessed poorly developed follicular cells around pre- or early vitellogenic oocytes, and rjeFSH did not induce sex steroid secretion. Testosterone (T) secretion was stimulated by rjeFSH in the type-B ovary with developed theca cells and undeveloped granulosa cells around early to mid-vitellogenic oocytes, whereas estradiol-17beta (E2) secretion was not enhanced. The rjeFSH stimulated both T and E2 secretion in a dose-dependent manner from the type-C ovary with fully developed theca and granulosa cells around mid-vitellogenic oocytes. Salmon GTH fraction (sGTH) and a membrane permeable cAMP analogue, 8-bromo-cAMP (8-Br-cAMP) also enhanced T and E2 secretion from the type-C ovary. Human chorionic gonadotropin (hCG) similarly enhanced T secretion, but failed to stimulate E2 secretion from the type-C ovary, suggesting different effects on steroidogenic activities between eel FSH and hCG in eel ovary. There was a positive correlation between the oocyte diameter and E2 secretion from eel ovaries stimulated by rjeFSH. These results suggest that aromatase activity is accelerated by eel FSH in the granulosa cells, which develop following theca cell development in this species.

Cloning of rainbow trout SLC26A1: involvement in renal sulfate secretion.

The kidney plays an important role in ion regulation in both freshwater and seawater fish. However, ion transport mechanisms in the teleost kidney are poorly understood, especially at the molecular level. We have cloned a kidney-specific SLC26 sulfate/anion exchanger from rainbow trout (Oncorhynchus mykiss) that is homologous to the mammalian SLC26A1 (Sat-1). Excretion of excess plasma sulfate concentration after Na2SO4 injection corresponded to significantly higher expression of the cloned SLC26A1 mRNA. Detailed morphological observation of rainbow trout renal tubules was also performed by light microscopy and transmission electron microscopy. According to the structure of brush border and tubular system in the cytoplasm, renal tubules of rainbow trout were classified into proximal tubule first and second (PI and PII) segments and distal tubules. In situ hybridization revealed that SLC26A1 anion exchanger mRNA is specifically localized in the PI segment of kidneys from both seawater- and freshwater-adapted rainbow trout. With immunocytochemistry, Na+-K+-ATPase and vacuolar-type H+-ATPase were colocalized to the same cells and distributed in the basolateral and the apical membranes, respectively, of the cells where the SLC26A1 mRNA expressed. These findings suggest that the cloned kidney-specific SLC26A1 is located in kidney proximal tubules and is involved in excretion of excess plasma sulfate in rainbow trout.

Effects of melatonin feeding on smoltification in masu salmon (Oncorhynchus masou).

Influences of photoperiod on plasma melatonin profiles and effects of melatonin administration on long-day-induced smoltification in masu salmon (Oncorhynchus masou) were investigated in order to reveal the roles of melatonin in the regulation of smoltification in salmonids. Under light-dark (LD) cycles, plasma melatonin levels exhibited daily variation, with higher values during the dark phase than during the light phase. The duration of nocturnal elevation under short photoperiod (LD 8:16) was longer than that under long photoperiod (LD 16:8). Melatonin feeding (0.01, 0.1 and 1 mg/kg body weight) elevated plasma levels of melatonin in a dose-dependent manner for at least 7 h but not for 24 h. When masu salmon reared under short photoperiod were exposed to long photoperiod (LD 16:8) and fed melatonin (1 mg/kg body weight) 7 hours before the onset of darkness, a significantly smaller proportion of smolts appeared in the melatonin-fed group after 32 days than in the control group. However, after 59 days of the treatment, there was no difference in the proportion of smolts between the control and melatonin-treated groups. Thus, melatonin feeding mimicked the effects of short photoperiod, which delays but does not completely suppress smoltification. These results indicate that the day length is transduced into changes in the duration of nocturnal elevation in plasma melatonin levels, and that artificial modification of the plasma melatonin pattern possibly delays the physiological processes of smoltification induced by long-day photoperiodic treatment.

The effects of crustacean hyperglycemic hormone-family peptides on vitellogenin gene expression in the kuruma prawn, Marsupenaeus japonicus.

In crustaceans, eyestalk ablation induces gonadal maturation of which vitellogenin gene expression is an essential step. However, the molecular mechanisms by which the hormones produced by the X-organ/sinus gland complex in the eyestalk regulate vitellogenesis remain poorly understood. We therefore investigated the effects of sinus gland extracts and certain sinus gland peptides belonging to the crustacean hyperglycemic hormone peptide family on vitellogenin gene expression in ovarian fragments of immature kuruma prawn, Marsupenaeus japonicus. Vitellogenin mRNA levels in incubated ovarian fragments were significantly higher than those in unincubated ovarian fragments prepared from the same animal. Sinus gland extracts and sinus gland peptide-III (type I peptide) both reduced vitellogenin mRNA levels in a dose-dependent manner. In contrast, neither molt-inhibiting hormone (sinus gland peptide-IV) nor molt-inhibiting hormone B, both of which are type II peptides, exerted significant effects on vitellogenin mRNA levels. These results suggest that, in the immature ovary, sinus gland peptide-III is involved in the suppression of vitellogenin gene expression. The existence of such a peptide in the X-organ/sinus gland complex provides a rationale for the significant increase in vitellogenin mRNA levels in the ovaries of eyestalk-ablated prawns.

Whole-cell electrophysiology of gonadotropin-releasing hormone neurons that express green fluorescent protein in the terminal nerve of transgenic medaka (Oryzias latipes).

Gonadotropin-releasing hormone (GnRH) controls reproduction in vertebrates. Most studies have focused on the population of GnRH neurons in the hypothalamus that ultimately controls gonadal function. However, all vertebrates studied to date have two to three anatomically distinct populations of GnRH neurons that express different forms of this hormone. The purpose of the present study was to develop a new model for studying the population of GnRH neurons in the terminal nerve (TN) associated with the olfactory bulb and then to characterize their pattern of action potential firing to provide a foundation for understanding the role of these neurons in regulating reproduction. A stable line of transgenic medaka (Oryzias latipes) was generated in which a DNA construct containing the salmon GnRH (Gnrh3) promoter linked to green fluorescent protein (GFP) was expressed in TN-GnRH3 neurons. This population of GnRH neurons is located at or near the ventral surface of the brain, making them ideally situated for electrophysiological analysis. Whole-cell and loose-patch recordings in current-clamp mode were performed on these neurons from excised, intact brains of adult males in which afferent and efferent neural connections remained intact. All TN-GnRH3-GFP neurons that we recorded showed a beating pattern of spontaneous action potential firing. Action potentials were blocked by tetrodotoxin, indicating they are generated by a voltage-sensitive Na+ current; however, an oscillation in subthreshold membrane potential persisted. The present results indicate that this transgenic fish will provide an excellent model for studying the cell physiology of an extrahypothalamic population of GnRH neurons.

Aquaporin-3 expressed in the basolateral membrane of gill chloride cells in Mozambique tilapia Oreochromis mossambicus adapted to freshwater and seawater.

We have cloned a homologue of mammalian aquaporin-3 (AQP3) from gills of Mozambique tilapia using a reverse transcription-polymerase chain reaction (RT-PCR). The deduced amino acid sequence shared 64-75% homology with other vertebrate AQP3 homologues. RT-PCR revealed that tilapia AQP3 was expressed in the brain, pituitary, kidney, spleen, intestine, skin, eye and gill in tilapia adapted to freshwater (FW) and seawater (SW). We also examined functional characteristics of tilapia AQP3 using Xenopus oocytes as an in vitro transcribed cRNA expression system. Osmotic water permeability (Pf) of Xenopus oocytes expressing tilapia AQP3 was about 30-fold higher than that of control oocytes, and was 80% inhibited by treatment with 0.3 mmol l(-1) HgCl2. Light-microscopic immunocytochemistry of branchial epithelia revealed that tilapia AQP3 was expressed in gill chloride cells of FW- and SW-adapted tilapia. Electron-microscopic immunocytochemistry further demonstrated that tilapia AQP3 was localized in the basolateral membrane of gill chloride cells. Basolateral localization of AQP3 in gill chloride cells suggests that AQP3 is involved in regulatory volume changes and osmoreception, which could trigger functional differentiation of chloride cells.

Localization and developmental expression of mRNA for cortical rod protein in kuruma prawn Marsupenaeus japonicus.

The mature penaeid oocytes possess cortical rods that contain two related cortical rod proteins (CRP, 28.6 kDa and 30.5 kDa). In the present study, localization of CRP mRNA and gene expression profiles of CRP and vitellogenin (Vg) during ovarian development were examined in kuruma prawn, Marsupenaeus japonicus, an economically important species for shrimp and prawn farming. Northern blot analysis revealed that CRP mRNA was expressed in the ovary. In situ hybridization showed strong signals for CRP transcripts in the oocytes at early developmental stages in both immature and mature ovaries. Quantitative analysis by real-time polymerase chain reaction revealed that CRP mRNA levels were higher in the previtellogenic and endogenous (primary) vitellogenic stages than in more advanced stages. Unlike CRP mRNA, Vg mRNA levels were low in the ovary and hepatopancreas in previtellogenic females. When the ovary developed into the endogenous vitellogenic stage, ovarian Vg mRNA levels increased significantly, followed by rapid decrease in more advanced stages. The Vg mRNA levels in the hepatopancreas, on the other hand, tended to be high in the exogenous (secondary) vitellogenic and maturation stages, in which ovarian Vg mRNA levels were decreased. Our findings indicate that CRP mRNA is highly expressed before the onset of vitellogenesis, suggesting that the transcription, translation, and cortical-rod formation of CRP occur at different phases of oocyte development. The endogenous vitellogenic stage is a crucial stage for the initiation of CRP and Vg syntheses. The coincidence of these protein syntheses suggests that CRP and Vg syntheses are regulated by closely-related mechanisms.

Existence of an extra-retinal and extra-pineal photoreceptive organ that regulates photoperiodism in gonadal development of an Osmerid teleost, ayu (Plecoglossus altivelis).

Ayu (Plecoglossus altivelis) is an Osmerid teleost whose gonadal development exhibits clear photoperiodism: it is stimulated and prevented under short and long photoperiod, respectively. However, the photoreceptor organ involved in this process remains to be identified. In the present study, we examined whether gonads of ophthalmectomized (Ex) and pinealectomized (Px) ayu respond to short photoperiod to test whether photoreceptors other than lateral eyes and pineal complex are involved in the photoperiodic response of gonadal development. Gonadosomatic index (GSI) and plasma levels of sex steroids (testosterone and 11-ketotestosterone for males and testosterone and estradiol 17-beta for females) were significantly increased in the Ex+Px ayu kept under short photoperiod in both males and females as compared with the initial control. On the other hand, there were no significant increases in GSI and sex steroids in the Ex+Px ayu kept under long photoperiod. Histological observation of gonads in the Ex+Px ayu revealed that oocytes undergoing final maturation in females and proliferation of germ cells in males were observed under short photoperiod but not under long photoperiod. These results indicate that extra-retinal and extra-pineal photoreceptive organ regulates photoperiodic gonadal development in this species.