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Kato A.,Tokyo Institute of Technology | Muro T.,Tokyo Institute of Technology | Kimura Y.,Tokyo Institute of Technology | Li S.,Tokyo Institute of Technology | And 4 more authors.
American Journal of Physiology - Regulatory Integrative and Comparative Physiology | Year: 2011

The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na+-K+-Cl- cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na +-Cl- cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7x decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater. Copyright © 2011 the American Physiological Society.


Motohashi E.,Kyushu University | Yoshihara T.,Kyushu University | Doi H.,Shimonoseki Academy of Marine Science | Ando H.,Kyushu University
Zoological Science | Year: 2010

The grass puffer (Takifugu niphobles) exhibits unique spawning behavior. Large numbers of fish aggregate to coastal spawning beds several hours before high tide during the spring tide. In order to examine the environmental and physiological regulation of this semilunar-synchronized spawning rhythm, the aggregating and spawning behaviors of the grass puffer were observed in the field, and in an aquarium without tidal changes. The fish aggregated to a spawning bed in the rising tidal phases both in the morning and evening during the spring tide, and several days after the spring tide. Spawning occurred on several days when large numbers of fish (200-1000) aggregated to the spawning bed. The timing of aggregation and spawning was tightly connected to the tidal changes; aggregation occurred 2-3 h before high tide, and spawning occurred 2 h before high tide. In the aquarium, in which a slope was constructed with pebbles, small groups of mature fish aggregated on the slope only in the rising tidal phases during and after the spring tide, when the fish aggregated in the field. However, there was no spawning in the aquarium. The aggregating behavior observed in the aquarium without tidal changes suggests that the semilunar reproductive rhythm is endogenously maintained with surprising precision during the spawning period in grass puffer. © 2010 Zoological Society of Japan.


Shahjahan M.,Niigata University | Doi H.,Shimonoseki Academy of Marine Science | Ando H.,Niigata University
General and Comparative Endocrinology | Year: 2015

Gonadotropin-inhibitory hormone (GnIH) plays as a multifunctional neurohormone that controls reproduction in birds and mammals. LPXRFamide (LPXRFa) peptide, the fish ortholog of GnIH, has been shown to regulate the secretion of not only gonadotropin (GTH) but also growth hormone (GH) and prolactin (PRL), which are potentially important for gonadal function. To investigate the role of LPXRFa peptide on reproduction of the grass puffer, which spawns in semilunar cycles, we examined changes in the levels of gh and prl expression over the several months during the reproductive cycle, and the effects of goldfish LPXRFa peptide-1 (gfLPXRFa-1) on their expression were examined using primary pituitary cultures. The expression levels of both gh and prl showed significant changes during the reproductive cycle in both sexes with one peak in the spawning and pre-spawning periods for gh and prl, respectively. Particularly, gh showed substantial increase in expression in the spawning and post-spawning periods, indicative of its essentiality in the advanced stage of reproduction. gfLPXRFa-1 stimulated the expression of both gh and prl but there was a marked difference in response between them: gfLPXRFa-1 stimulated gh expression at a relatively low dose but little effect was observed on prl. Combined with the previous results of daily and circadian oscillations of lpxrfa expression, the present results suggest that LPXRFa peptide is important in the control of the cyclic reproduction by serving as a multifunctional hypophysiotropic factor that regulates the expression of gh and prl as well as GTH subunit genes. © 2015 Elsevier Inc.


Shahjahan M.,Bangladesh Agricultural University | Doi H.,Shimonoseki Academy of Marine Science | Ando H.,Niigata University
General and Comparative Endocrinology | Year: 2015

Pain-modulatory neuropeptides, PQRFamide (PQRFa) peptides, have recently been implicated in the regulation of reproduction in fish. As a first step toward investigating the role of PQRFa peptides on reproductive function in the grass puffer Takifugu niphobles, which is a semilunar spawner, we cloned genes encoding PQRFa peptide precursor (pqrfa) and its two types of receptors (pqrfa-r1 and pqrfa-r2), and examined changes in their expression levels in the brain and pituitary over several months during the reproductive cycle. The grass puffer PQRFa peptide precursor of 126 amino acid residues contains two putative PQRFa peptides, PQRFa-1 and PQRFa-2, which correspond to NPFF and NPAF in other vertebrates, respectively. The grass puffer PQRFa-R1 and PQRFa-R2 consist of 426 and 453 amino acid residues, respectively, and contain distinct characteristics of G-protein coupled receptors. These three genes were exclusively expressed in the brain and pituitary. The expression levels of pqrfa and pqrfa-r1 were significantly increased during the late stage of sexual maturation, but low in the spawning fish just after releasing sperms and eggs. Therefore, the grass puffer PQRFa peptide may have a role in the late stage of sexual maturation before spawning via PQRFa-R1. In contrast, the pqrfa-r2 expression showed maximum levels in the spawning fish and in the post-spawning period. The present results provide fundamental data suggesting that the grass puffer PQRFa peptide may have multiple roles in the control of reproduction that are dependent on the reproductive stages. © 2014 Elsevier Inc.


Shahjahan M.,Kyushu University | Motohashi E.,Kyushu University | Doi H.,Shimonoseki Academy of Marine Science | Ando H.,Kyushu University
General and Comparative Endocrinology | Year: 2010

Kisspeptins are a family of neuropeptides encoded by Kiss1 and Kiss2 genes, and participate in neuroendocrine regulation of gonadotropin-releasing hormone (GnRH) secretion through activating their receptor, Kiss1r (or GPR54). Bioinformatic analyses have shown that there is a single gene for each kisspeptin (Kiss2) and its receptor (Kiss1r) in pufferfish, the function of which has yet to be elucidated. We cloned these two genes in grass puffer, which spawns on beach with semilunar cycles, and examined changes in their expression levels in the brain and pituitary at different reproductive stages over the spawning season. The Kiss2 precursor of 104 amino acid residues contains a putative kisspeptin peptide (SKFNLNPFGLRF). Kiss1r consists of 377 amino acid residues containing distinct characteristics of G-protein coupled receptors. Kiss2 and Kiss1r genes were expressed extensively in the brain, pituitary and gonads. The amounts of Kiss2 and Kiss1r mRNAs were significantly elevated during the spawning period in the brain and pituitary of both sexes. There were strong positive correlations between the amounts of Kiss2 and Kiss1r mRNAs in the brain and pituitary over the spawning season. Significant positive correlations were also observed between the amounts of Kiss2/Kiss1r mRNAs and GnRH1 mRNA in the brain. The present results indicate that the Kiss2/Kiss1r system most probably plays an important role in the regulation of reproductive function in the spawning period of grass puffer, possibly through the stimulation of GnRH1 secretion. Furthermore, Kiss2 may have a local action in the pituitary. © 2010 Elsevier Inc.


Shahjahan M.,Kyushu University | Hamabata T.,Kyushu University | Motohashi E.,Kyushu University | Doi H.,Shimonoseki Academy of Marine Science | Ando H.,Kyushu University
General and Comparative Endocrinology | Year: 2010

Grass puffer, Takifugu niphobles, has unique spawning behavior; spawning occurs on beach only for several days around new moon and full moon from spring to early summer. To investigate the role of gonadotropin-releasing hormone (GnRH) in the reproductive function, genes encoding three types of GnRHs, namely seabream GnRH (sbGnRH), chicken GnRH-II (cGnRH-II) and salmon GnRH (sGnRH), were cloned and changes in their mRNA amounts were examined over the spawning season. In addition, changes in the pituitary gonadotropin subunit mRNAs and the plasma steroid hormones were examined over the spawning season. Fishes were assessed at four reproductive stages, i.e., in December (early maturation), in April (maturing), in May (spawning), and in July (post-spawning). Moreover, spawning fish just after releasing eggs and sperm were taken at a spawning bed. The amounts of sbGnRH mRNA were substantially elevated in May and the spawning fish in both sexes, concomitant with considerable elevations of follicle-stimulating hormone and luteinizing hormone β subunit mRNAs and plasma estradiol-17β (E2) and testosterone (T) levels. There were strong positive correlations between the sbGnRH mRNA and the plasma E2 and T levels over the spawning season in both sexes. The amounts of cGnRH-II mRNA showed no noticeable changes except for an increase in the post-spawning females. The amounts of sGnRH mRNA in the males were significantly increased in May, but they were low in the spawning males. In the females, sGnRH mRNA increased from the maturing stage and reached a maximum in the post-spawning stage, in which a positive correlation with the plasma cortisol levels was observed. These specific changes suggest that the expression of three types of GnRH genes is differentially regulated during the spawning season, and sex steroids may be important for the differential expression of GnRH genes. © 2010 Elsevier Inc.


Shahjahan M.,Kyushu University | Ikegami T.,Kyushu University | Osugi T.,Waseda University | Ukena K.,Hiroshima University | And 4 more authors.
Journal of Neuroendocrinology | Year: 2011

Among the RFamide peptide family, the LPXRFamide peptide (LPXRFa) group regulates the release of various pituitary hormones and, recently, LPXRFa genes were found to be regulated by photoperiod via melatonin. As a first step towards investigating the role of LPXRFa on reproductive function in grass puffer (Takifugu niphobles), which spawns in semilunar cycles, genes encoding LPXRFa and its receptor (LPXRFa-R) were cloned, and seasonal, diurnal and circadian changes in their absolute amounts of mRNAs in the brain and pituitary were examined by quantitative real-time polymerase chain reaction. The grass puffer LPXRFa precursor contains two putative RFamide peptides and one possible RYamide peptide. LPXRFa and LPXRFa-R genes were extensively expressed in the diencephalon and pituitary. The expression levels of both genes were significantly elevated during the spawning periods in both sexes in the brain and pituitary, although they were low in the spawning fish just after releasing eggs and sperm. The treatment of primary pituitary cultures with goldfish LPXRFa increased the amounts of follicle-stimulating hormone β- and luteinising hormone β-subunit mRNAs. In the diencephalon, LPXRFa and LPXRFa-R genes showed synchronised diurnal and circadian variations with one peak at zeitgeber time 3 and circadian time 15, respectively. The correlated expression patterns of LPXRFa and LPXRFa-R genes in the diencephalon and pituitary and the possible stimulatory effects of LPXRFa on gonadotrophin subunit gene expression suggest the functional significance of the LPXRFa and LPXRFa-R system in the regulation of lunar-synchronised spawning of grass puffer. © 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.


Ando H.,Niigata University | Ogawa S.,University Institute of Health Sciences | Shahjahan M.,University Institute of Health Sciences | Shahjahan M.,Bangladesh Agricultural University | And 4 more authors.
Journal of Neuroendocrinology | Year: 2014

In seasonally breeding animals, the circadian and photoperiodic regulation of neuroendocrine system is important for precisely-timed reproduction. Kisspeptin, encoded by the Kiss1 gene, acts as a principal positive regulator of the reproductive axis by stimulating gonadotrophin-releasing hormone (GnRH) neurone activity in vertebrates. However, the precise mechanisms underlying the cyclic regulation of the kisspeptin neuroendocrine system remain largely unknown. The grass puffer, Takifugu niphobles, exhibits a unique spawning rhythm: spawning occurs 1.5-2 h before high tide on the day of spring tide every 2 weeks, and the spawning rhythm is connected to circadian and lunar-/tide-related clock mechanisms. The grass puffer has only one kisspeptin gene (kiss2), which is expressed in a single neural population in the preoptic area (POA), and has one kisspeptin receptor gene (kiss2r), which is expressed in the POA and the nucleus dorsomedialis thalami. Both kiss2 and kiss2r show diurnal variations in expression levels, with a peak at Zeitgeber time (ZT) 6 (middle of day time) under the light/dark conditions. They also show circadian expression with a peak at circadian time 15 (beginning of subjective night-time) under constant darkness. The synchronous and diurnal oscillations of kiss2 and kiss2r expression suggest that the action of Kiss2 in the diencephalon is highly dependent on time. Moreover, midbrain GnRH2 gene (gnrh2) but not GnRH1 or GnRH3 genes show a unique semidiurnal oscillation with two peaks at ZT6 and ZT18 within a day. The cyclic expression of kiss2, kiss2r and gnrh2 may be important in the control of the precisely-timed diurnal and semilunar spawning rhythm of the grass puffer, possibly through the circadian clock and melatonin, which may transmit the photoperiodic information of daylight and moonlight to the reproductive neuroendocrine centre in the hypothalamus. © 2014 British Society for Neuroendocrinology.


Islam Z.,Tokyo Institute of Technology | Hayashi N.,Tokyo Institute of Technology | Yamamoto Y.,Tokyo Institute of Technology | Doi H.,Shimonoseki Academy of Marine Science | And 4 more authors.
American Journal of Physiology - Regulatory Integrative and Comparative Physiology | Year: 2013

The second most abundant cation in seawater (SW), Mg2+, is present at concentrations of ~53 mM. Marine teleosts maintain plasma Mg2+ concentration at 1-2 mM by excreting Mg2+ into the urine. Urine Mg2+ concentrations of SW teleosts exceed 70 mM, most of which is secreted by the renal tubular epithelial cells. However, molecular mechanisms of the Mg2+ secretion have yet to be clarified. To identify transporters involved in Mg2+ secretion, we analyzed the expression of fish homologs of the Slc41 Mg2+ transporter family in various tissues of SW pufferfish torafugu (Takifugu rubripes) and its closely related euryhaline species mefugu (Takifugu obscurus). Takifugu genome contained five members of Slc41 genes, and only Slc41a1 was highly expressed in the kidney. Renal expression of Slc41a1 was markedly elevated when mefugu were transferred from fresh water (FW) to SW. In situ hybridization analysis and immunohistochemistry at the light and electron microscopic levels revealed that Slc41a1 is localized to vacuoles in the apical cytoplasm of the proximal tubules. These results suggest that pufferfish Slc41a1 is a Mg2+ transporter involved in renal tubular transepithelial Mg2+ secretion by mediating Mg2+ transport from the cytosol to the vacuolar lumen, and support the hypothesis that Mg2 + secretion is mediated by exocytosis of Mg2+-rich vacuoles to the lumen. © 2013 the American Physiological Society.


PubMed | Niigata University and Shimonoseki Academy of Marine Science
Type: | Journal: General and comparative endocrinology | Year: 2016

Gonadotropin-inhibitory hormone (GnIH) plays as a multifunctional neurohormone that controls reproduction in birds and mammals. LPXRFamide (LPXRFa) peptide, the fish ortholog of GnIH, has been shown to regulate the secretion of not only gonadotropin (GTH) but also growth hormone (GH) and prolactin (PRL), which are potentially important for gonadal function. To investigate the role of LPXRFa peptide on reproduction of the grass puffer, which spawns in semilunar cycles, we examined changes in the levels of gh and prl expression over the several months during the reproductive cycle, and the effects of goldfish LPXRFa peptide-1 (gfLPXRFa-1) on their expression were examined using primary pituitary cultures. The expression levels of both gh and prl showed significant changes during the reproductive cycle in both sexes with one peak in the spawning and pre-spawning periods for gh and prl, respectively. Particularly, gh showed substantial increase in expression in the spawning and post-spawning periods, indicative of its essentiality in the advanced stage of reproduction. gfLPXRFa-1 stimulated the expression of both gh and prl but there was a marked difference in response between them: gfLPXRFa-1 stimulated gh expression at a relatively low dose but little effect was observed on prl. Combined with the previous results of daily and circadian oscillations of lpxrfa expression, the present results suggest that LPXRFa peptide is important in the control of the cyclic reproduction by serving as a multifunctional hypophysiotropic factor that regulates the expression of gh and prl as well as GTH subunit genes.

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