Hiroyuki Doi

Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish

Marine teleost fish precipitate divalent cations as carbonate deposits in the intestine to minimize the potential for excessive Ca2+ entry and to stimulate water absorption by reducing luminal osmotic pressure. This carbonate deposit formation, therefore, helps maintain osmoregulation in the seawater (SW) environment and requires controlled secretion of HCO3(-) to match the amount of Ca2+ entering the intestinal lumen. Despite its physiological importance, the process of HCO3(-) secretion has not been characterized at the molecular level. We analyzed the expression of two families of HCO3(-) transporters, Slc4 and Slc26, in fresh-water- and SW-acclimated euryhaline pufferfish, mefugu (Takifugu obscurus), and obtained the following candidate clones: NBCe1 (an Na+-HCO3(-) cotransporter) and Slc26a6A and Slc26a6B (putative Cl(-)/HCO3(-) exchangers). Heterologous expression in Xenopus oocytes showed that Slc26a6A and Slc26a6B have potent HCO3(-)-transporting activity as electrogenic Cl(-)/nHCO3(-) exchangers, whereas mefugu NBCe1 functions as an electrogenic Na+-nHCO3(-) cotransporter. Expression of NBCe1 and Slc26a6A was highly induced in the intestine in SW and expression of Slc26a6B was high in the intestine in SW and fresh water, suggesting their involvement in HCO3(-) secretion and carbonate precipitate formation. Immunohistochemistry showed staining on the apical (Slc26a6A and Slc26a6B) and basolateral (NBCe1) membranes of the intestinal epithelial cells in SW. We therefore propose a mechanism for HCO3(-) transport across the intestinal epithelial cells of marine fish that includes basolateral HCO3(-) uptake (NBCe1) and apical HCO3(-) secretion (Slc26a6A and Slc26a6B).

Elevation of Kiss2 and its receptor gene expression in the brain and pituitary of grass puffer during the spawning season

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.

Explosive Speciation of Takifugu: Another Use of Fugu as a Model System for Evolutionary Biology

Although the fugu Takifugu rubripes has attracted attention as a model organism for genomic studies because of its compact genome, it is not generally appreciated that there are approximately 25 closely related species with limited distributions in the waters of East Asia. We performed molecular phylogenetic analyses and constructed a time tree using whole mitochondrial genome sequences from 15 Takifugu species together with 10 outgroups to examine patterns of diversification. The resultant time tree showed that the modern Takifugu species underwent explosive speciation during the Pliocene 1.8-5.3 Ma, which is comparable with that of the Malawi cichlids and tropheine cichlids in Lake Tanganyika. Considering their limited distributions and remarkable variations in coloration, morphology, and behavior, the results of the present study strongly suggest that Takifugu species are strong candidates as a model system for evolutionary studies of speciation mechanisms in marine environments where few such organisms are available.

Synchronised expressions of LPXRFamide peptide and its receptor genes: Seasonal, diurnal and circadian changes during spawning period in grass puffer

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.

Multiple Invasions into Freshwater by Pufferfishes (Teleostei: Tetraodontidae): A Mitogenomic Perspective

Pufferfishes of the Family Tetraodontidae are the most speciose group in the Order Tetraodontiformes and mainly inhabit coastal waters along continents. Although no members of other tetraodontiform families have fully discarded their marine lives, approximately 30 tetraodontid species spend their entire lives in freshwaters in disjunct tropical regions of South America, Central Africa, and Southeast Asia. To investigate the interrelationships of tetraodontid pufferfishes and thereby elucidate the evolutionary origins of their freshwater habitats, we performed phylogenetic analysis based on whole mitochondrial genome sequences from 50 tetraodontid species and closely related species (including 31 newly determined sequences). The resulting phylogenies reveal that the family is composed of four major lineages and that freshwater species from the different continents are independently nested in two of the four lineages. A monophyletic origin of the use of freshwater habitats was statistically rejected, and ancestral habitat reconstruction on the resulting tree demonstrates that tetraodontids independently entered freshwater habitats in different continents at least three times. Relaxed molecular-clock Bayesian divergence time estimation suggests that the timing of these invasions differs between continents, occurring at 0–10 million years ago (MA) in South America, 17–38 MA in Central Africa, and 48–78 MA in Southeast Asia. These timings are congruent with geological events that could facilitate adaptation to freshwater habitats in each continent.

Changes in cerebral blood flow under the prone condition with and without massage.

To investigate changes in regional cerebral blood flow (rCBF) under the prone condition with and without light massage on the back, we measured rCBF quantitatively in healthy human subjects using positron emission tomography with H(2)15O. Biochemical tests showed that the light massage (palm-pressure) reduced levels of stress-related serum cortisol and salivary stress protein chromogranin-A measured after the PET examination. Absolute rCBF significantly increased in the parietal cortex (precuneus) under the prone condition compared with the supine condition, and this rCBF increase was in parallel with comfortable sensation and slowing heart rate during the massage. Correlation analysis in statistical parametric mapping showed that the amygdalar and basal forebrain rCBF correlated with parasympathetic function (heart rate reduction), indicating involvement of the forebrain-amygdala system in mediating activities in the autonomic nervous system in the presence of comfortable sensation. To conclude, prone posture itself can stimulate the precuneus region to raise awareness, and the light massage on the back may help accommodate the brain to comfortable stimulation.

Differential expression of Na+-Cl- cotransporter and Na+-K+-Cl- cotransporter 2 in the distal nephrons of euryhaline and seawater pufferfishes

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 2.7× 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.

Differential expression of three types of gonadotropin-releasing hormone genes during the spawning season in grass puffer, Takifugu niphobles.

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 beta subunit mRNAs and plasma estradiol-17beta (E(2)) and testosterone (T) levels. There were strong positive correlations between the sbGnRH mRNA and the plasma E(2) 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.

Identification of renal transporters involved in sulfate excretion in marine teleost fish

Sulfate (SO(4)(2-)) is the second most abundant anion in seawater (SW), and excretion of excess SO(4)(2-) from ingested SW is essential for marine fish to survive. Marine teleosts excrete SO(4)(2-) via the urine produced in the kidney. The SO(4)(2-) transporter that secretes and concentrates SO(4)(2-) in the urine has not previously been identified. Here, we have identified and characterized candidates for the long-sought transporters. Using sequences from the fugu database, we have cloned cDNA fragments of all transporters belonging to the Slc13 and Slc26 families from mefugu (Takifugu obscurus). We compared Slc13 and Slc26 mRNA expression in the kidney between freshwater (FW) and SW mefugu. Among 14 clones examined, the expression of a Slc26a6 paralog (mfSlc26a6A) was the most upregulated (30-fold) in the kidney of SW mefugu. Electrophysiological analyses of Xenopus oocytes expressing mfSlc26a6A, mfSlc26a6B, and mouse Slc26a6 (mSlc26a6) demonstrated that all transporters mediate electrogenic Cl(-)/SO(4)(2-), Cl(-)/oxalate(2-), and Cl(-)/nHCO(3)(-) exchanges and electroneutral Cl(-)/formate(-) exchange. Two-electrode voltage-clamp experiments demonstrated that the SO(4)(2-)-elicited currents of mfSlc26a6A is quite large (approximately 35 microA at +60 mV) and 50- to 200-fold higher than those of mfSlc26a6B and mSlc26a6. Conversely, the currents elicited by oxalate and HCO(3)(-) are almost identical among mfSlc26a6A, mfSlc26a6B, and mSlc26a6. Kinetic analysis revealed that mfSlc26a6A has the highest SO(4)(2-) affinity as well as capacity. Immunohistochemical analyses demonstrated that mfSlc26a6A localizes to the apical (brush-border) region of the proximal tubules. Together, these findings suggest that mfSlc26a6A is the most likely candidate for the major apical SO(4)(2-) transporter that mediates SO(4)(2-) secretion in the kidney of marine teleosts.

Diurnal and Circadian Oscillations in Expression of Kisspeptin, Kisspeptin Receptor and Gonadotrophin‐Releasing Hormone 2 Genes in the Grass Puffer, A Semilunar‐Synchronised Spawner

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.