Gene duplication of C-type natriuretic peptide-4 (CNP4) in teleost lineage elicits subfunctionalization of ancestral CNP.

The diversified natriuretic peptide (NP) family, consisting of four CNPs (CNP1-4), ANP, BNP, and VNP, has been identified in the eel. Here, we successfully cloned additional cnp genes from the brain of eel (a basal teleost) and zebrafish (a later branching teleost). The genes were identified as paralogues of cnp4 generated by the third round of whole genome duplication (3R) in the teleost lineage, thereby being named eel cnp4b and zebrafish cnp4-like, respectively. To examine the histological patterns of their expressions, we employed a newly developed in situ hybridization (ISH) chain reaction using short hairpin DNAs, in addition to conventional ISH. Eel cnp4b was expressed in the medulla oblongata, while mRNAs of eel cnp4a (former cnp4) were localized in the preoptic area. In the zebrafish brain, cnp4-like mRNA was undetectable, while the known cnp4 was expressed in both the preoptic area and medulla oblongata. Together with the different mRNA distribution of cnp4a and cnp4b in eel peripheral tissues determined by RT-PCR and ISH, it is suggested that subfunctionalization by duplicated cnp4s in ancestral teleosts has been retained only in basal teleosts. Intriguingly, cnp4b-expressing neurons in the glossopharyngeal-vagal motor complex of the medulla oblongata were co-localized with choline acetyltransferase, suggesting an involvement of Cnp4b in swallowing and respiration functions that are modulated by the vagus. Since teleost Cnp4 is an ortholog of mammalian CNP, the identified localization of teleost Cnp4 will contribute to future studies aimed at deciphering the physiological functions of CNP.

Differences in the contributions of sex linkage and androgen regulation to sex-biased gene expression in juvenile and adult sticklebacks.

Different evolutionary interests between males and females can lead to the evolution of sexual dimorphism. However, intersex genetic correlations due to the shared genome can constrain the evolution of sexual dimorphism, resulting in intra-locus sexual conflict. One of the mechanisms resolving this conflict is sex linkage, which allows males and females to carry different alleles on sex chromosomes. Another is a regulatory mutation causing sex-biased gene expression, which is often mediated by gonadal steroids in vertebrates. How do these two mechanisms differ in the contributions to the resolution of intra-locus sexual conflict? The magnitude of sexual conflict often varies between the juvenile and adult stages. Because gonadal steroids change in titre during development, we hypothesized that gonadal steroids play a role in sexual dimorphism expression only at certain developmental stages, whereas sex linkage is more important for sexual dimorphism expressed throughout life. Our brain transcriptome analysis of juvenile and adult threespine sticklebacks showed that the majority of genes that were sex-biased in both stages were sex-linked. The relative contribution of androgen-dependent regulation to the sex-biased transcriptome increased and that of sex linkage declined in adults compared to juveniles. The magnitude of the sex differences was greater in sex-linked genes than androgen-responsive genes, suggesting that sex linkage is more effective than androgen regulation in the production of large sex differences in gene expression. Overall, our data are consistent with the hypothesis that sex linkage is effective in resolving sexual conflict throughout life, whereas androgen-dependent regulation can contribute to temporary resolution of sexual conflict.

Parallel transcriptome evolution in stream threespine sticklebacks.

Natural selection can cause similar phenotypic evolution in phylogenetically independent lineages inhabiting similar environments. Compared to morphological, behavioral, and physiological traits, little is known about the parallel evolution of transcriptome. Furthermore, the relative contribution of cis- and trans-regulatory changes to parallel transcriptome evolution largely remains unclear. The threespine stickleback fish (Gasterosteus aculeatus) is a great model for studying parallel evolution because its ancestral marine populations independently colonized freshwater habitats in multiple geographical regions, resulting in independent pairs of marine and freshwater ecotypes in each region. Here, we investigated transcriptomic parallelism among the marine and stream ecotypes of Japanese and Canadian threespine sticklebacks by conducting common garden experiments and microarray analysis of the brain, which controls several physiological and behavioral traits differing between these ecotypes. We found parallel expression differences in 103 genes, including those encoding the enzymes involved in taurine synthesis and glycoprotein hydrolysis. The number of genes differentially expressed in parallel was significantly larger than the number of genes showing an antiparallel pattern (71 genes). To investigate the genetic architecture underlying transcriptome divergence, we re-analyzed the previous expression quantitative trait locus (eQTL) data and found that most eQTLs were located on the same chromosome as the transcripts, possibly in cis-regulatory regions. Furthermore, the effect sizes of the eQTLs on the same chromosomes were larger than those on different chromosomes. Thus, we found that divergence in the brain transcriptome between the ecotypes shows parallelism and is mainly caused by genetic changes occurring on the same chromosome as the target genes.

[A Case of Survival Following Fourth-Line Outpatient Chemotherapy 19 Months after Surgery for Small Intestinal Cancer with Multiple Liver Metastases in a Patient with Good General Condition].

A 59-year-old woman who complained of melena and lightheadedness visited the outpatient clinic at our hospital.According to her blood test result, she had anemia, and her tumor marker levels were high.Enhanced computed tomography(CT) findings showed small intestinal cancer with multiple liver metastases.Partial resection of the small bowel for the small intestinal cancer was performed.Following the administration of fourth-line outpatient chemotherapy containing S-1 plus irinotecan( IRIS)and IRIS plus bevacizumab(IRIS plus Bev), S-1 plus oxaliplatin plus Bev(SOX plus Bev), and weekly paclitaxel (wPAC), she survived with good condition for 19 months after the surgery.

Distribution and co-localization of diversified natriuretic peptides in the eel heart.

Atrial and B-type natriuretic peptides (ANP and BNP) are cardiac hormones important for cardiovascular and body fluid regulation. In some teleost species, an additional member of the natriuretic peptide family, ventricular NP (VNP), has been identified. In this study, we examine tissue distribution of these three NPs in the eel heart. Quantitative real-time PCR showed that anp is almost exclusively expressed in atria, bnp equally in atria and ventricles and vnp three-fold more in ventricles than in atria. The amount of bnp transcript overall in the heart was 1/10 those of anp and vnp. There was no difference in transcript levels between freshwater and seawater-acclimated fishes. Immunohistochemistry using specific antisera and in situ hybridization using gene-specific probes showed that NP signals were detected in most atrial and ventricular myocytes with some regional differences in density. Because of high sequence similarity of the three NPs, each of the three NP antisera individually was pre-incubated with 10-8 M of the other two non-targeted cardiac NPs to increase the specificity. A few atrial myocytes contained all three NPs in the same cell. Immuno-electron microscopy identified many dense-core vesicles containing ANP in atria and VNP in ventricles and some vesicles contained both ANP and VNP as demonstrated using pre-absorbed antisera. Based on these data and those of previous studies, we suggest that in eels ANP is secreted from atria in a regulatory pathway and VNP from ventricles in a constitutive pathway. In addition, VNP, not BNP, is the principal ventricular hormone in eels.

Molecular mechanisms underlying active desalination and low water permeability in the esophagus of eels acclimated to seawater.

Marine teleosts can absorb imbibed seawater (SW) to maintain water balance, with esophageal desalination playing an essential role. NaCl absorption from luminal SW was enhanced 10-fold in the esophagus of SW-acclimated eels, and removal of Na+ or Cl- from luminal SW abolished the facilitated absorption, indicating coupled transport. Mucosal/serosal application of various blockers for Na+/Cl- transporters profoundly decreased the absorption. Among the transporter genes expressed in eel esophagus detected by RNA-seq, dimethyl amiloride-sensitive Na+/H+ exchanger (NHE3) and 4,4'-diisothiocyano-2,2'-disulfonic acid-sensitive Cl-/[Formula: see text] exchanger (AE) coupled by the scaffolding protein on the apical membrane of epithelial cells, and ouabain-sensitive Na+-K+-ATPases (NKA1α1c and NKA3α) and diphenylamine-2-carboxylic acid-sensitive Cl- channel (CLCN2) on the basolateral membrane, may be responsible for enhanced transcellular NaCl transport because of their profound upregulation after SW acclimation. Upregulated carbonic anhydrase 2a (CA2a) supplies H+ and [Formula: see text] for activation of the coupled NHE and AE. Apical hydrochlorothiazide-sensitive Na+-Cl- cotransporters and basolateral Na+-[Formula: see text] cotransporter (NBCe1) and AE1 are other possible candidates. Concerning the low water permeability that is typically seen in marine teleost esophagus, downregulated aquaporin genes (aqp1a and aqp3) and upregulated claudin gene (cldn15a) are candidates for transcellular/paracellular route. In situ hybridization showed that these upregulated transporters and tight-junction protein genes were expressed in the absorptive columnar epithelial cells of eel esophagus. These results allow us to provide a full picture of the molecular mechanism of active desalination and low water permeability that are characteristic to marine teleost esophagus and gain deeper insights into the role of gastrointestinal tracts in SW acclimation.

Genetic basis for variation in salinity tolerance between stickleback ecotypes.

Adaptation to different salinities can drive and maintain divergence between populations of aquatic organisms. Anadromous and stream ecotypes of threespine stickleback (Gasterosteus aculeatus) are an excellent model to explore the genetic mechanisms underlying osmoregulation divergence. Using a parapatric pair of anadromous and stream stickleback ecotypes, we employed an integrated genomic approach to identify candidate genes important for adaptation to different salinity environments. Quantitative trait loci (QTL) mapping of plasma sodium concentrations under a seawater challenge experiment identified a significant QTL on chromosome 16. To identify candidate genes within this QTL, we first conducted RNA-seq and microarray analysis on gill tissue to find ecotypic differences in gene expression that were associated with plasma Na+ levels. This resulted in the identification of ten candidate genes. Quantitative PCR analysis on gill tissue of additional Japanese stickleback populations revealed that the majority of the candidate genes showed parallel divergence in expression levels. Second, we conducted whole-genome sequencing and found five genes that are predicted to have functionally important amino acid substitutions. Finally, we conducted genome scan analysis and found that eight of these candidate genes were located in genomic islands of high differentiation, suggesting that they may be under divergent selection. The candidate genes included those involved in ATP synthesis and hormonal signalling, whose expression or amino acid changes may underlie the variation in salinity tolerance. Further functional molecular analysis of these genes will reveal the causative genetic and genomic changes underlying divergent adaptation.

Regulation of sex steroid production and mRNAs encoding gonadotropin receptors and steroidogenic proteins by gonadotropins, cyclic AMP and insulin-like growth factor-I in ovarian follicles of rainbow trout (Oncorhynchus mykiss) at two stages of vitellogenesis.

At the completion of vitellogenesis, the steroid biosynthetic pathway in teleost ovarian follicles switches from estradiol-17ß (E2) to maturational progestin production, associated with decreased follicle stimulating hormone (Fsh) and increased luteinizing hormone (Lh) signaling. This study compared effects of gonadotropins, human insulin-like growth factor-I (IGF1), and cAMP/protein kinase A signaling (forskolin) on E2 production and levels of mRNAs encoding steroidogenic proteins and gonadotropin receptors using midvitellogenic (MV) and late/postvitellogenic (L/PV) ovarian follicles of rainbow trout. Fsh, Lh and forskolin, but not IGF1, increased testosterone and E2 production in MV and L/PV follicles. Fsh increased steroidogenic acute regulatory protein (star; MV), 3ß-hydroxysteroid dehydrogenase/Δ(5-4) isomerase (hsd3b; MV) and P450 aromatase (cyp19a1a; MV) transcript levels. Lh increased star mRNA levels (MV, L/PV) but reduced cyp19a1a transcripts in L/PV follicles. At both follicle stages, IGF1 reduced levels of hsd3b transcripts. In MV follicles, IGF1 decreased P450 side-chain cleavage enzyme (cyp11a1) transcripts but increased cyp19a1a transcripts. In MV follicles only, forskolin increased star and hsd3b transcripts. Forskolin reduced MV follicle cyp11a1 transcripts and reduced cyp19a1a transcripts in follicles at both stages. Fsh and Lh reduced fshr transcripts in L/PV follicles. Lh also reduced lhcgr transcripts (L/PV). IGF1 had no effect on gonadotropin receptor transcripts. Forskolin reduced MV follicle fshr transcript levels and reduced lhcgr transcripts in L/PV follicles. These results reveal hormone- and stage-specific transcriptional regulation of steroidogenic protein and gonadotropin receptor genes and suggest that the steroidogenic shift at the completion of vitellogenesis involves loss of stimulatory effects of Fsh and Igfs on cyp19a1a expression and inhibition of cyp19a1a transcription by Lh.

Relaxin-related gene expression differs between anadromous and stream-resident stickleback (Gasterosteus aculeatus) following seawater transfer.

Relaxin (RLN) is a hormone that was originally identified as a regulator of pregnancy and reproduction. However, recent mammalian studies have demonstrated that relaxins also have potent osmoregulatory actions. In mammals, six relaxin family peptides have been identified: RLN1/2, RLN3, insulin-like peptide (INSL) 3, INSL4, INSL5, and INSL6. Previous genome database searches have revealed that teleosts also possess multiple relaxin family genes. However, the functions of these relaxin family peptides in teleosts remain unclear. In order to gain insight into the osmoregulatory functions of teleost relaxins, we studied the relaxin family peptides in euryhaline three-spined sticklebacks (Gasterosteus aculeatus), which have diversified into a variety of ecotypes. Rln3a, rln3b, and rln transcripts were abundant in the stickleback brain, whereas insl5b transcript levels were highest in the intestine among tissues. Seawater challenge experiments showed that transcript levels of rln3a, rln3b, and rln in the brain changed significantly after seawater transfer. Particularly, rln3b showed different patterns of temporal changes between anadromous and stream-resident morphs. The transcript levels of relaxin family peptide receptors, rxfp1, rxfp2b, rxfp3-2a, and rxfp3-2b, did not exhibit substantial changes in the brain, although these were constantly higher in the anadromous morph than the stream-resident morph. These results suggest that stickleback relaxin systems are differentially regulated by salinity signals, at least at the transcriptional level, and anadromous and stream-resident morphs differ in relaxin signaling pathways. The differences in the expression of relaxin-related genes between these two morphs provide a foundation for further exploration of the osmoregulatory function of relaxins in teleosts.

Convergent gene losses and pseudogenizations in multiple lineages of stomachless fishes.

The regressive evolution of independent lineages often results in convergent phenotypes. Several teleost groups display secondary loss of the stomach, and four gastric genes, atp4a, atp4b, pgc, and pga2 have been co-deleted in agastric (stomachless) fish. Analyses of genotypic convergence among agastric fishes showed that four genes, slc26a9, kcne2, cldn18a, and vsig1, were co-deleted or pseudogenized in most agastric fishes of the four major groups. kcne2 and vsig1 were also deleted or pseudogenized in the agastric monotreme echidna and platypus, respectively. In the stomachs of sticklebacks, these genes are expressed in gastric gland cells or surface epithelial cells. An ohnolog of cldn18 was retained in some agastric teleosts but exhibited an increased non-synonymous substitution when compared with gastric species. These results revealed novel convergent gene losses at multiple loci among the four major groups of agastric fish, as well as a single gene loss in the echidna and platypus.

Morphological and functional characterization of a novel Na+/K+-ATPase-immunoreactive, follicle-like structure on the gill septum of Japanese banded houndshark, Triakis scyllium.

In teleost fishes, it is well-established that the gill serves as an important ionoregulatory organ in addition to its primary function of respiratory gas exchange. In elasmobranch fish, however, the ionoregulatory function of the gills is still poorly understood. Although mitochondria-rich (MR) cells have also been found in elasmobranch fish, these cells are considered to function primarily in acid-base regulation. In this study, we found a novel aggregate structure made up of cells with basolaterally-expressed Na(+)/K(+)-ATPase (NKA), in addition to NKA-immunoreactive MR cells that have already been described in the gill filament and lamella. The cell aggregates, named follicularly-arranged NKA-rich cells (follicular NRCs), were found exclusively in the epithelial lining of the venous web in the cavernous region of the filament and the inter-filamental space of the gill septum. The follicular NRCs form a single-layered follicular structure with a large lumen leading to the external environment. The follicular NRCs were characterized by: (i) well-developed microvilli on the apical membrane, (ii) less prominent infoldings of the basolateral membrane and (iii) typical junction structures including deep tight junction between cells. In addition, large numbers of vesicles were observed in the cytoplasm and some of them were fused to the lateral membrane. The follicular NRCs expressed Na(+)/H(+) exchanger 3 and Ca(2+) transporter 1. The follicular NRCs thus have the characteristics of absorptive ionoregulatory cells and this suggests that the elasmobranch gill probably contributes more importantly to body fluid homeostasis than previously thought.

Salinity-dependent in vitro effects of homologous natriuretic peptides on the pituitary-interrenal axis in eels.

We examined the effects of atrial, B-type, ventricular and C-type natriuretic peptides (ANP, BNP, VNP and CNP1, 3, 4) on cortisol secretion from interrenal tissue in vitro in both freshwater (FW) and seawater (SW)-acclimated eels. We first localized the interrenal and chromaffin cells in the eel head kidney using cell specific markers (cholesterol side-chain cleavage enzyme (P450ssc) and tyrosine hydroxylase (TH), respectively) and established the in vitro incubation system for eel interrenal tissue. Unexpectedly, none of the NPs given alone to the interrenal tissue of FW and SW eels stimulated cortisol secretion. However, ANP and VNP, but not BNP and three CNPs, enhanced the steroidogenic action of ACTH in SW interrenal preparations, while CNP1 and CNP4, but not ANP, BNP, VNP and CNP3, potentiated the ACTH action in FW preparations. These salinity dependent effects of NPs are consistent with the previous in vivo study in the eel where endogenous ACTH can act with the injected NPs. 8-Br-cGMP also enhanced the ACTH action in both FW and SW eel preparations, suggesting that the NP actions were mediated by the guanylyl cyclase-coupled NP receptors (GC-A and B) that were localized in the eel interrenal. Further, ANP and CNP1 stimulated ACTH secretion from isolated pituitary glands of SW and/or FW eels. In summary, the present study revealed complex mechanisms of NP action on corticosteroidogenesis through the pituitary-interrenal axis in eels, thereby providing a deeper insight into the role of the NP family in the acclimation of this euryhaline teleost to diverse salinity environments.

Localization of diversified relaxin gene transcripts in the brain of eels.

Relaxin 3 (RLN3) is a newly-discovered member of the insulin superfamily. We isolated three RLN3-like cDNAs from the brain of the Japanese eel (Anguilla japonica). The deduced amino acid sequences of the RLN3-like cDNAs contained the two-chain structure common to relaxin including a RXXXRXXI/V motif in the B-chain. Phylogenetic analysis assigned the two prepropeptides into teleost/mammalian RLN3 group, which are a pair of duplicates generated by the teleost-specific third-round whole genome duplication, and the other one into teleost RLN group. Therefore, they have been named eel rln3a, rln3b and rln. rln3a transcripts were abundant in the middle-posterior region of the brain and detected at lower levels in the gills, head kidney and kidney. rln3b transcripts were also detected in the middle-posterior region of the brain, but the expression levels were lower than those of rln3a. Low levels of rln transcripts were detected in all brain areas, pituitary, digestive tract and gonad. Quantitative PCR analysis did not detect differences in expression of any rln3 or rln gene between freshwater- and seawater-acclimated eels. In situ hybridization showed that rln3a was expressed in neurons of the lateral lemniscus of the midbrain and of the griseum centrale (GC) of the hindbrain, while low amounts of rln transcripts were found in neurons of the periventricular nucleus of the posterior tuberculum of the diencephalon and the GC. These results suggest that the multiple RLN3-like peptides may play regulatory roles in the brain of euryhaline fish.

Effects of chronic manipulation of adrenocorticotropic hormone levels in Chinook salmon on expression of interrenal steroidogenic acute regulatory protein and steroidogenic enzymes.

The effects of chronic exposure to adrenocorticotropic hormone (ACTH) or the synthetic glucocorticoid dexamethasone (DEX) on the expression of genes involved in cortisol synthesis were examined using quantitative RT-PCR and immunohistochemistry. Juvenile Chinook salmon were treated with either ACTH via micro-osmotic pumps or with DEX via a lipid-based sustained release vehicle. Plasma cortisol levels were significantly elevated in ACTH-treated fish after 1 day, with a significant reduction in this effect with increasing treatment duration. ACTH also appeared to cause progressive hyperplasia of interrenal cells. Steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage enzyme (P450scc) transcripts but not 3ß-hydroxysteroid dehydrogenase-isomerase (3ß-HSD) or cytochrome P450 11ß-hydroxylase (P45011ß) transcripts in head kidneys significantly increased after 5 days of ACTH treatment. Significant linear relationships between plasma cortisol levels and transcript levels were identified at day 1 and day 5 for StAR, and day 5 for P450scc. Increased immunoreactivity for P450scc was observed in interrenal cells of ACTH-treated fish after 5 and 10 days. No effect of ACTH on 3ß-HSD immunoreactivity was apparent at any time point. P45011ß immunoreactivity was more intense after 5 days treatment with ACTH. DEX significantly reduced resting plasma cortisol levels and induced interrenal cell atrophy. Although no significant effect of treatment with DEX was found for any transcript, immunoreactivity for P450scc and P45011ß appeared to be reduced. These results indicate that StAR and P450scc are subject to transcriptional regulation by chronic changes in ACTH levels.

Whole-Genome Sequencing Highlights Conservative Genomic Strategies of a Stress-Tolerant, Long-Lived Scleractinian Coral, Porites australiensis Vaughan, 1918.

Massive corals of the genus Porites, common, keystone reef builders in the Indo-Pacific Ocean, are distinguished by their relative stress tolerance and longevity. In order to identify genetic bases of these attributes, we sequenced the complete genome of a massive coral, Porites australiensis. We developed a genome assembly and gene models of comparable quality to those of other coral genomes. Proteome analysis identified 60 Porites skeletal matrix protein genes, all of which show significant similarities to genes from other corals and even to those from a sea anemone, which has no skeleton. Nonetheless, 30% of its skeletal matrix proteins were unique to Porites and were not present in the skeletons of other corals. Comparative genomic analyses showed that genes widely conserved among other organisms are selectively expanded in Porites. Specifically, comparisons of transcriptomic responses of P. australiensis and Acropora digitifera, a stress-sensitive coral, reveal significant differences in regard to genes that respond to increased water temperature, and some of the genes expanded exclusively in Porites may account for the different thermal tolerances of these corals. Taken together, widely shared genes may have given rise to unique biological characteristics of Porites, massive skeletons and stress tolerance.

WITHDRAWN: Molecular cloning and expression analysis of relaxin 3c in Japanese eel, Anguilla japonica.

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Seawater transfer down-regulates C-type natriuretic peptide-3 expression in prolactin-producing cells of Japanese eel: Negative correlation with plasma chloride concentration.

In euryhaline fishes, atrial and B-type natriuretic peptides are important hormones in hypo-osmoregulation, whereas osmoregulatory functions of C-type natriuretic peptides (CNPs) remain to be investigated. Although four CNP isoforms (CNP1-4) are mainly expressed in the brain, multiorgan expression of CNP3 was found in euryhaline Japanese eel, Anguilla japonica. Here we identified the CNP3-expressing cells and examined their response to osmotic stress in eel. CNP3 was expressed in several endocrine cells: prolactin-producing cells (pituitary), glucagon-producing cells (pancreas), and cardiomyocytes (heart). Pituitary CNP3 expression was the highest among organs and was decreased following seawater transfer, followed by a decrease in the freshwater-adaptating (hyper-osmoregulatory) hormone prolactin. We also showed the negative correlation between CNP3/prolactin expression in the pituitary and plasma Cl- concentration, but not for plasma Na+ concentration. These results suggest that CNP3 in the pituitary (and pancreas) plays a critical role in freshwater adaptation of euryhaline eel together with prolactin.

Differential suppressive effects of low physiological doses of estradiol-17beta in vivo on levels of mRNAs encoding steroidogenic acute regulatory protein and three steroidogenic enzymes in previtellogenic ovarian follicles of rainbow trout.

Numerous recent reports have demonstrated effects of estrogenic chemicals on reproductive physiology of fish. However, there is little information available on the regulation of ovarian steroidogenesis by physiological levels of endogenous steroids in teleosts. Therefore, we analyzed the levels of mRNAs encoding steroidogenic proteins in ovaries of E2-treated rainbow trout Oncorhynchus mykiss). Previtellogenic (perinucleolar oocyte stage) trout received either blank or E2 implants (0.1 microg, 1 microg or 10 microg/g BW) for 7 days in order to achieve low, medium and high physiological levels of E2 in plasma. Plasma E2 levels were measured using radioimmunoassay. Levels of mRNAs encoding steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and P450 aromatase A (P450aromA) in the ovary were analyzed by real-time quantitative PCR. E2 levels in control animals were approximately 0.5 ng/ml. Levels in treated fish were approximately 1 ng/ml (0.1 microg implant), 2.6 ng/ml (1 microg implant) and 90 ng/ml (10 microg implant), within or just above the physiological range of immature and maturing female rainbow trout. StAR mRNA levels were significantly reduced by all E2 treatments. P450scc mRNA levels were not affected, but 3beta-HSD and P450arom mRNA levels were significantly decreased by the 1 and 10 microg E2/BW implants. These results indicate that E2, either directly or indirectly, downregulates expression of StAR and major steroidogenic enzyme genes in rainbow trout ovary. Furthermore, expression of the trout StAR gene seems particularly sensitive to E2.

Steroidogenic acute regulatory protein in white sturgeon (Acipenser transmontanus): cDNA cloning, sites of expression and transcript abundance in corticosteroidogenic tissue after an acute stressor.

The white sturgeon, Acipenser transmontanus, is a primitive bony fish that is recognized as an important emerging species for aquaculture. However, many aspects of its stress and reproductive physiology remain unclear. These processes are controlled by various steroid hormones. In order to investigate the regulation of steroidogenesis associated with acute stress in sturgeon, a cDNA-encoding steroidogenic acute regulatory protein (StAR) was isolated from white sturgeon. The putative amino acid sequence of sturgeon StAR shares high homology (over 60%) with other vertebrates. Phylogenetic analysis grouped sturgeon StAR within Actinopterygii, but it was clearly segregated from teleost StARs. RT-PCR analysis revealed that transcripts were most abundant in yellow corpuscles found throughout the kidney and weaker signals were detected in gonad and kidney. Very weak signals were also detected in brain and spleen by quantitative real-time PCR. In situ hybridization revealed that StAR is expressed in the cells of yellow corpuscles. No significant changes in StAR gene expression were detected in response to an acute handling stress. These results suggest that StAR is highly conserved throughout vertebrates, but the expression of the functional protein during the stress response may be partially regulated post-transcriptionally.

Hormonal regulation of thirst in the amphibious ray-finned fish suggests the requirement for terrestrialization during evolution.

Thirst has evolved for vertebrate terrestrial adaptation. We previously showed that buccal drying induced a series of drinking behaviours (migration to water-taking water into the mouth-swallowing) in the amphibious mudskipper goby, thereby discovering thirst in ray-finned fish. However, roles of dipsogenic/antidipsogenic hormones, which act on the thirst center in terrestrial tetrapods, have remained unclear in the mudskipper thirst. Here we examined the hormonal effects on the mudskipper drinking behaviours, particularly the antagonistic interaction between angiotensin II (AngII) and atrial natriuretic peptide (ANP) which is important for thirst regulation in mammalian 'forebrain'. Expectedly, intracerebroventricular injection of ANP in mudskippers reduced AngII-increased drinking rate. ANP also suppressed the neural activity at the 'hindbrain' region for the swallowing reflex, and the maintenance of buccopharyngeal water due to the swallowing inhibition may attenuate the motivation to move to water. Thus, the hormonal molecules involved in drinking regulation, as well as the influence of buccopharyngeal water, appear to be conserved in distantly related species to solve osmoregulatory problems, whereas hormonal control of thirst at the forebrain might have been acquired only in tetrapod lineage during evolution.