Cloning and sequence analyses of vasotocin and isotocin precursor cDNAs in the masu salmon, Oncorhynchus masou: evolution of neurohypophysial hormone precursors.

We have cloned and determined the nucleotide sequences of cDNAs encoding precursors of neurohypophysial hormones, vasotocin (VT) and isotocin (IT), from the hypothalamus of masu salmon, Oncorhynchus masou. The deduced amino acid sequences of masu salmon VT and IT precursors (proVT-I and proIT-I) are highly homologous to those of chum salmon proVT-I and proIT-I, respectively. The VT and IT precursors are composed of a signal peptide, hormone and neurophysin (NP), the middle portion of which is highly conserved among vertebrates. Both the NPs extend about 30 amino acids at the C-terminal. The extended C-terminals have a leucin-rich segment in the carboxyl-terminal, as copeptin of vasopressin precursor. Southern bot analysis showed the presence of two types of proVT genes (proVT-I and proVT-II) and proIT genes (proIT-I and proIT-II) in an individual masu salmon, as in a chum salmon. Southern blot analysis with proVT probes further suggested that at least two different types of proVT-I genes exist in a single masu salmon. Northern blot analysis indicated that proVT-I and proIT-I genes are expressed in the hypothalamus, whereas proVT-II and proIT-II genes are not expressed. Evolutionary distance between proVT-I and proIT-I genes was statistically estimated based on synonymous nucleotide substitution in the coding region of the cDNAs. The magnitude of distance between masu salmon proVT-I and proIT-I genes suggested that the highly conserved central portion of NPs resulted from a gene conversion event. Between masu salmon and chum salmon, evolutionary distance for proVT-I genes is about 6-fold larger than that for proIT-I genes.     

Vasotocin/isotocin-immunoreactive neurons in the medaka fish brain are sexually dimorphic and their numbers decrease after spawning in the female

In teleosts, the distribution of neurons in the preoptic-hypothalamic region and their associated neurohypophysial hormones, such as vasotocin (VT), appears to be different among species. This differential distribution is thought to reflect the social and/or sexual status of individuals within a species. In the present study, we analyzed the number, size and the distribution of vasotocin/isotocin (VT/IT) neurons in the brains of both male and female medaka (Oryzias latipes) using immunohistochemistry. VT/IT neurons were similarly located in an inverted L-shape in the nucleus preopticus in both gender, as has been already reported in salmonids. However, computer-assisted image analysis revealed sexual dimorphism in the number of VT/IT-immunoreactive (ir) neurons, with greater numbers found in males as compared to females. Further, in the female brain, the number of VT/IT-ir neurons decreased significantly after spawning. In pre-spawning compared to post-spawning females, the small-sized VT/IT-ir neurons dominated. Sexual differentiation of the medaka is fully dependent upon the steroid status during the early developmental stages and steroids are also known to trigger gender-specific behavior in the adult medaka. Our findings strongly suggest that VT and/or IT neurons may be functionally related to ovulation and/or the reproductive axes through connections to their steroidal status.     

Neuroanatomical Evidence That Kisspeptin Directly Regulates Isotocin and Vasotocin Neurons

Neuropeptide kisspeptin has been suggested to be an essential central regulator of reproduction in response to changes in serum gonadal steroid concentrations. However, in spite of wide kisspeptin receptor distribution in the brain, especially in the preoptic area and hypothalamus, the research focus has mostly been confined to the kisspeptin regulation on GnRH neurons. Here, by using medaka whose kisspeptin (kiss1) neurons have been clearly demonstrated to be regulated by sex steroids, we analyzed the anatomical distribution of kisspeptin receptors Gpr54-1 and Gpr54-2. Because the both receptors were shown to be activated by kisspeptins (Kiss1 and Kiss2), we analyzed the anatomical distribution of the both receptors by in situ hybridization. They were mainly expressed in the ventral telencephalon, preoptic area, and hypothalamus, which have been suggested to be involved in homeostatic functions including reproduction. First, we found gpr54-2 mRNA expression in nucleus preopticus pars magnocellularis and demonstrated that vasotocin and isotocin (Vasopressin and Oxytocin ortholog, respectively) neurons express gpr54-2 by dual in situ hybridization. Given that kisspeptin administration increases serum oxytocin and vasopressin concentration in mammals, the present finding are likely to be vertebrate-wide phenomenon, although direct regulation has not yet been demonstrated in mammals. We then analyzed co-expression of kisspeptin receptors in three types of GnRH neurons. It was clearly demonstrated that gpr54-expressing cells were located adjacent to GnRH1 neurons, although they were not GnRH1 neurons themselves. In contrast, there was no gpr54-expressing cell in the vicinities of neuromodulatory GnRH2 or GnRH3 neurons. From these results, we suggest that medaka kisspeptin neurons directly regulate some behavioral and neuroendocrine functions via vasotocin/isotocin neurons, whereas they do not regulate hypophysiotropic GnRH1 neurons at least in a direct manner. Thus, direct kisspeptin regulation of GnRH1 neurons proposed in mammals may not be the universal feature of vertebrate kisspeptin system in general.     

Localization of corticotropin-releasing factor immunoreactivity in the brain of the teleost Sparus aurata

The distribution of perikarya and fibers containing corticotropin-releasing factor (CRF) was studied in the brain of the teleost Sparus aurata by immunocytochemistry using the peroxidase-antiperoxidase method. Antisera against rat CRF, arginine vasotocin, and human adrenocorticotropin (ACTH) were used. Most CRF-immunoreactive neurons were located in the nucleus lateralis tuberis, but they were absent from the nucleus preopticus, which only contained arginine vasotocin neurons. Few CRF perikarya were identified in the nucleus preopticus periventricularis and in the mesencephalic tegmentum. A conspicuous bundle of immunoreactive fibers ran along the diencephalic floor and pituitary stalk to end near the cells of the hypophysial pars intermedia. No CRF was seen near the adenohypophysial rostral pars distalis. Our results suggest that, in Sparus aurata, CRF is a releasing factor for melanotropic cells. Its role as a releasing factor for ACTH is discussed.     

Fatty‐Acid Profile of Total and Polar Lipids in Cultured Rainbow Trout (Oncorhynchus mykiss) Raised in Freshwater and Seawater (Croatia) Determined by Transmethylation Method

Fatty acids from total lipids and polar lipids in cultured rainbow trout (Oncorhynchus mykiss) raised in seawater (SW) and freshwater (FW) were identified and quantified from the muscle samples in January, April, and July. The highest total lipid and polar lipid amounts were found in April. July contents of total lipids were low, but percent of the polyunsaturated fatty acids (PUFAs) was high in SW and FW environment (particularly n‐3 PUFAs). Variety of 17 fatty acids was identified by GC‐FID after transmethylation. The predominant fatty acids in rainbow trout from SW and FW were: docosahexaenoic acid among n‐3 PUFAs, palmitic acid among saturated fatty acids (SFAs), and oleic acid among monounsaturated fatty acids (MUFAs). Appreciably higher n‐3/n‐6 ratio was found in total lipids in April (6.40, FW fish) and in polar lipids in July (18.76; SW fish). High n‐3/n‐6 ratio in total lipids and polar lipids of rainbow trout from SW and FW, besides beneficial n‐3/n‐6 ratio in the commercial fish food, could be characteristic for the local environmental conditions (Croatia).     

Elevation of gene expression for salmon gonadotropin‐releasing hormone in discrete brain loci of prespawning chum salmon during upstream migration

Our previous studies suggested that salmon gonadotropin‐releasing hormone (sGnRH) neurons regulate both final maturation and migratory behavior in homing salmonids. Activation of sGnRH neurons can occur during upstream migration. We therefore examined expression of genes encoding the precursors of sGnRH, sGnRH‐I, and sGnRH‐II, in discrete forebrain loci of prespawning chum salmon, Oncorhynchus keta. Fish were captured from 1997 through 1999 along their homing pathway: coastal areas, a midway of the river, 4 km downstream of the natal hatchery, and the hatchery. Amounts of sGnRH mRNAs in fresh frozen sections including the olfactory bulb (OB), terminal nerve (TN), ventral telencephalon (VT), nucleus preopticus parvocellularis anterioris (PPa), and nucleus preopticus magnocellularis (PM) were determined by quantitative real‐time polymerase chain reactions. The amounts of sGnRH‐II mRNA were higher than those of sGnRH‐I mRNA, while they showed similar changes during upstream migration. In the OB and TN, the amounts of sGnRH mRNAs elevated from the coast to the natal hatchery. In the VT and PPa, they elevated along with the progress of final maturation. Such elevation was also observed in the rostroventral, middle, and dorsocaudal parts of the PM. The amounts of gonadotropin IIβ and somatolactin mRNAs in the pituitary also increased consistently with the elevation of gene expression for sGnRH. These results, in combination with lines of previous evidence, indicate that sGnRH neurons are activated in almost all the forebrain loci during the last phases of spawning migration, resulting in coordination of final gonadal maturation and migratory behavior to the spawning ground. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Teleost isotocin receptor: structure, functional expression, mRNA distribution and phylogeny

A cDNA encoding a receptor for the oxytocin-related peptide isotocin has been identified by screening a lambda gt11 library constructed from poly(A)⁺ RNA of the hypothalamic region of the teleost Catostomus commersoni. The probe used was obtained by PCR amplification of white sucker genomic DNA using degenerate primers based on conserved sequences in the mammalian receptor counterparts. The full-length cDNA specifies a polypeptide of 390 amino acid residues that displays the typical hydrophobicity profile of a seven transmembrane domain receptor and which exhibits greatest similarity to mammalian oxytocin receptors. Oocytes that express the cloned receptor respond to the application of isotocin by an induction of membrane chloride currents indicating that it is coupled to the inositol phosphate/calcium pathway. The isotocin receptor (ITR) can also be activated by vasotocin, mesotocin, oxytocin and Arg-vasopressin, although these have lower potencies than isotocin. ITR-encoding mRNA has been detected in brain, intestine, bladder, skeletal muscle, lateral line, gills and kidney indicating that this receptor may mediate a variety of physiological functions.     

Freshwater to seawater acclimation of juvenile bull sharks (<Emphasis Type="Italic">Carcharhinus leucas</Emphasis>): plasma osmolytes and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in gill,rectal gland,kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater (SW). Juvenile C. leucas captured in FW (3 mOsm l^–1 kg^–1) were acclimated to SW (980–1,000 mOsm l^–1 kg^–1) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l^–1 kg^–1. In SW, bull sharks had significantly higher plasma osmolarities (940 mOsm l^–1 kg^–1) than FW-acclimated animals and were slightly hypo-osmotic to the environment. Plasma Na⁺, Cl^–, K⁺, Mg²⁺, Ca²⁺, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity in the gills and intestine was less than 1 mmol Pi mg^–1 protein h^–1 and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na⁺/K⁺-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na⁺/K⁺-ATPase activity was 5.6±0.8 and 9.2±0.6 mmol Pi mg^–1 protein h^–1, respectively. Na⁺/K⁺-ATPase activity in the kidney of FW and SW acclimated animals was 8.4±1.1 and 3.3±1.1 Pi mg^–1 protein h^–1, respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.     

Are Membrane Lipids Involved in Osmoregulation? Studies in vivo on the European eel, Anguilla anguilla, After Reduced Ambient Salinity

Eel gill lipids were labelled in vivo with (³²P) phosphate and (¹⁴C) acetate as precursors added to the water in the incubation tank. We compared the transfer of fish from brackish water (BW) to fresh water (FW) and also the transfer from sea water (SW) to FW, with the corresponding transfer from FW to demineralised FW (soft fresh water, SFW). Results show a common (³²P) phosphatidylethanolamine (PE) dominated phospholipid incorporation pattern at steady state, whatever environmental salinity the eels are adapted to, be it SW, BW, FW or finally after about a week in SFW. A deviation from any established steady state, by lowering the environmental salinity, leads to a temporary loss of the (³²P) PE dominated pattern and this applies equally, whether fish are transferred from a hyper/iso- to a hypo-osmotic medium, or remain in a hypo-osmotic medium. After about 1 week in the transfer media, the original (³²P) PE dominated phospholipid pattern is restored. The concomitant incorporation of (¹⁴C) acetate into eel gill phospholipids is not affected by the induced environmental changes. It shows a (¹⁴C) phosphatidylcholine dominated incorporation pattern throughout.     

Na+ uptake through the brush border membranes of intestine from fresh water and sea-water adapted trout (Salmo gairdneri,R.)

Summary A comparative study of the mechanisms of Na⁺ absorption through brush border membranes of enterocytes from freshwater (FW) and seawater (SW) adapted trout were carried out using purified vesicle preparations. In contrast to FW trout, SW trout were found to possess a Na⁺–K⁺–Cl^– cotransport process. This finding is regarded as a major adaptation to SW since this cotransport allows an increase of ions and water absorption. Both FW and SW trout were equipped with a Na⁺–H⁺ exchange. In FW, the intestine of the trout had both a Na⁺–Na⁺ exchange and a Na⁺ conductance which may be responsible for enterocyte Na⁺ uptake along the potential gradient.     

Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis)

The time course of osmoregulatory adjustments and expressional changes of three key ion transporters in the gill were investigated in the striped bass during salinity acclimations. In three experiments, fish were transferred from fresh water (FW) to seawater (SW), from SW to FW, and from 15-ppt brackish water (BW) to either FW or SW, respectively. Each transfer induced minor deflections in serum [Na+] and muscle water content, both being corrected rapidly (24 hr). Transfer from FW to SW increased gill Na+,K+-ATPase activity and Na+,K+,2Cl- co-transporter expression after 3 days. Abundance of Na+,K+-ATPase alpha-subunit mRNA and protein was unchanged. Changes in Na+,K+,2Cl- co-transporter protein were preceded by increased mRNA expression after 24 hr. Expression of V-type H+-ATPase mRNA decreased after 3 days. Transfer from SW to FW induced no change in expression of gill Na+,K+-ATPase. However, Na+,K+,2Cl- co-transporter mRNA and protein levels decreased after 24 hr and 7 days, respectively. Expression of H+-ATPase mRNA increased in response to FW after 7 days. In BW fish transferred to FW and SW, gill Na+,K+-ATPase activity was stimulated by both challenges, suggesting both a hyper- and a hypo-osmoregulatory response of the enzyme. Acclimation of striped bass to SW occurs on a rapid time scale. This seems partly to rely on the relative high abundance of gill Na+,K+-ATPase and Na+,K+,2Cl- co-transporter in FW fish. In a separate study, we found a smaller response to SW in expression of these ion transport proteins in striped bass when compared with the less euryhaline brown trout. In both FW and SW, NEM-sensitive gill H+-ATPase activity was negligible in striped bass and approximately 10-fold higher in brown trout. This suggests that in striped bass Na+-uptake in FW may rely more on a relatively high abundance/activity of Na+,K+-ATPase compared to trout, where H+-ATPase is critical for establishing a thermodynamically favorable gradient for Na+-uptake.     

The hypothalamo-hypophyseal system of the white seabream Diplodus sargus: immunocytochemical identification of arginine-vasotocin,isotocin, melanin-concentrating hormone and corticotropin-releasing factor

The distribution of the neurosecretory hormones vasotocin, isotocin and melanin-concentrating hormone and the hypophysiotropic hormone corticotropin-releasing factor was studied in the hypothalamo-hypophyseal system of the white seabream (Diplodus sargus) using immunocytochemical techniques. Magnocellular and parvocellular perikarya immunoreactive for arginine-vasotocin and isotocin were present in the nucleus preopticus. Perikarya immunoreactive for arginine-vasotocin extended more caudally with respect to isotocin-immunoreactive perikarya. Parvocellular perikarya were located at rostroventral levels and magnocellular perikarya in the dorsocaudal portion of the nucleus. Arginine-vasotocin and isotocin did not coexist in the same neuron. Fibres immunoreactive for arginine-vasotocin and isotocin innervated all areas of neurohypophysis and terminate close to corticotropic and melanotropic cells. Perikarya immunoreactive for melanin-concentrating hormone and corticotropin-releasing factor were observed in the nucleus lateralis tuberis, with a few neurons in the nucleus periventricularis posterior. In addition, melanin-concentrating hormone immunoreactive perikarya were detected in the nucleus recessus lateralis. The preoptic nucleus did not show immunoreactivity for these antisera. Fibres showing melanin-concentrating hormone and corticotropin-releasing factor immunoreactivity ended close to the melanotropic and somatolactotrophic cells of the pars intermedia, and close to the corticotrophic cells of the rostral pars distalis.     

Vasotocin acts as a dipsogen in ducks at concentrations stimulating subfornical organ neurons in vitro

The effects of systemic infusions of the avian antidiuretic hormone arginine vasotocin on water intake of domestic ducks were investigated under steady conditions of water balance in which angiotensin II was effective as a dipsogen. The study proceeded from the consistent stimulatory effect of arginine vasotocin on angiotensin II-responsive neurons found in the subfornical organ of ducks, suggesting brain-intrinsic vasotocinergic control of these neurons which are also accessible to circulating agents because of the lacking blood-brain barrier. Levels of circulating arginine vasotocin of about 2700 pg·ml^-1 which were close to the threshold for activation of subfornical organ neurons in vitro, induced weak but significant drinking responses. Even at this high arginine vasotocin level circulatory effects were absent, thereby excluding their interference with water intake. Arginine vasotocin plasma levels of about 60 pg·ml^-1 significantly attenuated the dipsogenic action of angiotensin. While drinking in response to high pharmacological levels of arginine vasotocin is assumed to mimic a stimulatory innervation of angiotensin-responsive subfornical organ neurons by brain-intrinsic vasotocinergic axons, attenuation of angiotensin-induced drinking by high physiological arginine vasotocin levels cannot be explained by its action on central neurons, but may be secondary to body fluid retention caused by the antidiuretic action of arginine vasotocin.Abbreviations ADH antidiuretic hormone - ANGII angiotensin II - AVP arginine vasopressin - AVT arginine vasotocin - BBB blood-brain barrier - HR heart rate - ICV intracerebroventricular - IV intravenous - MAP mean arterial pressure - SFO subfornical organ     

Arterial hypotension in ducks adapted to high salt intake

Summary A homogeneous group of 8-week-old Pekin ducks was divided into two groups: saltwater (SW) ducks received salt water of gradually increasing salinity (200–600 mOsm·kg^-1) from the 8th to 20th week of age; freshwater (FW) ducks were maintained on fresh water but otherwise treated identically. During the course of salt-adaptation SW ducks increased plasma osmolality, Na⁺ and Cl^- levels, and concentrations of the osmoregulatory peptide hormones arginine vasotocin and angiotensin II. The apparent volume of inulin distribution decreased in SW ducks, but blood volume was not reduced. SW ducks also developed arterial hypotension, bradycardia, and reduced cardiac output in the course of salt adaptation. This depressed cardiovascular performance was associated with enhanced vagal restraint of cardiac function and reduced plasma concentrations of norepinephrine. Salt water adaptation did not alter the degrees to which mean arterial pressure and heart rate changed in response to intravenous bolus injections of catecholamines. The same applied to the osmoregulatory peptides which were, however, effective only at supraphysiological concentrations. The Pekin duck, as a bird predisposed for adaptation to high salt loads, presumably adapts to chronic hypertonic saline intake by resetting the central autonomic control of blood pressure to a lower level.Abbreviations FW ducks fresh water ducks - SW ducks salt water ducks - ANGI angiotensin II - AVT arginine vasotocin - MAP mean arterial pressure - HR heart rate - IV intravenous - CO cardiac output - SV stroke volume - TPR total peripheral resistance - ISp virtual inulin space - ECFV extracellular fluid volume     

Elevation of gene expression for salmon gonadotropin-releasing hormone in discrete brain loci of prespawning chum salmon during upstream migration

Our previous studies suggested that salmon gonadotropin-releasing hormone (sGnRH) neurons regulate both final maturation and migratory behavior in homing salmonids. Activation of sGnRH neurons can occur during upstream migration. We therefore examined expression of genes encoding the precursors of sGnRH, sGnRH-I, and sGnRH-II, in discrete forebrain loci of prespawning chum salmon, Oncorhynchus keta. Fish were captured from 1997 through 1999 along their homing pathway: coastal areas, a midway of the river, 4 km downstream of the natal hatchery, and the hatchery. Amounts of sGnRH mRNAs in fresh frozen sections including the olfactory bulb (OB), terminal nerve (TN), ventral telencephalon (VT), nucleus preopticus parvocellularis anterioris (PPa), and nucleus preopticus magnocellularis (PM) were determined by quantitative real-time polymerase chain reactions. The amounts of sGnRH-II mRNA were higher than those of sGnRH-I mRNA, while they showed similar changes during upstream migration. In the OB and TN, the amounts of sGnRH mRNAs elevated from the coast to the natal hatchery. In the VT and PPa, they elevated along with the progress of final maturation. Such elevation was also observed in the rostroventral, middle, and dorsocaudal parts of the PM. The amounts of gonadotropin IIbeta and somatolactin mRNAs in the pituitary also increased consistently with the elevation of gene expression for sGnRH. These results, in combination with lines of previous evidence, indicate that sGnRH neurons are activated in almost all the forebrain loci during the last phases of spawning migration, resulting in coordination of final gonadal maturation and migratory behavior to the spawning ground.     

Distinct Na+/K+/2Cl- cotransporter localization in kidneys and gills of two euryhaline species, rainbow trout and killifish

The kidney is an organ playing an important role in ion regulation in both freshwater (FW) and seawater (SW) fish. The mechanisms of ion regulation in the fish kidney are less well studied than that of their gills, especially at the level of transporter proteins. We have found striking differences in the pattern of Na⁺/K⁺/2Cl^- cotransporter (NKCC) expression between species. In the killifish kidney, NKCC is apically localized in the distal and collecting tubules and basolaterally localized in the proximal tubules. However, in the SW killifish gill, NKCC is basolaterally co-localized with Na⁺/K⁺-ATPase, whereas in FW, NKCC immunoreactivity is primarily apical, although still colocalized within the same mitochondria-rich cell with basolateral Na⁺/K⁺-ATPase. Rainbow trout kidney has NKCC only in the apical membrane of the distal and collecting tubules in both environments, with no signal being detected in the proximal tubule. On the other hand, in the trout gill, NKCC is found basolaterally in both FW and SW environments. An important observation is that, in the gills of rainbow trout, the trailing edge of the filament possesses mostly Na⁺/K⁺-ATPase-positive but NKCC-negative mitochondria-rich cells, whereas in the region between and at the roots of the gill lamellae, most mitochondria-rich cells exhibit both Na⁺/K⁺-ATPase- and NKCC-positive immunoreactivity. These results suggest that the differential localization of transporters between the two species represents differences in function between these two euryhaline fishes with different life histories and strategies. Funding for this research was provided by NSERC Discovery Grants to G.G.G. and W.S.M., an Alberta Ingenuity Fund PDF, and a fellowship from the NSERC Research Capacity Development Grant to F.K.     

Differential expression of branchial Na+/K(+)-ATPase of two medaka species, Oryzias latipes and Oryzias dancena, with different salinity tolerances acclimated to fresh water, brackish water and seawater

Previous studies on non-diadromous euryhaline teleosts introduced a hypothesis that the lowest level of gill Na⁺/K⁺-ATPase (NKA) activity occurs in the environments with salinity close to the primary natural habitats of the studied species. To provide more evidence of the hypothesis, two medaka species, Oryzias latipes and O. dancena, whose primary natural habitats are fresh water (FW) and brackish water (BW) environments, respectively, were compared from levels of mRNA to cells in this study. The plasma osmolalities of O. latipes and O. dancena were lowest in the FW individuals. The muscle water contents of O. latipes decreased with elevated external salinities, but were constant among FW-, BW-, and seawater (SW)-acclimated O. dancena. Expression of NKA, the primary driving force of ion transporters in gill ionocytes, revealed different patterns in the two Oryzias species. The highest NKA α-subunit mRNA abundances were found in the gills of the SW O. latipes and the FW O. dancena, respectively. The pattern of NKA activity and α-subunit protein abundance in the gills of O. latipes revealed that the FW group was the lowest, while the pattern in O. dancena revealed that the BW group was the lowest. Immunohistochemical staining showed similar profiles of NKA immunoreactive (NKIR) cell activities (NKIR cell number × cell size) in the gills of these two species among FW, BW, and SW groups. Taken together, O. latipes exhibited better hyposmoregulatory ability, while O. dancena exhibited better hyperosmoregulatory ability. Our results corresponding to the hypothesis indicated that the lowest branchial NKA activities of these two medaka species were found in the environments with salinities similar to their natural habitats.     

Colloid droplets in the magnocellular secretory neurons of the reptilian hypothalamus: An immunocytochemical and lectin-histochemical study

Summary The immunocytochemical and lectin-binding properties of the magnocellular neurosecretory neurons in the hypothalamus of 2 reptilian species, the snake Natrix maura and the lizard Liolaemus cyanogaster, were investigated. Particular attention was paid to the secretory droplets present in these neurons. Antisera against bovine neurophysins I+II, arginine-vasotocin, and mesotocin were used. The following lectins were applied: concanavalin A (Con A), wheat-germ agglutinin (WGA), and Limax flavus agglutinin (LFA). Adjacent 1-m-thick methacrylate sections were used to investigate the same secretory neuron and the same colloid droplets with all three antisera and all three lectins. Several sections were treated with trypsin and urea before immunostaining or lectin binding. Con A bound to both vasotocin- and mesotocin-immunoreactive neurons, WGA exclusively to vasotocin neurons; neither of these neurons reacted with LFA. The colloid droplets were present in vasotocin neurons but absent in the mesotocin neurons. These secretory droplets showed an affinity for Con A but not for WGA, and reacted with antisera against neurophysins and vasotocin. In Natrix maura, the colloid droplets became reactive with Con A and the antisera used only after pretreatment of the sections with trypsin and urea. Within the hypothalamo-neurohypophyseal system, antiserum against vasotocin and WGA revealed the same fiber bundles. It is concluded (i) that in reptiles the vasotocin-neurophysin precursor is glycosylated, (ii) that vasotocin neurons have the exclusive capacity to form colloid droplets, and (iii) that these droplets are an intracisternal (RER) storage form of the vasotocin-neurophysin precursor.This work was partially supported by Grants BOJA 27/9/88 from the Dirección General de Universidades e Investigación de Junta de Andalucía and DGICYT PB87-0710 from the Comisión Interministerial de Ciencia y Tecnología, Madrid, to P.F.-LL.; and Grant 89-01 from the Dirección de Investigaciones, Universidad Austral de Chile, to E.M.R.