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Regulatory peptides and physiological adaptations to the cold environment in Antarctic teleosts

Regulatory peptides and physiological adaptations to the cold environment in Antarctic teleosts Ital. J. Zool., SUPPLEMENT 1: 57-65 (2000) spermatogonia and Sertoli cells of Chionodraco hamatus and the Regulatory peptides and physiological lack of positivity in Trematomus bernacchii. adaptations to the cold environment in Antarctic teleosts KEY WORDS: Antarctic teleosts - Osmoregulation - Regulatory peptides - Brain - Intestine - Reproduction. BIANCA MARIA UVA ACKNOWLEDGEMENTS MAURO VALLARINO This study was supported by a grant from the Italian PNRA. GRAZIA TAGLIAFIERRO MARIO PESTARINO CARLA FALUGI INTRODUCTION ALBERTA MANDICH MARIA ANGELA MASINI Regulatory peptides are signal molecules involved in MADDALENA STURLA regulation of a variety of physiological activity. They PAOLA PRATO can regulate the activities of neurons involved in neuro- SIMONA CANDIANI transmission, or be involved in hormone secretion, Dipartimento di Biologia Sperimentale Ambientale ed Applicata, blood flow, fluid homeostasis, growth, and reproduc- Università di Genova, tion. The presence of regulatory peptides, their recep- viale Benedetto XV 5, I-16132 Genova (Italy) tors, and the enzymatic cascades that lead to the pro- duction of such peptides are of major importance in SILVANA FILOSA Antarctic fishes, whose survival, in an environment CHIARA CAMPANELLA characterised by low temperature, extreme changes in CHIARA MOTTA light intensity and availability of food, is founded on SILVANA FUSCO their capacity for adaptation. Dipartimento di Biologia Evolutiva e Comparata, Università di Napoli, via Mezzocannone 8, I-80100 Napoli (Italy) In this study, the presence of a number of regulatory peptides was investigated to discuss their involvement in cardiovascular and ion-water homeostasis, their char- ABSTRACT acterisation in the central and peripheral nervous sys- tems, and in the alimentary tracts. The male and female Physiological adaptation of Antarctic teleosts to cold environ- gonadal cycles, and the regulatory peptides involved ment was studied with regard to the presence of regulatory pep - tides involved in (i) cardiac and ion-water homeostasis, (ii) brain, wer e studied to show similarity and differences be- (iii) intestine, and (iv) gonads. Immunoreactivity for different re- tween the Antarctic species investigated. Comparisons gions of the Atrial Natriuretic Peptide was detected in the heart of were carried out with temperate marine teleosts. Trematomus bernacchii, Chionodraco hamatus, Cryodraco an- tarcticus, and Champsocephalus gunnari, while immunoreactivity for other regulatory peptides (i.e., endothelin-1, somatostatin-14, and galanin) appeared to be widely distributed in the cardio-vas- MATERIALS AND METHODS cular system. Osmoregulatory peptides (somatostatin-14, pro- lactin, atrial natriuretic peptide, galanin, and urotensin II) were lo- Animals calised by immunohistochemistry in the urinary bladder and gills. From studies on the distribution of the pituitary specific transcrip- Specimens of Antarctic teleosts, listed in Table I, were caught at tion factor 1 and adenohypophysial hormones (growth hormone, Terra Nova bay in the Ross Sea during the Italian Antarctic expe- prolactin, and gonadotropin releasing hormone) in the brain and ditions of 1997 and 1998, initially processed in the Italian Antarc- pituitary of Trematomus bernacchii and Pagothenia coriiceps, it tic station at Terranova Bay and subsequently analysed in the lab- was evident that the pituitary specific transcription factor 1 acts as oratories of Genova and Napoli. The fishes were housed in a re- a developmental regulator of the anterior pituitary, responsible for circulated sea water aquarium at 0° C, and within a few days growth hormone and prolactin cell commitment, differentiation, were killed with a blow on the head and spinal cord transection. and gene expression. This coexistence appeared to be a unique Tissues were immediately removed at 4° C, and processed for his- trait of Antarctic teleosts. Analyses of the distribution and localisa- tological, immunohistochemical and electron microscopy tech- tion of gastrointestinal hormones and neuropeptides (insulin, niques, or frozen at -80° C. Animal manipulation was performed glucagon, pancreatic polypeptide, somatostatin, vasoactive intesti- according to the recommendations of the Ethical Committee and nal polypeptide, pituitary adenylate cyclase activating peptide, under the supervision of authorised investigators. and peptide histidine isoleucine) in adults of Chionodraco hama- tus, Pagetopsis mascropterus, and Notothenia coriiceps, and larvae Histological and histochemical procedures of Pleuragramma antarcticum showed that most of the im- munoreactive nerve fibres in Notothenioids are of extrinsic type. Bouin-fixed paraffin-embedded sections, were processed for Ultrastructural analysis of Chionodraco hamatus, Champ- histological analysis using the Hematoxylin-Eosin technique; par- socephalus gunnari, and Notothenia coriiceps male gametes allel sections were treated with Alcian blue Periodic Acid-Schiff showed some peculiar aspects in the head of the spermatozoa. (PAS) for detection of mucous cells and mucus. Studies on the spermatogenesis indicated a shifted circannual cy- cle in the icefishes and in the red blooded fishes: when spermato- Scanning Electron Microscopy genensis is completed in the icefish, in the red blooded teleosts the testis is not in reproductive activity as shown by the positive The specimens were fixed in 2.5% glutaraldehyde and 2% immunoreaction for Fibroblast Growth Factor and its receptor 1 in paraformaldehyde in 0.1 M cacodilate buffer, pH 7.4, for 3 h at 4° Β. Μ. UVA, M. VALLARINO, G. TAGLIAFERRO, M. PESTARINO, C. FALUGI, A. MANDICH, M.A. MASINI, M. STURLA, P. PRATO, et alii TABLE I - List of the Antarctic teleosts examined. TABLE II - List of the antibodies used in the research. Family Species Antiserum Origin Dilution Source Nototheniidae Trematomus bernacchii Boulenger, 1902 ANP Rat 1-28 1:200 UCB Belgium Trematomus newnesi Boulenger, 1902 ANP Human 1-26 1:200 UCB Belgium Notothenia coriiceps Richardson, 1844 ET-1 Porcine 1:200 Peninsula USA Pleuragramma antarcticutn Boulenger, 1902 GAL 1:2000 Affinity UK Porcine 1-39 Channichthyidae Chionodraco hamatus Löennberg, 1905 SST-14 Synthetic 1:200 UCB Belgium Cryodraco antarcticus Dollo, 1900 PRL Ovine 1:200 UCB Belgium Pagetopsis macropterus Boulenger, 1907 Urotensin II Dogfish 1:200 Chartrel et al., 1996 Cbampsocephalus gunnari Löennberg, 1905 AMA Human 1:20 Medic Italy NaVK+ ATPase Chicken Univ. Iowa Pit-1 Rat 1:200 Santa Cruz Biotech s-GnRH Salmon 1:200-1:800 Biogenesis UK cII-GnRH Chicken 1:200-1:800 UCB Belgium C or in SPAF (2 g paraformaldehyde dissolved in saturated picric m-GnRH Mammal 1:200-1:800 • Sigma USA acid to 85 ml of 0.1 M cacodylate buffer, pH 7.4, and 35 mg of su- Sub-P Synthetic 1:800 Affinity UK crose) and identified by means of a Wild M3C stereomicroscope G H Human 1:200 Biomeda USA and a Leitz Diaplan. microscope . For the scanning electronic mi- INS Porcine 1:200 Biomeda USA croscopy, samples iwerç dehydrated in ethanol of increasing con- GLUC Human 1:200 Biomeda USA centration up to 100%, followed by critical-point drying in a CO 2 PP Human 1:200 Biomeda USA Pabish CPD apparatus. They were then mounted on stubs with VIP Human 1:200 Biomeda USA silver conducting paint, sputter-coated with gold-palladium in a PACAP 38 Human 1:200 Peninsula USA Balzer Union Evaporator, and observed with a Philips EM 515 PHI Porcine 1:200 Affinity UK electron microscope. BFGF Bovine 1:200 Sigma MO FGFR1 Human 1:200 Santa Cruz Biotech Transmission Electron Microscopy Small pieces (1 mm') of tissue were fixed in 2.5% glutaralde- hyde and 2% paraformaldehyde in 0.1 M cacodilate buffer, pH 7.4, for 3 h at 4° C. After a cacodilate buffer wash, the samples son, 1986; Uemura et al, 1991); Endothelin-1 (ET-1) hy- were fixed with 1% osmium tetroxide in distilled water, dehydrat- pertensive, shown to be secreted by a variety of tissues ed in ascending concentrations of ethanol and embedded in Epon 812. Ultrathin sections were collected on 150 mesh nickel grids such as brain, kidney, heart, intestine, adrenal, eyes (Le coated with a formvar film. The sections were stained with Monnier de Gouville et al., 1989; Yanagisawa & Masaki, Reinold's lead citrate, and finally lightly coated with carbon and 1989; Simonson & Dunn, 1990); Somatostatin-l4 (SST- examined under a transmission electron microscope. 14) whose osmotic regulatory actions have been docu- mented in amphibians and other vertebrates (Forrest et Immunob istochem istry al., 1980; Dobbins et al, 1981); Galanin (GAL) whose Sections (5 pm thick) from Bouin-fixed samples were subjected involvement in regulation of neurotransmission and in to the indirect immunofluorescence technique (Coons et al., 1955) ions-water balance has been hypothesised at least in and to the peroxidase-anti peroxidase (PAP) method (Stemberger et al., 1970). Sections were dewaxed and the endogenous peroxidase mammals (Rökaeus, 1994). was blocked with 1% H O in 0.1 M PBS, pH 7.4. Some Bouin- 2 2 Strong rANP-like immunoreactivity was detected in the fixed sections were treated with the alkaline phosphatase tech- atrial myocytes of Chionodraco hamatus, Cryodraco nique. After exposure in a moist chamber to Normal Swine Serum antarcticus, and Champsocephalus gunnari. Numerous (diluted 1:50; Dako, Santa Barbara, CA) at 20° C, the unwashed sections were incubated with the specific antibodies (Table II). The cardiocytes, in the subepicardial region and in the trabec- specificity of the immunostaining was verified by omitting one of ulae, were intensely immunostained, especially in their the steps of the immunohistochemical procedure, or by replacing perinuclear zone. The atrium of Trematomus bernacchii the primary antiserum with non-immune rabbit serum or PBS. gave weaker immunoreactivity, the cardiocytes were less numerous, but the localisation was similar in the two species. There was no immunostaining in the ventricles. RESULTS AND DISCUSSION The low or absent ANP-like immunostaining in the ventricles of Antarctic teleosts is in agreement with the Regulatory peptides and structures involved in cardiovas- observations made in mammals, in which the concen- cular and ion-water homeostasis tration of ANP in the ventricles is very low compared with that in the atria. It. may be that ANP, synthesised in Cardiovascular system the ventricle, is secreted without being concentrated in granules, while atrial cardiocytes store most of the ANP In the cardiovascular system, the presence of the fol- that is synthesised. Two molecular forms of natriuretic lowing regulatory peptides was observed: Atrial Natri- peptides have been identified in the eel ventricles and uretic Peptide (ANP) whose natriuretic and vasorelaxant named Ventricular Natriuretic Peptides (VNP 1-36 and activity is well known in teleostean fishes (Duff &Ό1- REGULATORY PEPTIDES IN ANTARCTIC TELEOSTS of both the species examined. In the cubic cells of C. VNP 1-25), the two forms differ slightly from the Atrial Natriuretic Peptide (Takei et al, 1990, 1991). The low hamatus it is localised in the whole cytoplasm. The im- immunoreactivity observed, in the ventricular chamber munoreaction in the columnar epithelium shows, in- of the teleosts investigated, may reflect the presence of stead, different localisation. Immunoreactivity for SST-14 a VNP with lower crossreactivity to the atrial natriuretic seems to be localised at the apical region of the colum- peptide antibody. ET-1 was observed in the wall of nar cells both in C. hamatus and T. bernacchii (Fig. blood vessels, SST-14 immunostaining was widely dis- 1A), while GAL immunoreactivity is present in the tributed in the cardio-vascular system, and GAL im- whole cytoplasm (Fig. IB). ANP is localised in the munoreactive cells were found in the endothelial layer columnar cells both at the apical and basal regions. PRL of the atrium. and Urotensin II show the same localisation as SST-14 in both species. Urinary bladder Gills The urinary bladder is known to be involved in ma- rine fishes in the control of water and salt homeostasis. We used transmission and scanning electron mi- Teleosts can modify the urine composition in order to croscopy and an antibody to the inner mitochondrial survive in hyperosmotic environments as a result of the membrane (ab-AMA) to study the presence and the lo- activity of the epithelial cells (Loretz & Bern, 1980; Fos- calisation of mitochondria-rich cells in the gills of C. sat & Lahlou, 1982). We investigated the occurrence of hamatus and T. bernacchii. The general morphology of immunohistochemical reactivity for the osmoregulatory the gills in the two species was slightly different (Fig. 1C, peptides SST-14, Prolactin (PRL), ANP, GAL, Urotensin D): in T. bernacchii the filament and secondary lamellae II, in the epithelial lining of the urinary bladders of the were short and thick, and mitochondria-rich cells were red blooded T. bernacchii and the icefish C. hamatus. less numerous than in C. hamatus. Immunolocalisation In mammals SST inhibits renin secretion following infu- using a specific antibody for the ot-subunit of Na+/K+-AT- sion into the renal artery (Gomez-Pan et al., 1976) and Pase was observed in numerous epithelial cells in the in- inhibits, in the toad urinary bladder, the hydro-osmotic terlamellar epithelium and on the secondary lamellae response to antidiuretic hormones. A SST immunofluo- (Fig. IE), suggesting an active sodium chloride secretion. rescence was identified in the urinary bladder epithelial In the two species, the mitochondria-rich cells, distrib- cells of the toad Bufo marinus (Bolaffi et al., 1980). In uted in the secondary and primary epithelium (Fig. IF), female mammals PRL is a well-known hormone in- were of the same morphological type, similar to the a- volved in milk production, but its function involves also chloride cells of temperate seawater teleosts. water and sodium metabolism. In teleosts, PRL regulates water and ion movements across the urinary bladder Brain and neuropeptides (Doñeen, 1976). Injections of PRL into fresh water adapted fish have been shown to reduce permeability The expression of the pituitary specific transcription to water reabsorption (Johnson et al., 1974; Hirano, factor (Pit-1) and the adenohypophyseal hormones 1975). Specific binding sites for PRL have been identi- [growth hormone (GH), prolactin (PRL), and a-melan- fied in the urinary bladder cells of the toad (Dunand et otropin (a-MSH)] was analysed by immunocytochem- al., 1985). ANP has been observed in lungfishes (Masini istry in the pituitary of T. bernacchii. Pit-1 (33 Kd), also et al, 1996) and freshwater teleosts (Kim et al, 199D as known as growth hormone factor-1 (GHF-1), is a mem- well as in heart, kidney, gills, and skin of Antarctic ber of the POU protein family. It acts as developmental teleost (Masini et al., 1998). ANP isolated from teleosts regulator of the anterior pituitary, and it is responsible is structurally similar to the mammalian ANP (Takei et for GH, PRL, and TSH cell commitment, differentiation, al., 1989). An involvement of Galanin in the regulation and gene expression in mouse (Rhodes et al, 1994). of fluid homeostasis has been postulated in mammals The Pit-1 gene has been cloned also in teleosts (Yama- (Koenig et al., 1989). Urotensin I and Urotensin II are da et al., 1993). Recently, Pit-1 protein has been lo- peptides produced by the caudal neurosecretory system calised in the proximal pars distalis, and in the pars in- (the urophysis) of the teleostean fishes. Urotensin II in- termedia of the rainbow trout pituitary (Takeo et al., fluences ion transport in teleost gill and urinary bladder 1996). It was found that Pit-1, GH, and PRL coexist in (Loretz & Bern, 1981). the pituitary of T. bernacchii, (Fig. 2A, B). Cells contain- ing PRL- and GH-like substances were, however, more There is a most striking histological difference be- widely distributed in T. bernacchii than in non Antarctic tween the urinary bladders of the two species exam- teleosts. Instead, cells containing α-MSH-like peptides ined. The epithelial layer comprises a single type of were confined to a pituitary region where PRL, GH, and cells, columnar in shape, in the whole urinary bladder Pit-1 cells were absent (Fig. 2C). of T. bernacchii, while in C. hamatus the columnar cells are restricted to the ventral region, and the dorso- Gonadotropin releasing hormones (GnRH) consist of lateral region is lined by cubic epithelial cells. a family of peptides that control many aspects of verte- brate reproduction. They regulate the reproductive sys- Immunoreactivity for SST-14, PRL, ANP, GAL, and tem by stimulating the release of gonadotropin hor- Urotensin II is present in the urinary bladder epithelium 60 Β. Μ. UVA, M. VALLARINO, G. TAGLIAFERRO, M. PESTARINO, C. FALUGI, A. MANDICH, M.A. MASINI, M. STURLA, P. PRATO, et alii Fig. 1 - Immunocytochemistry of urinary bladder of T. bernacchii and gills of C. hamatus and F bernacchii. A, Section of urinary blad- der of T. bernacchii. SST-14 immunoreactivity is localised at the apical region of the columnar cells, x 660. B, Section of urinary bladder of T. bernacchii. GAL immunoreactivity is present in the whole cytoplasm, x 600. C, D, Sections of gills respectively of C. hamatus and 7*. bernacchii, showing the difference in morphology. Η-E. (C) x 150, (D) X 180. E, Section of C. hamatus gill after incubation with the antiserum to Na+/K+ATPase. x 192. F, Section of C. hamatus gill: immunoreactivity after ab-AMA. χ 360. mones from the anterior pituitary gland. In tetrapods, pituitary gland, and two GnRH molecular forms are pre- GnRH are synthesised by hypothalamic neurons and re- sent: salmon (s-GnRH) (Sherwood et al., 1983) and leased in the median eminence-portal blood system. In- chicken II (cII-GnRH). Recently, GnRH variants were stead, in teleost fish GnRH are secreted directly into the found in catfish (cf-GnRH) (Bogerd et al, 1992), gilt- REGULATORY PEPTIDES IN ANTARCTIC TELEOSTS A Β Fig. 2 - Localisation of neuropeptides in the pituitary of T. bernacchii and TV. coriiceps. A, B, C, Transverse consecutive sections of the rostral part of the pituitary of T. bernacchii showing cells coexpressing, respectively, Pit-1 and GH in the dorsal portion, while the cells containing cx-MSH are confined ventrally. h, hypothalamus, χ 50. D, Section of brain of N. coriiceps. The outer plexiform layers of the optic tectum (ot) show an intense m-GnRH immunoreactivity. xl30. E, Section of brain of N. coriiceps. Immunofluorescence m-GnRH ependymal tanycytes (arrowheads) with long processes lining the diencephalic ventricle (dv). χ 250. head seabream (sb-GnRH) (Gothilf et al., 1995), tilapia GnRH immunoreactivity appears confined to cell bodies and sockeye salmon (Parhar et al., 1996), dogfish (df- located in the lateral hypothalamus, the ventral thala- GnRH) (Lovejoy et al., 1992) and lamprey (I-GnRH and mus, and the midbrain rostral tegmentum; immunoreac- III-GnRH) (Sherwood et al., 1986; Sower et al., 1993). tive fibres densely innervated the hypothalamic periven- The study on GnRH-like peptides in N. coriiceps was tricular region. By contrast, m-GnRH-like immunoreac- tive neurons are present exclusively in the torus semi- performed by immunocytochemistry using antisera raised against three variants of GnRH: mammalian (m- circularis of the mesencephalon and in the outer plex- iphorm layers of the optic tectum (Fig 2D). These find- GnRH), chicken (cII-GnRH), and salmon (s-GnRH). cll- Β. Μ. UVA, M. VALLARINO, G. TAGLIAFERRO, M. PESTARINO, C. FALUGI, A. MANDICH, M.A. MASINI, M. STURIA, P. PRATO, et alii ings suggest that cII-GnRH-like peptides appear to func- clonal antisera raised in rabbit, as listed in Table II. Da- tion as hypophysiotropic factors, as demonstrated in ta concerning the entero-pancreatic neuroendocrine other species of bony fish, whereas m-GnRH-like pep- system (NES) of non-Antarctic perciformes Serranus tides could be involved in modulatory pathways of cabrilla and Diplodus annularis are also reported for vestibular and visual functions of N. coriiceps. Incuba- comparison. The topographical distribution of Insulin tion with s-GnRH antiserum failed to prove the occur- (INS)-, Glucagon (GLU)-, Pancreatic Polypeptide (PP)- rence of immunoreactive elements, consequently, at and SST-14-ir cells in the Brockmann body in C. antarc- least two molecular forms related to cII-GnRH and m- ticus, P. macropterus, N. coriiceps, and P. antarcticum, GnRH seem to act as hypophysiotropic and neuromod- was similar to that found in other teleosts (Langer et al, ulatory factors in the brain of N. coriiceps. Moreover, m- 1979; Abad et al, 1987; Bjenning & Holmgren, 1988; GnRH immunoreactivity in ependymal tanycytes (Fig. Jonsson, 1991; Tagliafierro et al, 1994, 1996). GLU-ir 2E) suggests the involvement of such specialised glial cells were located at the periphery, while SST-14- and cells in neuroendocrine function by linking the cere- INS-ir cells were situated in the central portion. Differ- brospinal fluid and the median eminence, as demon- ent endocrine cell associations could be seen in smaller strated in mammals. islets: INS-ir cells could be found at the periphery, while SST- and PP-ir cells were distributed throughout the islet. In the large islet of C. antarcticus, PP-ir cells Intestine and gut peptides seemed to be fewer than what was observed in no- tothenioids and in Serranus cabrilla (Tagliafierro et al., The distribution and localisation of gastro-intestinal 1994, 1996). Pancreatic endocrine cells appeared very hormones and neuropeptides were studied using poly- Fig. 3 - Immunoreactivity in pancreatic endocrine islet of P. antarcticum, C. antarcticus and P. macropterus. A, SST immunoreactive cells in a P. antarcticum larva, x 200. B, GAL-immunoreactive nerve-fibers near GLU cells in C. antarcticus. x 400. C, Sub-P immunore- active nerve fibers near a blood vessel in the central portion of the islet in P. macropterus. χ 800. D, Sub-P immunoreactive nerve fibers in the périphérie portion of the islet in P. macropterus. χ 800. REGULATORY PEPTIDES IN ANTARCTIC TELEOSTS 63 Fig. 4 - Testis sections of C. hamatus and T. bernacchii caught in December. A, Section from C. hamatus testis. The immuoreaction with anti-FGF is present in the interstitial cells (arrow), in the Sertoli cells (arrowhead) and in the spermatogonia A (small arrow), χ 450. Β, Testis of C. hamatus. In a consecutive section, used as control, no stain is present after incubation with rabbit IgGs, used as primary antiserum, χ 300. C, Testis of C. hamatus incubated with Ab-FGFRl. The immunostaining is localised in the spermatogonia (arrowhead) and in the Sertoli cells (arrow), χ 300. D, Section of testis from T. bernacchii incubated with Ab-bFGF. No staining is present, χ 300. Β. Μ. UVA, M. VALLARINO, G. TAGLIAFERRO, M. PESTARINO, C. FALUGI, A. MANDICH, M.A. MASINI, M. STURLA, P. PRATO, et alii early during embryonic development an d one large islet tion prior to folliculogenesis, and then, during growth, with INS, GLU an d SST-ir cells could b e observed in 20- carries out a significant synthesis of both RNA and pro- cm-long P. antarcticum larvae (Fig. 3A) (Tagliafierro et teins. This determines the formation in the cytoplasm of al., unpublished results). a large Balbiani Body, comparable to that observed in GAL immunoreactive nerv e elements were detected other species, and of a 'spherical body' that, by con- trast, appears significantly different from any other both in the intestinal and pancreatic portion, showing a distribution similar to that found in non-Antarctic coun - known cell structure. At the electron microscope, the terparts but they were fewer in number (Karila et al., spherical bodies appeared organised in cords, arranged so as to form polygonal grids. The cords showed an in- 1993). In the exocrine pancreas, ir nerve fibres were found in the blood vessel wall, while in the endocrin e ner, denser portion and a more fibrillar, fuzzy edge. In situ hybridisation demonstrated that the edges were rich portion (Brockmann body ) they seemed to surroun d INS ir cells, and in particular to be close to the periph- in rRNA while immunocytochemical analysis indicated that the inner cords were rich in actin and vimentin erally located GLU ir cells (Fig. 3B). GAL is one of the regulatory peptides commonly detected in the entero- (Motta et al, 1999). pancreatic tract of different vertebrates, where it seems At ultrastructural level, the male gametes of C. hatna- to be widely distributed an d mainly localised in nervous tus, C. gunnari, and N. coriiceps, when analysed with elements. In the gut, it regulates motility and affects lu- scanning electron microscopy, showed a similar mor- minal secretion as well as plasma levels of the other gut phology, common to most perciform species. At trans- peptides and pancreatic hormones . mission electron microscopy, C. hamatus showed some In the Brockmann bod y of notothenioids only few Va- differences: two nuclear fossae, and abundant membra- neous ornamentations of the head (Falugi et al., 1999). soactive Intestinal Polypeptide (VIP), Pituitary Adenylate Cyclase Activating Peptide (PACAP), and Peptide Histi- Growth factors, Fibroblast Growth Factor (FGF) and dine Isoleucine (PHI)-ir nerve fibres were detected. Sur- its receptor Fibroblast Growth Factor Receptor 1 prisingly many sub-P-ir fibres (Fig. 3 O were seen, espe- (FGFR1) have a crucial role in the regulation of the cially in the outer portion of the large islet (Fig. 3D), spermatogenetic cycles in vertebrates, including fish. close to GLU- an d PP-ir cells. Many VIP-ir nerve fibres The immunocytochemical analysis, aimed at identifying and no Sub-P-ir fibres were immunodetected in the FGF (Fig. 4 A,B), and FGFR1 (Fig. 4C), revealed a posi- pancreatic endocrine portion of S. cabrilla, D. antlu- tive reaction both in Sertoli cells and spermatogonia in laris, and other non-Antarctic teleosts. VIP, PACAP, an d C. hamatus, indicating that this species was ready to PHI belong to the same family and are candidate mes- start a new spermatogenetic cycle. The weak reaction in sengers in the nonadrenergic, noncholinergi c nervou s T. bernacchii (Fig. 4D) suggested that the stage of cell control of pancreatic an d intestinal secretion, motility, division was over and that of meiosis and differentiation and blood flow; they appear to stimulate the release of was starting. These data may indicate that Antarctic islet hormones and the flow of pancreatic juice. Sub-P teleosts have an opportunistic spermatogenetic cycle. enhance s the blood flow but does not seem to have any influence on the release of mammalian pancreatic hormone s (Nilsson & Holmgren, 1993). No data are CONCLUDING REMARKS available about the role of this peptide in fish pancreat- ic function. Nevertheless, the finding of sub-P-ir nerve In conclusion, our data point to the important role fibres surrounding the endocrine cells of the Antarctic played in the organs and systems by a variety of regula- fish pancreatic islet suggests that this peptide might play tory peptides in the physiological adaptation of Antarctic some role in the release of pancreatic hormones. teleosts to the extreme environmental conditions. These findings confirm the role of neurotransmitter and/or neuromodulator of some neuropeptides in te- leosts and suggest that neurotransmitters, different from REFERENCES those usually found in temperate teleosts, are probably Abad M. E., Binkhorst F. M. 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C., Lovas S., Conlon J. M., 1993 - Primary cular cloning and characterization of a novel gonadotropin-re- structure and biological activity of a third gonadotropin-releasing leasing hormone from the gilthead seabream (Sparus aurata). hormone from lamprey brain. Endocrinology, 132: 1125-1131. Mol. Mar. Biol. Biotechnol., 4: 27-35. Sternberger L. Α., Hardy P. H., Cuculi J. J., Meyer H. C., 1970 - Hirano T., 1975 - Effects of prolactin on osmotic and diffusion The unlabeled antibody enzyme method in immunohistochem- permeability of the flounder Platichthys flesus. Gen. Comp. En- istry preparation and properties of soluble antigen-antibody docrinol., 27: 88-94. complex (horseradish peroxidase anti-horseradish peroxidase) Johnson D. W., Hirano T., Sage M., Foster R. C., Bern Η. Α., 1974 and its use in the identification of spirochetes. J. 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A biological overview. Springer-Verlag the smooth muscle of the gut. Cell Tissue Res., 271: 537-544. Italia, Milano, pp. 247-256. Kim S. Z., Cho K. W., Kim S. H., Ryu H., Hwang Y. H., Lee G. Y., Tagliafierro G., Faraldi G., Delù M., Morescalchi M. Α., 1995 - Gut 1991 - Identification of immunoreactive atrial natriuretic peptide regulatory peptides in some Antarctic notothenioids. Polar Bi- in bulbus arteriosus of freshwater teleostean fish. Comp. ol., 15: 429-435. Biochem. Physiol., 11B: 575-578. Tagliafierro G., Faraldi G., Delù M., Putti R., Carlini M., Della Koenig J. I., Hooi S., Gabriel S. M., Martin J. B., 1989 - Potential Rossa Α., 1994 - Islet hormones in the digestive system of bony involvement of galanin in the regulation of fluid homeostasis in fish. Abstracts 17th Conference European Comparative Endocri- the rat. Reg. Pept., 24: 81-86. nologists. Cordoba, Spain, September, 1994. Langer M., Van Noorden S., Polak J. M., Pearse A. G. 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I., Yamashita S., 1993 - Molecular cloning of localization of vasoactive hormones (atrial natriuretic peptide fish Pit-1 cDNA and its functional binding to promoter of gene and endothelin) in the heart of Protopterus annectens, an expressed in the pituitary. J. Biol. Chem., 268: 24361-24366. African lungfish. Cell Tissue Res., 284: 501-507. Yanagisawa M., Masaki T., 1989 - Endothelin, a novel endotheli- Masini Μ. Α., Sturla M., Pisano E., Koubbi P., Coradeghini R., Uva um-derived peptide. Biochem. Pharmacol., 38: 1877-1883. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Italian Journal of Zoology Taylor & Francis

Regulatory peptides and physiological adaptations to the cold environment in Antarctic teleosts

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Ital. J. Zool., SUPPLEMENT 1: 57-65 (2000) spermatogonia and Sertoli cells of Chionodraco hamatus and the Regulatory peptides and physiological lack of positivity in Trematomus bernacchii. adaptations to the cold environment in Antarctic teleosts KEY WORDS: Antarctic teleosts - Osmoregulation - Regulatory peptides - Brain - Intestine - Reproduction. BIANCA MARIA UVA ACKNOWLEDGEMENTS MAURO VALLARINO This study was supported by a grant from the Italian PNRA. GRAZIA TAGLIAFIERRO MARIO PESTARINO CARLA FALUGI INTRODUCTION ALBERTA MANDICH MARIA ANGELA MASINI Regulatory peptides are signal molecules involved in MADDALENA STURLA regulation of a variety of physiological activity. They PAOLA PRATO can regulate the activities of neurons involved in neuro- SIMONA CANDIANI transmission, or be involved in hormone secretion, Dipartimento di Biologia Sperimentale Ambientale ed Applicata, blood flow, fluid homeostasis, growth, and reproduc- Università di Genova, tion. The presence of regulatory peptides, their recep- viale Benedetto XV 5, I-16132 Genova (Italy) tors, and the enzymatic cascades that lead to the pro- duction of such peptides are of major importance in SILVANA FILOSA Antarctic fishes, whose survival, in an environment CHIARA CAMPANELLA characterised by low temperature, extreme changes in CHIARA MOTTA light intensity and availability of food, is founded on SILVANA FUSCO their capacity for adaptation. Dipartimento di Biologia Evolutiva e Comparata, Università di Napoli, via Mezzocannone 8, I-80100 Napoli (Italy) In this study, the presence of a number of regulatory peptides was investigated to discuss their involvement in cardiovascular and ion-water homeostasis, their char- ABSTRACT acterisation in the central and peripheral nervous sys- tems, and in the alimentary tracts. The male and female Physiological adaptation of Antarctic teleosts to cold environ- gonadal cycles, and the regulatory peptides involved ment was studied with regard to the presence of regulatory pep - tides involved in (i) cardiac and ion-water homeostasis, (ii) brain, wer e studied to show similarity and differences be- (iii) intestine, and (iv) gonads. Immunoreactivity for different re- tween the Antarctic species investigated. Comparisons gions of the Atrial Natriuretic Peptide was detected in the heart of were carried out with temperate marine teleosts. Trematomus bernacchii, Chionodraco hamatus, Cryodraco an- tarcticus, and Champsocephalus gunnari, while immunoreactivity for other regulatory peptides (i.e., endothelin-1, somatostatin-14, and galanin) appeared to be widely distributed in the cardio-vas- MATERIALS AND METHODS cular system. Osmoregulatory peptides (somatostatin-14, pro- lactin, atrial natriuretic peptide, galanin, and urotensin II) were lo- Animals calised by immunohistochemistry in the urinary bladder and gills. From studies on the distribution of the pituitary specific transcrip- Specimens of Antarctic teleosts, listed in Table I, were caught at tion factor 1 and adenohypophysial hormones (growth hormone, Terra Nova bay in the Ross Sea during the Italian Antarctic expe- prolactin, and gonadotropin releasing hormone) in the brain and ditions of 1997 and 1998, initially processed in the Italian Antarc- pituitary of Trematomus bernacchii and Pagothenia coriiceps, it tic station at Terranova Bay and subsequently analysed in the lab- was evident that the pituitary specific transcription factor 1 acts as oratories of Genova and Napoli. The fishes were housed in a re- a developmental regulator of the anterior pituitary, responsible for circulated sea water aquarium at 0° C, and within a few days growth hormone and prolactin cell commitment, differentiation, were killed with a blow on the head and spinal cord transection. and gene expression. This coexistence appeared to be a unique Tissues were immediately removed at 4° C, and processed for his- trait of Antarctic teleosts. Analyses of the distribution and localisa- tological, immunohistochemical and electron microscopy tech- tion of gastrointestinal hormones and neuropeptides (insulin, niques, or frozen at -80° C. Animal manipulation was performed glucagon, pancreatic polypeptide, somatostatin, vasoactive intesti- according to the recommendations of the Ethical Committee and nal polypeptide, pituitary adenylate cyclase activating peptide, under the supervision of authorised investigators. and peptide histidine isoleucine) in adults of Chionodraco hama- tus, Pagetopsis mascropterus, and Notothenia coriiceps, and larvae Histological and histochemical procedures of Pleuragramma antarcticum showed that most of the im- munoreactive nerve fibres in Notothenioids are of extrinsic type. Bouin-fixed paraffin-embedded sections, were processed for Ultrastructural analysis of Chionodraco hamatus, Champ- histological analysis using the Hematoxylin-Eosin technique; par- socephalus gunnari, and Notothenia coriiceps male gametes allel sections were treated with Alcian blue Periodic Acid-Schiff showed some peculiar aspects in the head of the spermatozoa. (PAS) for detection of mucous cells and mucus. Studies on the spermatogenesis indicated a shifted circannual cy- cle in the icefishes and in the red blooded fishes: when spermato- Scanning Electron Microscopy genensis is completed in the icefish, in the red blooded teleosts the testis is not in reproductive activity as shown by the positive The specimens were fixed in 2.5% glutaraldehyde and 2% immunoreaction for Fibroblast Growth Factor and its receptor 1 in paraformaldehyde in 0.1 M cacodilate buffer, pH 7.4, for 3 h at 4° Β. Μ. UVA, M. VALLARINO, G. TAGLIAFERRO, M. PESTARINO, C. FALUGI, A. MANDICH, M.A. MASINI, M. STURLA, P. PRATO, et alii TABLE I - List of the Antarctic teleosts examined. TABLE II - List of the antibodies used in the research. Family Species Antiserum Origin Dilution Source Nototheniidae Trematomus bernacchii Boulenger, 1902 ANP Rat 1-28 1:200 UCB Belgium Trematomus newnesi Boulenger, 1902 ANP Human 1-26 1:200 UCB Belgium Notothenia coriiceps Richardson, 1844 ET-1 Porcine 1:200 Peninsula USA Pleuragramma antarcticutn Boulenger, 1902 GAL 1:2000 Affinity UK Porcine 1-39 Channichthyidae Chionodraco hamatus Löennberg, 1905 SST-14 Synthetic 1:200 UCB Belgium Cryodraco antarcticus Dollo, 1900 PRL Ovine 1:200 UCB Belgium Pagetopsis macropterus Boulenger, 1907 Urotensin II Dogfish 1:200 Chartrel et al., 1996 Cbampsocephalus gunnari Löennberg, 1905 AMA Human 1:20 Medic Italy NaVK+ ATPase Chicken Univ. Iowa Pit-1 Rat 1:200 Santa Cruz Biotech s-GnRH Salmon 1:200-1:800 Biogenesis UK cII-GnRH Chicken 1:200-1:800 UCB Belgium C or in SPAF (2 g paraformaldehyde dissolved in saturated picric m-GnRH Mammal 1:200-1:800 • Sigma USA acid to 85 ml of 0.1 M cacodylate buffer, pH 7.4, and 35 mg of su- Sub-P Synthetic 1:800 Affinity UK crose) and identified by means of a Wild M3C stereomicroscope G H Human 1:200 Biomeda USA and a Leitz Diaplan. microscope . For the scanning electronic mi- INS Porcine 1:200 Biomeda USA croscopy, samples iwerç dehydrated in ethanol of increasing con- GLUC Human 1:200 Biomeda USA centration up to 100%, followed by critical-point drying in a CO 2 PP Human 1:200 Biomeda USA Pabish CPD apparatus. They were then mounted on stubs with VIP Human 1:200 Biomeda USA silver conducting paint, sputter-coated with gold-palladium in a PACAP 38 Human 1:200 Peninsula USA Balzer Union Evaporator, and observed with a Philips EM 515 PHI Porcine 1:200 Affinity UK electron microscope. BFGF Bovine 1:200 Sigma MO FGFR1 Human 1:200 Santa Cruz Biotech Transmission Electron Microscopy Small pieces (1 mm') of tissue were fixed in 2.5% glutaralde- hyde and 2% paraformaldehyde in 0.1 M cacodilate buffer, pH 7.4, for 3 h at 4° C. After a cacodilate buffer wash, the samples son, 1986; Uemura et al, 1991); Endothelin-1 (ET-1) hy- were fixed with 1% osmium tetroxide in distilled water, dehydrat- pertensive, shown to be secreted by a variety of tissues ed in ascending concentrations of ethanol and embedded in Epon 812. Ultrathin sections were collected on 150 mesh nickel grids such as brain, kidney, heart, intestine, adrenal, eyes (Le coated with a formvar film. The sections were stained with Monnier de Gouville et al., 1989; Yanagisawa & Masaki, Reinold's lead citrate, and finally lightly coated with carbon and 1989; Simonson & Dunn, 1990); Somatostatin-l4 (SST- examined under a transmission electron microscope. 14) whose osmotic regulatory actions have been docu- mented in amphibians and other vertebrates (Forrest et Immunob istochem istry al., 1980; Dobbins et al, 1981); Galanin (GAL) whose Sections (5 pm thick) from Bouin-fixed samples were subjected involvement in regulation of neurotransmission and in to the indirect immunofluorescence technique (Coons et al., 1955) ions-water balance has been hypothesised at least in and to the peroxidase-anti peroxidase (PAP) method (Stemberger et al., 1970). Sections were dewaxed and the endogenous peroxidase mammals (Rökaeus, 1994). was blocked with 1% H O in 0.1 M PBS, pH 7.4. Some Bouin- 2 2 Strong rANP-like immunoreactivity was detected in the fixed sections were treated with the alkaline phosphatase tech- atrial myocytes of Chionodraco hamatus, Cryodraco nique. After exposure in a moist chamber to Normal Swine Serum antarcticus, and Champsocephalus gunnari. Numerous (diluted 1:50; Dako, Santa Barbara, CA) at 20° C, the unwashed sections were incubated with the specific antibodies (Table II). The cardiocytes, in the subepicardial region and in the trabec- specificity of the immunostaining was verified by omitting one of ulae, were intensely immunostained, especially in their the steps of the immunohistochemical procedure, or by replacing perinuclear zone. The atrium of Trematomus bernacchii the primary antiserum with non-immune rabbit serum or PBS. gave weaker immunoreactivity, the cardiocytes were less numerous, but the localisation was similar in the two species. There was no immunostaining in the ventricles. RESULTS AND DISCUSSION The low or absent ANP-like immunostaining in the ventricles of Antarctic teleosts is in agreement with the Regulatory peptides and structures involved in cardiovas- observations made in mammals, in which the concen- cular and ion-water homeostasis tration of ANP in the ventricles is very low compared with that in the atria. It. may be that ANP, synthesised in Cardiovascular system the ventricle, is secreted without being concentrated in granules, while atrial cardiocytes store most of the ANP In the cardiovascular system, the presence of the fol- that is synthesised. Two molecular forms of natriuretic lowing regulatory peptides was observed: Atrial Natri- peptides have been identified in the eel ventricles and uretic Peptide (ANP) whose natriuretic and vasorelaxant named Ventricular Natriuretic Peptides (VNP 1-36 and activity is well known in teleostean fishes (Duff &Ό1- REGULATORY PEPTIDES IN ANTARCTIC TELEOSTS of both the species examined. In the cubic cells of C. VNP 1-25), the two forms differ slightly from the Atrial Natriuretic Peptide (Takei et al, 1990, 1991). The low hamatus it is localised in the whole cytoplasm. The im- immunoreactivity observed, in the ventricular chamber munoreaction in the columnar epithelium shows, in- of the teleosts investigated, may reflect the presence of stead, different localisation. Immunoreactivity for SST-14 a VNP with lower crossreactivity to the atrial natriuretic seems to be localised at the apical region of the colum- peptide antibody. ET-1 was observed in the wall of nar cells both in C. hamatus and T. bernacchii (Fig. blood vessels, SST-14 immunostaining was widely dis- 1A), while GAL immunoreactivity is present in the tributed in the cardio-vascular system, and GAL im- whole cytoplasm (Fig. IB). ANP is localised in the munoreactive cells were found in the endothelial layer columnar cells both at the apical and basal regions. PRL of the atrium. and Urotensin II show the same localisation as SST-14 in both species. Urinary bladder Gills The urinary bladder is known to be involved in ma- rine fishes in the control of water and salt homeostasis. We used transmission and scanning electron mi- Teleosts can modify the urine composition in order to croscopy and an antibody to the inner mitochondrial survive in hyperosmotic environments as a result of the membrane (ab-AMA) to study the presence and the lo- activity of the epithelial cells (Loretz & Bern, 1980; Fos- calisation of mitochondria-rich cells in the gills of C. sat & Lahlou, 1982). We investigated the occurrence of hamatus and T. bernacchii. The general morphology of immunohistochemical reactivity for the osmoregulatory the gills in the two species was slightly different (Fig. 1C, peptides SST-14, Prolactin (PRL), ANP, GAL, Urotensin D): in T. bernacchii the filament and secondary lamellae II, in the epithelial lining of the urinary bladders of the were short and thick, and mitochondria-rich cells were red blooded T. bernacchii and the icefish C. hamatus. less numerous than in C. hamatus. Immunolocalisation In mammals SST inhibits renin secretion following infu- using a specific antibody for the ot-subunit of Na+/K+-AT- sion into the renal artery (Gomez-Pan et al., 1976) and Pase was observed in numerous epithelial cells in the in- inhibits, in the toad urinary bladder, the hydro-osmotic terlamellar epithelium and on the secondary lamellae response to antidiuretic hormones. A SST immunofluo- (Fig. IE), suggesting an active sodium chloride secretion. rescence was identified in the urinary bladder epithelial In the two species, the mitochondria-rich cells, distrib- cells of the toad Bufo marinus (Bolaffi et al., 1980). In uted in the secondary and primary epithelium (Fig. IF), female mammals PRL is a well-known hormone in- were of the same morphological type, similar to the a- volved in milk production, but its function involves also chloride cells of temperate seawater teleosts. water and sodium metabolism. In teleosts, PRL regulates water and ion movements across the urinary bladder Brain and neuropeptides (Doñeen, 1976). Injections of PRL into fresh water adapted fish have been shown to reduce permeability The expression of the pituitary specific transcription to water reabsorption (Johnson et al., 1974; Hirano, factor (Pit-1) and the adenohypophyseal hormones 1975). Specific binding sites for PRL have been identi- [growth hormone (GH), prolactin (PRL), and a-melan- fied in the urinary bladder cells of the toad (Dunand et otropin (a-MSH)] was analysed by immunocytochem- al., 1985). ANP has been observed in lungfishes (Masini istry in the pituitary of T. bernacchii. Pit-1 (33 Kd), also et al, 1996) and freshwater teleosts (Kim et al, 199D as known as growth hormone factor-1 (GHF-1), is a mem- well as in heart, kidney, gills, and skin of Antarctic ber of the POU protein family. It acts as developmental teleost (Masini et al., 1998). ANP isolated from teleosts regulator of the anterior pituitary, and it is responsible is structurally similar to the mammalian ANP (Takei et for GH, PRL, and TSH cell commitment, differentiation, al., 1989). An involvement of Galanin in the regulation and gene expression in mouse (Rhodes et al, 1994). of fluid homeostasis has been postulated in mammals The Pit-1 gene has been cloned also in teleosts (Yama- (Koenig et al., 1989). Urotensin I and Urotensin II are da et al., 1993). Recently, Pit-1 protein has been lo- peptides produced by the caudal neurosecretory system calised in the proximal pars distalis, and in the pars in- (the urophysis) of the teleostean fishes. Urotensin II in- termedia of the rainbow trout pituitary (Takeo et al., fluences ion transport in teleost gill and urinary bladder 1996). It was found that Pit-1, GH, and PRL coexist in (Loretz & Bern, 1981). the pituitary of T. bernacchii, (Fig. 2A, B). Cells contain- ing PRL- and GH-like substances were, however, more There is a most striking histological difference be- widely distributed in T. bernacchii than in non Antarctic tween the urinary bladders of the two species exam- teleosts. Instead, cells containing α-MSH-like peptides ined. The epithelial layer comprises a single type of were confined to a pituitary region where PRL, GH, and cells, columnar in shape, in the whole urinary bladder Pit-1 cells were absent (Fig. 2C). of T. bernacchii, while in C. hamatus the columnar cells are restricted to the ventral region, and the dorso- Gonadotropin releasing hormones (GnRH) consist of lateral region is lined by cubic epithelial cells. a family of peptides that control many aspects of verte- brate reproduction. They regulate the reproductive sys- Immunoreactivity for SST-14, PRL, ANP, GAL, and tem by stimulating the release of gonadotropin hor- Urotensin II is present in the urinary bladder epithelium 60 Β. Μ. UVA, M. VALLARINO, G. TAGLIAFERRO, M. PESTARINO, C. FALUGI, A. MANDICH, M.A. MASINI, M. STURLA, P. PRATO, et alii Fig. 1 - Immunocytochemistry of urinary bladder of T. bernacchii and gills of C. hamatus and F bernacchii. A, Section of urinary blad- der of T. bernacchii. SST-14 immunoreactivity is localised at the apical region of the columnar cells, x 660. B, Section of urinary bladder of T. bernacchii. GAL immunoreactivity is present in the whole cytoplasm, x 600. C, D, Sections of gills respectively of C. hamatus and 7*. bernacchii, showing the difference in morphology. Η-E. (C) x 150, (D) X 180. E, Section of C. hamatus gill after incubation with the antiserum to Na+/K+ATPase. x 192. F, Section of C. hamatus gill: immunoreactivity after ab-AMA. χ 360. mones from the anterior pituitary gland. In tetrapods, pituitary gland, and two GnRH molecular forms are pre- GnRH are synthesised by hypothalamic neurons and re- sent: salmon (s-GnRH) (Sherwood et al., 1983) and leased in the median eminence-portal blood system. In- chicken II (cII-GnRH). Recently, GnRH variants were stead, in teleost fish GnRH are secreted directly into the found in catfish (cf-GnRH) (Bogerd et al, 1992), gilt- REGULATORY PEPTIDES IN ANTARCTIC TELEOSTS A Β Fig. 2 - Localisation of neuropeptides in the pituitary of T. bernacchii and TV. coriiceps. A, B, C, Transverse consecutive sections of the rostral part of the pituitary of T. bernacchii showing cells coexpressing, respectively, Pit-1 and GH in the dorsal portion, while the cells containing cx-MSH are confined ventrally. h, hypothalamus, χ 50. D, Section of brain of N. coriiceps. The outer plexiform layers of the optic tectum (ot) show an intense m-GnRH immunoreactivity. xl30. E, Section of brain of N. coriiceps. Immunofluorescence m-GnRH ependymal tanycytes (arrowheads) with long processes lining the diencephalic ventricle (dv). χ 250. head seabream (sb-GnRH) (Gothilf et al., 1995), tilapia GnRH immunoreactivity appears confined to cell bodies and sockeye salmon (Parhar et al., 1996), dogfish (df- located in the lateral hypothalamus, the ventral thala- GnRH) (Lovejoy et al., 1992) and lamprey (I-GnRH and mus, and the midbrain rostral tegmentum; immunoreac- III-GnRH) (Sherwood et al., 1986; Sower et al., 1993). tive fibres densely innervated the hypothalamic periven- The study on GnRH-like peptides in N. coriiceps was tricular region. By contrast, m-GnRH-like immunoreac- tive neurons are present exclusively in the torus semi- performed by immunocytochemistry using antisera raised against three variants of GnRH: mammalian (m- circularis of the mesencephalon and in the outer plex- iphorm layers of the optic tectum (Fig 2D). These find- GnRH), chicken (cII-GnRH), and salmon (s-GnRH). cll- Β. Μ. UVA, M. VALLARINO, G. TAGLIAFERRO, M. PESTARINO, C. FALUGI, A. MANDICH, M.A. MASINI, M. STURIA, P. PRATO, et alii ings suggest that cII-GnRH-like peptides appear to func- clonal antisera raised in rabbit, as listed in Table II. Da- tion as hypophysiotropic factors, as demonstrated in ta concerning the entero-pancreatic neuroendocrine other species of bony fish, whereas m-GnRH-like pep- system (NES) of non-Antarctic perciformes Serranus tides could be involved in modulatory pathways of cabrilla and Diplodus annularis are also reported for vestibular and visual functions of N. coriiceps. Incuba- comparison. The topographical distribution of Insulin tion with s-GnRH antiserum failed to prove the occur- (INS)-, Glucagon (GLU)-, Pancreatic Polypeptide (PP)- rence of immunoreactive elements, consequently, at and SST-14-ir cells in the Brockmann body in C. antarc- least two molecular forms related to cII-GnRH and m- ticus, P. macropterus, N. coriiceps, and P. antarcticum, GnRH seem to act as hypophysiotropic and neuromod- was similar to that found in other teleosts (Langer et al, ulatory factors in the brain of N. coriiceps. Moreover, m- 1979; Abad et al, 1987; Bjenning & Holmgren, 1988; GnRH immunoreactivity in ependymal tanycytes (Fig. Jonsson, 1991; Tagliafierro et al, 1994, 1996). GLU-ir 2E) suggests the involvement of such specialised glial cells were located at the periphery, while SST-14- and cells in neuroendocrine function by linking the cere- INS-ir cells were situated in the central portion. Differ- brospinal fluid and the median eminence, as demon- ent endocrine cell associations could be seen in smaller strated in mammals. islets: INS-ir cells could be found at the periphery, while SST- and PP-ir cells were distributed throughout the islet. In the large islet of C. antarcticus, PP-ir cells Intestine and gut peptides seemed to be fewer than what was observed in no- tothenioids and in Serranus cabrilla (Tagliafierro et al., The distribution and localisation of gastro-intestinal 1994, 1996). Pancreatic endocrine cells appeared very hormones and neuropeptides were studied using poly- Fig. 3 - Immunoreactivity in pancreatic endocrine islet of P. antarcticum, C. antarcticus and P. macropterus. A, SST immunoreactive cells in a P. antarcticum larva, x 200. B, GAL-immunoreactive nerve-fibers near GLU cells in C. antarcticus. x 400. C, Sub-P immunore- active nerve fibers near a blood vessel in the central portion of the islet in P. macropterus. χ 800. D, Sub-P immunoreactive nerve fibers in the périphérie portion of the islet in P. macropterus. χ 800. REGULATORY PEPTIDES IN ANTARCTIC TELEOSTS 63 Fig. 4 - Testis sections of C. hamatus and T. bernacchii caught in December. A, Section from C. hamatus testis. The immuoreaction with anti-FGF is present in the interstitial cells (arrow), in the Sertoli cells (arrowhead) and in the spermatogonia A (small arrow), χ 450. Β, Testis of C. hamatus. In a consecutive section, used as control, no stain is present after incubation with rabbit IgGs, used as primary antiserum, χ 300. C, Testis of C. hamatus incubated with Ab-FGFRl. The immunostaining is localised in the spermatogonia (arrowhead) and in the Sertoli cells (arrow), χ 300. D, Section of testis from T. bernacchii incubated with Ab-bFGF. No staining is present, χ 300. Β. Μ. UVA, M. VALLARINO, G. TAGLIAFERRO, M. PESTARINO, C. FALUGI, A. MANDICH, M.A. MASINI, M. STURLA, P. PRATO, et alii early during embryonic development an d one large islet tion prior to folliculogenesis, and then, during growth, with INS, GLU an d SST-ir cells could b e observed in 20- carries out a significant synthesis of both RNA and pro- cm-long P. antarcticum larvae (Fig. 3A) (Tagliafierro et teins. This determines the formation in the cytoplasm of al., unpublished results). a large Balbiani Body, comparable to that observed in GAL immunoreactive nerv e elements were detected other species, and of a 'spherical body' that, by con- trast, appears significantly different from any other both in the intestinal and pancreatic portion, showing a distribution similar to that found in non-Antarctic coun - known cell structure. At the electron microscope, the terparts but they were fewer in number (Karila et al., spherical bodies appeared organised in cords, arranged so as to form polygonal grids. The cords showed an in- 1993). In the exocrine pancreas, ir nerve fibres were found in the blood vessel wall, while in the endocrin e ner, denser portion and a more fibrillar, fuzzy edge. In situ hybridisation demonstrated that the edges were rich portion (Brockmann body ) they seemed to surroun d INS ir cells, and in particular to be close to the periph- in rRNA while immunocytochemical analysis indicated that the inner cords were rich in actin and vimentin erally located GLU ir cells (Fig. 3B). GAL is one of the regulatory peptides commonly detected in the entero- (Motta et al, 1999). pancreatic tract of different vertebrates, where it seems At ultrastructural level, the male gametes of C. hatna- to be widely distributed an d mainly localised in nervous tus, C. gunnari, and N. coriiceps, when analysed with elements. In the gut, it regulates motility and affects lu- scanning electron microscopy, showed a similar mor- minal secretion as well as plasma levels of the other gut phology, common to most perciform species. At trans- peptides and pancreatic hormones . mission electron microscopy, C. hamatus showed some In the Brockmann bod y of notothenioids only few Va- differences: two nuclear fossae, and abundant membra- neous ornamentations of the head (Falugi et al., 1999). soactive Intestinal Polypeptide (VIP), Pituitary Adenylate Cyclase Activating Peptide (PACAP), and Peptide Histi- Growth factors, Fibroblast Growth Factor (FGF) and dine Isoleucine (PHI)-ir nerve fibres were detected. Sur- its receptor Fibroblast Growth Factor Receptor 1 prisingly many sub-P-ir fibres (Fig. 3 O were seen, espe- (FGFR1) have a crucial role in the regulation of the cially in the outer portion of the large islet (Fig. 3D), spermatogenetic cycles in vertebrates, including fish. close to GLU- an d PP-ir cells. Many VIP-ir nerve fibres The immunocytochemical analysis, aimed at identifying and no Sub-P-ir fibres were immunodetected in the FGF (Fig. 4 A,B), and FGFR1 (Fig. 4C), revealed a posi- pancreatic endocrine portion of S. cabrilla, D. antlu- tive reaction both in Sertoli cells and spermatogonia in laris, and other non-Antarctic teleosts. VIP, PACAP, an d C. hamatus, indicating that this species was ready to PHI belong to the same family and are candidate mes- start a new spermatogenetic cycle. The weak reaction in sengers in the nonadrenergic, noncholinergi c nervou s T. bernacchii (Fig. 4D) suggested that the stage of cell control of pancreatic an d intestinal secretion, motility, division was over and that of meiosis and differentiation and blood flow; they appear to stimulate the release of was starting. These data may indicate that Antarctic islet hormones and the flow of pancreatic juice. Sub-P teleosts have an opportunistic spermatogenetic cycle. enhance s the blood flow but does not seem to have any influence on the release of mammalian pancreatic hormone s (Nilsson & Holmgren, 1993). No data are CONCLUDING REMARKS available about the role of this peptide in fish pancreat- ic function. Nevertheless, the finding of sub-P-ir nerve In conclusion, our data point to the important role fibres surrounding the endocrine cells of the Antarctic played in the organs and systems by a variety of regula- fish pancreatic islet suggests that this peptide might play tory peptides in the physiological adaptation of Antarctic some role in the release of pancreatic hormones. teleosts to the extreme environmental conditions. These findings confirm the role of neurotransmitter and/or neuromodulator of some neuropeptides in te- leosts and suggest that neurotransmitters, different from REFERENCES those usually found in temperate teleosts, are probably Abad M. E., Binkhorst F. M. 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Journal

Italian Journal of Zoology – Taylor & Francis

Published: Jan 1, 2000

Keywords: Antarctic teleosts; Osmoregulation; Regulatory peptides; Brain; Intestine; Reproduction

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