JOURNAL OF EXPERIMENTAL ZOOLOGY 311A:199–206 (2009) A Journal of Integrative Biology Localization and Role of Galanin in the Thyroid Gland of Podarcis sicula Lizard (Reptilia, Lacertide) ROSARIA SCIARRILLO1Ã, ANNA CAPALDO2, SALVATORE VALIANTE2, 2 2 VINCENZA LAFORGIA , AND MARIA DE FALCO 1Department of Biological and Environmental Sciences, University of Sannio, Benevento, Italy 2Department of Evolutive and Comparative Biology, University of Naples ‘‘Federico II,’’ Naples, Italy ABSTRACT Galanin (GAL) is a 29-amino acid residue neuropeptide, which was initially isolated from porcine intestine extracts and since then, widely found in a variety of vertebrate organs, in correlation with multiple neuro-hormonal actions exerted and so receiving a constantly growing attention. Moreover, although the studies undertaken so far suggest a local intrathyroidal peptidergic regulatory action, the exact role of GAL on thyroid gland remains to be established. The aim of this study was to determine in the lizard, Podarcis sicula, (1) the presence of GAL immunoreactivity in the thyroid gland and (2) the short- and long-term effects of in vivo GAL administration by intraperitoneal injection on thyroid gland physiology. First of all, the presence of GAL in the thyroid gland of P. sicula was demonstrated by immunohistochemical technique (avidin–biotin–peroxidase complex—ABC method). Second, the role of GAL in the control of thyroid gland activity was studied in vivo using light microscopy (LM) technique coupled to a specific radioimmunoassay for thyroid-stimulating hormone (TSH) and thyroid hormones (T4 and T3). Prolonged GAL administration [(0.4 mg/100 g body wt)/day] increased T4 and T3 release, but decreased the plasma concentration of TSH. In addition, using LM clear signs of stimulation of the thyroid gland were observed. These findings suggest that systemic administration of GAL was able to stimulate the thyroid gland of the lizard both at morphological and physiological level. J. Exp. Zool. 311A:199–206, 2009. r 2009 Wiley-Liss, Inc. How to cite this article: Sciarrillo R, Capaldo A, Valiante S, Laforgia V, Falco DM. 2009. Localization and role of galanin in the thyroid gland of Podarcis sicula lizard (reptilia, lacertide). J. Exp. Zool. 311A:199–206. Galanin (GAL) is a 29-amino acid residue extensive studies on GAL distribution have been neuropeptide, which was initially isolated from carried out on mammalian organs, in fact, high porcine intestine extracts (Tatemoto et al., ’83) concentrations of GAL were found in the nervous and since then widely found in a variety of system, especially in the hypothalamus, the vertebrate organs. The amino acid compositions median eminence and hypophyseal portal blood and sequences of GAL are known in mammals (Tatemoto et al., ’83; Ro¨kaeus et al., ’84; Skofitsch (Ro¨kaeus and Carlquist, ’88), in birds (Kohchi and and Jacobowitz, ’85; Lopez et al., ’90; Lopez et al., Tsutsui, 2000), amphibians (Chartrel et al., ’95) ’91; Giustina et al., ’94; Dutriez et al., ’97; Landry and fishes (Anglade et al., ’94). Moreover, GAL et al., 2000), in the endocrine system, particularly structure has also been identified in alligator and tortoise (Wang and Conlon, ’94; Wang et al., ’99), ÃCorrespondence to: Rosaria Sciarrillo, Department of Biological demonstrating that the structure of GAL in and Environmental Sciences, University of Sannio, 82100 Benevento, alligator was more similar to that of sheep (three Italy, Via Port’Arsa, 11- I-82100 Benevento, Italy. E-mail: [email protected] amino acid substitutions) (Wang and Conlon, ’94), Received 11 June 2008; Revised 2 December 2008; Accepted 4 whereas it differed from that of tortoise by five December 2008 Published online 7 January 2009 in Wiley InterScience (www. amino acid residues (Wang et al., ’99). The most interscience.wiley.com). DOI: 10.1002/jez.519 r 2009 WILEY-LISS, INC. 200 R. SCIARRILLO ET AL. in the adrenal gland (Rokaeus and Carlquist, ’88; (Grunditz et al., ’87; Ahre`n, ’91). Hence, although Holst et al., ’91; Mazzocchi et al., ’92; Malendowicz the studies undertaken so far suggest a local et al., ’94; Hocho´l et al., 2000), in the cardiovas- intrathyroidal peptidergic regulatory action, the cular (Revington et al., ’90; Johansson and exact role of GAL on thyroid gland remains to be Rokaeus, ’95), gastrointestinal (Melander et al., established. However, there is no knowledge of the ’85; Rattan, ’93; Botella et al., ’95) and in the physiological role played by GAL in the thyroid reproductive systems (Bauer et al., ’86; Bek et al., gland of reptiles. ’88; Papka and Traurig, ’89; Shew et al., ’92; The aim of this study was to determine in the Torsello et al., ’92; Fox et al., ’94; Lakomy et al., lizard, P. sicula, (1) the presence of GAL immu- ’95). In nonmammalian vertebrates, GAL-like- noreactivity in the thyroid gland and (2) the short- immunoreactivity (GAL-like ir) has been found and long-term effects of in vivo GAL administra- in the CNS of lamprey (Jime´nez et al., ’96), dogfsh tion by intraperitoneal injection on thyroid gland (Vallarino et al., ’91), teleosts (Batten et al., ’90a,b; physiology in winter when the gland is in func- Holmqvist and Ekstro¨m, ’91; Olivereau and tional stasis. Olivereau, ’91; Anglade et al., ’94; Rodrı´guez- Go´mez et al., 2000), amphibians (Wolfbauer and MATERIALS AND METHODS Skofitsch, ’89; McKeon et al., ’90; La´za´r et al., ’91;Olivereau and Olivereau, ’92; Pieribone et al., Animals and housing conditions ´ ´ ’94; Gonzalez-Nicolini et al., ’98), reptiles (Jime- Adult male lizards of P. sicula (weighing ´ nez et al., ’94; Alponti et al., 2006) and birds (Jozsa 13–15 g) were live-captured in the neighborhood and Mess, ’93). In the oviduct of the lizard of Naples both in June (n 5 20), when the thyroid P. sicula, GAL seemed to interact with estrogen, gland was in full functional activity and in vasoactive intestinal polypeptide and oviposition December (n 5 80), when the thyroid gland (Lamanna et al., ’99a,b). GAL caused vasoconstric- showed clear signs of functional stasis (Sciarrillo tion and occasionally activated the gut wall of the et al., 2000). After capture, the animals were estuarine crocodile, Crocodylus porosus (Kagstrom housed in large soil-filled terraria containing et al., ’98). There were no changes in the GAL heather, and exposed to natural temperature and innervation of the gut or on GAL-induced intest- photoperiod. Water dishes were present in the inal motility between fasting and digesting Bur- terraria, and the animals were fed on live fly mese python, Python molurus bivittatus larvae daily. Captivity lasted 20 days to reverse (Holmberg et al., 2003). Central injection of GAL capture-related stress (Manzo et al., ’94). All stimulates food intake in goldfish, Carassius animals have been captured with the authoriza- auratus (DePedro et al., ’95), while it does not tion of 06/01/2000 no. SCN/2D/2000/9213 of Italian affect feeding in the neonatal chick (Ando et al., Ministry of Environment. 2000). Present evidences assign a variety of physiolo- Experimental procedure gical functions to GAL in mammals, including the regulation of intestinal motility, the control of Only the animals live-captured in December, endocrine and exocrine pancreatic secretions and received i.p. injections of GAL (Rat, Sigma Chem. modulation of hormone secretion from pituitary Co., St. Louis, MO). GAL was dissolved in reptilian and adrenal glands. Besides, in the male and physiological solution (NaCl 0.75%) with an injec- female mammalian species, GAL seems to play an tion volume of 0.1 mL. Injections were adminis- estrogen-mediated role in reproductive functions. tered between 8.00 and 8.30 a.m.. The specimens Until now, comparative studies about the phy- were divided into four groups, each consisting of siological roles of GAL in nonmammalian verte- 20 animals in order to obtain an adequate plasma brates, instead, have been considerably less; our volume. understanding of GAL roles and its regulation in Group 1. The animals received a single i.p. nonmammalian vertebrates are very limited. At injection of GAL (0.4 mg/100 g wt) and were our knowledge the biological effects of GAL have sacrificed 2 hr later. not yet been studied in nonmammalian species. Group 2. Untreated animals (controls) received a The data on GAL effects on thyroid gland in single i.p. injection of reptilian physiological mammalian are largely still lacking. GAL-immu- solution and sacrificed 2 hr later. noreactive fibers were observed around blood Group 3. The animals received an i.p. injection vessels and thyroid follicles in mice and rats only of GAL (0.4 mg/100 g body wt)/day for four J. Exp. Zool. GAL AND THYROID GLAND 201 consecutive days and were sacrificed 2 hr after the Capture Reagent, a blend of ligand-tagged TSH- last injection. specific antibody and 125I labelled TSH (10 mCi) Group 4. Untreated animals (controls) received was added to each tube. A cubic spline function an i.p. injection of reptilian physiological solution with the zero standard as one of the standard for four consecutive days and were sacrificed 2 hr points was used for calculations. The minimum after the last injection. detectable dose was 0.01 mIU/mL, with an accuracy The animals were anaesthetized by hypother- close to 100%, and the mean intra and inter- mia, chilling them in chipped ice. Blood samples assay coefficients of variance of 5.0 and 7.5%, were collected by intracardiac puncture and put respectively. into heparinized tubes. Blood collection lasted less than 3 min; plasma obtained centrifuging (2,500 g Immunohistochemistry for 10 min at 41C) the blood samples, was stored at The sections of the thyroid gland of control À201C until assay. P. sicula animals live-captured in June were examined by immunohistochemistry with the Light microscopy avidin–biotin-peroxidase complex (ABC) method Immediately after collection of blood samples, (Hsu et al., ’81) to localize the GAL.