Expression of prolactin receptor mRNA in the abdominal gland of the newt Cynops ensicauda Hiroshi Matsukawa a , Itaru Hasunuma a , Takafumi Kato a , Kazutoshi Yamamoto a , Satoshi Miura b , Takashi Fujita c , Sakae Kikuyama a, * a Department of Biology, School of Education, Waseda University, Nishiwaseda 1-6-1, Shinju-ku, Tokyo 169-8050, Japan b Radioisotope Research Center, School of Medicine Yokohama City University, Fuku-ura 3-9, Yokohama 236-0004, Japan c Department of Tumor Cell Biology, Tokyo Metropolitan Institute for Medical Science, Honkomagome 3-18-22, Bunk-yo, Tokyo 113-8613, Japan Received 12 January 2004; received in revised form 29 February 2004; accepted 1 March 2004 Abstract To further the understanding that the structural development of the Cynops ensicauda abdominal gland and the synthesis of the pheromone silefrin in the gland are under the control of prolactin and androgen, we sought to demonstrate the presence of prolactin receptor (PRLR) mRNA in the gland. Firstly, PRLR cDNA was isolated from an abdominal gland cDNA library. A cDNA consisting of a 415-bp 5V - untranslated region, 1878-bp open reading frame and 175-bp 3V -untranslated region was obtained. The deduced amino acid sequence consisted of 626 amino acids with signal peptide and single transmembrane domain. By Northern blot analysis using partial C. ensicauda PRLR cDNA, two transcripts, of 3 and 10 kb, were detected for PRLR in the brain, liver, kidney, abdominal gland, oviduct and skin. RT-PCR coupled with Southern blot analysis showed that the PRLR gene was transcribed broadly in newt organs and revealed that PRLR mRNA levels in the abdominal gland were much higher in sexually developed newts than in the sexually undeveloped ones. By in situ hybridization, specific signals were detected in the epithelial cells of the abdominal gland of sexually developed newts, but much less in those of the sexually undeveloped ones. D 2004 Elsevier Inc. All rights reserved. Keywords: Newt; Prolactin; Prolactin receptor; Abdominal gland; cDNA cloning; Breeding season; Silefrin; Courtship behavior 1. Introduction Prolactin (PRL) is a pituitary hormone that regulates a variety of biological processes, including reproductive and immunological function, fluid balance, cellular growth and differentiation (Nicoll and Bern, 1972; Ensor, 1978; Kelly et al., 1993). In amphibians, PRL is involved in habitat preference, larval growth, metamorphosis, osmoregulation and various aspects of reproduction, all of which are essential for species survival (White and Nicoll, 1979; Carr and Jaffe, 1980; Bern, 1983; Kikuyama et al., 1993; Polzonetti-Magni et al., 1995). PRL receptor (PRLR) cDNAs have been cloned from several mammalian species (Boutin et al., 1988, 1989; Davis and Linzer, 1989; Edery et al., 1989; Shirota et al., 1990; Scott et al., 1992; Moore and Oka, 1993; Clarke et al., 1995; Bignon et al., 1997; Dalrymple et al., 2000), a few avian species (Tanaka et al., 1992; Chen and Horseman, 1994; Zhou et al., 1996), two amphibians (Yamamoto et al., 1998; Huang and Brown, 2000) and several teleosts (Sandra et al., 1995; Tse et al., 2000; Santos et al., 2001; Higashi- moto et al., 2001). These PRLRs belong to a class I cytokine receptor superfamily that includes receptors for growth hormone (GH) and several interleukins (Bole-Feysot et al., 1998). These receptors have several common structural features, including two pairs of cysteine residues, a con- served motif (WS motif) in the extracellular domain, a single transmembrane domain and a proline-rich region, termed box I, in the cytoplasmic domain (Bole-Feysot et al., 1998). Recently, we demonstrated that female-attracting phero- mones in two species of Japanese newt, Cynops pyr- rhogaster and C. ensicauda are secreted by the abdominal gland (Kikuyama et al., 1995; Yamamoto et al., 2000a) and are generated from their precursor proteins (Iwata et al., 1999, 2000). Furthermore, the synthesis of these pheromone 1095-6433/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2004.03.006 * Corresponding author. Tel.: +81-3-5286-1517; fax: +81-3-3207-9694. E-mail address: kikuyama@waseda.jp (S. Kikuyama). www.elsevier.com/locate/cbpa Comparative Biochemistry and Physiology, Part A 138 (2004) 79– 88