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K Sawada, K Ukena, S Kikuyama, and K Tsutsui

Recently, we identified in the bullfrog brain a novel neuropeptide with a C-terminal Leu-Pro-Leu-Arg-Phe-NH(2) sequence. This amphibian neuropeptide was shown to stimulate growth hormone (GH) release in vitro and in vivo and so was designated frog GH-releasing peptide (fGRP). In this study, we cloned a cDNA encoding fGRP from the bullfrog brain by a combination of 3' and 5' rapid amplification of cDNA ends (RACE). The deduced fGRP precursor consisted of 221 amino acid residues, encoding one fGRP and three putative fGRP-related peptides that included Leu-Pro-Xaa-Arg-Phe-NH(2) (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Northern blot analysis detected a single band of approximately 1.0 kb, indicating that no alternatively spliced forms were present. Such an apparent migration was in agreement with the estimated length of the cDNA, 902 bp. In situ hybridization further revealed the cellular localization of fGRP mRNA in the suprachiasmatic nucleus in the hypothalamus. In addition to fGRP, its related peptides may be hypothalamic factors involved in pituitary hormone secretion.

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H Yin, K Ukena, T Ubuka, and K Tsutsui

We recently identified a novel hypothalamic dodecapeptide inhibiting gonadotropin release in the Japanese quail (Coturnix japonica). This novel peptide was therefore named gonadotropin-inhibitory hormone (GnIH). The GnIH precursor encoded one GnIH and two GnIH-related peptides (GnIH-RP-1 and GnIH-RP-2) that shared the same C-terminal motif, Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa=Leu or Gln; LPXRF-amide peptides). Identification of the receptor for GnIH is crucial to elucidate the mode of action of GnIH. We therefore identified the receptor for GnIH in the quail diencephalon and characterized its expression and binding activity. We first cloned a cDNA encoding a putative GnIH receptor by a combination of 3′ and 5′ rapid amplification of cDNA ends (RACE) using PCR primers designed from the sequence for the receptor for rat RF-amide-related peptide (RFRP), an orthologous peptide of GnIH. Hydrophobic analysis revealed that the putative GnIH receptor possessed seven transmembrane domains, indicating a new member of the G protein-coupled receptor superfamily. The crude membrane fraction of COS-7 cells transfected with the putative GnIH receptor cDNA specifically bound to GnIH and GnIH-RPs in a concentration-dependent manner. Scatchard plot analysis of the binding showed that the identified GnIH receptor possessed a single class of high-affinity binding sites (K d=0.752 nM, B max=24.8 fmol/mg protein). Southern blotting analysis of reverse transcriptase-mediated PCR products revealed the expression of GnIH receptor mRNA in the pituitary gland and several brain regions including diencephalon in the quail. These results suggest that GnIH acts directly on the pituitary via GnIH receptor to inhibit gonadotropin release. GnIH may also act on the hypothalamus to inhibit gonadotropin-releasing hormone release.

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T Ubuka, M Ueno, K Ukena, and K Tsutsui

We previously isolated a novel dodecapeptide containing a C-terminal -Arg-Phe-NH(2) sequence, SIKPSAYLPLRF-NH(2) (RFamide peptide), from the Japanese quail (Coturnix japonica) brain. This novel quail peptide was shown to be located in neurons of the paraventricular nucleus (PVN) and their terminals in the median eminence (ME), and to decrease gonadotropin release from cultured anterior pituitary in adult birds. We therefore designated this peptide gonadotropin-inhibitory hormone (GnIH). Furthermore, a cDNA encoding the GnIH precursor polypeptide has been characterized. To understand the physiological roles of this peptide, in the present study we analyzed developmental changes in the expressions of GnIH precursor mRNA and the mature peptide GnIH during embryonic and posthatch ages in the quail diencephalon including the PVN and ME. GnIH precursor mRNA was expressed in the diencephalon on embryonic day 10 (E10) and showed a significant increase on E17, just before hatch. GnIH was also detected in the diencephalon on E10 and increased significantly around hatch. Subsequently, the diencephalic GnIH content decreased temporarily, and again increased progressively until adulthood. GnIH-like immunoreactive (GnIH-ir) neurons were localized in the PVN on E10, but GnIH-ir fibers did not extend to the ME. However, GnIH-ir neurons increased in the PVN on E17, just before hatch, and GnIH-ir fibers extended to the external layer of the ME, as in adulthood. These results suggest that GnIH begins its function around hatch and acts as a hypothalamic factor to regulate gonadotropin release in the bird.

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T Ubuka, H Sakamoto, D Li, K Ukena, and K Tsutsui

We recently found lumbosacral sympathetic ganglionic galanin neurons innervating the quail uterine oviduct. Galaninergic innervation of the uterine muscle may be essential for avian oviposition, as galanin evoked oviposition through a mechanism of induction of vigorous uterine contraction. The questions arising from these findings are: what changes occur in galanin expression in the sympathetic ganglionic galanin neuron during development, and what is the hormonal factor(s) that induces galanin expression in this neuron? Therefore, the present study examined the developmental changes in galanin of the quail sympathetic ganglionic neuron and uterus, and the effect of administration of ovarian sex steroids on galanin induction. Immature birds reared under long-day photoperiods from 4 weeks of age demonstrated progressive increases in galanin levels both per unit ganglionic protein (concentration) and per ganglia (content) concurrent with ganglionic development during weeks 4--13. The uterine galanin content and uterine weight also increased progressively during the same period, but the galanin concentration in the uterus at 4 weeks was high due to the much smaller tissue mass. Immunocytochemical analysis with anti-galanin serum showed that immunoreactive ganglionic cells were few and small at 4 weeks and increased progressively thereafter. Administration of oestradiol-17 beta to immature birds at 3 weeks of age for 1 week increased both the galanin concentration and content in the ganglia without ganglionic growth. A marked increase in galanin-immunoreactive ganglionic cells was detected following oestradiol treatment. In contrast, progesterone increased ganglionic galanin levels, but the effects were low. Expression of the mRNAs encoding oestrogen receptor-alpha and -beta (ER alpha and ER beta) in the ganglionic tissue was verified by RT-PCR/Southern blot analysis. Immunocytochemical staining with anti-ER serum further revealed an intense immunoreaction restricted to the nucleus of ganglionic neurons. These results suggest that ovarian sex steroids, in particular oestradiol-17 beta, contribute as hormonal factors to galanin induction, which takes place in the lumbosacral sympathetic ganglionic neurons innervating avian uterine oviduct during development. Oestradiol may act directly on this ganglionic neuron through intra-nuclear receptor-mediated mechanisms to induce galanin.

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T Osugi, K Ukena, GE Bentley, S O'Brien, IT Moore, JC Wingfield, and K Tsutsui

The neuropeptide control of gonadotropin secretion is primarily through the stimulatory action of the hypothalamic decapeptide, GnRH. We recently identified a novel hypothalamic dodecapeptide with a C-terminal LeuPro-Leu-Arg-Phe-NH2 sequence in the domestic bird, Japanese quail (Coturnix japonica). This novel peptide inhibited gonadotropin release in vitro from the quail anterior pituitary; thus it was named gonadotropin-inhibitory hormone (GnIH). GnIH may be an important factor regulating reproductive activity not only in domesticated birds but also in wild, seasonally breeding birds. Thus, we tested synthetic quail GnIH in seasonally breeding wild bird species. In an in vivo experiment, chicken gonadotropin-releasing hormone-I (cGnRH-I) alone or a cGnRH-I/quail GnIH cocktail was injected i.v. into non-breeding song sparrows (Melospiza melodia). Quail GnIH rapidly (within 2 min) attenuated the GnRH-induced rise in plasma LH. Furthermore, we tested the effects of quail GnIH in castrated, photostimulated Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), using quail GnIH or saline for injection. Again, quail GnIH rapidly reduced plasma LH (within 3 min) compared with controls. To characterize fully the action of GnIH in wild birds, the identification of their endogenous GnIH is essential. Therefore, in the present study a cDNA encoding GnIH in the brain of Gambel's white-crowned sparrow was cloned by a combination of 3' and 5' rapid amplification of cDNA ends and compared with the quail GnIH cDNA previously identified. The deduced sparrow GnIH precursor consisted of 173 amino acid residues, encoding one sparrow GnIH and two sparrow GnIH-related peptides (sparrow GnIH-RP-1 and GnIH-RP-2) that included Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa=Leu or Gln) at their C-termini. All these peptide sequences were flanked by a glycine C-terminal amidation signal and a single basic amino acid on each end as an endoproteolytic site. Although the homology of sparrow and quail GnIH precursors was approximately 66%, the C-terminal structures of GnIH, GnIH-RP-1 and GnIH-RP-2 were all identical in two species. In situ hybridization revealed the cellular localization of sparrow GnIH mRNA in the paraventricular nucleus (PVN) of the hypothalamus. Immunohistochemical analysis also showed that sparrow GnIH-like immunoreactive cell bodies and terminals were localized in the PVN and median eminence respectively. Thus, only the sparrow PVN expresses GnIH, which appears to be a hypothalamic inhibitory factor for LH release, as evident from our field injections of GnIH into free-living breeding white-crowned sparrows. Sparrow GnIH rapidly (within 2 min) reduced plasma LH when injected into free-living Gambel's white-crowned sparrows on their breeding grounds in northern Alaska. Taken together, our results indicate that, despite amino acid sequence differences, quail GnIH and sparrow GnIH have similar inhibitory effects on the reproductive axis in wild sparrow species. Thus, GnIH appears to be a modulator of gonadotropin release.

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A Kanda, K Takuwa-Kuroda, E Iwakoshi-Ukena, Y Furukawa, O Matsushima, and H Minakata

We reported that the common octopus, Octopus vulgaris, in common with vertebrates, possesses two members of the oxytocin/vasopressin superfamily: octopressin (OP) and cephalotocin (CT). This was the first observation of its kind in invertebrates. As OP and CT have different biological activities, the presence of specific receptors has been proposed. We cloned the cDNA of an orphan receptor from Octopus brain and found it to encode a polypeptide of 397 amino acids that displays sequences characteristic of G-protein coupled receptors. The orphan receptor showed high homology to receptors of the oxytocin/vasopressin superfamily and seemed to conserve the agonist-binding pocket common to the oxytocin and vasopressin receptors. Xenopus oocytes that express the orphan receptor responded to the application of CT by an induction of membrane Cl(-) currents coupled to the inositol phosphate/Ca(2+) pathway. OP and the other members of the oxytocin/vasopressin superfamily did not activate this receptor. HPLC fractionation of the Octopus brain extract combined with an oocyte assay yielded a single substance that was identical to CT. On the basis of these results, we conclude that the cloned receptor is the CT receptor (CTR). Expression of CTR mRNA in Octopus was detected in the central and the peripheral nervous systems, the pancreas, the oviduct and the ovary. This receptor may mediate physiological functions of CT in Octopus such as neurotransmission, reproduction and metabolism.