Gonadotropin-inhibitory hormone in Gambel's white-crowned sparrow (Zonotrichia leucophrys gambelii): cDNA identification, transcript localization and functional effects in laboratory and field experiments.

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.

Gonadotrophin inhibitory hormone depresses gonadotrophin alpha and follicle-stimulating hormone beta subunit expression in the pituitary of the domestic chicken.

Studies performed in vitro suggest that a novel 12 amino acid RF amide peptide, isolated from the quail hypothalamus, is a gonadotrophin inhibitory hormone (GnIH). The aim of the present study was to investigate this hypothesis in the domestic chicken. Injections of GnIH into nest-deprived incubating hens failed to depress the concentration of plasma luteinizing hormone (LH). Addition of GnIH to short-term (120 min) cultures of diced pituitary glands from adult cockerels depressed follicle-stimulating hormone (FSH) and LH release and depressed common alpha and FSHbeta gonadotrophin subunit mRNAs, with no effect on LHbeta subunit mRNA. Hypothalamic GnIH mRNA was higher in incubating (out-of-lay) than in laying hens, but there was no significant difference in the amount of hypothalamic GnIH mRNA in out-of-lay and laying broiler breeder hens at the end of a laying year. It is concluded that avian GnIH may play a role in controlling gonadotrophin synthesis and associated constitutive release in the domestic chicken.

Developmental changes in gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica) hypothalamo-hypophysial system.

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.

Gonadotropin-inhibitory peptide in song sparrows (Melospiza melodia) in different reproductive conditions, and in house sparrows (Passer domesticus) relative to chicken-gonadotropin-releasing hormone.

Gonadotropin-releasing hormone (GnRH) regulates reproduction in all vertebrates. Until recently, an antagonistic neuropeptide for gonadotropin was unknown. The discovery of an RFamide peptide in quail that inhibits gonadotropin release in vitro raised the possibility of direct hypothalamic inhibition of gonadotropin release. This peptide has now been named gonadotropin-inhibitory hormone (GnIH). We investigated GnIH presence in the hypothalamus of two seasonally breeding songbird species, house sparrows (Passer domesticus) and song sparrows (Melospiza melodia). Using immunocytochemistry (ICC), GnIH-containing neurones were localized in both species in the paraventricular nucleus, with GnIH-containing fibres visible in multiple brain locations, including the median eminence and brainstem. Double-label ICC with light microscopy and fluorescent ICC with confocal microscopy indicate a high probability of colocalization of GnIH with GnRH neurones and fibres within the avian brain. It is plausible that GnIH could be acting at the level of the hypothalamus to regulate gonadotropin release as well as at the pituitary gland. In a photoperiod manipulation experiment, GnIH-containing neurones were larger in birds at the termination of the breeding season than at other times, consistent with a role for this neuropeptide in the regulation of seasonal breeding. We have yet to elucidate the dynamics of GnIH synthesis and release at different times of year, but the data imply temporal regulation of this peptide. In summary, GnIH has the potential to regulate gonadotropin release at more than one level, and its distribution is suggestive of multiple regulatory functions in the central nervous system.

Activity and localization of 3beta-hydroxysteroid dehydrogenase/ Delta5-Delta4-isomerase in the zebrafish central nervous system.

Little information is available for neurosteroidogenesis in the central nervous system (CNS) of lower vertebrates. Therefore, in the present study, we examined the enzymatic activity and localization of 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3betaHSD), a key steroidogenic enzyme, in the CNS of adult male zebrafish to clarify central progesterone biosynthesis. Biochemical studies together with HPLC analysis revealed that the zebrafish brain converted pregnenolone to progesterone, suggesting the enzymatic activity of 3betaHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3betaHSD. By using Western immunoblotting with the polyclonal antiserum directed against purified bovine adrenal 3betaHSD, a 3betaHSD-like substance was found in homogenates of the zebrafish brain. Immunocytochemical analysis was then undertaken to investigate the localization of the 3betaHSD-like substance in the zebrafish brain and spinal cord. Clusters of immunoreactive cell bodies were localized in the dorsal telencephalic areas (D), central posterior thalamic nucleus (CP), preoptic nuclei (NPO), posterior tuberal nucleus (PTN), paraventricular organ (PVO), and nucleus of medial longitudinal fascicle (NMLF). 3betaHSD-like immunoreactivity was also observed in somata of cerebellar Purkinje neurons. A widespread distribution of immunoreactive fibers was found throughout the brain and spinal cord. In addition, positively stained cells were restricted to other organs, such as the pituitary and retina. Preabsorbing the antiserum with purified bovine adrenal microsome resulted in a complete absence of 3betaHSD-like immunoreactivity. These results suggest that the fish CNS possesses steroidogenic enzyme 3betaHSD and produces progesterone. The present study further provides the first immunocytochemical mapping of the site of 3betaHSD expression in the fish CNS.

Effects of progesterone synthesized de novo in the developing Purkinje cell on its dendritic growth and synaptogenesis.

De novo steroidogenesis from cholesterol is a conserved property of vertebrate brains, and such steroids synthesized de novo in the brain are called neurosteroids. The identification of neurosteroidogenic cells is essential to the understanding of the physiological role of neurosteroids in the brain. We have demonstrated recently that neuronal neurosteroidogenesis occurs in the brain and indicated that the Purkinje cell actively synthesizes several neurosteroids de novo from cholesterol in vertebrates. Interestingly, in the rat, this neuron actively synthesizes progesterone de novo from cholesterol only during neonatal life, when cerebellar cortical formation occurs most markedly. Therefore, in this study, the possible organizing actions of progesterone during cerebellar development have been examined. In vitro studies using cerebellar slice cultures from newborn rats showed that progesterone promotes dose-dependent dendritic outgrowth of Purkinje cells but dose not affect their somata. This effect was blocked by the anti-progestin RU 486 [mifepristone; 17beta-hydroxy-11beta-(4-methylaminophenyl)-17alpha-(1-propynyl) estra-4,9-dien-3 one-6-7]. In vivo administration of progesterone to pups further revealed an increase in the density of Purkinje spine synapses electron microscopically. In contrast to progesterone, there was no significant effect of 3alpha,5alpha-tetrahydroprogesterone, a progesterone metabolite, on Purkinje cell development. Reverse transcription-PCR-Southern and immunocytochemical analyses showed that intranuclear progesterone receptors were expressed in Purkinje cells. These results suggest that progesterone promotes both dendritic outgrowth and synaptogenesis in Purkinje cells through intranuclear receptor-mediated mechanisms during cerebellar development. Such organizing actions may contribute to the formation of the cerebellar neuronal circuit.

Developmental changes in galanin in lumbosacral sympathetic ganglionic neurons innervating the avian uterine oviduct and galanin induction by sex steroids.

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.

Developmental changes in progesterone biosynthesis and metabolism in the quail brain.

We have recently demonstrated that the quail brain possesses the cholesterol side-chain cleavage enzyme (cytochrome P450scc) and 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3beta-HSD) and produces pregnenolone, pregnenolone sulfate and progesterone from cholesterol. To elucidate the developmental changes in progesterone biosynthesis and its metabolism in the quail brain, we examined the expression and activity of 3beta-HSD and progesterone metabolite(s) during embryonic and post-hatched ages. Both the progesterone concentration and 3beta-HSD mRNA expression in the brain were almost constant during embryonic and post-hatched ages. The conversion of pregnenolone to progesterone (net 3beta-HSD enzymatic activity) was also constant during development and at maturity. However, without radioinert progesterone, the production of progesterone was drastically reduced in the embryonic brain, indicating active progesterone metabolism at the embryonic stage. Biochemical analysis together with HPLC and TLC revealed that only the embryonic brain actively produced 5beta-dihydroprogesterone from progesterone. Thus, progesterone production may be constant during embryonic and post-hatched development and in adulthood, whereas 5beta-dihydroprogesterone may be produced actively only in embryonic life due to 5beta-reductase.

Characterization of a cDNA encoding a novel avian hypothalamic neuropeptide exerting an inhibitory effect on gonadotropin release.

We previously isolated a novel dodecapeptide containing a C-terminal -Arg-Phe-NH(2) sequence, SIKPSAYLPLRF-NH(2) (RFamide peptide), from the quail brain. This quail RFamide peptide was shown to decrease gonadotropin release from the cultured anterior pituitary and to be located at least in the quail hypothalamo-hypophysial system. We therefore designated this RFamide peptide gonadotropin inhibitory hormone (GnIH). In the present study we characterized the GnIH cDNA from the quail brain by a combination of 3' and 5' rapid amplification of cDNA ends ('RACE'). The deduced GnIH precursor consisted of 173 amino acid residues, encoding one GnIH and two putative gene-related peptide (GnIH-RP-1 and GnIH-RP-2) sequences that included -LPXRF (X=L or Q) 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. Southern blotting analysis of reverse-transcriptase-mediated PCR products demonstrated a specific expression of the gene encoding GnIH in the diencephalon including the hypothalamus. Furthermore, mass spectrometric analyses detected the mass numbers for matured GnIH and GnIH-RP-2, revealing that both peptides are produced from the precursor in the diencephalon as an endogenous ligand. Taken together, these results lead to the conclusion that GnIH is a hypothalamic factor responsible for the negative regulation of gonadotropin secretion. Furthermore, the presence of a novel RFamide peptide family containing a C-terminal -LPXRF-NH(2) sequence has been revealed.

A novel avian hypothalamic peptide inhibiting gonadotropin release.

The neuropeptide control of gonadotropin secretion at the level of the anterior pituitary gland is primarily through the stimulatory action of the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), which was originally isolated from mammals and subsequently from non-mammals. To date, however, an inhibitory peptide of gonadotropin release is unknown in vertebrates. Here we show, in a bird, that the hypothalamus also contains a novel peptide which inhibits gonadotropin release. Acetic acid extracts of quail brains were passed through C-18 reversed-phase cartridges, and then the retained material was subjected to the reversed-phase and cation-exchange high-performance liquid chromatography (HPLC). The peptide was isolated from avian brain and shown to have the sequence Ser-Ile-Lys-Pro-Ser-Ala-Tyr-Leu-Pro-Leu-Arg-Phe-NH(2). Cell bodies and terminals containing this peptide were localized immunohistochemically in the paraventricular nucleus and median eminence, respectively. This peptide inhibited, in a dose-related way, gonadotropin release from cultured quail anterior pituitaries. This is the first hypothalamic peptide inhibiting gonadotropin release reported in a vertebrate. We therefore term it gonadotropin-inhibitory hormone (GnIH).

Comparison of precursor structures of the GGNG peptides derived from the earthworm Eisenia foetida and the leech Hirudo nipponia.

Earthworm and leech cDNAs encoding the GGNG peptides, a family of myotropic peptides, were cloned and examined in this study. Both of the predicted precursor proteins are of polyprotein structure and contain several putative peptides distinct from the GGNG peptides. However, the precursors show organizations distinct from each other and no sequence similarity except for the GGNG peptides.

Expression and activity of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase in the rat Purkinje neuron during neonatal life.

Recently, we demonstrated that cytochrome P450 side-chain cleavage enzyme (P450scc) occurs in the rat cerebellar Purkinje cell after differentiation and remains during neonatal development and into adulthood. 3Beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase (3betaHSD) is also an essential enzyme for progesterone biosynthesis not only in peripheral steroidogenic glands but also in the nervous system. In the present study, therefore, the expression of 3betaHSD in the rat cerebellum was investigated during neonatal development and in the adult. RT-PCR analysis showed that the expression of 3betaHSD messenger RNA (mRNA) in the cerebellum was higher at 7-14 days of age than at other times. Biochemical studies together with HPLC analysis revealed that cerebellar slices at 10 days of age converted pregnenolone to progesterone, suggesting enzymatic activity of 3betaHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3betaHSD. A specific RIA indicated that progesterone concentrations in the cerebellum were higher at 3 and 10 days of age than at 60 days of age. The progesterone level in the cerebellum was significantly higher than that in plasma at 10 days of age. In contrast, the concentrations in both cerebellum and plasma at 3 and 60 days of age were similar. In the present study, the site of 3betaHSD mRNA expression in the cerebellum was further examined in neonatal and adult rats using in situ hybridization. The cerebellar expression of 3betaHSD mRNA was obscure at 3 days of age, whereas intense expression occurred in Purkinje cells and external granule cells throughout the cerebellum at 10 days of age. 3BetaHSD mRNA was also expressed in Purkinje cells and granule cells at 60 days of age, but a restricted expression was observed along the cerebellar meninges. These results suggest that the steroidogenic enzyme 3betaHSD as well as P450scc are expressed at least in the cerebellar Purkinje cell. The expression of 3betaHSD, however, may increase for a limited period around 10 days of age, unlike P450scc.

Developmental changes in galanin receptors in the quail oviduct and the effect of ovarian sex steroids on galanin receptor induction.

We have recently isolated an oviposition-inducing peptide from mature quail oviducts identified as avian galanin. This peptide evoked vigorous contractions of the uterine oviduct through binding to receptors located in the uterus. The questions arising from these findings are: what changes occur in galanin receptors in the uterus during maturation, and what is the hormonal factor(s) that induces uterine galanin receptors? Therefore, the present study examined changes in uterine galanin receptors with age and the effect of administration of ovarian sex steroids on galanin receptors in the quail. Immature females reared under long day (LD) photoperiods from 4 weeks of age demonstrated a progressive increase in specific galanin binding per both unit uterine weight and per whole uterus concurrent with uterine development during 4-13 weeks. Scatchard plot analyses of the binding to the uterine preparation showed that the equilibrium dissociation constant (Kd) was about 0.30-0.34 nM regardless of age, and the change in galanin binding during uterine development was due to a change in the number of binding sites. Plasma 17beta-estradiol and progesterone concentrations were almost constant between 4-6 weeks and tended to increase thereafter. Administration of 17beta-estradiol to immature females for 1 week increased not only uterine weight but also specific galanin binding per unit uterine weight, whereas progesterone increased only the binding per unit uterine weight. Both sex steroids also induced an increase in total binding per uterus. Combined administration of 17beta-estradiol and progesterone induced marked increases in the galanin binding, and the effect was not additive but, rather, was synergistic. Scatchard plot analysis showed that the number of binding sites, but not the Kd, was increased by steroid treatment. Administration of 17beta-estradiol or progesterone increased each circulating steroid level to that relatively similar to the maximal levels observed in females exposed to LD. Thus, ovarian sex steroids may contribute at least in part as hormonal factors to galanin receptor induction, which takes place in the uterine oviduct during development.

Immunochemical demonstration of Eisenia tetradecapeptide, a bioactive peptide isolated from the gut of the earthworm Eisenia foetida, in tissues of the earthworm.

The quantity and localization of Eisenia tetradecapeptide which was isolated from the gut of the earthworm Eisenia foetida were examined in tissues of the same species by enzyme-linked immunosorbent assay and immunohistochemistry. Analysis by enzyme-linked immunosorbent assay showed that Eisenia-tetradecapeptide-like immunoreactivity was present in both the central nervous system (cerebral ganglion, subesophageal ganglion, ventral ganglia, and ventral nerve cord) and the gut (esophagus, crop, gizzard, and intestine). The central nervous system contained a higher amount of Eisenia-tetradecapeptide-like immunoreactivity (1.3 pmol/mg wet weight) than the gut (0.2-0.6 pmol/mg wet weight). Eisenia-tetradecapeptide-like immunoreactivity was scarcely detected in the body-wall muscle, nephridia, and sexual organs (testis, ovary, seminal vesicle, and ovisac). Immunohistochemical analysis demonstrated that intense Eisenia-tetradecapeptide-like immunopositive cells and nerve fibers were present in the central nervous system. Immunoreactivity was found in the epithelial cells lining the esophagus and in the submucous plexus in various parts of the gut. Thus, the present study suggests that Eisenia tetradecapeptide is a neuropeptide and/or peptide hormone present in both the central nervous system and the gut of the earthworm and that its role involves the regulation of gut motility.

A novel gut tetradecapeptide isolated from the earthworm, Eisenia foetida.

A novel bioactive tetradecapeptide, GFKDGAADRISHGFamide, was isolated from the gut of the oligochaete annelid, Eisenia foetida, using the isolated anterior gut (crop-gizzard) as a bioassay system. A highly homologous peptide, GFRDGSADRISHGFamide, was also purified from the whole body of another species of earthworm, Pheretima vittata. These peptides were termed Eisenia tetradecapeptide (ETP) and Pheretima tetradecapeptide (PTP), respectively. Both the peptides showed a potent excitatory action on spontaneous contractions of the anterior gut with a threshold as low as 10(-10)-10(-9) M. These peptides were significantly homologous to molluscan tetradecapeptides and, to a lesser extent, to arthropodan tridecapeptides that have been reported to date. All these peptides seem to be evolutionally related to each other.

Bioactive peptides of annelids are closely related to those of molluscs.

Several bioactive peptides were isolated from some annelids. Each of the annelidan peptides seemed to be a member of a previously found molluscan peptide family.