Masato Aoyama

The role of uropygial gland on sexual behavior in domestic chicken Gallus gallus domesticus.

Recent studies have indicated that avian social behavior is influenced by olfactory cues. During the reproductive season a change in the chemical composition of uropygial gland secretion has been reported in some species and the hypothesis that olfactory signals may be produced by this gland has been proposed. To examine this hypothesis we performed two behavioral experiments to determine whether a females uropygial gland produces chemical signals that stimulate mating behaviors in domestic chickens. In Experiment 1 the role of the females uropygial gland in male mating behavior was examined by removing and examining the females uropygial gland. The frequency of mounts and copulations of intact male birds with sham-operated female birds was significantly higher than with uropygial glandectomized female birds. With respect to the number of waltzing that is one of the courtship displays intact males showed no significant difference between sham-operated female birds and uropygial glandectomized female birds. In Experiment 2 the relationship between male olfaction and the females uropygial gland was investigated using olfactory bulbectomized male birds. The number of mounts and copulations of sham-operated male birds with sham-operated female bird was significantly higher than with uropygial glandectomized female birds. In contrast olfactory bulbectomized male birds showed no significant differences in the number of mounts and copulations between sham-operated female birds and uropygial glandectomized female birds. These results indicate that intact and sham-operated male birds prefer to mate with female birds with the uropygial gland. The number of courtship waltzing of sham-operated male birds showed no significant difference. However olfactory bulbectomized male birds significantly courted to uropygial glandectomized female birds. Summarizing our results show that while anosmic males did not have any preference, both intact and sham-operated male birds chose to mate with female birds having an intact uropygial gland, suggesting that mate preference involves in male olfaction and that the females uropygial gland acts as a source of social odor cues in domestic chickens.

Nasal oxytocin administration reduces food intake without affecting locomotor activity and glycemia with c-Fos induction in limited brain areas.

Recent studies have considered oxytocin (Oxt) as a possible medicine to treat obesity and hyperphagia. To find the effective and safe route for Oxt treatment, we compared the effects of its nasal and intraperitoneal (IP) administration on food intake, locomotor activity, and glucose tolerance in mice. Nasal Oxt administration decreased food intake without altering locomotor activity and increased the number of c-Fos-immunoreactive (ir) neurons in the paraventricular nucleus (PVN) of the hypothalamus, the area postrema (AP), and the dorsal motor nucleus of vagus (DMNV) of the medulla. IP Oxt administration decreased food intake and locomotor activity and increased the number of c-Fos-ir neurons not only in the PVN, AP, and DMNV but also in the nucleus of solitary tract of the medulla and in the arcuate nucleus of the hypothalamus. In IP glucose tolerance tests, IP Oxt injection attenuated the rise of blood glucose, whereas neither nasal nor intracerebroventricular Oxt affected blood glucose. In isolated islets, Oxt administration potentiated glucose-induced insulin secretion. These results indicate that both nasal and IP Oxt injections reduce food intake to a similar extent and increase the number of c-Fos-ir neurons in common brain regions. IP Oxt administration, in addition, activates broader brain regions, reduces locomotor activity, and affects glucose tolerance possibly by promoting insulin secretion from pancreatic islets. In comparison with IP administration, the nasal route of Oxt administration could exert a similar anorexigenic effect with a lesser effect on peripheral organs.

Peptidomic Analysis of the Central Nervous System of the Protochordate, Ciona intestinalis: Homologs and Prototypes of Vertebrate Peptides and Novel Peptides

The phylogenetic position of ascidians as the chordate invertebrates closest to vertebrates suggests that they might possess homologs and/or prototypes of vertebrate peptide hormones and neuropeptides as well as ascidian-specific peptides. However, only a small number of peptides have so far been identified in ascidians. In the present study, we have identified various peptides in the ascidian, Ciona intestinalis. Mass spectrometry-based peptidomic analysis detected 33 peptides, including 26 novel peptides, from C. intestinalis. The ascidian peptides are largely classified into three categories: 1) prototypes and homologs of vertebrate peptides, such as galanin/galanin-like peptide, which have never been identified in any invertebrates; 2) peptides partially homologous with vertebrate peptides, including novel neurotesin-like peptides; 3) novel peptides. These results not only provide evidence that C. intestinalis possesses various homologs and prototypes of vertebrate neuropeptides and peptide hormones but also suggest that several of these peptides might have diverged in the ascidian-specific evolutionary lineage. All Ciona peptide genes were expressed in the neural complex, whereas several peptide gene transcripts were also distributed in peripheral tissues, including the ovary. Furthermore, a Ciona neurotensin-like peptide, C. intestinalis neurotensin-like peptide 6, was shown to down-regulate growth of Ciona vitellogenic oocytes. These results suggest that the Ciona peptides act not only as neuropeptides in the neural tissue but also as hormones in nonneuronal tissues and that ascidians, unlike other invertebrates, such as nematodes, insects, and sea urchins, established an evolutionary origin of the peptidergic neuroendocrine, endocrine, and nervous systems of vertebrates with certain specific molecular diversity.

A Conserved Non-Reproductive GnRH System in Chordates

Gonadotropin-releasing hormone (GnRH) is a neuroendocrine peptide that plays a central role in the vertebrate hypothalamo-pituitary axis. The roles of GnRH in the control of vertebrate reproductive functions have been established, while its non-reproductive function has been suggested but less well understood. Here we show that the tunicate Ciona intestinalis has in its non-reproductive larval stage a prominent GnRH system spanning the entire length of the nervous system. Tunicate GnRH receptors are phylogenetically closest to vertebrate GnRH receptors, yet functional analysis of the receptors revealed that these simple chordates have evolved a unique GnRH system with multiple ligands and receptor heterodimerization enabling complex regulation. One of the gnrh genes is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord of vertebrates. Correspondingly, GnRH receptor genes were found to be expressed in the tail muscle and notochord of embryos, both of which are phylotypic axial structures along the nerve cord. Our findings suggest a novel non-reproductive role of GnRH in tunicates. Furthermore, we present evidence that GnRH-producing cells are present in the hindbrain and spinal cord of the medaka, Oryzias latipes, thereby suggesting the deep evolutionary origin of a non-reproductive GnRH system in chordates.

Insight into molecular and functional diversity of tachykinins and their receptors.

Tachykinins (TKs) and their structurally related peptides constitute the largest peptide superfamily in the animal kingdom. TKs have been shown to play various physiological roles not only as major brain/gut peptides but also as endocrine/paracrine hormones in chordates and exocrine factors in amphibians. Recent studies have also revealed that the biological roles of TKs as brain/gut peptides and endocrine/paracrine factors are essentially conserved in protochordates, and that alternative splicing mechanism in mammalian TK genes were established during the evolution of vertebrates. Protostomes possess two structurally and functionally different peptides; invertebrate TKs (inv-TKs) serve as toxin-like compounds secreted from the salivary gland of several organisms, whereas TK-related peptides (TKRPs) are functional counterparts for chordate TKs. Additionally, a TKRP-like sequence was detected in a diploblastic organism. The dramatic difference in structural organizations between TKRP precursors and chordate TK precursors clearly indicates the distinct evolutionary processes of TKs and TKRPs. Despite high sequence homology, TK receptors manifest selective affinity to their endogenous ligands, while TKRPs exhibit redundant activity at their receptors. Moreover, in addition to nociceptive, inflammatory, and contractile effects as brain/gut peptides, a number of studies have revealed novel biological effects of TKs on the hypothalamus and genital organs, revealing the biological roles of TKs as pivotal regulators of reproduction. These findings shed light on complicated evolutionary lineages of both structures and functions of the TK/TKRP superfamily and their receptors. In this review, we present basic and latest knowledge of the TK/TKRP superfamily with various points of view.

A novel tachykinin-related peptide receptor of Octopus vulgaris– evolutionary aspects of invertebrate tachykinin and tachykinin-related peptide

The tachykinin (TK) and tachykinin‐related peptide (TKRP) family represent one of the largest peptide families in the animal kingdom and exert their actions via a subfamily of structurally related G‐protein‐coupled receptors. In this study, we have identified a novel TKRP receptor from the Octopus heart, oct‐TKRPR. oct‐TKRPR includes domains and motifs typical of G‐protein‐coupled receptors. Xenopus oocytes that expressed oct‐TKRPR, like TK and TKRP receptors, elicited an induction of membrane chloride currents coupled to the inositol phosphate/calcium pathway in response to Octopus TKRPs (oct‐TKRP I–VII) with moderate ligand selectivity. Substance P and Octopus salivary gland‐specific TK, oct‐TK‐I, completely failed to activate oct‐TKRPR, whereas a Substance P analog containing a C‐terminal Arg‐NH2 exhibited equipotent activation of oct‐TKRPs. These functional analyses prove that oct‐TKRPs, but not oct‐TK‐I, serve as endogenous functional ligands through oct‐TKRPR, although both of the family peptides were identified in a single species, and the importance of C‐terminal Arg‐NH2 in the specific recognition of TKRPs by TKRPR is conserved through evolutionary lineages of Octopus. Southern blotting of RT‐PCR products revealed that the oct‐TKRPR mRNA was widely distributed in the central and peripheral nervous systems plus several peripheral tissues. These results suggest multiple physiologic functions of oct‐TKRPs as neuropeptides both in the Octopus central nervous system and in peripheral tissues. This is the first report on functional discrimination between invertebrate TKRPs and salivary gland‐specific TKs.

Neuropeptides, Hormone Peptides, and Their Receptors in Ciona intestinalis: An Update

The critical phylogenetic position of ascidians leads to the presumption that neuropeptides and hormones in vertebrates are highly likely to be evolutionarily conserved in ascidians, and the cosmopolitan species Ciona intestinalis is expected to be an excellent deuterostome Invertebrate model for studies on neuropeptides and hormones. Nevertheless, molecular and functional characterization of Ciona neuropeptides and hormone peptides was restricted to a few peptides such as a cholecystokinin (CCK)/gastrin peptide, cionin, and gonadotropin-releasing hormones (GnRHs). In the past few years, mass spectrometric analyses and database searches have detected Ciona orthologs or prototypes of vertebrate peptides and their receptors, including tachykinin, insulin/relaxin, calcitonin, and vasopressin. Furthermore, studies have shown that several Ciona peptides, including vasopressin and a novel GnRH-related peptide, have acquired ascidian-specific molecular forms and/or biological functions. These findings provided indisputable evidence that ascidians, unlike other invertebrates (including the traditional protostome model animals), possess neuropeptides and hormone peptides structurally and functionally related to vertebrate counterparts, and that several peptides have uniquely diverged in ascidian evolutionary lineages. Moreover, recent functional analyses of Ciona tachykinin in the ovary substantiated the novel tachykininergic protease-assoclated oocyte growth pathway, which could not have been revealed in studies on vertebrates. These findings confirm the outstanding advantages of ascidians in understanding the neuroscience, endocrinology, and evolution of vertebrate neuropeptides and hormone peptides. This article provides an overview of basic findings and reviews new knowledge on ascidian neuropeptides and hormone peptides.

Spontaneous discrimination of food quantities in the jungle crow, Corvus macrorhynchos

Despite considerable research on numeric judgements in animals, uncertainty remains about both the underlying mechanisms and the role of training. To address these issues, we study quantity discrimination in jungle crows that have previously been shown to select the larger of two quantities following training. In the current study, we examined whether jungle crows are able to discriminate between different quantities of food items without prior training. Using a simultaneous two-alternative test, we studied whether their performance reveals the underlying mechanism being used for discrimination of small and large quantities in other animals. First, jungle crows were tested with a choice between two discrete homogeneous quantities; one smaller and one larger, with ratios of 0.5 (1 versus 2, 2 versus 4 and 4 versus 8), 0.67 (2 versus 3, 4 versus 6 and 8 versus 12) and 0.75 (3 versus 4, 6 versus 8 and 12 versus 16). Then, we conducted a test using a non-numerical cue where the volume of comparison stimuli was equalized. Jungle crows selected the larger of the two quantities in all comparisons, except those when both quantities were large (6 versus 8, 8 versus 12 and 12 versus 16). Furthermore, accuracy of selection of the larger quantity declined with increasing numerical magnitude. These results suggest that in a spontaneous discrimination task, jungle crows use an object-file mechanism to compare quantities, even when the number of items in one of the arrays exceeds four. The crows showed no preference for the smaller or the larger quantity when the volume cue was removed. This lack of bias may suggest an ecological role of selection for a patch with higher amounts of food and not necessarily the exact number of food items for optimal foraging in jungle crows.

Evidence for Differential Regulation of GnRH Signaling via Heterodimerization among GnRH Receptor Paralogs in the Protochordate, Ciona intestinalis

The endocrine and neuroendocrine systems for reproductive functions have diversified as a result of the generation of species-specific paralogs of peptide hormones and their receptors including GnRH and their receptors (GnRHR), which belong to the class A G protein-coupled receptor family. A protochordate, Ciona intestinalis, has been found to possess seven GnRH (tGnRH-3 to -8 and Ci-GnRH-X) and four GnRHR (Ci-GnRHR1 to -4). Moreover, Ci-GnRHR4 (R4) does not bind to any Ciona GnRH and activate any signaling pathways. Here we show novel functional diversification of GnRH signaling pathways via G protein-coupled receptor heterodimerization among Ciona GnRHR. R4 was shown to heterodimerize with R2 specifically in test cells of vitellogenic oocytes by coimmunoprecipitation. The R2-R4 heterodimerization in human embryonic kidney 293 cells cotransfected with R2 and R4 was also observed by coimmunoprecipitation and fluorescent energy transfer analyses. Of particular interest is that the R2-R4 heterodimer decreases the cAMP production in a nonligand-selective manner via shift of activation of Gs protein to Gi protein by R2, compared with R2 monomer/homodimer. Considering that the R1-R4 heterodimer elicits 10-fold more potent Ca²⁺ mobilization than R1 monomer/homodimer in a ligand-selective manner but does not affect cAMP production, these results indicate that R4 regulates differential GnRH signaling cascades via heterodimerization with R1 and R2 as an endogenous allosteric modulator. Collectively, the present study suggests that the heterodimerization among GnRHR paralogs, including the species-specific orphan receptor subtype, is involved in rigorous and diversified GnRHergic signaling of the protochordate, which lacks a hypothalamus-pituitary gonad axis.

Localization and enzymatic activity profiles of the proteases responsible for tachykinin-directed oocyte growth in the protochordate, Ciona intestinalis

We previously substantiated that Ci-TK, a tachykinin of the protochordate, Ciona intestinalis (Ci), triggered oocyte growth from the vitellogenic stage (stage II) to the post-vitellogenic stage (stage III) via up-regulation of the gene expression and enzymatic activity of the proteases: cathepsin D, carboxypeptidase B1, and chymotrypsin. In the present study, we have elucidated the localization, gene expression and activation profile of these proteases. In situ hybridization showed that the Ci-cathepsin D mRNA was present exclusively in test cells of the stage II oocytes, whereas the Ci-carboxypeptidase B1 and Ci-chymotrypsin mRNAs were detected in follicular cells of the stage II and stage III oocytes. Double-immunostaining demonstrated that the immunoreactivity of Ci-cathepsin D was largely colocalized with that of the receptor of Ci-TK, Ci-TK-R, in test cells of the stage II oocytes. Ci-cathepsin D gene expression was detected at 2h after treatment with Ci-TK, and elevated for up to 5h, and then slightly decreased. Gene expression of Ci-carboxypeptidase B1 and Ci-chymotrypsin was observed at 5h after treatment with Ci-TK, and then decreased. The enzymatic activities of Ci-cathepsin D, Ci-carboxypeptidase B1, and Ci-chymotrypsin showed similar alterations with 1-h lags. These gene expression and protease activity profiles verified that Ci-cathepsin D is initially activated, which is followed by the activation of Ci-carboxypeptidase B1 and Ci-chymotrypsin. Collectively, the present data suggest that Ci-TK directly induces Ci-cahtepsin D in test cells expressing Ci-TK receptor, leading to the secondary activation of Ci-chymotrypsin and Ci-carboxypeptidase B1 in the follicle in the tachykininergic oocyte growth pathway.