Oxford University Press (OUP) Acta Medica Okayama 1743-6095 2025 Dual-action intranasal oxytocin enhances both male sexual performance and fertility in rats 1 3 EN Chica Enomoto Department of Biology, Faculty of Environmental, Life, Natural Science and Technology, Okayama University Takumi Oti Department of Biology, Faculty of Environmental, Life, Natural Science and Technology, Okayama University Takahiro Yamanaka Laboratory of Reproductive Endocrinology, Graduate School of Integrated Sciences for Life, Hiroshima University Masayuki Shimada Laboratory of Reproductive Endocrinology, Graduate School of Integrated Sciences for Life, Hiroshima University Hirotaka Sakamoto Department of Biology, Faculty of Environmental, Life, Natural Science and Technology, Okayama University No potential conflict of interest relevant to this article was reported.

Elsevier BV Acta Medica Okayama 0960-9822 35 12 2025 Oxytocin facilitates human touch-induced play behavior in rats 2916 2926.e3 EN Himeka Hayashi Department of Biology, Faculty of Environmental, Life, Natural Science and Technology, Okayama University, Sayaka Tateishi Ushimado Marine Institute (UMI), Faculty of Environmental, Life, Natural Science and Technology, Okayama University Ayumu Inutsuka Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University Sho Maejima Ushimado Marine Institute (UMI), Faculty of Environmental, Life, Natural Science and Technology, Okayama University Daisuke Hagiwara Department of Neuropeptide Research in Psychiatry, Central Institute of Mental Health, German Center for Psychiatry (DZPG), Medical Faculty Mannheim, University of Heidelberg Yasuo Sakuma Department of Anatomy and Neurobiology, Graduate School of Medical Sciences, Nippon Medical School Tatsushi Onaka Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University Valery Grinevich Department of Neuropeptide Research in Psychiatry, Central Institute of Mental Health, German Center for Psychiatry (DZPG), Medical Faculty Mannheim, University of Heidelberg Hirotaka Sakamoto Department of Biology, Faculty of Environmental, Life, Natural Science and Technology, Okayama University, Pleasant touch sensations play a fundamental role in social bonding, yet the neural mechanisms underlying affinity-like behaviors remain poorly understood. Here, we demonstrate that juvenile-adolescent rats, which naturally engage in social play with peers characterized by rough-and-tumble interactions and 50 kHz ultrasonic vocalizations indicating pleasant sensations, develop a strong affinity for human hands through similar playful contact achieved by repeated tickling with human hands. Using this rat with tickling-induced high affinity for human hands, we discovered that repeated tickling mimicking rough-and-tumble play led to increased oxytocin receptor (OTR) expression in the ventrolateral part of the ventromedial hypothalamus (VMHvl). Inhibition of oxytocin signaling in the VMHvl reduced affinity-like behaviors from rats to human hands. These findings suggest that OTR neurons in VMHvl play an important role in the increase in affinity for human hands induced by pleasant touch sensation with human touch-induced play behavior. Based on retrograde and anterograde tracing studies examining the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) as primary sources of oxytocin, we demonstrate that a subset of oxytocin fibers in the VMHvl originate from the SON, suggesting that affinity-like behavior from rats to human hands may be controlled by oxytocin signaling from magnocellular neurons. Together, this work advances our understanding of how oxytocin shapes social behavior and may inform the development of therapeutic strategies to promote positive social interactions. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2041-1723 16 1 2025 Keratinocyte-driven dermal collagen formation in the axolotl skin 1757 EN Ayaka Ohashi Graduate School of Environment, Life, Natural Science and Technology, Okayama University Hirotaka Sakamoto Graduate School of Environment, Life, Natural Science and Technology, Okayama University Junpei Kuroda Graduate School of Frontier Biosciences, Osaka University Yohei Kondo Center for One Medicine Innovative Translational Research (COMIT), Nagoya University Yasuhiro Kamei Laboratory for Biothermology, National Institute for Basic Biology Shigenori Nonaka The Graduate University for Advanced Studies (SOKENDAI) Saya Furukawa Graduate School of Environment, Life, Natural Science and Technology, Okayama University Sakiya Yamamoto Graduate School of Environment, Life, Natural Science and Technology, Okayama University Akira Satoh Graduate School of Environment, Life, Natural Science and Technology, Okayama University Type I collagen is a major component of the dermis and is formed by dermal fibroblasts. The development of dermal collagen structures has not been fully elucidated despite the major presence and importance of the dermis. This lack of understanding is due in part to the opacity of mammalian skin and it has been an obstacle to cosmetic and medical developments. We reveal the process of dermal collagen formation using the highly transparent skin of the axolotl and fluorescent collagen probes. We clarify that epidermal cells, not dermal fibroblasts, contribute to dermal collagen formation. Mesenchymal cells (fibroblasts) play a role in modifying the collagen fibers already built by keratinocytes. We confirm that collagen production by keratinocytes is a widely conserved mechanism in other model organisms. Our findings warrant a change in the current consensus about dermal collagen formation and could lead to innovations in cosmetology and skin medication. No potential conflict of interest relevant to this article was reported.

Zoological Society of Japan Acta Medica Okayama 0289-0003 41 3 2024 Volume X-Ray Micro-Computed Tomography Analysis of the Early Cephalized Central Nervous System in a Marine Flatworm, Stylochoplana pusilla 281 289 EN Takanori Ikenaga Graduate School of Science and Engineering, Kagoshima University Aoshi Kobayashi Ushimado Marine Institute (UMI), Faculty of Environmental, Life, Natural Science and Technology, Okayama University Akihisa Takeuchi Japan Synchrotron Radiation Research Institute/SPring-8 Kentaro Uesugi Japan Synchrotron Radiation Research Institute/SPring-8 Takanobu Maezawa Department of Integrated Science and Technology, National Institute of Technology, Tsuyama College Norito Shibata Department of Integrated Science and Technology, National Institute of Technology, Tsuyama College Tatsuya Sakamoto Ushimado Marine Institute (UMI), Faculty of Environmental, Life, Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Faculty of Environmental, Life, Natural Science and Technology, Okayama University Platyhelminthes are a phylum of simple bilaterian invertebrates with prototypic body systems. Compared with non-bilaterians such as cnidarians, the bilaterians are likely to exhibit integrated free-moving behaviors, which require a concentrated nervous system “brain” rather than the distributed nervous system of radiatans. Marine flatworms have an early cephalized ‘central’ nervous system compared not only with non-bilaterians but also with parasitic flatworms or freshwater planarians. In this study, we used the marine flatworm Stylochoplana pusilla as an excellent model organism in Platyhelminthes because of the early cephalized central nervous system. Here, we investigated the three-dimensional structures of the flatworm central nervous system by the use of X-ray micro-computed tomography (micro-CT) in a synchrotron radiation facility. We found that the obtained tomographic images were sufficient to discriminate some characteristic structures of the nervous system, including nerve cords around the cephalic ganglion, mushroom body-like structures, and putative optic nerves forming an optic commissure-like structure. Through the micro-CT imaging, we could obtain undistorted serial section images, permitting us to visualize precise spatial relationships of neuronal subpopulations and nerve tracts. 3-D micro-CT is very effective in the volume analysis of the nervous system at the cellular level; the methodology is straightforward and could be applied to many other non-model organisms. No potential conflict of interest relevant to this article was reported.

The Japan Society of Histochemistry and Cytochemistry Acta Medica Okayama 0044-5991 57 2 2024 Membrane-Targeted palGFP Predominantly Localizes to the Plasma Membrane but not to Neurosecretory Vesicle Membranes in Rat Oxytocin Neurons 85 88 EN Hirotaka Sakamoto Department of Biology, Faculty of Environmental, Life, Natural Science and Technology, Okayama University Ayumu Inutsuka Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University Recent advances in viral vector technology, specifically using adeno-associated virus (AAV) vectors, have significantly expanded possibilities in neuronal tracing. We have utilized the Cre/loxP system in combination with AAV techniques in rats to explore the subcellular localization of palmitoylation signal-tagged GFP (palGFP) in oxytocin-producing neurosecretory neurons. A distinctive branching pattern of single axons was observed at the level of the terminals in the posterior pituitary. Despite challenges in detecting palGFP signals by fluorescent microscopy, immunoelectron microscopy demonstrated predominant localization on the plasma membrane, with a minor presence on the neurosecretory vesicle membrane. These findings suggest that membrane-anchored palGFP may undergo exocytosis, translocating from the plasma membrane to the neurosecretory vesicle membrane. In this study, we observed characteristic axon terminal structures in the posterior pituitary of oxytocin neurons. This study indicates the importance of understanding the plasma membrane-specific sorting system in neuronal membrane migration and encourages future studies on the underlying mechanisms. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0953-8194 35 9 2023 Neuropeptidergic control circuits in the spinal cord for male sexual behaviour: Oxytocin–gastrin‐releasing peptide systems e13324 EN Takumi Oti Ushimado Marine Institute (UMI), Faculty of Environmental, Life, Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Faculty of Environmental, Life, Natural Science and Technology, Okayama University The neuropeptidergic mechanisms controlling socio-sexual behaviours consist of complex neuronal circuitry systems in widely distributed areas of the brain and spinal cord. At the organismal level, it is now becoming clear that “hormonal regulations” play an important role, in addition to the activation of neuronal circuits. The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord is an important component of the neural circuits that control penile reflexes in rats, circuits that are commonly referred to as the “spinal ejaculation generator (SEG).” Oxytocin, long known as a neurohypophyseal hormone, is now known to be involved in the regulation of socio-sexual behaviors in mammals, ranging from social bonding to empathy. However, the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system remains unclear. Oxytocin is known to be synthesised mainly in hypothalamic neurons and released from the posterior pituitary into the circulation. Oxytocin is also released from the dendrites of the neurons into the hypothalamus where they have important roles in social behaviours via non-synaptic volume transmission. Because the most familiar functions of oxytocin are to regulate female reproductive functions including parturition, milk ejection, and maternal behaviour, oxytocin is often thought of as a “feminine” hormone. However, there is evidence that a group of parvocellular oxytocin neurons project to the lower spinal cord and control male sexual function in rats. In this report, we review the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system and effects of these neuropeptides on male sexual behaviour. Furthermore, we discuss the finding of a recently identified, localised “volume transmission” role of oxytocin in the spinal cord. Findings from our studies suggest that the newly discovered “oxytocin-mediated spinal control of male sexual function” may be useful in the treatment of erectile and ejaculatory dysfunction. No potential conflict of interest relevant to this article was reported.

Public Library Science Acta Medica Okayama 1932-6203 17 12 2022 Behavioural osmoregulation during land invasion in fish: Prandial drinking and wetting of the dry skin e0277968 EN Yukitoshi Katayama Ushimado Marine Institute, Faculty of Science, Okayama University Takehiro Tsukada Department of Biomolecular Science, Toho University Susumu Hyodo Laboratory of Physiology, Atmosphere and Ocean Research Institute, University of Tokyo Hirotaka Sakamoto Ushimado Marine Institute, Faculty of Science, Okayama University Tatsuya Sakamoto Ushimado Marine Institute, Faculty of Science, Okayama University Osmoregulatory behaviours should have evolutionarily modified for terrestrialisation of vertebrates. In mammals, sensations of buccal food and drying have immediate effects on postprandial thirst to prevent future systemic dehydration, and is thereby considered to be 'anticipatory thirst'. However, it remains unclear whether such an anticipatory response has been acquired in the non-tetrapod lineage. Using the mudskipper goby (Periophthalmus modestus) as a semi-terrestrial ray-finned fish, we herein investigated postprandial drinking and other unique features like full-body 'rolling' over on the back although these behaviours had not been considered to have osmoregulatory functions. In our observations on tidal flats, mudskippers migrated into water areas within a minute after terrestrial eating, and exhibited rolling behaviour with accompanying pectoral-fin movements. In aquarium experiments, frequency of migration into a water area for drinking increased within a few minutes after eating onset, without systemic dehydration. During their low humidity exposure, frequency of the rolling behaviour and pectoral-fin movements increased by more than five times to moisten the skin before systemic dehydration. These findings suggest anticipatory responses which arise from oral/gastrointestinal and cutaneous sensation in the goby. These sensation and motivation seem to have evolved in distantly related species in order to solve osmoregulatory challenges during terrestrialisation. No potential conflict of interest relevant to this article was reported.

Nature Portfolio Acta Medica Okayama 2045-2322 12 1 2022 Mating experiences with the same partner enhanced mating activities of naive male medaka fish 19665 EN Masahiro Daimon Graduate School of Natural Science and Technology, Okayama University Takafumi Katsumura Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Graduate School of Natural Science and Technology, Okayama University Satoshi Ansai Graduate School of Life Sciences, Tohoku University Hideaki Takeuchi Graduate School of Natural Science and Technology, Okayama University Mating experience shapes male mating behavior across species, from insects, fish, and birds, to rodents. Here, we investigated the effect of multiple mating experiences on male mating behavior in "naive" (defined as sexually inexperienced) male medaka fish. The latency to mate with the same female partner significantly decreased after the second encounter, whereas when the partner was changed, the latency to mate was not decreased. These findings suggest that mating experiences enhanced the mating activity of naive males for the familiar female, but not for an unfamiliar female. In contrast, the mating experiences of "experienced" (defined as those having mated > 7 times) males with the same partner did not influence their latency to mate. Furthermore, we identified 10 highly and differentially expressed genes in the brains of the naive males after the mating experience and revealed 3 genes that are required for a functional cascade of the thyroid hormone system. Together, these findings suggest that the mating experience of naive male medaka fish influences their mating behaviors, with neural changes triggered by thyroid hormone activation in the brain. No potential conflict of interest relevant to this article was reported.

The Royal Society Acta Medica Okayama 0962-8452 289 1985 2022 Footedness for scratching itchy eyes in rodents 20221126 EN Yukitoshi Katayama Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi Ayane Miura Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi Keiko Takanami Mouse Genomics Resources Laboratory, National Institute of Genetics, Yata, Mishima Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado, Setouchi The neural bases of itchy eye transmission remain unclear compared with those involved in body itch. Here, we show in rodents that the gastrin-releasing peptide receptor (GRPR) of the trigeminal sensory system is involved in the transmission of itchy eyes. Interestingly, we further demonstrate a difference in scratching behaviour between the left and right hindfeet in rodents; histamine instillation into the conjunctival sac of both eyes revealed right-foot biased laterality in the scratching movements. Unilateral histamine instillation specifically induced neural activation in the ipsilateral sensory pathway, with no significant difference between the activations following left- and right-eye instillations. Thus, the behavioural laterality is presumably due to right-foot preference in rodents. Genetically modified rats with specific depletion of Grpr-expressing neurons in the trigeminal sensory nucleus caudalis of the medulla oblongata exhibited fewer and shorter histamine-induced scratching movements than controls and eliminated the footedness. These results taken together indicate that the Grp-expressing neurons are required for the transmission of itch sensation from the eyes, but that foot preference is generated centrally. These findings could open up a new field of research on the mechanisms of the laterality in vertebrates and also offer new potential therapeutic approaches to refractory pruritic eye disorders. No potential conflict of interest relevant to this article was reported.

CELL PRESS Acta Medica Okayama 2589-0042 25 7 2022 Lattice-patterned collagen fibers and their dynamics in axolotl skin regeneration 104524 EN Rena Kashimoto Division of Earth, Life, and Molecular Sciences, Graduate School of Natural Science and Technology, Okayama University Saya Furukawa Department of Biological Sciences, Faculty of Science, Okayama University Sakiya Yamamoto Department of Biological Sciences, Faculty of Science, Okayama University Yasuhiro Kamei National Institute for Basic Biology (NIBB), National Institutes for Natural Sciences Joe Sakamoto National Institute for Basic Biology (NIBB), National Institutes for Natural Sciences Shigenori Nonaka National Institute for Basic Biology (NIBB), National Institutes for Natural Sciences Tomonobu M. Watanabe Laboratory for Comprehensive Bioimaging, RIKEN Center for Biosystems Dynamics Research (BDR) Tatsuya Sakamoto Division of Earth, Life, and Molecular Sciences, Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Division of Earth, Life, and Molecular Sciences, Graduate School of Natural Science and Technology, Okayama University Akira Satoh Research Core for Interdisciplinary Sciences (RCIS), Okayama University The morphology of collagen-producing cells and the structure of produced collagen in the dermis have not been well-described. This lack of insights has been a serious obstacle in the evaluation of skin regeneration. We succeeded in visualizing collagen-producing cells and produced collagen using the axolotl skin, which is highly transparent. The visualized dermal collagen had a lattice-like structure. The collagen-producing fibroblasts consistently possessed the lattice-patterned filopodia along with the lattice-patterned collagen network. The dynamics of this lattice-like structure were also verified in the skin regeneration process of axolotls, and it was found that the correct lattice-like structure was not reorganized after simple skin wounding but was reorganized in the presence of nerves. These findings are not only fundamental insights in dermatology but also valuable insights into the mechanism of skin regeneration. No potential conflict of interest relevant to this article was reported.

American Association for the Advancement of Science (AAAS) Acta Medica Okayama 2375-2548 8 9 2022 Vasopressin-oxytocin–type signaling is ancient and has a conserved water homeostasis role in euryhaline marine planarians eabk0331 EN Aoshi Kobayashi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Mayuko Hamada Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Masa-aki Yoshida Oki Marine Biological Station, Shimane University Yasuhisa Kobayashi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Naoaki Tsutsui Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Toshio Sekiguchi Noto Marine Laboratory, Institute of Nature and Environmental Technology, Division of Marine Environmental Studies, Kanazawa University Yuta Matsukawa Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Sho Maejima Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Joseph J. Gingell Vertex Pharmaceuticals (Europe) Ltd. Shoko Sekiguchi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Ayumu Hamamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Debbie L. Hay School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland John F. Morris Department of Physiology, Anatomy, and Genetic, Le Gros Clark Building, University of Oxford Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University, Ushimado Vasopressin/oxytocin (VP/OT)–related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the “platytocin” system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an “antidiuretic hormone” and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 22 19 2021 Sexual Experience Induces the Expression of Gastrin-Releasing Peptide and Oxytocin Receptors in the Spinal Ejaculation Generator in Rats 10362 EN Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Ryota Ueda Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Ryoko Kumagai Department of Animal Sciences, Teikyo University of Science Junta Nagafuchi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Takashi Ito Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yasuhiko Kondo Department of Animal Sciences, Teikyo University of Science Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Male sexual function in mammals is controlled by the brain neural circuits and the spinal cord centers located in the lamina X of the lumbar spinal cord (L3-L4). Recently, we reported that hypothalamic oxytocin neurons project to the lumbar spinal cord to activate the neurons located in the dorsal lamina X of the lumbar spinal cord (dXL) via oxytocin receptors, thereby facilitating male sexual activity. Sexual experiences can influence male sexual activity in rats. However, how this experience affects the brain-spinal cord neural circuits underlying male sexual activity remains unknown. Focusing on dXL neurons that are innervated by hypothalamic oxytocinergic neurons controlling male sexual function, we examined whether sexual experience affects such neural circuits. We found that >50% of dXL neurons were activated in the first ejaculation group and similar to 30% in the control and intromission groups in sexually naive males. In contrast, in sexually experienced males, similar to 50% of dXL neurons were activated in both the intromission and ejaculation groups, compared to similar to 30% in the control group. Furthermore, sexual experience induced expressions of gastrin-releasing peptide and oxytocin receptors in the lumbar spinal cord. This is the first demonstration of the effects of sexual experience on molecular expressions in the neural circuits controlling male sexual activity in the spinal cord. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 22 17 2021 Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at-25 degrees C for Several Years 9180 EN Akito Otubo Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Sho Maejima Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Keita Satoh Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yasumasa Ueda Department of Physiology, Kyoto Prefectural University of Medicine John F. Morris Department of Physiology, Anatomy & Genetics, University of Oxford, South Parks Road Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at -25 degrees C for several years (4-6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research. No potential conflict of interest relevant to this article was reported.

Nature Research Acta Medica Okayama 2045-2322 11 1 2021 The gastrin-releasing peptide/bombesin system revisited by a reverse-evolutionary study considering Xenopus 13315 EN Asuka Hirooka Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Mayuko Hamada Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Daiki Fujiyama Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Keiko Takanami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yasuhisa Kobayashi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yukitoshi Katayama Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Bombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (Xenopus tropicalis), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand the derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in Xenopus. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of Xenopus. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the 'gut-brain peptide' systems. No potential conflict of interest relevant to this article was reported.

MDPI Acta Medica Okayama 1422-0067 22 7 2021 In Vivo Electrophysiology of Peptidergic Neurons in Deep Layers of the Lumbar Spinal Cord after Optogenetic Stimulation of Hypothalamic Paraventricular Oxytocin Neurons in Rats 3400 EN Daisuke Uta Department of Applied Pharmacology, Faculty of Pharmaceutical Sciences, University of Toyama Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University The spinal ejaculation generator (SEG) is located in the central gray (lamina X) of the rat lumbar spinal cord and plays a pivotal role in the ejaculatory reflex. We recently reported that SEG neurons express the oxytocin receptor and are activated by oxytocin projections from the paraventricular nucleus of hypothalamus (PVH). However, it is unknown whether the SEG responds to oxytocin in vivo. In this study, we analyzed the characteristics of the brain-spinal cord neural circuit that controls male sexual function using a newly developed in vivo electrophysiological technique. Optogenetic stimulation of the PVH of rats expressing channel rhodopsin under the oxytocin receptor promoter increased the spontaneous firing of most lamina X SEG neurons. This is the first demonstration of the in vivo electrical response from the deeper (lamina X) neurons in the spinal cord. Furthermore, we succeeded in the in vivo whole-cell recordings of lamina X neurons. In vivo whole-cell recordings may reveal the features of lamina X SEG neurons, including differences in neurotransmitters and response to stimulation. Taken together, these results suggest that in vivo electrophysiological stimulation can elucidate the neurophysiological response of a variety of spinal neurons during male sexual behavior. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0953-8194 32 8 2020 Vasopressin gene products are colocalised with corticotrophin‐releasing factor within neurosecretory vesicles in the external zone of the median eminence of the Japanese macaque monkey (Macaca fuscata) e12875 EN Akito Otubo Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Natsuko Kawakami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Sho Maejima Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yasumasa Ueda Department of Physiology, Kyoto Prefectural University of Medicine John F. Morris Department of Physiology, Anatomy & Genetics, University of Oxford Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Arginine vasopressin (AVP), when released into portal capillaries with corticotrophin‐releasing factor (CRF) from terminals of parvocellular neurones of the hypothalamic paraventricular nucleus (PVH), facilitates the secretion of adrenocorticotrophic hormone (ACTH) in stressed rodents. The AVP gene encodes a propeptide precursor containing AVP, AVP‐associated neurophysin II (NPII), and a glycopeptide copeptin, although it is currently unclear whether copeptin is always cleaved from the neurophysin and whether the NPII and/or copeptin have any functional role in the pituitary. Furthermore, for primates, it is unknown whether CRF, AVP, NPII and copeptin are all colocalised in neurosecretory vesicles in the terminal region of the paraventricular CRF neurone axons. Therefore, we investigated, by fluorescence and immunogold immunocytochemistry, the cellular and subcellular relationships of these peptides in the CRF‐ and AVP‐producing cells in unstressed Japanese macaque monkeys (Macaca fuscata). Reverse transcription‐polymerase chain reaction analysis showed the expression of both CRF and AVP mRNAs in the monkey PVH. As expected, in the magnocellular neurones of the PVH and supraoptic nucleus, essentially no CRF immunoreactivity could be detected in NPII‐immunoreactive (AVP‐producing) neurones. Immunofluorescence showed that, in the parvocellular part of the PVH, NPII was detectable in a subpopulation (approximately 39%) of the numerous CRF‐immunoreactive neuronal perikarya, whereas, in the outer median eminence, NPII was more prominent (approximately 52%) in the CRF varicosities. Triple immunoelectron microscopy in the median eminence demonstrated the presence of both NPII and copeptin immunoreactivity in dense‐cored vesicles of CRF‐containing axons. The results are consistent with an idea that the AVP propeptide is processed and NPII and copeptin are colocalised in hypothalamic‐pituitary CRF axons in the median eminence of a primate. The CRF, AVP and copeptin are all co‐packaged in neurosecretory vesicles in monkeys and are thus likely to be co‐released into the portal capillary blood to amplify ACTH release from the primate anterior pituitary. No potential conflict of interest relevant to this article was reported.

Cell Press Acta Medica Okayama 2589-0042 23 10 2020 Degradation of Mutant Protein Aggregates within the Endoplasmic Reticulum of Vasopressin Neurons 101648 EN Takashi Miyata Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Daisuke Hagiwara Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Yuichi Hodai Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Tsutomu Miwata Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Yohei Kawaguchi Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Junki Kurimoto Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Hajime Ozaki Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Kazuki Mitsumoto Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Hiroshi Takagi Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Hidetaka Suga Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Tomoko Kobayashi Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Mariko Sugiyama Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Takeshi Onoue Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Yoshihiro Ito Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Shintaro Iwama Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Ryoichi Banno Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Mami Matsumoto Section of Electron Microscopy, Supportive Center for Brain Research, National Institute for Physiological Sciences Natsuko Kawakami Section of Electron Microscopy, Supportive Center for Brain Research, National Institute for Physiological Sciences Nobuhiko Ohno Department of Anatomy, Division of Histology and Cell Biology, Jichi Medical University, School of Medicine Hirotaka Sakamoto Ushimado Marine Institute, Graduate School of Natural Science and Technology, Okayama University Hiroshi Arima Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine Misfolded or unfolded proteins in the ER are said to be degraded only after translocation or isolation from the ER. Here, we describe a mechanism by which mutant proteins are degraded within the ER. Aggregates of mutant arginine vasopressin (AVP) precursor were confined to ER-associated compartments (ERACs) connected to the ER in AVP neurons of a mouse model of familial neurohypophysial diabetes insipidus. The ERACs were enclosed by membranes, an ER chaperone and marker protein of phagophores and autophagosomes were expressed around the aggregates, and lysosomes fused with the ERACs. Moreover, lysosome-related molecules were present within the ERACs, and aggregate degradation within the ERACs was dependent on autophagic-lysosomal activity. Thus, we demonstrate that protein aggregates can be degraded by autophagic-lysosomal machinery within specialized compartments of the ER. No potential conflict of interest relevant to this article was reported.

Elsevier Acta Medica Okayama 0960-9822 31 1 2020 Oxytocin Influences Male Sexual Activity via Non-synaptic Axonal Release in the Spinal Cord 103 114.e5 EN Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Keita Satoh Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Daisuke Uta Department of Applied Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama Junta Nagafuchi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Sayaka Tateishi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Ryota Ueda Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Keiko Takanami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Larry J. Young Center for Translational Social Neuroscience, Silvio O. Conte Center for Oxytocin and Social Cognition, Department of Psychiatry and Behavioral Sciences, Yerkes National Primate Research Center, Emory University Antony Galione Department of Pharmacology, University of Oxford John F. Morris Department of Physiology, Anatomy & Genetics, University of Oxford Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Oxytocinergic neurons in the paraventricular nucleus of the hypothalamus that project to extrahypothalamic brain areas and the lumbar spinal cord play an important role in the control of erectile function and male sexual behavior in mammals. The gastrin-releasing peptide (GRP) system in the lumbosacral spinal cord is an important component of the neural circuits that control penile reflexes in rats, circuits that are commonly referred to as the “spinal ejaculation generator (SEG).” We have examined the functional interaction between the SEG neurons and the hypothalamo-spinal oxytocin system in rats. Here, we show that SEG/GRP neurons express oxytocin receptors and are activated by oxytocin during male sexual behavior. Intrathecal injection of oxytocin receptor antagonist not only attenuates ejaculation but also affects pre-ejaculatory behavior during normal sexual activity. Electron microscopy of potassium-stimulated acute slices of the lumbar cord showed that oxytocin-neurophysin-immunoreactivity was detected in large numbers of neurosecretory dense-cored vesicles, many of which are located close to the plasmalemma of axonal varicosities in which no electron-lucent microvesicles or synaptic membrane thickenings were visible. These results suggested that, in rats, release of oxytocin in the lumbar spinal cord is not limited to conventional synapses but occurs by exocytosis of the dense-cored vesicles from axonal varicosities and acts by diffusion―a localized volume transmission―to reach oxytocin receptors on GRP neurons and facilitate male sexual function. No potential conflict of interest relevant to this article was reported.

Wiley Acta Medica Okayama 0021-9967 529 7 2020 Variation of pro‐vasopressin processing in parvocellular and magnocellular neurons in the paraventricular nucleus of the hypothalamus: Evidence from the vasopressin‐related glycopeptide copeptin 1372 1390 EN Natsuko Kawakami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Akito Otubo Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Sho Maejima Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Ashraf H. Talukder Laboratory of Information Biology, Graduate School of Information Sciences, Tohoku University Keita Satoh Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Keiko Takanami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yasumasa Ueda Department of Physiology, Kyoto Prefectural University of Medicine Keiichi Itoi Laboratory of Information Biology, Graduate School of Information Sciences, Tohoku University John F. Morris Department of Physiology, Anatomy and Genetics, University of Oxford Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Arginine vasopressin (AVP) is synthesized in parvocellular‐ and magnocellular neuroendocrine neurons in the paraventricular nucleus (PVN) of the hypothalamus. Whereas magnocellular AVP neurons project primarily to the posterior pituitary, parvocellular AVP neurons project to the median eminence (ME) and to extrahypothalamic areas. The AVP gene encodes pre‐pro‐AVP that comprises the signal peptide, AVP, neurophysin (NPII), and a copeptin glycopeptide. In the present study, we used an N‐terminal copeptin antiserum to examine copeptin expression in magnocellular and parvocellular neurons in the hypothalamus in the mouse, rat, and macaque monkey. Although magnocellular NPII‐expressing neurons exhibited strong N‐terminal copeptin immunoreactivity in all three species, a great majority (~90%) of parvocellular neurons that expressed NPII was devoid of copeptin immunoreactivity in the mouse, and in approximately half (~53%) of them in the rat, whereas in monkey hypothalamus, virtually all NPII‐immunoreactive parvocellular neurons contained strong copeptin immunoreactivity. Immunoelectron microscopy in the mouse clearly showed copeptin‐immunoreactivity co‐localized with NPII‐immunoreactivity in neurosecretory vesicles in the internal layer of the ME and posterior pituitary, but not in the external layer of the ME. Intracerebroventricular administration of a prohormone convertase inhibitor, hexa‐d‐arginine amide resulted in a marked reduction of copeptin‐immunoreactivity in the NPII‐immunoreactive magnocellular PVN neurons in the mouse, suggesting that low protease activity and incomplete processing of pro‐AVP could explain the disproportionally low levels of N‐terminal copeptin expression in rodent AVP (NPII)‐expressing parvocellular neurons. Physiologic and phylogenetic aspects of copeptin expression among neuroendocrine neurons require further exploration. No potential conflict of interest relevant to this article was reported.

Japanese Society of Plant Physiologists Acta Medica Okayama 0032-0781 55 7 2014 Quality control of photosystem II: direct imaging of the changes in the thylakoid structure and distribution of FtsH proteases in spinach chloroplasts under light stress 1255 1265 EN Miho Yoshioka-Nishimura Graduate School of Natural Science and Technology, Okayama University Daisuke Nanba Graduate School of Natural Science and Technology, Okayama University Takashi Takaki Techinical support center, JEOL Chikako Ohba Graduate School of Natural Science and Technology, Okayama University Nodoka Tsumura Graduate School of Natural Science and Technology, Okayama University Noriko Morita Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute, Okayama University Kazuyoshi Murata National Institute for Physiological Sciences Yasusi Yamamoto Graduate School of Natural Science and Technology, Okayama University 　Under light stress, the reaction center-binding protein D1 of PSII is photo-oxidatively damaged and removed from PSII complexes by proteases located in the chloroplast. A protease considered to be responsible for degradation of the damaged D1 protein is the metalloprotease FtsH. We showed previously that the active hexameric FtsH protease is abundant at the grana margin and the grana end membranes, and this homo-complex removes the photodamaged D1 protein in the grana. Here, we showed a change in the distribution of FtsH in spinach thylakoids during excessive illumination by transmission electron microscopy (TEM) and immunogold labeling of FtsH. The change in distribution of the protease was accompanied by structural changes to the thylakoids, which we detected using spinach leaves by TEM after chemical fixation of the samples. Quantitative analyses showed several characteristic changes in the structure of the thylakoids, including shrinkage of the grana, outward bending of the marginal portions of the thylakoids and an increase in the height of the grana stacks under excessive illumination. The increase in the height of the grana stacks may include swelling of the thylakoids and an increase in the partition gaps between the thylakoids. These data strongly suggest that excessive illumination induces partial unstacking of the thylakoids, which enables FtsH to access easily the photodamaged D1 protein. Finally three-dimensional tomography of the grana was recorded to observe the effect of light stress on the overall structure of the thylakoids. No potential conflict of interest relevant to this article was reported.

Wistar Institute of Anatomy and Biology Acta Medica Okayama 0021-9967 525 7 2017 Identification of the sexually dimorphic gastrin-releasing peptide system in the lumbosacral spinal cord that controls male reproductive function in the mouse and Asian house musk shrew (Suncus murinus) 1586 1598 EN Kei Tamura Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Yasuhisa Kobayashi Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Asuka Hirooka Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Keiko Takanami Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Takumi Oti Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Takamichi Jogahara Laboratory of Animal Management and Resources, Department of Zoology, Okayama University of Science Sen-ichi Oda Laboratory of Animal Management and Resources, Department of Zoology, Okayama University of Science Tatsuya Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Hirotaka Sakamoto Ushimado Marine Institute (UMI), Graduate School of Natural Science and Technology, Okayama University Several regions of the brain and spinal cord control male reproductive function. We previously demonstrated that the gastrin-releasing peptide (GRP) system, located in the lumbosacral spinal cord of rats, controls spinal centers to promote penile reflexes during male copulatory behavior. However, little information exists on the male-specific spinal GRP system in animals other than rats. The objective of this study was to examine the functional generality of the spinal GRP system in mammals using the Asian house musk shrew (Suncus murinus; suncus named as the laboratory strain), a specialized placental mammal model. Mice are also used for a representative model of small laboratory animals. We first isolated complementary DNA encoding GRP in suncus. Phylogenetic analysis revealed that suncus preproGRP was clustered to an independent branch. Reverse transcription-PCR showed that GRP and its receptor mRNAs were both expressed in the lumbar spinal cord of suncus and mice. Immunohistochemistry for GRP demonstrated that the sexually dimorphic GRP system and male-specific expression/distribution patterns of GRP in the lumbosacral spinal cord in suncus are similar to those of mice. In suncus, we further found that most GRP-expressing neurons in males also express androgen receptors, suggesting that this male-dominant system in suncus is also androgen-dependent. Taken together, these results indicate that the sexually dimorphic spinal GRP system exists not only in mice but also in suncus, suggesting that this system is a conserved property in mammals. No potential conflict of interest relevant to this article was reported.