Sen-ichi Oda

Heterochrony and developmental modularity of cranial osteogenesis in lipotyphlan mammals

BackgroundHere we provide the most comprehensive study to date on the cranial ossification sequence in Lipotyphla, the group which includes shrews, moles and hedgehogs. This unique group, which encapsulates diverse ecological modes, such as terrestrial, subterranean, and aquatic lifestyles, is used to examine the evolutionary lability of cranial osteogenesis and to investigate the modularity of development.ResultsAn acceleration of developmental timing of the vomeronasal complex has occurred in the common ancestor of moles. However, ossification of the nasal bone has shifted late in the more terrestrial shrew mole. Among the lipotyphlans, sequence heterochrony shows no significant association with modules derived from developmental origins (that is, neural crest cells vs. mesoderm derived parts) or with those derived from ossification modes (that is, dermal vs. endochondral ossification).ConclusionsThe drastic acceleration of vomeronasal development in moles is most likely coupled with the increased importance of the rostrum for digging and its use as a specialized tactile surface, both fossorial adaptations. The late development of the nasal in shrew moles, a condition also displayed by hedgehogs and shrews, is suggested to be the result of an ecological reversal to terrestrial lifestyle and reduced functional importance of the rostrum. As an overall pattern in lipotyphlans, our results reject the hypothesis that ossification sequence heterochrony occurs in modular fashion when considering the developmental patterns of the skull. We suggest that shifts in the cranial ossification sequence are not evolutionarily constrained by developmental origins or mode of ossification.

Coordination of motilin and ghrelin regulates the migrating motor complex of gastrointestinal motility in Suncus murinus

Motilin and ghrelin are the gastrointestinal (GI) hormones released in a fasting state to stimulate the GI motility of the migrating motor complex (MMC). We focused on coordination of the ghrelin/motilin family in gastric contraction in vivo and in vitro using the house musk shrew (Suncus murinus), a ghrelin- and motilin-producing mammal. To measure the contractile activity of the stomach in vivo, we recorded GI contractions either in the free-moving conscious or anesthetized S. murinus and examined the effects of administration of motilin and/or ghrelin on spontaneous MMC in the fasting state. In the in vitro study, we also studied the coordinative effect of these hormones on the isolated stomach using an organ bath. In the fasting state, phase I, II, and III contractions were clearly recorded in the gastric body (as observed in humans and dogs). Intravenous infusion of ghrelin stimulated gastric contraction in the latter half of phase I and in the phase II in a dose-dependent manner. Continuous intravenous infusion of ghrelin antagonist (d-Lys3-GHRP6) significantly suppressed spontaneous phase II contractions and prolonged the time of occurrence of the peak of phase III contractions. However, intravenous infusion of motilin antagonist (MA-2029) did not inhibit phase II contractions but delayed the occurrence of phase III contractions of the MMC. In the in vitro study, even though a high dose of ghrelin did not stimulate contraction of stomach preparations, ghrelin administration (10(-10)-10(-7) M) with pretreatment of a low dose of motilin (10(-10) M) induced gastric contraction in a dose-dependent manner. Pretreatment with 10(-8) M ghrelin enhanced motilin-stimulated gastric contractions by 10 times. The interrelation of these peptides was also demonstrated in the anesthetized S. murinus. The results suggest that ghrelin is important for the phase II contraction and that coordination of motilin and ghrelin are necessary to initiate phase III contraction of the MMC.

Myenteric neural network activated by motilin in the stomach of Suncus murinus (house musk shrew)

Background  It has been shown in human and canine studies that motilin, a gastroprokinetic hormone, induces gastric phase III contractions via the enteric nervous; however, the center of motilin action in the stomach has not been clearly revealed. In the present study, we investigated the neural pathway of motilin‐induced gastric contraction by using Suncus murinus, a new animal model for motilin study.

Mechanism of Ghrelin-Induced Gastric Contractions in Suncus murinus (House Musk Shrew): Involvement of Intrinsic Primary Afferent Neurons

Here, we have reported that motilin can induce contractions in a dose-dependent manner in isolated Suncus murinus (house musk shrew) stomach. We have also shown that after pretreatment with a low dose of motilin (10−10 M), ghrelin also induces gastric contractions at levels of 10−10 M to 10−7 M. However, the neural mechanism of ghrelin action in the stomach has not been fully revealed. In the present study, we studied the mechanism of ghrelin-induced contraction in vitro using a pharmacological method. The responses to ghrelin in the stomach were almost completely abolished by hexamethonium and were significantly suppressed by the administration of phentolamine, prazosin, ondansetron, and naloxone. Additionally, N-nitro-l-arginine methylester significantly potentiated the contractions. Importantly, the mucosa is essential for ghrelin-induced, but not motilin-induced, gastric contractions. To evaluate the involvement of intrinsic primary afferent neurons (IPANs), which are multiaxonal neurons that pass signals from the mucosa to the myenteric plexus, we examined the effect of the IPAN-related pathway on ghrelin-induced contractions and found that pretreatment with adenosine and tachykinergic receptor 3 antagonists (SR142801) significantly eliminated the contractions and GR113808 (5-hydroxytryptamine receptor 4 antagonist) almost completely eliminated it. The results indicate that ghrelin stimulates and modulates suncus gastric contractions through cholinergic, adrenergic, serotonergic, opioidergic neurons and nitric oxide synthases in the myenteric plexus. The mucosa is also important for ghrelin-induced gastric contractions, and IPANs may be the important interneurons that pass the signal from the mucosa to the myenteric plexus.

Motilin Stimulates Gastric Acid Secretion in Coordination with Ghrelin in Suncus murinus

Motilin and ghrelin constitute a peptide family, and these hormones are important for the regulation of gastrointestinal motility. In this study, we examined the effect of motilin and ghrelin on gastric acid secretion in anesthetized suncus (house musk shrew, Suncus murinus), a ghrelin- and motilin-producing mammal. We first established a gastric lumen-perfusion system in the suncus and confirmed that intravenous (i.v.) administration of histamine (1 mg/kg body weight) stimulated acid secretion. Motilin (0.1, 1.0, and 10 μg/kg BW) stimulated the acid output in a dose-dependent manner in suncus, whereas ghrelin (0.1, 1.0, and 10 μg/kg BW) alone did not induce acid output. Furthermore, in comparison with the vehicle administration, the co-administration of low-dose (1 μg/kg BW) motilin and ghrelin significantly stimulated gastric acid secretion, whereas either motilin (1 μg/kg BW) or ghrelin (1 μg/kg BW) alone did not significantly induce gastric acid secretion. This indicates an additive role of ghrelin in motilin-induced gastric acid secretion. We then investigated the pathways of motilin/motilin and ghrelin-stimulated acid secretion using receptor antagonists. Treatment with YM 022 (a CCK-B receptor antagonist) and atropine (a muscarinic acetylcholine receptor antagonist) had no effect on motilin or motilin-ghrelin co-administration-induced acid output. In contrast, famotidine (a histamine H2 receptor antagonist) completely inhibited motilin-stimulated acid secretion and co-administration of motilin and ghrelin induced gastric acid output. This is the first report demonstrating that motilin stimulates gastric secretion in mammals. Our results also suggest that motilin and co-administration of motilin and ghrelin stimulate gastric acid secretion via the histamine-mediated pathway in suncus.

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)

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. J. Comp. Neurol. 525:1586–1598, 2017.

Factors Affecting the Distribution of the Japanese Weasel Mustela itatsi and the Siberian Weasel M. sibirica in Japan

Abstract. The vertical distribution of introduced Siberian weasels Mustela sibirica and endemic Japanese weasels M. itatsi in the Seburi Mountains in Kyushu, Japan, was examined from October 1996 to February 1998. Siberian weasels occurred near villages with paddy and cultivated fields, whereas Japanese weasels occurred in grasslands and plantations. The dispersion of yearlings destabilized the distributions of both weasel species. The horizontal distribution of both species throughout Japan was examined by means of collection of dead specimens and by trapping from March 1998 to March 2002. The eastern boundary of the distribution of the Siberian weasel was Fukui, Nagano, and Aichi prefectures; however, the distribution is expanding slowly eastward. The Siberian weasel cannot invade new habitats that lack nearby villages in Seburi, and cannot expand its range in the eastern area of Aichi, where Japanese weasels are dominant. The presence of the Japanese weasel likely prevents expansion of the distribution of the Siberian weasel.

Immunolocalization of Spetex-1 at the Connecting Piece in Spermatozoa of the Musk Shrew (Suncus murinus)

Spetex-1, which has been isolated by differential display and rat cDNA library screening as a haploid spermatid-specific gene, encodes a protein with two coiled-coil motifs that locates at both the segmented column in the connecting piece and outer dense fibers-affiliated satellite fibrils in rat sperm flagella. Orthologs of Spetex-1 are identified in many animal species, including human, chimpanzee, macaque, cow, dog, African clawed frog, green spotted puffer, and zebrafish. In this study, we used RT-PCR in combination with 5′ and 3′ RACE (Rapid Amplification of cDNA End) technique to isolate Spetex-1 ortholog of the musk shrew (Suneus murinus), which yielded a full-length Suncus Spetex-1 gene containing an open reading frame of 1,908 base pairs encoding a protein of 636 amino acids with the predicted molecular mass of 72,348 Da. Suncus Spetex-1 has two coiled-coil motifs at 118–184 and 242–276 amino acid residues, which is a characteristic shared by mammalian Spetex-1 proteins. To examine the subcellular localization of Spetex-1 in Suncus spermatozoa, we produced the anti-Suncus Spetex-1 antibody and carried out immunocytochemistry. In spite of that the primary structure of Suncus Spetex-1 is basically similar to that of rat and mouse Spetex-1, confocal laser scanning microscopy and immunoelectron microscopy revealed that Spetex-1 was restricted to the segmented column and capitulum in the connecting piece of Suncus spermatozoa and was not detected in other parts of flagella, suggesting a diversity of Spetex-1 localization in mammalian spermatozoa.

Expression patterns of Fgf8 and Shh in the developing external genitalia of Suncus murinus

Reciprocal epithelial-mesenchymal interactions and several signalling pathways regulate the development of the genital tubercle (GT), an embryonic primordium of external genitalia. The morphology of the adult male external genitalia of the Asian house musk shrew Suncus murinus (hereafter, laboratory name: suncus) belonging to the order Eulipotyphla (the former order Insectivora or Soricomorpha) differs from those of mice and humans. However, the developmental process of the suncus GT and its regulatory genes are unknown. In the present study, we explored the morphological changes and gene expression patterns during the development of the suncus GT. Morphological observations suggested the presence of common (during the initial outgrowth) and species-specific (during the sexual differentiation of GT) developmental processes of the suncus GT. In gene expression analysis, fibroblast growth factor 8 (Fgf8) and sonic hedgehog (Shh), an indicator and regulator of GT development in mice respectively, were found to be expressed in the cloacal epithelium and the developing urethral epithelium of the suncus GT. This pattern of expression specifically in GT epithelium is similar to that observed in the developing mouse GT. Our results indicate that the mechanism of GT formation regulated by the FGF and SHH signalling pathways is widely conserved in mammals.

Monoclonal Suncus Antibodies: Generation of Fusion Partners to Produce Suncus-Suncus Hybridomas

We used suncus (Suncus murinus; house musk shrew) to generate partner cells for cell fusion to produce suncus monoclonal antibodies. Suncus are insectivores that are genetically distant to rodents, and recognize antigens and epitopes that are not immunogenic in mice and rats, which are the animals most commonly used in basic life science research and from which monoclonal antibodies are usually produced. To date, monoclonal antibodies from suncus have not been generated due to the lack of a plasmacytoma fusion partner. To obtain suncus plasmacytoma cell lines suitable as a cell fusion partner, we injected suncus at both sides of the tail base with antigen emulsion, collected the lymph nodes and spleens, and cultured the cells to obtain immortalized lymphoid cell lines visually resembling mouse SP2/0-Ag14 myeloma cells. Three suncus immunized with the antigen provided 4 cell lines of suncus plasmacytoma, but they did not secrete immunoglobulins. Antibody-producing hybrid cells were generated from these cell lines using a cell fusion technique. Using one of the cell lines as a fusion partner, we obtained six lines of immunoglobulin-producing hybrid cells which secreted an unidentified monoclonal IgG. When these 6 lines were used as new fusion partners, we obtained several hybrid cell lines which secreted immunogen-specific monoclonal antibodies. These hybrid cells can be cloned and cryopreserved. We also obtained another good fusion partner which initially secreted antibody but later stopped doing so. These suncus-suncus hybrid cell lines will be useful for the production of suncus monoclonal antibodies.