Food habit of the juvenile of the Japanese newt Cynops pyrrhogaster

The previous study showed that the red coloration of the ventral skin of the Japanese newt Cynops pyrrhogaster was associated with the number of carotenoid vesicles and the content of carotenoid in the pigment cell of the skin. To elucidate the mechanism for the red coloration of the skin of the newt, we studied the food habit of the juvenile from the Japanese newt Cynops pyrrhogaster. Sixty-two juveniles were collected in Fukue Island in Nagasaki Prefecture from November 2000 to May 2002 and divided into 2 groups according to the snout-vent length (SVL). Over 400 prey animals were obtained from the juveniles by stomach flushing. In the larger group (SVL>30.0mm), Collembola (45.4%) and Acari (12.6%), which are very common species of soil animals, were the prey animals dominant in number. In the group with the smaller SVL (<29.9mm), Collembola (30.4%) and Acari (25.4%) were in number as well. We also studied the food habit of the Japanese clouded salamander, Hynobius nebulosus. In the salamander, Doratodesmidae (56.5%) and Amphipoda (13%) were the prey animals dominant in number. Our results, taken together, suggest that the Japanese juvenile C. pyrrhogaster does not change its food habit as it grows, and that it eats soil animals common in its habitat. Moreover, the food habit of juvenile C. pyrrhogaster differs from that of H. nebulosus, although the juveniles of both species live in the same area.     

'NUAGE'-CHROMOSOME ASSOCIATION IN METAPHASE SPERMATOGONIA OF THE NEWT, CYNOPUS PYRRHOGASTER

The 'nuage', an electron dense cytoplasmic component specific to the germ line cell, was found to be closely associated with a metaphase chromosome in the spermatogenic cell of the newt, Triturus (Cynops) pyrrhogaster. This finding suggests a possible relationship between both components at a certain stage of spermatogenesis.     

Peptide pheromones in newts

This article reviews the current state of understanding of reproductive pheromones in amphibians, focusing mainly on the purification and characterization of peptide pheromones in newts of the genus Cynops, molecular cloning of cDNAs encoding the pheromone molecules, and hormonal control of secretion of these pheromones. Pheromones that attract sexually developed female Cynops pyrrhogaster and C. ensicauda newts were isolated from the male abdominal glands. The C. pyrrhogaster and C. ensicauda pheromones are peptides, designated sodefrin and silefrin, with the amino acid sequences SIPSKDALLK and SILSKDAQLK, respectively. Each pheromone attracts only conspecific females. Molecular cloning of cDNAs encoding sodefrin and silefrin revealed the presence of precursor proteins that are considered to generate these pheromone peptides. Pheromone precursor mRNA levels and radioimmunoassayable pheromone concentrations in the abdominal glands were elevated by prolactin and androgen. Sexual dimorphism and hormone dependency of the responsiveness of vomeronasal epithelium to sodefrin were noted. Significance of pheromones in the form of peptide for those performing reproductive behavior in an aquatic environment was also discussed.     

Significance of egg-jelly substances in the internal fertilization of the newt, Cynops pyrrhogaster

Japanese newt, Cynops pyrrhogaster, undergoes internal fertilization as do most urodeles. In this study, we focused on the roles of egg-jelly in fertilization of C. pyrrhogaster and characterized the substances associated with those roles. When dry sperm were directly inseminated onto the egg, normal fertilization occurred without the presence of water. Egg-jelly extract (JE) prepared with Steinberg's salt solution contained the activity for the initiation of sperm motility. A substance of about 50 kDa in JE was significant for this activity; an inactive form of the substance probably exists in JE. Strong activity to induce acrosome reaction was detected in JE. It was inhibited by the treatment of JE with WGA, suggesting that carbohydrate in JE may be important for the induction of the acrosome reaction. This study suggests that two significant processes of fertilization are regulated by substances in the egg-jelly of the newt, C. pyrrhogaster.     

Isolation, characterization and bioactivity of a region-specific pheromone, [Val8]sodefrin from the newt Cynops pyrrhogaster

Previous analysis of PCR products derived from total RNA from the abdominal gland of the male newt, Cynops pyrrhogaster, inhabiting the Nara area of Japan led to the identification of a gene encoding [Val(8)]sodefrin, as well as the female-attracting peptide pheromone, sodefrin. In this study, purification of this sodefrin variant from the abdominal glands of male newts from the Nara area was accomplished using gel-filtration chromatography and reversed-phase HPLC. Amino acid sequence analysis and mass spectrometry confirmed that the final product was [Val(8)]sodefrin. A full-length cDNA encoding the biosynthetic precursor of [Val(8)]sodefrin was cloned and characterized. The deduced amino acid sequence of prepro[Val(8)]sodefrin showed 86.2% identity with that of the sodefrin precursor. The [Val(8)]sodefrin variant potently attracted females from the Nara area, but the variant was much less or not effective in attracting females captured in the Niigata and Chiba areas. The term aonirin ("aoni" from "aoni-yoshi", the conventional epithet of Nara) is proposed to designate this region-specific pheromone. It is speculated that the coevolution of a novel pheromone and its complementary receptor in the Nara newts may lead to reproductive isolation and eventual differentiation into a separate species.     

Scanning Electron Microscopy of the Extracellular Matrix of Amphibian Gastrulae by Freeze-drying

In order to demonstrate the extracellular matrix (ECM) which tends to be missing from specimens prepared by ordinary procedures, a freeze-drying method was applied to gastrulae of the newt ( Cynops pyrrhogaster ) without prefixation and the embryos were examined with a scanning electron microscope (SEM). It was revealed that a prominent amount of fibrillar ECM occupied the blastocoel and amorphous ECM covered the inner surface of the blastocoelic wall (BW) as well as the surface of migrating cells. These results were compared with those obtained from observations of specimens fixed chemically.     

Isolation and characterization of a gene expressed mainly in the gastric epithelium, a novel member of the ep37 family that belongs to the βγ-crystallin superfamily

EP37 family proteins are non-lens members of the βγ-crystallin superfamily, of which expression is observed in integumental tissues of the Japanese newt, Cynops pyrrhogaster . In the present study, a gene was isolated that has high homology with ep 37 and is transcribed mainly in the gastric epithelial cells and hence designated gep. The predicted amino acid sequence of the gep cDNA contains four βγ-crystallin motifs in the N-terminal half, as is the case in the integumental EP37 proteins. Immunohistochemical analysis showed that GEP protein was mainly localized on the luminal content of the surface mucous cells of the gastric epithelium in both premetamorphic larvae and adults. In addition, GEP protein was also expressed in fundic glands after metamorphosis. Considering the fact that β- and γ-crystallins are evolutionarily related to stress-induced proteins, this localization suggests that GEP protein may have an evolutionarily conserved role in protection against physicochemical stresses, such as physical abrasion and autodigestion, during assimilation.     

The Ca2+ increase by the sperm factor in physiologically polyspermic newt fertilization: its signaling mechanism in egg cytoplasm and the species-specificity

The newt, Cynops pyrrhogaster, exhibits physiological polyspermic fertilization, in which several sperm enter an egg before egg activation. An intracellular Ca(2+) increase occurs as a Ca(2+) wave at each sperm entry site in the polyspermic egg. Some Ca(2+) waves are preceded by a transient spike-like Ca(2+) increase, probably caused by a tryptic protease in the sperm acrosome at the contact of sperm on the egg surface. The following Ca(2+) wave was induced by a sperm factor derived from sperm cytoplasm after sperm-egg membrane fusion. The Ca(2+) increase in the isolated, cell-free cytoplasm indicates that the endoplasmic reticulum is the major Ca(2+) store for the Ca(2+) wave. We previously demonstrated that citrate synthase in the sperm cytoplasm is a major sperm factor for egg activation in newt fertilization. In the present study, we found that the activation by the sperm factor as well as by fertilizing sperm was prevented by an inhibitor of citrate synthase, palmitoyl CoA, and that an injection of acetyl-CoA or oxaloacetate caused egg activation, indicating that the citrate synthase activity is necessary for egg activation at fertilization. In the frog, Xenopus laevis, which exhibits monospermic fertilization, we were unable to activate the eggs with either the homologous sperm extract or the Cynops sperm extract, indicating that Xenopus sperm lack the sperm factor for egg activation and that their eggs are insensitive to the newt sperm factor. The mechanism of egg activation in the monospermy of frog eggs is quite different from that in the physiological polyspermy of newt eggs.     

Distribution of blue-sensitive photoreceptors in amphibian retinas

Previously, we reported that an opsin (Rc-MS) belonging to the SWS2 group opsins is expressed in bullfrog green rods [Hisatomi, O. et al., FEBS Lett., 1999, 447, 44-48]. An anti-Rc-MS antiserum recognized the cones of the Japanese common newt, Cynops pyrrhogaster, which has no green rods. We isolated a cDNA encoding an SWS2 group opsin (Cp-SWS2) from this newt and found that Cp-SWS2 is expressed in a small population of the cones. Our results suggest that SWS2 opsins can be expressed in either green rods or cones of caudata. It seems reasonable to suppose that green rods arose before amphibia were divided into caudata and anura.     

Development of gravity-sensing organs in altered gravity conditions: opposite conclusions from an amphibian and a molluscan preparation

Researchers examined early otolith development in microgravity using fertilized eggs of the Japanese newt, Cynops pyrrhogaster in space flight. Ground experiments examined statocyst, embryonic statolith volume, and statoconia in the post-metamorphic marine mollusk Aplysia californica reared at 1-g and 2-5.7-g. Results indicate that exposure to hypergravity decreased the otolith mass to compensate for increased weight in Aplysia. In the Cynops, there was no compensatory difference in otolith mass, though otoconia production in the endolymphatic system was enhanced.     

Sperm motility-initiating activity in the egg jelly of the externally-fertilizing urodele amphibian, Hynobius lichenatus

Low osmolality initiates sperm motility during the external fertilization of aquatic anuran amphibians. It is thought that this process occurs also in urodeles, but this has not been fully examined in these species. We report here that fertilization was achieved in the externally fertilizing hynobiid, Hynobius lichenatus, by direct insemination onto the egg jelly surface without initial exposure of the sperm to a hypoosmotic solution. To identify the factors in addition to low osmolality that initiate sperm motility in Hynobius, we suspended the sperm of this amphibian in egg jelly extract (JE), and about 90% began to move within 1 min. This indicated the presence of a substance in JE that promotes motility initiation, as is also the case in the newt, Cynops pyrrhogaster. To examine whether this JE factor is homologous to the sperm motility-initiating substance (SMIS) in the newt, we tested for possible inter-species cross-reactivity of the JE. The percentage of moving Cynops sperm was increased to 67% in Hynobius JE at 5 min, and 65% of the Hynobius sperm began to move in Cynops JE within 1 min, indicating that JE is indeed cross-reactive between these species of salamander and newt. Concomitantly, pretreatment of Hynobius JE with Fab fragments of a Cynops SMIS monoclonal antibody resulted in a decreased number of moving Hynobius sperm. Immunoblotting further suggested that the substance in Hynobius JE responsible for motility initiation has an 18 kDa molecular mass, with an isoelectric point at 7.5.     

Cell Culture of Spermatogenic Cells from Amphibians

Cell culture of spermatogenic cells from the primary spermatocyte stage to the early, midspermatid stage has been established for the Japanese newt, Cynops pyrrhogaster (1–4, 10, 11) and for Xenopus laevis (2, 3, 5, 17, 18). The results of many investigations suggest that sperm maturation in vitro for each species proceeds along the same pathway and under the same temporal control as in vivo . Since the shape and size of mature sperm of these two species are very different, comparative studies would enable us to elucidate the mechanisms by which sperm morphology is determined. The results obtained from in vitro studies of spermatogenesis in Cynops and Xenopus demonstrate that there are common and species-specific features.     

An ultrastructural and carotenoid analysis of the red ventrum of the Japanese newt,Cynops pyrrhogaster

The ventral skin of the wild Japanese newt Cynops pyrrhogaster is creamy at metamorphosis, but turns red when mature. The color of the ventral skin of laboratory (lab)-reared newts stays yellow throughout their life. However, the mechanism for the red coloration of this animal still remains unknown. In this study, we have performed ultrastructural and carotenoid analyses of the red ventrum of wild and lab-reared Japanese newts. Using electron microscopy, we observed a number of xanthophores having ring carotenoid vesicles (rcv) and homogenous carotenoid granules (hcg) in the ventral red skin of the wild newt. In the skin, beta-carotene and five other kinds of carotenoids were detected by thin-layer chromatography (TLC). In the ventral yellow skin of lab-reared newts, however, only beta-carotene and three other kinds of carotenoids were found. The total amount of carotenoids in the red skin of the wild adult newt was six times more than that of the yellow skin of the lab-reared newt. Moreover, rcv were more abundant in xanthophores in red skin, but hcg were more abundant in yellow skin. These results, taken together, suggest that the presence of carotenoids in rcv in xanthophores is one of the critical factors for producing the red ventral coloration of the Japanese newt C. pyrrhogaster.     

Morphological and biochemical changes in carotenoid granules in the ventral skin during growth of the Japanese newt Cynops pyrrhogaster

The color of the ventral skin of the Japanese adult newt Cynops pyrrhogaster is red, whereas that of the small juvenile newts at metamorphosis is creamy. Xanthophores in the red skin have many ring carotenoid vesicles (rcv) and a few homogenous carotenoid granules (hcg), as reported earlier. To understand the reason for this change in coloration of the ventral skin of the newt, we carried out histological and biochemical studies to see whether the size and the number of carotenoid granules (hcg and rcv) in the xanthophores and also carotenoid content in the ventral skin change during the growth of this animal. By electron microscopic observation, only hcg were observed in the creamy skin of larvae at stage 59. The diameter of the hcg in the skin of the larvae was approximately 0.85 microm, but significantly decreased to 0.35 microm in the skin of the small juvenile newt. However, the number of the hcg/100 microm (2) of a xanthophore in the ventral skin was very low in the larva at stage 59, but increased in the small juvenile. The carotenoid content was very low in the creamy skin of small juveniles, but dramatically high in the red skin of the adult newts. In the red skin of the adult newt, many rcv (85%) and a few hcg (15%) were observed. However, the number of carotenoid granules (rcv and hcg)/100 microm(2) of a xanthophore in the red skin of adult newts was not different from that of hcg/100 microm (2) of a xanthophore in the creamy skin of small juveniles. The results, taken together, suggest that the increase in the size and the number of carotenoid granules and also carotenoid content in the ventral skin is very important for red body coloration during the growth of the Japanese newt Cynops pyrrhogaster.     

Endoderm differentiation and inductive effect of activin-treated ectoderm in Xenopus

When presumptive ectoderm is treated with high concentrations of activin A, it mainly differentiates into axial mesoderm (notochord, muscle) in Xenopus and into yolk-rich endodermal cells in newt (Cynops pyrrhogaster). Xenopus ectoderm consists of multiple layers, different from the single layer of Cynops ectoderm. This multilayer structure of Xenopus ectoderm may prevent complete treatment of activin A and subsequent whole differentiation into endoderm. In the present study, therefore, Xenopus ectoderm was separated into an outer layer and an inner layer, which were individually treated with a high concentration of activin A (100 ng/mL). Then the differentiation and inductive activity of these ectodermal cells were examined in explantation and transplantation experiments. In isolation culture, ectoderm treated with activin A formed endoderm. Ectodermal and mesodermal tissues were seldom found in these explants. The activin-treated ectoderm induced axial mesoderm and neural tissues, and differentiated into endoderm when it was sandwiched between two sheets of ectoderm or was transplanted into the ventral marginal zone of other blastulae. These findings suggest that Xenopus ectoderm treated with a high concentration of activin A forms endoderm and mimics the properties of the organizer as in Cynops.     

Elephant prolactin: isolation and characterization

Prolactin was isolated from anterior lobes of elephant pituitary glands. It consisted of 199 amino acids with three disulfide bridges and two tryptophan residues as found in prolactin from other species. The sequence of the NH2-terminal 28 amino acids was determined and shown homologous with the ovine hormone. In comparison with ovine prolactin, a marked difference was seen in the methionine content; the elephant hormone possessed only 18-34% lactogenic potency. The conformation of elephant prolactin was examined by zero order, second order and circular dichroism spectroscopy. The alpha helical content was estimated to be about 60%. In comparison with prolactins from other species, the second order spectra of elephant prolactin suggest that the local microenvironment for one or both tryptophan residues is somewhat different.     

Subcortical Rotation and Specification of the Dorsoventral Axis in Newt Eggs

The specification of the dorsoventral axis in naturally polyspermic eggs of the Japanese newt, Cynops pyrrhogaster , was first examined by studies on the spatial relationship between the dorsal midline of the future body plan and the sperm entrance points (SEPs ¹ ). On local insemination, the dorsal blastopore lip was usually found to be formed opposite the SEPs, as in anuran monospermic eggs. Next the movements of the subcortical layer and the cortex were analyzed. "Subcortical rotation" was observed, similar to that of Xenopus laevis eggs with respect to its timing and extent, and its direction was shown to predict the embryonic axis of the eggs. Thus, the dorsoventral axis was concluded to be determined by essentially the same mechanism in the newt as in Xenopus .
Owing to their large size and long first cell cycle, newt eggs appear to be suitable material for study of subcortical rotation, but their behavior is unique in that subcortical rotation occurs in only the vegetal hemisphere so that the subcortical layer stretches in the future dorsal side. Studies on the movement of Nile blue spots suggested that the cytoplasm under the cortex in newt eggs consists of two layers.     

Presence of D-aspartate oxidase and free D-aspartate in amphibian (Xenopus laevis, Cynops pyrrhogaster) tissues.

1. This paper is the first report on the presence of D-aspartate oxidase activity and free D-aspartate in the amphibian tissues. 2. The presence of D-aspartate oxidase activity in tissues of clawed toad (Xenopus laevis) and Japanese newt (Cynops pyrrhogaster) was demonstrated by requirements for enzyme activity, selective inhibition with meso-tartrate and substrate specificity. 3. In each animal, the highest activity was found in kidney, followed by liver and brain, and no gender difference in the specific activity was observed in each tissue. 4. A small but significant amount of D-aspartate was detected in liver and kidney, irrespective of species. 5. In the newt, there was a gender difference in the hepatic and renal content of D-aspartate and not in the D-/D+L-aspartate ratio.     

Extracellular Matrix in the Blastocoel of Newt Gastrula: its Effects on Dissociated Embryonic Cells and some Aspects of its Biochemical Nature

The extracellular matrix (ECM) was removed manually from the blastocoel of freeze-dried embryos of the newt, Cynops pyrrhogaster . In vitro examination demonstrated that the substratum of the blastocoelic ECM (B–ECM) promoted adhesion and spreading of dissociated gastrula cells, while little effect was shown on dissociated blastula cells under the same conditions. Furthermore, the B–ECM promoted locomotion of the spreading gastrula cells. SDS-polyacrylamide gel electrophoretic analysis revealed that the B–ECM contained three major proteins (108, 97 and 30 kilodaltons), which co-migrate with yolk proteins, and a 190-kilodalton protein. The yolk proteins extracted from yolk platelets were found also to act effectively as an strong adhesive sub-stratum for dissociated gastrula cells, however they did not promote cell locomotion. These results suggested that the yolk proteins in the B–ECM may act as an effective adhesion substance for dissociated gastrula cells.     

Cell death during normal gastrulation in the newt, Cynops pyrrhogaster

Cells falling off from ectoderm were observed in normally developing gastrulae of the newt, Cynops pyrrhogaster, in light microscopic examination. These cells eventually died. The number of such necrotic cells per embryo varied from a few dozen to a few thousand. In embryos with many necrotic cells, the ectoderm was thin, the yolk plug large, and establishment of the neural plate was delayed compared with embryos with fewer necrotic cells. A neural tube of normal size was formed at the tail bud stage irrespective of the number of necrotic cells. A new hypothesis to explain these observations is presented.