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.     

Characteristics of sperm motility induced on the egg-jelly in the internal fertilization of the newt,Cynops pyrrhogaster

Most urodeles undergo internal fertilization and sperm are directly inseminated onto the surface of egg-jelly. Feature of sperm motility induced on the egg-jelly was examined in the newt, Cynops pyrrhogaster. When sperm were directly inseminated onto an egg-jelly, sperm motility was immediately induced on its surface. The egg-jelly of C. pyrrhogaster was composed of six sublayers that were added by turns in oviduct. When the eggs with various sets of the sublayers were obtained and sperm were inseminated onto the egg-jelly, the immediate activity for the initiation of sperm motility was observed only on the outermost sublayer. Similarly, the immediate initiation of sperm motility was induced in the sperm suspended in the extract of the egg-jelly (JE). The initiation of sperm motility was affected by the external pH, and the motility was activated in the moving sperm. A K(+)-channel antagonist, charybdotoxin (CTX), or a Ca(2+)-channel antagonist, gallopamil inhibited the initiation of sperm motility in a dose dependent manner. These results demonstrated the feature of the mechanism regulating sperm motility under stable surroundings in the internal fertilization of amphibians.     

Initiation of sperm motility in the newt, Cynops pyrrhogaster, is induced by a heat-stable component of egg-jelly

Sperm motility in amphibians is thought to be initiated by a decrease in environmental osmolarity. However, fertilisation in the newt, Cynops pyrrhogaster, is achieved in an environment without osmotic change. We show here that sperm motility initiating activity is present in jelly layer extract (JE). JE was gel-filtrated and a single peak with sperm motility initiating activity was detected in the fraction corresponding to about 50 kDa. The activity was strengthened by heat treatment of JE at 100 degrees C for 30 min. This suggests that JE includes the inactive form of sperm motility inducing substance (SMIS) in addition to active substance. Thus JE was fractionated before and after the heat treatment. When JE was fractionated first and then each fraction was heated, the activity was detected in the fraction both above 500 kDa and below 500 kDa. When heat-treated JE was fractionated, the activity was detected only in the fraction below 500 kDa. These results suggest that JE includes the inactive form of SMIS of more than 500 kDa in molecular weight. A regulatory mechanism for the initiation of sperm motility in C. pyrrhogaster is proposed according to the results of the present study.     

The activity for the induction of the sperm acrosome reaction localises in the outer layers and exists in the high-molecular-weight components of the egg-jelly of the newt, Cynops pyrrhogaster

Localisation of the acrosome reaction inducing activity in egg-jelly was examined in the newt, Cynops pyrrhogaster. The jelly has six layers: the J0, J1, J2, J3, J4 and st layers. Jelly was mechanically dissected and placed on a Millipore filter. When sperm were added from the outer surface side of the jelly, most of them exhibited the acrosome reaction after passing through the jelly. When egg-jelly was divided into four layers, strong activity for the induction of acrosome reaction was detected in the outer layers, J4+st. These findings suggest that the acrosome reaction is induced by a substance in the outer layers of the egg-jelly. Among jelly components separated by SDS-PAGE, a fraction of more than 500 kDa in molecular weight induced the acrosome reaction. Wheat germ agglutinin (WGA), Griffonia simplicifoliar agglutinin 1 (GS-1), Maclura pomifera agglutinin (MPA) and Arachis hypogaea agglutinin (PNA) inhibited the induction of the acrosome reaction by jelly extract, and WGA did so in a dose-dependent manner. Those lectins precipitated some molecules of over 500 kDa. These results suggest that the acrosome reaction is induced by the high molecular-weight components of egg-jelly in C. pyrrhogaster.     

The outermost layer of egg-jelly is crucial to successful fertilization in the newt, Cynops pyrrhogaster

The significance of egg-jelly layers in internal fertilization was evaluated in the newt, Cynops pyrrhogaster. In this species, six egg-jelly layers, J1, J2, J3, J4, J5 and the outermost J6 layers, are accumulated on the surface of the fertilizable eggs in pars convoluta of the oviduct. When a large number of sperm (about 6 x 10(5)) were placed on eggs having different numbers of jelly layers, all the eggs were fully fertilized, although many of the eggs developed abnormally. Upon insemination using about 600 sperm, only eggs with the full set of jelly layers were fertilized at a high rate with normal development. Since around 300 (the range of 48-1,192) sperm were observed on and in the egg-jelly in naturally spawned eggs, we conclude that the J6 layer must be present on the outermost surface of the egg-jelly for successful internal fertilization of the newt. Previous studies have suggested that the J6 layer is a prerequisite for the initiation of sperm motility and the acrosome reaction. In the present study, the fertilization rate decreased in eggs with a full set of jelly layers when inseminated using acrosome-reacted and motile sperm. However, the fertilization rate was high when motile sperm with intact acrosome was used. These results suggest that induction of the sperm acrosome reaction in the J6 layer is an important step in the internal fertilization of the newt.     

Injection of a sperm extract triggers egg activation in the newt Cynops pyrrhogaster

Unfertilized eggs of the newt Cynops pyrrhogaster are arrested at the second meiotic metaphase. The primary signal for egg activation is a transient increase in [Ca2+](i), which is triggered by the fertilizing sperm and propagates over the egg cortex as a Ca2+ wave. We injected an extract of Cynops sperm (SE) into unfertilized eggs and induced a wave-like [Ca2+](i) increase which resulted in activation and resumption of meiosis. The SE-injected eggs showed degradation of cyclin B1 and DNA replication. When SE was boiled or treated with proteinase K before injection, it was unable to cause egg activation. Preinjection of Ca2+ -chelator BAPTA before SE injection inhibited egg activation. These results indicate that a heat-labile and proteinaceous factor in the sperm cytoplasm induces a transient increase in [Ca2+](i) which is required for egg activation. Injection of IP3 into unfertilized eggs caused an increase in [Ca2+](i) and egg activation, but injection of cADP-ribose did not. These results support the hypothesis that Ca2+ release at fertilization occurs via IP3 receptors.     

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.     

Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster

Eggs of the newt, Cynops pyrrhogaster, arrested at the second meiotic metaphase are activated by sperm at fertilization and then complete meiosis to initiate development. We highly purified a sperm factor for egg activation from a sperm extract with several chromatographies. The purified fraction containing only a 45 kDa protein induced egg activation accompanied by an intracellular Ca2+ increase when injected into unfertilized eggs. Although injection of mouse phospholipase C (PLC) zeta-mRNA caused a Ca2+ increase and egg activation, partial amino acid sequences of the 45 kDa protein were homologous to those of Xenopus citrate synthase, but not to PLCs. An anti-porcine citrate synthase antibody recognized the 45 kDa protein both in the purified fraction and in the sperm extract. Treatment with the anti-citrate synthase antibody reduced the egg-activation activity in the sperm extract. Injection of porcine citrate synthase or mRNA of Xenopus citrate synthase induced a Ca2+ increase and caused egg activation. A large amount of the 45 kDa protein was localized in two lines elongated from the neck to the middle piece of sperm. These results indicate that the 45 kDa protein is a major component of the sperm factor for egg activation at newt fertilization.     

Endocrine regulation of reproductive behavior in the newt Cynops pyrrhogaster

Hormonal control of the expression of courtship behavior and of secretion of the female-attracting pheromone sodefrin by the male red-bellied newt, Cynops pyrrhogaster, together with the hormonal influence on the responsiveness to the pheromone in the female, is reviewed.Expression of the initial stage of the courtship behavior, i.e., tail vibration by the male in front of the female, is dependent on prolactin (PRL) and androgen. During the courtship, sodefrin seems to be released from the cloaca through the ducts of the abdominal gland. Both content of immunoreactive sodefrin and preprosodefrin mRNA levels in the abdominal gland are elevated by a combination of PRL and androgen, indicating that the pheromone synthesis is stimulated by these two hormones. On the other hand, the discharge of sodefrin is accelerated by AVT, its action being mediated by V1 receptor. In female newts, responsiveness of the vomeronasal epithelium to the pheromone is elevated by a combination of PRL and estrogen. Thus, it can be concluded that PRL, AVT, and sex steroids are key hormones for the reproductive performance in the red-bellied newt. In this article, the significance of the structure of the pheromone molecule as a peptide is also discussed in terms of its species-specificity and its effectiveness in an aquatic environment.     

Evidence for true fall-mating in Japanese newt Cynops pyrrhogaster

The mating season of Japanese newt Cynops pyrrhogaster is generally thought to occur once a year in spring to early summer, during the months of April to June, as in many other Japanese amphibians. However, in fall, from September to October, we often observed breeding colored males demonstrating a mating behavior with females in the field. In this study, in order to identify their true mating season, we anatomically and histologically investigated the annual maturation cycle of gonads and reproductive organs, including cloacal spermathecae in females, and, using a molecular marker, identified the seasonal origins of sperm, which are released in spring to perform insemination. We found that, in fall, ovaries are somewhat immature, while the testes were mature and the sperm already stored in the deferent ducts. Females stored a significant amount of sperm in around 80% of the spermatechae examined in October and 100% in December. When artificially ovulated in March before contact with male partners after hibernation, the females spawned fertilized eggs and these developed normally. Finally, we identified heterozygous genotypes of the visual pigment gene for the two different population types in the embryos, which were derived from a female who established contact with males of the same population in fall and then switched to males from another population until oviposition in spring. We therefore, conclude that the true mating season of this species occurs from fall to early summer, interrupted only by winter, and lasts six months longer (from October to June) than generally believed.     

Magnetic shielding induces early developmental abnormalities in the newt, Cynops pyrrhogaster

Developing larvae of the Japanese newt, Cynops pyrrhogaster, were subjected for 5 days to a shielded environment in which the static magnetic field was about 10,000 times weaker (5 nT) than the geomagnetic norm, which ranges between 30 and 60 microT at the earth's surface. Larvae from non-cleavage to neurula stages were exposed under shielded or normal (control) conditions and then examined for evidence of developmental abnormalities either 1 day or 20 days after treatment. The magnetic shielding was associated with an increased incidence of somatic defects, especially in larvae that were examined 20 days after shielding. Bi-headedness and intestinal protrusion were observed in magnetically shielded larvae but not in controls. Other abnormalities more frequently observed in shielded larvae were spinal curvature, malformed eyes, and retarded or blocked development. These data are among the first to illustrate the effects of magnetic-field deprivation on a developing animal.     

Generation of transgenic newt Cynops pyrrhogaster for regeneration study

To take advantage of the ample potential for tissue regeneration by the newt, a technique to create transgenic newt was developed. The technique was based on a procedure for producing transgenic Xenopus, but modified to adapt to the different sperm morphology and to overcome the refractoriness of newt eggs to activation by normal cleavage. Sperm was collected from mature testes early in winter, permeabilized with digitonin, but without treatment of egg extract. Efficient egg activation was achieved by coinjection of inositol 1,4,5-trisphosphate (IP3) with DNA-sperm nucleus complex. Transgenic Cynops for EGFP/DsRed2 genes under the control of cytomegalovirus (CMV) enhancer/promoter showed nonmosaic widespread expression of reporter genes in embryos, swimming larvae, and adults after metamorphosis. Transgenic newt carrying EGFP gene under regulation of betaB1-crystallin promoter expressed the transgene uniquely in the lens. During lens regeneration after lens removal, EGFP expression occurred, reflecting the lens regeneration process. The newt transgenesis technique described here is likely to be of wide use in monitoring and manipulating gene expression in the study of molecular mechanisms underlying tissue regeneration.     

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.     

Egg-jelly structure promotes efficiency of internal fertilization in the newt, Cynops pyrrhogaster

Egg-jelly is composed of a network of fibrous components and contains substances regulating the sperm-egg interaction. Many studies on the latter have been conducted, whereas the role of the egg-jelly structure in fertilization has not yet been fully assessed. In this study, we examined the fertilization efficiency in the presence and absence of the structure around the egg of the newt, Cynops pyrrhogaster, using a gelatin gel system. Although de-jellied eggs of C. pyrrhogaster can be fertilized with an adequate number of sperm, the fertilization rate was dramatically increased through the use of the gelatin gel. Sperm showed forward motility with straight morphology in the gel, whereas they swam in circles in solution. This result indicates that the gel structure is significant for sperm guidance to the egg surface, and its presence raises the fertilization efficiency in C. pyrrhogaster. When sperm were entangled in the gel structure, they were immediately folded and never showed any forward motility. Sperm with zigzag morphology were observed in the gelatin gel as well as in the egg-jelly, indicating the elimination of sperm by the gel structure. The effect of sperm elimination on successful fertilization was estimated using gelatin gels of different thickness. Though the variation did not affect the fertilization rate, the rate of normal development gradually increased in the thicker gels. This result indicates that sperm elimination in egg-jelly can function in the fertilization system. The roles of sperm guidance and sperm elimination under the physiological condition of internal fertilization of the newt are discussed.     

Egg jelly of the newt, Cynops pyrrhogaster contains a factor essential for sperm binding to the vitelline envelope

The acrosome reaction of newt sperm is induced at the surface of egg jelly and the acrosome-reacted sperm acquire the ability to bind to the vitelline envelope. However, because the substance that induces the acrosome reaction has not been identified, the mechanism by which the acrosome-reacted sperm bind to the vitelline envelope remains unclear. We found here that a Dolichos biforus agglutinin (DBA) specifically mimicked the acrosome reaction immediately upon its addition in the presence of milimolar level Ca(2+). Fluorescein isothiocyanate-labeled DBA bound specifically to the acrosomal cap of the intact sperm in the presence of a Ca(2+)-chelating agent, EDTA, suggesting that binding of DBA to the native receptor for the egg jelly substance on the acrosomal region took the place of the egg jelly substance-induced acrosome reaction. In contrast, the sperm that had been acrosome reacted by DBA treatment did not bind to the vitelline envelope of the egg whose jelly layers were removed. Subsequent addition of jelly extract caused the sperm binding to vitelline envelope, indicating that the egg jelly of the newt contains substances that are involved in not only inducing the acrosome reaction but also binding to the vitelline envelope. This is the first demonstration of the involvement of egg jelly substance in the binding of acrosome-reacted sperm to the vitelline envelope.     

Hormonal control of meiosis initiation in the testis from Japanese newt, Cynops pyrrhogaster

Meiosis is an event that occurs prerequisitely and specifically in gametogenesis. However, the mechanisms of conversion from mitosis to meiosis are poorly understood. I will review the results so far obtained by us using newt testis as a model system, and discuss about the extrinsic mechanism(s) controlling the conversion from mitosis to meiosis. In the newt spermatogonia enter meiosis in the 8th generation after 7 mitotic divisions. We developed organ and reaggregate culture systems with a chemically defined medium in which porcine follicle-stimulating hormone (pFSH) promotes spermatogonial proliferation and differentiation into primary spermatocytes. Human recombinant stem cell factor (RhSCF) in vitro stimulates the spermatogonial proliferation and progression to the 7th generation, but not the differentiation into primary spermatocytes; instead they die of apoptosis. The reason why rhSCF does not stimulate meiosis entrance seems to be due to the low level expression of c-kit protein at the 7th generation of spermatogonia. Ovine PRL induces apoptosis in the 7th generation of spermatogonia in vivo and in vitro. Incubation of newts at low temperature causes spermatogonial apoptosis by the elevation of plasma PRL titer. In the absence of FSH in organ culture spermatogonia can progress until the 7th generation, but the 8th generation never appear due to the apoptosis. Altogether there seems to be a regulatory checkpoint for entrance into meiosis in the 7th generation. Spermatogonia could circumvent the checkpoint by the influence of some factor(s) produced by Sertoli cells upon activation by FSH. Trial to isolate factor(s) responsible for the meiosis-initiation is now underway.     

Metamorphosis inhibition: an alternative rearing protocol for the newt, Cynops pyrrhogaster

The newt is an indispensable model animal, of particular utility for regeneration studies. Recently, a high-throughput transgenic protocol was established for the Japanese common newt, Cynops pyrrhogaster. For studies of regeneration, metamorphosed animals may be favorable; however, for this species, there is no efficient protocol for maintaining juveniles after metamorphosis in the laboratory. In these animals, survival drops drastically after metamorphosis as their foraging behaviour changes to adapt to a terrestrial habitat, making feeding in the laboratory with live or moving foods more difficult. To elevate the efficiency of laboratory rearing of this species, we examined metamorphosis inhibition (Ml) protocols to bypass the period (four months to two years after hatching) in which the animal feeds exclusively on moving foods. We found that approximately 30% of animals survived after 2-year Ml, and that the survivors continuously grew, only with static food while maintaining their larval form and foraging behaviour in 0.02% thiourea (TU) aqueous solution, then metamorphosed when returned to a standard rearing solution even after 2-year-MI. The morphology and foraging behavior (feeding on static foods in water) of these metamorphosed newts resembled that of normally developed adult newts. Furthermore, they were able to fully regenerate amputated limbs, suggesting regenerative capacity is preserved in these animals. Thus, controlling metamorphosis with TU allows newts to be reared with the same static food under aqueous conditions, providing an alternative rearing protocol that offers the advantage of bypassing the critical period and obtaining animals that have grown sufficiently for use in regeneration studies.     

Differential expression of annexin V during spermatogenesis in the newt Cynops pyrrhogaster

 In order to isolate genes whose expression is up-regulated after the initiation of meiosis, we screened a cDNA expression library of newt testes with antiserum against homogenates of testes derived from the spermatogonial and spermatocyte stages. We report the isolation of spermatocyte-specific cDNA clones encoding a newt homologue of the calcium-dependent phospholipid-binding protein, annexin V. Northern blot analysis showed that newt annexin V mRNA was 1.7 kb in length and was expressed strongly in testes, but weakly in other organs. In situ hybridization revealed that the expression of newt annexin mRNA was barely observed in spermatogonia, but increased significantly in leptotene-zygotene primary spermatocytes and reached a maximum level in pachytene spermatocytes and round spermatids. The newt annexin V cDNA predicted a 323-amino acid protein and had a 68% homology to human annexin V. The predicted amino acid sequence contained a conserved 4-fold internal repeat of approximately 70 residues like other annexin proteins. Immunoblot analysis using the monoclonal antibody against newt annexin V showed that the protein was expressed scarcely in spermatogonia but was abundantly expressed in stages from primary spermatocytes to spermatids; this pattern was consistent to that of the mRNA. Immunohistochemical analysis revealed that newt annexin V was localized in the cytoplasm of the spermatogenic cells, but not in somatic cells such as Sertoli cells or pericystic cells. These results indicate that the expression of newt annexin V is up-regulated in the spermatogenic cells after the initiation of meiosis and suggest that newt annexin V plays an important role in spermatogenesis. Received: 8 December 1995 / Accepted: 12 February 1996 Edited by H. Shimada/D. Tautz     

Cloning of dynein intermediate chain cDNA of the newt Cynops pyrrhogaster.

To isolate genes whose expression is up-regulated after initiation of meiosis, we employed an mRNA differential display method using RNA extracted from newt testis fragments in the spermatogonial and spermatocyte stages. We report here isolation of a spermatocyte stage-specific cDNA clone encoding a newt homologue of dynein intermediate chain (IC). The newt dynein IC cDNA was found to encode a polypeptide consisting of 694 amino acid residues with 66.8% and 45.8% amino acid sequence similarity to sea urchin dynein IC3 and Chlamydomonas IC69, respectively. The predicted protein contains five WD repeats and a novel repeated motif in the C-terminal region. Northern blot analysis revealed that newt dynein IC mRNA was expressed in the spermatocyte and round spermatid stages, suggesting that dynein IC plays a role in formation of flagella as well as in meiotic events.