Identification of egg-jelly substances triggering sperm acrosome reaction in the newt, Cynops pyrrhogaster

Our previous studies have shown that the acrosome reaction (AR) occurs in egg-jelly of the Japanese newt, Cynops pyrrhogaster. This is analogous to the substances of echinoderms but distinct from those of many other vertebrates derived from the egg envelope or its derivative, the zona pellucida. To identify the AR-inducing substances in newt egg jelly, a monoclonal antibody (mAb) was generated against the jelly by screening the culture supernatants to find the one that best neutralized the AR-inducing activity of the jelly substance. The mAb specifically reacted to protein bands in the jelly. These proteins, with apparent molecular weights of 122 and 90 kDa, exhibited AR-inducing activity, indicating that they are definitely AR-inducing substances. Western blotting using the mAb indicated that the 122 and 90 kDa proteins are present only in the egg jelly's outermost layer, where AR-inducing activity is known to occur. Both proteins were recognized with wheat germ agglutinin (WGA), a lectin that inhibits AR-induction in egg jelly extract. Taken together, these findings indicate that the 122 and 90 kDa proteins are the AR-inducing substances in the egg jelly of C. pyrrhogaster. The WGA recognition of the proteins was lost by N-glycosidase digestion, suggesting that N-linked carbohydrate moieties in these proteins may be responsible for the AR-inducing activity.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The effects of pinealectomy and blinding on the circadian locomotor activity rhythm in the Japanese newt,Cynops pyrrhogaster

We examined the effects of pinealectomy and blinding (bilateral ocular enucleation) on the circadian locomotor activity rhythm in the Japanese newt, Cynops pyrrhogaster. The pinealectomized newts were entrained to a light-dark cycle of 12 h light and 12 h darkness. After transfer to constant darkness they showed residual rhythmicity for at least several days which was gradually disrupted in prolonged constant darkness. Blinded newts were also entrained to a 12 h light/12 h dark cycle. In subsequent constant darkness they showed free-running rhythms of locomotor activity. However, the freerunning periods noticeably increased compared with those observed in the previous period of constant darkness before blinding. In blinded newts entrained to the light/dark cycle the activity rhythms were gradually disrupted after pinealectomy even in the presence of the light/dark cycle. These results suggest that both the pineal and the eyes are involved in the newt's circadian system, and also suggest that the pineal of the newt acts as an extraretinal photoreceptor which mediates the entrainment of the locomotor activity rhythm.Abbreviations circadian period - DD constant darkness - LD cycle, light-dark cycle - LD 12:12 light-dark cycle of 12 h light and 12 h darkness     

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.     

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.     

Development of cell surface activity and cell surface adhesiveness in early embryos of the newt,Cynops pyrrhogaster

The development of cell surface activity and adhesiveness was examined in relation to cleavage number in early embryos of the newt, Cynops pyrrhogaster. Both large hyaline bleb formation and surface adhesiveness to substratum were manifested in presumptive ectodermal cells isolated from embryos after the eleventh cleavage (mid-blastula stage). Scanning electron microscopy of the inner surface of the blastocoelic wall (presumptive ectodermal cell layer) revealed the formation of large blebs after the eleventh cleavage. Treatment with alcian blue and lanthanum nitrate demonstrated the accumulation of an extracellular matrix (ECM) on the surface of large blebs.     

Sperm surface heparin/heparan sulfate is responsible for sperm binding to the uterine envelope in the newt, Cynops pyrrhogaster

The sperm-binding properties of egg envelopes are investigated in the newt, Cynops pyrrhogaster. Sperm binding was only seen on the uterine envelope when acrosome-reacted sperm were inseminated. No acrosome-intact sperm bound to the envelopes. By scanning electron microscopic observation, acrosome-reacted sperm were revealed to bind to a seat-like structure present on the surface of the uterine envelope. Sperm binding to the uterine envelope was inhibited by treatment of eggs with heparin or heparan sulfate, or treatment of acrosome-reacted sperm with heparinase prior to insemination. A molecule with a molecular mass of 75 kDa was purified from the uterine envelope by affinity chromatography with heparin-Sepharose. These results indicated that sperm binding was mediated by heparin-like molecules expressed on the surface of acrosome-reacted sperm and the 75 kDa molecule was present as a constituent of uterine envelopes.     

Isolation and characterization of four VIP-related peptides from red-bellied newt, Cynops pyrrhogaster

Four novel bioactive peptides were isolated from the red-bellied newt, Cynops pyrrhogaster, using a bioassay system monitoring the rectum contraction of the Japanese quail, Coturnix japonica. As these peptides are structurally related to vasoactive intestinal polypeptide (VIP), we termed these peptides newt VIP-related peptides 1, 2, 3, and 4 (NVRP-1, -2, -3, and -4). The primary sequences of these peptides were determined to be HSDAVFTDNYSRLLGKTALKNYLDGALKKE (NVRP-1), HSDAVFTDNYSRLLAKTALKNYLDGALKKE (NVRP-2), HSDAVFT-DNYSRLLGKIALKNYLDEALKKE (NVRP-3), and HSDAVFTDNYSRLLGKT-ALKNYLDSALKKE (NVRP-4). The N-terminal regions of these NVRPs possessed homology at the amino-acid level to various VIP, while the NVRP C-termini differed from VIPs significantly. All of the VIP consist of 28 amino-acid residues with amidated forms at the C-termini, whereas NVRPs possess 30 amino-acid residues and have free forms at the C-termini. NVRPs exert relaxant activities on isolated quail rectums in a dose-dependent manner, with threshold concentrations between 1 x 10(-8) and 3 x 10(-8) M. NVRPs also exhibited potent relaxant activities acting on the newt duodenum at 3 x 10(-8) M. As yet, this is the first isolation of biologically active VIP-related peptides from urodele.     

Differentiation and distribution of annulate lamellae in growing oocytes in the newt, Cynops pyrrhogaster

Stages of oocyte development in Cynops pyrrogaster are defined, and changes of annulate lamellae in their fine structure, number, sizes and locations during oogenesis are described. The results show that two different types of annulate lamellae occur during oogenesis. One type differentiates in or at the periphery of vesicle-rich cytoplasm at the early stages of vitellogenesis and increases in number and size. The maximum number of about 40 stacks per median section of oocyte is reached at the stage of complete differentiation of the animal and the vegetal hemispheres. In these growing oocytes, all the stacks show elongate appearances and tetragonal arrangements of annuli as common characteristics. A second type of stacks of annulate lamellae is added anew in full-grown oocytes, increasing the number of stacks per median section of the oocyte to about 90. The new stacks occur in close contact with electron-dense bodies in the cytoplasm and have a massive appearance and hexagonal array of annuli. It is suggested that they appear coincidentally with the onset of oocyte maturation. The possible significance of the observed results is discussed.     

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.