True enamel matrix of the newt,Triturus pyrrhogaster,contains no sulfated glycoconjugates

Summary The ultrastructural distibution and histochemical properties of sulfated glycoconjugates were investigated in the developing enamel of the adult newt, Triturus pyrrhogaster, by use of the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion methods. Development and ultrastructure of the enamel were also studied. After deposition of the mantle dentin matrix to a certain thickness, the first enamel matrix, globular in shape, appeared in juxtaposition to the dental basement membrane and tended to be intermixed with the previously deposited dentin matrix. Subsequently, enamel matrix was deposited outside (ameloblastic side) of the dental basal lamina and formed a true enamel layer. Thus, developing enamel of the newt consists of two layers: (1) an inner layer made up of a dentin-enamel mixed matrix and (2) an outer layer composed of only true enamel matrix. HID-TCH-SP precipitates resulting from the abovementioned studies were found in the mixed matrix and were identified as chondroitin sulfates; in contrast, the true enamel matrix contained no sulfated glycoconjugates.     

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.     

Axon regeneration across the site of injury in the optic nerve of the newt Triturus pyrrhogaster

Summary The process by which axons regenerate following a freeze injury to the optic nerve of the newt was analyzed by light and electron microscopy. Freezing destroys cellular constituents in a one millimeter segment of the nerve, leaving intact the basal lamina and the blood supply to the eye. No axons are seen at the site of injury one to seven days post lesion. This contrasts with the persistence of normal-appearing but severed unmyelinated axons within the cranial stump which thus give a false appearance of early regeneration. The first axon sprouts traverse the lesion and enter the cranial stump by ten days. The number of regenerating axons increases rapidly thereafter with no signs of random growth at the site of injury. These axon sprouts tend to be somewhat larger than normal unmyelinated axons and contain dense core vesicles and abnormal organelles similar to those in growing axons in tissue culture. The persisting basal lamina inside the optic sheath appears to provide continuity across the site of injury, to orient axon sprouts, and to favor an orderly process of axon regeneration without neuroma formation.The authors wish to express their gratitude to Barbara Heindel and Jill Jones for extremely helpful technical assistance. This work was supported by grants NS 10864 and NS 05666 from the U.S. Public Health Service and by the Medical Research Service of the Veterans Administration     

Promotion of spermatogonial proliferation by neuregulin 1 in newt (Cynops pyrrhogaster) testis

We have previously shown that mammalian follicle-stimulating hormone (FSH) promotes the proliferation of spermatogonia and their differentiation into primary spermatocytes in organ culture of newt testis. In the current study, we performed microarray analysis to isolate local factors secreted from somatic cells upon FSH treatment and acting on the germ cells. We identified neuregulin 1 (NRG1) as a novel FSH-upregulated clone homologous to mouse NRG1 known to control cell proliferation, differentiation and survival in various tissues. We further isolated cDNAs encoding two different clones. Amino acid sequences of the two clones were 75% and 94% identical to Xenopus leavis immunoglobulin (Ig)-type and cysteine-rich domain (CRD)-type NRG1, respectively, which had distinct sequences in their N-terminal region but identical in their epidermal growth factor (EGF)-like domain. Semi-quantitative and quantitative PCR analyses indicated that both clones were highly expressed at spermatogonial stage than at spermatocyte stage. In vitro FSH treatment increased newt Ig-NRG1 (nIg-NRG1) mRNA expression markedly in somatic cells, whereas newt CRD-NRG1 (nCRD-NRG1) mRNA was only slightly increased by FSH. To elucidate the function of newt NRG1 (nNRG1) in spermatogenesis, recombinant EGF domain of nNRG1 (nNRG1-EGF) was added to organ and reaggregated cultures with or without somatic cells: it promoted spermatogonial proliferation in all cases. Treatment of the cultures with the antibody against nNRG1-EGF caused remarkable suppression of spermatogonial proliferation activated by FSH. These results indicated that nNRG1 plays a pivotal role in promoting spermatogonial proliferation by both direct effect on spermatogonia and indirect effect via somatic cells in newt testes.     

Differentiation of liquor contacting neurons in the regenerating spinal cord of the newt Triturus

This paper reports a detailed electron microscopic study of the ventricular cells in the growing spinal cord during the first two months of tail regeneration in Triturus vulgaris and T. cristatus. In the proliferating ependyma some cells undergo a neural differentiation in order to produce "Cerebro-Spinal-Fluid-Contacting-Neurons (CSFCNs). These round or pear-like cells store dense core vesicles, 80-100 nm in diameter, and show less electron-density than ependymal elements. The stereociliar apparatus is the last cytological differentiation of these elements and after two months of regeneration, synapse-like endings were occasionally found.     

Chromosome polymorphism in the Italian newt,Triturus italicus

A chromosomal variation, changing shape and C-banding pattern of chromosome XII of Triturus italicus was detected among the offspring of two F₁ hybrid families of T. italicus × T. vulgaris meridionalis . In both families a number of individuals appeared to have a metacentric instead of the expected subtelocentric chromosome XII of T. italicus. — Investigations in three well separated localities in the range of the species showed the polymorphism to have a wide distribution and to be part of a complex pattern involving at least two inversions and (presumably) deficiencies of large C-bands. At meiosis, the shape of bivalent XII, and the location and frequency of chiasmata in the bivalent varied with the karyomorph involved. It is suggested that large rearrangements may still play an important role in the karyological evolution of Triturus.     

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.     

Function of the hepatic melanogenesis in the newt, Triturus carnifex

Like the majority of lower vertebrates, the newt Triturus carnifex holds varying quantities of melanin and hemosiderin in the Kupffer cells of the liver. Following hypoxic treatment, the amount of these two pigments can increase to such an extent that they can occupy nearly a quarter of the surface of histological sections. A group of six specimens, anesthetised with chlorbutol, were subjected to hypoxic treatment by keeping them in a respiratory chamber containing degassed water under vacuum, with only 1.1 ppm of residual oxygen, until they had consumed the oxygen completely (4 hours, at a temperature of 18 degrees C). Using hematological and histochemical techniques and computerised image analysis, it has been shown that hypoxic animals not only increase the extent of the melanic areas of the liver from about 5-7% to almost 24% compared to control groups kept under two different respiratory conditions (6 anesthetised specimens exposed to the air and 6 submerged in normoxic water), they also went through a remarkable hemolytic process to justify a parallel increase in hemosiderin deposits. Melanin was extracted from the liver by keeping fragments of the organ for one hour at 37 degrees C in an oxidising solution (20 mL of benzyl alcohol, 10 mL of acetone, 5 mL of 10% hydrogen peroxide, and 4 drops of concentrated ammonia solution), then quickly rinsing them in 50% acetone and subsequently letting them stand for 6 hours in 10 mL of distilled water alkalised to pH 12 with a drop of ammonia solution. The extract was then left to sediment at pH 2.5 and the black precipitate washed and dried under vacuum. Elemental and spectrophotometric analyses revealed a significant presence of purines in the melanic pigment. This phenomenon can be explained by the animals' need under hypoxic crisis to rapidly neutralise purines resulting from lysis of the nucleated red blood cells by introducing them into an inert molecular complex. A partial model of structure is proposed here. Synthesis of the mixed polymer is possible through the well-known capacity of ferrous iron to activate tyrosinase (the enzyme responsible for melanogenesis) even in the absence of DOPA.     

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.     

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.     

Cellular cooperation in hapten-carrier responses in the newt, Triturus viridescens

Heterologous erythrocyte responses in the three hematopoietic organs, spleen, liver and kidney, were studied in the American Common Newt, Triturus (Diemictylus) viridescens. These responses were measured by immunocytoadherence. Horse (HRBC) and sheep (SRBC) red cells were used as immunogens. Background and response values to HRBC were consistent in all three organs. Activity levels and their kinetics were greater in spleen than in liver or kidney. Anti-SRBC activity levels proved to be too variable and as a consequence HRBC's were used in subsequent studies. Secondary responses in the newt showed modest increases in the spleen, a decrease in liver activity levels and a proportionately large (5×) increase within kidney. While splenectomy prior to immunization did not effect primary response activity in liver and kidney, it caused a three-fold enhancement of the usual loss of activity in secondary responses within liver. Kidney secondary response levels were unaffected by prior splenectomy. Spleen-liver lymphoid traffic may be involved in anamnestic responses in the newt.The main question had to do with whether one could demonstrate cellular cooperation in immune responses in so primitive a vertebrate. Chicken (CRBC) and toad (TRBC) red cells were used as carriers for the hapten trinitrophenol (TNP). Pre-immunization with the carrier immunogen led to the development of rosette forming cells (RFC) specific for the hapten in spleen, liver, and kidney after challenge with the TNP on the same carrier. Injection of the TNP carrier alone or pre-immunization with a different carrier caused no TNP-specific RFC's to be generated. The anti-TNP responses were assayed with HRBC and TNP-HRBC. Prior treatment with TNP-HSA (human serum albumin), before TNP-HRBC assay, blocked the RFC with TNP-HRBC and helped to establish the specificity of the hapten-specific RFC's. These kinds of immune responses in the newt require at least two cooperating cellular populations. Cellular cooperation may have been an early phylogenetic feature of immune responses.     

Cell proliferation in vivo and in vitro in visceral organs of the adult newt, Triturus cristatus carnifex

The mitotic and labelling incidence of intestine, liver, spleen and pancreas cells of Triturus cristatus carnifex adults kept at 15°C, 20°C, 25°C and 30°C were examined. Intestine mitotic and labelling incidences were highest at 25°C and lowest at 30°C. There was no significant difference between 15°C and 20°C. No such relationship could be shown for liver, spleen or pancreas, which had very much lower mitotic and labelling incidences. In culture, intestine mitotic and labelling incidences fell significantly within the first four hours, and maintained these low levels for the next five days. In contrast, liver mitotic and labelling incidences rose for 9–11 days, and then began to fall, while pancreas mitotic and labelling incidences reached peak values at day 5, and were kept in good condition for up to 14 days.     

Chromosome and C-heterochromatin polymorphisms in the Italian newt,Triturus italicus

A combined chromosome and C-heterochromatin polymorphism in pair 12 in the complement of the newt species, T. italicus is described. The C-heterochromatin polymorphism is presumably due to a loss in the proximal C-band, whereas the chromosomal polymorphism has its origin in two different independent pericentric inversions both including the centromere and the proximal C-band of chromosome 12. The double-inversion polymorphism has a wide distribution over the range and follows a clear bipolarity between a northern area where the karyotype is homomorphic for the standard type of pair 12 (ST/ST) and an opposite area where the ST type is completely replaced by variant M₁ and M₂ metacentric chromosomes 12. Various karyophylogenies are possible, but the simplest and the most probable presumes an ancestral karyotype of ST/ST and a mechanism of gradual replacement of the heterobrachial chromosome ST by two independent pericentric inversions. The present data are discussed in relation to existing theories on karyological evolution of Urodeles and the functional significance of telocentric chromosomes suggested by Sessions et al. (1982).     

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.     

Melatonin,melanogenesis, and hypoxic stress in the newt,Triturus carnifex

Groups of 6 specimens each of the newt Triturus carnifex were treated with melatonin to see if the hormone inhibited melanogenesis in the Kupffer cells of the liver (melanomacrophages), a process markedly stimulated by hypoxia. A dose of 500 microg/g in 27% ethanol, injected intraperitoneally, induced loss of consciousness and tetany of all the skeletal muscles, which on the contrary appeared relaxed in animals pre-anesthetised by immersion in chlorbutol at 0.2%. Anesthetised specimens injected with melatonin showed a significantly lower increase in hepatic pigmentation after acute hypoxia, a condition attained by sealing each specimen in a 620 mL respiratory chamber with water containing 1.1 ppm of oxygen for the time needed to consume it all (about two hours). If hypoxia is reached gradually, beginning with 8 ppm of oxygen (normoxic condition), the increase in hepatic pigmentation after melatonin injection does not differ significantly from that of non-hormone treated specimens: thus melatonin does not seem to play a direct part in controlling hepatic melanogenesis. Instead, the hormone induces significant increase in oxygen consumption, marked general steatosis of the liver and the almost total disappearance of glycogen. Intraperitoneal injection of 500 microg/g of melatonin in anesthetised animals exposed to the air (normoxic) also causes severe steatosis and an unexpected increase in the hepatic deposits of melanin, as after hypoxic treatment. A dose of 100 ng/g in 1% ethanol, ineffective when injected intraperitoneally, also induces these effects if injected directly into the arterial blood-stream through the conus arteriosus, thus avoiding the hepatic filter. The phenomena observed appear to be induced by a powerful endocrine mechanism that provokes metabolic hypoxia by consuming all the available ATP for synthesizing fat. A less intense form of steatosis can also be observed in animals subjected to hypoxia but without prior hormone treatment, indicating that a natural process triggered by hypoxic stress is pushed to the extreme by exogenous melatonin: the hormone changes the entire energy metabolism of the organism so that it can survive for a long time under adverse environmental conditions.