Zoological Science

Embryonic induction plays an important role in establishing the fundamental body plan during early amphibian development. The factors mediating this embryonic induction have, however, only recently been discovered. In the mid-1980's, certain peptide growth factors belonging to the FGF and TGF-β families were found to have a mesoderm-inducing effect on isolated Xenopus blastula ectoderm. The study of embryonic induction subsequently expanded rapidly and knowledge at the molecular level has gradually accumulated. One of these peptide growth factors, activin, a member of the TGF-β superfamily, is present maternally in the Xenopus early embryo and induces various mesodermal and endodermal tissues in isolated presumptive ectoderm. After exposure of presumptive ectoderm to activin, many genes are expressed in the same manner as in normal embryogenesis. Ectoderm treated with activin can induce a complete secondary embryo, the same as the organizer does in transplantation experiments. These findings suggest that activin is one of the first induction signals responsible for establishing the embryonic body plan in early amphibian development. In this article we shall review to what extent we can control the embryonic body plan in vitro, referring to some significant findings in this field.

Three types of descending interneurones (type A, B and C) that project into the terminal abdominal ganglion and control the movements of the uropod have been classified according to their physiological and morphological characters in the crayfish Procambarus clarkii (Girard) using intracellular recording and staining. They were activated by electrical stimulation of a small bundle of isolated fibres in a lateral portion of the ventral nerve cord containing the extension evoking fibres. The type A and B interneurones responded to each stimulus pulse with a spike in one-to-one fashion. The type A interneurones had antagonistic effects on uropod closer and opener motor neurones, while the type B interneurones coactivated both motor neurone types. The type C interneurones were activated indirectly by electrical stimulation and had antagonistic effects on the uropod motor neurones. The axons of the three types of interneurones descended in a dorso-lateral regions of the abdominal fifth–sixth connective, and entered the neuropil in the lateral dorsal tract. Type A and B interneurones were restricted to different regions of the bundle. Within the terminal abdominal ganglion, their branches were restricted to the dorsal neuropil, where numerous branches of premotor nonspiking local interneurones and motor neurones controlling the uropod movements project.

Random Amplified Polymorphic DNA (RAPD) fingerprinting offers a rapid and efficient method for generating a new series of DNA markers in fishes. Three oligonucleotide primers (two 10-mers and one 9-mer) and their paired combinations were found to generate different but reproducible RAPD fingerprints in the guppy. Of these, a 10-mer primer (designated S3D2) was used to detect DNA polymorphisms in two guppy varieties, Green Snakeskin (GSS) and ¾ Black (¾B). High Genetic Similarity (SI) was found among individuals of the GSS and ¾B varieties indicating low intra-variety genetic variability. The average SI values for the Green Snakeskin and ¾ Black varieties were 0.78 ± 0.104 and 0.81 ± 0.083, respectively. The average SI value between individuals of the GSS and ¾B varieties was 0.66 ± 0.066, indicating higher genetic variability between the two varieties. To study the inheritance of RAPD markers, single-pair crosses were set up between males of the GSS variety and females of the ¾B variety. The S3D2 primer was used to generate RAPD fingerprints of the parents and their F₁ offsprings. A total of 14 RAPD markers were scored from these crosses. Of these markers, eight (60.0%) of them were polymorphic. The RAPD markers were shown by the F₁ to exhibit dominant Mendelian inheritance and could thus be used for subsequent genetic linkage mapping of the guppy.

In order to study the pathogenesis of the testicular autoimmunity induced by the immunization with allogeneic testis homogenate (ATH) mixed with Freund's complete adjuvant (FCA) in the Nile tilapia Oreochromis niloticus, the initiation of inflammatory reactions in the testis was ultrastructurally investigated and seminal plasma was analyzed for presence of autoantigens. In the seminal plasma, mainly 120 kD and 80 kD autoantigens were identifiable by Western blotting with tilapia antisperm autoantibody. Eight weeks after injection with FCA, globate structures, possibly originating from FCA, were large and distended the interstitium where the inflammatory reactions were more frequently recognizable. The globate structures may be one of causes to break the blood-testis barrier, and the inflammatory reaction may be due to the leakage of soluble autoantigens from the lumen. In the interstitium, several kinds of immunocompetent cells formed masses which were mainly composed of lymphocytes, macrophages, eosinophils and plasma cells. We suggest that the inflammatory reactions in fish caused by the immunization with ATH + FCA are initiated by sensitization of the immunocompetent cells with testicular autoantigens, the 120 kD and 80 kD autoantigens in the seminal plasma are two of them, leaking from the lumen because of the provable effect of FCA. These initial reactions were then amplified by cellular and humoral immunity.

A method is described for culturing embryonic cells of the starfish Asterias amurensis. A network composed purely of mesenchyme cells can be obtained by treating dissociated cells of embryos at the mesenchyme migration stage with 0.6 M glycine in half strength Ca²⁺, Mg²⁺ free Jamarin (artificial sea water) for 24 hr and subsequently culturing them for 24 hr. When the treatment period is shortened to 12 hr, aggregates of epithelial cells come to coexist with the network. After 3 days of culture, these aggregates form monolayer sheets constituted of four types of cells which can be identified morphologically and by 8-anilino-1-naphthalenesulfonic acid (1, 8-ANS) stainability. These cell types correspond to cells composing the esophagus, the stomach, the intestine and the coelomic pouch. No ectodermal cells were found in the sheet. These results suggest that there is an order of resistibility to 0.6 M glycine treatment among cells of the three germ layers, i. e. mesoderm, endoderm and ectoderm in descending order.

We have devised a simple method for introducing macromolecules into the blastocoel of living starfish embryos. In this method, embryos are treated for 15 min with Ca²⁺ free sea water (CFSW) containing 0.1–5.0 mg/ml of enzymes or immunoglobulins. These molecules are considered to flow through the septate junction, the septa of which are made diffuse by deprivation of Ca²⁺ ions from the environmental sea water.

Introduction of enzymes which affect the component molecules of the extracellular matrix (ECM) drastically deformed the larval morphology. Introduction of a monoclonal antibody recognizing an ECM component prevented the mesenchyme cells from stretching and migrating normally. These results show that our method can be used to study the function of various ECM components in embryonic development.

Neural differentiation in amphibian embryos had been thought to start after gastrulation because of vertical neural induction from the underlying dorsal involuting marginal zone (DIMZ). However, recent studies show that another mode of induction, namely the planar induction, also directs neural differentiation in Xenopus laevis. Here, we attempted to specify when the planar induction occurs. From middle blastula to early gastrula stages, explants that included animal cap (AC) and dorsal noninvoluting marginal zone (DNIMZ), but not DIMZ were prepared. Neural differentiation was detected in such explants (named AC-DNIMZ explants) from the early gastrula (St. 10 +) but not from the late blastula (St. 9) or earlier embryos, indicating that the planar induction occurs at about St. 10+. To assess the further effect of planar induction on neural differentiation, Keller sandwiches were prepared from St. 10+ embryos, and cultured for 2∼6 hr in vitro, after which the explants were separated into AC-DNIMZ and DIMZ parts. AC-DNIMZ part differentiated a little larger neural tissues, suggesting that the planar neural induction during the gastrula stages promote neural differentiation. Neural tissues in these explants could be detected by histology and immunohistochemistry using a monoclonal antibody specific for neural tissues, as opposed to earlier studies that indicated the planar induction to be insufficent for inducing morphological neural structures.

When unfertilized Xenopus eggs were treated by Cynops sperm extract in 10% Steinberg's solution (SB), egg's membranes hyperpolarized to about −37 mV and then depolarized to elicit a positive-going potential amounting to about +34 mV. The eggs underwent cortical contraction and resumption of meiosis. Activation of eggs in various external solutions indicates that the hyperpolarization is due mainly to opening of Na channels, but the positive-going potential is due to Cl channels on the egg's plasma membranes. Since the activation was inhibited by CdCl₂, CoCl₂, or NiCl₂ as well as by amiloride, Ca influx through Ca channels is necessary for the activation by the sperm extract. A propagative intracellular Ca release was induced not only by Cynops sperm, but also by their sperm extract. Injection of BAPTA or heparin into the eggs completely inhibited activation, indicating that egg activation requires an intracellular Ca release dependent upon receptors for inositol 1,4,5-trisphosphate.

The sea urchin, Hemicentrotus pulcherrimus, spawns during winter and spring in Kagoshima, Aomori and Kanagawa. The seawater temperature during the spawning season ranges from about 16 to 19°C at Sendai, Kagoshima, from about 12 to 15°C at Misaki, Kanagawa, and from about 5 to 7°C in Mutsu Bay, Aomori. Among the groups of this species differing in their habitats, optimal temperature ranges for development were found to differ significantly and the difference corresponded to the difference in seawater temperature during the spawning season. Despite the difference in the optimal temperature range for development among the groups, the relationship between developmental speed and temperature within the common optimal temperature range for these groups was nearly constant.

The thermotolerance of embryos produced by cross-fertilization between the group from Kagoshima and that from Aomori was also examined. These embryos showed maternal inheritance of embryonic thermotolerance.

The sugar chain expression in germ cells of a cricket, Gryllus bimaculatus, during spermatogenesis was cytochemically followed using soybean agglutinin (SBA) as a probe. Affinities to SBA were tested on paraffin sections of the testes fixed in Bouin's solution with biotinylated SBA and streptavidin-peroxidase. SBA showed spermatogenesis stage-specific binding. The most intense binding activity appeared at the early meiotic prophase of the spermatocytes, with the activity decreasing at later stages of spermatocytes. Through electron microscopic analysis, it was elucidated that SBA bound to the cell surface and some clusters of dense bodies with a multilaminar structure. At the stage of round spermatids, SBA bound to Golgi apparatus. Treatment of the section with α-N-acetylgalactosaminidase before cytochemistry resulted in the disappearance of most of the binding activity. These results suggest that glycoconjugates with terminal α-N-acetylgalactosamine take part in cricket spermatogenesis during meiotic prophase and acrosome formation.

Oocyte maturation is promoted by the sequential actions of several kinases, of which MPF (a histone H1 kinase) and MAP kinase (a myelin basic protein (MBP) kinase) are known to play pivotal roles. However, other kinases responsible for inducing oocyte maturation have yet to be characterized. To identify these kinases, we examined phosphorylation activities toward 44 exogenous substrate proteins during oocyte maturation in goldfish and Xenopus. We found that 4 substrates in goldfish and 6 in Xenopus were phosphorylated and their phosphorylation states changed during oocyte maturation. Among them, only 3 substrates (histone H1, MBP and pepsin) were common to both species. Precipitation of cdc2 showed that, like histone H1, pepsin was also phosphorylated by cdc2 (MPF). These results suggest that different kinase cascades are involved in goldfish and Xenopus oocyte maturation, although MPF and MAP kinases are common to both species. We also found novel phosphorylation activities that precede the activation of MPF and MAP kinases using deoxyribonuclease I, casein, pepsin and protamine as exogenous substrates.

Cyclostomes have been regarded as having no ultimobranchial gland. However, cells producing immunoreactive calcitonin (CT) were recently found in cyclostome brains. In the present study, we examined using biochemical and biological methods whether there is a CT-like substance in the plasma of the hagfish, Eptatretus burgeri. Hagfish plasma was first subjected to reverse-phase high-performance liquid chromatography (RP-HPLC) and then separated into twenty fractions. The presence of immunoreactive CT in each fraction was then investigated by Western blotting with salmon CT antiserum. Two fractions (36–39% CH³CN; 39–42% CH³CN) showed positive immunoreactivity. Hypocalcemic and hypophosphatemic activities were detected by a rat bioassay only in the former fraction (36–39% CH³CN). The molecular weight (MW) of the CT-like substance in this fraction was 3.5 kDa, which is equal to that of genuine CT. Furthermore, when hagfish plasma was examined by enzyme-linked immunosorbent assay using antisalmon CT, the dilution curve of the plasma paralleled the standard curve of salmon CT. The CT-like substance detected here was present at a high concentration (14 ng/ml) in the plasma. From the results of the present study, the CT-like substance present in hagfish plasma appears to be very similar to salmon CT.

In order to extend our knowledge on the occurrence of neuropeptide Y (NPY)-like-immunoreactive cells in the vitellointestinal duct and yolk sac of oviparous elasmobranchs, we performed immunohistochemical examination on two species of elasmobranchs, Cephaloscyllium isabellum and Raja kenojei. In both species, NPY-positive cells were demonstrated in the epithelial layer of the vitellointestinal duct and internal yolk sac, as previously reported in Scyliorhinus torazame. In the Cephaloscyllium embryo, NPY-positive cells were sporadically encountered even in the endodermal layer of the external yolk sac proximal to the vitellointestinal duct. These findings support the view that the NPY-positive cell is a common endocrine component in the vitellointestinal duct of oviparous elasmobranchs.

We compared the adrenocortical responses to acute stress in two free-living populations of bush warblers, Cettia diphone by measuring the increase in circulating levels of corticosterone in response to capture, handling and restraint over one hour. We wished to test the hypothesis that populations living in more severe habitats with shorter breeding seasons are likely to suppress the adrenocortical response to acute stresses during the nesting phase. We chose the bush warbler because males show no parental care which also has been shown to be an ecological correlate of stress modulation. Male bush warblers responded to playback of songs and long calls with aggressive behavior that was identical at the two sites (Furano, Hokkaido and Chichibu, Honshu). Plasma levels of testosterone were also similar in the two populations suggesting that they were in the same reproductive state when tested. Males of both populations showed marked increases in circulating corticosterone levels during the capture stress protocol. We assumed that at the northern site, Furano, bush warblers would have a reduced adrenocortical response to stress compared with Chichibu (the southern site). Males at Furano, however, showed higher initial plasma levels of corticosterone and showed a greater response compared with males at Chichibu. Relatively less individual variation in the adrenocortical response to stress was seen at Chichibu compared with Furano, and less than in other passerine species studied to date. There was no relationship of body mass or fat score to initial corticosterone level, maximum level generated over one hour of capture stress, or the rate and percent increases of corticosterone. These data indicate that stress modulation may not be a simple consequence of shorter breeding season and more severe climate. Other ecological bases, as yet unknown, are involved.

The skin glands of Rana cancrivora, the euryhaline crab-eating frog, were examined using histochemistry and immunohistochemistry. The skin contained three different glands: a mucous gland, a mixed gland and a vacuolated gland. The acinar cells of the mucous gland stained strongly with alcian blue at pH 2.5 (AB) and weakly with periodic acid-Schiff stain (PAS). In the acinus of the mixed glands, several kinds of cells could be identified: (1) cells with abundant cytoplasm which stained with PAS in the transition region between acinus and duct; (2) cuboidal or squamous cells in the apical portion of the acinus; (3) cells filled with carminophilic granules; (4) cells containing many large vacuoles which stained by PAS and AB. In the acinus of the vacuolated glands, the nuclei of the epithelical cells were located peripherally and the large lumen was filled with vacuoles. In all gland types, no specific immunoreactivity was detected by immunohistochemistry for thyrotropin releasing hormone, cholecystokinin 10, neuropeptide Y, neurotensin, somatostatin and 5-hydroxytryptamine. These results indicate that the skin glands of Rana cancrivora have a different structure and properties from those of other amphibians studied so far.

This study offers an example of the attempts to correlate the shape of an organ to the nucleus size. The pituitary glands of gobiid teleosts are partly embedded in the hypothalamus in contrast to those of non-gobiid teleosts. The pituitaries of the embryonic smelt, catfish, and guppy are partly embedded in the hypothalamus as in the embryonic and adult goby, Rhinogobius flumineus. The nucleus size of cerebellar granular cells, that may represent the genome size, is variable in gobies (16 species studied) as in non-gobiid perciforms (63 species studied). However, the ratio of the nucleus size of cerebellar Purkinje's cells to that of granular cells is consistently low in gobiids compared with non-gobiid perciforms. This ratio is a sort of peramorphic (morphologically advanced) index, and the low value may represent paedomorphosis (juvenile morphology). The gobiid pituitary gland partly embedded in the hypothalamus may be the result of paedomorphosis that could be reflected in the relatively poor increase rate in the size of Purkinje's cells as against the genome size.

The genitalia of the Japanese species of Anthrax (5 species) and Brachyanax (1 species) are studied for the first time. The male genitalia of Anthrax are furnished with useful specific characters in many structures. One of them is a dorsal sclerite just before the gonostyli, and this sclerite varies markedly between species. Besides the spermatheca and genital furca, which are peculiar to each species, various segments of the female genitalia (e. g., tergum 8, sternum 8, etc.) vary considerably with species and it is taxonomically desirable to study them in every species of Anthrax.

The first stage zoeas of two box crabs, Calappa japonica Ortmann, 1892 and Calappa gallus (Herbst, 1803), are described and illustrated from laboratory-hatched material. The main morphological characters are compared with those of previously described species within the Calappidae. The first stage zoeas of all Calappa species so far described are very similar except in overall size and the number of spinules on rostral spine. However, there are conspicuous differences among the early zoeal stages of the calappid genera.

We previously revealed, based on mitochondrial DNA sequence analysis, that the Iriomote cat is very closely related to the leopard cat Felis bengalensis, which is widespread in Asia [24]. In this study, in order to understand the phylogenetic status of the Tsushima cat which is the other wildcat in Japan, partial sequences (402 bases) of the mitochondrial cytochrome b region were determined and compared with those of the Iriomote cat and other feline species. The phylogenetic tree of the cytochrome b sequences indicated that the Tsushima cat and the Iriomote cat have the same mitochondrial DNA lineage as the leopard cat. One or two transitional substitutions were observed among the two Japanese wildcats and the leopard cat. The divergence time (approximately 100,000 years ago) of the Tsushima cat and the leopard cat, estimated by sequence data, was in concordance with the formation date of the Tsushima Island. These results suggest that genetic drift after geographic isolation has brought fixation of some genetic and morphological characters to the Tsushima cat and the Iriomote cat, while these two Japanese wildcats are still genetically close to the continental leopard cat. Considering morphological differences and molecular phylogeny, it is reasonable for the two Japanese wildcats to be classified as two subspecies of F. bengalensis.

About 83% of the amino acid sequence of hemocyanin subunit HR6 from the Southeast Asian horseshoe crab, Carcinoscorpius rotundicauda, has been determined. There is a difference of about 43% between HR6 and complete sequences of chelicerate hemocyanin subunits from the American horseshoe crab, Limulus polyphemus, and a tarantula, Eurypelma californicum. However, the immunologically identical subunits HR6 and HT6 from Tachypleus tridentatus (Japanese horseshoe crab) show 2.7% sequence difference. Based on the amino acid sequences of HR6 and HT6, the divergence between C. rotundicauda and T. tridentatus occurred about 9.6 million years ago. In the case of horseshoe crab hemocyanin subunits, it seems that the orthologous homologues in many homologous subunits between species are immunologically detectable.

Random amplified polymorphic DNA (RAPD) was exploited as a genetic marker to assess the level of genetic variation in populations of Sika deer, Cervus nippon, in Japan. DNA samples were collected from three local populations in Japan, namely, Kinkazan, Goyozan and Ashoro populations. Four arbitrary primers, when used individually, amplified an average of five RAPD fragments in the polymerase chain reaction (PCR). The number of polymorphic bands was scored to calculate band-sharing coefficients within populations. Average band-sharing coefficients revealed a higher degree of homogeneity in the Kinkazan population. Samples collected from larger populations, namely, Ashoro and Goyozan, revealed greater polymorphism than samples from the Kinkazan deer. Our data suggest that RAPD is useful as a marker for detecting genetic variations in populations of Sika deer with reduced levels of genetic diversity.