Zoological Science

Ascidians are sessile marine animals known to accumulate high levels of vanadium selectively in vanadium-containing blood cells (vanadocytes). Almost all the vanadium accumulated in the vacuoles of vanadocytes is reduced to the +3 oxidation state via the +4 oxidation state, although vanadium is dissolved in the +5 oxidation state in sea water. Some of the reducing agents that participate in the reduction have been proposed. By chemical study, vanadium in the +5 oxidation state was reported to be reduced to the +4 oxidation state in the presence of NADPH. The present study revealed the existence of glucose-6-phosphodehydrogenase (G6PDH), the first enzyme to produce NADPH in the pentose phosphate pathway, in vanadocytes of a vanadium-rich ascidian. The results suggested that G6PDH conjugates the reduction of vanadium from the +5 through to the +4 oxidation state in vanadocytes of ascidians.

To investigate the involvement of the yolk-sac membrane in ion absorption, developmental changes in whole-body cation contents, cellular localization of vacuolar-type H⁺-ATPase (V-ATPase), and size and density of pavement and chloride cells in the yolk-sac membrane were examined in tilapia (Oreochromis mossambicus) larvae in fresh water (FW) and those transferred to seawater (SW) at 2 days before hatching (day-2). The whole-body content of Na⁺ in embryos and larvae adapted to both FW and SW increased constantly from day-2 to day 10, although they were not fed through the experiment. The yolk-sac membrane of FW larvae at days 0 and 2 showed V-ATPase immunoreactivity in pavement cells, but not in chloride cells. No positive immunoreactivity was detected in SW larvae. Whole-mount immunocytochemistry showed that some pavement cells were intensively immunoreactive, whereas others were less or not immunoreactive. Electron-microscopic immunocytochemistry revealed that V-ATPase immunoreactivity was present in the apical regions of pavement cells in FW larvae, especially in their ridges. The pavement cells in FW larvae were significantly smaller in size but higher in density than those in SW. These results suggest that pavement cells are the site of active Na⁺ uptake in exchange for H⁺ secretion through V-ATPase in FW-adapted tilapia during early life stages.

The developmental sequence of chloride cells was examined in both the body skin and gills of Japanese flounder (Paralichthys olivaceus) larvae by whole-mount immunocytochemistry using an antiserum specific for Na⁺,K⁺-ATPase. In premetamorphic larvae at 0 and 4 days after hatching (days 0 and 4), immunoreactive chloride cells were distributed only in the yolk-sac membrane and body skin. Premetamorphic larvae at days 8–18 possessed both cutaneous and branchial chloride cells. Large chloride cells in the skin of premetamorphic larvae often formed multicellular complexes, suggestive of their ion-secreting function. Cutaneous chloride cells decreased in size and density at the beginning of metamorphosis (days 21 and 24), and disappeared at the metamorphic climax (days 28 and 33). In contrast, branchial chloride cells first appeared at day 8, and increased during metamorphosis. These results indicate that the site for ion secretion in seawater may shift from cutaneous to branchial chloride cells during metamorphosis. The appearance of branchial chloride cells before the differentiation of gill lamellae suggests that the primary function of the gills during the early development is ion regulation rather than gas exchanges.

The developmental and the tissue-specific expression of glucosephosphate isomerase (GPI), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) multilocus isozymes were analyzed in samples of Leuciscus cephalus and the adult patterns compared with those of 8 additional Italian cyprinid species: Alburnus alburnus alborella, Chondrostoma genei, L. lucumonis, L. souffia, Rutilus rubilio, R. erythrophthalmus, Scardinius erythrophthalmus and Tinca tinca, the taxonomic status of many of them being uncertain and highly debated. The spatial and temporal patterns of expression obtained generally agree with literature data. Main exceptions are the single expression of GPI-A* and MDH-A* loci of the liver in L. cephalus and the GPI pattern of the eye in all species examined. Since delayed appearence of the subunits coded by the GPI-B* locus and the very early ontogenetic expression of the sMDH-B* locus were found in L. cephalus, the onset of expression of orthologous loci can vary in related species. Genetic structure comparisons support a high genetic divergence of T. tinca from all other species.

Buergeria buergeri is female heterozygous in sex determination; chromosome pair No. 7 in this species is a pair of sex chromosomes of the ZZ/ZW type. Genetic analysis of AAT-1 variants was carried out to elucidate the mode of inheritance of this locus by starch-gel electrophoresis using field-caught females and males and their offspring produced by artificial crossings. The results showed that the AAT-1 locus is sex-linked and that alleles are expressed on the Z chromosome, but not the W chromosome. It is evident that the AAT-1 gene of female offspring is hemizygous and that the allele present is on the Z chromosome, which is derived from the male parent.

γ-Tubulin is an ubiquitous MTOC (microtubule-organizing center) component essential for the regulation of microtubule functions. A 1.8 kb cDNA coding for γ-tubulin was isolated from CHO cells. Analysis of nucleotide sequence predicts a protein of 451 amino acids, which is over 97% identical to human and Xenopus γ-tubulin. When CHO cells were transiently transfected with the γ-tubulin clone, epitope-tagged full-length, as well as truncated polypeptides (amino acids 1-398 and 1-340), resulted in the formation of cytoplasmic foci of various sizes. Although one of the foci was identified as the centrosome, the rest of the dots were not associated with any other centrosomal components tested so far. The pattern of microtubule organization was not affected by induction of such γ-tubulin-containing dots in transfected cells. In addition, the cytoplasmic foci were unable to serve as the site for microtubule regrowth in nocodazole-treated cells upon removal of the drug, suggesting that γ-tubulin-containing foci were not involved in the activity for microtubule formation and organization. Using the monomeric form of Chlamydomonas γ-tubulin purified from insect Sf9 cells (Vassilev et al., J. Cell Sci. 108: 1083, 1995), interaction between γ-tubulin and microtubules was further investigated by immunoelectron microscopy. Microtubules incubated with γ-tubulin monomers in vitro were associated with more gold particles conjugated with γ-tubulin than in controls where no exogenous γ-tubulin was added. However, binding of γ-tubulin to microtubules was not extensive and was easily lost during sample preparation. Although γ-tubulin was detected at the minus end of microtubules several times more frequently than the plus end, the majority of gold particles were seen along the microtubule length. These results contradict the previous reports (Li and Joshi, J. Cell Biol. 131: 207, 1995; Shu and Joshi, J. Cell Biol. 130: 1137, 1995), which might be ascribed to the difference in the level of protein expression in transfected cells.

The colonial ascidian Botrylloides simodensis displays multiple body colors—yellow, orangered, violet, black and white—in a clonal colony. The colors are due to pigmented blood cells that exist particularly around a branchial siphon or on an atrial languet of individual zooids. These pigment cells are distributed in mesenchymal space or vascular lumen, and many of them are loosely bound to the epithelium. In the space, there are also colorless blood cells that are circulating with blood. When the colorless blood cells are isolated and cultured, some of the cells produce colored substances and change to pigment cells. Therefore, it is presumed that the pigment cells are derived from colorless blood cells. The pigment cells in this ascidian have a spherical shape with no dendrites and contain many granules. Electron microscopic observation showed that there are several different types of granules, and all types of granules are similarly packed in a large vacuole in the cytoplasm. Chemical analysis disclosed that the pigmentary tissues contain carotenoids, pteridine, and purines that are known pigments in vertebrates. The main components of black and violet pigment cells are still unidentified. The former may be a melanin-like substance, but a significant dose of eumelanin or phaeomelanin was not obtained. Although ascidian pigment cells show a certain extent of similarity to vertebrates in their pigments, their cell structure is quite different from that in vertebrates. Because of the phylogenetic relationship between ascidians and vertebrates, it is assumed that ascidian pigment cells might be a primitive type of those in chordates, although these ascidian cells seem to have a unique origin and function.

Human single-chain tissue-type plasminogen activator (tPA) was activated by the culture media of mouse B16 melanoma and Lewis lung carcinoma cells. This activation was due to the proteolytic conversion of single-chain tPA to two-chain tPA. Typical serine proteinase inhibitors, such as diisopropylfluorophosphate and aprotinin, strongly inhibited the proteolytic activation, suggesting that the enyzme responsible for this activation is a serine proteinase. Through a process of gel filtration, the molecular weight of the putative tPA-activating enzyme was estimated to be approximately 35 kDa. Expression of the tPA mRNA was demonstrated by Northern blot analysis both for the melanoma and carcinoma cells. Zymographic experiments showed that the culture medium of the melanoma cells contained active two-chain tPA. These results indicate that a common serine proteinase may be involved in the proteolytic activation of single-chain tPA in these cancer cells.

We have developed a large-scale purification method of the phosphorylated form (131 kDa) of membrane-bound guanylyl cyclase (mGC) from Hemicentrotus pulcherrimus spermatozoa. The purified mGC contained 26.0 ± 1.3 moles of phosphate/mol enzyme (mean ± S.D., n = 6). Phosphorylated peptides were isolated from the trypsin digest of the carboxymethylated H. pulcherrimus sperm mGC by affinity chromatography on a Chelating Sepharose Fast Flow column, and the peptides were then subjected to mass spectrometric analysis and determination of phosphoserines, after the conversion of phosphoserines to Sethylcysteines by amino acid analysis. Based on the observed mass number and the content of phosphoserine, serine residues at positions 561, 565, 652, 722, 740, 755, 894, 897, 914, 918, 927, 930, 951, and 985, in addition to two residues among those at positions 666, 670, and 671, were shown to be phosphorylated. They are all located in the intracellular region (kinase-like and catalytic domains). Notably, serine residues at positions 894, 918, 927, and 930, that are conserved in the sequence of mammalian mGCs and medaka fish-eye-specific mGCs, are phosphorylated in the sea urchin sperm mGC.

The levels of cathepsins D and E in various rat tissues during development were determined with the sensitive assay method we have developed. The level of cathepsin D increased gradually in each tissue during fetal development suggesting the gradual maturation of the lysosomal system in a cell. The level of cathepsin E differed significantly between tissues at various developmental stages. The level in liver increased rapidly from 13-day-gestation fetal stage and decreased gradually at later fetal stages. The level in other tissues such as stomach and spleen began to increase at later fetal stages or the infant stage. Cathepsin E was found in fetal hepatocytes and its gene was hypomethylated when the expression of the gene was elevated. The enzyme was found to be present mainly as a proform suggesting that, after working, an active form is rapidly inactivated.

Beginning 15 days after ovariectomy (OVX), a high mammary tumor strain of SHN virgin mice at 3 months of age received subcutaneous injections of danazol (0.5 μg / 0.1 ml olive oil, once a day), perphenazine (0.05 mg / 0.1 ml saline, twice a day) or ovine prolactin (oPRL: 0.25 mg / 0.05 ml buffer, twice a day) for 3 days to modulate their circulating PRL levels. The serum PRL level was significantly decreased by danazol and increased by perphenazine compared to the intact and OVX-control groups. The expression of both transforming growth factor α (TGFα) mRNA and epidermal growth factor receptor (EGFR) mRNA in the mammary gland was increased by danazol. However, TGFα mRNA expression was decreased by perphenazine. Meanwhile, mammary end-bud formation was inhibited in danazol-treated group. All findings suggest that the manifestation of the effect of TGFα on mammary gland is rather suppressed by PRL, while mammary gland growth needs the participation of PRL; in other words, PRL is dominant to TGFα on the mammary gland growth. OVX resulted in a significant decrease of TGFα mRNA expression in the mammary gland despite of little alteration in serum PRL, confirming the previous observations. The similar trend was observed in ICR mice; however, the response to hormonal modulation is generally less susceptible than SHN mice.

The distribution of fibroblast growth factor (FGF) was investigated in developing and matured ovaries of the medaka, Oryzias latipes. In the fry, FGF localized in the cytoplasmic region of all oocytes in the ovary at the pre-vitellogenic stage. Before the initiation of vitellogenesis, it disappeared in the cytoplasmic region and newly appeared around each oocyte, and then it localized around all oocytes in the ovary at the vitellogenic stage. Interestingly, the change in FGF distribution was orderly occurring from the posterior to anterior region of the ovary. In the adult, FGF was detected by immunofluorescence staining around the oocytes. These results suggest that FGF plays a significant role in the initiation of oocyte development through follicle cells, and the expression of FGF is rigidly regulated in the developing ovary of O. latipes.

The effect of slightly acidic intracellular pH (pHi) on the development of the sea urchin, Hemicentrotus pulcherrimus was investigated. At first cleavage, the fertilized eggs were treated with artificial sea water containing sodium acetate (Ac-pHSW) at pH 6.8 or 7.0 at the onset of nuclear envelope breakdown, and their pHi decreased from 7.30 to 6.68 or 6.78, respectively. When the eggs were observed after fixation by indirect immunofluorescence and differential interference contrast microscopy, the mitotic stage of the treated eggs was arrested at metaphase and the mitotic apparatus was maintained until more than 50 min after the treatment, although it was smaller in size than that of non-treated eggs. On the other hand, the number of the mitotic asters increased from 2 to 3-4, and further to 6-8 following prolonged exposure, suggesting that the centrosomes had divided and replicated. These results suggest that the centrosome cycle advanced at slightly acidic pHi, even when the mitotic cycle did not advance beyond metaphase.

Mesodermal cell differentiation in echinoid embryos derived from the animal cap recombined with micromeres was examined. An animal cap consisting of mesomere-descendants was isolated from a 32-cell stage embryo, and recombined with a quartet of micromeres isolated from a 16-cell stage embryo. The recombined embryos were completely depleted of the progenitors of an archenteron, pigment cells, blastocoelar cells and muscle cells. Secondary mesenchyme-like cells (induced SMC) were released from the archenteron derived from the animal cap cells in the recombined embryos. Some induced SMC differentiated into pigment cells, confirming previous data for another echinoid species. Moreover, three different kinds of mesodermal cells–blastocoelar, muscle and coelomic pouch cells–were formed in the recombined larvae. Experiments using a fluorescent probe confirmed that the pigment, blastocoelar, muscle cells and cells in part of the coelomic pouches in the recombined larvae were derived from the animal cap mesomeres. These results indicated that the animal cap mesomere had the potential to differentiate through cell fate regulation into four mesodermal cell types–pigment, blastocoelar, muscle and coelomic pouch cells–.

The spatial and temporal expression pattern of cyclophilin (Cyp) was examined during the embryonic development of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus using Western blot analysis and indirect immunofluorescence microscopy. In this study, affinity-purified anti-human Cyp A antibody was used as the primary antibody. Western blot analysis revealed that a single 17.5 kDa immunoreactive band of Cyp was present in unfertilized eggs, in embryos during several stages of development, and in ovaries and testes of adult sea urchins. Cyp was also recognized in unfertilized eggs and embryonic sea urchin cells by indirect immunofluorescence microscopy, but its concentrations within the embryonic tissues varied significantly during embryogenesis. Expression of Cyp during the cleavage stage was thought to be attributable to maternal Cyp products, with zygotic expression appearing after gastrulation. Cyp expression appears to increase depending on the Cyp concentration in the vegetal and apical plates and primary mesenchyme cells in mesenchyme blastulae, and in the oral ectodermal ridge, gut and skeletogenetic mesenchyme cells in pluteus larvae. These results suggest that widespread embryonic distribution and an increased Cyp content occur during the gastrulation in sea urchin development.

Mechanisms of cell specification of mesenchyme during ascidian embryogenesis are poorly understood. This is because no good molecular markers have been available to evaluate differentiation of the mesenchyme cells. To obtain molecular markers of mesenchyme differentiation, we established monoclonal antibodies, Mch-1 and Mch-3, that recognize antigens present in the mesenchyme cells of the larva of Halocynthia roretzi. The antigens recognized by both antibodies start to be detectable in the mesenchyme cells at the late tailbud stage. The Mch-3 antibody specifically recognized all mesenchyme cells of the larva, whereas the Mch-1 antibody stained the cells only in the anterior portions of mesenchyme clusters in the trunk region of the larva. The Mch-1 antibody also stained trunk lateral cells. In addition, both antibodies recognized the mesenchyme cells in the ventro-lateral boundary between endoderm and epidermis that are migrating to the anterior head region of the larva. The partial embryos that originated from the mesenchymelineage cells at the 8-cell stage expressed the Mch-1 and Mch-3 antigens. The Mch-1 and Mch-3 antibodies will be useful as immunological probes for studying the specification mechanisms of mesenchyme cells.

The effects of temperature, photoperiod and aging on eupyrene sperm movement from the testis to the duplex in Polygonia c-aureum male adults were examined in relation to seasonal form and imaginal diapause. In males of both summer and autumn forms obtained under long-day and short-day conditions, respectively, the number of eupyrene sperm bundles in the duplex increased linearly with age during the early stage of adult life at 21°C, and no marked difference was observed between the two seasonal forms. Photoperiod during the adult stage did not influence the rate of sperm movement in autumn forms with diapause. The lower the temperature, the smaller the number of sperm bundles moved from the testis to the duplex. Sperm movement did not appear to occur during a period of chilling at 5°C. Males of the autumn form which were pre-incubated for 30 days and chilled for 60 days failed to resume rapid sperm movement at the final incubation temperature of 21°C. Those which were pre-incubated only for 15 days at 21°C had much fewer sperm bundles in the duplex after chilling than those pre-incubated for 45 days. These results suggest the possibilities that eupyrene sperm movement is suppressed strongly during and after overwintering in the field, and that the eupyrene sperm to be used for reproduction in spring are transferred from the testis to the duplex before overwintering.

Suckling- and estrogen-induced prolactin release from the anterior pituitary is mediated by α-melanocyte stimulating hormone (α-MSH) secreted by the intermediate lobe of the pituitary in the rat. Melanocortin 5-receptors are expressed in the anterior pituitary and probably mediate the α-MSH function. In contrast, the mouse anterior pituitary does not express the receptor. To examine whether or not α-MSH regulates prolactin release in mice, we performed cell immunoblot assay using anterior pituitary cells from adult female mice. We found that α-MSH acted on mammotrophs (prolactin-secreting cells) and stimulated prolactin release in a dose dependent manner. A series of RT-PCR using oligonucleotide primer pairs specific for each subtypes of melanocortin receptors revealed that the melanocortin 3-receptor is the sole receptor expressed in the mouse anterior pituitary. These results suggest that α-MSH-induced prolactin release is mediated by melanocortin 3-receptors in female mice.

Insulin-like growth factor-I (IGF-I) is produced in the liver and other peripheral tissues in response to growth hormone (GH) stimuli. IGF-I regulates diverse physiological functions in an autocrine and/or paracrine manner. IGF-I and IGF-I receptor (type-I receptor) are expressed in human and rat pituitary glands. However, the cell types of IGF-I-expressing cells and target cells of IGF-I in the pituitary glands are not known. The present study was aimed to identify the cell types of IGF-I-expressing cells and of its type-I receptor-expressing cells in mouse pituitary glands. In the mouse pituitary glands, IGF-I mRNA and IGF-I receptor mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR). IGF-I-expressing cells and its receptor-expressing cells were detected by non-radioisotopic in situ hybridization using mouse IGF-I cDNA and IGF-I receptor cDNA probes, and their cell types were immunocytochemically determined using antibodies raised against pituitary hormones. We found that somatotrophs expressed both IGF-I and IGF-I receptors, and some of corticotrophs expressed IGF-I receptors. Co-localization of IGF-I and GH in the same cultured pituitary cells was observed by dual-labelling immunocytochemistry. The present study demonstrated that pituitary IGF-I produced in somatotrophs regulated functions of somatotrophs and corticotrophs in an autocrine and/or paracrine manner.

The ultimobranchial glands of juvenile African lungfish (Protopterus dolloi) (14 individuals; total body length 25–205 mm) were immunohistochemically examined. In individuals larger than 36 mm, one ultimobranchial gland was close to the left afferent branchial arteries. The topography of the ultimobranchial gland was similar to that of salamanders and sharks, but not to teleosts. With body growth, the ultimobranchial gland was vascularized and the parenchymal cells were gradually immunostained with anti-calcitonin antibody. In all individuals examined, the ultimobranchial gland existed only on the left side of the pharynx. These observations are discussed from a phylogenetic viewpoint.

Microporella speciosa sp. nov. is described from Alaska, USA. The new species is characterized by the following combination of features: 1) semicircular orifice with slightly curved and faintly denticu-late proximal edge having a straight, shallow groove, 2) two distal oral spines, 3) laterally directed avicularian rostrum with a broad, channeled tip, 4) open frontal pores, and 5) completely calcified basal walls. Opercula, avicularian mandibles, the ancestrula, and early ontogeny are also described.

Mitinokuidrilus excavatus n. g., n. sp. is described from shallow subtidal coarse sands in northern Japan (NW Pacific). The species is characterized by a unique mode of sexual reproduction. Each of the two types of mature worms represents the opposite sex: “male” worms have testes, seminal vesicles, male ducts and small spermathecae; “female” individuals possess clitellum, ovaries, ovisac, female ducts and fully developed spermathecae. No mature worms with intermediate sexual condition were collected. The taxonomic position and the possibility of consecutive hermaphroditism of the present species are discussed.

A repetitive DNA family (phBglII400) was characterized in the clam species Tapes philippinarum (Veneridae Tapetinae). The tandemly repeated sequences are AT-rich and show a mainly pericentromeric localization, as most satellite DNAs do. Sequence analysis of phBglII400 DNA family showed a high level of intraspecific homogeneity. Furthermore a 200 bp subunit motif within the 400 bp monomer was apparent as well as the existence of two main “open reading frames” along the 400 bp sequence.

In order to investigate the possible distribution of this DNA family among Veneridae, Southern blot analyses were performed on genomic DNAs of Tapes decussatus, Venerupis aurea and Paphia undulata (Tapetinae), Callista chione (Pitarinae), Chamelea gallina (Chioninae) and Venus verrucosa (Venerinae). The phBglII400 family has been found in two additional Tapetinae, namely V. aurea and P. undulata, but not in T. decussatus or other analyzed species. This pattern of sat-DNA distribution supports the high level of differentiation of T. decussatus observed in the previous gene-allozyme analysis. All of these suggest a better allocation of T. decussatus to a genus different from that of T. philippinarum.

An electrophoretic survey was conducted for the Southeast and East Asian populations of Rana limnocharis. Results indicated that specimens from a single locality in Java derived from two genodemes showing complete allelic displacement at eight out of 25 presumptive loci examined. Within each of these genodemes, genotype frequencies at all polymorphic loci were well concordant with the Hardy-Weinberg expectation. These results strongly suggest that those specimens actually represent two biological species. Comprehensive distance analyses suggested that one of the two species from Java is genetically most divergent among all samples examined, whereas the other is most similar to the Laos sample and then to a group consisting of samples from Hongkong, western China, and the southern Ryukyus. Large values of genetic distances obtained for each combination of allopatric samples imply the presence of additional cryptic species in this nominal species.

Hiroshima and Aomori populations of Buergeria buergeri (hereinafter abbreviated as HIROSHIMA and AOMORI, respectively) were morphologically differentiated in both sexually matured frogs and tadpoles. The mean snout-vent lengths of females were 67.4 mm in HIROSHIMA and 50.4 mm in AOMORI, and those of males were 42.9 mm in HIROSHIMA and 37.2 mm in AOMORI. The mean body weights of females of HIROSHIMA and AOMORI just after spawning were 18.0 g and 7.2 g, and those of males of HIROSHIMA and AOMORI were 4.9 g and 3.8 g, respectively. AOMORI tadpoles were rather stocky and their appearance seemed to be more adaptive to lentic water than HIROSHIMA tadpoles. The lower lip of the tadpoles at stage XIII consisted of 2 continuous and 1 broken teeth rows in HIROSHIMA, whereas 3 continuous and 1 broken ones in AOMORI. The growth rate of the embryos was higher in AOMORI than in HIROSHIMA. The embryos of AOMORI were more tolerant to high temperature and less tolerant to low temperature than those of HIROSHIMA. Hybrids between these two populations showed considerably reduced viability in either combination of reciprocal crosses.