Vertebrate oocytes arrested at meiotic prophase I are still immature even when they reach their fully grown stage. For the acquisition of fertilizability, the fully grown oocytes must undergo oocyte maturation, during which the meiosis is released from prophase I arrest and stops again at metaphase II until inseminated. The resumption of meiosis from prophase I to metaphase II is induced by the action of maturation-promoting factor (MPF). The molecular structure of MPF is common to all eukaryotes, but the mechanisms of its formation and activation vary in cell types and in species. In this review, I summarize the mechanisms of MPF formation during oocyte maturation in two amphibian species, Xenopus laevis and Rana japonica. In Xenopus, immature oocytes are equipped with inactive MPF (pre-MPF) sufficient for completing oocyte maturation, and therefore only its activation is required after hormonal stimulation. In contrast, immature Rana oocytes contain no pre-MPF. Therefore, MPF must be newly formed during oocyte maturation, as is the case in fishes and other amphibians (toads and newts). The mechanism of MPF formation in Xenopus therefore seems to be different to that in other lower vertebrates. However, I wish to propose a new mechanism of MPF formation that might be appropriate for all species of lower vertebrates, inclusive of Xenopus, based on the novel concept that pre-MPF is an artifact produced under unnatural conditions and is not an essential molecule for initiating oocyte maturation, in contradiction to the generally believed notion that pre-MPF is actively stocked for the initiation of oocyte maturation. The standpoint that pre-MPF is not indispensable for initiating oocyte maturation might provide a new insight into the mechanism of MPF formation during oocyte maturation, allowing us to model a comprehensive mechanism applicable to all vertebrates, including mammals.

Mammalian endothelins (ETs) -1, -2 and -3 effectively aggregated pigmentary organelles in xanthophores of the medaka, Oryzias latipes. IRL 1620 and sarafotoxin S6c, both being selective agonists of the mammalian ET_B receptor, were also found to aggregate xanthosomes in those cells. Quantitative studies on the action of ET-1, ET-3 and IRL 1620 on xanthophores indicated that the responses were concentration-dependent, and that they may act directly on xanthophores, because denervated cells responded to the peptides quite similarly. Blockers of some receptors known to mediate motile activity of chromatophores were ineffective in inhibiting the action of ETs. In addition, other blockers of mammalian ET_A receptors (BQ-123 and TTA-386) or ET_B receptors (BQ-788) were also disclosed to be ineffective in interfering with the action of ET. Therefore, ETs may act through the mediation of ET receptors on the medaka xanthophores that do not resemble mammalian ET receptors pharmacologically. Thus, along with their effects on other kinds of chromatophores, ETs may take part in the delicate and exquisite control of integumentary hues and patterns.

The effects of testosterone (T) on downstream migratory behavior of yearling masu salmon, Oncorhynchus masou, were studied during its downstream migratory period using artificial raceways. In experiment 1, 22 and 19 smolts were implanted with a medical silicone tube capsules containing 500μg of T or vehicle only. These groups were transferred together to the upper pond of artificial raceway which was connected to the lower pond through fish-way. Downstream migratory behavior was then observed for 1 week. In experiment 2, T500μg-, T50μg-, and T5μg-treated smolts, control smolts, and precociously mature males were transferred to the upper pond of the raceway and downstream migratory behavior was observed for 2 months. In experiment 3, 40 smolts were implanted with capsules containing 500μg of T or vehicle only. These groups were transferred separately to the upper ponds of raceways and downstream migratory behavior was observed for 3 weeks. In each experiment, injection of T caused increases in plasma T levels within physiological levels. In experiment 1, frequency of downstream migratory behavior was 89.5% in the control group and 31.8% in the T500μg-treated group. In experiment 2, the frequency was lower in the T500μg- and T50μg-treated groups than in the control group. T5μg implantation failed to inhibit downstream migratory behavior. Precocious males were not observed to go down the raceway. In experiment 3, frequency of downstream migratory behavior of the control group and the T500μg-treated group was 100 and 40%, respectively. In each experiment, plasma levels of T in T500μg-treated smolts which did not show downstream migratory behavior was higher than those of migrants. These results indicate that the downstream migratory behavior of masu salmon smolts is inhibited by physiological levels of T.

Tropomyosin (TM) is one of the actin-regulatory proteins and stabilizes the filamentous structure of F-actin. Although it has been suggested that multiple TM isoforms play important roles in nonmuscle cells and several TM-binding proteins have been identified and characterized, the functional properties of nonmuscle TM isoforms are not well understood. In order to investigate the roles of TM in nonmuscle cells, we searched for novel nonmuscle TM-binding proteins from the chicken liver total acetone powder extract by TM-affinity column chromatography. As a consequence, a protein of 57 kDa was mainly eluted from the column and its partial amino acid sequence of the protease-digested fragment was determined. Homology search using SWISS-PLOT showed that the 57 kDa protein was nearly identical to chicken glutamate dehydrogenase (GDH: EC 1.4.1.3). Purified GDH had the ability to bind to the TM-affinity column, indicating that GDH associated directly with liver TM. The binding of GDH to TM was abolished in the presence of 0.1 mM ATP, that is known to decrease the rate of the GDH enzymatic activity at pH 7.5. Thus, we have demonstrated the interaction between nonmuscle TM and GDH for the first time.

The colony specificity of colonial animals involves allorejection reactions, which are the defense reactions for allogeneic tissues that occur naturally. In colonial botryllid ascidians, all of the species already studied have colony specificity, and their allorejection modes differ from one another. However, in most of these botryllids, morula cells (MCs) always participate in the allorejection reactions, and the prophenoloxidase (proPO) system of MCs is considered to contribute to the allorejection reaction. The present study was performed using five botryllids and Symplegma reptans, which is closely related to botryllids, in an effort to clarify the relationship between the modes of allorejection and the characteristics of MCs, such as the ratio of MCs to total hemocytes and the phenoloxidase (PO) activity levels in the MCs. The MCs of these six ascidians resembled one another morphologically and the MCs of all species showed PO activity. In Botryllus scalaris, PO activity was also found in granular leukocytes, but the level of activity was much lower than that in MCs. The PO of these species resembles one another, at least in terms of their sensitivity to inhibition by common inhibitors of the proPO system. The PO activity per fixed number of hemocytes varied among these ascidians. This variation was due to a difference in the ratio of MCs to total hemocytes and/or a difference in PO activity per MC. In most ascidians, except for B. scalaris, the rejection reaction area showed a higher level of PO activity than the fusion area of the syngeneic colonies. These results suggest that the characteristics of MCs including their PO activity are closely correlated with the mode of the allorejection reaction.

Peripheral nerve development was studied in the cat shark, Scyliorhinus torazame, using whole-mount and sectioned embryos. Nerve fibers were immunohistochemically stained using a monoclonal antibody against acetylated tubulin and, in early embryos, cephalic crest cells were observed by scanning electron microscopy. The initial distribution patterns of crest cells were identical to the typical vertebrate embryonic pattern, in that three crest cell populations were associated with even-numbered rhombomeres, prefiguring the pattern of the branchiomeric nerve roots. In older pharyngula, however, the trigeminal and postotic branchiomeric nerve roots were found to have shifted caudally along the neuraxis: the trigeminal nerve root finally arose from r3, while the glossopharyngeal nerve root arose from the presumptive region r7 of the hindbrain. The shift apparently takes place between the root fibers and the dorsolateral fasciculus. From observing the topographical relationships between the peripheral nerves and other epithelial structures (for example, the otocyst and the mesodermal head cavities - the anlagen of extrinsic ocular muscles), it was assumed that the shift was the result of an epigenetic effect caused by allometric growth of the otocyst, the mandibular cavity, and the spiracle epithelia anchoring the mandibular branch of the trigeminal nerve. It was concluded that the deviated morphological pattern of elasmobranch cranial nerves is a secondary phenomenon caused by the well-developed head cavities. In those animals whose head cavities are degenerated, the original pattern of the cranial nerve-rhombomere assignment, which is intact in lamprey embryos, is retained.

As sturgeons are considered to represent a basal group of Osteichthyes, it is necessary to evaluate their developmental features to understand the evolution, not only of bony fishes, but also of tetrapods in general. Using Besters, commercially established hybrid sturgeons, the neural crest cell distribution pattern, mesodermal epithelium, and peripheral nerves were observed based on whole-mount immunostained and -sectioned embryos, from the pre-hatching embryonic stage to a late swimming larval stage. At the early pharyngula stage, the hindbrain exhibits at least six rhombomeres. These have a typical arrangement of neuroepithelial cells, and segmentally distributed cephalic crest cell populations associated with even-numbered rhombomeres medially, and single pharyngeal arches laterally. The head cavities first arise as a pair of epithelial primordia in the prechordal region. Secondarily, the cavity is subdivided mediolaterally into the premandibular and mandibular cavities. These mesodermal components never affect the segmental pattern of cranial nerve roots as seen in the shark embryo (Kuratani and Horigome, 2000), probably due to the early degeneration of the cavities. The hyoid cavity never appears. As observed in several teleosts, the newly hatched Bester larva possesses extensive neurites in the epidermis, originating from both trigeminal placodes and Rohon-Beard cells. This neurite network diminishes during development, in concordance with the appearance of lateral line nerves. All the epibranchial placodes are seen as focal, HNK-1-positive epidermal thickenings and give rise to inferior ganglia of the branchiomeric nerves. Metameric morphology of the branchiomeric nerve innervation is secondarily disturbed through modification of the head region, involving the expansion of the operculum and modification of the jaw.

While the signal of insulin-like growth factors (IGF) in skeletal muscle cells is known to be mediated by IGF receptor (IGFR) 1, not IGFR 2, there is no report even on the expression of IGFRs during tongue myogenesis. Here we examined changes in the mRNA level of IGFR 1 and 2, in addition to IGF-I and II, during mouse tongue myogenesis between embryonic day (E) 11 and newborn by competitive reverse-transcriptase polymerase chain reaction. Immunolocalization of IGF-I, II, IGFR 1 and 2 was examined using confocal laser scanning microscope at E13, E15 and newborn stages. Immunolocalization of fast myosin heavy chain was also examined to detect differentiating myoblasts, myotubes and myofibers. IGF-I, II and IGFR 1 mRNAs were highly expressed between E13 and E15 during differentiation of myoblasts and formation of myotubes. IGF-I and II proteins were co-localized to differentiating myoblasts, myotubes and myofibers with IGFR 1 protein. High level expression of IGFR 2 mRNA was also observed between E13 and E15. However, the expression of IGFR 2 protein was sparsely observed throughout the whole tongue tissues and not restricted to the striated muscle tissue. These data suggest that IGFR 1 is related to the IGF signal transduction and the differentiation of mouse tongue striated muscle, whereas IGFR 2 is not directly involved in them.

We observed spermatozoa of five freshwater Japanese unionid mussel species under a light microscope. Males discharged spermatozoa as spherical masses. Spermatozoa embedded their heads into a spherical, colorless body with their tails extended around the sphere. We termed this spherical mass of sperm as a “sperm sphere”. Just after release the sperm spheres rotated rapidly by synchronous movement of the sperm tails. By the next day, the sperm spheres increased in size and some spermatozoa had detached from the sperm spheres. Spermatozoa embedded in the sperm sphere were active at least forty-eight hours after being discharged. Single spermatozoa in a balanced salt solution were also active until forty-eight hr after extraction from the gonads, while they lost their motility within a few minutes in freshwater. Spermatozoa may maintain their motility for a long time in the form of a sperm sphere and the use of sperm spheres may allow the fertilization of eggs discharged into the gill chambers of females far away from the male.

A hybrid medaka between Oryzias latipes and O. curvinotus spawns diploid eggs. We examined the cytological mechanisms of diploid egg formation in this hybrid. Oocytes in the hybrid passed through the first and second meiotic divisions and excluded two polar bodies, associated with reduction of the DNA content in oocytes. Each germinal vesicle in vitellogenic oocytes of the hybrid had 48 chromosomes with bivalent chiasmata, precisely twice the number of chromosomes in normal oocytes. These results suggest that before meiosis the chromosomes are doubled by mitosis without cytokinesis, that is, endomitosis, and that the resulting tetraploid oogonia undergo normal meiosis to produce diploid eggs. Except for a few vitellogenic oocytes that are probably derived from endomitotic oogonia, most oocytes were arrested at the zygotene stage in the hybrid ovary, suggesting the existence of checkpoint control that ensures the pairing of homologous chromosomes at prophase I, a situation differing from that in the male in which the checkpoint is at metaphase I. A sac-like structure was characteristic of the hybrid ovary. Although this structure was observed only in the hybrid, it may be a native component of the medaka ovary but difficult to observe because of its deformed outer structure due to enlargement of the inside oocytes. The present study confirms that this hybrid medaka provides a useful experimental system for investigations into the mechanisms of oogenesis and basic architecture of the ovary, which are difficult to analyze by using normal medaka.

We analyzed postembryonic neurogenesis in zebrafish brain using the thymidine analogue 5-bromo-2′-deoxyuridine (BrdU). At 1 and 3 weeks after fertilization, BrdU-labeled cells were detected at the brain midline, especially in the telencephalon, optic tectum, hypothalamus, and cerebellum. Cell replication ceased in the telencephalon and hypothalamus by 6 weeks after fertilization, but continued in the optic tectum and cerebellum even in adult fish. Although the area of replication was widely distributed in the tectum and cerebellum of young fish, it seemed to gradually become restricted to the medial and lateral margin of the tectum and the dorsal region of the cerebellum. These areas of replication showed common morphological features, with small, tightly packed spherical cells which were stained very densely by toluidine blue.

In the optic tectum and cerebellum, the areas which were BrdU-positive (or densely stained by toluidine blue) gradually decreased in size relative to the growing brain, but the total volume of these areas in each individual remained unchanged. These results indicate that there are two different neurogenic systems in teleost fish. One system, found in the telencephalon and the hypothalamus, stops replication at an early stage of development; dividing cells in these areas are repressed or removed. The other system, found in the tectum and cerebellum, retains its replicative activity even in adult fish; the number of dividing cells in these areas seems to be maintained during development of the brain.

In the polygynous coral-reef fish, Labroides dimidiatus, the largest female may complete sex change within a few weeks if the male disappears. We conducted male removal experiments just prior to spawning time. The largest female possessing ovulated eggs spawned in the male role with smaller females, 1–2 hr after the male removal, suggesting that ovulation could not prevent male sexual behavior. During the female-female spawning eggs were actually released, but of course unfertilized. The largest female subsequently spawned in the female role when the male was immediately returned. If the male was never returned, such spawning behavior was repeated almost every day until the largest fish was able to release sperm. Smaller females will participate in such fruitless spawning according to their preference for larger mates. The largest females should perform male sexual behavior to secure future mates, even before completion of gonadal sex change.

The effects of activin A and follistatin on the release of follicle-stimulating hormone (FSH), luteinizing hormone (LH), growth hormone (GH) and prolactin (PRL) from dispersed pituitary cells of the bullfrogs Rana catesbeiana were studied. Activin A stimulated the release of FSH, GH, and PRL dosedependently, but not that of LH. Follistatin suppressed the activin-induced FSH, GH, and PRL release, but did not affect the basal secretion of those hormones. From the results obtained in this experiment, together with the previously obtained findings that activin B enhanced the release of FSH, LH, GH, and PRL, we conclude that activin A, in addition to activin B, influences the function of multiple types of pituitary cells in the bullfrog.

We examined the diet of Hyla japonica, a prolonged breeder, in the reproductive season (early May to late July). This species breeds in rice fields. Females attended the breeding site only at the time of spawning, but males remained and foraged in rice fields for their continuous advertising during the prolonged season. A high frequency of empty stomachs and fewer and less stomach contents observed at the beginning of the breeding season, may result from energetic constraints associated with reproduction in males. Diverse ground-dwelling invertebrates predominated in the diet, but aquatic organisms were quite few. We estimated the prey availability in the environment by sweeps. The most easily available prey such as ants, beetles, dipterans, caterpillars, and spiders were numerously consumed by frogs. Significant correlations between the diet compositions and prey availability suggest that H. japonica is an opportunistic predator.

We investigated the endosymbiotic microbiota of the citrus psyllid Diaphorina citri that vectors Liberobacter sp., the causative agent of citrus greening disease. Eubacterial 16S rDNA fragments were amplified by PCR and cloned from the whole DNA of D. citri. RFLP analysis showed that the PCR products contained five major sequence types. Nucleotide sequences of the five types were determined and subjected to molecular phylogenetic analysis. The first sequence was a member of the γ-Proteobacteria, closely related to mycetocyte symbionts of other psyllids. The second sequence belonged to the β-Proteobacteria, related to Oxalobacter and Herbaspirillum. The third sequence, belonging to the γ-Proteobacteria, showed a significant similarity to the sequences of Arsenophonus spp., known as endosymbionts of a triatomine bug and a parasitoid wasp. The fourth sequence was almost identical to the sequences of Liberobacter spp. in the α-Proteobacteria. The fifth sequence showed a high similarity to the sequences of Wolbachia spp. in the α-Proteobacteria. In situ hybridization experiments using specific oligonucleotide probes confirmed that the first and second sequences originated from the mycetocyte and syncytium symbionts, respectively. The presence/absence of the five endosymbiotic bacteria in adult individuals of D. citri from an Indonesian natural population was examined by diagnostic PCR analysis. The mycetocyte symbiont and the syncytium symbiont exhibited 100% infection, whereas the Arsenophonus, Liberobacter and Wolbachia showed partial infections at a rate of 83.3%, 45.2% and 76.2%, respectively.

A new species of oribatid mite belonging to the genus Eporibatula Sellnick is described from litter of larch plantation in Mt. Yatsugatake, Yamanashi Prefecture, Central Japan. The new species, Eporibatula variabilis sp. nov. differs from other species of the genus by the presence of ten pairs of notogastral setae, the short median and posterior notogastral setae, the wide lamellae, the well-developed humeral projection, and the weakly arched dorsosejugal suture. Two known species, E. tuberosa Fujikawa, 1972 and E. prominens Bayartogtokh et Aoki, 1998 from Japan and Mongolia, respectively are redescribed. In addition a revised diagnosis of the genus Eporibatula is proposed, and the taxonomic problems of some species belonging to this genus are discussed. The following new combinations are proposed: Eporibatula venusta (Berlese, 1908) comb. nov. and Eporibatula sakamorii (Aoki, 1970) comb. nov. A key to the known species of the genus Eporibatula is given.

The mourning gecko, Lepidodactylus lugubris, is an all-female parthenogenetic species widely distributed in tropical and subtropical regions. We examined clonal diversity of L. lugubris in southern Japan, the northernmost extremity of the species' range. Results indicated that the assemblages of the Ogasawara Islands are composed solely of diploid clone A, which may have originated from artificial transportation after World War II. Assemblages of L. lugubris in the Ryukyu Archipelago were composed solely of triploid clone C. This, along with literature records, suggests that the gecko recently colonized the Ryukyu Archipelago on limited opportunities from limited sources. Samples from the Daito Islands included both diploid and triploid individuals representing one and 11 different clones, respectively. Except for one triploid clone (clone B), these clones were most likely to be endemic to the Daito Islands. Analyses of genotypic pattern suggest that most of the putative endemic triploid clones of the Daito Islands originated from iterative crosses between sympatric diploid clones and males of closely related bisexual species that were most likely extirpated subsequently.

There is little information on the larvae of squat lobsters of the family Galatheidae in spite of their abundance in mesopelagic and abyssal fauna. Larvae of two galatheid species, Agononida incerta (Henderson, 1888) and Munida striola Macpherson and Baba, 1993, are described and illustrated for the first time from the northwest Pacific region based on laboratory-hatched material. The zoeas of these two species are easily distinguished from each other by their size and the morphology of the antenna and maxillule. Previously-known zoeas of the family are compared and a provisional key for 6 galatheid genera is presented.