Enzymatic hatching of fish embryos is caused by a sequential occurrence of many elementary processes from the commitment of the hatching gland cells to the emergence of the embryos. Molecular biological approaches to the formation, properties and function of the hatching enzyme should be required for elucidation of the enzymatic hatching, since this enzyme is a key molecule to analyze these processes. Besides them, there are some other processes indirectly related to hatching, e.g., formation and hardening of the egg envelope. The present article describes the results of our studies on some of the above-mentioned problems in the fish, Oryzias latipes, which have been obtained mostly in the early 1990s.

A neuropeptide Y (eNPY) was isolated from the intestinal extract of eels. This peptide enhanced significantly the serosa-negative transepithelial potential difference (PD) and short-circuit current (l_sc) across the intestine of the seawater eel after pretreatment with isobutylmethylxanthine, serotonin and methacholine. The effects of eNPY on the l_sc were concentration-dependent with a threshold concentration of 3×10⁻⁹ M and a maximal effect at 3×10⁻⁷ M. Similar concentration-responce curve was obtained by porcine peptide YY (pPYY). Since 9 amino acid residues are replaced in the pPYY, this result indicates that these substitutions do not change the potency and the efficacy. These stimulatory actions of eNPY were not blocked by tetrodotoxin, an inhibitor of neural firing, or yohimbine, an α₂-adrenoceptor antagonist, indicating that eNPY acts without enteric neural firing or catecholamine release. When eNPY and adrenaline (AD) were applied simultaneously, the effects were additive only at lower dosage (3×10⁻⁸ M for eNPY, 3×10⁻⁸ M for AD), but not at high dosage (10⁻⁶ M eNPY, 10⁻⁷ M AD). The ceiling effect at high dosage suggests that these two regulators act through common signal transduction systems and affect the Na -K -Cl⁻ cotransport system, since both effects were completely blocked by bumetanide, a specific inhibitor of Na -K -Cl⁻ cotransporter.

The distribution of perikarya and fibers containing neuropeptide Y (NPY)-like immunoreactant was studied in the saccus vasculosus of cartilaginous and bony fishes by immunohistochemistry using streptavidin-biotin technique. NPY-positive fibers were demonstrated in most of the 20 species examined, although the density of the fibers varied among the species. In the white sturgeon, Acipenser transmontanus, NPY-positive varicose fibers formed a supraependymal plexus. On the other hand, NPY-positive perikarya could be demonstrated in the ayu, Plecoglossus altivelis, in which an NPY-positive, presumed afferent fiber fascicle was encountered. These results suggest an involvement of NPY or related substance in the saccus vasculosus function and its regulation.

Using split-fin preparations of the tilapia, Oreochromis niloticus, the effects of various drugs affecting the signal transduction pathway on MCH-induced aggregation of melanosomes were examined. A phospholipase C inhibitor (neomycin sulphate), protein kinase C (PKC) inhibitors (H-7 and staurosporine), PKC activators (SC-9 and TPA) and calmodulin inhibitors (W-7 and W-5) did not block melanophore response to MCH. However, forskolin, an adenylate cyclase stimulating drug, diminished MCH-induced aggregation of melanosomes in a dose-dependent manner. In fact, intracellular concentration of cyclic AMP (cAMP) in MCH (10 nM)-treated melanophores was found to be about 54% of the control level in melanophores immersed in physiological saline. These results suggest that cAMP is the second messenger for MCH action. Since okadaic acid at 10 μM perfectly inhibited melanosome-aggregating response to MCH, the involvement of protein phosphatase 2B in the response was also implied. In addition, the effects of several drugs on NE-induced aggregation were studied, and the possible signal transduction responsible for melanosome aggregation is comparatively discussed.

The mechanism of C-banding was analyzed on the basis of the structural changes of the 30 nm chromatin fibre using scanning electron microscopy (SEM). SEM of non-banded metaphase spreads of L-cells revealed chromosomes consisting of 30 nm chromatin fibre loops along the entire length. No marked difference in both the dimension and appearance of such looped structures was discernible between the centromeric region and the rest of the chromosome. In contrast, C-banded chromosomes exhibited a conspicuous alteration of the fibre conformation in the centromeric region. The looped, fibrous structures were almost completely lost from this region, while the non-centromeric region still exhibited fibrous structures with slightly different appearances compared with those observed in the control chromosomes. On the other hand, results obtained using fluorescence microscopy showed that more DNA retained in the centromeric region than in the non-centromeric region. Since the analytical experiments exhibited that the characteristic collapsed state of the centromeric region occurred only with the alkali treatment but neither with the 2 × SSC nor acid treatments, the centromeric heterochromatin seemed to contain some specific protein which should be sensitive to alkali. The structurally collapsed but subsecuently compact centromeric region may become more, or still, resistant to the DNA extraction due to the 2 × SSC treatment and the centromeric chromatin thus retained may be visualized as the C-band.

We have previously demonstrated that calcium ionophore induced the release of a novel metallo-protease from hemocytes of a solitary ascidian, Halocynthia roretzi. Here, we isolated the enzymes, PI and PII, from the culture media of H. roretzi hemocytes, which had been treated with calcium ionophore, A23187. The purification procedure included hydorophobic and anion-exchange chromatographies, and gel filtration. The molecular weights of the enzymes were estimated to be 11,000 by gel filtration, but the apparent sedimentation coefficients were 5.0 S, which suggests that the H. roretzi enzymes are of larger proteins with molecular weights of 80,000-90,000. The most susceptible substrate was succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, and the optimum pH was 8.0, in either case of PI or PII. The activities of PI and PII enzymes were strongly inhibited by metal-chelating agents and propioxatin A, but not by phosphoramidon, a typical metallo-protease inhibitor. Zinc and calcium ions were found to be essential for the maximum expression of protease activity in both enzymes. Thus, the isolated enzymes are characterized as phosphoramidon-insensitive metallo-proteases, which are inhibited by propioxatin A. Extracellular roles of these enzymes were also discussed.

Elongation factor 1α (EF-1α) is an essential factor for protein synthesis in eukaryotes. Here, we demonstrated that Tetrahymena EF-1α induced bundles of rabbit skeletal muscle F-actin as well as Tetrahymena F-actin in vitro, although Tetrahymena and skeletal muscle actins are different in some parts of their primary structures and in the binding abilities to some actin-binding proteins. Co-sedimentation experiments showed that the binding ratio of Tetrahymena EF-1α to skeletal muscle F-actin in the bundles was 1:1 . Electron microscopic observation showed that alkaline pH or high ionic strength reduced the bundling activity of Tetrahymena EF-1α to some extent, although the EF-1α seemed to be able to induce bundling of the F-actin within the range of physiological condition.

Intact sea urchin spermatozoa were successfully biotinylated with NHS-LC-Biotin and the biotinylated spermatozoa retained the viability. Analysis of the membrane prepared from the biotinylated spermatozoa of the sea urchin Hemicentrotus pulcherrimus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that several proteins such as wheat-germ agglutinin (WGA)-binding protein (220 kDa), guanylyl cyclase (131 kDa), sperm-activating peptide-l (SAP-l)-crosslinking protein (71 kDa), GPI-anchored protein (63 kDa) and functionally unknown proteins (50 kDa and 30 kDa) were specifically biotinylated. Experiments using spermatozoa of sea urchins, Anthocidaris crassispina and Clypeasterjaponicus showed that several proteins similar to those of H. pulcherrimus spermatozoa were also labeled with NHS-LC-Biotin.

Fucose sulfate glycoconjugate (FSG) isolated from the jelly coat of H. pulcherrimus was mixed with solubilized biotinylated sperm membrane proteins of H. pulcherrimus, A. crassispina or C. japonicus and then subjected to gel filtration chromatography on a Sepharose 2B column, indicating that only two biotinylated H. pulcherrimus sperm proteins were coeluted with H. pulcherrimus FSG.

Previously we examined that inhibin-α subunit, transforming growth factor-β₁ (TGF-β₁) and epidermal growth factor (EGF) were expressed in sex-, cell- and stage-specific manners in perinatal rat gonads. To clarify effects of these growth factors on the rat gonadal differentiation and development, indifferent gonadal primordia with mesonephric tubules on gestational day 13 were cultured in vitro for 4 days in serum-free CMRL-1066 medium with inhibin, TGF-β₁, EGF, anti-sera against these growth factors, testosterone or estradiol-17β, and then morphologically examined with reference to seminiferous tubule formation, germ cell division, Wolffian and Müllerian duct development. In male gonads, anti-inhibin-α serum suppressed the seminiferous tubule formation but inhibin, TGF-β₁, EGF or steroid hormones did not affect on the tubule formation, germ cell proliferation nor gonoduct development. Seminiferous tubules in male gonads cultured in the medium containing anti-inhibin-α serum were incomplete and irregular in shape. On the other hand, in female gonads, inhibin suppressed the germ cell division and anti-inhibin-α serum led to the necrosis of germ cells, but other factors affected to neither sex cord formation nor germ cell division. Testosterone and estradiol-17β stimulated female Wolffian and Müllerian duct development, respectively. These results indicate that inhibin induces the seminiferous tubule formation and suppresses the female germ cell division in developing rat gonads in vitro.

Gametogenesis of one year-old induced triploid mussels, Mytilus galloprovincialis, was examined histologically and compared to sibling diploid mussels. Histological analysis revealed that triploid mussels developed a number of primary spermatocytes that were arrested at prophase I. Late in the reproductive season, triploid mussels produced an extremely small number of spermatozoa (9/10000 μm² gonadal section) compared to diploid mussels (1072/10000 μm² gonadal section). All triploid mussels were identified as males, whereas the sex ratio of diploid mussels was almost equal (1.12:1.0, male:female), indicating that sex determination for this species may follow a Z:W model. Sertoli cells in triploid mussels were prominent, had an enlarged cytoplasm, and were easily seen using light microscopy. In comparison, Sertoli cells in diploids were thin and could only be seen by electron microscopy. Sertoli cell hypertrophy in triploid mussels probably reflects their role in eliminating abnormal and degenerating germ cells.

We have been investigating the possible relationship between the teleost caudal neurosecretory system and osmoregulation, by comparing immunostaining intensities of the caudal neuropeptides, urotensins I (UI) and II (UII), in fish sequentially following transfer to different water salinities. Freshwater trout (Oncorhynchus mykiss) were transferred from fresh water (FW) to new FW and from FW to 100% seawater (SW). After 2, 10 and 48 hr posterior spinal cords were removed, fixed and double sequentially immunostained. The 2 hr SW urophyses exhibited more UII and less UI intensity than FW ones. Perikarya anterior to the SW urophyses had less UII and more UI intensity. The 10 hr SW spinal cords showed lower intensity of UI and UII in urophyses and higher intensity of both in anterior perikarya than FW spinal cords. The 48 hr spinal cords did not show any difference in intensity for either UI or UII. We conclude that UI and UII are differentially regulated, that urophysial UI release and perikaryal synthesis are stimulated 2 and 10 hr following transfer to seawater, and that there is an initial inhibition followed within 10 hr by a stimulation of urophysial UII release and perikaryal synthesis following transfer to seawater. By 48 hr the caudal neurosecretory response to SW challenge appears to have subsided, and we hypothesize that the caudal system's role in osmoregulation may be only acute (i.e. within 48 hr following a challenge).

Carassin is a 21-amino-acid tachykinin-related peptide originally isolated from the goldfish brain. Carassin-immunoreactive (ir) perikarya were restricted to the nucleus preopticus periventricularis (NPP); immunoreactive perikarya were distributed sparsely in the rostral and caudal NPP, and were comparatively more in the lateral areas of the mid-NPP. Most perikarya showed thick axonal processes that extended into the rostral area of the Organum vasculosum lamina terminalis (OVLT) and terminated extensively in the vicinity of cells and blood capillaries in its ventral area below the preoptic recess; the OVLT may play a role in release of the carassin-ir material into the general circulation. The axonal terminals diminished caudally and were almost absent in the posterior region of the OVLT. Most epithelial cells of the olfactory organ were carassin-ir positive. Several fibers showing carassin-ir were also present in the olfactory bulb and are presumed to originate from the olfactory epithelium. Carassin-like ir granules were found in some cells of the proximal pars distalis (PPD) of the pituitary gland. Adjacent sections reacted with growth hormone (GH) and gonadotropin (GtH) antibodies, revealed that carassin coexists with GtH in a small percentage of cells. Further, there is sexual dimorphism in the nature and distribution pattern of carassin-ir granules in the PPD. In males, GtH cells contained a few carassin-ir granules; whereas, in females, GtH cells frequently had clusters of carassin-ir granules. These carassin-containing granules may participate in auto- and/or paracrine regulation of the pituitary. The ir-perikarya of the NPP may influence hypophysial function through a multisynaptic pathway. The functions of carassin in goldfish remain to be investigated.

To investigate regulatory mechanisms of proopiomelanocortin (POMC) gene expression in sockeye salmon, we have isolated and characterized cDNAs encoding two types of sockeye salmon POMC, which are referred to as ssPOMC-A and -B. Two types of PCR products were amplified from total RNA of sockeye salmon pituitaries by use of rainbow trout sequences. Full length cDNA clones encoding ssPOMCA and ssPOMC-B were obtained from a pituitary cDNA library of sockeye salmon using the PCR products as probes. The ssPOMC-A and -B cDNAs have a length of 1072 and 1709 bps, respectively. Northern blot analysis showed that both ssPOMC-A and -B mRNAs were expressed only in the pituitary, and their sizes were about 1.2 kb and 1.8 kb, respectively. The presence of two ssPOMC genes was confirmed by Southern blot analysis of genomic DNA obtained from a single sockeye salmon. The deduced amino acid sequences of the ssPOMC-A and -B contained 230 and 226 residues, respectively. The amino terminal of β-endorphin in ssPOMC-B which corresponds to Met-enkephalin domain is YSGFM, which is different from YGGFM of Met-enkephalin found in many other vertebrate species. The homology of nucleotide sequences between ssPOMC-A and -B is 59% in the entire coding region, whereas α-MSH coding regions are highly homologous (91 %). Although the deduced amino acid sequences of ssPOMCs show 43% overall similarity, their hydropathy profiles are coincident with those of several other vertebrate species, particularly the amino terminal of N-terminal peptide (NPP) shows almost the same pattern with other vertebrate NPPs.

Androgen receptor immunoreactivity was examined in the abdominal glands of the cloaca in adult male red-bellied newts, Cynops pyrrhogaster, using a polyclonal anti-androgen receptor antibody, PG21. In castrated males treated with saline, prolactin, testosterone propionate or both prolactin and testosterone propionate, all displayed androgen receptor immunostaining of nuclei in the epithelial cells of the glands. Androgen receptor-immunoreactive signals were distributed uniformly in the nuclei in the castrates treated with saline or prolactin. On the other hand, the signals were distributed reticulately in the nuclei in the castrates treated with testosterone propionate or both prolactin and testosterone propionate. Following treatment with testosterone propionate or both prolactin and testosterone propionate, shape of the androgen receptor-immunoreactive nuclei was altered from nonspherical profiles toward spherical profiles and its size became increased. There were no differences in these changes between the testosterone propionate- and both prolactin and testosterone propionate-treated castrates. These findings suggest that androgen participates in regulating size and shape of the nuclei and distribution of androgen receptor in the nuclei of the epithelial cells of the abdominal glands of male newts and that the structural reorganization is necessary for gene expression under the influence of androgen.

We developed new means of measuring the ratio of the short to the long form (S/L ratio) of the mouse prolactin receptor (mPRL-R) cDNA by PCR using a primer common to the two forms and two specific primers. A means of estimating the amount of mPRL-R cDNA by competitive PCR was also established. We confirmed that these procedures were valid, since the S/L ratio of standard DNA was unaltered by one-sided cPCR amplification under the following conditions: the ratio was between 0.1 and 4, and the amount of cDNA was between 10³ and 10⁷ molecules/tube.

The result of one-sided cPCR showed that the short form was dominant in the mouse liver, while the long form was dominant in other tissues. In addition, pituitary grafting increased the S/L ratio in the liver, implying that prolactin down-regulated the functional long form of PRL-R and lowered tissue sensitivity to prolactin itself by modifying the post-transcriptional regulation of PRL-R.

When mature chum salmon (Oncorhynchus keta) caught in Otsuchi Bay (salinity 34 ppt), northern Honshu Island, Japan, were transferred directly to fresh water, they attained normal plasma ion levels within 24 hr. Plasma prolactin remained low in the fish kept in seawater. On transfer to fresh water, a significant increase in plasma prolactin concentration was seen only in females, but not in males. Metabolic clearance rate (MCR) and secretion rate of prolactin were calculated from its plasma levels after intra-arterial injection of chum salmon prolactin into chronically-cannulated fish. In both males and females, a significant increase in MCR was seen after transfer to fresh water, indicating that prolactin is involved in freshwater adaptation in both sexes. In females, the secretion rate increased significantly after 6 days in fresh water. No such change in the secretion rate was seen in males. It seems likely that prolactin secretion is affected by reproduction-related changes occurring in mature females in fresh water.

To investigate whether four Papilio species, Papilio xuthus, P. machaon, P. bianor and P. helenus, have two molecular forms of the prothoracicotropic hormones (PTTHs), referred to as big- and small-PTTHs, the PTTHs were extracted and fractionated from their pupal brains. The activating ability of big- and small-PTTH fractions was examined by the in vitro assay using the prothoracic glands (PGs) of 2-day-old 5th-instar larvae of their own and several other papilionid species.

Big- and small-PTTH fractions activated the larval PGs of their own species to increase the ecdysteroid secretion in vitro. The doses of small-PTTH fractions for activating the larval PGs were 8- to 10-times larger than those of big-PTTH fractions.

The big- and small-PTTH fractions as well as those of P. machaon activated the PGs of 2-day-old 5thinstar larvae of several heterogeneous papilionids, but the activating ability did not always decrease with the distance of the genetic (or phylogenetic) relationships.

The results indicate that P. machaon, P. bianor and P. helenus may have two molecular forms of the PTTHs, both of which activate the larval PGs of the same species in vitro as in the case of P. xuthus. The bigand small- PTTHs of P. xuthus as well as those of several other Papilio species may retain an ability to activate the 5th-instar larval PGs of numbers of heterogeneous papilionids in vitro.

Compiling all the previous and new records, the most recent list of drosophilid species from East Siberia (56 spp.) and Russian Far East (120 spp.) is provided along with descriptions of five new species, supplementary descriptions of two known species, some nomenclatural changes (five new synonymies, a new homonymy, and a change in status of a taxon from the specific to the subspecific rank), and a key to all the species. Drosophilid faunas of these two regions are compared with those of surrounding six regions in the Northern Hemisphere. Russian Far East constitutes the northeastern Asiatic realm in drosophilid biogeography together with northeastern China and Hokkaido, northern Japan. On the other hand, East Siberia belongs to the northern Palearctic realm extending to northern Europe. The border between these two biogeographic realms lies on the Stanovoy Mts.

Animals in each subgroup of the phylum Chordata exhibit a similar process by which they form a tubular central nervous system (CNS). However, little is known about spatial relationship among the CNSs of chordates; vertebrates, cephalochordates and urochordates (tunicates). Ascidians constitute a major animal group in the subphylum Urochordata. In the present study, we examined the expression patterns of labial and orthodenticle related genes of the ascidian, Halocynthia roretzi, in the developing larval CNS. These homeobox genes exhibited region-specific expression patterns that are strikingly similar to those of murine Hoxb-1 and Otx2. The regionalization as characterized by the expression of these genes supports the division of the ascidian larval CNS suggested by the previous morphological studies. Furthermore, conservation of the expression pattern of the homeobox genes suggests that such regionalization occurred in the CNS of a putative common ancestor of chordates.

Heterochronic expressions of several larval and adult phenotypes in Hynobius retardatus, which had been reported to show neotenic reproduction, were described in normally metamorphosing animals (controls) and goitrogen-induced, metamorphosis-arrested larvae. External gills, dorsal tailfins and Leydig's cells in the epidermis were completely diminished in the controls during and after metamorphosis, but fully remained in the metamorphosis-arrested larvae. Two types of dermal glands, mucous and serous glands, however, behaved differently from Leydig's cells, even though they constituted the same skin. The dermal gland cells in the controls appeared at a premetamorphic stage, gradually increased in number during the metamorphosis and fully developed after the metamorphosis. Those in metamorphosis-arrested larvae appeared much later than in the controls. Thus, aged, metamorphosis-arrested larvae had skin which consisted of larval type epidermis (Leydig's cells) and adult type dermis (mucous and serous glands). Contrary to these, a transition of globin subunits from larval to adult types occurred practically on the same time schedule in both the controls and metamorphosis-arrested larvae. These observations suggest that there are at least three types of organs or cells which behave differently during and after metamorphosis in Hynobius retardatus.