Hormonal control of the expression of courtship behavior and of secretion of the female-attracting pheromone sodefrin by the male red-bellied newt, Cynops pyrrhogaster, together with the hormonal influence on the responsiveness to the pheromone in the female, is reviewed.

Expression of the initial stage of the courtship behavior, i.e., tail vibration by the male in front of the female, is dependent on prolactin (PRL) and androgen. During the courtship, sodefrin seems to be released from the cloaca through the ducts of the abdominal gland. Both content of immunoreactive sodefrin and preprosodefrin mRNA levels in the abdominal gland are elevated by a combination of PRL and androgen, indicating that the pheromone synthesis is stimulated by these two hormones. On the other hand, the discharge of sodefrin is accelerated by AVT, its action being mediated by V₁ receptor. In female newts, responsiveness of the vomeronasal epithelium to the pheromone is elevated by a combination of PRL and estrogen. Thus, it can be concluded that PRL, AVT, and sex steroids are key hormones for the reproductive performance in the red-bellied newt. In this article, the significance of the structure of the pheromone molecule as a peptide is also discussed in terms of its species-specificity and its effectiveness in an aquatic environment.

We have isolated a new microglial gene, mrf-1, which is upregulated on microglia in response to apoptosis of granule neurons in cerebellar cell cultures. We examined whether or not upregulation of MRF-1 is observed in response to necrotic neuronal death both in vivo and in vitro. Though MRF-1 was detected on ramified/resting microglia in the brain of normal adult rats, activated microglia in the region of the brain where neuronal damage was induced by ischemia were strongly immunostained with anti-MRF-1 anti-body. In the in vitro system, we confirmed, with immunocytochemistry or RT-PCR, that MRF-1 or mrf-1 mRNA were constitutively expressed in ramified microglia at significant but lower levels than in amoeboid one. Moreover, by Northern blot, it was ascertained that expression level of mrf-1 mRNA on microglia was markedly upregulated in response to glutamate-induced death of granule cells in a cerebellar cell culture. These results indicate the following: 1) expression of mrf-1 in microglia may be markedly enhanced upon not only apoptotic but also necrotic neuronal death, and 2) MRF-1 is, thus, an useful marker for identifying all types of microglia in vivo and in vitro.

Sulfate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Sulfate transport can be coupled to proton, sodium symport or antiport processes involving chloride or bicarbonate. It had previously been observed in Aplysia gut that sulfate was actively absorbed. To understand the mechanism for this transport, short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and sulfate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na was enhanced by the presence of sulfate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of sulfate was dependent upon the presence of Na and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetanide, added to either side, had no effect on transepithelial potential difference or short-circuit current in the Aplysia gut bathed in a Na₂SO₄ seawater medium. However, mucosal thiosulfate inhibited the net mucosal-to-serosal fluxes of both sulfate and Na and the thiosulfate-sensitive Na flux to that of sulfate was 2:1. These results suggest the presence of a Na-SO₄ symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.

A slow transient electric potential change (electro-olfactogram, EOG) can be recorded through an Ag-AgCl electrode placed on the olfactory epithelium in response to stimulation with an air stream to the tissue in toads (Bufo japonicus). During the breeding season, oscillatory potential changes (OSC) superimpose on the EOG. In the present study the OSC amplitude was found to be highly correlated with the migratory behavior. Since toads track the route to and from the breeding pond using olfactory cues along the migration route, the enhanced OSC should be responsible for the breeding migration.

A significant positive correlation was found between plasma gonadotropin levels and the OSC amplitude in males captured during the breeding migration. There was no significant relationship between plasma gonadotropin levels and the OSC amplitude in female toads during the breeding season, but there was a significant correlation between plasma progesterone levels and the OSC amplitude. In males, hypophysectomy just before the breeding season decreased the OSC amplitude. And testis weight was also positively correlated with the OSC amplitude in January. These results suggest that the appearance of the OSC is related to the timing of the activation of the reproductive system. However, treatment of toads with hCG (human chorionic gonadotropin), testosterone, estradiol or progesterone in the non-breeding season did not induce a significant change in the OSC amplitude. Other factor or factors may be required in activation of the olfactory system of the toad in a non-reproductive stage together with the hormones of the gonadal axis.

The aim of this study was to present evidence for prolonged spontaneous firing in an anucleate axonal segment of an identifiable crayfish anal motoneuron L (AML) and to determine the initiation site of this firing. AML has its soma in the 6th abdominal ganglion (A6). By separating a nerve with the AML axon from A6 and the target muscle, various lengths of an anucleate AML axonal segment were procured. Then, AML activity was recorded extracellularly for 14–26 hr from the distal end of this axonal segment. This segment (n=19) exhibited spontaneous firing, which occurred without any stimulation 0.03–5.13 hr after the A6-cut and persisted tonically for 0.20–19.98 hr. During firing, the frequency augmented gradually, whereas the amplitude decreased gradually. There was no significant correlation between latency and duration of the firings. No correlation was noted between latency and length of the axonal segment or its size, or between duration and length or size. These results revealed that the anucleate AML axon itself can inherently generate prolonged firing. The delay in the appearance of AML impulses between the proximal and distal regions at the same axonal segment proved that the firing occurred proximally. There was no significant difference in delays between firing following the A6-cut and the spontaneous firing observed after the A6-cut. This suggests that the initiation site of the spontaneous firing is at the proximal end of the AML axonal segment, since the AML firing following the A6-cut occurs at its cut end.

Sea urchin spinal ligaments (the catch apparatus) were extracted with glycerin, and electron microscopic observations comfirmed that no cell membranes remained intact after glycerination. We studied the effects of cations (Na , K , Ca² , Mg² ) on the mechanical properties of the glycerinated ligaments. Monovalent cations decreased whereas divalent cations increased the viscosity of the ligaments. The ion dependencies were similar to previous results with detergent-extracted holothurian dermis, which suggests that the echinoid ligament shares a similar mechanism for changes in mechanical properties with other catch connective tissues. This provides evidence against the hypothesis of del Castillo et al (1995) that muscles in the catch apparatus are responsible for the changes in mechanical properties of the ligament. Fine projections cross-bridging collagen fibrils were observed in the glycerin-extracted ligaments as well as in the intact ligaments. They were found in all the ionic conditions studied.

A model system for the formation of astral-shaped microtubules (Mts) consisting of Latex beads (diameter of 0.2 μm), a protein fraction (p51) comprised of MTOGs (microtubule-organizing granules) and tubulin was established. The Latex beads were first incubated with p51 in the presence of GTP at 0°C, then the purified tubulin dimer fraction was added, resulting in the formation of an aster-like structure observed by dark-field microscopy. On preincubation of the Latex beads with GDP instead of GTP, the asters did not form. Unhydrolyzable GTP analogues such as GTP-γS and GMP-PNP promoted aster formation as did GTP as observed by dark-field microscopy. Polylysine, as representative of basic polymers capable of binding to the surface of the Latex beads, promoted spontaneous Mt assembly, and eventually an aster-like structure without Latex beads in the center formed. Further analyses made by measuring the optical density of the aster-forming system produced the following results. 1) preincubation of the Latex beads with GTP or GMP-PNP supported Mt assembly from the beads showing profiles typical for a sitedirected assembly without the lag phase. 2) GTP-γS and GDP inhibited the turbidity increase of the system, causing a decrease in both the initial velocity and the level of steady state of Mt assembly. 3) Anti-p51 monoclonal antibody (HP1) substantially inhibited the aster formation, while anti-γ-tubulin antibody only slightly inhibited assembly.

Mechanical stress causes various responses in cells both in vivo and in vitro. Realignment of cells and stress fibers is one of the remarkable phenomena that are induced by the stress. However, the mechanism by which their realignment is controlled is largely unknown. In this study, effects of mechanical stretch on the morphology of cultured cells were examined using a cyclic and reciprocal cell stretching apparatus. A10 cells, a cell line derived from rat aortic smooth muscle, were used as a model, since they are spindle-shaped and have remarkable stress fibers aligned along the longitudinal cell axis. Therefore, the orientation of the cell and stress fibers could be easily identified. When the cells were cultured on elastic silicone membranes and subjected to cyclic and reciprocal stretch with an amplitude of 20% at a frequency of 60 cycles per minute, actin stress fibers were aligned obliquely to the direction of stretching with angles of 50 to 70 degrees within about 15 min after the onset of stretching. Then, after 1–3 hr of cyclic stretching, the long axes of a majority of the cells were also reoriented to similar directions to the stress fibers. The stretch-induced cell reorientation was blocked by 1 μM cytochalasin B, but not by colcemid. These results indicate that the orientation of cells and actin filaments are closely related and actin filaments play a critical role in the early step of the cell reorientation.

To clarify the molecular mechanisms of phagocytosis, we have been preparing monoclonal antibodies that inhibit phagocytosis by the hemocytes of the ascidian Halocynthia roretzi. A monoclonal antibody, RA5, inhibited the phagocytosis of non-treated sheep red blood cells (SRBCs) and yeast cells. It was demonstrated that the phagocytosis by the hemocytes was enhanced by pretreatment of target cells, SRBCs or yeast cells, with H. roretzi plasma. However, the RA5 antibody was unable to inhibit the phagocytosis of plasma-treated target cells. These results strongly suggest that the molecule recognized with the RA5 antibody is involved in the opsonin-independent phagocytosis. Western blot analysis showed that this antibody recognized a 200 kDa protein in H. roretzi hemocytes. On the other hand, flow cytometry analyses showed that a galactose-specific lectin (Gal-lectin) and complement C3 (AsC3), present in H. roretzi plasma, can bind to SRBCs and yeast cells, respectively, to enhance the phagocytosis of the respective target cells. Thus, H. roretzi hemocytes undergo opsonin-independent and -dependent phagocytosis, and Gal-lectin and AsC3 both function in the opsonin-dependent phagocytosis.

Previous studies with tissue recombination experiments demonstrated that the splanchnic mesenchymes, including hepatic, pulmonary and stomach mesenchymes can support hepatocyte differentiation from the hepatic endoderm in 9.5-day mouse embryos. This phenomenon corresponds to the second hepatic induction. The present study was undertaken to determine whether direct cell-cell contacts between the hepatic endoderm and mesenchyme are required for hepatocyte differentiation, using transfilter experiments in which membrane filters with various pore sizes were inserted between the endoderm and the hepatocyte-inducing mesenchyme (the chick lung mesenchyme). Hepatocyte differentiation occurred even when the direct cell-cell contacts between the hepatic endoderm and the mesenchyme were absent, suggesting that humoral factors may work in this interaction. However, growth of hepatocytes was most prominent in the transfilter experiments with filters having pore sizes of 0.2 and 0.8 μm, which permitted mesenchymal cells or their cell processes to penetrate to the side of the endoderm. These results suggest that two types of tissue interactions, including humoral mesenchymal factors and very local tissue interactions such as direct cell-cell contacts, may be involved in the second step of hepatic induction.

Genomic and complementary DNA fragments encoding an estrogen receptor (ER) homolog were cloned from the medaka fish, Oryzias latipes. The genomic DNA contains nine introns. The cDNA encodes a protein of 620 amino acids that is composed of the complete set of the conserved ER domains. Sequence comparison among fish and human ER genes suggests that the human gene may have lost two introns during the evolution. ER-specific mRNA was expressed in large amounts in adult tissues such as brain, ovary, testis, and female liver. As expected by the fact that embryos and fry are highly sensitive to and sex-reversed by estrogen, ER mRNA was detected in them but at a basal level. Oral administration of estrogen elevated the hepatic expression of ER mRNA, suggesting an autoregulatory loop for transcriptional activation. However, estrogen did not affect the expression in embryos and fry, suggesting that the positive loop is silent at the juvenile stage and that the basal expression is not due to the absence of estrogen. We also found that estrogen inhibits the embryonic development of blood vessels, providing a new, simple method of bioassay for estrogenic activity.

In Crustacea, reproductive function and mechanisms regulating vitellogenesis have not been fully elucidated. This is due in great part to a lack of information concerning the biochemical nature of the vitellogenin molecule, the hemolymph precursor of yolk protein, vitellin, as well as the functional expression of the vitellogenin-encoding gene. We have therefore cloned a cDNA encoding vitellogenin in the kuruma prawn, Penaeus japonicus based on the N-terminal amino acid sequence of the 91 kDa subunit of vitellin. The open reading frame of this cDNA encoded 2,587 amino acid residues. This is the first investigation reporting a full-length cDNA and its corresponding amino acid sequence for vitellogenin in any crustacean species.

Northern blot analysis and in situ hybridization have revealed that mRNA encoding vitellogenin was expressed in both the follicle cells in the ovary and the parenchymal cells in the hepatopancreas. In nonvitellogenic females, vitellogenin mRNA levels were negligible in both the ovary and hepatopancreas, but in vitellogenic females, levels were dramatically increased in both tissues. In the ovary, highest levels were observed during the early exogenous vitellogenic stage, and thereafter rapidly decreased, whereas in the hepatopancreas, high levels were maintained until the onset of the late vitellogenic stage. Differing profiles of vitellogenin mRNA levels in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenin synthesis harbor separate and complementary roles during vitellogenesis.

Epidermal growth factor (EGF) is one of growth factors that are thought to mediate the stimulatory effects of estrogen on the proliferation of uterine epithelial cells. The present study was attempted to obtain direct evidence for the mitogenic effects of EGF on uterine epithelial cells, and to prove that EGF and EGF receptors are expressed in these cells. Mouse uterine epithelial cells were isolated from immature female mice and cultured with or without EGF for 5 days. EGF (1 to 100 ng/ml) significantly increased the number of uterine epithelial cells, and the maximal growth (141.9± 8.3% of controls) was obtained at a dose of 10 ng/ml. In addition, EGF (0.1 to 100 ng/ml) increased the number of DNA-synthesizing cells immunocytochemically detected by bromodeoxyuridine uptake to the nucleus. Northern blot analysis revealed that the uterine epithelial cells expressed both EGF mRNA (4.7 kb) and EGF receptor mRNAs (10.5, 6.6, and 2.7 kb) These results suggest that the proliferation of uterine epithelial cells is regulated by the paracrine and/or autocrine action of EGF. Our previous study demonstrated the mitogenic effect of IGF-I on uterine epithelial cells. To examine whether the EGF- and IGF-I signaling act at the same level in the regulation of the proliferation of uterine epithelial cells, the cultured cells were simultaneously treated with IGF-I and EGF. IGF-I was found to additively stimulate the mitogenic effects of EGF, suggesting that the EGF-induced growth of uterine epithelial cells is distinct from IGF-I-induced growth.

Sperm displacement is a sperm competition avoidance mechanism that reduces the paternity of males that have already mated with the female. Direct anatomical sperm removal or sperm flushing is known to occur in four insect orders: Odonata, Orthoptera, Coleoptera and Hymenoptera. In a fifth order, Dermaptera (earwigs), I found that the virga (the elongated rod of the male genitalia) of Euborellia plebeja seems to be used to remove rival sperm from the spermatheca (a fine-tubed female sperm storage organ). In this species, copulation lasted on average 4.6 minutes, during which time the male inserted the virga deep into the spermatheca, and then extracted it ejaculating semen from the opening of the virgal tip. The extraction of virgae (with its brim-like tip) appeared to cause removal of stored sperm in the spermatheca. The virga was as long as the body length of males, and the spermatheca was twice the female body length. The long length of the spermatheca and the possible removal function of the virga may select for virgal elongation.

To illuminate molecular phylogenetic relationships among Eurasian species of the genus Mustela (Mustelidae, Carnivora), we determined nucleotide sequences of the complete mitochondrial cytochrome b gene region (1,140 base pairs). Molecular phylogenetic trees, constructed using the neighbor-joining and the maximum likelihood methods, showed the common topology of species relationships to each other. The American mink M. vison first branched off and was positioned very remotely from the other species of Mustela. Excluding M. vison, the ermine M. erminea first split from the rest of the species. Two small body-sized weasels, the least weasel M. nivalis and the mountain weasel M. altaica, comprised one cluster (named “the small weasel group”). The other species formed another cluster, where the remarkably close relationships among the domestic ferret M. furo, the European polecat M. putorius, and the steppe polecat M. eversmanni were noticed with 87–94% bootstrap values (named “the ferret group”), supporting the history that the ferret was domesticated from M. putorius and/or M. eversmanni. The European mink M. lutreola was the closest to the ferret group. The genetic distance between the Siberian weasel M. sibirica and the Japanese weasel M. itatsi corresponded to differences of interspecific level, while the two species were relatively close to M. lutreola and the ferret group. These results provide invaluable insight for understanding the evolution of Mustela as well as for investigating the hybridization status between native and introduced species for conservation.

A new species of scopelocheirid amphipod, Scopelocheirus onagawae, was found during an ecological study of the marine scavenging amphipods in Onagawa Bay, Northeastern Japan. This species is closely similar to S. hopei and S. polymedus in the elongate gnathopod 1 and the broad merus of pereopod 5, but clearly distinguished from them by lateral cephalic lobe, maxilla 2, pereopod 6, epimeron 2 and uropods 1-3. This paper also describes some taxonomic commentary on the generic identity of the genus Scopelocheirus and a related genus Aroui.

Phylogenetic relationships within the dominant reef coral family Acroporidae were inferred from the mitochondrial genes cytochrome b and ATPase 6. The rate of nucleotide substitution in the genes gave proper resolution to deduce genetic relationships between the genera in this family. The molecular phylogeny divided this family into three major lineages: the genera Astreopora, Montipora and Acropora. The genus Anacropora was included in the same clade as the genus Montipora, suggesting its recent speciation from Montipora. The subgenus Isopora was significantly distant from the subgenus Acropora. Taken together with morphological and reproductive differences, we propose that these two subgenera be classified as independent genera. The divergence times deduced from the genetic distances were consistent with the fossil record for the major genera. The results also suggest that the extant reef corals speciated and expanded very recently, probably after the Miocene, from single lineage which survived repeated extinction by climate changes during the Cenozoic era.