Dynamics of changes in physiology and morphology were studied in Hermissenda photoreceptors after in vitro conditioning with paired light and vibration. An increase in input resistance of the type B photoreceptor was observed following 5 paired presentations of light and vibration. It peaked at 10 min after in vitro conditioning, then decreased to a level twice the pre-conditioning level for more than 60 min. Contraction of the terminal branches along centro-lateral direction was initiated 5 min after conditioning and reached its final state at 10 min after conditioning. The pairing specific contraction of the axon terminal was not observed in ASW containing anisomycin. The dynamics in physiology and morphology were completely parallel 30 min after conditioning. These findings suggested that in vitro conditioning induced contraction was dependent on protein synthesis dependent process initiated within 5 min after training trials and that the change of cell morphology is a form of short-term synaptic plasticity that involves changes in macromolecular synthesis. Present findings that functional remodeling at the terminal branch of the type B photoreceptor occurred within 10 min after conditioning was the fastest modification process reported so far.

The ischemia-induced synaptic potentiation (ISP) during and/or after brain ischemia has been suggested to be one of the crucial factors responsible for irreversible neuronal damage of hippo-campal CA1 pyramidal neurons. However, the presynaptic modulation mechanism that leads to neuronal damage during and/or after ischemia was still unknown. By combining electrophysiological methods and infra-red differential interference contrast (IR-DIC) imaging procedures, we showed for the first time that ISP is the result of extraordinary presynaptic depolarization in association with the suppression of 4-amino-pyridine (4-AP) sensitive K channels at the presynaptic sites. Furthermore, we also showed that the 4-AP sensitive presynaptic K channels played a crucial role in inducing neuronal damage at a very acute phase of ischemia-induced neuronal damage and would be a therapeutic target against the neuronal damage after brain ischemia.

Ascidian larvae of Ciona intestinalis change their photic behavior during the course of development. Newly hatched larvae show no response to a light stimulus at any intensity. At 4 hr after hatching, larvae were induced to start to swimming upon the cessation of illumination, and to stop swimming upon the onset of illumination. At a weaker light intensity (5.0×10⁻³ J/m²·s), the larvae showed similar responses to either a single stimulus or repeated stimuli of onset and cessation of light until 10 hr after hatching. At a stronger light intensity (3.2×10⁻¹ J/m²·s), when the stimulus was repeated, they showed sensitization and habituation of the swimming response.

At 3 hr after hatching the larvae failed to show any response to an initial stimulus at any intensity of light, but after several repeated stimuli (sensitization) they showed a swimming response at light intensities above 4.0×10⁻² J/m²·s. At 5 hr and with intensity above 1.0×10⁻² J/m²·s, the larvae showed photoresponses to the first stimulus, but after several repetitions the larvae failed to stop swimming upon the onset of light (habituation). A repeated series of stimuli at stronger intensities of light caused greater habituation; this habituation was retained for about 1 min. Since the larval central nervous system in Ciona is comprised of only about 100 neurons, learning behavior in ascidian larvae should provide insights for a minimal mechanism of memory in vertebrates.

The physiological significance of the cooperativity of human hemoglobin (Hb) is considered from the viewpoint of the effectiveness of the Bohr shift at the sites of O₂ release and uptake across the placental membrane. The effects of the Bohr shift was examined by changing the O₂ saturation of Hb (S(pO2)) per unit change in P₅₀, -dS(PO2)/dP₅₀, where P₅₀ is partial pressure of O₂ at half saturation. The Bohr shift at the sites of O₂ uptake and release was found to be highly effective in both fetal and maternal bloods at physiological degree of cooperativity (Hill's coefficient, n=2.65). From the results obtained in this paper, it is concluded that the positions of OECs of fetal and maternal Hbs are regulated to receive a maximal benefit from the Bohr shift, and that a relatively low n value of human tetrameric Hb is adequate for the O₂ and CO₂ exchange across the placental membrane.

Na,K-ATPase (sodium pumps) provide the primitive driving force for ion transport in bran-chial epithelial cells. Immunoblots of epithelial homogenates of both seawater (SW)- and freshwater (FW)-adapted tilapia gills as well as rat brain homogenate, a positive control, revealed one major band with a molecular weight of about 100 kDa. SW-adapted tilapia gills possessed larger (about 2-fold) amounts of sodium pumps compared with FW-adapted tilapia gills. ³H-ouabain binding representing functional binding sites of Na,K-ATPase was also higher (about 3.5-fold) in gills of SW-adapted tilapia compared to that of FW-adapted fish. Moreover, specific activities of SW fish were higher (about 2-fold) than those of FW fish. Double labeling of Na,K-ATPase and Con-A, a fluorescent marker of MR cells, in tilapia gills followed by analysis with confocal microscopy showed that sodium pumps were localized mainly in MR cells, including the SW type and different FW types. Although more-active expression of Na,K-ATPase was demonstrated in gills of SW-adapted tilapia, no significant differences in densities of apical openings of MR cells were found between SW- and FW-adapted fish. These results indicate that, during salinity challenge, tilapia develop more “functional” Na,K-ATPase in SW-type MR cells to meet physiological demands.

To elucidate the molecular mechanisms involved in oogenesis, we applied a differential display method to identify genes whose expression was detected only in ovaries containing oocytes. One of the cDNA fragments isolated by mRNA differential display was similar in structure to vitellogenin. Using this fragment, a full-length cDNA encoding putative vitellogenin in the Pacific oyster Crassostrea gigas was cloned by RACE (rapid amplification of cDNA ends), and its amino acid sequence was deduced. The open reading frame predicted 1583 amino acid residues. The deduced primary structure of putative vitellogenin in C. gigas was shown to be similar to vitellogenins of various other mollusk, fish, crustacean and nematode species, especially in the N-terminal region. Reverse transcription-mediated PCR revealed that mRNA encoding putative vitellogenin was expressed only in the ovary. In situ hybridization analysis revealed that putative vitellogenin mRNA was expressed strongly in the follicle cells in the ovary. It is concluded that the follicle cells are the site of putative vitellogenin synthesis.

In order to elucidate a possible role of estrogen receptor in the gonadal sex differentiation and the sex reversal with sex steroids, we examined for the formation of testis or ovary in transgenic medaka fish overexpressing the medaka estrogen receptor under the constitutive medaka β-actin promoter. The transgenic fish underwent the genetically determined gonadal differentiation and showed the same sex-reversal rates as those of wild-type non-transgenic fish after treatments with estrogen and androgen. These results present invaluable data to reconsider the role of estrogen receptor in the gonadal sex determination.

A strain of carnivorous tardigrade, Milnesium tardigradum, was reared in water on agar plates at 25°C. The monogonont rotifer Lecane inermis was presented as a food source. This rearing system permitted detailed observation of tardigrade behaviour. Daily measurements of body length allowed the growth rate and moulting cycle of this species to be determined. The life history of M. tardigradum raised under these conditions included up to seven periods of moult. The first and second moults occurred at intervals of 4–5 days, and individuals reached reproductive maturity at the 3rd-instar stage; the first period of egg laying accompanied the third moult. The most rapidly developing animal in the study population laid eggs 12 days after hatching. The egg-laying intervals or moulting intervals of adult animals were around 6-10 days. The mean clutch size was 6.9 eggs. All tardigrades in this laboratory population were female and reproduced by parthenogenesis. The duration of the embryonic stage ranged from 5–16 days. The most long-lived female survived for 58 days after hatching, and laid a total of 41 eggs in 5 separate clutches. The entire life cycle of tardigrades reared under these conditions was recorded and photographed. A brief description of the embryonic development of M. tardigradum was also reported.

The Sox family is a large group of transcription factors that are characterized by the presence of a DNA-binding HMG domain. We isolated HrSoxB1, an ascidian homolog of the Sox gene that belongs to the B1 subclass of the Sox family, from Halocynthia roretzi. Expression was initiated as early as the 8-cell stage. During cleavage stages, HrSoxB1 was expressed in three quarters of embryonic blastomeres but not in posterior-vegetal (B-line) blastomeres. Misexpression of mRNAs of HrPEM but not of macho-1, whose maternal mRNAs are localized to the posterior-vegetal cytoplasm of eggs and early embryos, repressed the anterior-vegetal expression of HrSoxB1. This result suggests that the zygotic expression of HrSoxB1 is controlled by the localized maternal mRNA. When HrSoxB1 was overexpressed in early embryos, ectopic expression of HrBra, a gene for a transcription factor expressed in notochord blastomeres, occurred in the most posterior blastomeres (B7.5), although these blastomeres did not eventually differentiate into notochord but developed into muscle, as they do in normal embryogenesis. In later embryogenesis, HrSoxB1 was specifically expressed in neural plate cells. However, overexpression of HrSoxB1 did not affect the expression of a neural plate marker gene, HrETR-1.

An organ culture method for pituitary glands isolated from immature Japanese eels (Anguilla japonica) was developed. This method could conserve the histological features of the pituitary glands for at least 21 days. The ability to synthesize gonadotropic hormone (GTH) in cultured eel pituitary glands was examined by detecting luteinizing hormone (LH) β protein immunohistochemically. In a basal medium (Leibovitz L-15), LH β-immunoreactive cells were very scarce, but after addition of estradiol-17β (E2) a large number of immunoreactive cells appeared, particularly in the proximal pars distalis. The stimulatory effects of E2 on LH β synthesis were dose (1–100 ng/ml)- and time (1.5–7 days)-dependent. Thus, in contrast with previous reports of the lack of a direct effect of E2 on GTH synthesis in primary cultured eel pituitary cells, the present results clearly indicate that E2 can stimulate GTH synthesis in immature eel pituitary glands. This organ culture method is useful to examine the actions of steroids and also other endocrine factors on the eel pituitary gland.

Male sexual characteristics in Crustacea are induced by androgenic gland hormone (AGH), which is produced by the male-specific androgenic gland. Recently, AGH in the terrestrial isopod Armadillidium vulgare was characterized and its cDNA cloned, the first example in which the structure of AGH was elucidated. We report here the molecular cloning of cDNAs encoding AGH precursors from two additional terrestrial isopods, Porcellio scaber and P. dilatatus. cDNA fragments encoding Porcellio scaber AGH (Pos-AGH) and P. dilatatus AGH (Pod-AGH) were amplified by RT-PCR using degenerate oligonucleotide primers designed based on the amino acid sequence of A. vulgare AGH (Arv-AGH). Subsequently, full length cDNAs were obtained by 5′- and 3′-RACE. Both AGH precursors consisted of a signal peptide, B chain, C peptide and A chain, and exhibited the same organization as that of Arv-AGH. The amino acid sequences of the A and B chains, which comprise mature AGH peptide, were highly conserved among the three species, while that of the C peptide showed only low sequence similarity. In Northern blot analysis, each cDNA fragment used as a probe specifically hybridized with a single band (0.75 kb) in mRNA extracted from whole male reproductive organs. In analysis of the tissue-specific gene expression of these two AGHs by RT-PCR, it was revealed that both AGH transcripts were detected only in cDNA synthesized using total RNA from the testis carrying the androgenic glands, but not in that from testis only, seminal vesicle, vas deferens, or hepatopancreas.

Transforming growth factor-α (TGF-α), a member of the epidermal growth factor (EGF) family, is produced within the mouse anterior pituitaries. However, the cell types of TGF-α-expressing cells and the physiological roles of TGF-α within mouse pituitary glands remain unclear. The aim of the present study was to localize TGF-α mRNA-expressing cells, and to clarify the involvement of TGF-α in estrogen-induced DNA replication in mouse anterior pituitary cells. Northern blot analysis demonstrated TGF-α mRNA expression in adult male and female mouse anterior pituitaries. In situ hybridization analysis of the pituitaries in these mice showed that TGF-α mRNA-expressing cells in the anterior pituitary are round, oval, and medium-sized. TGF-α mRNA was colocalized in most of the growth hormone (GH) mRNA-expressing cells, while only some of the prolactin (PRL) mRNA-expressing cells. DNA replication in the anterior pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine (BrdU) in a primary serum-free culture system. Estradiol-17β (E2) and TGF-α treatment increased the number of BrdU-labelled mammotrophs, indicating that E2 and TGF-α treatment stimulates the DNA replication in mammotrophs. Immunoneutralization of TGF-α with anti-TGF-α-antibodies nullified the E2-induced increase in DNA replication. RT-PCR analysis of TGF-α mRNA expression in ovariectomized female mice revealed that E2 increases TGF-α mRNA levels. These results indicate that the TGF-α produced primarily in the somatotrophs mediates the stimulatory effects of estrogen on the DNA replication of pituitary cells in a paracrine or autocrine manner.

Phylogenetical positions of Sorex specimens unassigned to species from Cheju Island, Korea, and S. caecutiens from southern Korean Peninsula were investigated based on full nucleotide sequences (1,140 bp) of the mitochondrial cytochrome b gene, comparing specimens of the S. caecutiens/shinto group from locations throughout its range. In the phylogenetic tree obtained, S. caecutiens were separated into two main groups: Hokkaido and Continent-Sakhalin-Cheju clusters. Shrews from Cheju and Korean Peninsula were included in the latter cluster. Thus, we suggest that the shrew on Cheju Island should be ranked as S. caecutiens, although taxonomic description of the shrew has to be conducted elsewhere. The Cheju shrews formed a single sub-cluster while the peninsular shrews were not included in a single sub-cluster. The clustering of individuals in Continent-Sakhalin-Cheju cluster did not always reflect the geographical proximity of their capture locations. We interpret these findings as indicating ancestral isolation of a Hokkaido population and recent rapid range expansion of the modern population in Eurasian Continent-Sakhalin-Cheju.

The genetic variation in Eumeces latiscutatus from the main islands of Japan and the northern Ryukyus was investigated and compared with that of its close relatives (E. okadae and E. marginatus) using allozyme data. All three samples from the northern Tokara Island Group, currently identified as E. latiscutatus, were shown to belong to E. marginatus and not to E. latiscutatus. The non-monophyly of the northern Tokara samples and the great genetic differences within these samples may have resulted from colonization from more than one source population by northeastward overseas dispersal. The samples from the Izu Peninsula were genetically much closer to E. okadae than the other conspecific ones. This indicates that the samples from the Izu Peninsula and the other samples of E. latiscutatus should be treated as distinct species, and that E. latiscutatus from the Izu Peninsula and E. okadae from the Izu Island Group may be treated as conspecific. Samples from western Japan were genetically well differentiated from those of eastern Japan. Within the western group, the samples from the Osumi Island Group was genetically distinct from those from the other regions, by possessing unique alleles. Our phenograms also reveal a distant affinity between samples from the Danjo Island Group and the main islands of Japan. This may be the result of long geographic isolation of the Osumi and Danjo Island Groups from Kyushu. By contrast, samples from Sapporo and Aomori were poorly differentiated genetically in spite of the long separation of these two localities by the Tsugaru Strait. This suggests that overseas dispersal of E. latiscutatus occurred across this strait after its formation.