The incomplete morphological characterization of type 2 astrocytes is in part responsible for the slow progress of studies on these cells. To examine and characterize type 2 astrocytes morphologically, three-dimensional fine structures of the surfaces of type 2 astrocytes cultured from rat cerebella were studied by a combination of atomic force microscopic and immunocytochemical techniques. Atomic force microscopy (AFM) revealed irregular ridge-like structures that form a meshwork distributed throughout the cell body surfaces and the thick processes. These ridges were found to be of two heights (31 nm and 82 nm). This finding indicates two possible configurations responsible for shaping the meshwork: (1) two structures of different thickness are beneath the cell membrane; and (2) two structures are located at two different depths from the cell membrane. On the other hand, immunocytochemical studies for tubulin and glial fibrillary acidic protein (GFAP) revealed that these cytoskeletal filaments are similarly distributed within the resolution power of a light microscope. However, no detectable structures were obtained by actin staining. The immunocytochemical findings suggest that the AFM-revealed ridges forming the irregular meshwork on the cell surfaces may reflect very fine bundles of tubulin and/or GFAP. Therefore, AFM study, with the help of immunocytochemical study, is a powerful tool for characterizing cell morphology. The results of the present study reveal the first morphological characterization of type 2 astrocytes.

As the first step to study relationships between development and learning in the molluscan central nervous system, we examined developmental changes in acquisition and retention of a conditioned taste aversion (CTA) in the pond snail, Lymnaea stagnalis. We found that snails developed ability of a CTA as a long-term memory through three critical stages. Embryos in veliconcha started to respond to appetitive sucrose at the first critical stage. This response was in good agreement with morphological observations that embryos at this developmental stage seemed to be physically ready to eat. However, they could not associate this appetitive stimulus (conditioned stimulus: CS) with an aversive stimulus of KCl (unconditioned stimulus: UCS). At the second critical stage, embryos just before hatching acquired the CTA, but the conditioned response did not persist. Through this stage, they may acquire learning ability to safely seek out food in an external environment. At the third critical stage, immature snails with a 10 mm shell could use a long-term memory to maintain the conditioned response. This memory persisted for at least a month, showing that now they are able to maintain a long-term memory so that they can safely eat a variety of food when they cover wide territory to search for a mate. The present findings indicate that the development of learning ability in snails, which secures acquisition of better survival ability, is coincident with the major changes in their life cycle.

The compound eye of the Japanese yellow swallowtail butterfly, Papilio xuthus, consists of different types of ommatidia characterized by the pigmentation around the rhabdom. About 75% of the ommatidia harbor red pigment, whereas the other 25% contain yellow pigment. We find that the pigments function as spectral filters for the proximal photoreceptor cells. Intracellular recordings of the proximal cells yielded spectral sensitivities peaking in the red (λ_max = 600 nm) and in the green (λ_max = 520 nm), respectively. Staining of the recorded cells and subsequent histology demonstrated that the red receptors contain red pigment and that the green receptors contain yellow pigment. The sensitivity spectrum of the red receptors was considerably narrower compared to the absorption spectrum of a visual pigment peaking at 600 nm. The sensitivity spectrum can be calculated with an optical model for the butterfly rhabdom by incorporating measured absorbance spectra of the red pigments, yielding that the cell contains a visual pigment peaking at about 575 nm. The model also indicated that the spectral sensitivity of the green receptors is produced by the combination of the yellow lateral filter and a visual pigment peaking at 515 nm.

This paper presents immunocytochemical, freeze-fracture, and fine-structural evidence for the hypothesis that the precursors of the rhabdomeric membranes are vesicles in the photoreceptors of the crab Hemigrapsus sanguineus. The number of vesicles starts to increase in the photoreceptor cell body at midday and peaks at approximately one hour before light-off. The vesicles move toward the rhabdom: they almost disappear from the cell body within the first hour after light-off. As they move, the rhabdom area increases. Electron microscopic immunocytochemistry and freeze-fracture EM revealed that the vesicles contain the visual pigment opsin as an integral membrane protein. Based on detailed observation at the microvillar base by conventional electron microscopy, we present a model of how the vesicles are incorporated into the rhabdom to elongate the rhabdomeric microvilli.

The longitudinally striped pattern of the threeline pencilfish, Nannostomus trifasciatus, is observed only in the daytime, and changes into a different pattern with three dark spots at night. In this study, microscopic examinations and photoelectric measurements revealed that melanophores in the greater part of the integument respond to melatonin by the aggregation of melanosomes. By contrast, larger melanophores existing in dark spots respond to the amine by the dispersal of pigment. Physiological tests indicated that the nervous mechanisms controlling these melanophores are practically identical to each other, and also with those existing in many teleost species known hitherto. It is further shown that membrane-permeating analogues of 3′,5′-cyclic nucleotides effectively disperse melanosomes in all these melanophores. We conclude that melanophores in the spots possess melatonin receptors that mediate melanosome dispersion, which we have recently described as “β-melatonin receptors” in some melanophores of another pencilfish species, N. beckfordi. Stimulation of the regulatory subunit of these receptors may be signaled via the G_s subunit, which activates the catalytic subunit, nucleotide cyclase, to increase the cytosolic concentration of cyclic nucleotide(s) as the second messenger. These results demonstrate that the threeline pencilfish affords an excellent model for studying signaling mechanisms through β-melatonin receptors.

Distributional changes in branchial chloride cells were examined in Japanese sea bass (Lateolabrax japonicus) juveniles transferred from seawater (SW) to fresh water (FW) during their migration season toward low salinity habitat in nature. Chloride cells were identified by immunocytochemistry with a specific antiserum for Na ,K -ATPase. In fish reared in SW as controls, branchial chloride cells were localized exclusively in the filaments and absent in the lamellae. When sea bass were transferred from SW to FW, chloride cells emerged in gill lamellae, starting at the proximal part of the lamellae and thereafter spread over the lamellar epithelium. On 7th and 15th days after FW transfer, chloride cells were mostly found on the lamellae, whereas the number of filament chloride cells was decreased. These results suggest that, in Japanese sea bass juveniles, chloride cells in the gill lamellae are important in FW adaptation, and that lamellar chloride cells originated from the filaments and migrated to the lamellae during FW adaptation.

Innervated and denervated erythrophores of the tilapia, Oreochromis niloticus, responded directly to light of defined wavelength by pigment aggregation or dispersion, although similar effects did not occur in melanophores. In spectral regions between 400–440 nm and 550–600 nm, pigment aggregation took place, while the dispersion response was accelerated at wavelengths between 470–530 nm. The progress of the aggregation or dispersion response depended on the light intensity. These results may imply the coexistence of three kinds of visual pigments in tilapia erythrophores.

A one-day old virgin male of the swallowtail butterfly, Papilio xuthus, requires about 50 min (at ca. 26.5°C) to transfer a spermatophore into the bursa copulatrix of a virgin female. Under such conditions, the spermatophore mass averages 4.7 mg, but is much smaller at the male's second mating (about a half). The energy stored during the larval stage is only enough to produce 1.5 spermatophores of the average size of the first mating. There is a positive correlation between adult male sugar intake and spermatophore mass at the second mating. To produce a spermatophore in a second mating that is the size typically produced by virgin males, a male must ingest 390 mg of sucrose solution (20%), which would take an estimated 4.3 days. If a large spermatophore is advantageous for mating, males are expected to have a period after mating when they feed but do not search for mates.

In behavioral investigations examining mechanisms and functions of inter- and intra specific communications, whether one can manipulate stimulus properties is a critical factor. If we can substitute a real animal with an artificial model, that should greatly advance the research. Here we tested whether male zebra finches (Taeniopygia guttata castanotis) and male Bengalese finches (Lonchura striata var. domestica) emit natural behavior of directed singing to video images of conspecific females. When a conventional cathode ray tube (CRT) monitor was used, birds showed few signs of behavioral responses. However, when a thin film transistor (TFT) liquid crystal display was used, several behavioral responses, mostly sexual displays, to the images were observed. The amount of directed singing emitted towards the TFT projected images were comparable to that emitted to the live female birds in both species of birds. The reason why TFT monitor is much more powerful than CRT monitor in eliciting natural behavior from these birds may lie in the fact that TFT monitor is flickerless while CRT monitor might produce some flickers to the eye of birds that has high critical flicker frequency. TFT monitors should be better substitute of real objects than CRT monitors in behavioral investigations. This technique, combined with modern image processing techniques, should be useful for neuroethological studies of bird behavior.

The air-puff evoked escape behavior of the cricket, Gryllus bimaculatus, was investigated. Crickets almost always escaped away from the stimulus source. In an optimal condition, the mean escape direction was 162° opposite to the stimulus source. Stronger (higher velocity) air-puff elicited an escape in larger number of crickets. However, the escape direction became incorrect when the stimulus was too strong.

Crickets with bilateral cercal ablation did not show any escape to an air-puff, while unilaterally ablated ones did respond to the same stimulus with an escape. However, the response rate of animals with unilateral cercal ablation was lower than that in intact animals. Although the mean escape direction of the crickets with unilateral cercal ablation was still opposite to the stimulus source, the direction was not so accurate as in intact animals.

About 6 days after the unilateral cercal ablation, the response rate showed a statistically significant compensational recovery. On the other hand, 14 days were necessary for the recovery of the escape direction. Information which regulating the response rate and the behavioral orientation is likely being processed in different neural pathways.

The intertidal snail Thais clavigera (Küster, 1858) has been believed to show a wide variability in shell morphology. However, the population in Tanabe Bay, on the west coast of the Kii Peninsula, is known to consist of only two forms, which are distinguishable by the shape of nodules on the shell. As the first step toward a better understanding of shell morphology variation, the existence of reproductive isolation between these two forms was tested in Tanabe Bay using allozyme analysis. The two forms were confirmed to be isolated genetically; there are significant differences between them in allele frequencies at the 6 analysed loci.

The chromosome number of the andromerogones obtained by fertilizing non-nucleate egg fragments of Hemicentrotus pulcherrimus was examined by an air-drying method during early development. The non-nucleate egg fragments were prepared by centrifuging unfertilized eggs in a stepwise saccharose density gradient with a purity of 99.9%, comprising from about one-tenth to one-fourth of the total egg volume. The andromerogones cleaved, hatched and then actively swam. Though their development tended to be slower compared with that of control whole embryos after hatching, most of them developed into larvae and the remainder developed as permanent blastulae. While the larvae had differentiated gut and pigment cells, they also had various skeleton types which varied from almost normal to irregular. The chromosome preparations were made from blastomeres dissociated from many andromerogones. The rate of cells having a haploid chromosome number of 21 was 73% at the two cell stage, 84% at the 8 cell stage, 75% at the morula stage, 76% at the hatching blastula stage and 87% at the swimming blastula stage, while that of cells having a diploid number of 42 was 2% if averaged out for total cells examined. A total of 73–87% of the andromerogones was found to develop with a haploid number of chromosomes.

Under a phase-contrast microscope, the infectious form of Holospora obtusa appears to consist of three regions in tandem: a dark region, a refractile region, and a small dark tip in the refractile region. By electron microscopy, this bacterium appears to consist of a cytoplasmic region, a periplasmic region, and a small electron-translucent tip in the periplasmic region. It had previously been thought that these three regions in each microscopy respectively corresponded to one another. However, indirect immunofluorescence microscopy with monoclonal antibodies, IF-3-1 and IF-3-2, specific for periplasmic proteins of H. obtusa and an antiserum raised against a cytoplasmic stress protein, the GroEL homologue of aphid endosymbiotic bacterium, showed that the dark region was the periplasm and the refractile region was the cytoplasm. This shows that the small dark tip does not correspond to the small electron-translucent tip, and that the electrontranslucent tip should be present at the opposite end. We found that this special tip could be identified with Nomarski- and phase-contrast optics at the end of the dark region when the infectious form was embedded in 30% gelatin. The same correspondences between the light and the electron microscopic structures were also confirmed in the infectious form of the micronucleus-specific bacterium H. recta.

Ascorbic acid (AsA) is highly concentrated in vitreous in bovine, human as well as in other species. In order to evaluate the role of ascorbic acid as an ocular neovascularization inhibitor, we examined the effect of ascorbic acid on growth and survival of cultured vascular endothelial cells. When added to culture medium, high concentration of ascorbic acid (0.3 mM<) reduced viability of bovine aortic endothelial cells (BAEC) within 24 hr. Morphology of ascorbic acid treated endothelial cells demonstrated that fragmentation of nuclei does not accompany during this incubation period, suggesting that ascorbic acid induces vascular endothelial cell death in a non-apoptotic manner. To further confirm that this event was not specific on BAEC, bovine retinal endothelial cells (BREC) and human aortic endothelial cells (HAEC) were tested for ascorbic acid cytotoxicity. Ascorbic acid induced cell death in all three types of cells, but the dose required for induction of cell death differed, human endothelial cells were apparently more resistant to ascorbic acid cytotoxicity than bovine cells. Decrease in viability of BAEC exposed to ascorbic acid were partially inhibited by exposure to low oxygen concentration (O₂ = 1%). Addition of vascular endothelial growth factor (VEGF) stimulated proliferation of both BAEC and BREC, but co-addition of ascorbic acid reduced VEGF-induced endothelial cell proliferation. These results show that ascorbic acid modulates endothelial cell behavior in vitro and suggest that it is a negative regulator for ocular neovascularization.

We found three distinct shell color types in samples of the freshwater clam Corbicula fluminea Müller collected at Hou Don, Keelung, Taiwan. DNA microfluorometric analysis revealed that these three types consisted of both diploids and triploids. DNA microfluorometry on sperm and somatic cells showed that both diploid and triploid produced non-reductional spermatozoa. These characteristics are similar to triploid C. leana Prime sampled in Japan. These findings suggest that Corbicula fluminea at different ploidy levels may be reproducing by androgenesis as already shown in C. leana from Japan.

To reveal the fertilization process of arrow worms, oocytes from fixed specimens of Spadella cephaloptera were isolated and observed by staining fluorescent nucleic acid dyes. Fully grown oocytes with a germinal vesicle (GV oocytes) are associated with an accessory fertilization cell (AFC), which is easily removed from the oocyte during isolation. Oocytes after germinal vesicle breakdown (GVBD oocytes) do not have an AFC and are at the metaphase of the first meiosis. In most cases GVBD oocytes contain a small bright spot that is regarded as a condensed sperm chromatin. In rare cases the sperm chromatin in GVBD oocytes has the form of a thin-thread (20 μm long) which corresponds to the sperm nucleus immediately after fertilization. Therefore, fertilization occurs at the first meiotic metaphase after disappearance of the AFC. Numerous zygotes from a single ovary have an hourglass shape, indicative of passage of ovulating eggs into the oviducal complex. Thus, ovulation occurs after fertilization in S. cephaloptera as reported in the genus Sagitta.

The inductive interactions between activin-induced and non-induced cells were investigated in newt animal cap explants. A wide range of concentrations of activin A (0.1–100 ng/ml) induced mesodermal tissues in the animal caps, but at generally low frequencies. Animal caps treated with 100 ng/ml of activin A, on the other hand, differentiated solely into nonspecific endoderm. At this concentration, various mesodermal tissues were induced in addition to endoderm as the animal caps increased in size. They were more frequently induced in explants in which the activin-treated animal caps were combined with untreated animal caps. Central nervous systems were frequently induced in sandwich explants with larger amounts of untreated animal caps. Differentiation of endodermal organs such as the liver, the pancreas and the intestine in the long-term cultured sandwich explants was confirmed by electron microscopy. Lineage tracing of the combination and sandwich explants revealed that the activin-treated animal caps mainly formed the endoderm and induced mesodermal and neural tissues in the untreated animal caps. These results suggest that activin A is capable of inducing the endoderm that can act as an initiator of further inductive interactions in early newt development.

In order to identify the maternal mRNAs which have important roles in the very early stage of embryogenesis, a Ciona intestinalis 64-cell stage cDNA library was subtracted from an unfertilized egg cDNA library. We thereby cloned Cipros1, which encodes the protochordate homologue of the proteasome regulatory subunit Rpn12. Neither Cipros1 mRNA nor Cipros1 protein showed any spatial localization. However, Cipros1 mRNA was expressed at a level at least five-times higher in unfertilized eggs and about two-times higher in cleavage stage embryos, than in other embryonic stages. In unfertilized eggs, Cipros1 protein was expressed at a level about twice as higher as during the other stages. Moreover, minor, smaller isoforms of Cipros1 were expressed specifically in unfertilized eggs and during early cleavage stages. Since a single Cipros1 transcript was detected throughout the development, these smaller isoforms might be generated post-translationally.

To cytochemically demonstrate the accumulation of sodium ions in decomposing yolk platelets, we first aimed to develop a method of sodium detection by administrating the magnesium uranyl acetate (double acetate) reagent, which throws down the sodium as triple acetate, to sciatic nerves of Xenopus laevis. By observing the cell contour and the volume of the precipitates produced in the intercellular and interlamellar spaces of the myelin sheath around the nerves, we determined that a 15-times dilution of the original double acetate reagent (Caley and Foulk, 1929) was the best concentration for biological usage. The validity of the double acetate method was assessed by observing the specific localization of triple acetate precipitates in the skeletal muscle and kidney; In addition to their localization in intercellular spaces, the precipitates were preferentially present in the transverse system of skeletal muscle cells and present in the ground cytoplasm as well as in the organelles other than vacuoles of proximal convoluted tubule cells of the kidney. By applying this method to developing embryos, it was found that the sodium ions are stored in the vesicles during the cleavage stages of development, are apparently transported by the vesicles to decomposing yolk platelets at the early neural plate and later stages, and are accumulated in these platelets. Those results would satisfy the prediction that the sodium concentration must be increased in decomposing yolk platelets, since yolk solubilization by high salt concentrations is prerequisite for amphibian yolk digestion to occur.

Expression of prolactin receptor (PRLR) and cortisol receptor (CR) mRNAs was examined during early-life stages of euryhaline Mozambique tilapia (Oreochromis mossambicus) by competitive reverse transcription-polymerase chain reaction (cRT-PCR). Concentration of prolactin receptor mRNA was higher in the gills of mature fish reared in fresh water (FW) than in those reared in seawater (SW), whereas no difference was seen in CR mRNA. Whole eggs just after fertilization contained the receptor mRNAs for both prolactin and cortisol. The concentration of PRLR mRNA increased gradually as the embryo grows both in FW and in SW. On the other hand, the concentration of CR mRNA was highest in the egg just after fertilization, decreased rapidly toward hatching, and increased slightly thereafter. When embryos 3 days before hatching were transferred to SW, the levels of PRLR mRNA were significantly lower at the time of hatching and also 3 days after hatching than in the embryo and larvae maintained in FW. Environmental salinity did not affect CR mRNA content at any stage examined. Both PRLR and CR mRNAs were identified in the yolk-sac membrane and in the embryonic body. Significantly more PRLR gene was expressed in the embryonic body developing in FW than in SW, whereas no difference was seen in the yolk-sac membrane. The greater expression of PRLR gene in embryos and larvae developing in FW than in those in SW clearly indicates the presence of regulatory mechanisms of gene expression in early-life stages of tilapia.

The pineal gland (PG) possesses receptor proteins capable of binding androgen with high affinity and specificity. However, the sensitivity of PG to exogenous testosterone (T) in term of biochemical constituents during different reproductive phases is still unknown. Hence, an attempt was made to study the effect of testosterone propionate (TP; 20 μg/animal/day; i.m. injection) on the biochemical constituents of PG i.e. protein, cholesterol, serotonin and plasma melatonin (MEL) level of the subtropical zone rodent, Funabulus pennanti during reproductive active (RAP) and inactive phases (RIP). During RAP, TP increased the plasma MEL level and prostate gland weight while, it had no effect on other biochemical constituents of PG, plasma T level and testes weight. It may be suggested that during the RAP, exogenous TP initiates the negative feed back mechanism at leydig cell level. Further, TP is known to affect directly the MEL synthesis by increasing the activity of PG. During RIP, TP decreased the PG weight, plasma MEL level, serotonin and protein content and increased the testes, accessory sex organ's weight and plasma T level. This decrease in PG activity may be due to the decrease in enzyme activity by TP which in turn inhibited the MEL synthesis and initiated the gonadal function.

Therefore, in this squirrel PG biochemical constituents presented a reproductive phase dependent variation modulated by T. Further, active PG of RIP appeared to be more sensitive to exogenous TP treatment in reducing the biochemical constituents and increasing MEL synthesis.

In order to determine what growth-promoting factors may be involved in androgen-induced male reproductive tract development, the newborn mouse urogenital sinus region with seminal vesicles attached was cultured for 5 days on collagen gel matrix in a serum-free medium composed of DMEM and Ham's F-12 (1:1) supplemented with BSA, insulin, cholera toxin and transferrin. Testosterone and 5 α-dihydrotestosterone (DHT) stimulated development of seminal vesicle (SV), coagulating gland (CG), prostate (P) and bulbourethral gland (BG). Epidermal growth factor (EGF) stimulated development of CG, P and BG, but inhibited that of SV. Transforming growth factor-α (TGF-α) inhibited SV development, but had stimulatory effects on both CG and P. Addition of anti-EGF antibody significantly inhibited the DHT-induced development of CG and BG, but not of SV and P. These findings suggest that both EGF and TGF-α have organspecific regulatory actions on male reproductive tract development and that EGF may mediate the action of DHT in the development of CG and BG.

We report alterations in serum estradiol-17β (E₂) and hepatic estrogen receptor (ER) DNA-binding activity in female rainbow trout given a single ip injection of 0, 6.25, 12.5, 25, or 50 mg ß-naphthoflavone (BNF)/kg body weight, followed by sacrifice at 24 or 48 hr. BNF affected E₂ in a dichotomous fashion at 24 hr, and reduced E₂ at 48 hr with increasing BNF. DNA binding by ER was decreased in an apparently dosedependent manner after 24 hr and increased after 48 hr. These data suggest that the regulation of ER by AHR agonists may occur both at the ER promoter, and also secondarily via E₂, and that the differential effects observed are both time and dose dependent.

Details of the karyological relationship between the lesser Japanese shrew-mole Dymecodon pilirostris (2n = 34) and the greater Japanese shrew-mole Urotrichus talpoides (2n = 34) were examined by five differential-staining techniques, namely, G-, C-, NOR-, Q- and CMA-banding. Staining revealed that thirteen autosomal pairs and the sex chromosomes of the two species exhibited strong homology in terms of banding patterns. The remaining three autosomal pairs, nos.13, 14 and 15, exhibited distinct interspecific differences both in banding patterns and in the morphology of the respective chromosomes. These interspecific differences can be explained by the presence or absence of an unusual (G-, C-, NOR- and Q-band-negative, but CMA-band-positive) region of chromatin, by the pericentric inversion inv (14) (p13q31), and by duplication of C-heterochromatin, respectively. It has been suggested that the unusual chromatin found in U. talpoides might contain highly repetitive GC-rich sequences, even though its staining properties appear to be unique and are unlike those of so-called C-heterochromatin. Detailed pair-matching analyses of banded chromosomes led us to revise the relationship, proposed previously by Hamada and Yosida, between D. pilirostris and U. talpoides (Hamada and Yosida, La Kromosomo II-20, 585-590, 1980).

Phylogenetic relationships among 18 species of orthopteroid insects (Blattaria: cockroaches, Isoptera: termites, Mantodea: mantids, Grylloblattodea: grylloblattids, Phasmatodea: stick-insects, Orthoptera-Caerifera: locusts, Orthoptera-Ensifera: crickets, and Dermaptera: earwigs), were estimated based on DNA sequencing of the mitochondrial cytochrome oxidase II gene. Our results drew attention to the need for caution in using third codon positions for tree construction, since it was likely that base pair substitutions of third codon positions in the COII gene were saturated among taxa used in the present study. We also detected that there were many phylogenetically informative sites in first codon positions. Phylogenetic trees using first and second codon positions based on both the neighbor-joining method and parsimony analysis indicated that the topology was nearly identical to each other. The phylogenetic relationships among these taxa differ from the current classification based on morphological characters. The inferred trees showed that grylloblattids were not a primitive group, but closely related to the Dictyoptera. Stick-insects were closely related to the Dictyoptera and grylloblattids, not to crickets. Locusts and crickets formed a monophyletic group. Earwigs were only distantly related to the Dictyoptera. Within the Dictyoptera, cockroaches and termites constituted a monophyletic group, with mantids as a sister group to that complex.