Sex determination is the product of coordinated gene expression. Mutational analyses have yielded great progress in our understanding of mammalian sex determination, and insight into the evolution of this sex chromosome system would be valuable. Mammals arose from turtle-like reptiles, and in many turtles the incubation temperature of the egg determines the sex of the offspring, a process known as temperature-dependent sex determination. There is mounting evidence that sex steroid hormones are the physiological equivalent of incubation temperature and serve as the proximate trigger for male and female sex determination. Temperature appears to accomplish this end by acting on genes coding for steroidogenic enzymes and sex steroid hormone receptors. The ability to manipulate sex determination in turtles both by temperature and by sex steroid hormones extends our understanding of the evolution as well as the physiology and molecular biology of sex determination.

The suboesophageal ganglion of the silkworm, Bombyx mori, contains three clusters of neurosecretory cells that are imrnunoreactive with antisera against the diapause hormone (DH) and the pheromone biosynthesis-activating neuropeptide (PBAN), the two neurohormones that are generated from a common precursor protein. The cells lie on the ventral midline of the ganglion. Neurosecretory cell clusters responsible for the diapause induction activity and the pheromonotropic activity of females were determined by surgically removing one or two of the three clusters of the DH/ PBAN imrnunoreactive cells. A potent diapause induction activity was obtained in females retaining a posterior cluster of cells while a strong pheromonotropic activity was obtained in case of females with a medial cluster. The functional differentiation of these cells may relate to different biochemical and/or physiological natures.

Pheromone content in the pheromone gland of the female moth, Bombyx mori, declines after mating with a time course closely resembling that of decapitated females. The inactivation of pheromone production after mating was prevented when the ventral nerve code or a pair of peripheral nerves (N4) extending from the terminal abdominal ganglion was severed before mating. In contrast, the post-mating inactivation of pheromone production was not prevented when the ventral nerve cord was transected 1 hr after the initiation of mating. Although females produced only a small amount of pheromone when the connection between the brain and the suboesophageal ganglion (SG) was cut at an early pupal stage, mating did not induce a significant decline of pheromone production in these females. The results suggest that inactivation of pheromone production is mediated by a neural signal, originating from a peripheral receptor, that is sent via the ventral nerve cord to the brain-SG complex to suppress activity of the neurosecretory cells responsible for the release of pheromonotropic neuropeptides, such as a pheromone biosynthesis activating neuropeptide (PBAN).

Response properties of six giant interneurons (GIs) 8-1, 9-1, 9-2, 9-3, 10-2 and 10-3 of the cricket, Gryllus bimaculatus, were investigated with a short duration unidirectional air-current stimulus which elicits escape behavior of the insects effectively. Air-currents were presented to the insects from 12 different directions in the horizontal plane in order to explore the directional characteristics of the GIs. The frequency dependency of velocity thresholds (threshold curves) of the GIs changed with the direction of stimulus air-current. Preferential directions of the GIs to the air-currents were ipsilateral-rear in Gl 8-1, ipsilateral-front in Gl 9-1, ipsilateral-rear in Gl 9-2, ipsilateral-front in Gl 9-3, ipsilateral-rear in Gl 10-2 and ipsilateral-front in Gl 10-3, with respect to the ventral nerve cord containing the axons. Intensity-response curves suggest that GIs 8-1 and 9-1 show the maximum response to air-current velocities lower than 300 mm/sec regardless of the stimulus direction. On the other hand, response magnitudes of GIs 9-2 and 9-3 increased proportional to the logarithm of the stimulus velocity up to 300 mm/sec and did not show a plateau even at 300 mm/sec regardless of the stimulus direction. Based on the directional characteristics revealed in the present study, a possible neural mechanism for encoding the direction of air-current by the combinational activity of GIs 9-2 and 9-3 is discussed.

We cultured retinal pigment epithelial (RPE) cells dissociated from the adult newt eye and examined changes in the voltage-gated ionic currents with time in culture using whole-cell patch-clamp techniques. RPE cells allowed to adhere onto a concanavalin A-coated substrate began to flatten within 1-2 days and some of them started to divide on the 6th day of culturing. Freshly dissociated RPE cells exhibited only voltage-gated outward K currents. Voltage-gated Na currents associated with the initiation of immature action potentials were expressed in more than 50% of sufficiently stretched RPE cells before the first mitosis was observed. This fact may suggest that its expression is not coupled with the cell division. The Na current was insensitive to the Na channel blockers TTX and STX. Trichostatin A (TSA) arrested the cell division of cultured RPE cells reversibly. However, the Na current expression was not blocked by this drug. Since TSA has been shown to act as an inhibitor of the cell cycle at G1 phase, the Na current expression may be regulated by certain factors appearing between GO and G1 phases before entering S phase. However, this does not mean that the current expression is directly coupled with the cell cycle, because about half of stretched cells do not express the current. We hypothesize that the Na current expression and the presence of immature action potentials may represent a tendency of RPE cells to dedifferentiate towards embryonic neuroepithelial or more neuronal phenotypes in culture.

The isolated heart or pericardium of the chiton Acanthopleura japonica each showed rhythmic automaticity, even after they were isolated from the body. Intracellular action potentials could be recorded from spontaneously active myocardial cells in the isolated heart and pericardium. They always appeared to be preceded by pacemaker potentials much as has been found for myocardial cells of other molluscs. These show that both heart and pericardial beats of Acanthopleura have a myogenic nature. Stimuli applied to each of the lateral and ventral nerve cords produced both excitatory and inhibitory effects on the heart and the pericardium. Both the heart and the pericardium in Acanthopleura may receive excitatory and inhibitory control from each of the lateral and ventral nerve cords. The dual innervation of the heart which is seen commonly in molluscs, at higher stages of evolution, may have been established in Acanthopleura at the stage of the phylogenetic beginning of the Mollusca. On the contrary, the dual innervation which is required for acceleratory and inhibitory control of the spontaneously rhythmically active pericardium is different from the innervation seen in molluscs at the higher stages.

Off-responses in frog taste cells evoked by a Ringer rinse following 1 mM quinine-HCI (Q-HCl) stimulation were investigated with an intracellular recording technique. Three types of off-responses were found; a transient off-depolarization, a rebound-type off-depolarization and a transient off-hyperpolarization. The time to peak and duration of off-responses were in the order of rebound type-off-depolarization > transient off-depolarization > transient off-hyperpolarization, The reversal potential for the rebound-type off-depolarization existed in more positive level than the resting potential. The reversal potential for the transient off-depolarization was around 0 mV, and that for the transient off-hyperpolarization was −58 mV. These three off-responses in frog taste cells may be initiated by an increase in permeability of the apical receptive membrane to Na , K and Cl⁻. A kind of principal ion is dependent on off-response types.

We have quantitatively analyzed the three-dimensional dendritic structure of an identified nonspiking interneuron, Local Directionally Selective (LDS) interneuron in the terminal abdominal ganglion of the crayfish, Procambarus clarkii Girard, using a confocal laser scanning microscopic system. Comparison of the cell size before and after fixation followed by dehydration and clearing revealed that the diameter of dendritic branches shrank by about 15% while the branch length showed no significant change. The soma and dendritic diameter in vivo were obtained from the measurement in the cleared preparation multiplied by the shrinkage factor.

The total length of dendrites and their membrane area as well as their electrotonic structure were found to vary from preparation to preparation. The spatial distribution of fine dendritic branches was variable, but that of major branches was constant among preparations. The interneuron further shared bilaterally asymmetrical dendritic structure: those branches on the soma side commonly showed larger membrane area and larger number of branching than those on the opposite side. The results indicate that the functional difference in the synaptic activity so far reported between dendrites on the soma side and those on the opposite side is paralleled by their differences in the dendritic morphology and electrotonic structure.

Male silkworm moths, Bombyx mori, exhibited sidewise movements of the head when they showed zigzagging walking in response to pheromonal stimulation. When the moth changed the direction of walking, the head angles also changed to those associated with the walking direction. The sidewise movements of the head were thought to be regulated by neck motor neurons which innervated the first cervical ventral muscles and the ventral muscles through a second cervical nerve. It has been reported that the state transition, resembling the ‘flipflop’ operation of an electrical circuit, in the spike activity of descending interneurons running in the ventral nerve cord to a thoracic motor system appears to be important in the pheromone-modulated turning of male B. mori. We recorded the activity of neck motor neurons in the second cervical nerve and the flipflopping activity in the ventral nerve cord simultaneously with multiple suction electrodes, in order to clarify the physiological functions of such flipflopping signals involved in the behavior. We demonstrated that the activity pattern of some neck motor neurons was correlated with the state transition of a flipflopping activity pattern triggered by the pheromones. The result suggests that the flipflopping activity pattern is correlated with the instruction of the zigzag turning during the pheromone-mediated walking of B. mori males.

We previously isolated and purified Paramecium growth factor (ParGF) from a cell-free fluid of an early stationary mass culture of Paramecium tetraurelia (Tanabe et al., 1990). The mitogenic activity of the purified ParGF and of the crude sample (ca. a 100-fold concentrate obtained by ultrafiltration of cell-free fluid) has been assessed based on restoration of the fission rate of the jumyo mutant of P. tetraurelia in daily reisolation cultures. With this assay system, we found that crude samples of Tetrahymena pyriformis and T. thermophila showed mitogenic activity. This suggests that Tetrahymena cells secrete a mitogenic factor(s) like ParGF. To some extent, fetal bovine serum (FBS) and calf serum (CS) also acted as mitogens on the jumyo mutant. Of nine mammalian growth factors assayed for their mitogenic effects on the jumyo mutant, epidermal growth factor (EGF) and transforming growth factor α (TGFα) were slightly and occasionally effective. These results support the idea of actual use of similar kind of growth factors to control cell divisions from protozoa to mammals.

Fluorescent granules that are stained with Nile blue sulfate are present in larval Malpighian tubules in the wild type strain of Drosophila melanogaster, Oregon-R. These granules emit a weak blue fluorescence and most of them are about 2 μm or more in diameter. The ommochrome precursor 3-hydroxy-kynurenine (3-HK) is actively transferred into the tubules of Oregon-R. Changes in the fluorescent granules in the Malpighian tubules on administration of ommochrome precursors were investigated in eye color mutants of Drosophila. The fluorescent granules in the tubules of the nonautonomous mutants v;bw and cn bw emit a strong blue fluorescence and most of them are about 1μm or less in diameter. When v;bw and cn bw larvae were cultured on medium supplemented with kynurenine or 3-HK, respectively, the fluorescence intensity of their granules decreased, and their size increased. These additions resulted in almost equal accumulation of 3-HK to that in Oregon-R. On the other hand, no 3-HK accumulated in the tubules of larvae of the autonomous mutants bw;st, ltd bw and w, which lack the fluorescent granules. These findings indicate that the fluorescent granules are an important intracellular site for uptake or storage of ommochrome precursors in larval Malpighian tubules of Drosophila.

Previously we reported that a long (522 bp) polypyrimidine: polypurine stretch in the 5′ flanking region of the arylsulfatase gene of the sea urchin, Hemicentrotus pulcherrimus, took an unusual, perhaps triplex, DNA structure, when subjected to an acidic pH (pH 5) (Yamamoto et al., 1994). In the present study we have isolated a polypyrimidine: polypurine containing fragment from the arylsuifatase gene and surveyed the sensitivities of the polypyrimidine: polypurine stretch to base modification by diethylpyrocarbonate and osmium tetroxide under various levels of negative supercoiling. Based on the sensitivity of highly negatively supercoiled DNA to these base-modifying reagents, we conclude that, when highly negatively supercoiled, the polypyrimidine: polypurine stretch can take a triplex DNA structure even at a neutral pH and under physiological ionic strength in the presence of Mg² .

Escherichia coli injection rapidly induced bactericidal activity in the hemolymph of a hemipteran insect, Riptortus clavatus. This activity reached its maximum at 9 hr after injection and thereafter declined slowly. Two types of cDNA clones involved in this response were isolated by differential screening. The predominant type encoded for an open reading frame of 678 amino acids, which consisted of fourteen tandem repeats. Each repeat was rich in charged residues and had a proline-rich region which had striking sequence similarities to proline-rich antibacterial peptides from other insect species, indicating these clones encode a multipeptide precursor of antibacterial peptides. The other type encoded for a glycine-rich peptide similar to a known antibacterial peptide as well. Northern blot analyses revealed rapid induction of mRNAs corresponding to these clones after the injection. To our knowledge, this is the first report on the mRNA sequences of antibacterial peptides of hemimetabolous insects, and the second report on the occurrence of multipeptide precursor structure in insect antibacterial peptides.

The cDNA sequence encoding the C terminal region of chicken skeletal muscle connectin was described. Its predicted amino acid sequence had 1,021 amino acids comprising six motif lls (immunoglobulin C2 domain) and five interdomains. The sequence showed 70–75% homology with that of human cardiac connectin, but 168 amino acids including one motif II were missing in chicken skeletal muscle connectin. The C terminal sequence of chicken skeletal muscle connectin reported by the previous work (Maruyama et al., 1994) was erroneous due to the accidental ligation of the cDNA clone encoding a N terminal region of connectin with a partial porin cDNA clone.

Isolated two dorsal animal blastomeres of 8-cell-stage Xenopus embryos differentiated in about 24% of the cases into mesodermal structures, while the two ventral animal ones formed exclusively atypical epidermis. Of special interest is the fact that most of the dorsal animal blastomeres without mesodermal structures, differentiated into atypical epidermis with large parts of cement gland. Cement glands could not be detected in the derivatives of the ventral animal blastomeres. In earlier concepts it has been suggested that the animal hemisphere of the amphibian egg is an uncommitted area, which receives its instructions for further determination and differentiation from the vegetal part of the embryo. However, the results, shown in this paper, support the view that the developmental determinants are distributed in distinct gradients already in the early cleavage stages. Although the highest concentrations of these putative determinants are located in the dorsal vegetal area (Melton, 1995) and the dorsal marginal zone, lower concentrations of these substances are thought to be present in the animal part, especially in the dorsal animal hemisphere of the egg and the early embryo. Our results indicate that dorsal animal blastomeres of early cleavage stage embryos possess the capacity to form mesodermal derivatives after their separation from the vegetal hemisphere.

We present the nucleotide sequence of a cDNA coding for cyclophilin homologue of the sea urchin Hemicentrotus pulcherrimus. The 1,755-nucleotide sequence contains a 492-bp open reading frame corresponding to a translation product of 164 amino acids. Comparison of the deduced amino acid sequence with the previous data shows a high degree of conservation (∼80% homology). Southern blot analysis of genomic DNA suggests the presence of a multi-gene for sea urchin cyclophilin. Northern blot analysis indicates a mRNA size of ∼3 kb and that message is accumulated at blastula stage.

A consistent defect in follicle-stimulating hormone (FSH) secretion is seen in humans with Polycystic Ovarian Syndrome (PCOS); therefore, we evaluated whether Metrodin (a highly purified urinary FSH) administration concurrent with cyst induction or following cyst induction inhibits estrogen-induced cyst development and augments ovarian follicular growth in an established guinea pig model. All animals in these studies received subcutaneous implants containing oestradiol-17β (E₂)-filled Silastic capsules for a 48-hour period. Guinea pigs in study #1 were administered four 0.25 ml injections of FSH or placebo at twelve-hour intervals simultaneously with the E₂ treatment; guinea pigs assigned to study #2 were administered four 0.25 ml injections of FSH or placebo at twelve-hour intervals following the induction of the cystic condition by E₂. Exogenous FSH appears to negate cyst formation when superimposed upon the cyst-inducing agent (E₂). Further, treatment with FSH augmented the number of mid-sized follicles in both paradigms. This study is the first to establish evidence of an anti-cystic effect of FSH in an animal model.

The ontogenic expression of progesterone and estrogen receptors (PR and ER) and effect of estrogen on these receptors were investigated immunohistochemically in rat uterus from the day of birth (=0 day) to 30 days of age. Uterine epithelial and stromal cells showed a negative PR immunoreaction at 0 day. The PR in the epithelial cell nuclei appeared by 5 days, while the stromal cells showed a negative PR reaction until 12 days. The staining of the stromal cells appeared from 12 to 15 days. In both the epithelial and stromal cells, the initiation of the PR appearance was not affected by ovariectomy performed at 0 day or 5 days prior to the appearance of PR in the epithelial and stromal cells. Estrogen injections from 0 day failed to initiate the appearance of PR in the epithelial cells, regardless of doses of estradiol-17β(0.1, 1 and 10μg daily), but induced PR in the stromal cells. The staining of ER appeared at 5 days in the epithelial cells and at 1 day in the stromal cells, respectively. ER appeared after 2–3 daily injections of estrogen from 0 day depending upon the doses. These results suggest that steroid hormones secreted from neonatal ovary do not play any important role in ontogenic expression of PR during the postnatal uterine maturation.

Amplexus and mating behavior in the horseshoe crab, Tachypleus tridentatus, were studied at imari Bay and Kitsuki Bay, Kyushu, Japan, during Summer, 1994. The pairwise size distribution of mated pairs (n =28) showed a lack of size-assortative mating. Long-term amplexus is primarily maintained by the male's pair of posterior claspers, which is significantly larger than the anterior claspers. The posterior claspers always attach directly to the female's opisthosoma, just lateral to the terminal spines, but the anterior claspers attach in various anteriorward positions on the lateral edges of the female's opisthosoma. We conclude that the mating system of T. tridentatus is fundamentally similar to the American horseshoe crab (Limulus polyphemus), despite the >100 million years of isolation between the two groups. T. tridentatus morphologies, however, show more adaptations to long-term amplexus than those of L. polyphemus.

In the present experiments, we examined the changes in cell size (profile area) of oxytocinergic magnocellular neurons during reproductive states and dehydration with quantitative immunohistochemistry. During lactation, hypertrophy was observed in oxytocinergic magnocellular neurons but not in vasopressinergic ones in the supraoptic nucleus. In virgin rats, chronic dehydration increased the cell size in both oxytocinergic and vasopressinergic neurons. After normal weaning time, the cell size decreased, returning to virgin level within 20 days. However, if the mothers were deprived of their litters immediately after parturition, the cell size rapidly returned to virgin level within 5 days. Furthermore, the increase in the cell size of the mothers was not affected by the size of their nursing litters.

A new cercaria, Cercaria kuwaitae VII, is described from the intertidal prosobranch gastropod Clypeomorus bifasciata in Kuwait Bay. It is a heterophyid cercaria of the magnacercous type with its oral sucker modified into a penetration organ, eyespots, seven pairs of penetration glands, spherical epithelial excretory bladder and large pigmented tail. Probably it belongs to the heterophyid genus Condylocotyla or Galactosomum. Scanning electron microscopy was used to study the surface topography of the redial and cercarial stages.

The genetic relationships and taxonomic status of 7 taxa belonging to the genus Tridentiger (Pisces, Gobiidae) were investigated by means of analysis of allozymic variation at 14 loci. The results suggest that the two taxa “T. obscurus” and “T. brevispinis” which are sympatric and morphologically similar are reproductively isolated and are highly divergent from each other (the genetic distance values are 0.501-0,707). It is also suggested that “T. brevispinis” and “T. kuroiwae” are genetically different enough from each other to deserve subspecies at least. The other 4 taxa, “T. barbatus”, “T. nudicervicus”, “T. trigonocephalus” and “T. bifasciatus”, are genetically divergent each and are considered to be 4 biological species. A dendrogram showing the phylogenetic relationships of the 7 taxa was constructed from the genetic distances.

The preys of three flabellinid nudibranchs and the distribution of their pediveliger larvae were investigated to reveal the relation between the width of dietary spectrum and the larval recruitment site. Hundreds of pediveligers were found in total from the field. Flabellina amabilis and Flabellina sp. were specialists consuming exclusively one hydroid species, while Flabellina athadona was a generalist eating five hydroid species. Not only in the specialists but in the generalist, the pediveligers were associated with their adult diet hydroids, although there was a hydroid species on which the larvae of the generalist could hardly succeed to settle. As to microdistribution of pediveligers on the hydroids, there was a significant difference between the specialists and the generalist; pediveligers of F. amabilis and F. sp. were mostly found to directly adhere to the naked hydranths, while those of F. athadona were found away from the naked hydranths. The causes of these phenomena are discussed.

It is generally accepted that there are 3 subspecies of Oncorhynchus masou in Japan, namely, Masu salmon (Oncorhynchus masou masou (Brevoort)), Amago salmon (O. masou ishikawae Jordan & McGregor), and Biwa salmon (O. masou rhodurus Jordan & McGregor or O. masou subsp. Kimura). Since the genetic relationship of these three taxa is not well known, there has been considerable confusion over their nomenclature. We have clarified the genetic relationship among these three taxa by partially sequencing their mitochondrial DNA. Sequences of 948 base pairs from the 3′ region of the ATPase subunit 6 gene to the 5′ region of the cytochrome oxidase subunit 3 gene were obtained for 20 individuals including wild Biwa salmon, wild and farmed Amago and Masu salmon. Furthermore, 2,162 base pairs from the 3′ region of ATPase subunit 6 gene to the 5′ region of NADH dehydrogenase subunit 4L gene were determined in 4 individuals. In total, there were 26 sites of base substitutions. The haplotypes of Masu salmon and Amago salmon were stmilar.On the other hand, 17 of the 26 sites had substitutions characteristic of Biwa salmon. A matrix of genetic distances and maximum parsimony analysis among the haplotypes indicated that Biwa salmon is genetically more distant from Masu and Amago salmon, than Masu salmon is from Amago salmon. This means that Biwa salmon diverged from the common ancestor of the Oncorhynchus masou complex before the divergence between Masu salmon and Amago salmon.

Intracellular recordings from photoreceptor cells of all eyes of the spider Cupiennius salei Keys reveal 3 groups of cells with spectral sensitivity maxima in the blue (480 nm). green (520 nm), and UV (340 nm), respectively. The blue and green cells show secondary peaks in the UV at 340-380 nm. In the posterior median, posterior lateral and anterior median eyes, the majority of photoreceptor cells are the blue and green cells in roughly equal numbers. In the anterior lateral eye, however, the green cells dominate. UV cells were only found in the secondary eyes and only once in each of them.