Zoological Science

The distribution of neurons which express the gene for the growth-associated protein, GAP-43, in the adult carp central nervous system (CNS) was studied by in situ hybridization using newly formed RNA probes for carp GAP-43 mRNA. A great number of neurons heavily labeled by the ³⁵S-labeled antisense probe were found in the telencephalon, diencephalon, mesencephalon, optic tectum, pontine area, medulla oblongata and spinal cord. Motoneurons of the cranial nerves, i.e., the oculomotor, trochlear, trigeminal and spinal motor nerves, also strongly expressed GAP-43 mRNA, in contrast to the low level of GAP-43 signals in the motoneurons in the adult mammalian CNS. These results suggested that synaptogenesis and continuous synaptic reorganization might normally occur in the adult carp nervous system, since GAP-43 protein is generally accepted to be essential for the dynamic growth of axonal processes which leads to synaptogenesis.

In the mature skeletal muscle of the adult carp, a number of small-sized neuromuscular junctions (NMJs), which were visualized with acetylcholinesterase (AchE) histochemistry, were detected on each muscle fiber. This polyinnervation pattern was similar to that of the immature muscle of mammalian embryos. These findings indicate that, unlike mammalian muscles, maturation of carp muscles is not accompanied by the synapse elimination which is thought to be coupled with the down-regulation of motoneuron GAP-43. NMJs of the adult carp muscle are supposed to be continuously reorganized, keeping the motoneurons expressing GAP-43.

The expression of GAP-43 under physiological conditions in the adult carp CNS may facilitate axonal regeneration in various kinds of carp CNS neurons.

In order to determine the contribution of each cercus and its receptors in organizing the receptive field of four air-motion sensitive giant interneurons (GIs 8–1, 9–1, 9–2 and 9–3) of the cricket Gryllus bimaculatus, effects of removing the ipsilateral or contralateral cercus (referred to the side of the axons) on specific parameters of the wind-evoked responses of these neurons were investigated. All 4 GIs received only excitatory inputs from a group of filiform hairs on the ipsilateral cercus. In addition to the ipsilateral excitatory inputs, GIs 8–1 and 9–1 received weak excitatory and strong inhibitory inputs from a group of filiform hairs on the contralateral cercus. GI 9–2 received only inhibitory inputs from filiform hairs on the contralateral cercus. GI 9–3 received excitatory inputs from filiform hairs on the contralateral cercus and no inhibitory input was confirmed. In addition to such simple excitatory and inhibitory connections, the rebound motion of cercal filiform hairs had some role in organizing the receptive fields of GIs 9–2 and 9–3. Furthermore, the possibility of using a rebound depolarization of the membrane potential for mediating the long latency response in GIs 8–1 and 9–2 will be discussed.

In order to explore the functional recovery of the cercal sensory system of the cricket Gryllus bimaculatus, changes in response properties of four air-motion sensitive giant interneurons (GIs 8–1, 9–1, 9–2 and 9–3) were investigated after partial sensory deprivations. Velocity thresholds, response magnitudes and response latencies of each GI to a directional air current stimulus were investigated 21 days after ipsilateral or contralateral cercal ablations. The results were compared to those measured 1 day after the same treatment (Matsuura and Kanou, 1998) in order to specify the changes which occurred during the first 20 days. Each GI showed a different pattern of change in responses according to the direction of stimulus air current; i.e. the changes observed were direction dependent regardless of whether they were compensational or not. Compensational changes in response magnitudes and/or response latencies were observed in GIs 8–1, 9–1 and 9–2 when air currents were applied from particular directions. As there was no regeneration of cercal filiform hairs, those changes must be caused by the changes in synaptic strength between GIs and particular sensory afferents associated with cercal filiform hairs. Such neural compensation must underlie the basis of behavioral compensation when the insects have damaged sensory apparatus.

Novel clonidine binding sites were characterized in the intestinal membrane isolated from seawater eels. The specific clonidine binding sites consisted of at least two classes, high affinity (K_d = 1.4 ± 0.3 nM, n = 5) and low affinity (K_d = 175 ± 34 nM, n = 5) sites. The specific binding of 2 nM [³H]clonidinewas most enhanced at 20°C and at pH 7.5, and reversed by unlabelled clonidine. Such binding was hardly inhibited by adrenaline, yohimbine or rauwolscine, indicating that most binding sites are distinct from α₂-adrenoceptor. The specific clonidine binding was inhibited by various imidazoline/guanidinium drugs, indicating existence of imidazoline/guanidinium receptive sites (IGRS) or imidazoline receptors in the eel intestine. Competition experiments revealed that rank order to displace 2 nM [³H]clonidine from their binding sites was as follows: guanabenz > cirazoline = naphazoline = UK14304 = ST587 ≥ clonidine ≥ idazoxan = RX821002 = tolazoline > ST93 = oxymetazoline = amiloride = ST91 > yohimbine = efaroxan = rauwolscine ≥ adrenaline = ST567 = histamine = agmatine. The rank order was different from those in I₁ or I₂ sites of IG RS reported in various mammalian tissues, suggesting existence of new IGRS, non I₁ and non I₂ sites, in the eel intestine. In addition, structure-affinity relationships are discussed from the results of competition experiments. Although physiological role of IGRS is not clear yet even in mammalian cells/tissues, eel intestine may be a good model to elucidate how the IGRS act in the cell and to decide what is the endogenous ligand for the IGRS, since eel intestine contains great amount of IGRS and it responds to guanabenz, an exogenous clonidine derivative.

The proliferation of aortic endothelial cells of 10,14, and 20 day-old embryonic and 3 day post hatch chicks was studied by vital labeling with bromodeoxyuridine (BrdU) and immunofluorescent visualization using anti-BrdU. We modified the DNA denaturation step using 0.2% Triton X-100 and incubation with 0.1 M hydrogen chloride for 15 min at 30 to 35°C. These modifications were effective to stain the chromosomes with anti-BrdU in the whole mounted tissues. The BrdU-positive endothelial cells were present in the whole aortic areas without showing any mitotic hot spots. The average labeling index of the endothelial cells was high in the young stages; 3.7% at 10 day-old embryos, and then it reduced to 1.4% at 3 day-old chicks.

β-Actinin (CapZ) is a heterodimeric actin-binding protein which is localized in the Z-bands of myofibril. It caps the barbed end of actin filaments and nucleates actin-polymerization in a Ca²⁺-independent manner. As judged by these properties of β-actinin, it is conceivable that β-actinin is involved in the regulation of actin assembly, especially in the formation of I-Z-I complex during myofibrillogenesis. In this study, to examine the function of β-actinin in myofibrillogenesis, recombinant β-actinin (r-β-actinin) produced in an E. coli expression system was introduced into cultured myogenic cells by a microinjection method. Stress fibers in C2 myoblasts were disrupted soon after microinjection of recombinant β-actinin, but nascent as well as well-organized myofibrils were scarcely affected by exogenous β-actinin. Based on these observations, we suggest that in myoblasts where actin filaments are dynamically reorganized, reassembly process of actin filaments may be affected by the exogenous β-actinin, whereas actin filaments become more stable and less sensitive to exogenous β-actinin, when they are organized into myofibrillar structures and anchored to Z-lines in myotubes.

The abnormal furrow (af) gene was identified through a maternal effect mutation that causes a defect in cell division in Xenopus embryos. In eggs obtained from mutant females (af eggs), polar body formation and cytokinesis are completely inhibited while nuclear division continues uninterrupted. Cleavage furrows are recognized as unpigmented narrow bands on the egg surface but they do not contract. Transfer of cytoplasm from wild-type eggs into af eggs partially rescues contraction of the cleavage furrow. The factor responsible for contraction promoting activity was present in wild-type eggs throughout the first cell division cycle and induced contraction in a dose dependent manner. The factor was characterized as a high molecular weight protein complex that was associated with particulate fraction in the extract. The recovery of contraction was associated with accumulation of filamentous actin and WGA-binding sites within the cleavage furrow of the af eggs which were normally not enriched for these components. Formation and contraction of a filamentous actin ring during cortical wound healing in af eggs was indistinguishable from that of wild-type eggs. From these results, the af gene product may be specifically required for reorganization of the actin filaments and WGA-binding sites in the cleavage furrow and contraction through these structures during cytokinesis.

In beginning of muscle development, determination is induced in the mesoderm, and then differentiation occurs with accumulation of muscle structural proteins. Mesoderm cells differentiate to many type cells, but the direct signaling activator for muscle determination is still unknown. In this paper we report some of the conditions required for determination of muscle. Muscle determination during Xenopus development was found to be marked by Xmyf-5 and XmyoD expression, but not by expression of Xmyogenin or Xmrf4. Xmyf-5 and XmyoD expression was first detected in the early gastrula stage. Xmyf-5 expression was first detected on the dorsal side, whereas XmyoD was initially expressed on the ventral side. Subsequently, expression of both genes was strongly induced on the dorsal side. The expression of Xmyf-5 and XmyoD did not continue to increase on the ventral side when it was separated from the dorsal side, although muscle originates from the both sides. These findings suggest that a continuous increase in expression of both genes require the dorsalizing signal. The mesoderm inducers bFGF and Activin A induced both genes in animal caps, and the inductive activity of Activin A was stronger than that of bFGF. Overexpression of Xbra, a pan-mesoderm marker, alone induced both genes, but weakly. The inductive activity of Xbra was enhanced by co-injection with noggin. This suggests that inhibition of BMP4 by noggin in the mesoderm mediates dorsalizing signal, and may induce the direct dorsalizing activator genes of Xmyf-5 and XmyoD.

Ascidian eggs and embryos have provided an appropriate experimental system to explore the cellular and molecular mechanisms involved in the embryonic cell specification and pattern formation of the embryo. In Japan, most of the studies of ascidian embryology have been carried out with the large eggs of Halocynthia roretzi. However, for future studies, Ciona species may provide a better experimental system, in particular with respect to the incorporation of genetic approaches. In order to establish Ciona as an experimental system, molecular markers with which to examine cellular differentiation are required. In the present study, we isolated and characterized cDNA clones for two epidermis-specific genes (CsEpi-1 and CsEpi-2) and for two muscle-specific genes (CsMA-1 and CsMu-1). CsEpi-1 encodes a polypeptide with three trefoil domains, while CsMA-1 encodes a muscle-type actin from C. savignyi. Although CsEpi-2 and CsMu-1 transcripts seem to have a poly(A) tail at the 3′ end, we could not find a distinct open reading frame in the sequences. Probes for CsEpi-1, CsMA-1 and CsMu-1 cross-reacted with C. intestinalis embryos. These cDNAs are useful as molecular markers for the specification of epidermis and muscle of Ciona embryos.

We have previously reported that glia-like folliculo-stellate (FS) cells in the rat anterior pituitary may be involved in heterotopic differentiation of striated muscles which appear during pituitary cell culture. In the present immunocytochemical study, cytological alterations of FS cells in vitro were investigated by using antibodies to marker proteins (i.e., S-100 and vimentin) for FS cells. Expression of skeletal muscle-specific MyoD1 detected by immunocytochemistry, enabled identification of myogenic cells at a stage when they were still unicellular. Elongated myoblasts containing MyoD1-positive nuclei were found as early as the fifth day of monolayer culture of pituitary cells. The double immunostaining technique showed that some of these myoblasts reacted with antiserum to S-100. Although all of the myoblasts were immunoreac-tive to vimentin, this marker protein was unable to identify FS cells in vitro because rapidly proliferating fibroblasts were also immunoreactive to vimentin. Since the antiserum to S-100 that we used did not react with already differentiated striated muscle fibers, the demonstration of both MyoD1 and S-100-immunoreac-tive myoblasts in our pituitary cultures suggests that these myogenic cells are derived from FS cells. The present study does not rule out the possibility that fibroblasts, whose origin is presently unknown, are also involved in myogenesis in pituitary cultures.

Previously we reported that the experimental unilateral cryptorchidism altered the expression pattern of LH receptor mRNA in abdominal testis. To clarify whether the change was due to exposure of Leydig cells to high temperature in the abdomen, we examined the effect of unilateral efferent duct ligation on the expression. Two weeks after the operation, a relatively decreased expression of 1.8 kb transcript, increased expression of other transcripts, and degeneration of germ cells were observed in the efferent duct-ligated testes. As these changes were similar to those seen in abdominal testes of cryptorchid rats, exposure of Leydig cells to high temperature was not responsible for the changes in expression pattern of LH receptor mRNA. To examine the correlation between the changes in the receptor mRNA expression and the degeneration of seminiferous epithelium, expression of the mRNA was analyzed in unilaterally cryptorchid rats for 28 days after the operation. In scrotal testes which showed no histological changes, a major, 1.8 kb and several minor, 6.5, 2.6,1.4,1.1 and 0.9 kb, transcripts were detected throughout the experimental period. In abdominal testes, the expression of 1.8 kb transcript declined rapidly within three days, while that of 6.5 kb and 2.6 kb was increased, reaching a maximum 14 days after the operation. Histological observations of cryptorchid and efferent duct-ligated testes revealed that these changes in the receptor mRNA expression paralleled the successive disappearance of spermatids and spermatocytes. These results suggest that the changes in expression pattern of LH receptor mRNA are closely related with degeneration of germ cells and that intratesticular paracrine factor(s) from seminiferous tubules might be concerned to the phenomenon.

Protogynous wrasses (Thalassoma duperrey): females (F), primary males (PM) along with a few terminal-phase males (TM) and sex-changed males (SM), were used to characterize the topographical organization of the pituitary. In general, immunocytochemical and ultrastructural features of the adenohypo-physeal cell types of the saddleback wrasse pituitary resemble those of other teleosts. In the rostral pars distalis (RPD), corticotropic cells were found bordering the neurohypophysis (NH) and surrounding the centroventrally located prolactin cells. Thyrotropic cells formed a small group in the anteriodorsal part of the rostral and proximal pars distalis (PPD). The somatotropic cells were distributed in large clusters, mostly organized in cell cords around the interdigitations of the NH of the dorsal PPD. Cells containing gonadotropin Ib subunit were localized in the dorsal parts of the PPD, in close association with somatotropic cells and gonadotropin IIb subunit containing cells were seen in the centroventral parts of the PPD and along the periphery of the pars intermedia (PI). The pars intermedia was composed of melanotropic cells and somatolactin cells that lined the neurohypohysis.

Distinct ultrastructural differences in corticotropic and somatotropic cells were not observed between the four groups. In all groups, prolactin cells in the ventral-most RPD could be immature cells or actively secreting prolactin. Gonadotropic II cells of PM and F had relatively higher incidence of “nuclear budding” and cell organelles compared to TM and SM. Besides gonadotropic, the active melanotropic and somatolactin cells might be associated with some aspect(s) of reproduction.

UE8 is one of several uterine cell lines established from p53-deficient fetal female mice. UE8 exhibits a typical epithelial morphology in culture; it is strongly positive for cytokeratin, but negative for vimentin. Immunoblot analysis confirmed that UE8 has glucocorticoid receptors. It grows actively in medium supplemented with 3% heat-inactivated, dextran-coated charcoal-treated fetal calf serum. Dexamethazone (DEX) inhibited its growth dose-dependently at concentrations between 10⁻⁶and 10⁻⁹ M. The inhibition by DEX was further examined in chemically defined medium (CDM): DMEM/F12 containing transferrin (10 μg/ml), selenium (10⁻⁸ M) and 0.1% bovine serum albumin. DEX at 10⁻⁶ M inhibited the growth of UE8 in CDM or in CDM supplemented with insulin-like growth factor-1 (IGF-1: 10 ng/ml), but there was no inhibition by DEX(10⁻⁶M) in the presence of epidermal growth factor (10 ng/ml). Thus, DEX inhibits the growth of a uterine epithelial cell line derived from p53-deficient mice in vitro, and also suppresses IGF-1-induced proliferation.

In order to assess phylogenetic relationships of Taiwanese brown frogs (Rana longicrus and the R. sauteri complex), the partial sequences (587 base pairs) of the mitochondrial cytochrome b genes were compared with six brown frogs from Japan (R. pirica, R. ornativentris, R. japonica, R. tagoi tagoi, R. tsushimensis, and R. okinavana). Resultant phylogenetic trees indicated a considerable genetic differentiation between R. longicrus and R. japonica in spite of their close morphological and ecological similarities. The R. sauteri complex includes two genetically distinct groups that are not consistent with current classification. One group including populations of Alishan (central Taiwan) and Sanyi (western Taiwan) seemed to be closest to R. tagoi and the presumptive common ancestor of these frogs is thought to have diverged very early. Another group including a population from Wulai (northern Taiwan) showed a sister relationship with R. tsushimensis and R. okinavana, both isolated on small islands of Japan. These Taiwanese and Japanese brown frogs as a whole form a monophyletic group, and separation of the R. sauteri complex as a distinct genus or subgenus Pseudorana was not supported.

Comparisons of nucleotide sequences of mitochondrial cytochrome b gene revealed that the brown frog from Sakhalin has genetically little differentiated from Rana pirica from Hokkaido. Although they show some differences in adult morphology, they are considered to be conspecific. These frogs have sequences substantially different from R. dybowskii from the Maritime territory of Russia, and the current taxo-nomic idea to specifically separate them is genetically supported. On the other hand, R. dybowskii from the Maritime territory is genetically well differentiated from a conspecific population from Tsushima.

Each hemocyanin of the 3 Asian horseshoe crabs is composed of 6 immunologically different subunits, each subunit of which is immunologically identical with the comparable subunits of 2 other species. These immunologically identical subunits show 0-15% N-terminal sequence differences between Carcinoscorpius rotundicauda and Tachypleus gigas, while the immunologically different subunits show 25-74% sequence differences within species and 25-72% between C. rotundicauda and T. gigas. From the N-terminal sequence comparison and immunological comparison of hemocyanin subunits, it is evident that Asian horseshoe crabs share 6 orthologous hemocyanin subunits which are immunologically identical. We may produce 6 phylogenetic trees of Asian horseshoe crabs using the 6 sets of orthologous hemocyanin subunits and an evolutionary tree of horseshoe crab's hemocyanin molecules.

Two new species of vespertilionid bats (Vespertilionidae: Chiroptera) were described on the basis of materials collected from Yanbaru, northern part of Okinawa Island, Okinawa Pref., Japan. These are bats of genera Myotis and Murina.