A synthesis of developmental genetics with evolutionary genetics is now making possible to understand significant evolutionary changes in multicellular organisms. The key concept for unifying the two must be heterochrony. Heterochrony causes evolutionary modifications due to changes in timing and/or rate of development. The heterochrony is conventionally categorized into three patterns as neoteny (retardation in somatic development), progenesis (acceleration in gonadal development), and direct development (acceleration in somatic development, resulting in lack of larval or tadpole stages). A lot of species showing neoteny are known in urodeles, but not in anurans. Neotenic urodeles are also divided into three categories; permanent or obligate, “inducible” obligate, and facultative neotenies. Hynobius retardatus, a specific population of which had been reported to show neoteny but is believed to be extinct at present, has become to be used for experimental analysis of heterochronic expression of several adult characters during its ontogeny. Gonadal maturation and a transition of globin subunits from larval to adult types have been shown to occur independently on the morphological metamorphosis in H. retardatus. Mechanisms underlying the heterochrony, including morphogenetic clock, heterochronic genes in Drosophila and C. elegans, temporal colinearity in Hox gene complex in mice, and atavistic transformation induced by altered expression of Hox genes are discussed in terms of current molecular biology.

Effects of the external pH on the voltage-dependent Na conductance in the marine dinoflagellate Noctiluca miliaris were examined under voltage-clamp conditions. The depolarizing Na spike (positive spike) in the tentacle regulating potentials became attenuated when the externa! pH was lowered from 8 (normal value) to less than 5. Under voltage-clamp conditions, a depolarization-activated transient inward current corresponding to the positive spike decreased with lowering the external pH to less than 5, and entirely disappeared at pH 3. Threshold for the inward current shifted towards the depolarizing direction while the reversal potential of the current shifted towards the hyperpolarizing direction with lowering the external pH. Rate of increase in the inward current reduced whereas rate of decrease did not change at low pH. Lowering of the external pH did not affect the steady state inactivation of the inward current. It is concluded that the external pH controls appearance of the positive spike through modification of activation kinetics and selectivity of the voltage-dependent Na conductance responsible for the spike.

Limited numbers of melanophores and xanthophores exist in the skin and also inside the body of the ice goby, Leucopsarion petersii. These chromatophores show fundamentally identical fine structural features to those described in many other fishes examined hitherto. When the ice goby is adapted to a dark or white background, chromatosomes in the cells disperse or aggregate, respectively. Interestingly, the melanophores existing inside the body, namely, those in the peritoneum and those close to the vertebrae, are likewise responsive. Studies on chromatophores in the excised tissue pieces show that they are responsive to various agents known to affect chromatophores. Although the population of the chromatophores in the ice goby is much smaller than that of many other fish, these cells may bear functional significance in the strategy for survival.

Immunohistochemical localization of Mytilus inhibitory peptides (MIPs) in the anterior byssus retractor muscle (ABRM) of Mytilus edulis was investigated by using the anti-MIP polyclonal antibody. The antibody was shown to recognize the seven members of the MIP family that had been previously isolated from the ABRM extract. The MlP-like immunoreactivity was found abundantly in neuronal fiber-like structures in the ABRM and in the connective tissue sheath covering it. The immunoreactive fibers in both areas were rich in varicosities. In addition, it was demonstrated that the MlP-like immunoreactivity was released from the neuromuscular preparation to the bathing solution in response to the repetitive electrical pulses applied to the pedal ganglion. The release was Ca² dependent. These findings suggest that the seven MlP-family peptides, originally isolated from the muscle extract, are the inhibitory neuropeptides involved in physiological regulation of the ABRM contraction.

It is necessary to determine whether, in the same species and for the same behavior, aversive and appetitive conditioning yield different strengths and periods of either acquisition or retention. To this end, we first examined the effects of various chemo-sensory and physical stimuli on feeding and avoidance behavioral responses in the pond snail, Lymnaea stagnalis. Then, using these findings, we constructed classical-conditioning paradigms with aversive and appetitive stimuli. In the aversive conditioning paradigm, an appetitive stimulus (sucrose), which increased the feeding response, was paired with an aversive stimulus (KCI, quinidine sulfate or electric shock), which inhibited the feeding behavior. Upon presentation of KCI, the first type of aversive conditioning, which is generally called “taste-aversion learning with cessation of feeding response”, was acquired quickly and persisted for up to a month. When using a noxious stimulus (quinidine sulfate or electric shock) inducing pain we additionally found the second type of aversive conditioning, in which the previously appetitive stimulus (sucrose) not only failed to increase the feeding response, but came to elicit an avoidance response. This second type conditioning took longer to acquire and persisted for a shorter period of time than the first type. On the other hand, the appetitive conditioning paradigm paired a neutral stimulus (vibratory) with an appetitive stimulus (sucrose). The strength and period for acquisition and retention of this appetitive learned response were very similar to those of the second type aversive conditioning but not to the first one. On the basis of these behavioral analyses, the neuronal mechanisms of the two types of aversive and appetitive conditioning were discussed.

Contactin (F3/F11) is an immunoglobulin superfamily cell surface glycoprotein predominantly expressed in the nervous system. To examine the structure and tissue distribution of Xenopus contactin, a cDNA clone was isolated based on the amino acid sequences conserved among chicken and mammalian contactin proteins. The conceptual translate of the cDNA consists of 1005 amino acid residues that have 70% identity to those of chicken and mammalian contactin. Northern blot hybridization using a labeled cDNA fragment revealed specific expression of 6.5 kb mRNA in the brain. Monoclonal and polyclonal antibodies prepared to the recombinant Xenopus contactin peptides detected a single 135 kD band on Western blots of the brain and spinal cord extracts. Differential extraction and phosphatidylinositol-specific phospholipase C (PI-PLC) digestion experiments showed that the immunoreactive 135 kD proteins bind, at least in part, to the membrane by GPI anchor. On brain tissue sections, strong contactin immunoreactivities were detected on nerve fibers of a subset of cerebral and cerebellar neurons. These results suggest that the basic structure and tissue distribution of Xenopus contactin are similar to those in other vertebrates.

The effect of melanotic tumour formation on the survival rate of female flies was investigated for a period of five weeks after eclosion in the C-104 mutant strain of Drosophila melanogaster, found in a natural population in Budapest. Melanotic tumours developed only in female flies in the vicinity of spermathecae. The incidence of tumour formation was higher in the dead females than in the living ones. This fact explicitly indicated that tumour formation has a deleterious effect on the survival rate and thus the life span of its carriers. St also implied that non-self recognition or self-defense reaction is accomplished at the expense of the organisms. The reason for the maintenance of this melanotic tumour gene in a natural population is discussed.

The melanogenic gene-transfected cell system serves as a useful tool for the study of the symphonic relation between melanin synthesis and intracellular organelles such as melanosomes in melanocytes. We constructed melanin-producing mouse fibroblasts by transfection of human tyrosinase cDNA to investigate the intracellular changes caused by tyrosinase expression. DHICA-oxidase (5,6-dihydroxyindole-2-carboxylic acid oxidase) activity without TRP-1 (Tyrosinase Related Protein-1) expression in the cells suggested that human tyrosinase also possesses a DHICA-oxidase activities different from mouse tyrosinase, Electron microscopic observation indicated that melanin-deposit organelles have some lysosomal features. These properties of melanin-deposit organelles in tyrosinase expressing fibroblasts provide one evidence for the hypothesis that melanosome is the specialized lysosome in melanocytes.

Na , K -ATPase α-subunit cDNA of the sea urchin, Hemicentrotus pulcherrimus, was obtained by twice screening prism and gastrula λgt10 cDNA libraries using an oligonucleotide probe derived from a mostly conserved region, FSBA (5′-p-(fluorosulfonyl)-benzoyladenosine) binding site of cation transport ATPases. The 5′-end of the non-coding region was determined by primer extension and the region was amplified by 5′-RACE method. The sea urchin α-subunit cDNA consists of 4401 nucleotides and encodes 1038 amino acid residues (MW, 114 kDa). The predicted primary structure, except N-terminal region, has similar degree of high homology to various metazoan Na , K -ATPase α-subunits. Alignment of amino acid sequence and a hydropathy profile also predicts eight putative transmembrane segments at least. The phylogenetic tree suspected from alignment of amino acid sequences of 21 species suggests that sea urchin and vertebrate Na , K -ATPase α-subunits seem to have evolved from a common origin, before vertebrate α-subunit divided into three isoforms.

Fluorescence from the wings of male Morpho sulkowskyi and Papilio xuthus butterflies has been investigated for the first time using time-resolved fluorescence spectroscopy. Intensity of emission spectra from the wings of Papilio xuthus decreased with increasing delay time after the application of exciting laser pulse and the intensity peak showed an interesting red shift from 480 to 520 nm. In contrast, such a peak shift was not observed from the wings of Morpho sulkowskyi. The decay times obtained from the wings of Morpho sulkowskyi and Papilio xuthus were given by two components, respectively; those of the former at 510 nm were about 0.545 ns and 2.74 ns and those of the latter at 480 nm were about 0.490 ns and 2.10 ns. Based on these results, the possibility of using time-resolved spectroscopy as a tool for the classification of butterflies is described along with a brief discussion of the origin of the 520 nm peak observed for Papilio xuthus.

The monomer subunits of giant extracellular hemoglobins from earthworms Pontodrilus matsushimensis and Pheretima communissima that belong to the family Megascolecidae, Oligochaeta, were purified by a reversed-phase column, Resource RPC, and sequenced. The complete amino acid sequences of the two monomeric globin chains were determined: 141 amino acid residues with a molecular weight of 16,366 Da for Pontodrilus matsushimensis and 140 amino acid residues with a molecular weight of 16,000 for Pheretima communissima, respectively. The Pontodrilus matsushimensis monomer globin has three cysteine residues, and the two located at positions 2 and 131 are conserved as those observed in all annelids and contribute to form a disulfide-bonded interchain. The third cysteine residue at position 73 is the first evidence for the annelid monomer globin subunits. The physiological functions of the third cysteine residue, however, are still unknown. The monomer sequences of the two species were aligned with those of five known sequences from annelids, including a polychaete, Tylorrhynchus heterochaetus, and four oligochaetes, Pheretima hilgendorfi, Pheretima sieboldi, Lumbricus terrestris and Tubifex tubifex. Using computer analysis, a 87.9% identity of the amino acid sequences between two monomeric subunits of Pheretima communissima and Pheretima hilgendorfi hemoglobins showed the highest degree of sequence similarity. A molecular phylogenetic tree of seven species of annelids has constructed, suggesting that the divergence times among the three species of Pheretima and between Pheretima and Pontodrilus were 50 to 100 and about 209 million years ago, respectively.

Motility and respiration were examined in spermatozoa of the sea urchin Hemicentrotus pulcherrimus after dilution and incubation in seawater at pH 8.2 at 20°C. Almost all spermatozoa were motile during 12 hr of incubation, but their respiratory rate decreased gradually. The acrosome reaction was also induced by egg jelly solution during 12 hr of incubation in seawater. However, the ratio of spontaneous acrosome reacted spermatozoa was quite low during the same period. An intracellular pH (pHi) of spermatozoa was about 7.5 just after dilution in seawater and was almost constant during 12 hr of incubation. Upon dilution and incubation in seawater, activity of NADH-cytochrome c reductase decreased in propotion to the decrease in the respiration in spermatozoa, whereas cytochrome c oxidase activity was hardly changed. These suggest that the degeneration of respiratory system during 12 hr of incubation in seawater is due to the decrease in the NADH-cytochrome c reductase activity. In energy production system, phosphatidylcholine as a preferred substrate was efficiently hydrolyzed during 4 hr of incubation and then the activity of the energy metabolism decreased gradually. Beyond 12 hr incubation in seawater, the number of immotile spermatozoa increased and respiratory rate declined rapidly. Also, the percentage of the acrosome reaction induced by the egg jelly solution decreased. These are probably due to the increase in the number of dead spermatozoa after 12 hr of incubation in seawater. It is thus concluded that the life-span of H. pulcherrimus spermatozoa is about 12 hr after dilution in seawater.

We have isolated three embryonic stem (ES) cell lines from C3H/He mice using mouse STO cells as a feeder layer. One ES cell line (H-1) was male, and two (H-2 and H-3) were female, as determined by polymerase chain reaction, in situ hybridization, and karyotype analyses. All were immunocytochemically reactive with a C3H strain-specific antibody. Injection of cells from the female ES H-3 line into C57BL/6 blastocysts yielded four chimeras with slight coat color chimerism. All chimeras were male, and as expected, no germline-transmission was observed. By contrast, when male ES H-1 cells were injected into the perivitelline space of 8-cell C57BL/6 embryos, one male mouse with overt coat color chimerism was recovered, and it produced ES H-1-derived offspring exclusively. This germline-transmissible C3H/He cell line represents a novel addition to those ES lines currently employed for gene manipulation studies of development.

In previtellogenic oocytes of the freshwater teleost, Oryzias latipes, the yolk nucleus is a prominent structure in the cytoplasm. The process by which the animal-vegetal (A-V) axis is established in these oocytes was observed by focusing on the yolk nucleus, granulosa cells and attaching filaments using light and electron microscopy. Prior to appearance of the rudiments of attaching filaments a special area was recognized near the yolk nucleus of early stage III oocytes by gathering of granulosa cells with irregular-shaped nuclei, The rudiments of attaching filaments clearly appeared in the area where granulosa cells gathered. The area with a cluster of granulosa cells and the rudiments of attaching filaments corresponded to the position of the yolk nucleus. In late stage III, the future vegetal pole area (VPA) was easily recognizable by the presence of the rudiments of attaching filaments. The present results suggest that the vegetal pole may be primarily determined by the random position of the yolk nucleus and established by reciprocal interactions between the cortical cytoplasm containing the yolk nucleus and its surrounding granulosa cells. It was postulated that the animal pole is determined by differentiation of the micropylar cell from a granulosa cell and by locomotion of granulosa cells around the axis connecting the centers of the germinal vesicle and the yolk nucleus before the initiation of vitellogenesis.

As the first step to understand the mechanism of pharyngeal cartilage development in the embryo of flounder, Paralichthys olivaceus, we studied the process of cartilage formation in the pharynx histologically, then analyzed the role of fibroblast growth factor (FGF) during the process. At hatching (56-62 hr post-fertilization), each mandible, hyoid and gill cartilage existed as a pair of blastemas composed of prechondroblasts. Paired blastemas fused at medial line at 1.5 days post-hatching, forming primordia. Labeling with 5-bromodeoxyuridine indicated that proliferation of prechondroblasts was highly active at this stage. Deposition of cartilage matrix began from the mandible and hyoid primordia at 2.5 days post-hatching, and at 4.0 days the basic patterning of the pharyngeal cartilage was complete.

Immunohistochemical analyses with an antiserum against 22.5 kD FGF (anti-22.5 kD FGF) isolated from swimbladder of red seabream, Pagrus major, revealed that the surface ectoderm, gut, lateral line blastema and otic vesicle show FGF-like immunoreactivity. Immunoreactivity signals persisted through the stages when the proliferation of prechondroblasts was active. In order to estimate possible function of 22.5 kD FGF, the blocking effect of anti-22.5 kD FGF towards pharyngeal cartilage formation was assayed using cranial explants dissected from hatched embryos. When incubated with anti-22.5 kD FGF, cartilaginous nodules formed bilaterally at the position of the original blastemas, while in the control treatment, incubation with normal rabbit IgG, cartilages connecting the two lateral sides were formed. Thus, development of pharyngeal cartilage was severely curtailed in the presence of anti-22.5 kD FGF. These results suggest the possibility that 22.5 kD FGF is produced in the pharynx and that it takes part in the regulation of pharyngeal cartilage development, possibly as a signal to stimulate the growth of prechondroblasts.

Massive accumulation of glycogen in the follicles of Scotophilus heathi during the period of delayed ovulation was noticed. The follicles which survive for prolonged periods are morphologically specialized. The accumulation of glycogen was noticed in the granulosa cells. The egg cytoplasm, thecal cells and interstitial cells were almost devoid of glycogen. The first sign of a glycogen deposit in the ovary was noticed during the recrudescence phase (October) in late pre-antral follicles. Most of the morphologically healthy late pre-antral and antral follicles showed a positive reaction with PAS and Best carmine from October to early February. Morphologically atretic follicles showed only a mild glycogen accumulation. Little or no glycogen accumulation was noticed in some of the healthy late-antral follicles before ovulation during late February and early March. HCG-induced antral follicles during quiescence also did not show any accumulation of glycogen. The study suggests that glycogen laden follicles are not suitable for ovulation and may be the reason for the occurrence of delayed ovulation in S. heathi.

The organization of cytoplasmic microtubules during hormone-induced meiotic maturation of goldfish oocytes in vitro was examined by confocal immunofluorescence microscopy using an anti-tubulin antibody. The microtubule network was well distributed in fully grown immature oocytes. Once goldfish oocytes resumed meiotic maturation by a proposed maturation-inducing hormone of this species (17α, 20β-dihydroxy-4-pregnen-3-one, 17α, 20β-DP), cytoplasmic microtubules serially re-organized. Soon after the onset of the germinal vesicle (GV) migration towards the animal pole, the former microtubule network disappeared, followed by the appearance of a long perinuclear tail with high ordered microtubules extending from the vegetal surface of the GV. Incubation of fully grown immature follilces in colcemid, an inhibitor of tubulin polymerization, caused the disappearance of microtubules. However, this treatment did not prevent either the 17α, 20β-DP-induced migration of the GV or GVBD. Coincident with the breakdown of the GV (GVBD), numerous microtubules intruded into the GV from its vegetal surface. Soon after GVBD, a disk-shaped ring consisting of microtubule asters and a small ring with a radial array of microtubules in its center was observed at the animal pole region. In mature oocytes with meiotic spindles at the animal pole surface, cytoplasmic microtubules were concentrated in a small region around the animal pole showing complicated microtubule arrays. The results presented define distinct changes in microtubule organization during the 17α, 20β-DP-induced meiotic maturation of goldfish oocytes.

We have produced polyclonal antibodies against two oligopeptides corresponding to middle and c-terminal regions of amino acid sequences predicted from rainbow trout (Oncorhynchus mykiss) 3β-hydroxysteroid dehydrogenase (3β-HSD) cDNA (Sakai et al., 1994, FEBS Letters 350, 309-313). Both antibodies (α-tr3β-M and α-tr3β-C) recognized recombinant rainbow trout 3β-HSD protein derived from rabbit reticulocyte lysate system and non-steroidogenic mammalian COS-1 cell lysate. Immunoblot analysis of rainbow trout ovarian follicle homogenates revealed specific recognition of 3β-HSD protein, in rainbow trout testis, furthermore, immunoreactive 3β-HSD localized in Leydig cells in the interstitium of immature testes and interrenal cells in the head kidney. These results indicate that both α-3β-HSD antibodies recognized rainbow trout 3β-HSD protein at the level of immunoblot and immunohistochemical analyses. Furthermore, both antibodies also recognized immunohistochemically 3β-HSD in various steroidogenic organs (ovary, testis, and interrenal glands) of several teleost fishes.

We examined the structural stabilities after heat treatment of 22 mutants of rat prolactin (rPRL) with amino acid replacements at 15 different positions and recombinant wild-type rPRL (WT-PRL) as part of our series of studies on site-directed mutagenesis of rPRL. When WT-PRL at low concentrations (0.1 ∼ 10 ng/ ml) was heated at 100°C for 20 min, it lost its Nb2 proliferation activity, whereas at high concentrations (above 1 fig/ml), its activity remained. Temperature-dependent loss of the proliferation activity of 10 ng/ml WT-PRL after heat treatment for 5 min was observed. Next, we examined the proliferation activities of the 22 mutants heated at 60 and 70°C for 5 min. After treatment at 60°C, all the mutants retained their initial proliferation activities, whereas treatment at 70°C reduced their activities to about 63%, except for one in which glutamic acid at position 118 was replaced by lysine (E118K), suggesting that the mutations did not induce structural instability. The mutant E118K retained 84% of its initial activity after treatment at 70°C, significantly (P < 0.01) higher than the WT-PRL value. The temperature-dependency profile of the Nb2 proliferation activity of E118K also showed it had significantly increased thermo-stability. Meanwhile another mutant (E118Q) at the same residue showed no increased thermo-stability, suggesting that changing a negative charge (E) to a positive one (K) at position 118 induces ionic bond formation with a neighboring negative charge, resulting in thermostabilization of the structure of PRL.

Changes in the steroidogenic pathway in medaka (Oryzias latipes) ovarian follicles during vitellogenesis and oocyte maturation were investigated in vitro by incubation of follicles with several radiolabeled steroid precursors, followed by thin layer chromatography (TLC) fractionation and recrystallization. When vitellogenic follicles collected at 18 hr before the expected time of spawning were incubated with ³H-labeled pregnenolone, the major metabolites were 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, and androstenedione. Incubations of vitellogenic follicles with androstenedione produced testosterone and estradiol-17β By contrast, when maturing follicles (postvitellogenic follicles undergoing maturation) collected at 10 hr before spawning were incubated with ³H-labeled pregnenolone, the major metabolites were 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, and 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-DP, maturation-inducing hormone of medaka); androstenedione was not detected. Neither vitellogenic and maturing follicles produced progesterone when they were incubated with ³H-labeled pregnenolone, suggesting that in medaka ovarian follicles both estradiol-17β and 17α, 20β-DP are synthesized by the ⁵Δ-steroid pathway. Thus, there is a distinct shift in the steroidogenic pathway from estradiol-17β to 17α, 20β-DP production in medaka ovarian follicles, and it is suggested that the decrease in C17, 20-lyase activity is responsible for this shift.

The phosphodiesterase inhibitor IBMX enhanced androstenedione production in incubations of vitellogenic follicles with ¹⁴C-labeled progesterone. Calcium ionophore A23187 and the phorbol ester TPA (a protein kinase C activator) blocked the stimulatory actions of IBMX on androstenedione production. These findings suggest that multiple signalling pathways may participate in the regulation of ovarian steroidogenesis, and further emphasize the importance of calcium as a regulator of P-450c17 activity.

A unique specimen of the family Stylodactyiidae (Crustacea: Decapoda) collected from a great depth, 3436-3452 m off the east coast of Taiwan, northwestern Pacific, is described as representative of a new species close to Stylodactylus bathyalis Cleva, 1994 from the Coral Sea. These two species are characterized by several specialized features which warrant the establishment of a new genus Bathystylodactylus. The new species named B. inflatus differs from B. bathyalis in having the carapace markedly swollen at the posterodorsal part, a greater number of rostral spines, and the rounded third abdominal pleuron.