Zoological Science

Two main hormones regulate water balance in amphibian. First, mesotocin (MT) acting as a diuretic agent, and second arginine vasotocin (AVT) being an anti-diuretic hormone. In addition, prolactin (PRL), aldosterone, corticosterone, angiotensin II and atriunatriuretic hormones, play a role too in regulating water and ion balance. The hormones affect the epidermis and bladder permeability to water and ions as well as the kidney through the control of the glomerular filtration rate (GFR). The main questions concern the presence and action of these hormones during the amphibian's life history. Are they present in both larval and adult stages? Are these hormones being synthesized in both aquatic and terrestrial adult phases? Under what circumstances are they being stored or released? Would the target organs (epidermis, bladder, kidney) respond in a similar way during all periods? The problem is the fact that under most circumstances an amphibian while in an aquatic environment responds physiologically differently than when on land. Only partial information concerning hormone presence, release and control of water balance is available at the moment, and even that is fragmentary and based on only a very small number of amphibian species.

A decapeptide (GFDLNGGGVG) isolated from the solubilized jelly layer of the sea urchin Hemicentrotus pulcherrimus stimulates the respiration and motility of H. pulcherrimus spermatozoa and, in addition, produces a number of biological effects on H. pulcherrimus spermatozoa including increases in cAMP and cGMP levels, activation of a Na⁺/H⁺ exchange system, and increases in intracellular pH (pH_i) and [Ca²⁺] ([Ca²⁺]ⁱ). The peptide activates the metabolism of endogenous phosphatidylcholine and promotes the acrosome reaction as a specific co-factor of a major acrosome reaction-inducing substance, fucose sulfate glycoconjugate. The peptide also induces an electrophoretic mobility change in the guanylate cyclase of the sperm plasma membrane with concomitant dephosphorylation and inactivation of the enzyme. Seventy-four peptides producing similar biological effects, named sperm-activating peptide (SAP), have since been purified from the solubilized jelly layer of seventeen species of sea urchins distributed over five taxonomic orders. These peptides show essentially the same biological effects on sea urchin spermatozoa although their activity and structures are specific at the ordinal level. Equilibrium binding experiments using a radioiodinated SAP-I analogue [GGGY(¹²⁵I)GFDLNGGGVG] to H. pulcherrimus spermatozoa suggests the presence of two classes of receptors (high affinity and low affinity) specific for SAP-I binding. Based on the Kd values and EC₅₀'s for SAP-I's biological activity, we presume that the high affinity receptor is associated with respiration-stimulating activity and elevations in pH_i, while the low affinity receptor is coupled to elevations in cGMP and [Ca²⁺]_i. The radioiodinated SAP-I analogue crosslinks to a 71 kDa protein which contains a single membrane-spanning domain at almost near C-terminus. A SAP-I precursor which is synthesized in the accessory cells contains five SAP-I and seven SAP-I-like decapeptides, each separated by a single lysine residue.

Isolated cone-driven horizontal cells dissociated from catfish retina were voltage-clamped using the whole-cell patch-clamp technique. The effects of acidification and alkalinization on an anomalous type, inwardly-rectifying K⁺ current (IRK⁺) were investigated. The magnitude of IRK⁺ was enhanced by raising the intracellular pH above 7.4, however, in contrast, intracellular acidification had little effect on this current. The range over which intracellular pH ([pH]_i) modulates IRK⁺ is different from that for modulation of a sustained high-voltage activated calcium current in these same cells and also for proton-sensitive, inward rectifier currents in starfish oocytes, skeletal muscle and heart myocytes.

Modulation of a cloned Aplysia K⁺ channel, AKv1.1a, by protein kinase C (PKC) activators was examined in Xenopus oocytes expression system. Following the application of phorbol esters (phorbol 12-myristate 13-acetate, PMA; phorbol 12,13-dibutyrate, PDBu), or a diacylgrycerol analogue (1-oleoyl-2-acetyl-sn-glycerol, OAG), the fast inactivation of the AKv1.1a became slower and the peak current increased (up-modulation). However, the effect was transient. The expressed current was decreased even below control level about 15 to 20 min after the treatment (down-modulation). Both effects by PMA was blocked by the kinase inhibitor, H7, suggesting that phosphorylation by PKC is involved. The amino acid sequence of AKv1.1a contains three putative phosphorylation sites by PKC (Ser²⁴, Thr³⁴⁵, Ser³⁴⁹). We tested their contributions to the PMA-induced modulation by site-directed mutagenesis. The results suggest that the up-modulation by PKC activators is due to the inhibition of the fast inactivation by the amino-terminal domain (N-type inactivation), thereby increase the time the channels are conductive. Phosphorylation of Ser²⁴ may enhance the PKC-induced down-modulation, while phosphorylation of Thr³⁴⁵ may inhibit the down-modulation. By contrast, mutation of Ser³⁴⁹ did not affect the modulation. The N-type inactivation were not indispensable for the down-modulation because the amino-terminal deletion mutant also showed some down-modulation although its onset was quite slow. Thus, the down-modulation of AKv1.1a may be heterogeneous. Because some modulation was still observed even in a mutant which lacks all putative phosphorylation sites mentioned above, additional mechanisms such as the regulation by other phosphorylated protein(s) exist endogenously in oocytes and/or recruitment of other kinases by PKC-activation may also be involved in the observed modulation of the AKv1.1a.

After frog taste cells were adapted to 1 mM quinine-HCl (Q-HCl) for 10 sec, modification of receptor potentials in the taste cells induced by salt, acid, sugar and bitter stimuli was studied with microelectrodes. The phasic component of receptor potentials induced by 0.1 M NaCl, KCl, NH₄Cl and MgCl₂ was enhanced following adaptation to Q-HCl. The rate of rise of receptor potentials in response to the salts was increased after Q-HCl adaptation. The amplitude and the rate of rise of receptor potentials induced by 1 mM acetic acid were larger after Q-HCl adaptation than after water adaptation. The amplitude of phasic component and rate of rise of receptor potentials for 0.5 M sucrose after Q-HCl were the same as those after water. The amplitudes of tonic receptor potentials for 1 mM Q-H₂SO₄, brucine and picric acid after Q-HCl adaptation were the same as those after 1 mM NaCl adaptation. Correlation coefficient between taste cell responses induced by 1 mM Q-HCl and 1 mM Q-H₂SO₄ was very high, but those between 1 mM Q-HCl and 1 mM brucine responses and between 1 mM Q-HCl and 1 mM picric acid responses were low. This indicates that Q-HCl and Q-H₂SO₄ bind to the same receptor site, but brucine and picric acid bind to different receptor sites to which Q-HCl does not bind.

The circadian photoaccumulation rhythm of thirty strains of Paramecium bursaria collected at different places in Japan and China were measured with a microcomputer assisted data collection apparatus. Although most strains showed a period of 23–26 hours in LL, we found two strains of conspicuously different periods; a short period strain (UK1, 21.8 hr) and a long period strain (T316, 28.7 hr). F1 progeny from a cross between the short and the long period strains showed an intermediate period of about 24.7 hours (range 22.5–25.8 hr). The character was not distributed in a Mendelian ratio among the F1 progeny. We isolated a mutant (E2) with short period (21.8 hr) from the stock strain Kz1 by treatment with nitrosoguanidine (MNNG). The progeny of crosses between E2 and UK1, and between E2 and T316 exhibited the short period and the normal period phenotype respectively. Moreover, the progeny from a cross between E2 and a wild type strain (Sj2w) became sexually mature about 25 fissions after conjugation. This length of immaturity is much shorter than that of the progeny from wild type strains (about 50 fissions). This early maturation character was inherited to progeny in a Mendelian ratio. Homozygotes for the early maturation allele (EM2) exhibited mating ability about 15 fissions after conjugation. These data suggest that there is a correlation between the period length of the circadian rhythm and the length of immaturity after conjugation in Paramecium bursaria.

In general, cell differentiation and proliferation are mutually exclusive. Transition of the cellular slime mold Dictyostelium discoideum from growth to differentiation is triggered mainly by a secreted factor(s) in addition to nutritional deprivation. To purify and identify the factor required for the growth/differentiation transition, a new assay system was designed. Under low-nutrient conditions, cells could grow to multiply, but never developed. The cellular development including aggregation, however, was induced by the addition of conditioned medium (CM) in which growing or starving Dictyostelium cells had been cultured. The CM inhibited the synthesis of nuclear DNA and induced the cells to acquire chemotactic competence to cAMP, thus suggesting the presence of a secreted factor(s) required for growth/differentiation transition in the CM. The active factor(s) in CM (referred to as CMF⁴⁵⁰; conditioned medium factor) was found to be sensitive to heat and have a large molecular size. The CMF⁴⁵⁰ was purified using FPLC through a gel filtration column, and was identified to be a proteinous macromolecule of Mr 450 kDa, which was mainly composed of 94 kDa, 79 kDa, and 49 kDa subunits under a native condition.

Genetic differentiation and evolutionary relationships were surveyed on 12 species of the Old and seven species of the New World Emberizidae by allozyme electrophoresis of 20 loci. Genetic variability of the Emberizidae is similar to those of the other Passeriformes. The degree of genetic differentiation in the family were large among species of the genus Emberiza of the Old World, and also among genera of the New World Emberizidae. Evolutionary relationships of the Emberizidae based on these genetic distances differed considerably from those of previous publications on some points: (1) Emberiza tristrami, E. elegans, E. bruniceps and E. schoeniclus were genetically much diverged from the other Emberiza as a species of the same genus. (2) Some genetic distances between Emberiza were larger than distances between subfamilies of the New World Emberizidae. (3) Species of the Cardinarinae examined genetically, belonged to the Emberizinae of the New World. Genetic data did not support the current classification that the Old World buntings arose from the New World forms by recent colonization. Discussion was made on the evolution of the Old and New World Emberizidae from the genetic view point.

The expression of PNA-binding glycoproteins on lizard lymphocytes was investigated by studying the reactivity of FITC-PNA towards lizard lymphocytes obtained from the different lymphoid organs. Direct immunofluorescence assays have demonstrated that the majority of lizard thymocytes (70%) and only a fraction of lymphocytes in the spleen, peripheral blood and bone marrow were PNA-positive. This positivity was selectively inhibited by galactose as well as lactose, indicating the specificity of binding. Putative PNA receptors were purified from lizard thymocytes and splenocytes by affinity chromatography on a PNA-Sepharose 4B column and resulted in fractions enriched 1,792-fold and 3,141-fold for the PNA-binding component expressed on lizard thymocytes and splenocytes, respectively. Analysis on reducing and non-reducing SDS-PAGE revealed that both thymic and splenic PNA-binding glycoproteins migrated as a single component of 35 KDa, with no evidence for the association into higher multimers in both tissues. Analyses for amino acid and carbohydrate compositions indicated that the thymic and splenic glycoproteins have similar amino acid composition and differed in the content of neutral and amino-sugars as well as sialic acid. The content of the latter residue was relatively higher in the splenic form of the receptor compared to its thymic counterpart, and was inversely correlated with the content of galactosyl residues in both forms of the receptor. The functional significance of PNA-binding glycoproteins during vertebrate evolution is discussed.

Porcine ovary folliclular fluid contains a latent form of a protease which is activatable with trypsin. The active enzyme hydrolyzed peptide 4-methylcoumaryl-7-amide (MCA) substrates with a preference for the Arg-MCA bond. The enzyme was strongly inhibited by diisopropylfluorophosphatc, aprotinin, leupeptin and antipain, but not by soybean trypsin inhibitor. The apparent molecular weight of the enzyme was approximately 630,000 as estimated by gel filtration. No significant difference in molecular size was seen between the inactive precursor and trypsin-activated enzyme. The results suggest that the present enzyme is a novel type of serine protease.

The Fourspine sculpin (Cottus kazika) is a catadromous fish which is widely distributed in the rivers of Japan. The fish was used to examine the relationship between the migration behavior and hormonal control by studying the histological changes in the pituitary gland, thyroid gland and gonads during its downstream migration. By use of the immonocytochemical and histochemical techniques, 7 types of cells were identified in the pituitary gland namely; immunoreactive (ir)-PRL, GH, TSH, GTH, ACTH, MSH and SL cells. From among the first 4 types of the aforementioned cells, remarkable histological changes were observed in cells containing ir-GTH during the downstream migration. At this time also, the gonads were obsereved to be well developed, while the thyroid glands did not show clear changes morphologically. These results suggest that the gonadotropin regulates gonadal development in the Fourspine sculpin during downstream migration and possibly sex hormones synthesized by the gonads cause the downstream migration of this catadromous fish.

The present study was made to clarify the relationship between functions of the pars intermedia of the mouse pituitary gland and hypoglycemic stress. Morphometrical analysis of the ultrastructures of the pars intermedia cells showed (1) a rise in the percentage volume of rough endoplasmic reticulum (r-ER) indicative of an increase in protein synthesis, (2) an increase in the number of Golgi granules per unit Golgi area showing an induction of granule-forming activity and (3) a decrease in the numerical density of secretory granules reflecting a release of the secretory granules. These findings suggest that hypoglycemic stress induced by daily treatment with insulin or restriction of food intake was able to elicit heightened secretory activity of the pars intermedia cells of the mouse pituitary gland. However, acute hypoglycemic stress induced by food deprivation did not cytologically affect the pars intermedia. These observations suggest that repeated hypoglycemic stress, rather than acute hypoglycemic stress, may be a natural physiological stimulus of the pars intermedia of the mouse pituitary gland.

Metamorphosis of the sea urchin, Hemicentrotus pulcherrimus, can be induced by L-glutamine as reported previously for Pseudocentrotus depressus [15]. To analyze more precisely the process of metamorphosis induced by L-glutamine, the development of the echinus rudiment (ER) was classified into six stages. The stage at which the larvae underwent the normal metamorphosis by glutamine treatment was confirmed. The time of the glutamine treatment required for metamorphosis (the eversion of ER) was over 10 hr but treatment for more than 25 hr tended to decrease the number of metamorphosed larvae, although the larval arms had mostly been resorbed. The time of glutamine treatment to induce the metamorphosis, depended on the development of ER; more time was required for the younger larvae and less time for the older. The high mitotic activities observed in the cells of the ciliary bands were markedly decreased in the glutamine-treated larvae to metamorphose. These findings suggested that a degenerative process of metamorphosis including cell death is induced by L-glutamine.

We report here on the amplectic behavior of the Japanese treefrog (Hyla japonica) in microgravity. Treefrogs were exposed to 35 cycles of altered gravity, including ≈ 1.5 sec of G < 0.1 every 3 min and 15 sec, on the FreeFall “G.0” ride at Space World amusement park in Kitakyushu, Japan. During this period a pair of frogs spontaneously entered and maintained amplexus for 1 hr 20 min, before being removed from the ride. In freefall, the pair extended their hindlimbs in the characteristic posture of treefrogs in microgravity.

This is the first report of a vertebrate entering and sustaining a copulatory or amplectic posture under gravitational extremes, including true freefall. These observations bode well for the potential of anurans to breed in microgravity and to be used for biological research in space.

The numbers of olfactory receptor cells from electron micrographs in various species of birds were counted and the fine structure of their olfactory nerves was observed using electron micrographs. The birds were domestic ducks, a slay-backed gull, quails, budgerigars and bengalees. Data from pigeons obtained from a previous study were also included for comparison. The approximate numbers of olfactory cells on each side were 5,800,000 in the duck, 2,700,000 in the gull, 570,000 in the quail, 130,000 in the budgerigar, and 110,000 in the bengalee. From a cross section of an olfactory nerve, the nerve was observed to be divided roughly into several fascicles by perineurium. Each fascicle was composed of many small bundles which were surrounded by endoneurium. Each small bundle was separated into several divisions by mesaxon which originated from Schwann cells. In the anterior part of the nerve, the number of axons surrounded by mesaxon ranged between one and several dozen. The number in the middle part was much larger than in the anterior part. In the posterior part the number decreased again. The diameter of an axon was 0.21–0.26 μm on average. The axon contained neurotubules, neurofilaments, mitochondria and axonal smooth ER. The fine structure of the olfactory nerve and the numbers of olfactory cells in these birds are discussed and compared with those of other vertebrates.

To find how larval fish sense mechanical stimuli via their free neuromasts, we examined morphological changes in free neuromasts in the larval willow shiner, Gnathopogon elongatus caerulescens. Free neuromasts were found on the body surface of newly hatched larvae and their number increased on both the head and trunk with larval growth. The apical surface of free neuromasts changed in outline from a circle to a lozenge shape as the number of sensory cells increased in the prelarval stage, and then the cupulae of the free neuromasts changed from a stick-like to a blade-like shape. Seven-day-old larvae were at the postlarval stage and had many free neuromasts that were nearly mature. All free neuromasts contained sensory cells of opposing polarity. The orientation of the maximum sensitivity of free neuromasts, decided from the polarity of the sensory cells, coincided with the minor axis of the lozenge-shaped outline of the apical surface of the free neuromasts, and was in the same axis as the direction in which the blade-like cupulae bent. The change to a blade-like shape would cause a stimulus parallel to the minor axis to be perceived as being stronger than the same stimulus from other directions. The polarity of trunk neuromasts was usually oriented along the antero-posterior axis of the fish body, but a few had dorso-ventral orientation. On the head, free neuromasts were oriented on lines tangential to concentric circles around the eye.

A division arrest mutant, cdaA, of Tetrahymena thermophila is known to have a temperature sensitive-defect in the determination of the division plane, and its gene product had been shown to be a protein designated as p85 (Mr=85,000; pI=4.7). Here the localization of p85 was shown to be the accessary structure of specialized basal body close to the division furrow by immunoelectron microscopy using anti-p85 antiserum.