Localization and diurnal variations of carbonic anhydrase mRNA expression in the inner ear of the rainbow trout Oncorhynchus mykiss

Physiological studies have suggested that carbonic anhydrase (CA) plays a central role in otolith biomineralization via ion transport. However, the presence and exact function of CA in the inner ear have not been determined. In the present study, to investigate the localization of CA and its involvement in otolith calcification, we cloned two cDNAs encoding CAs from the rainbow trout sacculus. These two cDNAs, designated rainbow trout CAa (rtCAa) and rtCAb, both had an open reading frame encoding 260 amino acids with a sequence identity of 78%. Remarkably, rtCAb has a high degree of homology (82%) with “high activity CA” in the zebrafish, and its mRNA expression showed variation in the range 1.9–11.4 × 10⁴ copies/ng total RNA in the sacculus. In contrast, rtCAa mRNA was constantly expressed at approximately 3 × 10⁴ copies/ng total RNA. In situ hybridization revealed that rtCAb mRNA was strongly expressed in the distal squamous epithelial cells and transitional epithelial cells, except the mitochondria-rich cells, whereas, rtCAa was localized in extrasaccular tissue. These results suggest that the rtCAb isozyme is involved in the daily increment formation and calcification of otoliths via phase and spatial differences of the bicarbonate supply to the endolymph.     

Enzyme-histochemical localization of carbonic anhydrase in the inner ear of the guinea pig and several improvements of the technique

We have made several improvements in the method of fixation of the inner ear and the enzyme-histochemical technique for carbonic anhydrase (CA) detection. The results confirmed that CA is localized in the hair cells of the organ of Corti, Deiters' cells or nerve endings, inner pillar cells, Boettcher's cells, stria vascularis, spiral ligament, spiral limbus, and spiral ganglion cells. These results generally agree with previous histochemical observations but showed some differences. Our method preserved tissue morphology and showed more detailed localization of CA activity in the inner ear. In particular, the marginal zone of stria vascularis and the epithelial cells of spiral prominence, facing the endolymph, showed no CA activity, while the suprastrial region of the spiral ligament and the supralimbal region of the spiral limbus, juxtaposed to the perilymph, showed CA activity. In outer hair cells, the cuticular plate, which faces the endolymph showed CA activity, but the lateral membrane, which faces the perilymph showed no CA activity. In contrast, the inner hair cell cytoplasm showed diffuse CA activity. These results will be useful in considering ion exchange between endolymph and its adjacent cells, and between perilymph and its adjacent structures.     

Purification and properties of carbonic anhydrase from salmon erythrocytes

Carbonic anhydrase (CA) from erythrocytes of the pink salmon, Onchorhyncus gorbushka, was purified using chloroform-ethanol extraction and Sephadex G-75 gel filtration. A single, high specific-activity CA isozyme having a molecular weight of 29,000 was found. The enzyme sedimented as a single boundary at a sedimentation velocity of 2.9S. Amino acid analysis revealed a composition similar to other submammalian CAs with the exception that the cysteine content was low (1 mol cysteine/mol enzyme). Like other submammalian CAs, the presence of a sulfhydryl reducing agent was required to maintain full activity and to prevent structural changes in the enzyme.     

Gaseous intermediates in the brain of the salmon Oncorhynchus masou

Distribution of nitroxidergic and H2S-producing neurons in the brain of the salmon Oncorhynchus masou was studied by methods of histochemical markering of NADPH-diaphorase and by immunohistochemical markering of the neuronal nitric oxide synthase and cystathionin beta-synthase (CBS). The established distribution of CBS and nNOS/NADPH-d of neurons and fibers in the salmon telencephalon, optic tectum, and cerebellum allows suggesting that the NO- and H2S-producing systems, represent individual, non-overlapping neuronal complexes performing specialized functions in the activity of local neuronal networks. In the brainstem part, the nNOS-ir and NADPH-d-positive neurons were detected in the composition of viscerosensor (V, VII, and IX-X) and visceromotor (III, IV, and VI) nuclei of craniocerebral nerves, octavolateral afferent complex, reticulospinal neurons, and medial reticular formation. CBS in the salmon medulla was revealed in neurons of the X nerve nucleus, reticulospinal neurons, and ventrolateral reticular formation. Distribution of NO-ergical and H2S-producing neurons in the salmon medulla nuclei indicates that NO in salmon is the predominant neuromodulator of medulla viscerosensory systems, while H2S seems to modulate only the descending motor systems. The results of the performed study allow suggesting that NI in the descending motor systems. The results of the performed study allow suggesting that NO in the salmon medulla periventricular area can act as a regulator of postnatal ontogenesis.     

Morphological changes in the parr masou salmon Oncorhynchus masou retina in experimental change of geomagnetic field

Topography of photoreceptor cells in young salmon Oncorhynchus masou retina, their properties and morphology of cellular organelles in external and internal segments of photoceptors have been first described. Morphological changes of retina cells were analysed in day and night time, and also in the experiment for indemnification of geomagnetic field (GMF) in the body of the aquarium. A comparison of retina structure in fishes of night and day time controls with that in experimental fishes has shown that the external cone segments in the latter occupy, in relation to the external limiting membrane, an intermediate position, characteristic of retinae exposed to twilight lighting. It is supposed that GMF indemnification was equivalent to weak light pulse, which, however, could considerably change melatonin production by retina photoreceptor cells. Thus, at experimental indemnification of GMF, retina sensitive cells demonstrate typical retinomotor response. Some ultrastructural changes in retina cells were also detected, in particular, size changes in ribbon synapses in rod and cone terminations. In addition, nematosomes appeared in the internal nuclear layer, and in the spinules, i.e. digitiform invaginations of terminal dentrites of horizontal cells into cone nervous terminations, the quantity of an electron dense material was noticeably magnified in comparison with a night control. The noted changes testify, in our opinion, to essential modifications in metabolic processes of retina photoreceptors under effect of GMF variations, in particular, to changes in retinal melatonin synthesis.     

The lateral line system and its innervation in the masu salmon Oncorhynchus masou masou (Salmonidae)

Ichthyological Research - The lateral line system and its innervation were examined in the masu salmon Oncorhynchus masou masou. The species has 8 cephalic canals (supraorbital, infraorbital, otic,...     

Purification and localization of human carbonic anhydrase

Summary Three different isoenzymes of human carbonic anhydrase are now well characterized. Carbonic anhydrase I and II have been known for several years and are located in high amounts in red blood cells as well as in many other tissues.Carbonic anhydrase III, a protein showing CO₂ hydratase and p-nitrophenylphosphatase activity was isolated from skeletal muscle some years ago. Earlier observations based on enzyme activity and radioimmunoassay studies have suggested that this protein is present in greater quantities in red skeletal muscles than in white ones. We have purified CA III from human soleus muscle and using obtained monospecific polyclonal antibody localized this protein in the same muscle fibers which show acid resistant ATPase activity. Using this protein as a marker for type I muscle fibers, fiber classification into type I and II could now be done also from paraffin embedded sections.This study is supported by the Research Council of Physical Education and Sport, Ministry of Education, Finland     

Ultrastructural immunocytochemical localization of carbonic anhydrase

Summary Specific antibodies against human erythrocyte carbonic anhydrase isozyme C were used to determine the ultrastructural localization of this enzyme in the collecting ducts of rat kidney. Using a pre-embedding labeling technique, carbonic anhydrase C was found in the cytoplasm of intercalated cells, whereas the principal cells were negative.     

Ultrastructure of the retinal pigment epithelium in the young masu salmon Oncorhynchus masou

The ultrastructure of retinal pigment epithelium (RPE) cells in the masu salmon Oncorhynchus masou fry was studied. The physiological state of the pigment was dependent on the level of light/dark adaptation. Similar morphological properties were observed in the response of the RPE cells to light and a magnetic field.     

Interpopulation variation in reproductive traits of female masu salmon, Oncorhynchus masou

Several life history models predict that larger eggs and lower fecundity should be favored in a low-growth environment. We applied the model of Sibly et al. to seven Japanese populations of masu salmon ( Oncorhynchus masou ) in order to test the predictions that populations in which individual growth rate is low are characterized by larger eggs and lower fecundity. Two populations, Shumarinai and Shikaribetsu, had the lowest growth rates of individuals, largest egg sizes and lowest fecundities of all populations examined. In contrast, the Shiribetsu, Chitose and Uono populations had highest growth rates of individuals, smallest egg sizes and highest fecundities. The Shibetsu and Toya populations were intermediate between these two groups. The gonadsomatic index (GSI) of the Shumarinai and Shikaribetsu populations was smaller than that of all other populations. Correlation analysis indicated that populations with lower individual growth rates had larger eggs and lower fecundities. The results were consistent with the predictions of the Sibly et al. model: increased egg size which results in decreased fecundity is probably an adaptation to low-growth environments. Therefore, in masu salmon, growth differences among populations may explain interpopulation variation in egg size and fecundity.     

Proteomic analysis of inviable salmonid hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss during early embryogenesis

Early embryos of inviable hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss at 9, 12, 15 and 20 days after fertilization were examined for protein expression profiles. A total of 44 proteins, mostly down-regulated products of house-keeping genes and those involved in nucleic acid metabolism or chromatin replication, were identified in hybrid embryos by mass spectrometry analysis and protein database searching. The identified down-regulated proteins may be responsible for the inviability in the hybrids.     

Latitudinal variation in egg size and number in anadromous masu salmon Oncorhynchus masou

Latitudinal variation in egg size and number in anadromous masu salmon Oncorhynchus masou was examined. Relatively greater variation in egg size occurred among rivers than among females within rivers or within females. Egg size was generally greater and egg number generally lower at more northerly latitudes.     

Autoradiographic localization of carbonic anhydrase in the rabbit ciliary body

The high binding affinity of acetazolamide for carbonic anhydrase (K1 congruent to 10(-8) M) was employed to demonstrate the distribution of the enzyme in the rabbit ciliary body by incubating the tissue with 3H-acetazolamide (1.5 Ci/mmol). Specificity of binding was ascertained by displacing 3H-acetazolamide with a high concentration of unlabeled ethoxzolamide (K1 congruent to 10(-9) M). Wedges of the globe anterior to the ora serrata were incubated in bicarbonate buffered physiological saline, 95% O2/5% CO2, at 0 degrees C for 2 hr with either 3H-acetazolamide (0.2 microM), 3H-acetazolamide and unlabeled ethoxzolamide (100 microM), or physiological saline alone. They were then washed for 2 hr in fresh physiological saline and processed for autoradiography. The autoradiographs showed the label localized in both pigmented and nonpigmented layers of ciliary body epithelium in the pars plicata and in the iridial processes. The epithelia of both crests and troughs showed localization of label. In contrast, no concentration of label was found in the stroma of the ciliary body, including vascular endothelium, and in the epithelia of the pars plana. In sections that were incubated with 3H-acetazolamide in the presence of an excess of unlabeled ethoxzolamide, no localization of label occurred. These findings suggest that the epithelia of the pars plicata, but not those of the pars plana, contain carbonic anhydrase. This is consistent with hypotheses restricting aqueous humor formation to the pars plicata.     

Molecular cloning and characterization of three distinct choriogenins in masu salmon, Oncorhynchus masou

Three cDNAs, each encoding a different choriogenin (Chg), were isolated from a female masu salmon (Oncorhynchus masou) liver cDNA library. Two of the cDNA clones, Chg Halpha and Chg Hbeta, showed a close relationship and contained the typical domains of zona pellucida (ZP) B genes in fish, namely proline and glutamine rich repeats, a trefoil factor family domain, and a ZP domain. Specific antibodies against recombinant Chg H products (rmHalpha and rmHbeta) were generated to elucidate the relationship between the Chg H cDNAs and two types of serum Chg H protein, which were previously purified and characterized, and designated as very-high-molecular-weight vitelline envelope-related protein (vhVERP) and Chg H of masu salmon. The immunobiochemical analyses revealed that the Chg Halpha and Chg Hbeta clones encoded vhVERP and Chg H proteins, respectively. The third cDNA clone (Chg L) appeared to be a ZPC gene and, by mapping the N-terminal sequence of purified Chg L, was shown to encode serum Chg L protein. Various types of heteromultimer of the three Chgs were identified immunologically as high molecular weight chorion components, indicating the involvement of complex heterodimerization of multiple Chgs in the construction of chorion architecture in masu salmon.     

Effects of melatonin feeding on smoltification in masu salmon (Oncorhynchus masou)

Influences of photoperiod on plasma melatonin profiles and effects of melatonin administration on long-day-induced smoltification in masu salmon (Oncorhynchus masou) were investigated in order to reveal the roles of melatonin in the regulation of smoltification in salmonids. Under light-dark (LD) cycles, plasma melatonin levels exhibited daily variation, with higher values during the dark phase than during the light phase. The duration of nocturnal elevation under short photoperiod (LD 8:16) was longer than that under long photoperiod (LD 16:8). Melatonin feeding (0.01, 0.1 and 1 mg/kg body weight) elevated plasma levels of melatonin in a dose-dependent manner for at least 7 h but not for 24 h. When masu salmon reared under short photoperiod were exposed to long photoperiod (LD 16:8) and fed melatonin (1 mg/kg body weight) 7 hours before the onset of darkness, a significantly smaller proportion of smolts appeared in the melatonin-fed group after 32 days than in the control group. However, after 59 days of the treatment, there was no difference in the proportion of smolts between the control and melatonin-treated groups. Thus, melatonin feeding mimicked the effects of short photoperiod, which delays but does not completely suppress smoltification. These results indicate that the day length is transduced into changes in the duration of nocturnal elevation in plasma melatonin levels, and that artificial modification of the plasma melatonin pattern possibly delays the physiological processes of smoltification induced by long-day photoperiodic treatment.     

Latitudinal variation in sexual size dimorphism of sea-run masu salmon, Oncorhynchus masou

Sexual size dimorphism (SSD), a difference in body size between the sexes, occurs in many animal species. Although the larger sex is often considered invariable within species, patterns of selection may result in interpopulation variation or even reversal of SSD. We evaluated correlations between latitude and female body size, male body size, and relative body size (male body size/female body size) in 22 populations (ranging from 37 degrees N to 49 degrees N) of sea-run masu salmon (Oncorhynchus masou) that spawn in rivers along the Sea of Japan coast. Male size and the relative body size increased with latitude, but female size did not correlate with latitude. In addition, increase in male size with latitude was sufficient to result in a reversal of SSD, the switch-point being around 45 degrees N. We suggest that the positive correlation between latitude and male size is due to increasing operational sex ratios or sexual selection on sea-run male body size that result from sex-biased patterns of anadromy. In conclusion, our study provides the first example of predictable geographic variation in SSD shaped by apparent patterns of sexual selection.     

Identification and localization of carbonic anhydrase in two chlorella species

Carbonic anhydrase (CA) activity and localization have been examined in two species of the eukaryotic green alga Chlorella. Mass spectrometric and potentiometric assays of CA activity indicate that C. ellipsoidea contains very little extracellular CA activity whereas C. saccharophila exhibits significant extracellular activity when grown at alkaline pH values. Extracellular CA activity appears to be correlated with the presence of a 36 kilodalton polypeptide that was detected immunologically using a polyclonal antibody directed against the 37 kilodalton Chlamydomonas CA monomer. Both Chlorella species and enzymatically isolated C. ellipsoidea chloroplasts also contain an immunologically similar 38 kilodalton polypeptide that may be a cytosolic or chloroplastic form of CA.     

Immunohistochemical localization of human carbonic anhydrase isoenzyme C

Summary Methods for immunohistochemical localization of human carbonic anhydrase isoenzyme C (HCA C) with indirect fluorescent antibody and immunoperoxidase techniques are described. Both methods revealed large amounts of this high activity isoenzyme in the mucosae of human stomach and appendix. With the indirect immunofluorescent method the presence of the enzyme in human erythrocyte cytoplasm was also demonstrated. Correlations of present findings with those obtained with the traditional histochemical methods for demonstration of carbonic anhydrase activity are discussed.