Modifications in the neurosecretory system of the fish,Misgurnus fossilis,induced by lasting inanition

Summary The reaction of the hypothalamic neurosecretory system to prolonged starvation was examined in the fish,Misgurnus fossilis L. Two and four months of starvation resulted in storage of neurosecretory substance in the perikarya of the nucleus preopticus and in the preoptico-hypophysial tract. Further starvation, prolonged to five or seven months, caused degenerative changes in the nucleus preopticus. However, there were no drastic changes in the amount of stainable substance in the neurohypophysis. The examination of control animals revealed that the nucleus preopticus undergoes seasonal changes which differ from those brought about by starvation.     

Transplantation of somatic nuclei into activated teleost eggs (the example of the loach, Misgurnus fossilis L.)]

Some peculiarities of the technique of nuclear transplantation for teleostean fishes are described, the loach (Misgurnus fossilis) taken as an example. The cells of the late high blastula were used as donors and the activated non-enucleated and enucleated (by X-rays, 20 kR) eggs of the loach as recipients. Following the nuclear transplantation in the activated non-enucleated eggs, 33 (out of 128) normal blastulae were obtained, one of which developed until hatching. Following the nuclear transplantation in the activated enucleated eggs, 34 (out of 251) blastulae were obtained, 6 embryos hatched and 2 larvae attained the stage of active feeding.     

Replication of mitochondrial DNA in the early development of Misgurnus fossilis

Mitochondria isolated from Misgurnus fossilis embryos at various developmental stages were incubated with ³H-dTTP in vitro and the incorporation into mtDNA was determined. It has been found that the rate of mtDNA labeling increases exponentially with a doubling time of 7 hr from 0.01 pmole of ³H-dTMP/mg protein/hr in mitochondria from unfertilized eggs to 0.4 pmoles of ³H-dTMP/mg protein/hr in mitochondria of 35 hr embryos. The pool of intramitochondrial dTTP decreases 2.5 times during the first 10 hr after fertilization, then remains practically constant up to 35 hr of development. The rate of exogenous ³H-dTTP incorporation into the acid soluble pool of isolated mitochondria at two stages is approximately proportional to the pool size. Thus identical specific activities of ³H-dTTP inside mitochondria would be obtained even with pools of different sizes. We conclude that the increase of ³H-dTMP incorporation into mtDNA in development reflects genuine activation of mtDNA synthesis. As early as 6 hr after fertilization the bulk of the label incorporated into mtDNA is found in the fraction associated with covalently closed molecules. This pattern of labeling characteristic for replicating mtDNA is maintained throughout early development. In contrast such preferential label incorporation into the closed circular fraction was not found with mitochondria of unfertilized eggs. Closed mtDNA from unfertilized eggs contains not more than 1% of molecules with D-loops. In 35 hr embryos the corresponding value is equal to about 4%. Activation of mtDNA replication in embryogenesis is probably due to the activation of mechanisms responsible for the generation of primers for replication. DNA polymerase activity solubilized from mitochondria remains unchanged in the course of embryogenesis.     

Cyclic changes of membrane conductivity in fertilized and activated eggs of teleost (Misgurnus fossilis) and their relation to the cell shape.

Oscillations of membrane ionic conductivity, with a period similar to cell cycle duration, were observed in fertilized and activated loach (Misgurnus fossilis) eggs. In cleaving eggs the decrease in conductivity coincided with mitosis. Synchronously with the oscillations of membrane conductivity in activated as well as in fertilized eggs, rhythmic changes in blastodisc shape occurred. The blastodisc rounded up during the period of increasing membrane conductivity and flattened while conductivity decreased. Scanning microscopy of fertilized and activated eggs revealed differences in the surface relief of rounded and flattened blastodiscs.     

Calcium in myocardial tissue and isolated mitochondria from a teleost

Summary A method is presented for isolating cardiac mitochondria from bony-fish. Calcium levels in ventricular whole tissue and isolated mitochondria of Gadus virens L. are determined by atomic absorption flame spectroscopy, and were found to be about 8 and 16 nmolCa/mg prot., respectively. In conclusion, the calcium concentration within the myocardial mitochondria in this species may be nearly three times higher than at the outside, and probably these structures serve as a calcium sink. The results are compared with those previously reported for mammals.     

Calcium in myocardial tissue and isolated mitochondria from a teleost

A method is presented for isolating cardiac mitochondria from bony-fish. Calcium levels in ventricular whole tissue and isolated mitochondria of Gadus virens L. are determined by atomic absorption flame spectroscopy, and were found to be about 8 and 16 nmolCa/mg prot., respectively. In conclusion, the calcium concentration within the myocardial mitochondria in this species may be nearly three times higher than at the outside, and probably these structures serve as a "calcium sink". The results are compared with those previously reported for mammals.     

Isolation and properties of lactate dehydrogenase isoenzymes from loach (Misgurnus fossilis) skeletal muscles and eggs

The main lactate dehydrogenase (EC 1.1.1.27) isozyme from loach (Misgurnus fossilis) skeletal muscles was purified to homogeneity by polyacrylamide gel electrophoresis. The main group of lactate dehydrogenase isozymes which predominate in their activity in the unfertilized eggs of this teleost species and are stable to AgNO3 inhibition were partially purified. The effects of various concentrations of pyruvate, oxalate and urea on the activities of these purified enzyme preparations and their pH optima were studied. The antiserum for the purified lactate dehydrogenase isozyme from loach skeletal muscle was obtained. The decrease of the activity of this isozyme and that of the investigated group of isozymes from the eggs in the presence of increasing concentrations of antiserum was estimated.     

Organization of the surface and adhesive properties of cleavage furrows in loach (Misgurnus fossilis) eggs

The experiments with chimerous embryos of teleost loach (Misgurnus fossilis) have shown that before furrows of the 1st cleavage appeared, the eggs were completely nonadhesive. At the stages of cleavages II-IV whole eggs were able to adhere and then make extended junctions in the blastodisc region. Adhesive domains of blastomeres were discovered by using carmine dye particles which attached primarily in the region of blastomere cleavage furrows. Scanning electron microscopy (SEM) showed that the cells in the depth of the cleavage furrows have domains with a smoother relief than their outer surface with numerous folds. Moreover, most adhesive flat lamellar formations (ruffles) with microvilli at their ends were discovered in the furrow region, particularly in its apical part (in the sites of intercellular contact formation and carmine absorption). Colchicine (5 X 10(-4) M) treatment affects the reorganization of the cleavage furrow surface, and the eggs, one by one, lose their ability to adhere, and then to attach dye particles. Mechanisms of formation of an adhesive contact between divided cells are discussed.     

Repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes

To study the repair capacity of fertilized mouse eggs for X-ray damage induced in sperm and mature oocytes, the potentiating effects of 3 well-known repair inhibitors, arabinofuranosyl cytosine (ara-C), 3-aminobenzamide (3AB) and caffeine, on the frequency of induced chromosome aberrations were examined in eggs fertilized with X-irradiated sperm or in eggs irradiated with X-rays at the mature oocyte stage immediately before fertilization. Gametic treatment, fertilization and embryo culture were carried out in vitro. Ara-C treatment was done only in the pre-DNA replication period, while treatment with 3AB and caffeine was continuous from fertilization to the first-cleavage metaphase. The induction of chromosome aberrations by exposing sperm or oocytes to X-rays was remarkably potentiated by post-treatment incubation in the presence of each of the 3 inhibitors. This result indicates the possibility that X-ray damage induced in sperm or oocytes is reparable in the fertilized eggs and that various types of repair processes are involved.     

A novel rod-like opsin isolated from the extra-retinal photoreceptors of teleost fish

We have isolated a novel opsin from the pineal complex of Atlantic salmon (Salmo salar) and from the brain of the puffer fish (Fugu rubripes). These extra-retinal opsins share approximately 74% identity at the nucleotide and amino acid level with rod-opsins from the retina of these species. By PCR, we have determined that the novel rod-like opsin is not expressed in the salmon retina, and the retinal rod-opsin is not expressed in the salmon pineal. Phylogenetic analysis suggests that the rod-like opsins arose from a gene duplication event approximately 205 million years ago, a time of considerable adaptive radiation of the bony fish. In view of the large differences in the coding sequences of the pineal/brain rod-like opsins, their extra-retinal sites of expression, and phylogenetic position we have termed these novel opsins 'extra-retinal rod-like opsins' (ERrod-like opsins). We speculate that the differences between retinal rod-opsins and ERrod-like opsins have arisen from their differing photosensory roles and/or genetic drift after the gene duplication event in the Triassic.     

Thermo- and radioinduced enzymatic relaxation of superhelical DNA from mud loach Misgurnus fossilis L. spermatozoa

Actions of environmental impacts on mud loach spermatozoa were studied using various model systems: a) temperature stress, b) X-ray irradiation in vivo only of the animal head (a condition to trigger stress reaction), c) X-ray irradiation in vivo only of the animal body (a condition to exclude a direct activation of principal stress-realizing organism systems), d) gamma-irradiation in vitro of the cell suspension. It has been demonstrated that the temperature stress or X-ray irradiation of the mud loach head induced three lines of effects: 1) significant decrease in DNA superhelical density, 2) activity redistribution (functional activation) of DNase II between chromatin subfractions (with the increase of its association to chromatin), and 3) intracellular acidification up to pH value to satisfy the DNase II initiation. The obtained facts allow to suggest that, first, DNase II participates in the presented temperature- and radio-induced supercoiled DNA relaxation in spermatozoa, and, second, DNase II is involved in physiological (season elimination of spermatozoa that remained within male gonads after fertilization) or environmentally-induced DNA degradation.     

Microcalorimetric study of mitochondria isolated from fish liver tissue

We determined the thermogenesis curves of mitochondria isolated from fish liver tissue by using an LKB 2277 Bioactivity Monitor. After isolation from the fish liver, mitochondria still have activity and can live for a long time by using the stored nutrients. We calculated the recovery rate constants of mitochondria. We found that the thermogenesis curves of mitochondria are similar to those obtained from prokaryotic cells, but not similar to those obtained from eukaryotic cells. We determined the metabolic thermogenesis curves of mitochondria isolated from two kinds of carp liver tissue, scattered-scaled mirror carp and harvest carp. There are some important similarities and some important differences between these thermogenesis curves.     

Metabolic-morphologic characteristics of the integument of teleost fish with mature lymphocystis nodules

Summary Normal and virus-infected (lymphocystis disease) integument from five species of teleosts was examined by light and TEM autoradiography and SEM to establish metabolic-morphologic characteristics of integument with mature lymphocystis cells (LC's). LC's with numerous morphologic attributes of a late developmental stage showed highest incorporation of [³H]-thymidine in vivo (1–91 h) above the intracytoplasmic inclusion body (ci) with little radiolabel in nuclei, cytoplasmic icosahedral deoxyriboviruses (ICDVs) or capsule. Analysis by quantitative autoradiography revealed that the % total cell label in ci and cytoplasm did not vary appreciably from 1–91 h and was corroborative with morphologic criteria of maturity. A possible phylogenetic difference was noted between teleosts, wherein normal integument showed uptake of [³H]-thymidine in vivo (1 h) by cells at all levels of the epidermis, and cyclostomes (Spitzer et al. 1979) wherein labeling was confined to the basal third of the epidermis. Among four infected teleost species, the mean diameters of the ICDVs measured under the same conditions, ranged from 259.5 nm to 290.0 nm with the mean for each species differing significantly (p < 0.01) from each of the other means. Ruptured LC's were shown by TEM and SEM to have released ICDVs onto the lesions and integument. Various stages of LC degeneration, host response, and integumental repair processes were documented. An evaluation of labeling in vivo of the capsular matrix was compatible ([³H]-D-galactose> [³H]-L-lysine [³H]-L-fucose) with a glycosaminoglycan-protein structure.     

Changes in enzyme activity of glycogen and hexose metabolism during oocyte maturation in a teleost,Misgurnus fossilis L.

Summary The oocyte at the end of oogenesis, mature egg and developing embryo of the loachMisgurnus fossilis L.) are characterized by indentical enzyme profiles of the Embden-Meyerhof chain, pentose phosphate cycle and key gluconeogenic enzymes. However, the carbohydrate metabolism in the oocyte differs substantially from that in the embryo.Oocyte maturation is followed by a complete loss of hexokinase (EC2.7.1.1),2-fold decrease of glycogen synthetase (EC 2.4.1.11) and 10-fold increase of glycogen phosphorylase (EC2.4.1.1) activity. This process is correlated with a gradual decrease of the ATP/(ADP+AMP) ratio from 41 to 21 and increase of the Fructose-6-Phosphate/Fructose-1,6-Diphosphate ratio from 0.27 to 2.0. Thus, oocyte maturation involves a number of changes in control mechanisms resulting in cessation of glycogen accumulation and a transition of carbohydrate metabolism from gluconeogenesis to glycogenolysis.

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Zusammenfassung Die Oozyte und das reife Ei vonMisgurnus fossilis L. besitzen die gleiche Enzymzusammensetzung der Embden-Meyerhof Wege, unterscheiden sich aber wesentlich in den Enzymen des Metabolismus des Glykogens und der Hexosemonophosphate. Beim Reifen der Oozyte erfolgt eine 10fache Zunahme der Aktivität der Phosphorylase (EC 2.4.1.1.), völliger Verlust der Hexokinase (EC 2.7.1.1.) und eine wesentliche Abnahme der Glycogensynthetase (EC 2.4.1.11).Das Verhältnis ATP/(ADP+AMP)geht von 4l auf 2-1 zurück, dasjenige von Fructose-6-Phosphat/Fructose-1,6-Diphosphat nimmt von 0,27 auf 2,0 zu.Änderungen der regulatorischen Mechanismen beim Reifen der Oozyte setzen die Geschwindigkeit der Glykogensynthese und Glukoneogenese scharf herab, unterbinden die Ausnutzung der freien Glukose bei der Glykolyse und sichert eine allmähliche Zunahme der Glykogenolyse während der frühen Entwicklung des Embryos.