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PubMed1991

Further investigation on nuclear transplantation in different orders of teleost: the combination of the nucleus of Tilapia (Oreochromis nilotica) and the cytoplasm of Loach (Paramisgurnus dabryanus).

The nucleus of a blastula cell from Tilapia (Oreochromis nilotica, family Cichlidae, order Perciformes) was transplanted into an enucleated egg of Loach (Paramisgurnus dabryanus, family Cobitidae, order Cypriniformes). From among 3747 nucleo-cytoplasmic hybrid (NCH) eggs two NCH larval fish (0.05%) were obtained; one died on the 6th day and the other died on the 12th day after the operation. Morphological examinations showed that both NCH larval fish had developed normally with an opened mouth except they could not take food after complete utilization of their egg yolk on the 5th day of development. The possible mechanisms for obtaining such inter-order NCH larval fish are discussed. This is the first report indicating that inter-order NCH larval fish can be obtained in spite of their evolutionary divergence.

Animals↗
PubMed1990

The cranial cartilages of teleosts and their classification.

The structure and distribution of cartilages has been studied in 45 species from 24 families. The resulting data have been used as a basis for establishing a new classification. A cartilage is regarded as 'cell-rich' if its cells or their lacunae occupy more than half of the tissue volume. Five classes of cell-rich cartilage are recognised (a) hyaline-cell cartilage (common in the lips of bottom-dwelling cyprinids) and its subtypes fibro/hyaline-cell cartilage, elastic/hyaline-cell cartilage and lipo/hyaline-cell cartilage, (b) Schaffer's Zellknorpel, typified by the cartilage in the gill filaments of most teleosts examined, (c) elastic/cell-rich cartilage, such as that which supports the barbels and oral valves of catfish, e.g. Corydoras metae, (d) fibro/cell-rich cartilage, as in the submaxillary meniscus of Sphaerichthys osphromenoides, (e) cell-rich hyaline and (f) matrix-rich hyaline cartilage--both of which are common in the neurocranium and gill arches of most teleosts. The range of cartilages seen, and the predominant cartilage type, is recorded for each species and a list is provided of the tissues that most typify different organs or regions of the head. As a preliminary pointer to developmental relationships between the cartilages, note was taken of gradual transitions between one cartilage and another. It is suggested that hyaline-cell cartilage occupies a key position in teleosts as the most labile of the supporting tissues and is highly characteristic of Cypriniformes. The cartilage that best resembles mammalian hyaline cartilage (matrix-rich hyaline cartilage) has a very conservative distribution in different skeletal elements and the least number of associations with other tissues. It is well represented in Siluriformes.

Animals↗
PubMed1989

Comparative study of the anatomy and laminar organization in the olfactory bulb of three orders of freshwater teleosts.

The anatomy and lamination of the olfactory bulb in Cyprinus carpio, Tinca tinca, Barbus bocagei (Fam. Cyprinidae, Or. Cypriniformes); Salmo gairdneri (Fam. Salmonidae, Or. Salmoniformes); and Gambusia affinis (Fam. Poeciliidae, Or. Cyprinodontiformes), all of them freshwater teleosts, are studied. These species show significative differences on the location, size, morphology, and lamination of their olfactory bulbs. The presence of a new stratum in the olfactory bulb of Salmo gairdneri and a completely different laminar organization in the olfactory bulb of Gambusia affinis are described for the first time. The anatomical and histological peculiarities of this structure in the orders studied could be the basis for different experimental approaches.

Animals↗
PubMed1999

Exclusively juvenile centrioles in Xenopus laevis oocytes injected with preparations of mature centrioles.

Activated oocytes of Xenopus laevis were injected with centriole preparations isolated either from spermatozoa of loach fish Misgurnus fossilis or from rat liver. These injections induced the development of cytasters in the ooplasm and egg cleavage. Electron microscopic study of cytasters was made at the stage that corresponded to interphase between first and second cleavage divisions. This study revealed in cytasters singleton centrioles surrounded by pericentriolar material and numerous microtubules. Surprisingly, the ultrastructure of centrioles in cytasters corresponded to that of juvenile, newly formed vertebrate centrioles, whereas the injected preparations contained only adult mature centrioles. We suggested that xenogenic centrioles injected to Xenopus laevis oocytes could dissolve after formation of centrioles made from molecules of oocyte origin. A special mechanism that eliminates male centrioles after egg fertilization is speculated.

Animals↗
PubMed2000

Electroporated sperm mediation of a gene transfer system for finfish and shellfish.

We investigated gene transfer in finfish and shellfish via electroporated sperm. The mobility of sperm, the fertilization rate, the hatching rate, gene transfer rate, and abnormality rate of derived embryos were primarily dependent on the voltage level and concentration of DNA during electroporation. Optimal conditions for sperm of each species of aquatic animals can be reached. Genome of the electroporated sperm was analyzed by PCR, and it was shown that an expected-sized product was amplified, corresponding to that of the transgene's amplification. Southern blotting also showed that a positive band located at the same position as the DNA fragment used for the transfer was found in the electroporated sperm after DNase treatment. When the genome isolated from embryos, larvae, juvenile, and adult individuals, all derived from sperm electroporated with foreign DNA molecules, was analyzed by PCR, the existence of foreign DNA was detected in some samples. The integration of the transferred DNA into the genome of transgenic samples was also shown by Southern blot analysis. There was a mosaic distribution of exogenous DNA in a wide variety of tissues analyzed. In addition to CAT activity being positive for the experimental larvae, the transferred GH gene was functional in transgenic finfish and shellfish and resulted in fast-growing transgenic varieties. The overall evidence strongly suggests that the use of electroporated sperm is the simplest yet most efficient approach to perform mass gene transfer in aquacultural animals, including marine mollusks.

Animals↗
PubMed1990

Ultrastructural features of mitochondria-rich cells in stenohaline freshwater and seawater fishes.

In order to elucidate the functional significance of accessory cells in freshwater fishes, such as the rainbow trout, which displays a poor adaptability to seawater life, a search for such cells was performed in two stenohaline freshwater fishes: the loach and the gudgeon. Accessory cells were never encountered in these species; but, in contrast, two types of chloride cells were observed consistently that strikingly resembled the alpha- and beta-cells previously described in the guppy, a freshwater-adapted euryhaline fish. The alpha-cell, a pale and elongated chloride cell, was located at the base of the secondary lamellae in close contact with the arterioarterial pillar capillary. Darker, ovoid chloride cells resembling the beta-cell were found exclusively in the interlamellar region of the primary epithelium facing the central venous sinous. The latter cells frequently formed multicellular complexes linked together by deep, narrow, apical junctions. In another experiment, a stenohaline seawater fish, the turbot, was adapted to diluted 5% saltwater and to fresh water. In seawater, the gill epithelium contained only one type of chloride cell, always associated with accessory cells. Due to numerous cytoplasmic interdigitations between the accessory cells and the apical portion of the chloride cell, there was a noticeable increase in the length of the shallow apical junction, sealing off the intercellular space between the two cell types. In 5% saltwater, there was a decrease in the number of these interdigitations and a concomitant decrease in the length of the shallow apical junction. In fresh water, chloride cells were partially or completely separated from the outside medium by modified accessory cells. It is thus concluded that accessory cells are found exclusively in fish living in seawater or preadapted to seawater and that they probably are involved in the formation and modulation of paracellular pathways for ionic excretion. In contrast, the respective roles of the two types of chloride cells observed in freshwater fishes are still to be determined.

Adaptation, Physiological↗
PubMed1989

Two isotocin genes are present in the white sucker Catostomus commersoni both lacking introns in their protein coding regions.

Two genes each encoding a distinct precursor protein to the hormone isotocin and a neurophysin-related protein are present in the teleost fish Catostomus commersoni. These precursors are referred to as isotocin 1 and 2. As shown by the polymerase chain reaction technique, both genes lack introns in their protein-coding sequences. Both genes are transcribed giving rise to mRNAs of 920 (isotocin 1) and 1020 (isotocin 2) bases, respectively. Based on the nucleotide sequences, the predicted isotocin precursors contain, besides the hormone moiety, a neurophysin-like protein that, in contrast to its mammalian counterpart, is extended at its C-terminus by a peptide which includes a leucine-rich core segment. This segment shows similarities to the copeptin of the mammalian vasopressin precursor that is known to possess prolactin-releasing activity. The data imply that the mammalian copeptin sequence was initially part of a larger ancestral neurophysin molecule.

Amino Acid Sequence↗
PubMed2002

Production of germline chimera in loach (Misgurnus anguillicaudatus) and proposal of new method for preservation of endangered fish species.

As part of the technique for preservation of endangered fish species, germline chimeras using the loach (Misgurnus anguillicaudatus) model were generated by embryonic cell manipulation. Transplanting cells from early-stage embryos of a wild-type strain to the blastoderm of an orange-type strain generated chimeras. Within three days, many of the resulting individuals exhibited a dark pigment on several parts of their body, characteristic of the donor but not of the orange strain. At one year of age, four chimeras (one female and three males) were pair-mated with the orange type, and F(1) was obtained. One of the four F(0) chimeras produced progeny similar to the wild type at a frequency of 0.24% (2/841). The results suggest that this method is a promising technique for the preservation of endangered fish species.

Animals↗
PubMed2006

Premeiotic endomitosis produces diploid eggs in the natural clone loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae).

The natural clone loach produces unreduced eggs genetically identical to somatic cells of the mother fish and such diploid eggs normally develop as a clone without genetic contribution of sperm. Following the identification of clonal nature and diploidy of eggs, we conducted cytological studies to determine the mechanisms responsible for this unusual oogenesis. Cytolological observation of full-grown oocytes cultured in vitro revealed that oocytes of both the clone and the control loach underwent two successive meiotic divisions: formation of a bipolar spindle and metaphase in meiosis I and equal segregation of chromosomes, extrusion of the first polar body and the appearance of metaphase of meiosis II. However, spindle size of the clone was larger than that of the control. Bivalent chromosome number of germinal vesicle of oocytes was 25 in the control diploid, whereas 50 in the clone. The results suggest that chromosomes are duplicated by mitosis without cytokinesis before meiosis, i.e. premeiotic endomitosis and then oocytes differentiated from tetraploid oogonia undergo a quasinormal meiosis followed by two successive divisions to produce diploid eggs.

Animals↗
PubMed2007

Diploid sperm produced by artificially sex-reversed clone loaches.

Clone loaches reproduce unisexually in a wild population of Hokkaido Island, Japan. These clone loaches produce genetically identical unreduced eggs which develop to diploid individuals without any genetic contribution of sperm donors. In the present study, sex reversal of clone loaches was attempted and the reproductive potential of resultant clone males was examined. Clone loaches administered 0.5 ppm of 17-alpha methyltestosterone (MT) for 30 days from 1 month after hatching differentiated into physiological males. These sex-reversed clone males produced fertile spermatozoa with a diploid DNA content. Diploid spermatozoa had significantly larger heads than normal haploid sperm, but had a normal shape showing a head, mid-piece, and tail. The motility of diploid spermatozoa was low after ambient water was added. Concentration of diploid spermatozoa per unit of sperm was lower than that of control haploid spermatozoa. Microsatellite genotyping revealed that triploid progeny from the cross between a normal diploid female and a sex-reversed clone male had two alleles specific to the diploid clone male and one allele of the mother loach. These results indicated that the sex-reversed clone males produced fertile diploid spermatozoa genetically identical to the clone lineage.

Animals↗
PubMed2007

Cytological mechanisms of gynogenesis and sperm incorporation in unreduced diploid eggs of the clonal loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae).

The loach Misgurnus anguillicaudatus comprises diploid clonal, triploid and diploid-triploid mosaic individuals in a wild population on Hokkaido island, Japan. When diploid eggs of clonal loaches are fertilized by haploid sperm of normal bisexual loaches, both diploid clonal and non-diploid aclonal individuals occur in the progeny. Flow cytometry and microsatellite analyses revealed that the occurrence of triploid, diploid-triploid and other progeny was essentially due to the genetic incorporation of sperm to diploid clonal genomes of unreduced eggs. In this study, we examined the influence of water temperature from fertilization to early embryogenesis on frequencies of diploid clonal and other progeny and observed that progeny of three out of four clonal females examined exhibited approximately constant rates of diploid clonal individuals (54.2-68.9%) at hatching stage. Thus, no drastic increase of non-diploid progeny was detected. However, the 28 degrees C group of the fourth clonal female gave significantly lower rate (28.1%) of diploid clonal progeny, suggesting that this temperature might be a critical or a borderline temperature inducing sperm incorporation. We also examined the cytological process by which diploid clonal and other aclonal progeny develop after fertilization. In some fertilized eggs, the sperm nucleus remained condensed throughout fertilization and early embryogenesis and never fused with the female pronucleus. This cytological observation concludes that clonal eggs develop by the mechanism of gynogenesis. However, some other eggs showed the cytological process of syngamy between the female pronucleus and an accidentally formed male nucleus, suggesting the formation of triploid progeny. The syngamy between an accidentally activated sperm nucleus with a male pronucleus-like structure and nucleus of a blastomere of gynogenetically developing clonal diploid embryo might produce a diploid-triploid mosaic individual.

Animals↗
PubMed2004

Clonal diploid sperm of the diploid-triploid mosaic loach, Misgurnus anguillicaudatus (Teleostei:Cobitidae).

The loach Misgurnus anguillicaudatus comprises diploid, triploid and diploid-triploid mosaic individuals in a wild population of the Hokkaido island, Japan. Previous studies revealed the presence of a cryptic clonal lineage among diploid loaches, which is maintained by uniparental reproduction of genetically identical diploid eggs. In the present study, we analyzed distribution and genetic status of diploid and triploid cells in infrequent mosaic males. Flow cytometry, microsatellite genotyping and DNA fingerprinting verified that mosaic males consisted of diploid cells with genotypes identical to the natural clone and triploid cells with diploid genomes of the clonal lineage plus haploid genome from sperm nucleus of the father. Thus, the occurrence of diploid-triploid mosaicism might be caused by accidental fertilization of a diploid blastomere nucleus with haploid sperm after the initiation of clonal development of unreduced eggs. Such mosaic males produced fertile sperm with diploid DNA content. The experimental cross between normal diploid female and diploid-triploid mosaic male gave rise to the appearance of triploid progeny which exhibited two microsatellite alleles identical to the clonal genotype and one allele derived from the normal female. In DNA fingerprinting, such triploid progeny gave not only all the DNA fragments from the clone, but also other fragments from the normal female. Induced androgenesis using UV irradiated eggs and sperm of the mosaic male gave rise to the occurrence of diploid individuals with paternally derived microsatellite genotypes and DNA fingerprints, absolutely identical to the natural clonal lineage. These results conclude that the diploid-triploid mosaic male produced unreduced diploid sperm with genetically identical genotypes. The spermatogenesis in the clonal diploid cells under the mosaic condition suggests that triploid male somatic cells might transform genetically all-female germ cells to differentiate into functionally male gametes. The discovery of the mosaic male producing unreduced sperm suggests the theoretical occurrence of triploids and other polyploids by the syngamy of such paternally derived diploid gametes.

Animals↗
PubMed1994

Formation of new sensory cells in deafferented tuberous organs of the gymnotid fish Eigenmannia virescens.

The cutaneous electroreceptor "tuberous organs" of the lateral line system of Eigenmannia virescens were studied at light and electron microscopic levels with immunohistochemical and autoradiographic techniques after sectioning of the posterior branch of the lateral line nerve. After deafferentation total degeneration of the sensory cells was observed. The accessory cells of the basal platform, however, remain intact and undergo a process of differentiation. The cytoplasms and nuclei of these cells increase in volume, and the nuclei incorporate tritiated thymidine. In control tuberous organs with intact innervation, tritiated thymidine is absorbed by the nuclei of the elongated epidermal cells surrounding the sensory cavity. The newly formed sensory cells, but not those of the intact organs, are substance P immunoreactive. They have synaptic bars surrounded by vesicles and their free membrane surface is covered with microvilli. The new sensory cells are fully differentiated 35 days after the lateral line was cut. These results demonstrate that in the tuberous organs of E. virescens new sensory cells are formed in the absence of an innervation.

Afferent Pathways↗
PubMed2004

Proteomic approach to identify acute phase response-related proteins with low molecular weight in loach skin following injury.

Proteome analysis by two-dimensional gel electrophoresis (2-DE) together with mass spectrometry was applied to screen acute phase response (APR)-related proteins with low molecular weight in loach skin following injury. Furthermore, Western blotting and function tests were applied to confirm the results obtained from the proteomic study. Fifteen APR-related proteins with sixteen spots (PLA with two spots) on a 2-DE map were identified in this study. Furthermore, six were known acute phase proteins including galactose-binding lectin (GBL), lysozyme, C3, CD59, double PLA and 50s ribosomal protein; while ATP kinase, zinc finger protein 183, alpha-neurotoxin homology, angiostatin, serine/threonine kinase, metalloproteinase inhibitor, regulator of G-protein 4, cryptdin-9 and disintegrin trigranin were found by our lab to be APR-related proteins. In addition, our results suggest that proteomes with low molecular weight can be characterized by 2-DE with a Tris-tricine system followed by mass spectrometry.

Acute-Phase Proteins↗
PubMed2002

Impact of selenium and other trace elements on the endangered adult razorback sucker.

A study was conducted with endangered the razorback sucker (Xyrauchen texanus) to determine if environmental exposure to selenium in flooded bottomland sites affected survival, growth, and egg-hatching success. Adults were stocked at three sites adjacent to the Colorado River near Grand Junction, Colorado, in July 1996: hatchery ponds at Horsethief Canyon State Wildlife Area (referred to here as Horsethief; the reference site), a diked tertiary channel at Adobe Creek, and North Pond at Walter Walker State Wildlife Area (WWSWA). Fish were collected in April 1997 and spawned. After two spawnings adults from the three sites were held at Horsethief for an 86-day selenium depuration period. Selenium concentrations at Horsethief were 1.4-3.0 microg/L in water, 0.8-0.9 microg/g in sediment, 4.5 microg/g in muscle plug, and 6.0 microg/g in eggs; at Adobe Creek, <0.7-4.5 microg/L in water, 1.2-2.5 microg/g in sediment, 16-20 microg/g in zooplankton, 9.6 microg/g in muscle plug, and 40 microg/g in eggs; and at North Pond, 3.2-17 microg/L in water, 16-94 microg/g in sediment, 32-48 microg/g in zooplankton, 14 microg/g in muscle plug, and 55 microg/g in eggs. During the depuration period, when adults from Adobe Creek and North Pond were held at Horsethief, the fish lost 7%-13% of their selenium burden in 59 days and 14%-21% in 86 days. Larvae from North Pond adults had the most deformities, followed by Adobe Creek adults, with the fewest deformities found in the Horsethief adults.

Animals↗
PubMed1994

A novel sialidase capable of cleaving 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN).

We have examined the tissues of several species of fish and found that the liver of the loach (Misgurnus fossilis) contains a novel sialidase capable of efficiently hydrolyzing 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (KDN) from the 4-methylumbelliferyl alpha-ketoside of KDN, KDN alpha 2-->3Gal beta 1-->4GlcCer and KDN alpha 2-->6 N-acetylgalactosaminitol as well as Neu5Ac from the 4-methylumbelliferyl alpha-ketoside of Neu5Ac and GM3. The pH optimum for this enzyme was determined to be 4.6, and the Km using the 4-methylumbelliferyl alpha-ketoside of KDN and 4-methylumbelliferyl alpha-ketoside of Neu5Ac as substrates were 0.07 and 0.12 mM, respectively. The enzyme was stable in the pH range of 4 to 5 but very unstable above pH 6. This is the first report of a sialidase capable of efficiently cleaving glycosidically linked KDN.

Animals↗
PubMed1994

Impact of low-level sampling stress on interpretation of physiological responses of white sucker exposed to effluent from a bleached kraft pulp mill.

In this study the authors evaluated the effects of handling and confinement stress on a number of biochemical parameters in prespawning male white sucker collected from a bleached kraft mill effluent (BKME) exposed and a reference site. There were no effects of site or stress level on plasma cholesterol or glutamic oxalacetic transaminase activity; all other parameters were affected by either sampling stress or overnight confinement. Unstressed BKME fish reflected higher levels of plasma lactate, and lower levels of plasma testosterone, 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, and cortisol. Acute handling stress or overnight confinement created a site difference in plasma glucose, protein, and 11-ketotestosterone, reversed the site effect for plasma lactate, and eliminated site differences for 17 alpha,20 beta-dihydroxy-4-pregnen-3-one. Since it is very difficult to standardize capture and handling stress in field studies impacted by industrial effluents, biochemical differences must be interpreted carefully. This study has also demonstrated for the first time that BKME exposure resulted in reduced circulating levels of the stress steroid cortisol, similar to the decreases in reproductive sex steroids previously reported.

Animals↗