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1.
We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz’s L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.  相似文献   

2.
A novel cell line, Epinephelus moara kidney cell line (EMK), was established from kidneys of kelp grouper E. moara. Cells were cultured at 24 °C in Leibovitz’s L-15 medium (L15) supplemented with antibiotics, basic fibroblast growth factor (bFGF), foetal bovine serum (FBS) and 2-mercaptoethanol (2-ME). EMK cells, fibroblastic in morphology, proliferated to 100% confluency in 3–4 days and were subcultured for over 50 passages. The cells could grow from 18 to 30 °C, with optimal growth at 24 °C. Chromosome analysis indicated that the modal chromosome number was 48 in the cells at passage 42. Green fluorescent signals could be observed in EMK cells when the cells were transfected with pEGFP-N3 plasmid. Moreover, a significant cytopathic effect (CPE) was observed in the cells after infection with Singapore grouper iridovirus (SGIV) or nervous necrosis virus (NNV), and viral replication was confirmed by quantitative real-time PCR (qPCR). These results suggested the potential of the EMK cell line for studies of transgene and pathogenesis of SGIV and NNV.  相似文献   

3.
This study established and characterized a new cell line (MAF) from the fin of blunt snout bream (Megalobrama amblycephala), a freshwater fish cultivated in China. MAF cells proliferated well in medium 199 supplemented with 10 % fetal bovine serum at 28 °C and have been subcultured more than 95 times in almost a year. MAF cells were revived at 90–95 % viability after 3–6 months of storage in liquid nitrogen. Karyotyping indicated that the modal chromosome number of MAF cells was 48. The MAF cell line consisted predominantly of fibroblastic and epithelial-like cells from M. amblycephala, which was confirmed by immunofluorescence and mitochondrial 12s rRNA sequencing. Viral susceptibility tests showed that MAF cells were susceptible to infection by snakehead rhabdovirus, spring viremia carp virus, and channel catfish virus, which was demonstrated by the presence of cytopathic effect, high viral titers, and PCR products. Bacterial cytotoxicity studies showed that extracellular products from Aeromonas hydrophila were toxic to MAF cells. Cu2+ was also cytotoxic to MAF cells, and the 24-h IC50 value was 144.48 μmol/l. When MAF cells were transfected with pEGFP-N1 plasmid, bright fluorescent signals were observed, and the transfection efficiency reached up to 5 %. These results suggest that the MAF cell line may provide a valuable tool for studying virus pathogenesis, as well as cytotoxicity testing and genetic manipulation studies.  相似文献   

4.
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5.
Establishment and characterization of two cobia, Rachycentron canadum, cell lines derived from cobia brain (CB) and cobia fin (CF) are described. Caudal fin and brain from juvenile cobia were dissociated for 30 and 10 min, respectively, in phosphate‐buffered saline containing 0.25% trypsin at 25 °C. The optimal culture condition for both dissociated cells (primary cell culture) was at 28 °C in Leibovitz‐15 medium containing 10% foetal bovine serum. The cells have been sub‐cultured at a ratio of 1:2 for more than 160 passages over a period of 3 years. Origin of the cultured cells was verified by comparison of their sequences of mitochondrial cytochrome oxidase subunit I genes (cox I) with the cox 1 sequence from cobia muscle tissue. The cell lines showed polyploidy. No mycoplasma contamination was detected. Susceptibility to grouper iridovirus was observed for the CB cell line but not the CF cell line. Both cell lines expressed green fluorescent protein after being transfected with green fluorescent reporter gene driven by the cytomegalovirus promoter.  相似文献   

6.
A new cell line (TSHKC) derived from half-smooth tongue sole (Cynoglossus semilaevis) head kidney was developed. The cell line was subcultured for 40 passages over a period of 360?days. The cell line was optimally maintained in minimum essential medium supplemented with HEPES, antibiotics, fetal bovine serum, 2-Mercaptoethanol (2-Me), sodium pyruvate and basic fibroblast growth factor. The suitable growth temperature for TSHKC cells was 24?°C, and microscopically, TSHKC cells were composed of fibroblast-like cells. Chromosome analysis revealed that the TSHKC cell line had a normal diploid karyotype with 2n?=?42, contained the heterogametic W chromosome. The TSHKC cell line was found to be susceptible to lymphocystis disease virus. The fluorescent signals were observed in TSHKC when the cells were transfected with green fluorescent protein and red fluorescent protein reporter plasmids.  相似文献   

7.
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8.
The expression profiles of several mRNAs were characterized during follicular maturational competence (FMC) acquisition. Post-vitellogenic female rainbow trout (Oncorhynchus mykiss) were assayed in vitro for follicular maturational competence (FMC). Ovarian follicles were stimulated in vitro for 60 h at 12 °C with a range of concentrations of partially purified gonadotropin and the efficient concentration for 50% germinal vesicle breakdown was calculated and used as an indicator of follicular maturational competence. Before in vitro assay, ovarian tissue was sampled in order to quantify mRNA abundance in the ovarian follicle by real-time PCR. The mRNA expression of Luteinizing Hormone receptor (LH-r), Follicular Stimulating Hormone receptor (FSH-r), Insulin-like Growth Factor 1 (IGF1), Insulin-like Growth Factor 2 (IGF2), Insulin-like Growth Factor receptor 1a (IGF-r1a) and 20β-hydroxysteroid dehydrogenase (20β-HSD), that are putatively expressed in the preovulatory ovary, was thus studied in females of varying FMC.  相似文献   

9.
In this study, we investigated the effects of hyperthermia and recovery on cell viability, lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, malondialdehyde (MDA), total antioxidant capacity (T-AOC), and heat shock protein (HSP60, 70, and 90) mRNA expression in the hepatic cells of the grass carp, Ctenopharyngodon idellus. Triplicate groups of cultured cells were exposed to 30, 32, or 34 °C for 0.5 h and then immediately incubated at 27 °C in 5 % CO2 for 6, 12, 24, or 48 h. Hyperthermia stress greatly reduced cell viability and increased LDH release. Cell damage declined after recovery. Hyperthermia stress increased the lipid peroxide levels and reduced the antioxidant capacity (e.g., reduced SOD and T-AOC) of the cells. However, oxidative damage declined as the recovery period increased, and the levels of MDA, SOD, and T-AOC were restored. After cells were exposed to 32 °C, the expression of HSP60 after recovery for 1, 2, and 4 h (P < 0.05), the expression of HSP70 after recovery for 0.5 and 1 h (P < 0.01), and the expression of HSP90 throughout recovery were significantly higher (P < 0.01) than the prestress levels. During the recovery period, the variations in HSP gene expression reflected the transition period from a state of cellular growth to one of the cellular repairs. In conclusion, hyperthermia depresses cell viability, induces oxidative damage, and increases HSP expression, which plays an important role during hyperthermic stress in grass carp hepatic cells.  相似文献   

10.
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11.
The goldfish Carassius auratus, a freshwater fish in the family Cyprinidae, was one of the earliest fish to be domesticated for ornamental purposes. A cell line was established from goldfish heart (GH) tissue to create a biological monitoring tool for viral diseases. The GH cell line was optimally maintained at 25 °C in M199 medium supplemented with 10–20% fetal bovine serum. A chromosomal analysis indicated that the cell line remained diploid, with a mean chromosomal count of 100. In viral inoculation assays, significant cytopathic effects (CPEs) were caused by epizootic hematopoietic necrosis virus (EHNV), Andrias davidianus iridovirus (ADIV), and Bohle iridovirus (BIV) infections in the fish cells and the viral titers (average value) of EHNV, ADIV, and BIV in GH cells reached 105.0, 104.5, and 105.0 TCID50/0.1 mL, respectively, within 7 days. However, no CPE was observed in the cells infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), or grass carp reovirus (GCRV). These results suggest that the GH cell line is a valuable tool for studying viral pathogenesis.  相似文献   

12.
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13.
Bioaccumulation of mercury and histomorphological changes in the olfactory epithelium of Labeo rohita were investigated after exposing the fish to two sublethal concentrations of HgCl2 (66 and 132 μg/L) for 15 and 30 days. Mercury deposition increased in the tissue significantly (p < 0.05) with dose- and duration-dependent manner. Severe damage to the olfactory epithelium was evident. When fish exposed to 66 μg/L for 15 days, the histology of olfactory epithelium exhibited that mucous cell proliferation was upregulated and cell size was significantly increased from the control. Similar trends were found in 30 days exposure in both treated groups. Histology showed that mercury induced degeneration of columnar sensory cells, supporting cells and ciliated non-sensory cells and induced basal cell proliferation. Basal cell hyperplasia led to form intraepithelial proliferative lesion, thickening of epithelium, basal lamina disruption and cyst formation. Scanning electron microscopy revealed that mercury exposure at 66 μg/L caused clumping and loss of cilia, erosion in microridges on the supporting cells and proliferation of mucous cell opening. Complete degeneration of ciliated cells and cyst formation was observed in the fish when exposed to 132 μg/L HgCl2. This result suggests that prolonged exposure to mercury might cause irreversible damage to the olfactory epithelium and impair the olfactory function of fish.  相似文献   

14.
A continuous cell line has been developed from thymus explants of Catla catla and the cells have been subcultured for 63 passages. The cells exhibited optimum growth at 30°C in L‐15 medium containing 15% foetal bovine serum. The cultured cells engulfed yeast cells and fluorescent latex beads. These cells produced reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharide and phorbol esters. The culture supernatant from the cultured cells had lysozyme activity and these cells demonstrated Fc receptors. Almost all the cells were positive for alpha‐naphthyl acetate esterase enzyme suggesting that the cells are of macrophage lineage and therefore, the cell line was designated as catla thymus macrophage (CTM) cell line. CTM cells formed aggregates around zoospores of Aphanomyces invadans, but were unable to inhibit the germination of spores. The karyotype analysis of CTM cells at 25th passage revealed a typical diploid model with 50 chromosomes per cell. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and cytochrome c oxidase subunit 1 confirmed that the CTM cell line originated from C. catla. This cell line should be useful for studying the role of macrophages in differentiation and maturation of thymocytes and can be a source of macrophage‐specific enzymes and cytokines.  相似文献   

15.
A new marine fish cell line, derived from the heart of giant grouper, Epinephelus lanceolatus (Bloch), was established and characterized. The cell line was designated as ELGH and subcultured with more than 60 passages. The ELGH cells were mainly composed of fibroblast-like cells and multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum (FBS) at 28 °C. Chromosome analysis indicated that the modal chromosome number was 48. The fluorescent signals were detected in ELGH when transfected with green fluorescent protein reporter plasmids. The 50% cytotoxic concentration (CC50) of the extracellular products (ECPs) from Streptococcus iniae and Vibrio alginolyticus E333 on ELGH cells was 60.02 and 12.49 μg mL−1, respectively. Moreover, the ELGH cells showed susceptibility to Singapore grouper iridovirus (SGIV), but not to soft-shelled turtle iridovirus (STIV), red-spotted grouper nervous necrosis virus (RGNNV) and spring viremia of carp virus (SVCV), which was demonstrated by the presence of a severe cytopathic effect (CPE) and increased viral titres. In addition, electron microscopy observation showed that abundant virus particles were present in the infected cells. Taken together, our data above provided the potential utility of ELGH cells for transgenic and genetic manipulation, as well as cytotoxicity testing and virus pathogenesis.  相似文献   

16.
The establishment and partial characterization of a continuous cell line from the dorsal fin of red sea bream, Pagrus major, are described. The cell line, designated RSBF‐2, has been subcultured for more than 100 passages since its initiation in November 2000. It was optimally maintained at 28 °C in Leibovitz L‐15 medium with 10% foetal bovine serum. Propagation of RSBF‐2 cells was serum dependent and exhibited low plating efficiency (<1.7%). Aside from long‐term cryopreservation, the cells could also be kept at 4 °C for 72 days. The distribution of the chromosome number was 38–98 with a mode of 48. The RSBF‐2 cell line was susceptible to red sea bream iridovirus but only produced a few rounded and refractory cells. Virus‐inoculated RSBF‐2 cells were then subcultured to generate a persistently infected cell line. RSBF‐2 was also very sensitive to the extracellular products of Photobacterium damselae ssp. piscicida and produced significant fluorescent signals after transfection with pEGFP‐C3. Analysis of mitochondrial cytochrome b gene sequences revealed 99% identity between the cell line and Pagrus major.  相似文献   

17.
High-mobility group box 2 (HMGB2) is a nonhistone architectural protein that plays important roles in many biological processes. In this study, we cloned a homologue of the HMGB2 from the lymphocyte-like cells of Lampetra japonica (L. japonica). Sequence analysis reveals that L. japonica HMGB2 contains two highly conserved motifs and shares more than 70 % identity with the homologues from other vertebrate species. Subsequently, Lj-HMGB2 was subcloned into the pET-28a(+) and pIRES2 AcGFP1-Nuc vector and expressed in Rosetta blue (DE3) and Hela cell lines, respectively. The recombinant L. japonica HMGB2 (rLj-HMGB2) with apparent molecular mass of 22 kDa was further purified by His-Bind affinity chromatography. Real-time quantitative PCR indicates that the expression level of Lj-HMGB2 was particularly up-regulated in intestines after challenged with lipopolysaccharide, while up-regulated in lymphocyte-like cells and heart after challenged with concanavalin A in vivo. In addition, rLj-HMGB2 could induce the generation of proinflammatory mediators in the activated human acute monocytic leukemia cell line (THP1), which suggested that Lj-HMGB2 may participate in the immune response of the lampreys.  相似文献   

18.
In India, many of the fish farmers stock 1-year-old stunted fishes (stunted yearlings) of Indian major carps (IMC) for enhancing fish production through compensatory growth, but many of them observed problems of early maturation in these fishes. Application of aromatase inhibitors for deceleration of ovarian maturation is one of the probable solutions to mitigate this issue. In the present study, a synthetic aromatase inhibitor letrozole [25 (L25) and 50 (L50) mg kg?1 feed] and a plant-derived aromatase inhibitor, grape seed extract [100 (G100) and 200 (G200) mg kg?1 feed], were fed to stunted yearlings of rohu (Labeo rohita) for 45 days well before the onset of breeding season. Maturation indices such as gonadosomatic index (GSI) and serum oestradiol (E) levels indicated a dose-dependent inhibition of ovarian development in the aromatase-inhibitor-treated fish. Higher dose of letrozole (GSI, 15.12 ± 0.18; E, 3.19 ± 0.42) and grape seed extract (GSI, 16.90 ± 0.40; E, 3.60 ± 0.75) were found to be more effective since control fish showed further advancement in maturation (GSI, 21.20 ± 1.10; E, 7.33 ± 0.74) during the peak breeding season (15th June). Histological observations also confirmed the results revealing a delayed initiation of ovarian development in the case of higher doses of letrozole and grape seed extracts. These results indicate the possible use of aromatase inhibitors in arresting the early maturation process in IMC.  相似文献   

19.
Plasma sex steroid hormonal profile and gonad histology were correlated to study the annual reproductive cycle of Hemibagrus nemurus. Hormones were measured by Enzyme Linked Immunosorbent Assay. Gonad tissues were observed by using light microscopy. The highest testosterone (T) value for male was observed in November and that of female was in October. 11-ketotestosterone (11-KT) and 17β-estradiol (E2) levels were highest in June and November, respectively. Hormonal profiles of T, 11-KT and E2 showed several peaks which indicated a non-seasonal pattern. There were significant differences (p < 0.05) in the monthly levels of T, 11-KT and E2. Gonadosomatic index of H. nemurus ranged from 1.14 ± 0.02 % to 7.06 ± 1.40 %, and high gonadosomatic indices were recorded in May, August and November. Gonad histology revealed that spermatozoa were always present in the testes which implied continuous spermatogenesis and asynchronous ovarian development pattern was observed in the ovaries. The annual reproductive cycle of H. nemurus did not show a seasonal pattern and this indicate that H. nemurus is a non-seasonal breeder with several spawning cycles and can be referred to as indeterminate batch spawner. The major significances of this study are annual sex steroid hormonal profile and asynchronous ovarian development of H. nemurus. This information will contribute to our knowledge of reproductive biology of H. nemurus.  相似文献   

20.
In this study, the effects of dietary tryptophan (a precursor of 5-hydroxytryptamine, 5-HT, serotonin) on the agonistic behavior, growth, and survival of freshwater crayfish were investigated. For this aim, a control diet (D1) and three experimental diets (D2, D3, and D4) were prepared. D1 contained no additional tryptophan (TRP), but D2, D3, and D4 diets were supplemented with TRP at 0.50, 0.75, and 1.00 % in dry diet, respectively. The control contained 0.33 % TRP. Results revealed that higher supplemental dietary TRP levels caused a significant decrease in the aggressive behavior (P < 0.05), but an increase in the calmness of crayfish. In addition, the results showed that 5-HT levels in the hemolymph before the fight (after 15 days of feeding; resting) were significantly different (P < 0.05) between D1 and D4. There was a significant difference (P < 0.05) in the 5-HT level of hemolymph between the crayfish fed control and D4 after 15 days of feeding. However, after the fight, 5-HT concentration was significantly higher (P < 0.05) in TRP-supplemented A. leptodactylus as compared with the control. The findings of this study showed that supplemental dietary TRP caused a significant increase (P < 0.05) in the growth and survival rate of A. leptodactylus. The crayfish fed D4 had the best survival rate at the end of the experiment (91.60 % in August). The findings of this study also showed that difference in survival rate between the crayfish fed control and D2 in June, July, and August was not significant (P > 0.05). In addition to these, supplemental dietary TRP gave rise to significantly higher specific growth rate (SGR) values in June and July (P < 0.05). For example, in June, it was 0.38 for the crayfish fed D4, 0.28 for the crayfish fed D3, 0.18 for the crayfish fed D2, and 0.13 for the crayfish fed control. However, in August, the highest SGR (0.56) was obtained from the crayfish fed control. The results also showed that the SGRs of females were lower than the males in June, July, and August (P < 0.05). In conclusion, this research shows that the aggressive behavior of A. leptodactylus can be suppressed, and rearing efficiency (i.e., increased growth and high survival rate) of this species can be improved by increasing TRP levels to 1.00 % in its diet.  相似文献   

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