Numbers of muscle nuclei and myosatellite cell nuclei in red and white axial muscle during growth of the carp (Cyprinus carpio)

We determined the percentages of muscle fibie nuclei and satellite nuclei over a growth range of carp ( Cyprinus carpio ), as the increase in the number of muscle fibre nuclei is an important aspect of the increase in muscle mass, and myosatellite cells are believed to be the source of new muscle fibre nuclei. In white as well as in red axial muscle the percentage of the nuclei present in muscle that are muscle nuclei (muscle fibre nuclei+myosatellite nuclei) remained constant during growth (54 and 32% respectively). The difference in the percentage of non-muscle nuclei between white and red axial muscle is mainly caused by the higher content of endothelial nuclei in red axial muscle.
In white axial muscle the DNA/protein ratio (nucleus/sarcoplasm ratio) decreased between 3 and 15 cm S.l. In red axial muscle we found a continuous decrease in DNA/protein ratio over the entire investigated size range (3–50 cm s.l.). This may be related to a longer occurrence of hyperplasia in red than in white axial muscle.
In both fibre types the percentage of muscle nuclei being myosatellite nuclei decreased with increasing length, In white axial muscle it decreased from about 5% in carp of 5 cm s.l. to less than 1% in carp of 20 cm S.L.; for red muscle these values were 11 and 3% respectively.
For white axial muscle we calculated that, especially in larger fish, the myosatellite ceils alone cannot account for the increase in the number of muscle fibre nuclei during growth. The percentage of proliferating nuclei in muscle tissue, measured by the uptake of 5-bromo-2'-deoxy-uridine, is high enough to account for the total increase in nuclei. So indirect evidence is available that another cell type present in the muscle tissue may also be involved in the formation of additional muscle fibre nuclei.     

Influence of fish size on proliferation and differentiation of cultured myosatellite cells of white axial muscle of carp (Cyprinus carpio L.)

Abstract. The in vitro proliferation [uptake of 5-bromo-2'-deoxyuridine (BrdU)] and the degree of differentiation (presence of desmin) of myosatellite cells isolated from white axial muscle of carp between 3 cm and 27 cm standard length (SL) were examined 17 h after isolation. The fraction of the myosatellite cells that were both desmin positive and BrdU positive never exceeded 2% of the total number of isolated myosatellite cells, irrespective of the standard length of the donor(s). This indicates that, for carp, the temporal relationship between replication and desmin expression of myosatellite cells is different from that described for myogenic cells of mammals and birds. The percentage of BrdU positive myosatellite cells was significantly correlated with standard length: it increased from 10% for carp of about 5 cm SL to 40–50% for carp between 20 cm and 27 cm SL. The percentage of desmin positive myosatellite cells was about 50–60%; it was not significantly correlated with standard length. The percentage of myosatellite cells that were both BrdU negative and desmin negative showed a stepwise difference in this percentage with increasing length. Fish smaller than 10 cm SL, had more of these cells (10–40%), than larger fish (which had 0–12%). So, apparently the composition of the myosatellite cell population changes during growth. The low percentage of proliferating cells, and the relatively high percentage of differentiated (desmin positive) myosatellite cells obtained from 3–6 cm large carp, suggests that, in these small fish, muscle growth strongly depends on the use of a pool of myogenic cells that has been formed at an earlier stage of their development.     

Growth of carp (Cyprinus carpio) white axial muscle; hyperplasia and hypertrophy in relation to the myonucleus/sarcoplasm ratio and the occurrence of different subclasses of myogenic cells

In white axial muscle of carp addition of new fibres to the muscle mass (hyperplasia) decreased with increasing length of the fish. This was deducted from the decrease in the amount of small fibres. In carp larger than about 40 cm standard length (s.l.) hyperplasia no longer occurred (small fibres were absent) and muscle growth only occurred by means of hypertrophy (growth of existing fibres). The stage of growth in which many new fibres were added showed a relatively fast increase in trunk weight, as calculated from growth curves. During the stage of fast growth with a high occurrence of hyperplasia, the DNA/protein ratio decreased. The high percentage of postmitotic myosatellite cells isolated from carp of 5 cm s.l. suggests that in hyperplasia a subpopulation of already differentiated myosatellite cells formed in an earlier stage of development is incorporated in new muscle fibres. The increase of the relative importance of hypertrophy appears to be correlated to an increase in the percentage of proliferating myosatellite cells 17 h after isolation in vitro . This suggests that in hyperplasia and in hypertrophy different subpopulations of myosatellite cells are involved.     

Uptake of tritiated thymidine in muscle of juvenile carp

In juvenile carp (4.5–6 cm s.l .) labelled myosatellite cell nuclei are found 24 h after injection of tritiated thymidine, but labelled myonuclei after 48 h, indicating that fish myonuclei do not proliferate but originate from undifferentiated myogenic cells. The time pattern accords with the estimated cell-cycle time of adult myogenic cells of carp.     

Myosatellite cells of Cyprinus carpio (Teleostei) in vitro: isolation,recognition and differentiation

Summary We have developed a method for the dissociation and purification of myosatellite cells from white epaxial muscle of carp. The dissociated myosatellite cells were identified by their morphology, their ultrastructure, the formation of multinucleated myotubes containing myofibrils and the immunocytochemical demonstration of desmin. Desmin and 5-bromo-2-deoxyuridine (BrdU) were used to identify terminally differentiated and proliferating myosatellite cells, respectively. The in vitro behavior of myosatellite cells dissociated from carp of 5 cm standard length differed from that described for myosatellite cells of mammals and birds. No substantial proliferation of the myosatellite cells could be observed. Most cells were differentiated (desmin-positive, BrdU-negative) 17 h after plating, regardless of the medium used. This indicates that the investigated white epaxial muscle of carp of 5 cm standard length contains subpopulations of myosatellite cells, arrested at various stages of differentiation.     

Transient neonatal denervation alters the proliferative capacity of myosatellite cells in dystrophic (129ReJdy/dy) muscle.

It has been previously shown that transiently denervated, neonatal dystrophic muscle fails to undergo the degeneration-regeneration cycle characteristic of murine dystrophy (Moschella and Ontell, 1987). Thus, the myosatellite cells (myogenic stem cells) in these muscles have been spared the mitotic challenge to which dystrophic myosatellite cells are normally subjected early in the time course of the disease. By in vitro evaluation of the proliferative capacity of myosatellite cells derived from extensor digitorum longus (EDL) muscles of 100-day-old genetically normal (+/+) and genetically dystrophic [dy/dy (129ReJdy/dy)] mice and from muscles of age-matched mice that had been neonatally denervated (by sciaticotomy) and allowed to reinnervate, it has been possible to directly determine whether the cessation of spontaneous regeneration in older dy/dy muscles in vivo, is due to an innate defect in the proliferative capacity of the myosatellite cells or exhaustion of the myosatellite cells' mitotic activity during the regenerative phase of the disease. This study demonstrates that transient neonatal denervation of dystrophic muscle (Den.dy/dy) increases the number of muscle colony-forming cells (MCFs) per milligram of wet weight muscle tissue, increases the plating efficiency, and significantly increases the in vitro mitotic activity of dystrophic myosatellite cells toward normal values. The increased mitotic capability of myosatellite cells derived from Den.dy/dy muscle as compared to unoperated dy/dy muscle suggests that there is no innate defect in the proliferative capacity of the myosatellite cells of dy/dy muscles and that the cessation of spontaneous regeneration in the dy/dy muscles is related to the exhaustion of their myosatellite cells' mitotic capability.     

Transient neonatal denervation alters the proliferative capacity of myosatellite cells in dystrophic (129ReJdy/dy) muscle

It has been previously shown that transiently denervated, neonatal dystrophic muscle fails to undergo the degeneration–regeneration cycle characteristic of murine dystrophy (Moschella and Ontell, 1987). Thus, the myosatellite cells (myogenic stem cells) in these muscles have been spared the mitotic challenge to which dystrophic myosatellite cells are normally subjected early in the time course of the disease. By in vitro evaluation of the proliferative capacity of myosatellite cells derived from extensor digitorum longus (EDL) muscles of 100-day-old genetically normal (+/+) and genetically dystrophic [dy/dy (129ReJ^dy/dy)] mice and from muscles of age-matched mice that had been neonatally denervated (by sciaticotomy) and allowed to reinnervate, it has been possible to directly determine whether the cessation of spontaneous regeneration in older dy/dy muscles in vivo, is due to an innate defect in the proliferative capacity of the myosatellite cells or exhaustion of the myosatellite cells' mitotic activity during the regenerative phase of the disease. This study demonstrates that transient neonatal denervation of dystrophic muscle (Den.dy/dy) increases the number of muscle colony-forming cells (MCFs) permilligram of wet weight muscle tissue, increases the plating efficiency, and significantly increases the in vitro mitotic activity of dystrophic myosatellite cells toward normal values. The increased mitotic capability of myosatellite cells derived from Den.dy/dy muscle as compared to unoperated dy/dy muscle suggests that there is no innate defect in the proliferative capacity of the myosatellite cells of dy/dy muscles and that the cessation of spontaneous regeneration in the dy/dy muscles is related to the exhaustion of their myosatellite cells' mitotic capability. © 1992 John Wiley & Sons, Inc.     

蛋白磷酸酶-2Ac在不同倍性鱼6种组织中的分化表达模式

蛋白磷酸酶-2A是最重要的丝氨酸/苏氨酸蛋白磷酸酶之一,对于调控多细胞的生命活动起着非常重要的作用.以异源四倍体鲫鲤及其二倍体父/母本(湘江野鲤/红鲫)和子代三倍体湘云鲫等为实验材料,运用Westernblot技术及荧光免疫组织化学技术等实验手段,得到了Protein PJhosphatase-2A(PP2A)的催化亚基在上述不同倍性鱼体内6种不同组织的表达模式:Protein Phosphatase-2Ac(PP2Ac)在异源四倍体鲫鲤及其二倍体父/母本及子代三倍体湘云鲫不同组织中蛋白水平均有表达,而且出现了明显的种属特异性和组织特异性,如在大脑、肌肉、肝脏三组织中,三倍体湘云鲫中PP2Ae的表达相对最高.而在肾脏组织中,PP2Ac在异源四倍体鲫鲤中的表达水平最高,父本与三倍体湘云鲫中的表达比较相近,且最低;而在性腺组织中则是父本精巢中的表达最高;在心脏组织中,PP2Ae在母本红鲫中的表达相对较高.这种明显的种属之间组织特异性可能说明了子代与父母本之间的变异性.荧光免疫组化实验结果显示,从整体水平来看,4种不同鱼的同一组织中,PP2Ac的相对定位是非常相似的,这可能说明了异源四倍体鲫鲤与其二倍体父/母本及子代三倍体湘云鲫之间的遗传相似性.研究结果为进一步探索PP2Ac在脊椎动物不同组织中的功能提供了实验依据.     

Insulin-like growth factor as a novel stimulator for somatolactin secretion and synthesis in carp pituitary cells via activation of MAPK cascades

Somatolactin (SL), a member of the growth hormone/prolactin family, is a pituitary hormone unique to fish models. Although SL is known to have diverse functions in fish, the mechanisms regulating its secretion and synthesis have not been fully characterized. Using grass carp pituitary cells as a model, here we examined the role of insulin-like growth factor (IGF) in SL regulation at the pituitary level. As a first step, the antisera for the two SL isoforms expressed in the carp pituitary, SLα and SLβ, were produced, and their specificity was confirmed by antiserum preabsorption and immunohistochemical staining in the carp pituitary. Western blot using these antisera revealed that grass carp SLα and SLβ could be N-linked glycosylated and their basal secretion and cell content in carp pituitary cells could be elevated by IGF-I and -II treatment. These stimulatory effects occurred with parallel rises in SLα and SLβ mRNA levels, and these SL gene expression responses were not mimicked by insulin but blocked by IGF-I receptor inactivation. In carp pituitary cells, IGF-I and -II could induce rapid phosphorylation of IGF-I receptor, MEK1/2, ERK1/2, MKK3/6, and p38 MAPK; and SLα and SLβ secretion, protein production, and mRNA expression caused by IGF-I and -II stimulation were negated by inactivating MEK1/2 and p38 MAPK. Parallel inhibition of PI3K and Akt, however, were not effective in these regards. These results, taken together, provide evidence that IGF can upregulate SL secretion and synthesis at the pituitary level via stimulation of MAPK- but not PI3K/Akt-dependent pathways.     

Grass carp somatolactin: I. Evidence for PACAP induction of somatolactin-alpha and -beta gene expression via activation of pituitary PAC-I receptors

Somatolactin (SL), the latest member of the growth hormone/prolactin family, is a novel pituitary hormone with diverse functions. At present, SL can be identified only in fish but not in tetrapods and its regulation at the pituitary level has not been fully characterized. Using grass carp as a model, we examined the direct effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on SL secretion and synthesis at the pituitary cell level. As a first step, the structural identity of grass carp SL, SLalpha and SLbeta, was established by 5'/3'-rapid amplification of cDNA ends. These two SL isoforms are single-copy genes and are expressed in two separate populations of pituitary cells located in the pars intermedia. In the carp pituitary, PACAP nerve fibers were detected in the nerve tracts of the neurohypophysis and extended into the vicinity of pituitary cells forming the pars intermedia. In primary cultures of grass carp pituitary cells, PACAP was effective in stimulating SL release, cellular SL content, and total SL production. The increase in SL production also occurred with parallel rises in SLalpha and SLbeta mRNA levels. With the use of a combination of molecular and pharmacological approaches, PACAP-induced SL release and SL gene expression were shown to be mediated by pituitary PAC-I receptors. These findings, as a whole, suggest that PACAP may serve as a hypophysiotropic factor in fish stimulating SL secretion and synthesis at the pituitary level. Apparently, PACAP-induced SL production is mediated by upregulation of SLalpha and SLbeta gene expression through activation of PAC-I receptors.     

Effects of thermal acclimation on the relaxation system of crucian carp white myotomal muscle.

Many cyprinid fish are able to compensate for a decrease in ambient temperature by process of physiological adaptation in the function of muscles. In the winter habitat of crucian carp (Carassius carassius L.), low temperature is associated with simultaneous oxygen shortage. Because of the oxygen deprivation, there is probably little space for compensatory adaptation because positive thermal compensation would increase energy demand and accelerate depletion of glycogen reserves. Thus, we assumed that the crucian carp, unlike many other cyprinid fish, would not show positive thermal compensation but either no compensation or inverse compensation in muscle function. To test this hypothesis in the relaxation system of skeletal muscles, we determined the parvalbumin content and the activity of sarcoplasmic reticular (SR) Ca-ATPase in white myotomal muscle of winter- and summer-acclimated crucian carp. In the laboratory, the winter fish were kept at 2 degrees C and the summer fish at 22 degrees C for a minimum of 3 weeks before the experiments. The specific activity of SR Ca-ATPase at low experimental temperature (2 degrees C) was similar in summer- and winter-acclimated fish (0.26 +/- 0.04 vs. 0.25 +/- 0.04 mM/mg/min; P > 0.05). Because of the bigger Q(10) of cold-acclimated carp, the enzyme activity at 30 degrees C was higher in cold-acclimated winter fish than in warm-acclimated summer fish (7.42 +/- 0.90 vs. 5.18 +/- 0.53 mM/mg/min; P < 0.05). In contrast, the yield of SR protein was 70% higher in summer than winter fish (0.315 +/- 0.045 vs. 0.187 +/- 0.017 mg/g; P < 0.001). Because of these opposing changes, total Ca-ATPase activity of SR (per gram muscle weight) remained relatively constant. Similarly, the parvalbumin content of the myotomal muscle was not different between summer (4.09 +/- 0.95 mg/g) and winter (3.70 +/- 0.60 mg/g) fish. Although there were no seasonal changes in the total relaxing system of the crucian carp white myotomal muscle, the same activity of SR Ca-ATPase in winter fish was obtained with less amount of SR pump protein, owing to the increased catalytic activity of the enzyme. The higher catalytic activity of winter fish SR Ca-ATPase might be caused by differences in fatty acid composition noted in membrane lipids; i.e., fewer saturated fatty acids and more n-6 polyunsaturated fatty acids (PUFAs), at the expense of n-3 PUFAs, were present in the SR of cold-acclimated winter fish. Temperature-induced changes in enzyme protein, however, cannot be excluded. Thus, the present results indicate the absence of positive thermal compensation in the relaxing system of crucian carp white muscle. It seems, however, that lipid composition of SR membranes and temperature dependence of SR Ca-ATPase are altered by seasonal acclimation.     

Cytoplasmic impact on cross-genus cloned fish derived from transgenic common carp (Cyprinus carpio) nuclei and goldfish (Carassius auratus) enucleated eggs

In previous studies of nuclear transplantation, most cloned animals were obtained by intraspecies nuclear transfer and are phenotypically identical to their nuclear donors; furthermore, there was no further report on successful fish cloning since the report of cloned zebrafish. Here we report the production of seven cross-genus cloned fish by transferring nuclei from transgenic common carp into enucleated eggs of goldfish. Nuclear genomes of the cloned fish were exclusively derived from the nuclear donor species, common carp, whereas the mitochondrial DNA from the donor carp gradually disappeared during the development of nuclear transfer (NT) embryos. The somite development process and somite number of nuclear transplants were consistent with the recipient species, goldfish, rather than the nuclear donor species, common carp. This resulted in a long-lasting effect on the vertebral numbers of the cloned fish, which belonged to the range of goldfish. These demonstrate that fish egg cytoplasm not only can support the development driven by transplanted nuclei from a distantly related species at the genus scale but also can modulate development of the nuclear transplants.     

A quantitative study of testicular germ cell populations in masculinized neomale common carp (Cyprinus carpio L.)

The main objective of the present study was to investigate the effects of 17 alpha-methyltestosterone treatment upon the testicular germ cells of gynogenetic masculinized neomale common carp (Cyprinus carpio L.) in comparison with diploid carp. Gynogenetic common carp progeny (mean body weight, BW, 2.6+/-0.3g; mean total length, 10.4+/-0.5 cm) were treated for a period of 40 days with 17 alpha-methyltestosterone (MT) at a dose of 100mg kg(-1). The oral administration of MT resulted in 61.5-100% of fish exhibiting male gonads. The masculinized neomales exhibited reduced (P<0.05) body weight (BW=22.9+/-0.8) but significantly increased (P<0.05) mean testis weight (2.1+/-0.3) and mean gonadosomatic index (GSI=9.5+/-0.2%) in comparison with fish not treated with MT (BW 54.8+/-1.3; GSI=0.61%). Furthermore, treatment with MT also resulted in an increased number of fish exhibiting abnormal gonads. However, neomales did not exhibit abnormalities in the development of sperm ducts. MT treatment significantly increased germ cell volume, nuclear diameter, nuclear volume and cyst volume (P<0.01 in all cases) in MT-treated fish compared to untreated fish. The area occupied by seminiferous tubules, the number of Sertoli cells and germ cells per cyst, and the number of Leydig cells were significantly (P<0.05) greater in fish treated with MT. The carp neomales exhibited approximately 20-60% more Sertoli cells per cyst (P<0.05). Leydig cell nuclear volume and Leydig cell individual volume were significantly reduced in MT-treated groups (P<0.05) compared with untreated groups. In conclusion, our study strongly suggests that the abnormal gonadal structure evident in masculinized neomales could be explained by a combination of MT-induced genetic (homozygosity) and anabolic effects (upon germ and somatic cells).     

Spontaneous regeneration of older dystrophic muscle does not reflect its regenerative capacity

Young dystrophic (dy) murine muscle is capable of "spontaneous" regeneration (i.e., regeneration in the absence of external trauma); however, by the time the mice are 8 weeks old, this regeneration ceases. It has been suggested that the cessation of regeneration in dystrophic muscle may be due to exhaustion of the mitotic capability of myosatellite cells during the early stages of the disease. To test this hypothesis, orthotopic transplantation of bupivacaine treated, whole extensor digitorum longus muscles has been performed on 14 to 16-week-old 129 ReJ/++ and 129 ReJ/dydy mice. The grafted dystrophic muscle is able to produce and maintain for 100 days post-transplantation 356 +/- 22 myofibers, a number similar to that found in age-matched dystrophic muscle. The ability of old dystrophic muscle to regenerate subsequent to extreme trauma indicates that the cessation of "spontaneous" regeneration is due to factor(s) other than the exhaustion of mitotic capability of myosatellite cells. Moreover, there is no significant difference in myosatellite cell frequencies between grafted normal and dystrophic muscles (100 days post-transplantation). Myosatellite cell frequencies in grafted muscles are similar to those in age-matched, untraumatized muscles. While grafting of young dystrophic muscle modifies the phenotypic expression of histopathological changes usually associated with murine dystrophy, grafts of older dystrophic muscle show extensive connective-tissue infiltration and significantly fewer myofibers than do grafts of age-matched normal muscle. As early as 14 days post-transplantation, it is possible to distinguish between grafts of old, normal and dystrophic muscles. It is suggested that the connective tissue stroma, present in the dystrophic muscle at the time of transplantation, may survive the grafting procedure.     

Effect of incorporating probiotics into the diet of matrinxã (Brycon amazonicus) breeders

The aim of the present study was to evaluate the influence of probiotics prepared from Bacillus subtilis (strain C‐3102‐Calsporin^®; Calpis, Tokyo, Japan) as a dietary supplement on several reproductive aspects of female ‘matrinxã’(Brycon amazonicus), including hematological parameters, immunological characteristics and total lipid concentrations in muscle and liver tissue. The broodstock were kept in two ponds of 600 m², at a density of 50 fish per pond, from March to November 2008. The fish were divided into two treatment groups: untreated control fish (T1), and fish fed with 10 g of probiotics per kg of ration (T2). Fish were fed a diet containing 32% crude protein (CP), 7% crude fat (CF), and 6.5% ether extract (EE) twice daily at a rate of 1% of the total biomass per day during the coldest months (May through August) and 3% during the warm season (March, April and September through November). During the reproductive period, 20 individuals from each treatment group were selected for the experiments; two doses of crude carp pituitary extract were given to females in 10‐h intervals, and one dose was given to the males at the same time the females were given the second dose. A total of 20 fish were used for the hematological analyses. After anesthetization, blood samples were withdrawn by caudal puncture to determine the total number of cells, differential and total leukocyte counts, thrombocyte count, hematocrit, hemoglobin rate and plasma cortisol and glucose levels. The hematimetric index mean corpuscular volume (VCM) and mean corpuscular hemoglobin concentration (MHCM) were calculated. Ten other ‘matrinxãs’ from the same groups were used to assess macrophage phagocytic activity and lipid levels. The results showed that females fed a probiotics‐supplemented diet exhibited an increase in numbers of oocytes and, consequently, had higher rates of fertilization and hatching of larvae. Probiotic‐treated fish also exhibited a significant increase in the phagocytic activity of macrophages, indicating an improvement in the immune system of breeders. Hematologic parameters were different in comparison to the time‐zero group, and plasma cortisol and glucose levels were higher in the females. The total lipids levels in muscle and liver tissue were lower in B. amazonicus that received feed supplemented with probiotics.     

萍乡肉红鲫的性腺发育研究

萍乡肉红鲫(Pingxiang red-transparent crucian carp,Carassius auratus L.)是在江西省萍乡地区分布的天然三倍体鲫突变体经人工选育后获得的遗传性状基本稳定的后代,具有两性生殖和雌核生殖两种生殖方式.研究以F5代萍乡肉红鲫为材料,自孵化后每满1个月开始取性腺,观察了其卵巢1周年性成熟和精巢的发育过程,结果表明萍乡肉红鲫的性腺为1年成熟类型.卵巢发育进程町以分为6个时期,卵母细胞发育相应可分为6个时相.统计了卵巢成熟系数周年变化,体重为95 g左右的雌性萍乡肉红鲫,其成熟卵巢的成熟系数约为(11.73±2.8)%,成熟的卵母细胞内充满卵黄,相对怀卵量为(3018±310)粒/g.萍乡肉红鲫精巢属于小叶型,在精小叶中可观察到不同发育阶段的生殖细胞.由精原细胞分裂而来的仞级精母细胞经分裂增殖,产生次级精母细胞并最终发育成为精子.萍乡肉红鲫的精巢发育程序与普通鲫鱼和鲤鱼相似,卵巢和精巢的发育过程基本同步,孵化后50日龄内性腺分化不明显,到70日龄左右开始出现雌雄分化,3月龄发育为第1期,4-5月龄发育为第2期,6-7月龄发育至第3期,7-10月龄可见第4期卵巢,1年即可成熟产卵,精巢可排出精液.结果表明,该鲫鱼突变体的性腺发育与普通二倍体鲤(鲫)鱼的性腺发育方式类似.     

Frequency analyses of active NORs in nuclei of artificially induced triploid fishes

Summary The correspondence between increased numbers of both chromosomal and nuclear NORs and artificially induced triploidy in three fish species (rainbow trout, Oncorhynchus mykiss; common carp, Cyprinus carpio; and tench, Tinea tinea) has been confirmed by CMA₃ fluorescence and Ag-staining. The frequencies of cell nuclei with one, two and three active NORs, as revealed by Ag-staining, has been analyzed statistically to find the minimum cell number which verifies the increased ploidy level. A minimum sample size of about 80 cells exhibiting three active NORs is sufficient to confirm triploidy in all three species and may be of use for categorising other ploidy-manipulated fish species.     

Changes in Tissue Cellularity Are Associated with Growth Enhancement in Genetically Modified Arctic Char (Salvelinus alpinus L.) Carrying Recombinant Growth Hormone Gene

Biochemical and histological analyses were used to study the number and size of cells (cellularity) in tissues of fast-growing, genetically modified Arctic char (Salvelinus alpinus L.), overexpressing sockeye salmon (Oncorhynchus nerka) growth hormone gene (OnGH1). DNA contents of muscle, heart, and liver were compared in transformed, sibling (age control) and 1 year older (size control) char. Total white muscle cross-sectional area, white muscle fiber number, and total nuclei number within the muscle tissue were determined from one complete half-section of each fish. The analyzed tissues responded differently to growth hormone overproduction. In muscle tissue of OnGH1-transformed char, the enhanced growth was clearly associated with proliferation of muscle cells (hyperplasia), whereas in heart tissue both cell proliferation and increase in cell size (hypertrophy) were enhanced. The relative DNA concentration in the liver of transformed char was significantly greater than that of control fish, suggesting reduction in size of hepatic cells. Received June 29, 2000; accepted October 25, 2000     

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.