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.

The Chinese rare minnow (Gobiocypris rarus) as an in vivo model for endocrine disruption in freshwater teleosts: a full life-cycle test with diethylstilbestrol

Chinese rare minnow (Gobiocypris rarus), a freshwater teleost,. was exposed to diethylstilbestrol (DES) at 0.05, 0.5, 1 and 5 mug/L from fertilized eggs for up to mature period under flow-through condition. Several endpoints that related to development, reproductive fitness and transgenerational effects were evaluated. It was found that body length and body weight were significantly reduced and vitellogenin (Via) levels were significantly increased for fish exposed to DES. Histological examination showed that the sex ratios of F-0 fish skewed to female and about 2% of the fish exposed to 0.05 mug/L DES developed testes-ova. The reproductive success, as determined from data on egg production, was reduced in female fish exposed to 0.05, 0.5, 1 and 5 mug/L DES. The lowest-observed-effect concentrations (LOEC) for chances of sex ratios, reproductive success and histology alteration of F-0 are 0.05 mug/L. In the offspring, transgenerational effects on egg hatching rate. egg fertilization and Vtg levels of juvenile individuals were not observed. However. survival of F, generation fry significantly declined. The analysis of sex steroid levels revealed a significant decrease of testosterone (T) in the whole body homogenates (WBH) of male progeny and somewhat elevation of estradiol (E-T) in the WBH of female offspring. These findings indicate that exposure to DES causes a variety of developmental, reproductive and transgenerational effects. (C) 2004 Elsevier B.V. All rights reserved.

Identification of a novel C1q family member in color crucian carp (Carassius auratus) ovary

Potential roles of Clq/tumor necrosis factor (TNF) superfamily proteins have been observed in vertebrate oogenesis and oocyte maturation, but no ovary-specific member has been identified so far. In this study, we have cloned and identified a novel member of Clq family with a Clq domain in the C-terminal from fully grown oocyte cDNA library of color crucian carp and demonstrated that the gene might be specifically expressed in ovary and therefore designated as Carassius auratus ovary-specific Clq-like factor, CaOClq-like factor. It encodes a 213 amino acid protein with a 17 amino acid signal peptide. There is only one protein band of about 24.5 kDa in the extracts from phase I to phase IV oocytes, but two positive protein bands are detected in the extracts of mature eggs and fertilized eggs. Furthermore, the mobility shift of the smaller target protein band cannot be eliminated by phosphatase treatment, but the larger protein band increases its mobility on the gel after phosphatase treatment, suggesting that the larger protein might be a phosphorylated form. Immunofluorescence localization indicates that the CaOClq-like proteins localize in cytoplasm, cytoplasm membrane and egg envelope of the oocytes at cortical granule stage and vitellogenesis stage, whereas they were compressed to cytoplasm margin in ovulated mature eggs and discharged into perivitelline space between cytoplasm membrane and egg envelope after egg fertilization. Further studies on distribution and translocation mechanism of the CaOClq-like factor will be benefit to elucidate the unique function in oogenesis, oocyte maturation and egg fertilization. (C) 2004 Elsevier Inc. All rights reserved.

The reproductive strategies of an endemic Tibetan fish, Gymnocypris selincuoensis

The reproductive traits of Gymnocypris selincuoensis from Selincuo Lake and its tributaries were investigated in 1997 and 1998. The youngest mature male was age 7 with a standard length (SL) of 172.0 mm, and the youngest mature female was age 8 with a SL of 194.0 mm. The L(50)s Of SL and age at first maturity were respectively 250.32 mm and age 9 for males and 224.71 mm and age 8 for females. The gonadosomatic index (GSI) significantly changed with seasons for mature individuals but not for immature individuals. GSIs of mature females at stages IV and V of ovary development increased with SL and reached a maximum value at the SL range from 370 mm to 390 mm; the GSIs of mature males were negatively correlated with SL. The breeding season lasted from early April to early August. Egg size did not significantly change with SL but increased with the delay of spawning. The individual absolute fecundity varied from 1,341 to 28,002 eggs (mean 12,607+/-7,349), and the individual relative fecundity varied from 6.4 to 42.0 eggs.g(-1) (mean 25.5+/-9.7). The individual fecundity increased with total body weight; it also increased with SL for those of SL less than 370 mm. There was a rest of spawning for mature individuals.

Two unisexual artificial polyploid clones constructed by genome addition of common carp (Cyprinus carp) and crucian carp (Carassius auratus)

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.

Molecular cytogenetic detection of paternal chromosome fragments in allogynogenetic gibel carp, Carassius auratus gibelio Bloch

In gynogenesis, sperm from related species activates egg and embryonic development, but normally does not contribute genetically to the offspring. In gibel carp, Carassius auratus gibelio Bloch, however, gynogenetic offspring often show some phenotypes apparently derived from the heterologous sperm donor. This paternal effect of allogynogenesis is outstanding in an artificial clone F produced by cold treatment of clone E eggs after insemination with blunt-nose black bream (Megaloabrama amblycephala Yin) sperm. Karyotype analysis revealed 5-15 supernumerary microchromosomes in different individuals of clone F in addition to 156 normal chromosomes inherited from the maternal clone E. A painting probe was prepared from the microdissected microchromosomes, and used to investigate the origin of these microchromosomes. Strong positive signals were detected on each microchromosomes of clone F and on 4 pairs of chromosomes in blunt-nose black bream, whereas no signals were detected on the chromosomes of clone E. This result indicates that some paternal chromosome fragments of blunt-nose black bream have been incorporated into the artificial clone F. Therefore, the manipulation of allogynogenesis may provide a unique method to transfer DNA between diverse species for fish breeding.

Factors affecting the efficiency of somatic cell nuclear transplantation in the fish embryo

Procedures to improve somatic cell nuclear transplantation in fish were evaluated. We reported effects of nonirradiated recipient eggs, inactivated recipient eggs, different combinations between recipient eggs and donor cells, duration of serum starvation, generation number, and passage number of donor cells on developmental rates of nuclear transplant (NT) embryos. Exposure to 25,000 R of gamma-rays inactivated recipient eggs. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transplanted into nonirradiated or genetically inactivated unfertilized egg of gibel carp (Carassius auratus gibelio). There was no significant difference in developmental rate between nonirradiated and inactivated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. It showed that most NT embryos came from donor nuclei of bighead carp, which was supported by microsatellite analysis of NT embryos. But 23.53% of NT embryos contained more than 48 chromosomes. It was presumed that those superfluous chromosomes came from nonirradiated recipient eggs. Besides, 5.88% of NT embryos were chimeras. Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgurnus anguillicaudatus) (25% and 18.03% vs. 8.43%). Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei of three-day serum starvation was the highest, reaching 25.71% compared to 14.14% (control), 20% (five-day), and 21.95% (seven-day). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei. Developmental rate of fourth generation was the highest (54.83%) and the lowest for first generation (14.14%) compared to second generation (38.96%) and third generation (53.01%). (C) 2002 Wiley-Liss, Inc.

Nuclear transplantation in different strains of zebrafish

Single later blastula nuclei from AB strain of zebrafish (Danio rerio) were transplanted into enucleated unfertilized eggs of Long fin strain. Of 1119 cloning embryos, 14 reconstructed embryos developed into fry. DNA fingerprinting systems of the cloned fish were similar to those of the nuclear donor fish, but were distinctly different from those of the unclear recipient fish. It confirmed that the genetic material originated from nuclear donor cell other than from nuclear recipient egg. The research suggested that the basic technique for nuclear transplantation performed with different strains of zebrafish has made a breakthrough. It should be helpful for the study of some important developmental problems such as gene function, the regulation of gene expression during animal development, the developmental potential of a nucleus and the interactions between the donor nucleus and the recipient cytoplasm, etc.

Manipulation of host density affects infestation of a peritrich ciliate (Epistylis lacustris) on a calanoid copepod (Leptodiaptomus minutus) in Crystal Lake, Wisconsin, USA

Specimens of the calanoid copepod, Leptodiaptomus minutus, collected in June 1994 in oligotrophic: north temperate Crystal Lake, were infested with the stalked ciliate Epistylis lacustris. E. lacustris was highly specific to L. minutus and no other coexisting zooplankters were infested. Excluding nauplii, nearly 70% of copepods carried 1-20 ciliates, although the maximum load was as high as 250 ciliates. A lower percentage of nauplii were infested by the ciliate; those that were infested had a lower ciliate load than other copepod stages. Infestation by ciliates had no significant influence on the average egg number of female copepods. In a field experiment, higher copepod densities in enclosures resulted in a significantly higher infestation rate, but the ciliate load per individual copepod did not differ significantly among treatments.

Genome segment S8 of grass carp hemorrhage virus encodes a virion protein

The complete nucleotide sequence of the genome segment S8 of grass carp hemorrhage virus (GCHV) was determined from cDNA corresponding to the viral genomic RNA. It is 1,287 nucleotides in length and contains a large open reading frame that could encode a protein of 409 amino acids with a predicted molecular mass of 44 kD. The S8 was expressed using the pET fusion protein vector and detected by Western blotting analysis using the chicken egg IgY against intact GCHV particles, indicating that S8 encodes a virion protein. Amino acid sequence comparisons revealed that the protein encoded by S8 is closely related to protein alpha2 of mammalian reovirus, suggesting that the deduced protein of S8 is an inner capsid protein. Copyright (C) 2001 S. Karger AG, Basel.

Growth and feed utilization in two strains of gibel carp, Carassius auratus gibelio: paternal effects in a gynogenetic fish

Gibel carp (Carassius auratus gibelio Bloch) is a natural gynogenetic fish which requires sperm of the same or related species to activate egg development. The eggs of one gibel carp were divided into two batches. One batch was 'fertilized' with sperm from gibel carp (strain DD), and the other 'fertilized' with sperm from red common carp (Cyprinus carpio red variety) (strain DR). The juveniles were transferred to the laboratory 36 days post-hatch. Triplicate groups of each strain were fed a formulated diet at either 3% or satiation ration for 8 weeks. At both the restricted and satiation rations, specific growth rate was significantly higher in strain DR than in strain DD. At the 3% ration, there was no significant difference in feeding rate or feed conversion efficiency between the two strains. At the satiation ration, strain DR had a significantly lower feeding rate but higher feed conversion efficiency than strain DD. At the satiation ration, strain DR had a significantly lower intake protein, but higher recovered protein than strain DD. There was no significant difference in faecal protein loss between the two strains. At the 3% ration, strain had no significant effects on intake protein, faecal protein or recovered protein. Neither faecal energy loss nor recovered energy was affected by strain or ration. At both the 3% and satiation ration, final body contents of dry matter and lipid were significantly lower in strain DR than strain DD, while there was no significant difference in protein and energy content between the two strains at either ration level. The results suggested that gibel carp 'fertilized' with sperm of common carp grew faster than those 'fertilized' with sperm of gibel carp through increased feed conversion efficiency and protein retention.

The onset of foreign gene transcription in nuclear-transferred embryos of fish

The transcriptional onset of hGH-transgene in fish was studied in the following three cases: the first is in MThGH-transgenic F-4 common carp (Cyprinus carpio) embryos, the second is in nuclear-transferred embryos supported by the transgenic F-4 embryonic nuclei, and the third is in nuclear-transferred embryos supported by the transgenic F-4 tail-fin nuclei. RT-PCR results show that the hGH-transgene initiates its transcriptional activity from early-gastrula stage, the early blastula stage and even 16-cell stage in the first, second and third cases, respectively. it looks like that fish egg cytoplasm could just offer a very restricted reprogramming on transcriptional activity of specific gene in differentiated cell nuclei by nuclear transplantation.

Distribution, transformation, and long-term accumulation of polychlorinated dibenzo-p-dioxins and dibenzofurans in different tissues of fish and piscivorous birds

The present study monitored 10-year-old fish and piscivorous birds from sites contaminated for many Stars. The data reflected the results of actual, long-term environmental exposures, The results demonstrate that different tissues of fish have quite different concentrations of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F), The concentration order of PCDD/F within fish is liver congruent to egg congruent to intestines kidney congruent to hearts gill congruent to bladders > muscle > brain. The concentration order of PCDD/F within piscivorous birds was livers egg congruent to hearts muscle congruent to stomachs brain, The results obtained also demonstrate that the accumulation patterns of piscivorous birds and fish are quite different. The tissues of fish and piscivorous birds have different capacities for bioaccumulation and biotransformation of PCDD/F; variable proportions of TEQs were also found throughout their bodies. In fish, toxic equivalency quotient (TEQ): PCDD/F ratios in various tissues ranged from 0.01 to 0.07, whereas in birds the ratios ranged from 0.07 to 0.43. If the concentrations are normalized with lipid content, the results vary less. The effect of different lipid properties is obvious in the case of brain tissue, which is richer in phospholipids. (C) 2000 Academic Press.

Effect of available food and temperature on the growth and reproduction of Daphnia rosea

Population parameters of Daphnia rosea were studied at various concentrations of Chlorella sp. (0.25, 0.75 and 3.0 mg C l(-1)) at several temperatures (20, 25, 28, and 30 degrees C) in the laboratory. Although there were some differences in the degrees of the effects of the various temperature-food combinations, both food and temperature exerted influences on almost all of the main population parameters of D. rosea. At a water temperature of 28 degrees C, growth and reproduction were reduced, and at the lowest food level (0.25 mgC l(-1)), reproduction failed. D, rosea did not survive at 30 degrees C in spite of abundant food supply, indicating that 30 degrees C is a physiological limit. A positive relationship between body length and brood size was recognized at high and medium food levels. The slope of the regression was the highest at the highest food level and at the lowest temperature (20 degrees C). The low food level exerted a negative influence on the net reproductive rate by lowering the size of egg-bearing females, by decreasing the brood size of each size class, by decreasing the brood number per female, and by increasing the period of empty brood chamber. High water temperature (28 degrees C) also exerted a negative influence on the net reproductive rate in a similar way. For the better understanding of the key factors driving the midsummer dynamics of daphnids in the field, it may be of crucial importance to compare the population parameters of the field populations with experimentally derived values under controlled conditions of food concentration and temperature.