Molecular characterization and expression pattern of AFPIV during embryogenesis in gibel carp(Carassiu auratus gibelio)

As a new type of AFPs, AFPIV has been firstly identified in longhorn sculpin (Myoxocephalus octodecimspinosus), and in recent years, its cDNA and amino acid sequence have been reported, and its pancreatic synthesis has been firstly reported in polar fish. However, its expression patterns during fish embryogenesis have not been elucidated yet. By differential screening, we cloned the CagAFPIV in gibel carp, Carassius auratus gibelio, demonstrated its predominant expression during embryogenesis. RT-PCR detection revealed that CagAFPIV was first transcribed from blastula stage and kept a high level during embryogenesis and declined remarkably in hatched larva. In situ hybridization revealed that CagAFPIV transcripts were firstly distributed over the margin and marginal blastomere in blastula stage embryos, at the early-gastrula stage the positive signals distributed in the marginal cells and the internalization cells, and later restricted to the cells the yolk syncytial layer (YSL) from later gastrula stage to larva stage. Consistently, the CagAFPIV protein also kept a high level during embryogenesis, and the high protein level retained some days after the larva hatched. Our work, for the first time, revealed the dynamic expression and distribution of CagAFPIV during embryogenesis.

Molecular characterization and expression pattern of fetuin-B in gibel carp (Carassius auratus gibelio)

Fetuin-B has recently been cloned and identified from rats, mice, and humans; their expression patterns, however, have not been elucidated yet. In this study, Cagfetuin-B has been cloned in gibel carp. RT-PCR and Western blot detection revealed that Cagfetuin-B is first transcribed from the blastula stage and at a relatively stable level afterward during embryogenesis and the larval stage. Cagfetuin-B transcripts are predominantly distributed over the yolk syncytial layer in the early embryos and later restricted to the cells of liver and brain in newly hatched larvae. Moreover, a dynamic distribution of Cagfetuin-B protein was observed in brain, kidney, liver, and skin during morphogenesis. In adult fish, Cagfetuin-B transcripts are restricted in liver and ovary. Our work, for the first time, revealed the extra-hepatic transcription and a dynamic distribution of fetuin-B during embryogenesis and in adults, which indicates the potential roles of fetuin-B in fish organogenesis.

Identification of a novel C2 domain factor in ovaries of orange-spotted grouper (Epinephelus coioides)

Follicle consists of an oocyte and a lot of surrounding follicular cells, and significant interactions exist between the oocyte and the somatic cells. In this study, a novel cDNA has been screened from a subtractive cDNA library between tail bud embryos and blastula embryos in the protogynous hermaphrodite orange-spotted grouper (Epinephelus coioides). Its full-length cDNA is 821 bp, and has an ORF of 414 by for encoding a peptide of 137 aa, which shows 38%, 37%, 33%, and 33% homology with 4 putative proteins screened from zebrafish (Danio rerio). Conserved domain search in NCBI reveals a single C2 domain existing in the C2 domain superfamily proteins, and has only 7 beta strands in comparison with 8 beta strands of C2 domains in other C2 domain superfamily proteins. Artificial sex reversal, RT-PCR analysis and Western blot detection demonstrated ovary-specific expression of the C2 domain factor, and therefore the novel gene was designated as E. coioides ovary-specific C2 domain factor, EcOC2 factor. Moreover, predominant expression of EcOC2 factor was further revealed in grouper mature ovary, and its strong immunofluorescence signals were located between granulosa cells and oocyte zona radiata in grouper mature follicles. The data indicate that the novel EcOC2 factor might be a main component that associates between granulosa cells and the oocyte during oocyte maturation, and might play significant roles in regulating oocyte maturation and ovulation. Further studies on its developmental behaviour and physiological functions will elucidate the interactions between oocyte and the surrounding somatic cells and the underlying molecular mechanisms. (C) 2005 Elsevier Inc. All rights reserved.

Zebraflsh z-otu, a novel Otu and Tudor domain-containing gene, is expressed in early stages of oogenesis and embryogenesis

Several studies have suggested that Otu domain had de-ubiquitinating activity and Tudor domain was important for the formation of germ cells. Here, we reported a novel zebrafish ovary-specific gene containing Otu and Tudor domain, z-otu, which was expressed at stages I-III oocytes and embryonic stages from zygotes to early blastula during embryonic cells maintained their totipotency. Therefore, z-otu might link the ubiquitin signaling pathway to early oogenesis and maintaining the totipotency of embryonic cell. (c) 2005 Elsevier B.V. All rights reserved.

Identification of a Spindlin homolog in gibel carp (Carassius auratus gibelio)

Spindlin has been suggested to play an important role during the transition from oocyte maturation to embryo development in mouse, but its homolog similar to the mouse Spindlin in molecular and expression characterization has not been identified up to now in other vertebrates. In this study, a full length of cDNA sequence is cloned and sequenced from the gibel carp (Carassius auratus gibelio). It contains 1240 nucleotides with an open reading frame of 771 nt encoding 257 amino acids. Based on its amino acid sequence alignment and comparison analysis with the known Spin family proteins, the newly cloned Spin is named Carassius auratus gibelio Spindlin (CagSpin). Its product could be detected from mature eggs to blastula embryos, but its content decreased from the two-cell stage, and could not be detected after the gastrula stage. It suggests that the CagSpin should be a maternal protein that is expressed during oocyte maturation, and plays a crucial role in early cleavage of embryogenesis. CagSpin is the first homolog similar to mouse spindlin identified in fish, and also in other vertebrates. GST pull-down assay reveals the first biochemical evidence for the association of CagSpin and p-tubulin, the microtubule component. Therefore, CagSpin may play important functions by interacting with beta-tubulin and other spindle proteins during oocyte maturation and egg fertilization. (c) 2005 Elsevier Inc. All rights reserved.

Effects of cell-cycle arrest agents on cleavage and development of loach (Misgurnus anguillicaudatus) embryos

The aim of this study was to determine the lowest concentration of nocodazole and colchicine to arrest blastomere division during the cleavage stage of loach embryos and to assess the reversibility and toxicity of the treatments in the treated embryos. Eight-cell loach embryos were incubated for 4, 8, 12, or 16 h in 1/10x Holtfreter supplemented with either nocodazole, an inhibitor of tubulin polymerization, or colchicine, an inhibitor of tubulin assembly. Complete arrest of cell cycle was observed, at a colchicine concentration of 0.996 mM and at a nocodazole concentration of 0.275 muM, respectively (the lowest effective concentration). No major morphological alteration in chromatin was observed. Reversibility and toxicity of both agents were dose and exposure period dependent. For both agents, prolonging cleavage arrest for more than 4 h (at the effective concentrations) is detrimental to development of embryos. Nocodazole treatment was less cytotoxic, whereas the concentrations of colchicine which induce cleavage arrest were detrimental to development beyond the blastula stage. Toxic effects beyond the blastula stage could be minimized for both agents by reducing the period of treatment and concentration.

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.

Nuclear transfer in leach (Paramisgurnus dabryanus Sauvage) by cell-to-cell electrofusion

To study nuclear transfer in the leach (Paramisgurnus dabryanus Sauvage), blastula and gastrula cells were fused with UV-inactivated oocytes by cell-to-cell electrofusion. To facilitate nuclear transfer, blastula and gastrula cells were cultured or incubated at 4 degreesC in different solutions. TC-199 medium supplemented with 20% calf serum was the best culture solution, and effectively retained the totipotence of blastula or gastrula cells for up to 10 days, It was found that gastrula cells incubated at 4 degreesC had the same totipotence as blastula cells, The optimal UV dosage for inactivation of the oocyte chromatin was 180-240 mJ cm(-2). Electrofusion was carried out in a cone-shaped fusion chamber, which permitted the recipient oocyte and the donor blastula cell to contact one another. The electrofusion procedure resulted in a 10% success rate of normal-appearing fish. Genetic analysis indicated that the nuclear material originated from the donor cell (blastomere) and the oocyte pronucleus did not take part in development.

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.

Nuclear transplantation with early-embryonic cells of transgenic fish

Like other transgenic animals, transgenic fishes produced by microinjection are transgenic mosaics. In order to produce homogenous transgenic fish, the transgenic blastula or gastrula cells were dissociated from Carassius auratus, Pengze var, and Cyprinus carpio, Huanghe var., and the nuclei were transferred into the mature eggs of the same species via microinjection or electro-fusion. Five nuclear-transferred Carassius auratus, Pengze var. and one Cyprinus carpio, Huanghe var. were obtained and the existence of the transgene was detected. The possibility of generating homogenous strain of transgenic fish by nuclear transplantation with transgenic early-embryonic cells is discussed.

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic leach (Misgurnus anguillicaudatus Cantor) have been studied. The GFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of the GFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.

Embryonic and genetic manipulation in fish

Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An "all-fish" gene construct CAgcGH has been made by splicing the common carp beta-actin gene (CA) promoter onto the grass carp growth hormone gene (gcGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21(st) century.

Improvement of polyploidy induction in Eriocheir sinensis

An incubating temperature of 15 degreesC is used to induce triploidy in Etiocheir sinensis through inhibition of the release of polar body H, and that of 18 degreesC to induce tetraploidy through inhibition of the first cleavage. Flow cytometry is used to identify the ploidy in different developmental stages. For induction of triploidy in fertilized eggs in vitro, the highest induction rate observed in blastula by cytochalasin B, 6-DMAP and KCI is 49.1%, 51.7% and 77.5%, respectively. In the KCI treatment of pregnant crabs with the fertilized eggs, the highest triploid induction rate observed in the zoea is 85.3%. For induction of tetraploidy, the highest induction rate observed in the blastula by cytochaslasin 13, 6-DMAP and KCI is 50.3%, 54.9% and 79.8% respectively. In the KCI treatment of pregnant crabs with the fertilized eggs, the highest induction rate in zoea is 27.3%. Through this study such difficulty as in vitro culture is overcome. Triploid zoea Etiocheir sinensis has been developed for the first time. The induction rate of tetraploid zoea has also been greatly improved.

Inhibition of egg hatching success and larvae survival of the scallop, Chlamys farreri, associated with exposure to cells and cell fragments of the dinoflagellate Alexandrium tamarense

We report an apparently novel toxic effect of the dinoflagellate Alexandrium tamarense, manifested by inhibition of the egg hatching success of the scallop, Chlamys farreri. The hatching rate of C. farreri approached only 30% of controls when its fertilised eggs were exposed for 36 h to A. tamarense cells or cellular fragments at a concentration of 100 cells/ml, and the hatching rate was just 5% after exposure to A. tamarense of 500 cells/ml. Similar exposures of the fertilised scallop eggs to two other algal species, the diatom Phaeodactylum tricornutum and the raphidophyte Heterosigma carterae, resulted in no such toxicity or inhibitory effects.. Likewise, exposure of eggs to standard STX toxin. as well as to A. tamarense cell contents (supernant of re-suspended algal cells following ultrasonication and centrifugation), did not elicit this inhibitory response. However, exposure of the scallop eggs to cell cultures, intact algal cells, or cell fragments of A. tamarense produced marked toxicity. The alga also influenced larvae at early D-shape stage of scallop. The survival rates began to decrease significantly after exposed for 6 days at concentration of 3000 cells/ml and above: no larvae could survive after 14-day exposure to A. tamarense at 10,000 cells/ml or 20-day at 5000 cells/ml. The results indicated the production of novel substances from A. tamarense which can cause adverse effects on egg hatching and survival of the scallop larvae, The experiment also found that the developmental stages before blastula was the developmental period most sensitive to the A. tamarense toxin(s) and the alga at early exponential stage had the strongest effect on egg hatching comparing with other growth phases. The adverse effect of A. tamarense on early development of scallops may cause decline of shellfish population and may have further impact on marine ecosystem. (C) 2001 Elsevier Science Ltd. All rights reserved.