不同供核细胞、处理方法及激活时间对猪核移植重构胚发育的影响

在不同的外界因素下,使用胞质内体细胞核注射的方法,为进一步提高猪体细胞核移植的效率来寻找新途径。利用屠宰场卵巢采集的卵母细胞经过去核,注核及激活等操作后,研究猪颗粒细胞和胎儿成纤维细胞,血清饥饿处理法及不同的激活时间等因素对猪核移植重构胚体外发育的影响.结果表明,猪颗粒细胞和胎儿成纤维细胞用作核供体与猪卵母细胞构建重构胚,在卵裂率上无显著性差异。作为供核细胞的猪成纤维细胞,经过血清饥饿与不经过血清饥饿培养,核移植重构胚的卵裂率无显著性差异。在进行核注射后,使供核细胞与胞质受体作用适当的时间(1～6h)在进行激活,有利于重构胚的发育。提示颗粒细胞和猪胎儿成纤维细胞用作核供体与猪卵母细胞构建重构胚以及作为供核细胞的猪成纤维细胞,经过血清饥饿与不经过血清饥饿培养处理后,这二者对猪核移植重构胚发育的均无明显影响。但是使供核细胞与胞质受体作经过适当的时间(1～6h)的激活,却有利于重构胚的发育。     

Artificially induced tetraploid masu salmon have the ability to form primordial germ cells

Diploid gametes generated with tetraploid animals are a stepping stone to improving chromosome manipulation techniques. However, artificially induced tetraploid individuals generally die soon after hatching. Diploid gametes could be induced by in vivo cultures of tetraploid primordial germ cells (PGCs) through germ-line chimera. In the present study, characteristics of PGCs were studied in inviable tetraploid masu salmon, Oncorhynchus masou. Histological observation of tetraploid embryos revealed that the same or smaller numbers of PGCs were observed and they migrate into the genital ridges as did diploid PGCs during gonadogenesis. By whole-mount in situ hybridization using vasa messenger RNA (mRNA), 4–35 vasa-positive signals were detected in a pair of genital ridges of tetraploids. By cytological observation of genital ridge cell suspensions, several large round cells were observed, some of which extended pseudopodia. They also contained large nuclei and round granules in their cytoplasm, characteristics of PGCs. As the results suggest that inviable artificial tetraploids have PGCs, we expect to achieve diploid gamete production through surrogate propagation and tetraploid fish production.     

鱼类生殖细胞移植的研究进展及应用前景

生殖细胞移植是指将供体的生殖细胞移植到同种或异种受体体内,供体生殖细胞嵌合到受体性腺,经过增殖、分化并最终发育为功能性配子的过程。作为辅助生殖技术,它不仅为珍稀濒危动物的繁育和保护提供了新途径,同时也为生殖干细胞的功能研究提供了有效手段。鱼类生殖细胞移植研究首先在模式鱼类斑马鱼中开展,经过十多年的发展,取得了一系列突破性的进展:主要包括先后建立了以胚胎、仔鱼和成鱼为受体的生殖细胞移植体系,精原和卵原干细胞的发现拓宽了供体生殖细胞的选择,受体的选择与制备方法的完善。该技术在缩短鱼类性成熟周期、性控育种、珍稀濒危鱼类保护等方面具有巨大的应用前景,已成功在多种淡水和海水鱼类中开展了研究和应用。本文结合作者的研究实践和经验,系统地梳理和总结了鱼类生殖细胞移植的研究进展,指出了该技术实践应用的关键问题,并探讨了其应用前景。     

Optimization of hydrostatic pressure,timing, and duration parameters for the induction of tetraploidy in turbot,Scophthalmus maximus

Aquaculture International - Tetraploid fish are the key source of diploid gametes in polyploid breeding, and they can be induced by disrupting the first mitotic cell cleavage. In this study, the...     

Embryonic stem cells in fish: current status and perspectives

Totipotent embryonic stem (ES) cells represent a bridge that links in vitro and in vivo manipulations of animal genomes and have enormous potential for genetic engineering of livestock. We have recently established feeder cell-free conditions for culturing cells of midblastula embryos (MBE) of the medaka (Oryzias latipes) and obtained several stable cell lines that show all features of mouse ES cells in vitro. One of these lines, MES1, has been demonstrated to retain a diploid karyotype and can be induced to differentiate into various cell types in vitro. Upon microinjection into albino host blastulae, MES1 cells are able to form pigmented chimeras. Genotype-specific PCR analysis revealed that 90% of host blastulae transplanted with MES1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. Transplantation of genetically labelled MES1 cells revealed a wide contribution to numerous organs derived from all three germ layers and differentiation into various types of functional cells. These ES properties of MES1 line was not abolished by stable gene transfer and long-term selection. Thus MES1 cells may represent a first promising cellular vehicle for the production of genetically modified fish. The genetic background has been found to have a profound effect on the efficacy of ES cell derivation and of chimera formation.     

利用鳍条组织进行流式细胞实验的可行性研究

流式细胞术能快速检测生物细胞的DNA含量和细胞周期,在鱼类遗传育种方面已得到广泛运用。目前的研究多基于鱼类血液和生殖细胞开展实验,存在周期性和操作难度的限制。该研究利用草鱼(Ctenopharyngodon idellus)不同组织进行流式分析,在鳍条组织中得到了明显的峰图,并在4种鱼类的重复实验中得到了稳定结果,且不同鱼类的鳍条组织均存在细胞周期,大部分细胞停留在G_0/G_1期。利用斑马鱼(Danio rerio)作为内标测定出团头鲂(Megalobrama amblycephala)鳍条组织的相对DNA含量(C值)为(1.25±0.07)pg,与相关研究结果基本吻合,认为利用鳍条组织作为血液和生殖细胞的替代材料进行流式细胞实验是科学可行的。     

Heat-shock-induced tetraploid and diploid/tetraploid mosaic in pond loach, Misgurnus anguillicaudatus

Tetraploid fish, which are considered as key resources of diploid gametes for further breeding and ploidy manipulation, can be artificially induced by inhibition of the mitotic cell division with hydrostatic pressure or temperature treatments. Although many attempts have been made to induce artificial tetraploid strains, successful establishment of viable and fertile tetraploid strains are rare. In pond loach, Misgurnus anguillicaudatus, natural tetraploid individuals are distributed in wild populations and diploid gametes from the tetraploid fish have been used for the induction of polyploid individuals, but artificially induced tetraploid strains have not been established yet. In the present study, we optimised starting timing of the heat-shock treatment (41 °C for 2 min) to inhibit a mitotic cell division in fertilised eggs of the normal diploid pond loach between 21 and 51 min after insemination at 20 °C. After the treatment, we observed external appearance of hatching larvae and flow cytometrically determined ploidy status of the resultant larvae. Although tetraploid and diploid/tetraploid mosaic larvae were obtained, the optimum timings for induction of tetraploidy varied amongst crosses. Various kinds of ploidy such as haploidy, diploidy, triploidy, pentaploidy, hexaploidy, aneuploidy and mosaic were detected in non-optimum heat-shock timings for tetraploidisation. Survivors, a tetraploid and a diploid/tetraploid mosaic male, matured at the age of 1-year-old, but they produced functional haploid spermatozoa.     

Progress towards the identification of the sex-determining mechanism of the sole,Solea solea (L.), by the induction of diploid gynogenesis*

Methods were determined for the induction of diploid gynogenesis in the sole, Solea solea (L.), in order to investigate the sex-determining mechanism. The experiments utilized gametes obtained by dissection of fish caught at sea. Activation of eggs with UV-irradiated sole sperm was not feasible but development was initiated by exposure to halibut, Hippoglossus hippoglossus (L.), sperm. These embryos displayed the typical characteristics of haploids and few survived beyond hatching. High levels (> 90%) of diploidy were induced in such eggs by subjecting them to a cold shock (2°C or 4°C) for 1-2 h about 10-15 min after activation at 12°C. The resultant embryos were indistinguishable from those developing from normally fertilized eggs and were considered to be diploid gynogenomes. Fifteen of these were reared to a length of 5-10 cm by which time gonad differentiation had been initiated. Both sexes were represented, indicating that sex in this species is not determined by a simple XX-XY system with female homogamy.     

Application of silver staining to the identification of triploid fish cells

A simple inexpensive method of determining level of ploidy in fish using silver-stained cell preparations is evaluated. The method involves determining the maximum number of nucleoli per cell on silver-stained slides. Any tissue can be used since whole cells, rather than chromosomes are required for analysis. Since the amount of tissue needed to make slides is very small, samples may be obtained from fish about 7–8 cm long without sacrificing the animal. Results from three salmonid species (rainbow trout, chinook salmon and coho salmon) showed that haploid individuals had one nucleolus/cell, diploid individuals had one or two nucleoli/cell and triploid individuals had 1, 2 or 3 nucleoli/cell. The fraction of cells with three nucleoli/cell was higher in triploid embryos (averaging 75%) than in gill tissue from triploid fish of 6 months or 2 years of age (averaging 43% and 36%, respectively). However, no cells with three nucleoli were ever found in diploid individuals, making identification of triploids unambiguous. This method should be applicable to most fish species since the majority of those examined have been shown to have only one chromosome with a nucleolar organizer region (NOR) per haploid genome.     

Germ cell transplantation as a potential biotechnological approach to fish reproduction

Although the use of germ cell transplantation has been relatively well established in mammals, the technique has only been adapted for use in fish after entering the 2000s. During the last decade, several different approaches have been developed for germ cell transplantation in fish using recipients of various ages and life stages, such as blastula-stage embryos, newly hatched larvae and sexually mature specimens. As germ cells can develop into live organisms through maturation and fertilization processes, germ cell transplantation in fish has opened up new avenues of research in reproductive biotechnology and aquaculture. For instance, the use of xenotransplantation in fish has lead to advances in the conservation of endangered species and the production of commercially valuable fish using surrogated recipients. Further, this could also facilitate the engineering of transgenic fish. However, as is the case with mammals, knowledge regarding the basic biology and physiology of germline stem cells in fish remains incomplete, imposing a considerable limitation on the application of germ cell transplantation in fish. Furthering our understanding of germline stem cells would contribute significantly to advances regarding germ cell transplantation in fish.     

Establishment and characterization of two new cell lines derived from flounder, Paralichthys olivaceus (Temminck & Schlegel)

Two new cell cultures from flounder, Paralichthys olivaceus (Temminck & Schlegel), flounder fin (FFN) cells from fin tissue and flounder spleen (FSP) cells from spleen tissue, were established and characterized. The cells multiplied well in Eagle's minimum essential medium, supplemented with 10% foetal bovine serum, and have been subcultured more than 100 times, becoming continuous cell lines. Modal diploid chromosome number of FFN and FSP cells was 64 and 62, respectively. Polymerase chain reaction products were obtained from FFN and FSP cells with primer sets ofmicrosatellite markers of flounder. Optimal growth temperature was 20 degrees C and consisted of epithelioid cells. FFN and FSP cells showed cytopathic effects after inoculation of infectious pancreatic necrosis virus, marine birnavirus, chum salmon virus, infectious haematopoietic necrosis virus, spring viraemia of carp virus and hirame rhabdovirus. Thus these new cell lines may be useful for studying a wide range of fish viruses.     

Gonadal development in early life stages of <Emphasis Type="Italic">Spratelloides gracilis</Emphasis>

ABSTRACT:   The gonad of Spratelloides gracilis was not sexually differentiated in the yolk-sac, preflexion, flexion and postflexion stages. Sexual differentiation and development of the ovary and testis started in the transition stage from larva to juvenile. In juveniles at the fin ray completion stage, the ovary and testis could be distinguished because the ovary contained germ cells initiating meiosis and the testis had blood vessels and a high density of somatic cells. The ovary further developed in larger juveniles to have oocytes of perinucleolus stage together with those of the chromatin nucleolus stage, and oogonium. However, in the testis of larger juveniles, primary spermatogonium began proliferation by meiosis. Sexual differentiation may be regarded as one of morphological and functional changes accompanying metamorphosis in S. gracilis . Some fish larger than the mature size of 60 mm standard length had advanced germ cells and functional gonads, others did not have functional gonads. The distal end of the immature gonads did not connect with a genital duct near the anus. These observations indicate that S. gracilis has large variability in size-at-maturity. The variability in size-at-maturity in S. gracilis , together with large variability in age-at-maturity, may constitute an ecological basis for an extended spawning season in S. gracilis .     

Chromosome set manipulation and sex control in common carp: a review

The development of techniques for production of gynogenetic, androgenetic, polyploid, and monosex progenies in common carp (Cyprinus carpio L.) is described from a chronological perspective. Gynogenetic progenies were obtained either by suppression of the second meiotic division in eggs (meiotic gynogenesis) or by suppression of the first mitotic division in haploid embryos (mitotic gynogenesis). As a rule, gynogenetic progenies of common carp were all-female, revealing female homogamety (females—XX, males—XY) in this species. Induced gynogenesis results in increased homozygosity; the rate of increase depends on the type of gynogenesis. Inbreeding coefficient (F) for one generation of meiotic gynogenesis in common carp is about 0.6, while diploids obtained by mitotic gynogenesis are homozygous for all genes (F = 1.0). Mitotic gynogenesis was used for production of clones in common carp. In androgenetic progenies of common carp, YY males were identified, that after crossing with normal females (XX) produced all-male progenies. Triploids of common carp are characterized by a significant reduction in gonad development (especially ovaries). However, the reduction in gonad development did not result in an increase of somatic growth rate of fish. The procedure for androgen treatment to induce phenotypic sex reversal in genotypic females (XX) was elaborated. All-female progenies of common carp were produced on a large scale by crossing normal females (XX) with hormonally sex-reversed males (XX). Rearing of all-female progenies in conditions when fish normally reach sexual maturity before reaching of market size increased production yield by 7–8%. In a few cases distant hybridization resulted in polyploidy of fish without application of any physical treatment. The ability of hybrid females between crucian carp (Carassius auratus) and common carp to produce diploid (with unreduced chromosome number) gametes resulted in opportunities to produce triploid and tetraploid hybrid progenies.     

The sexually dimorphic adipose fin is an androgen target tissue in the brown trout (Salmo trutta fario)

An investigation has been described on the relationship of body length, age and sex with adipose fin length and the number of androgen receptor (AR)-containing cells in the adipose fin as a secondary sexual characteristic for brown trout (Salmo trutta fario). Firstly, body and adipose fin lengths of 2- to 5-year-old brown trout were measured. Thereafter, these fish were killed by decapitation, then their sexes were determined, and adipose fins were excised. The cellular bases of AR binding activities in the adipose fins were analyzed with an antibody against human/rat AR peptide. Immunocytochemistry and western blotting techniques were performed with this antibody. Analysis of morphological measurements indicated that body length and age had a linear relationship with adipose fin length. The coefficients of determination for the body length and age were 0.92 and 0.85 in the male fish and 0.76 and 0.73 in the female fish against the adipose fin length, respectively. At 2 years of age, cells in the adipose fin did not exhibit AR immunoreactivity. However, AR-immunopositive cells were abundant in the adipose fin of 3- to 5-year-old fish. Moreover, the number of AR-immunopositive cells was significantly (P < 0.05) high in males and increased with age. These observations indicate that the adipose fin in the brown trout is a probable target for androgen action and that tissue function or development may to some extent be androgen dependent. In addition, it is likely that such an effect will be mediated by specific androgen receptors.     

波纹唇鱼染色体制备及核型的初步研究

出于对波纹唇鱼这一濒危物种资源调查和保护的目的,探讨了活体状况下波纹唇鱼染色体制备的方法,并对其染色体进行分析。剪取1龄波纹唇鱼的微量尾部鳍条及对应部位的愈合增生组织为材料,采用热滴片法制备染色体标本。核型分析结果表明,波纹唇鱼二倍体染色体数目2n=48,核型为2n=4M+10SM+32ST+2T,因所取鱼的条数有限,且均为雌性,所以尚不能确定是否有异型性染色体的存在。     

Ethidium Bromide Nuclear Staining and Fluorescence Microscopy: An Alternative Method for Triploidy Detection in Fish

Abstract.– Erythrocyte nuclei from normal diploid (2N) and cold-shocked triploid (3N) Asian catfish Clarias macrocephalus were comparatively stained with fluorescent stain, ethidium bromide and with a bright-field stain, Giemsa. Stains were applied to dried blood smears on glass slides, with blood taken from fingerling fish without sacrificing the fish. Both stains produced comparable quality visual comparisons, with significant differences ( P < 0.001) between nuclear dimensions of 2N and 3N cells. Triploid nuclei were larger and more elongate than diploid nuclei. Karyotypic comparisons revealed that diploid fish had 54 chromosomes, while triploid fish had 81. Ethidium bromide nuclear staining required only 3 min after slides were air-dried and fixed, while Giemsa staining required more than 50 min.     

Establishment of a novel fin cell line from Brown-marbled grouper, Epinephelus fuscoguttatus (Forsskål), and evaluation of its viral susceptibility

To lay a solid foundation of in vitro investigations of fish viral diseases, cytotechnology and cytotoxicology, a novel fin cell line from brown-marbled grouper, Epinephelus fuscoguttatus , was established and its viral susceptibility was evaluated. The fin tissues, digested with hyaluronidase and collagenase II, were used to initiate primary culture at 24 °C by using 20% foetal bovine serum-Dulbecco's modified Eagle medium/F12 medium, which was further supplemented with carboxymethyl–chitooligosaccharide, basic fibroblast growth factor and insulin-like growth factor-I. The fibroblastic fin cells grew at a steady rate during subsequent subculture and had a population doubling time of 50.6 h at passage 60. The modal diploid chromosome number was 48. A brown-marbled grouper fin cell line (bmGF-1) has been established and subcultured to passage 75 by now. Viral susceptibilities revealed that typical cytopathic effects of bmGF-1 cells emerged after being infected by turbot reddish-body iridovirus (TRBIV) or lymphocystis disease virus (LCDV). However, a large number of TRBIV and LCDV particles were also found in infected bmGF-1 cells. All these indicate that the bmGF-1 cell line has good susceptibility to TRBIV and LCDV, which may serve as a valuable tool for studies of cell–virus interactions and have potential applications in fish virus propagation and vaccine development.     

Mottled coloration of haploid-diploid and diploid-triploid mosaic amago salmon Oncorhynchus masou ishikawae

ABSTRACT:   Mottled amago salmon Oncorhynchus masou ishikawe with both yellow- and dark-pigmented skin occurred together with typical albino individuals in a commercial farm. Out of 12 mottled fish examined by DNA content flow-cytometry and erythrocytic nucleus size, three were diploid, eight were haploid-diploid mosaic and one was diploid-triploid mosaic. This fact indicates that the mottled coloration might link to polyploid mosaicism. Genotype of diploid and non-diploid cells at the albino locus was estimated in nine mature mottled fish by observing the frequency of wild-type and albino progeny when mating to homozygous albino ( aa ). One diploid and three haploid-diploid mosaic mottled fish were presumed to have mosaic genotype with both hemizygous ' a ' and heterozygous ' Aa ' cells ( a/Aa ), because the segregation ratio between two phenotypes was 1 : 1. Three other haploid-diploid mosaic fish were presumed to have mosaic genotype with both hemizygous ' a ' and homozygous ' AA ' cells ( a/AA ), because of exclusive occurrence of wild-type phenotype in the progeny. The diploid-triploid mosaic mottled fish was presumed to have mosaic genotypes ' aa / AAA ', ' aa/AAa ' or ' aa / Aaa ', because this fish yielded only albino progeny. One diploid mottled fish produced both two phenotypes but albino embryos appeared with much more frequency than the expectation, when assuming the genotype ' Aa '. Thus, this fish was considered to have mosaic genotype ' Aa/aa '.