Developmental incompatibility between cell nucleus and cytoplasm as revealed by nuclear transplantation experiments in teleost of different families and orders

Teleosts from different families and orders were used as materials for nuclear transplantation experiments. (1) The nuclei of goldfish (Carassius auratus, family Cyprinidae, order Cypriniformes) were transplanted into the enucleated egg cytoplasm of loach (Paramisgurnus dabryanus, family Cobitidae, order Cypriniformes) and vice-versa. (2) The nuclei of Tilapia (oreochromis nilotica, order Perciformes) were transplanted into the enucleated egg cytoplasm of goldfish (Carassius auratus, order Cypriniformes). The chromosome number of the nucleus donor fish is different from that of the cytoplasmic recipient fish in each of the two combinations. In the first case, only a few early nucleo-cytoplasmic hybrid (NCH) larval fish were obtained in each combination. In second case, even though a high percentage of NCH blastulas were also obtained, the majority of them died at the same developmental stage, except a few which survived until early gastrula stage. The examination of the metaphase chromosome figures of the NCH blastulas or embryos obtained in all three combinations indicated that they were of nucleus-donor type. The developmental rates of all the NCH eggs were similar to those of cytoplasmic-recipient type. Scanning electronmicroscopy examination showed that the morphology of NCH blastula cells, which were obtained from the combination of Tilapia nucleus and goldfish cytoplasm, manifested obviously abnormal features and the cells were arrested at different stages of cell disintegration. Two-dimension polyacrylamide gel electrophoretograms of the homogenates of Tilapia, goldfish and their NCH blastula cells showed that the protein synthetic pattern of NCH blastula was similar to that of Tilapia nucleus type. The results of experiments which failed to obtain NCH adult fish in all three combinations can be explained as a result of developmental incompatibility between the donor nucleus and the enucleated recipient egg cytoplasm, which were from distantly related fish species. And the chromosome numbers of all the component fish of the three combinations which were examined in the experiment and shown to be quite different from each other in the tested fish, should not be overlooked as one of the essential factors causing the developmental incompatibility in NCH fish in this experiment.     

硬骨鱼类体细胞核移植的研究

本文用不同属、科、目的硬骨鱼类作材料进行体细胞核移植研究。鲫鱼（Ｃａｒａｓｓｉｕｓａｕｒａｔｕｓ）、鲮鱼（Ｃｉｒｒｈｉｎｕｓｍｏｌｉｔｏｒｅｌｌａ）和尼罗罗非鲫（Ｔｉｌａｐｉａｎｉｌｏｔｉｃａ）的体细胞核（头肾细胞）移植到鲤鱼（Ｃｙｐｒｉｎｕｓｃａｒｐｉｏ）的成熟去核卵中,通过继代核移植,在鲫鱼体细胞核和鲤鱼去核卵的属间组合中,获得发育到血液循环期的幼鱼;在鲮鱼体细胞核和鲤鱼去核卵的亚科间组合中,获得发育到心脏跳动期的晚期胚胎;在尼罗非鲫体细胞核和鲤鱼去枚卵的目间组合中,获得发育到肌肉效应期的胚胎。由于是直接用成鱼体细胞核作供核体进行核移植,因而能够克服供体鱼和受体鱼不同步产卵的困难。实验结果表明,这对进行硬骨鱼类核质杂交研究无疑是一种简便而又有效的方法。     

The carp–goldfish nucleocytoplasmic hybrid has mitochondria from the carp as the nuclear donor species

It is widely accepted that mitochondria and its DNA (mtDNA) exhibit strict maternal inheritance, with sperm contributing no or non-detectable mitochondria to the next generation. In fish, nuclear transfer (NT) through the combination of a donor nucleus and an enucleated oocyte can produce fertile nucleocytoplasmic hybrids (NCHs) even between different genera and subfamilies. One of the best studied fish NCHs is CyCa produced by transplanting the nuclei plus cytoplasm from the common carp (Cyprinus carpio var. wuyuanensis) into the oocytes of the wild goldfish (Carassius auratus), which has been propagated by self-mating for three generations. These NCH fish thus provide a unique model to study the origin of mitochondria. Here we report the complete mtDNA sequence of the CyCa hybrid and its parental species carp and goldfish as nuclear donor and cytoplasm host, respectively. Interestingly, the mtDNA of NCH fish CyCa is 99.69% identical to the nuclear donor species carp, and 89.25% identical to the oocyte host species goldfish. Furthermore, an amino acid sequence comparison of 13 mitochondrial proteins reveals that CyCa is 99.68% identical to the carp and 87.68% identical to the goldfish. On an mtDNA-based phylogenetic tree, CyCa is clustered with the carp but separated from the goldfish. A real-time PCR analysis revealed the presence of carp mtDNA but the absence of goldfish mtDNA. These results demonstrate – for the first time to our knowledge – that the mtDNA of a NCH such as CyCa fish may originate from its nuclear donor rather than its oocyte host.     

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.     

Somatic lamins in germinal vesicles of goldfish (Carassius auratus) vitellogenic oocytes

In fish and amphibians, B-type lamins are divided into somatic (B1, B2) and oocyte-type (B3) lamins. In this study, we purified nuclear lamins from rainbow trout erythrocytes, raised an anti-lamin monoclonal antibody (L-200) that recognizes goldfish somatic-lamins, and isolated cDNAs encoding goldfish B-type lamins (B1 and B2) from a goldfish cell culture cDNA library. Goldfish B-type lamins are structurally similar to lamins found in other vertebrates with minor amino acid substitutions in the conserved region. Western blot analysis showed that goldfish oocytes contained mainly GV-lamin B3 as well as some somatic lamins. Laser-confocal microscope observations revealed that lamin B3 was present only in GV nuclear lamina, whereas somatic lamins were present in dense fibrillar structures throughout nuclear gels of isolated GVs. Similar nuclear filamentous structures were also observed in GVs of paraffin embedded oocytes. Epitope mapping indicated that L-200 recognized a conserved region containing a short stretch of the alpha-helix coiled-coil rod domain (Y(E/Q)(Q/E)LL). A similar motif is also present in other cytoplasmic intermediate filaments (i.e., vimentin, desmin, peripherin and GFAP). Taken together, these findings suggest that lamins or lamin-related intermediate filaments are an important component of the interior architecture of goldfish vitellogenic oocyte nuclei (GVs).     

鱼类培养细胞核发育潜能的研究

本文用细胞核连续移植方法,从鲫鱼囊胚细胞的继代培养细胞,获得一尾存活达三年之久的移核鱼,但性腺未分化,不育。并从性成熟的鲫鱼短期培养肾细胞,获得一尾完成发育的性成熟的成鱼。实验结果提示鱼类囊胚细胞的继代培养细胞核和已分化的成鱼体细胞核仍具发育的全能性,为用细胞核移植方法进行鱼类体细胞育种的可能性提供了依据。     

Diploidized eggs reprogram adult somatic cell nuclei to pluripotency in nuclear transfer in medaka fish (Oryzias latipes)

Reprogramming of adult somatic cell nuclei to pluripotency has been unsuccessful in non-mammalian animals, primarily because of chromosomal aberrations in nuclear transplants, which are considered to be caused by asynchrony between the cell cycles of the recipient egg and donor nucleus. In order to normalize the chromosomal status, we used diploidized eggs by retention of second polar body release, instead of enucleated eggs, as recipients in nuclear transfer of primary culture cells from the caudal fin of adult green fluorescent protein gene (GFP) transgenic medaka fish (Oryzias latipes). We found that 2.7% of the reconstructed embryos grew into adults that expressed GFP in various tissues in the same pattern as in the donor fish. Moreover, these fish were diploid, fertile and capable of passing the marker gene to the next generation in Mendelian fashion. We hesitate to call these fish 'clones' because we used non-enucleated eggs as recipients; in effect, they may be chimeras consisting of cells derived from diploid recipient nuclei and donor nuclei. In either case, fish adult somatic cell nuclei were reprogrammed to pluripotency and differentiated into a variety of cell types including germ cells via the use of diploidized recipient eggs.     

Cryobanking of fish somatic cells: optimizations of fin explant culture and fin cell cryopreservation

When gametes or embryos are not available, somatic cells should be considered for fish genome cryobanking of valuable or endangered fish. The objective of this work was to develop a method for fin explant culture with an assessed reliability, and to assess fin cells ability to cryopreservation. Anal fins from goldfish (Carassius auratus) were minced and gently loosened with collagenase before explants were plated at 20 degrees C in L-15 medium supplemented with fetal bovine serum and pH buffering additives. Quantification of cell-donor explants per fin rated the culture success. Cells were successfully obtained from every cultured anal fin (mean = 65% cell-donor explant per fin). All other fin types were suitable except the dorsal fin. Explant plating could be deferred 3 days from fin collecting. Fins from seven other fish species were successfully cultured with the method. After 2-3 weeks, sub-confluent fin cells from goldfish were cryopreserved. Cryopreservation with dimethyl sulfoxide and sucrose at a slow freezing rate allowed the recovery of half the goldfish fin cells. Cells displayed the same viability as fresh ones. 1,2-propanediol was unsuitable when a fast freezing rate was used. The procedure could now be considered for cryobanking with only minimal adaptation to each new species.     

Studies on the Developmental Potentiality of Cultured Cell Nuclei of Fish

By means of the serial nuclear transplantation technique, the authors obtained a nuclear transplant fish from subcultured cell originated from the blastula cells of the crucian carp (Carassius auratus Linnaeus). This nuclear transplant fish survived for three years, but its sexual glands were undifferentiated. The authors have also obtained a sexually mature adult fish from short-term cultured kidney cell nucleus of an adult crucian carp.Results of the experiment implied that the subcultured cell nuclei of fish blastula cells and the specialized somatic cell nuclei of adult fish still retained their developmental totipotency, and thus, it indicated that there is a possibility of fish somatic cell breeding through the use of nuclear transplantation.     

Morphometric characteristics and reproductive capacity of nuclear transplants derived from embryonic cells of loach, Misgurnus anguillicaudatus

Nuclear transplants of loach were produced by transplantating blastula cell nuclei into nonenucleated unfertilized eggs, using recipient eggs and donor cells distinguished by different polymorphic microsatellites. Of the total of 2,847 operated eggs, 143 hatched and 119 developed to the feeding larval stage. For 15 nuclear transplants (i.e., 11.1-year-old fish and 4.2-year-old fish) that survived up to the adulthood, DNA analysis and karyotype analysis were performed. Results showed that, of the 15 fish, 14 had only a nucleus derived from the donor; moreover, 12 were diploids, 1 was a triploid, 1 was a tetraploid, and 1 was a diploid-tetraploid mosaic with both donor and recipient nuclei. For the 12 fish with only a 2n donor nucleus, morphometric analysis was performed, and two female fish and two male fish were mated with normal fish. The fish with only a 2n donor nucleus were determined to be morphometrically identical to normal fish: they had normal gametogenesis and were able to reproduce. Currently, nuclear transplantation technology is beginning to be adopted in fisheries. Biological information on nuclear transplants obtained in this study can be used in the development of nuclear transplantation technology.     

电脉冲介导金鱼囊胚细胞融合及其发育能力的研究

本实验首次成功地利用电脉冲介异法使金鱼的囊胚细胞融合,融合率高于95％,并通过细胞核移植方法,将融合细胞的细胞核移入金鱼成熟未受精的去核卵内,以了解融合后细胞核的发育能力。实验中共移植111个细胞核,得44个囊胚、7个原肠胚和1条活了8天的幼鱼(因不进食而死亡)。并对移核后发育至囊胚的胚胎用静态光度计测定了DNA含量,共测定了11个移核胚胎的细胞,其中9个移核囊胚细胞核的DNA含量增加,这一结果证明:利用电脉冲介导法能有效地转移外源染色体,供体核有促进个体发育的能力。为人工干与鱼类染色体组的组成,进一步研究鱼类个体发育对染色体倍性的依赖关系以及体细胞遗传提供了一条新途径。     

Recovery of Fertility in Male Hybrids of a Cross between Goldfish and Common Carp by Transplantation of PGC (Primordial Germ Cell)-Containing Graft

In germ-line chimera, gametes originate from both the donor and recipient. In order to increase the proportion of gametes from the donor, the elimination or reduction of primordial germ cells (PGCs) from the recipient is required. In the present study, histological and genetic analyses were performed in the chimeric fish obtained when sterile goldfish × common carp hybrid and fertile goldfish embryos were used as a recipient and donor, respectively. Chimerism was induced by transplantation of the lower part of the goldfish blastoderm into the hybrid blastoderm at the blastula stage. Neither spermatid nor spermatozoa were observed in the testis of the male hybrid. Motile sperm were obtained from 15 chimeric males by human chorionic gonadotropin (HCG) injection. When the sperm of chimeric fish were genetically analyzed, only goldfish-specific repetitive DNA sequences were detected. These results revealed that chimeric fish of the cross between a sterile male hybrid and fertile goldfish produced sperm exclusively derived from the donor goldfish.     

Identification and molecular cloning of germinal vesicle lamin B3 in goldfish (Carassius auratus) oocytes.

A bulk isolation method was developed to collect a large number of germinal vesicles (GV) from postvitellogenic oocytes of goldfish (Carassius auratus). Using this method, we obtained GV lamina which are resistant to high salt and nonionic detergent. 2D PAGE revealed that the goldfish GV lamina contained several spots with similar molecular masses (67 kDa) and slightly different neutral isoelectrofocusing values (pI 5.8-6.2). After trypsin digestion and extraction of a major spot (pI 6.1), the peptide was subjected to RP-HPLC and sequenced. A homology search identified this spot as a nuclear lamin. A cDNA encoding goldfish GV lamin was isolated by RT-PCR using degenerate primers designed from the GV lamin tryptic peptide sequence. The goldfish GV lamin cDNA encodes a predicted molecular mass of 67 455 Da with a pI of 5.84. Phylogenetic analysis indicates that the amino-acid sequence is most similar to Xenopus oocyte-specific GV lamin B3, but differs from somatic lamins (A, B1 or B2). In contrast to somatic lamins, neither goldfish nor Xenopus GV lamin contain conserved phosphorylation sites for nuclear transport, except the nuclear localization sequence. Therefore, we conclude that the goldfish oocyte GV is mainly comprised of GV-type lamin (the homolog of Xenopus lamin B3).     

Trypanosoma danilewskyi: host specificity and host's effect on morphometrics

Carassius auratus, Barbus conhus, Danio malabaricus, Catostomus commersoni, Notropis cornutus, Etheostoma caeruleum, and Ictalurus nebulosus were susceptible to Trypanosoma danilewskyi by intraperitoneal inoculation. Trypanosomes isolated from all species of susceptible fishes were infective to goldfish. No trypanosomes were detected in inoculated Semotilus atromaculatus, Ambloplites rupestris, Lepomis gibbosus and Perca flavescens. Fifty specimens were measured from each of 3 C. auratus, 2 B. conhus, 2 C. auratus inoculated with an isolate from a C. commersoni, 2 C. commersoni, and 2 E. caeruleum, for statistical analyses. The distance of the kinetoplast from the posterior end, length of free flagellum, nuclear length, and area of nucleus were similar among samples from the same host but differed significantly among samples from different host species. After discriminate analysis, samples from goldfish clustered into a tight group while those from the other fishes formed a loose cluster along the first canonical axis. Using the jacknife method of correct classification, 93% of trypanosomes from C. auratus, 36% from C. commersoni, 61% from C. commersoni-C. auratus, 25% from B. conhus, and 40% from E. caeruleum, could be correctly assigned to host species. However, only 1% of trypanosomes from C. commersoni would be classified as coming from C. auratus, 4% for C. commersoni-C. auratus, 0% for B. conhus, and 7% for E. caeruleum.     

Novel method for the nuclear transfer of adult somatic cells in medaka fish (Oryzias latipes): use of diploidized eggs as recipients

Until recently, the nuclear transfer of adult somatic cell nuclei in fish has been unsuccessful. This is primarily because of chromosomal aberrations in nuclear transplants, which are thought to arise due to asynchrony between the cell cycles of the recipient egg and donor nucleus. We recently succeeded in circumventing this difficulty by using a new nuclear transfer method in medaka fish ( Oryzias latipes ). Instead of enucleated eggs, the method uses non-enucleated and diploidized eggs, obtained by retention of the second polar body release, as recipients in the nuclear transfer of primary culture cells from the caudal fin of an adult green fluorescent protein gene ( GFP )-transgenic strain. We found that 2.7% of the reconstructed embryos grew into diploid and fertile adults exhibiting donor expression characteristics and transmission of the GFP marker gene to progeny. The mechanism underlying the generation of nuclear transplants using this method is unknown at present; however, analyses of donor and recipient nuclei behavior and the cytoskeletal mechanisms involved in the early developmental stages, as well as the special ability of diploidized eggs to facilitate reprogramming of the donor nuclei will result in elucidation of the mechanism.     

Identification of the goldfish 20S proteasome beta6 subunit bound to nuclear matrix

Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most of the regulated intracellular protein breakdown in eukaryotic cells. Proteasomes are present in both the nucleus and cytoplasm. When we analyzed the molecular composition of protein constituents of the nuclear matrix preparation of goldfish oocytes by two-dimensional polyacrylamide gel electrophoresis followed by sequence analysis, we found a 26 kDa spot identical in amino acid sequence to the beta6 subunits of the 20S proteasome. No spot of other subunits of 20S proteasome was detected. Here we describe the cloning, sequencing and expression analysis of Carassius auratus, beta6_ca, which encodes one of the proteasome beta subunits from goldfish ovary. From the screening of an ovarian cDNA library, two types of cDNA were obtained, one 941 bp and the other 884 bp long. The deduced amino acid sequences comprise 239 and 238 residues, respectively. These deduced amino acid sequences are highly homologous to those of beta6 subunits of other vertebrates. Immunoblot analysis of nuclear matrix using anti-proteasome antibodies showed only a spot of beta6_ca. These results suggest that the beta6 subunit of the goldfish 20S proteasome, beta6_ca, is responsible for anchoring proteasomes in the nucleus.     

Short-term cell culture technique for obtaining chromosomes in marine and freshwater fish

In this paper we describe a simple and reliable short-term fish cell culture technique, for obtaining chromosome spreads with minimum stress involving the specimens sampled. Fibroblasts were derived from primary cultures from the caudal fin of some economically important freshwater species: northern pike, Esox lucius L. , goldfish, Carassius auratus L., and marine species: gilthead sea bream, Sparus aurata L. and cardinal fish, Apogon imberbis L.     

Nuclear transfer of embryonic cell nuclei to non-enucleated eggs in zebrafish, Danio rerio

We previously established a novel method for nuclear transfer in medaka (Oryzias latipes) using non-enucleated, diploidized eggs as recipients for adult somatic cell nuclei. Here we report the first attempt to apply this method to another fish species. To examine suitability of using non-enucleated eggs as recipients for nuclear transfer in the zebrafish (Danio rerio), we transferred blastula cell nuclei from a wild-type donor strain to non-enucleated, unfertilized eggs from a golden recipient strain. As a result, 31 of 184 (16.8%) operated eggs developed normally and reached the adult stage. Twenty-eight (15.2%) of these transplants showed wild-type phenotype and the remaining three (1.6%) were golden. Except for one individual that exhibited diploid/tetraploid mosaicism, all of the wild-type nuclear transplants were either triploid or diploid. While all of 19 triploid transplants were infertile, a total of six transplants (21.4%) were fertile (five of the eight diploid transplants and one transplant exhibiting ploidy mosaicism). Except for one diploid individual, all of the fertile transplants transferred both the wild-type golden gene allele (slc24a5) as well as the phenotype, the wild-type body color, to their F(1) and F(2) progeny in a typical Mendelian fashion. PCR analysis of slc24a5 suggested that triploidy originated from a fused nucleus in the diploid donor and haploid recipient nuclei, and that the sole origin of diploidy was the diploid donor nucleus. The results of the present study demonstrated the suitability of using non-enucleated eggs as recipients for nuclear transfer experiments in zebrafish.     

家畜体细胞克隆中核重编程机理研究进展

体细胞克隆在绵羊、山羊、牛、猪等家畜中获得了成功,但目前的克隆效率非常低。克隆效率低使家畜体细胞克隆技术在畜牧业生产及其他领域的应用受到极大的限制,问题的根源在于对体细胞克隆中核重编程的分子机理缺乏了解。供体细胞核移入去核的卵母细胞后,必须经过后成表观遗传修饰的重编程,从而恢复供体细胞核的全能性,才能保证重构胚的正常发育及个体的正常生长。本文从移植核的重构、DNA甲基化总体改变、组蛋白修饰、X染色体失活、端粒长度和端粒酶活性恢复、印迹基因及其他与发育相关基因的表达及核重编程的影响因素等几个方面探讨了体细胞克隆中的核重编程机理,为克隆效率提高的方法研究提供理论依据。     

Two-staged nuclear transfer can enhance the developmental ability of goat�Csheep interspecies nuclear transfer embryos in vitro

The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animal oocytes as nuclear recipients and endangered animal or human somatic cells as nuclear donors, can afford more opportunities in endangered animal rescue and human tissue transplantation, but the application of this technique is limited by extremely low efficiency which may be attributed to donor nucleus not fully reprogrammed by xenogenic cytoplasm. In this study, goat fetal fibroblasts (GFFs) were used as nuclear donors, in vitro-matured sheep oocytes were used as nuclear recipients, and a two-stage nuclear transfer procedure was performed to improve the developmental ability of goat-sheep interspecies clone embryos. In the first stage nuclear transfer (FSNT), GFFs were injected into the ooplasm of enucleated sheep metaphase-II oocytes, then non-activated reconstructed embryos were cultured in vitro, so that the donor nucleus could be exposed to the ooplasm for a period of time. Subsequently, in the second stage nuclear transfer, FSNT-derived non-activated reconstructed embryo was centrifuged, and the donor nucleus was then transferred into another freshly enucleated sheep oocyte. Compared with the one-stage nuclear transfer, two-stage nuclear transfer could significantly enhance the blastocyst rate of goat-sheep interspecies clone embryos, and this result indicated that longtime exposure to xenogenic ooplasm benefits the donor nucleus to be reprogrammed. The two-stage nuclear transfer procedure has two advantages, one is that the donor nucleus can be exposed to the ooplasm for a long time, the other is that the problem of oocyte aging can be solved.