Germ-line chimera by lower-part blastoderm transplantation between diploid goldfish and triploid crucian carp

Germ-line chimerism was successfully induced by blastoderm transplantation from donor triploid crucian carp, which reproduces gynogenetically, to recipient diploid goldfish, which reproduces bisexually. Lower part of donor blastoderm including primordial germ cells (PGCs) was sandwiched between recipient blastoderm at the mid- to late-blastula stage. When donor grafts were prepared from intact embryos or ventralized ones by removing vegetal yolk hemisphere at the 1- to 2-cell stage, malformations including double axes were observed in the resultant chimeras transplanted with grafts from intact embryos at the hatching stage, while a few malformations in those from ventralized embryos. PGCs originated from donor grafts were observed around the gonadal anlage at 10 days post-fertilization in chimeras. When ploidy of erythrocytes and epidermal cells in chimeric fish was examined by flow-cytometry, no triploid cells were detected at 1- and 5-year-old chimeras. Three-year-old chimeric fish (n=5) laid eggs originated from the donor together with those from the recipient. The frequency of eggs from the donor crucian carp blastoderm varied from 3.1 to 89.3% between chimeras.     

Zebrafish germline chimeras produced by transplantation of ovarian germ cells into sterile host larvae

High frequency production of zebrafish germline chimeras was achieved by transplanting ovarian germ cells into sterile Danio hybrid recipients. Ovarian germ cells were obtained from 3-mo-old adult Tg(vasa:DsRed2-vasa);Tg(bactin:EGFP) double transgenic zebrafish by discontinuous Percoll gradient centrifugation. An average of 755 ± 108 DsRed-positive germ cells was recovered from each female. For transplantations, a total of approximately 620 ± 242 EGFP-positive cells of which 12 ± 4.7 were DsRed-positive germ cells were introduced into the abdominal cavity under the swim bladder of 2-wk-old sterile hybrid larvae. Six weeks after transplantation, a total of 10 recipients, obtained from 2 different transplantations, were examined, and 2 individuals (20%) were identified that possessed a large number of DsRed- and EGFP-positive cells in the gonadal region. The transplanted ovarian germ cells successfully colonized the gonads and differentiated into sperm in the male hybrid recipients. Of 67 adult recipients, 12 (18%) male chimeric fish reproduced and generated normal offspring when paired with wild-type zebrafish females. The fertilization efficiency ranged from 23% to 56%. Although the fertile male chimeras were generated by transplantation of ovarian germ cells, the F1 generation produced by the male chimeras contained both male and female progeny, indicating that male sex determination in zebrafish is not controlled by sex chromosome heterogamy. Our findings indicate that a population of ovarian germ cells that are present in the ovary of adult zebrafish can function as germline stem cells, able to proliferate and differentiate into testicular germ cells and functional sperm in male recipients. The high frequency of germline chimera formation achieved with the ovarian germ cells and the convenience of identifying the chimeras in the sterile host background should make this transplantation system useful for performing genetic manipulations in zebrafish.     

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 hybridization in Nicotiana: mitochondrial DNA analysis in progenies resulting from fusion between protoplasts having different organelle constitutions

Summary Our previous studies indicated that fusion products with one functional nucleus but organelles of the two fusion partners (i.e. heteroplastomic cybrids) could be obtained by fusing X-irradiated (cytoplasmic donor) with non-irradiated (recipient) Nicotiana protoplasts. The present report deals with the analysis of mitochondria in cybrid populations resulting from the fusion of donor Nicotiana tabacum protoplasts with recipient protoplasts having a N. Sylvestris nucleus but chloroplasts of an alien Nicotiana species, and exhibiting cytoplasmic male sterility. The two fusion parents showed significant differences in restriction patterns of their chloroplast and mitochondrial DNA. Four groups of cybrid plants were obtained by this fusion. All had N. sylvestris nuclei but contained either donor or recipient chloroplasts and had either sterile or fertile anthers. There was no correlation between anther fertility and chloroplasts type. The mitochondrial DNA restriction patterns of sterile cybrids were similar to the respective patterns of the sterile fusion partner while the mitochondrial DNA restriction patterns of the fertile cybrids were similar to the respective patterns of the fertile fusion partner. The results indicate an independent assortment of chloroplasts and mitochondria from the heteroplastomic fusion products.     

Production of germ-line chimeras in rainbow trout by blastomere transplantation

We describe a technique for producing germ-line chimeric rainbow trout, Oncorhynchus mykiss, by microinjection of the isolated blastomeres. FITC-labeled donor cells and non-labeled recipient embryos at various developmental stages between the early blastula and early gastrula stages were used for cell transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40-somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient embryos, the combination of midblastula (2.5 dpf) donor cells and early blastula (1.5 dpf) recipient embryos gave the highest chimera formation rate and the best distribution pattern of donor cells. Using this combination, chimeric rainbow trout were produced with donor blastomeres from dominant orange-colored mutant embryos and wild-type recipient embryos. Of the 238 chimeric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor-derived orange body color. To test for germ-line transmission of donor cells, gametes obtained from the matured chimeras were fertilized with gametes from wild-type fish. Of the 19 matured chimeras, 6 (32%) yielded donor-derived orange-colored progeny, in addition to wild-type siblings. The contribution rates of donor cells in the germ-line ranged from 0.3 to 14%. This technique for producing germ-line chimeras should be a powerful tool for cell-mediated gene transfer in rainbow trout. Especially, if body color mutants are used for either donor cells or the host embryos, it will be possible to easily concentrate F1 transgenic embryos derived from transplanted donor cells by body color screening. Mol. Reprod. Dev. 59: 380-389, 2001.     

Differentiation of donor primordial germ cells into functional gametes in the gonads of mixed-sex germline chimaeric chickens produced by transfer of primordial germ cells isolated from embryonic blood

This study was carried out to elucidate whether primordial germ cells, obtained from embryonic blood and transferred into partially sterilized male and female recipient embryos, could differentiate into functional gametes and give rise to viable offspring. Manipulated embryos were cultured until hatching and the chicks were raised until maturity, when they were mated. When the sex of the donor primordial germ cells and the recipient embryo was the same, 15 out of 22 male chimaeric chickens (68.2%) and 10 out of 16 female chimaeric chickens (62.5%) produced donor-derived offspring. When the sex of the donor primordial germ cells and the recipient embryo was different, 4 out of 18 male chimaeric chickens (22.2%) and 2 out of 18 female chimaeric chickens (11.1%) produced donor-derived offspring. The rates of donor-derived offspring from the chimaeric chickens were 0.6-40.0% in male donor and male recipient and 0.4-34.9% in female donor and female recipient. However, the rates of donor-derived offspring from the chimaeric chickens were 0.4-0.9% in male donor and female recipient and 0.1-0.3% in female donor and male recipient. The presence of W chromosome-specific repeating sequences was detected in the sperm samples of male chimaeric chickens produced by transfer of female primordial germ cells. These results indicate that primordial germ cells isolated from embryonic blood can differentiate into functional gametes giving rise to viable offspring in the gonads of opposite-sex recipient embryos and chickens, although the efficiency was very low.     

Strain preference in donor and recipient for production of W-bearing sperm in mixed-sex germline chimeric chickens

To elucidate the strain preference in donor and recipient for the production of W-bearing sperm, mixed-sex germline chimeric chickens were produced. The combination of donor and recipient was White Leghorn (WL) and Barred Plymouth Rock (BPR), and vice versa. Four sets of mixed-sex chimeras that had the male phenotype at sexual maturity were subjected to analysis: group 1, a female WL donor and a male BPR recipient; group 2, a male WL donor and a female BPR recipient; group 3, a female BPR donor and a male WL recipient; group 4, a male BPR donor and a female WL recipient. The mean number of W-bearing sperm detected by in situ hybridization among 10000 sperm observed was 135, 158, 26 and 71 in groups 1, 2, 3 and 4, respectively. The number in group 1 was significantly higher than that of group 3 (P<0.05). And the number in group 2 was significantly higher than those of groups 3 and 4 (P<0.05). It is suggested that the combination of a WL donor and a BPR recipient produced W-bearing sperm more efficiently than the reverse combination.     

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.     

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 developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken

A novel system has been developed to determine the origin and development of primordial germ cells (PGCs) in avian embryos directly. Approximately 700 cells were removed from the center of the area pellucida, the outer of the area pellucida, and the area opaca of the stage X blastoderm (Eyal-Giladi and Kochav, 1976; Dev Biol 49:321–337). When the cells were removed from the center of the area pellucida, the mean number of circulating PGCs per 1 μl of blood was significantly decreased to 13 (P < 0.05) in the embryo at stage 15 (Hamburger and Hamilton, 1951: J Morphol 88:49–92) as compared to intact embryos of 51. When the removed recipient cells from the center of the area pellucida were replenished with 500 donor cells, no reduction in the PGC number was observed. The removal of cells from the outer of area pellucida or from the area opaca had no effect on the number of PGCs. When another set of the manipulated embryos were cultured ex vivo to hatching and reared to sexual maturity, the absence of germ cells and the degeneration of seminiferous tubules were observed in resulting chickens derived from the blastoderm from which the cells were removed from the center of the area pellucida. Chimeric embryos produced by the male donor cells and the female recipient contained the female-derived cells at 97.2% in the whole embryo and 94.3% in the erythrocytes at 5 days of incubation. At 5–7 days of incubation, masculinization was observed in about one half of the mixed-sex embryos. The proportions of the female-derived cells in the whole embryo and in the erythrocytes were 76.5% and 80.2% at 7 days to 55.7% and 62.5% at 10 days of incubation, respectively. When the chimeras reached their sexual maturity, they were test mated to assess donor contribution to their germline. Five of six male chimeras (83%) and three of five female chimeras (60%) from male donor cells and a female recipient embryo from which 700 cells at the center of area pellucida were removed were germline chimeras. Three of the five male germline chimeras (60%) and one of the three female germline chimeras (33%) transmitted exclusively (100%) donor-derived gametes into the offspring. When embryonic cells were removed from the outer of area pellucida or area opaca, regardless of the sex combination of the donor and the recipient, the transmission of the donor-derived gametes was essentially null. The findings in the present studies demonstrated, both in vivo and in vitro, that the PGCs originate in the central part of the area pellucida and that the developmental fate to germ cell (PGCs) had been destined at stage X blastoderm in chickens. Mol. Reprod. Dev. 48:501–510, 1997. © 1997 Wiley-Liss, Inc.     

SPERM PRODUCTION AND STERILITY IN HYBRIDS BETWEEN TWO SUBSPECIES OF DROSOPHILA PSEUDOOBSCURA

Subspecies of Drosophila pseudoobscura, one occurring in the United States and the other in Bogota, Columbia, exhibit Haldane's Rule in one direction of the cross. Additionally, D. pseudoobscura produces two sperm types: short, sterile sperm and long, fertile, sperm. Here I examine the relationship between the production of short and long sperm and hybrid sterility. Fertile and sterile hybrid males produce a greater proportion of short sperm compared to parental males with sterile hybrids producing mainly short, immotile sperm. Sperm transfer and storage patterns were similar between fertile hybrid and parental strains; and unexpectedly, short, immotile sperm from sterile hybrids were stored. These findings raise the question of whether different genetic mechanisms disrupt both sperm heteromorphic production and sperm motility and whether this indicates that females exert some control over sperm storage.     

Generation of germ-line chimera zebrafish using primordial germ cells isolated from cultured blastomeres and cryopreserved embryoids

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. In our previous study, a single PGC transplanted into a host differentiated into fertile gametes and produced germ-line chimeras of cyprinid fish, including zebrafish. In this study, we aimed to induce germ-line chimeras by transplanting donor PGCs from various sources (normal embryos at different stages, dissociated blastomeres, embryoids, or embryoids cryopreserved by vitrification) into host blastulae, and compare the migration rates of the PGCs towards the gonadal ridge. Isolated, cultured blastomeres not subject to mesodermal induction were able to differentiate into PGCs that retained their motility. Moreover, these PGCs successfully migrated towards the gonadal ridge of the host and formed viable gametes. Motility depended on developmental stage and culture duration: PGCs obtained at earlier developmental stages and with shorter cultivation periods showed an increased rate of migration to the gonadal ridge. Offspring were obtained from natural spawning between normal females and chimeric males. These results provide the basis for new methods of gene preservation in zebrafish.     

Analysis of the Drosophila maternal effect mutant MAT(3)1 by pole cell transplantation experiments

Summary Pole cell transplantations were used to construct germ line mosaics of the Drosophila melanogaster maternal effect mutant mat(3)1. The mutant is of particular interest since the development of embryos derived from homozygous mat(3)1 females is arrested at the pole cell stage. Such embryos form exclusively pole cells and no blastoderm cells. By means of germ line mosaics we could demonstrate the primary target tissue of mutant gene expression. For normal development the mat(3)1 ⁺gene has to be expressed in the germ line. Pole cells formed in defective embryos derived from homozygous mutant mothers were transplanted into normal recipient embryos to test their developmental potential. Heterozygous mat(3)1 pole cells were found to form fertile gametes in both sexes whereas homozygous mat(3)1 pole cells form fertile gametes only in males. The lack of progeny derived from homozygous mat(3)1 donor pole cells in recipient females further demonstrates the germ line autonomy of the mat(3)1 mutation. Pole cells from defective embryos that are transplanted into normal hosts colonize the gonads with the same frequency as donor pole cells derived from normal embryos. This indicates that mat(3)1 derived pole cells are normal with respect to their function as germ cells and that the mat(3)1 mutant might therefore offer a convenient source for the mass isolation of functional pole cells.     

提高光敏感雄性不育水稻花粉单倍体育种效率及不育花粉植株育性转换特点的研究

比较了(光敏s/正常品种)F_1及F_2为供体亲本,对在花药培养时所获得的花粉植株中不育个体/全部花粉植株之比例的影响。结果表明,以F_1为供体亲本,在所获得的二倍体花粉植株(A_1)中,不育株(长日下)约占20%左右;而从F_2分离的不育株为供体亲本,相应的比例为90%左右。对获得不育的花粉植株而言,供体亲本经过F_2的选择,在花粉一代中可以提高育种效率3—4倍。指出,以培育光敏感雄性不育系为目的的花药培养,与一般育种之花药培养采用杂种F_1为供体亲本不同,不仅应对杂种F_2代在长日照条件下进行不育株的选择,而且应在短日照下对这种不育株作育性转换的双重选择。以这种个体作为花药培养的供体亲本,可以大大提高育种效率。 在长日照下表现不育的花粉植株的育性转换具多样性。来自同一组合的不育花粉植株在晚造(短日照)条件下,其花粉有的染色,频率高且稳定;有的虽然可变为染色,但频率不高或不稳定或二者兼有;有些却一直不为Ⅰ-KⅠ染色,或即使染色频率也在10%以下。这一结果与收集全国各地15个光敏核不育系在本昕同期种值条件下的反应十分吻合。这说明通过花药培养,从特定的组合培育出所需要的光敏/光温互作或温敏型的核不育系的可能性是存在的。     

Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation

The surrogate reproduction technique, such as inter-specific spermatogonial stem cells (SSCs) transplantation (SSCT), provides a powerful tool for production of gametes derived from endangered species or those with desirable traits. However, generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily, by SSCT, has not succeeded. Here, using two small cyprinid fishes from different subfamilies, Chinese rare minnow (gobiocypris rarus, for brief: Gr) and zebrafish (danio rerio), we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure. The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis, resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells. Interestingly, the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior. When pou5f3 and chd knockout Gr SSCs were transplanted, Gr-derived genome-edited sperm was successfully produced in zebrafish. This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT, and surrogate production of genome-edited gametes from another species as well. This method is feasible to be applied to future breeding of commercial fish and livestock.

     

Spermatogonial transplantation in fish: A novel method for the preservation of genetic resources

Recent progress in genome-based breeding has created various fish strains carrying desirable genetic traits; however, methods for the long-term preservation of their genetic resources have not yet been developed, mainly due to the lack of cryopreservation techniques for fish eggs and embryos. Recently, we established an alternative cryopreservation technique for fish spermatogonia using a slow-freezing method. Furthermore, we developed a transplantation system to produce functional eggs and sperm derived from spermatogonia. Spermatogonia isolated from the testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of triploid masu salmon (Oncorhynchus masou) hatchlings of both genders. The transplanted trout spermatogonia migrated towards the gonadal anlagen of the recipient salmon, into which they were subsequently incorporated. We confirmed that the donor-derived spermatogonia resumed gametogenesis, and produced sperm and eggs in male and female recipient salmon, respectively. Fertilization of the resultant eggs and sperm produced only rainbow trout in the first filial (F₁) generation, suggesting that the sterile triploid recipient salmon produced functional eggs and sperm derived from the trout donors. A combination of spermatogonial transplantation and cryopreservation could be a powerful tool for preserving valuable fish strains with desirable genetic traits and endangered species.     

离体受精作为技术平台在被子植物有性生殖研究中的应用

被子植物的离体受精10a前在玉米中已获得成功,尽管目前只在玉米获得完全成功和小麦获得部分成功,但离体受精技术的研究成果非常显著。目前离体受精技术已被用于其他的研究,如用分离的精细胞和卵细胞筛选配子细胞的特异基因和蛋白质：研究合子细胞被激活的机理：用不同种植物的精、卵细胞体外融合进行新的远缘杂交尝试;利用合子细胞易分裂和胚胎发生特征探索用其作为转基因研究的受体细胞等。以离体受精技术为基础在高等植物发育生物学和生殖生物学领域的基础研究和应用探索显示了巨大潜力。介绍了离体受精技术在被子植物有性生殖的研究成果和应用前景,为研究和利用被子植物有性生殖过程中的生殖细胞特征提供线索。     

Male sterility caused by p6H and qk mutations is not corrected in chimeric mice

It is not known if the male sterility caused by the pleiotropic mutations p6H (pink-eyed 6H) and qk (quaking) is intrinsic or extrinsic to spermatogenic cells. This question was addressed by juxtaposing mutant and normal cells in the testes of chimeric mice and determining whether the mutant germ cells could form functional sperm. Twenty-one male chimeras consisting of normal cells and p6H/p6H or qk/qk cells were analyzed. For each, breeding productivity and testicular and sperm morphology were determined. Karyotypes and isozyme analyses were performed to identify the two cellular components of each chimera. All male chimeras that contained p6H/p6H, XY cells were sterile. Although some chimeras with a qk/qk, XY mutant component were fertile, none produced offspring from the homozygous qk component. Spermatids of the sterile chimeras showed abnormalities characteristic of the mutations. We conclude from this study that the presence of normal XY germ and somatic cells in the testis did not rescue the male sterile phenotype of homozygous p6H or qk XY germ cells. Therefore, the action of these mutant genes in causing sperm abnormalities and sterility is autonomous to the germ cells.     

Production of chimeric loach by cell transplantation from genetically pigmented to orange embryos

To establish techniques for chimera formation and to obtain further knowledge of chimerism, chimeric loach were produced using the wild strain as the donor and the orange strain as the recipient by cell transplantation. Transplantation between embryos at two different stages was performed to achieve efficient chimera formation. In the combination of the early-mid-blastula as the donor and the late-blastula as the recipient, 100-150 blastomeres were injected into the blastoderm of the recipient and the rate of chimera formation was 46.2%. On the other hand, in the combination of early-mid-blastula and early-gastrula, only 30 blastomeres were injected and the rate of chimera formation was 80.0%. These results demonstrating the combination of embryonic stages may provide a key for efficient chimera formation. We also compared the number of melanophores on chimeric larvae with that on donor cells labelled with latex beads; it was found that the number of transplanted cells has a profound effect on chimerism, whereas the site of pigmentation is not always in agreement with the site of actual transplantation of donor cells.     

Genetic and reproductive potential of spermatozoa of diploid and triploid males obtained from interspecific hybridization of Misgurnus anguillicaudatus female with M. mizolepis male

Diploid and triploid interspecific hybrid male progeny obtained from mating Misgurnus anguillicaudatus with M. mizoleis were reported to have histologically fertile and sterile testes, respectively. However, their reproductive capacity is still unclear because mating tests have not been examined using mature hybrids. Here, we examined physiological and genetic characteristics of spermatozoa of diploid and triploid hybrids. In diploid hybrid males, 1n, 2n and 4n spermatozoa showing low motility were detected. However, spermatozoa of three diploid hybrid males could generate 2n larvae. Therefore, only 1n spermatozoa of diploid hybrid males was fertile to produce larva. The chromosomes of diploid hybrid males were transmitted to spermatozoa by random segregation between the homologous chromosomes because most larvae had one allele derived from both M. anguillicaudatus and M. mizolepis at all loci examined. In triploid hybrid males, spermatozoa could be categorized to three different types based on their ploidy status. Type 1: In the first and second males, sperm samples mainly comprised 6n spermatozoa. Motility and fertility were not recorded. Type 2: The third male gave a large proportion of 6n spermatozoa as well as a small proportion of 1n spermatozoa. Although no motility was observed, larvae arose from eggs inseminated with such spermatozoa. Type 3: In the fourth male, only 1n spermatozoa were detected and their motility was vigorous. When eggs were fertilized with such 1n spermatozoa, normal larvae hatched. 1n spermatozoa of the triploid hybrid male only included the M. anguillicaudatus genome. In Misgurnus fishes, diploid hybrid males exhibited semi-sterility or slight fertility. On the contrary, triploid hybrid males were sometime fertile due to the production of 1n spermatozoa by a kind of transformation of meiosis like meiotic hybridogenesis.