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.     

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.     

Expression of mRNA for 3HADH in manipulated embryos to produce germline chimeric chickens

Germline chimeric chickens were produced by the transfer of primordial germ cells (PGCs) or blastoderm cells. The hatchability of eggs produced by transfer of exogenous PGCs is usually low. The purpose of the present study was investigated to express (3-hydroxyacyl CoA dehydrogenase) 3HADH which is a limiting enzyme in the beta-oxidation of fatty acids for hatching energy. Manipulations of both donor and recipient eggshells were as follows. A window approximately 10 mm in diameter was opened at the pointed end of the eggs at stage 12–15 days incubation. Donor PGCs, taken from the blood vessels of donor embryos from fertilized eggs at the same stage of development, were injected into the blood vessels of recipient embryos. The muscles of chicks in the eggs with transferred PGCs were removed after 20 days of incubation. A cDNA was prepared from the total RNA. The expression of 3HADH in the manipulated embryos was investigated using real-time PCR analysis. Real-time PCR analysis showed that expression of 3HADH was reduced in the muscles of manipulated embryos.     

Green fluorescent protein labeling of primordial germ cells using a nontransgenic method and its application for germ cell transplantation in salmonidae

Transplanting primordial germ cells (PGCs) has a number of potential applications in fish bioengineering. Previously, we established a system to visualize live PGCs in the rainbow trout by introducing the green fluorescent protein (Gfp) gene driven by rainbow trout vasa gene regulatory regions. However, for PGC transplantation to be practically useful in aquaculture, visualization of PGCs using a nontransgenic technique is required. In this study, we demonstrate a method for labeling PGCs from various fish species by introducing chimeric RNAs composed of the Gfp coding region and vasa gene 3'-untranslated regions (UTRs); these sequences play a critical role in stabilizing mRNA in zebrafish PGCs. The GFP chimeric RNAs, including vasa 3'-UTR RNAs from rainbow trout, Nibe croaker, and zebrafish, were microinjected into the cytoplasm of fertilized eggs of several Salmonidae species. All the resulting embryos showed specific labeling in PGCs after the somatogenesis stage, which continued to be visible for at least 50 days. To apply this technique to PGC transplantation, PGCs labeled with chimeric RNA were microinjected into the peritoneal cavity of newly hatched salmonid embryos. The GFP labeling was sufficiently long-lived for the initial stage of donor PGC behavior to be followed in the recipient embryos. Importantly, donor PGCs from brown trout and masu salmon were incorporated into xenogeneic genital ridges in recipient rainbow trout. This nontransgenic method for labeling fish PGCs should be extremely useful for applications of PGC transplantation where the resulting progeny are to be released into the environment, such as PGC cryopreservation for fish stocks and surrogate brood stock technology.     

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.     

Pluripotency of cryopreserved blastomeres of the goldfish

To examine the pluripotency of cryopreserved blastomeres, we transplanted them into blastula. Donor blastomeres were prepared from blastula of goldfish (Carassius auratus) and cryopreserved in liquid nitrogen for two months. Fifty-five percent and 44% of blastomeres survived after thawing. Cryopreserved blastomeres were transplanted to the blastula of triploid crucian carp (C. a. longsdorfii), which reproduces gynogenetically in nature. At four days after the operation, resultant chimeric embryos transplanted with cryopreserved blastomeres showed a survival rate (41.6%) lower than that of embryos transplanted with unfrozen blastomeres (57.1%). Transplanted blastomeres were histologically identified in various organs derived from all three germ layers. A primordial germ cell differentiated from a cryopreserved blastomere was detected in one of the 32 chimeric fish examined. These results suggest blastomeres that survive after cryopreservation retain their pluripotency and are able to differentiate into both somatic and germ cell lines.     

四类不同倍性鲫鱼在胚胎发育期间同工酶基因表达的比较分析

Isozyme zymograms of esterase (EST), lactate dehydrogenase (LDH), malate dehydrogenase (MDH) and superoxide dismutase (SOD) were analysed by polyacrylamide gradient gel electrophoresis at different developmental stages of embryogenesis in 4 types of various ploidy crucian carp embryos, including haploids, diploids, natural triploids, and multiple tetraploids, and 2 types of haploid and diploid common carp embryos. Haploid embryos of crucian carp (Carassius auratus) and common carp (Cyprinus carpio) were produced by treating eggs with UV-irradiated milt from blunt snout bream (Megalobrama amblycephala). Natural triploid embryos were obtained from the eggs of gynogenetic silver crucian carp (Carassius auratus gibelio) inseminated with milt from red common carp. Multiple tetraploid embryos were also produced by gynogenesis from eggs of the newly discovered multiple tetraploid females inseminated with milt from red common carp. Gradient gel electrophoresis indicated that the band types and staining intensity of 4 isozymes expressed in haploid embryos of crucian carp and red common carp were similar to that in the correlative diploid embryos. In natural triploid silver crucian carp embryos, the zymograms of MDH and SOD isozymes were identical with that of diploid crucian carp embryos, but the EST and LDH isozymes manifested more new enzyme bands in comparison with diploid embryos. The corresponding expressed products of some bands in the triploid embryos, such as EST5 and EST6, could be observed also in red common carp embryos, which provided evidence for hybrid origin about the gynogenetic fish. The multiple tetraploids incorporated one foreign genome of red common carp, therefore, the effects of genes from the foreign genome could be observed in the multiple tetraploid embryos. Gene expression of the isozymes in the tetraploid embryos was somewhat similar to that in hybrids. Owing to interaction of triploid silver crucian carp genomes and common carp haploid genome, some isozyme bands, such as EST5 and EST6, changed in quantity, and some bands increased, such as s-SOD1, s-SOD2, s-SOD3 and s-SOD4 in the tetraploid embryos. Moreover, the heterogeneity was revealed among embryos developed from gynogenetic eggs of 3 different multiple tetraploid individuals.     

四类不同倍性鲫鱼在胚胎发育期间同工酶基因表达的比较分析

用聚丙烯酰胺梯度凝胶电泳比较分析了单倍体、二倍体、三倍体和复合四倍体4类不同倍性鲫鱼以及单倍体和二倍体鲤鱼在胚胎发育时期4种同工酶(EST,LDH,MDH,SOD)酶谱。结果表明,单倍体鲫鱼和单倍体鲤鱼胚胎与各自的二倍体胚胎相比,同工酶酶谱看不出差异;天然三倍体银鲫胚胎的MDH和SOD同工酶酶谱与二倍体鲫相似,但EST和LDH同工酶比二倍体增多了酶带,有的酶带如EST5和EST6还可在鲤鱼胚胎中找到相应的表达产物,提供了天然雌核发育三倍体银鲫杂交起源的证据;复合四倍体由于含有鲤鱼的一个外来基因组,其胚胎的基因表达有些与杂种类似,在所分析的4种同工酶酶谱中,都可观察到来自鲤鱼基因的影响。此外,在由源于不同复合四倍体个体的卵子发育形成的胚胎间,还观察到同工酶基因表达的异质性。     

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.     

Production of germline chimeric chickens following the administration of a busulfan emulsion

Busulfan (1,4-butanediol dimethanesulfonate) was used to deplete endogenous germ cells for the enhanced production of chicken germline chimeras. Utilizing immunohistochemical identification of primordial gem cells (PGCs) in Stage 27 chicken embryos, two delivery formulations were compared relative to the degree of endogenous PGC depletion, a busulfan suspension (BS) and a solublized busulfan emulsion (SBE). Both busulfan treatments resulted in a significant reduction in PGCs when compared to controls. However, the SBE resulted in a more consistent and extensive depletion of PGCs than that observed with the BS treatment. Repopulation of SBE-treated embryos with exogenous PGCs resulted in a threefold increase of PGCs in Stage 27 embryos. Subsequently, germline chimeras were produced by the transfer of male gonadal PGCs from Barred Plymouth Rock embryos into untreated and SBE-treated White Leghorn embryos. Progeny testing of the presumptive chimeras with adult Barred Plymouth Rock chickens was performed to evaluate the efficiency of germline chimera production. The frequency of germline chimerism in SBE-treated recipients increased fivefold when compared to untreated recipients. The number of donor-derived offspring from the germline chimeras also increased eightfold following SBE-treatment of the recipient embryos. These results demonstrated that the administration of a busulfan emulsion into the egg yolk of unincubated eggs improved the depletion of endogenous PGCs in the embryo and enhanced the efficiency of germline chimera production.     

鸡Ⅹ期胚盘细胞体外培养

为证实经遗传修饰的鸡X期胚盘细胞具有参与受体胚胎发育和形成嵌合体的能力 ,本研究将由鸡X期胚盘制成的细胞悬液与经脂质体包埋的抗鸡传染性支气管炎病毒基因重组质粒PGS1共孵育后 ,直接显微注入同期受体胚盘 (14 0枚 ) ;或对转染后供体细胞进行G418抗性筛选后显微注入同期受体鸡胚盘 (14 0枚 ) ;或将供体细胞体外培养 4 8h ,再与脂质体 PGS1复合物共孵育后显微注入同期受体鸡胚盘 (190枚 ) ,制备转基因嵌合体鸡 ,并应用PCR和RAPD方法 ,对鸡胚和雏鸡不同组织或血液中的DNA进行检测。结果表明 :直接注射组孵化率(5 7% )显著 (P <0 0 1)高于G418筛选处理组 (1 4 % )和培养 4 8h处理组 (2 1% ) ;G418筛选处理组不同胚龄鸡胚组织、器官中外源DNA的PCR检测阳性率均高于其它二个组。实验结果证明 ,体外培养 4 8h并经遗传修饰的胚盘细胞仍然具有形成嵌合体的能力 ,利用早期胚盘细胞途径制备转基因鸡是可行的。     

PRODUCTION OF GERMLINE CHIMERIC CHICKENS BY TRANSFER OF CULTURED PRIMORDIAL GERM CELLS

Primordial germ cells (PGCs) from stage 27 (5.5-day-old) Korean native ogol chicken embryonic germinal ridges were cultured in vitro for 5 days. As in in vivo culture, these cultured PGCs were expected to have already passed beyond the migration stage. Approximately 200 of these PGCs were transferred into 2.5-day-old white leghorn embryonic blood stream, and then the recipient embryos were incubated until hatching. The rate of hatching was 58.8% in the manipulated eggs. Six out of 60 recipients were identified as germline chimeric chickens by their feather colour. The frequency of germline transmission of donor PGCs was 1.3–3.1% regardless of sex. The stage 27 PGCs will be very useful for collecting large numbers of PGCs, handling of exogenous DNA transfection during culture, and for the production of desired transgenic chickens.     

Inter embryonic (homo- or hetero-sexual) transfer of primordial germ cells between chicken embryos

Chicken primordial germ cells (PGCs) collected from thecirculating blood in embryonic vessels at stage 13–15 were inter-embryonically, homo- or hetero-sexually,transferred to the blood vessels of recipient embryosat the same stage of development. Approximately 30%of the embryos treated with hetero-sexual transfer of PGCs had abnormal gonads, showing ovotestis likeorgans. In this case, some of these reversed gonadswere considered to be dependent upon the ratio of thenumber of PGCs from donor to recipient embryos. Oneof the treated embryos possessed completely reversedorgans. Therefore, the introduction of exogenousembryonic vessels was thought to be also useful forproducing transgened gonads.     

The fate of female donor blastodermal cells in male chimeric chickens.

In previous experiments in our laboratories, chickens that are chimeric in their gamete, melanocyte, and blood cell populations have been produced by injection of dispersed stage X blastodermal donor cells into the subgerminal cavity of stage X recipient embryos. In some experiments, donor cells were transfected with reporter gene constructs prior to injection as a preliminary step in the production of transgenic birds. Chimerism was assessed by test mating, observation of plumage, and DNA fingerprinting. Methods were sought that would provide a relatively rapid analysis of the spatial distribution of descendants of donor cells in chimeras to assess the efficacy of various methods of chimera construction. To date, the sex of donor and recipient embryos was not known and, therefore, numerous mixed sex chimeras must have been constructed by chance, since donor cells were usually collected from several embryos rather than from individual embryos. The presence of female-derived cells was determined by in situ hybridization using a W-chromosome-specific DNA probe, using smears of washed erythrocytes from 16 phenotypically male chimeric chickens ranging in age from 4 days to 42 months posthatching. The proportion of female cells detected in the erythrocyte samples was zero (eight samples) or very low (0.020-0.083%), although 1% of the erythrocytes from a phenotypically male chick that was killed 4 days after hatch were female-derived. The low proportions of female-derived cells were surprising, considering that most of these chimeras had been produced by the injection of cells pooled from several donor embryos and most recipients had been exposed to gamma irradiation prior to injection, thus dramatically enhancing the level of incorporation of donor cells into the resulting chimeras. By contrast, 0-100% of the erythrocytes were female-derived in blood samples taken at 10 days of incubation from the chorioallantois of seven phenotypically normal male embryos that resulted from the injection of blastodermal cells pooled from five embryos into irradiated recipient embryos. Approximately 70% of the erythrocytes in a blood sample from a phenotypically normal female chimeric embryo were female-derived, and 100% of the erythrocytes examined from an intersex embryo bearing a right testis and a left ovary were female-derived. These results indicate that female-derived cells can contribute to the formation of erythropoietic tissue during the early development of what will become a phenotypically male chimeric embryo. It would appear, therefore, that female-derived cells are blocked in development or destroyed, or certain male-female combinations of cells may be lethal prior to hatching.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12–15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.     

Germ line chimera produced by transfer of cultured chick primordial germ cells.

Intrinsic primordial germ cells (PGCs) from stage 27 (5-day-old) chick embryonic germinal ridges were cultured in vitro for a further 5 days, and shown to proliferate on stroma cells derived from the germinal ridge. To determine whether these cultured PGCs could colonize and contribute to the germ-line, PGCs were isolated by gentle pipetting, labeled with PKH26 fluorescent dye and injected into the blood stream of stage 17 (2.5-day-old) chick embryos. The recipient embryos were incubated until they reached stage 28. Thin sections of these embryos were analysed by fluorescent confocal laser microscopy. These analyses showed that the labeled donor PGCs had migrated into the germinal ridges of the recipient embryos, and transplanted PGCs had undergone at least 3-7 divisions. These results suggest that PGCs that had passed far beyond the migration stage in vivo were still able to migrate, colonize and proliferate in recipient chick embryonic gonads.     

Production of donor-derived offspring by transfer of primordial germ cells in Japanese quail.

We transfused concentrated primordial germ cells (PGCs) of the black strain (D: homozygous for the autosomal incomplete dominant gene, D) of quail into the embryos of the wild-type plumage strain (WP: d+/d+) of quail. The recipient quail were raised until sexual maturity and a progeny test of the putative germline chimeras was performed to examine the donor gamete-derived offspring (D/d+). Thirty-one percent (36/115) of the transfused quail hatched and 21 (13 females and 8 males) of them reached maturity. Five females and 2 males were germline chimeras producing donor gamete-derived offspring. Transmission rates of the donor derived gametes in the chimeric females and males were 1.8-8.3% and 2.6-63.0%, respectively. Germline chimeric and the other putative chimeric males were also test-mated with females from the sex-linked imperfect albino strain (AL: d+/d+, al/W, where al indicates the sex-linked imperfect albino gene on the Z chromosome, and W indicates the W chromosome) for autosexing of W-bearing spermatozoa: No albino offspring were born.     

Reproduction of wild birds via interspecies germ cell transplantation

The present study was conducted to apply an interspecies germ cell transfer technique to wild bird reproduction. Pheasant (Phasianus colchicus) primordial germ cells (PGCs) retrieved from the gonads of 7-day-old embryos were transferred to the bloodstream of 2.5-day-old chicken (Gallus gallus) embryos. Pheasant-to-chicken germline chimeras hatched from the recipient embryos, and 10 pheasants were derived from testcross reproduction of the male chimeras with female pheasants. Gonadal migration of the transferred PGCs, their involvement in spermatogenesis, and production of chimeric semen were confirmed. The phenotype of pheasant progenies derived from the interspecies transfer was identical to that of wild pheasants. The average efficiency of reproduction estimated from the percentage of pheasants to total progenies was 17.5%. In conclusion, interspecies germ cell transfer into a developing embryo can be used for wild bird reproduction, and this reproductive technology may be applicable in conserving endangered bird species.     

Distribution analysis of transferred donor cells in avian blastodermal chimeras.

Blastodermal chimeras were constructed by transferring quail cells to chick blastoderm. Contribution of donor cells to host were histologically analyzed utilizing an in situ cell marker. Of the embryos produced by injection of stage XI-XIII quail cells into stage XI-2 chick blastoderm, more than 50 percent were definite chimeras. The restriction on the spatial arrangement of donor cells was induced by varying the stage of host. Ectodermal chimerism was limited to the head region and no mesodermal chimerism was shown when the quail cells were injected into stage XI-XIII blastoderm. Mesodermal and ectodermal chimerisms were limited to the trunk, not to the head region, when the quail cells were injected into the stage XIV-2 blastoderm. In these chimeras, however, some of the injected quail cells formed ectopic epidermal cysts. Consequently, the stage XIV-2 blastoderm may become intolerant of the injected cells. Our results suggest that it is possible to obtain chimeras that have chimerism limited to a particular germ layer and region by varying the stage of donor cell injection. Injected quail cells contributed to endodermal tissues and primordial germ cells regardless of the injection site. The quail-chick blastodermal chimeras could be useful in the production of a transgenic chicken and in the investigation of immunological tolerance.     

Survival capacity of haploid-diploid goldfish chimeras

In teleosts, haploidy has been considered to be inviable due to the expression of abnormalities during embryogenesis, but the recent report of live haploid-diploid mosaic fish suggests the probable improvement of survival capacity by adding diploid cells or tissues to haploid embryos. In order to examine such possibilities, two types of haploid-diploid goldfish chimeric embryos were produced by transplantation of blastoderm between the normally fertilized diploid and the artificially induced gynogenetic haploid: the haploid-base chimera with the diploid upper half on the haploid lower half blastoderm and the diploid-base chimera with the haploid upper half on the diploid lower half blastoderm. Fluorescent detection of FITC-labeled cells, subsequent histochemical detection of biotin-labeled haploid cells and flow-cytometrical detection of both haploid and diploid cells proved successful induction of the haploid-diploid chimera. Both types of chimeric embryos demonstrated much better survival capacity than pure haploid individuals, but all the haploid-base chimeras died before 10 days after fertilization due to the expression of edema, whereas several diploid-base chimeras survived until 16 months after fertilization when the experiment was ended. This concluded diploid-base chimeras became viable by adding diploid cells to haploid embryos. However, the proportion of transplanted haploid cells was reduced and the distribution of these cells was limited to certain organs because survivors exhibited haploid cells only in brain, eye and/or skin. These results suggest possible elimination of haploid cells from the organs originated from ectoderm.