Donor Primordial Germ Cells Research Articles

Successful formation of sperm cells from transplanted primordial germ cells in sterile interspecific avian recipients.

Primordial germ cells (PGCs) are stem cells, from which only gametes develop. In birds, the female sex is heterogametic, thus female gene conservation necessitates preservation of PGCs. PGC transplantation can generate germline chimeras in a host organism and develop into gametes. However, competition between host and transplanted PGCs hinder efficiency of germline chimera generation. We hypothezised that in sterile hybrid recipients with no germ cells of its own, transplanted donor PGCs may exclusively form gametes. Advantages of sterile hybrids as host for PGCs is compliant with many national regulations on genetically modified organisms and technically simpler procedure than the use of busulphan. Therefore, we investigated whether sterile interspecific hybrids may be suitable as recipients for supporting donor PGCs by injecting green fluorescent protein-labelled chicken PGCs into 3-day-old Guinea fowl and domestic fowl hybrid embryos and monitoring PGC development. The injected PGCs colonized almost 100% of the recipient gonads and produced mature spermatozoa after 44 weeks. However, gamete production in these hybrids was initiated much slower than in domestic fowls. This delay may be caused by suboptimal hormonal regulation of gametogenesis in the hybrids. Our results suggest that sterile interspecific hybrids may be suitable hosts for PGCs for efficient gamete production.

PGC-based cryobanking, regeneration through germline chimera mating, and CRISPR/Cas9-mediated TYRP1 modification in indigenous Chinese chickens

Primordial germ cells (PGCs) are vital for producing sperm and eggs and are crucial for conserving chicken germplasm and creating genetically modified chickens. However, efforts to use PGCs for preserving native chicken germplasm and genetic modification via CRISPR/Cas9 are limited. Here we show that we established 289 PGC lines from eight Chinese chicken populations with an 81.6% success rate. We regenerated Piao chickens by repropagating cryopreserved PGCs and transplanting them into recipient chickens, achieving a 12.7% efficiency rate. These regenerated chickens carried mitochondrial DNA from female donor PGC and the rumplessness mutation from both male and female donors. Additionally, we created the TYRP1 (tyrosinase-related protein 1) knockout (KO) PGC lines via CRISPR/Cas9. Transplanting KO cells into male recipients and mating them with wild-type hens produced four TYRP1 KO chickens with brown plumage due to reduced eumelanin production. Our work demonstrates efficient PGC culture, cryopreservation, regeneration, and gene editing in chickens.

Establishing an induced infertile chicken line for efficient germline transmission of exogenous PGCs

Primordial germ cells (PGCs) are the stem-cell population of adult animal gametes, which develop into sperm or eggs. It can be propagated in vitro and injected into the host chicken for genome editing to obtain germline chimeric chicken. However, it has the limitation that the host embryo contains endogenous PGCs, which raises complications, resultantly donor PGCs fail to compete, and transmission efficiency reduced. Therefore, to increase the transmission efficiency, we generated a novel sterile chicken with the inducible elimination of endogenous PGCs in the host. This is the first study that applied the herpes simplex virus thymidine kinase (HSV-TK) cell ablation system in avian. CRISPR/Cas9-mediated homology-directed repair was performed to localize the HSV-TK suicide gene to the last exon of the deleted in azoospermia-like (DAZL) gene, and ganciclovir (GCV) was added to induce the apoptosis in the germ cells of the host embryo. The sterilized host embryo introduced genome-edited PGCs to produce chimeric chicken carrying exogenous germ cells only. It was observed that the germline transmission efficiency was 100% achieved, and the obtained chicks were purely from donor breeds. The technologies established in the current study have important applications in germplasm conservation and gene editing in chicken.

Induced formation of primordial germ cells from zebrafish blastomeres by germplasm factors

The combination of genome editing and primordial germ cell (PGC) transplantation has enormous significance in the study of developmental biology and genetic breeding, despite its low efficiency due to limited number of donor PGCs. Here, we employ a combination of germplasm factors to convert blastoderm cells into induced PGCs (iPGCs) in zebrafish and obtain functional gametes either through iPGC transplantation or via the single blastomere overexpression of germplasm factors. Zebrafish-derived germplasm factors convert blastula cells of Gobiocypris rarus into iPGCs, and Gobiocypris rarus spermatozoa can be produced by iPGC-transplanted zebrafish. Moreover, the combination of genome knock-in and iPGC transplantation perfectly resolves the contradiction between high knock-in efficiency and early lethality during embryonic stages and greatly improves the efficiency of genome knock-in. Together, we present an efficient method for generating PGCs in a teleost, a technique that will have a strong impact in basic research and aquaculture.

Offspring production from cryopreserved primordial germ cells in Drosophila

There is an urgent need to cryopreserve Drosophila stocks that have been maintained as living cultures for a long time. Long-term culture increases the risk of accidental loss and of unwanted genetic alteration. Here, we report that cryopreserved primordial germ cells (PGCs) can produce F1 progeny when transplanted into hosts. The cryopreserved donor PGCs could form germline stem cells in host gonads and contributed to continuous offspring production. Furthermore, the ability to produce offspring did not appear to vary with either differences between donor strains or cryopreservation duration. Therefore, we propose that our cryopreservation method is feasible for long-term storage of various Drosophila strains. These results underscore the potential usefulness of our cryopreservation method for backing up living stocks to avoid either accidental loss or genetic alteration.

Investigation of the Guinea fowl and domestic fowl hybrids as potential surrogate hosts for avian cryopreservation programmes

In the last decade, avian gene preservation research has focused on the use of the early precursors of the reproductive cells, the primordial germ cells (PGCs). This is because avian PGCs have a unique migration route through the vascular system which offers easy accessibility. Furthermore, culturing of the cells in vitro, freezing/thawing, reintegration into a recipient embryo and the development of the germ cells can be carried out in well-defined laboratory circumstances. The efficient recovery of the donor genotype and the frequency of germline transmission from the surrogate host animals are still areas which need further development. Thus, the aim of the present study was to investigate an infertile interspecific hybrid (recipient) as an appropriate host for primordial germ cells from native poultry breeds. Guinea fowl × chicken hybrids were produced, the crossing was repeated inversely. The phenotype, the hatching time, the hatching rate, the sex ratio, the presence of own germ cells, the fertility and the phenotype of viable hybrids and the incidence of chromosomal abnormalities of dead hybrid embryos were described. 6.65% viable offspring was obtained with crossing of Guinea fowl females with domestic fowl males. Crossing of domestic fowl hens with Guinea fowl male resulted in lower fertility, 0.14% viable offspring. Based on the investigations, the observed offspring from the successful crossing were sterile male hybrids, thus an extreme form of Haldane’s rule was manifested. The sterile hybrid male embryos were tested by injecting fluorescently labeled chicken PGCs. The integration rate of labeled PGCs was measured in 7.5-day, 14.5-day and 18.5-day old embryonic gonads. 50%, 5.3% and 2.4% of the injected hybrid embryos survived and 40%, 5.3% and 2.4% of the examined gonads contained fluorescent labeled donor PGCs. Therefore, these sterile hybrid males may be suitable recipients for male PGCs and possibly for female PGCs although with lower efficiency. This research work shows that the sterility of hybrids can be used in gene conservation to be a universal host for PGCs of different avian species.

Germ Cell Transplantation in Avian Species.

Germcell transplantation technology has played a critical role in germline modification and preservation of genetic resources. Several germcell transplantation systems have been developed, including sperm, oocyte, or germline stemcell transplantation systems in mammals. Meanwhile, in avian species, this has mostly relied on primordial germcell (PGC) transplantation for efficient germline transmission. In this chapter, we describe how to isolate PGCs from avian embryos and produce germline chimeras through transplantation of donor PGCs to recipient embryos.

200 Optimization of Individual Stages of Chicken Transgenesis to Increase Efficiency

Primordial germ cells (PGC) are the precursors of male and female progenitor cells. The cells are considered a valuable genetic material for the production of transgenic poultry. This technology includes isolation of the PGC from chick donor embryos, transformation of the cells, and injection into the dorsal aorta of recipient embryos. After injection, the PGC are involved in the process of embryo development and differentiate into male or female sex cells. The aim of the research was to optimize the individual stages of this technology to increase the efficiency of transgenesis. The PGC were extracted from embryo gonads at stage 26 to 27 (H&H) using the trypsinization process. The trypsin concentration and incubation time were determined experimentally. Treatment of chick embryos with a 0.05% trypsin solution for 5 min was optimal for obtaining culture of embryonic cells. Separation of the PGC from other types of embryonic cells was based on a differential adhesive capacity. The maximum homogeneity of the cell population for further cultivation was established by transfer (twice) of the supernatant containing unattached cells after 1 h of cultivation in a new culture dish. The cell population is represented mainly by the PGC (81 ± 4%). Additional purification of the PGC from other cell types using magnetic-activated cell sorting (MACS) increased the proportion of these cells up to 93 ± 2%. The lentiviral transduction (pHAGE vector, ZsGreen under CMV promotor) was used to transform the resulting culture of the PGC. The efficiency of infection of PGC with lentiviral particles (TU/mL = 2.5 × 108) was 70 ± 3%. The transformed cells were injected into the dorsal aorta of recipient embryos on Day 2.5 (n = 80). Before injecting donor PGC, recipient embryos were treated with busulfan to remove the endogenous PGC. The optimal dose of busulfan was selected experimentally. A series of experiments introducing busulfan in concentrations from 50 to 250 μg into chick embryos at 24 h of incubation showed that the optimal dose was 100 μg/embryo. The efficacy of colonization of gonads with donor PGC was assessed on Day-10 embryos (n = 32) and 4-week-old hatched chickens (n = 12). Cells from gonads were studied using fluorescence microscopy, fluorescence-activated cell sorting (FACS) and qPCR. The presence of fluorescent cells in the gonads of recipients was established in both embryos and hatched chickens. The relative number of the recombinant DNA copies and the relative level of expression were confirmed by qPCR. The FACS analysis of sex cells isolated from gonads of recipients showed that the percentage of transformed germ cells reached 55.8% in females (n = 5) and 31.9% in males (n = 7). Thus, the effectiveness of poultry transgenesis can be enhanced by preparation of donor PGC for injection into embryo recipients and elimination of endogenous PGC in recipients. Both the purification of PGC from other cell types based on adhesive capacity as well as treatment of embryo recipients at 24 h incubation with busulfan (100 μg/embryo) increased the effectiveness of transgenesis. Study supported by the RSF within project No. 16-16-10059.

Inserting random and site-specific changes into the genome of chickens

During the past decade, modifications to the chicken genome have evolved from random insertions of small transgenes using viral vectors to site-specific deletions using homologous recombination vectors and nontargeted insertions of large transgenes using phi-31 integrase. Primordial germ cells (PGC) and gonocytes are the germline-competent cell lines in which targeted modifications and large transgenes are inserted into the genome. After extended periods of in vitro culture, PGC retain their capacity to form functional gametes when reintroduced in vivo. Rates of stable germline modification vary from 1 × 10−5 for nontargeted insertions to 1 × 10−8 for targeted insertions. Following transfection, clonally derived cell lines are expanded, injected into Stage 13–15 Hamburger and Hamilton embryos, and putative chimeras are incubated to term in surrogate shells. Green fluorescent protein (GFP) is incorporated into transgenes to reveal the presence of genetically modified PGC in culture and the extent of colonization of the gonad during the first week posthatch. If the extent of colonization is adequate, cohorts of putative chimeras are reared to sexual maturity. Semen is collected and the contribution from donor PGC is estimated by evaluating GFP expression using flow cytometry and PCR. The most promising candidates are selected for breeding to obtain G1 heterozygote offspring. To date, this protocol has been used to (1) knockout the immunoglobulin heavy and light chain genes and produce chickens lacking humoral immunity, (2) insert human V genes and arrays of pseudo V genes into the heavy and light immunoglobulin loci to produce chickens making antibodies with human V regions, (3) insert GFP into nontargeted locations within the genome to produce chickens expressing GFP, and (4) insert Cre recombinase into the genome to produce chickens that excise sequences of DNA flanked by loxP sites.

Inserting random and site-specific changes into the genome of chickens.

During the past decade, modifications to the chicken genome have evolved from random insertions of small transgenes using viral vectors to site-specific deletions using homologous recombination vectors and nontargeted insertions of large transgenes using phi-31 integrase. Primordial germ cells (PGC) and gonocytes are the germline-competent cell lines in which targeted modifications and large transgenes are inserted into the genome. After extended periods of in vitro culture, PGC retain their capacity to form functional gametes when reintroduced in vivo. Rates of stable germline modification vary from 1×10(-5) for nontargeted insertions to 1×10(-8) for targeted insertions. Following transfection, clonally derived cell lines are expanded, injected into Stage 13-15 Hamburger and Hamilton embryos, and putative chimeras are incubated to term in surrogate shells. Green fluorescent protein (GFP) is incorporated into transgenes to reveal the presence of genetically modified PGC in culture and the extent of colonization of the gonad during the first week posthatch. If the extent of colonization is adequate, cohorts of putative chimeras are reared to sexual maturity. Semen is collected and the contribution from donor PGC is estimated by evaluating GFP expression using flow cytometry and PCR. The most promising candidates are selected for breeding to obtain G1 heterozygote offspring. To date, this protocol has been used to (1) knockout the immunoglobulin heavy and light chain genes and produce chickens lacking humoral immunity, (2) insert human V genes and arrays of pseudo V genes into the heavy and light immunoglobulin loci to produce chickens making antibodies with human V regions, (3) insert GFP into nontargeted locations within the genome to produce chickens expressing GFP, and (4) insert Cre recombinase into the genome to produce chickens that excise sequences of DNA flanked by loxP sites.

PiggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens

Transgenic birds embody one of the most potent and exciting research tools in biotechnology for agriculture, medicine, and model animals. To date, retrovirus- or lentivirus-mediated transgenesis has been established in chickens and quail. However, despite having a valid technique for viral transduction to achieve transgenic birds, many obstacles exist for practical applications because of relatively low and variable rates of germ-line transmission and transgenic offspring showing transgene silencing, as well as safety issues related to viral vector use. Thus, the generation of transgenic poultry by nonviral integration is a prerequisite for the introduction of biotechnology to practical applications. Herein, we show that a germ-line-competent chicken primordial germ-cell (PGC) line was established with high efficiency of transmission to offspring and that piggyBac transposition into PGCs improved the efficiency of transgenic chicken production and led to high-level transgene expression. GFP transgene-expressing donor PGC-transferred recipient chickens produced donor-derived progenies, and the germ-line transmission efficiency of donor PGCs was 95.2% on average. Subsequently, half of the donor-derived offspring (52.2%) were transgenic chicks because GFP-expressing donor PGCs, in which a transgene was inserted into one chromosome 20, were heterozygous. In all of the transgenic chickens, GFP expression was constant and strong, regardless of age. Our results demonstrate that piggyBac transposition into the chicken PGC line could be the surest way to generate transgenic chickens safely for practical applications.

Interspecific Germline Transmission of Cultured Primordial Germ Cells

In birds, the primordial germ cell (PGC) lineage separates from the soma within 24 h following fertilization. Here we show that the endogenous population of about 200 PGCs from a single chicken embryo can be expanded one million fold in culture. When cultured PGCs are injected into a xenogeneic embryo at an equivalent stage of development, they colonize the testis. At sexual maturity, these donor PGCs undergo spermatogenesis in the xenogeneic host and become functional sperm. Insemination of semen from the xenogeneic host into females from the donor species produces normal offspring from the donor species. In our model system, the donor species is chicken (Gallus domesticus) and the recipient species is guinea fowl (Numida meleagris), a member of a different avian family, suggesting that the mechanisms controlling proliferation of the germline are highly conserved within birds. From a pragmatic perspective, these data are the basis of a novel strategy to produce endangered species of birds using domesticated hosts that are both tractable and fecund.

X-irradiation Removes Endogenous Primordial Germ Cells (PGCs) and Increases Germline Transmission of Donor PGCs in Chimeric Chickens

Primordial germ cells (PGCs) are embryonic precursors of germline cells with potential applications in genetic conservation, transgenic animal production and germline stem cell research. These lines of research would benefit from improved germline transmission of transplanted PGCs in chimeric chickens. We therefore evaluated the effects of pretransplant X-irradiation of recipient embryos on the efficacy of germline transmission of donor PGCs in chimeric chickens. Intact chicken eggs were exposed to X-ray doses of 3, 6 and 9 Gy (dose rate = 0.12 Gy/min) after 52 h of incubation. There was no significant difference in hatching rate between the 3-Gy-irradiated group and the nonirradiated control group (40.0 vs. 69.6%), but the hatching rate in the 6-Gy-irradiated group (28.6%) was significantly lower than in the control group (P<0.05). No embryos irradiated with 9 Gy of X-rays survived to hatching. X-irradiation significantly reduced the number of endogenous PGCs in the embryonic gonads at stage 27 in a dose-dependent manner compared with nonirradiated controls. The numbers of endogenous PGCs in the 3-, 6- and 9-Gy-irradiated groups were 21.0, 9.6 and 4.6% of the nonirradiated control numbers, respectively. Sets of 100 donor PGCs were subsequently transferred intravascularly into embryos irradiated with 3 Gy X-rays and nonirradiated control embryos. Genetic cross-test analysis revealed that the germline transmission rate in the 3-Gy-irradiated group was significantly higher than in the control group (27.5 vs. 5.6%; P<0.05). In conclusion, X-irradiation reduced the number of endogenous PGCs and increased the germline transmission of transferred PGCs in chimeric chickens.

Visualization and motility of primordial germ cells using green fluorescent protein fused to 3'UTR of common carp nanos-related gene

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. We previously visualized the PGCs of several teleostean embryos by injecting RNA synthesized from constructs encoding green fluorescent protein (GFP) fused to the 3'UTR of the zebrafish ( Danio rerio) nanos1 gene ( nos1). However, this technique was not always suitable for visualizing PGCs in embryos from all teleost species. In this study, we compared the visualization of PGCs in common carp ( Cyprinus carpio) embryos using two artificial constructs containing GFP fused to the 3'UTR of nanos from either common carp or zebrafish. Visualization was better using GFP fused to the 3'UTR of the nanos gene from common carp, compared with that from zebrafish. The visualized PGCs successfully migrated toward the gonadal ridge after transplantation into goldfish host embryos, suggesting that they maintained normal migratory motility. These techniques could be useful for the production of inter-specific germline chimeras using common carp donor PGCs.

메추리 원시생식세포 감소를 위한 감마선 조사의 효과

조류의 원시생식세포는 수용체 배자로의 주입을 통해서 생식선 카이메라 생산을 가능하게 하기 때문에 유전자 도입에 매우 효율적인 도구이다. 특히, 메추리는 성성숙이 빠르며 산란능력이 뛰어 나기 때문에 형질전환 조류 생산과 유전자 기능 연구에 매우 적합하다. 형질전환 조류 생산 효율을 높이기 위해서는 수용체 배자 내의 원시생식세포의 수를 줄이는 것이 필수적인 요소이지만, 아직까지 메추리에서 이러한 시도를 했다는 보고는 없다. 본 연구는 감마선 조사가 수용체 내의 원시생식세포의 수를 감소시킬 수 있는지 알아보았다. 먼저 0, 250, 500, 750, 1,000 rads 강도의 감마선을 갓 산란된 메추리알에 조사 후 5일령 배자에서 기형 발생빈도를 측정하였고, 0과 500 rads에서 17일째 부화율을 검정하였다. 그리고 500 rad의 감마선을 산란된 알에 73초간 조사 후 5일간 배양시킨 뒤 원시생식세포의 수를 측정하였다. 그결과, <TEX>$1{\times}10^4$</TEX>개의 세포 당 원시생식세포의 수는 수컷은 <TEX>$107.75{\pm}3.96$</TEX>에서 <TEX>$80.30{\pm}4.34$</TEX>로, 암컷에서 <TEX>$99.56{\pm}3.22$</TEX>에서 <TEX>$81.67{\pm}3.72$</TEX>로 원시생식세포수가 감소되었다. 이상의 연구 결과는 감마선 조사가 수용체 배자내의 원시생식세포를 감소시킬 수 있고, 이를 형질 전환 기술에 접목시켜서 생식선 카이메라 효율을 높일 수 있는 가능성을 보여 주고 있다. Quail is a very useful animal model for studying vertebrate development because of its small body size and unique reproductive traits. This species is also ideal model for producing germline chimeras via transferring exogenous primordial germ cells (PGCs) into the recipient embryo. To increase the contribution efficiency of donor PGCs into recipients' tissues, decreasing the population of endogenous PGCs has been rate-limiting factor. We therefore conducted this study to investigate if gamma (<TEX>$\gamma$</TEX>)-irradiation depletes endogenous PGCs in developing quail embryo. Firstly, freshly laid stage X quail embryos were irradiated with various output of <TEX>$\gamma$</TEX>-irradiation and its teratogenic effect on the embryo was evaluated. Although a dose-dependent increase in the number of embryo showing malformation was found as the output increased (0, 250, 500, 750, and 1,000 rads), only a maximum of 10.1% of embryos were abnormal in 1,000 rads. Immunocytochemical analysis using the QCR1 antibody, which is specific marker for quail PGCs, was conducted to analyze the effect of sterilization. As results, <TEX>$\gamma$</TEX>-rays at a dose-rate of 500 rads/73 sec onto undeveloped stage X embryo significantly reduced the number of germ cells to an average of 75.55 % and 82.03 % in male and female embryos, respectively. We conclude that <TEX>$\gamma$</TEX>-ray selectively targets PGCs while affects minimally to the somatic development in quail embryo. Our results will not only provide important data for germline chimera production but can be used for analyzing the effect of ionized rays on the differentiating germ cells in various stages during animal development.

Germline Replacement by Transfer of Primordial Germ Cells into Partially Sterilized Embryos in the Chicken1

We report a novel technique for almost complete replacement of the recipient germline with donor germ cells in the chicken. Busulfan solubilized in a sustained-release emulsion was injected into the yolk of fertile eggs before incubation. A dose of 100 microg was found to provide the best outcome in terms of reducing the number of endogenous primordial germ cells (PGCs) in embryonic gonads (0.6% of control numbers) and hatchability (36.4%). This was applied for preparing partially sterilized embryos to serve as recipients for the transfer of exogenous PGCs. Immunohistochemical analysis showed that the proportion of donor PGCs in busulfan-treated embryos was significantly higher than in controls (98.6% vs. 6.4%). Genetic cross-test analysis revealed that the germline transmission rate in busulfan-treated chickens was significantly higher than in controls (99.5% vs. 6.0%). Of 11 chimeras, 7 produced only donor-derived progenies, suggesting that these produced only donor-derived gametes in the recipient's gonads. This novel germline replacement technique provides a powerful tool for studying germline differentiation, for generating transgenic individuals, and for conserving genetic resources in birds.

Inter-species transplantation and migration of primordial germ cells in cyprinid fish

Primordial germ cells (PGCs) are the only cells in developing embryos that can transmit genetic information to the next generation. PGCs therefore have considerable potential value for gene banking and cryopreservation, particularly via production of donor gametes using germ-line chimeras. In some animal species, including teleost fish, the feasibility of using PGC transplantation to obtain donor-derived offspring, within and between species, has been demonstrated. Successful use of PGC transplantation to produce germ-line chimeras is absolutely dependent on the migration of the transplanted cells from the site of transplantation to the host gonadal region. Here, we induced germ-line chimeras between teleost species using two different protocols: blastomere transplantation and single PGC transplantation. We evaluated the methods using the rate of successful migration of transplanted PGCs to the gonadal region of the host embryo. First, we transplanted blastomeres from zebrafish, pearl danio, goldfish, or loach into blastula-stage zebrafish embryos. Some somatic cells, derived from donor blastomeres, were co-transplanted with the PGCs and formed aggregates in the host embryos; a low efficiency of PGC transfer was achieved. Second, a single PGC from the donor species was transplanted into a zebrafish embryo. In all inter-species combinations, the donor PGC migrated toward the gonadal region of the host embryo at a comparatively high rate, regardless of the phylogenetic relationship of the donor and host species. These transplantation experiments showed that the mechanism of PGC migration is highly conserved beyond the family barrier in fish and that transplantation of a single PGC is an efficient method for producing inter-species germ-line chimeras.

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.

Germ Cell Depletion During Embryogenesis Does Not Affect Subsequent Gonadal Differentiation in Goldfish.

In mammals, the germ line is essential for early folliculogenesis, and thus germ cell deficient animals fail to maintain ovarian differentiation. However, in teleosts, the roles of germ cells in the regulation of sex determination and differentiation are less well understood. Recent reports suggest that germ cell numbers influence ovary fate determination in zebrafish. Fish that lack germ cells or have only a single transplanted primordial germ cell (PGC) develop as phenotypic males. However, as sex determination and gonadal differentiation differ among teleost species, this phenomenon may be restricted to zebrafish or juvenile undifferentiated gonochoristic species. Here, we examined whether germ cells are necessary for gonadal differentiation in goldfish, a gonochoristic species that possesses XX-XY genetic sex determination. Our first step was to isolate the dead end (dnd) gene, which is necessary for the proper migration and survival of PGCs, and to design an anti-sense morpholino (MO) to this gene. Then, we compared germ cell development at 5 months in uninjected controls and in fish derived from embryos injected with dnd-MO at the 1-4 cell stage. In contrast to zebrafish, goldfish without germ cells developed either an ovary or a testis. Two types of gonad were identified: one was flat, wide and transparent, the other was narrow and string-like. Histological analysis showed the former to be an "empty ovary" with a clear ovarian cavity, while the latter was an "gempty testis" with a tubular structure. Thus, germ cells are not essential for gonadal fate determination in goldfish. In a subsequent experiment, we tested whether function was restored to these empty gonads in germline chimeras produced by transplanting a single PGC. Donor PGCs from embryos with an albino phenotype were labeled using gfp-nos1 3'UTR mRNA injected at the 1-4 cell stage. The embryos were enzymatically dissociated at the early somite stage and a single PGC was transplanted into each host embryo (wild type phenotype), which had been injected with dnd-MO. These germline chimeras developed either an ovary or a testis with a fully mature gonad on one side and an empty gonad on the other, showing the typical features of single PGC-transplanted chimeras. Cross breeding with male chimeric and female albino fish yielded 100% albino offspring, indicating that complete germ line replacement had occurred and the PGC-transplanted chimeras had recovered gonadal function. These findings suggest that, in goldfish, phenotypic sex is determined by somatic cells that express the relevant genes under genetic sex control rather than an interaction between germ cells and somatic cells. The generation of germline chimeras provides a valuable approach to investigating the contribution of germ cells to sex determination and differentiation, gonad formation and gametogenesis in teleosts, because of the ability to completely replace the germ line. (platform)

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.