Yoshiaki Nakamura

National Institutes Of Natural Sciences, Edo, Tōkyō, Japan

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Publications (20)31.93 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The production method of germline chimeras by transfer of primordial germ cells (PGCs) can be used in gene banking of chickens. However, little is known about the reproductive capability of offspring obtained by mating of male and female germline chimeras. To cryopreserve chicken genetic resources at the cellular level in future, it is necessary to demonstrate whether offspring originating from donor-derived PGCs have normal reproductive capability. In this study, we attempted to obtain the pure Hinai-droi offspring by mating of male and female germline chimeras produced by the transplantation of Hinai-dori PGCs into the Akita White Leghorn line (WL) embryos. Hinai-dori offspring from germline chimeras were screened using the Hinai-dori-specific autosomal microsatellite marker, namely, ADL0315, which allows distinction between the Hinai-dori breed and Akita WL. After sexual maturity of the Hinai-dori offspring originating from donor-derived Hinai-dori PGCs, the reproductive capability (egg production rate, fertilization rate and hatchability) was examined. Percentage hen-day egg production was investigated between 21 and 60 wk of age, and fertilization rate and hatchability were investigated between 43 and 60 wk of age, respectively. Among the 105 progeny obtained by mating of male and female germline chimeras, a single brown-colored female hatchling was obtained. The results from molecular genotyping using the Hinai-dori-specific microsatellite marker ADL0315 were consistent with results from morphological identification, and the brown-colored female hatchling was identified as the pure Hinai-dori breed. The mean percentage hen-day egg production, fertilization rate and hatchability of the Hinai-dori hen were 36.4%, 58.1% and 53.2%, which were similar to those of normal Hinai-dori hens. These results indicated that the pure Hinai-dori offspring originating from donor-derived Hinai-dori PGCs has normal reproductive capability.
    The Journal of Poultry Science 07/2014; 51(3):297-306. DOI:10.2141/jpsa.0130102 · 0.79 Impact Factor
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    ABSTRACT: Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGC-LCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline.
    The Journal of Poultry Science 01/2014; 51(1):87-95. DOI:10.2141/jpsa.0130077 · 0.79 Impact Factor
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    ABSTRACT: The Japanese quail (Coturnix japonica) is a valuable bird as both an experimental animal, for a wide range of scientific disciplines, and an agricultural animal, for the production of eggs and meat. Cryopreservation of PGCs would be a feasible strategy for the conservation of both male and female fertility cells in Japanese quail. However, the effects of freeze-thaw treatment on viability, migration ability and germline transmission ability of quail PGCs still remain unclear. In the present study, male and female PGCs were isolated from the blood of 2-day-old embryos, which were cooled by slow freezing and then cryopreserved at -196 C for 77-185 days, respectively. The average recovery rate of PGCs after freeze-thawing was 47.0%. The viability of PGCs in the frozen group was significantly lower than that of the control group (P<0.05) (95.1% vs. 85.5%). Both fresh and Frozen-thawed PGCs that were intravascularly transplanted into recipient embryos migrated toward and were incorporated into recipient gonads, although the number of PGCs settled in the gonads was 48.5% lower in the frozen group than in the unfrozen control group (P<0.05). Genetic cross analysis revealed that one female and two male recipients produced live progeny derived from the frozen-thawed PGCs. The frequency of donor-derived offspring was slightly lower than that of unfrozen controls, but the difference was not significant (4.0% vs. 14.0%). These results revealed that freeze-thaw treatment causes a decrease in viability, migration ability and germline transmission ability of PGCs in quail.
    Journal of Reproduction and Development 09/2013; 59(6). DOI:10.1262/jrd.2013-065 · 1.64 Impact Factor
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    Yoshiaki Nakamura, Hiroshi Kagami, Takahiro Tagami
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    ABSTRACT: Germ cells are the only cell type capable of transmitting genetic information to the next generation. During development, they are set aside from all somatic cells of the embryo. In many species, germ cells form at the fringe of the embryo proper and then traverse through several developing somatic tissues on their migration to the emerging gonads. Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. Unlike other species, in avian embryos, PGCs use blood circulation for transport to the future gonadal region. This unique accessibility of avian PGCs during early development provides an opportunity to collect and transplant PGCs. The recent development of methods for production of germline chimeras by transfer of PGCs, and long-term cultivation methods of chicken PGCs without losing their germline transmission ability have provided important breakthroughs for the preservation of germplasm , for the production of transgenic birds and study the germ cell system. This review will describe the development, migration, differentiation and manipulation of germ cells, and discuss the prospects that germ cell technologies offer for agriculture, biotechnology and academic research.
    Development Growth and Regeneration 01/2013; 55(1):20-40. DOI:10.1111/dgd.12026 · 2.18 Impact Factor
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    ABSTRACT: 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.
    Journal of Reproduction and Development 04/2012; 58(4):432-7. DOI:10.1262/jrd.2012-045 · 1.64 Impact Factor
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    ABSTRACT: Somatic cell nuclear transfer (SCNT) has been exploited in efforts to clone and propagate valuable animal lineages. However, in many instances, recipient oocytes are obtained from sources independent of donor cell populations. As such, influences of potential nuclear-cytoplasmic incompatibility, post SCNT, are largely unknown. In the present study, alterations in mitochondrial protein levels were investigated in adult SCNT pigs produced by microinjection of Meishan pig fetus fibroblast cells into enucleated matured oocytes (maternal Landrace genetic background). Mitochondrial fractions were prepared from liver samples by mechanical homogenization and differential centrifugation. Liver mitochondria were then subjected to two-dimensional difference gel electrophoresis (2-D DIGE). Protein expression changes were confirmed with a volume ratio greater than 2 fold (P<0.05). 2-D DIGE analysis further revealed differential expression of three proteins between the Meishan (n=3) and Landrace (n=3) breeds. Differential expression patterns of 16 proteins were detected in SCNT pig liver tissue (n=3) when compared with Meishan control samples. However, none of the 16 proteins correlated with the three differentially expressed Meishan and Landrace liver mitochondrial proteins. In summary, alteration of mitochondrial protein expression levels was observed in adult SCNT pigs that did not reflect the breed difference of the recipient oocytes. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.
    Journal of Reproduction and Development 12/2011; 58(2):248-53. DOI:10.1262/jrd.11-074A · 1.64 Impact Factor
  • Takahiro Tagami, Yoshiaki Nakamura
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    ABSTRACT: The administration of a busulfan solubilised sustained-release emulsion was used to improve the efficiency of endogenous primordial germ cell (PGC) depletion in early chicken embryos. Busulfan was solubilised in N,N-dimethylformamide and diluted 10-fold in phosphate-buffered saline [PBS(–)]. The sustained-release emulsion was prepared by mixing equal amounts of busulfan solubilised solution and sesame oil using a filter. The appropriate time and dose of busulfan sustained-release emulsion administration were established to optimise our novel germline replacement technique. Injecting busulfan sustained-release emulsion into the yolk of recipient embryos at zero hours of incubation did not disturb the movement and proliferation of donor cells during the generation of germline chimeras. A dose of 100 μg of busulfan resulted in the greatest reduction of endogenous PGCs in embryonic gonads and the most successful hatchability of sterilised embryos. Test-cross analysis revealed that the germline transmission rate in busulfan treated chickens was significantly higher than in non-treated controls (99.5 versus 6.0%). This unique and efficient germline transmission system has potential applications in the conservation of endangered and y or rare avian species.
    Avian biology research 07/2011; 4(2). DOI:10.3184/175815511X13085702790617 · 0.90 Impact Factor
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    ABSTRACT: Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for such a developmental deficiency. However, detailed knowledge of protein expression during somatic cell nuclear transfer (SCNT) in cattle is lacking. In the present study, variations in mitochondrial protein levels between SCNT-derived and control cattle, and from calves derived by artificial insemination were investigated. Mitochondrial fractions were prepared from frozen liver samples and subjected to two-dimensional (2-D) fluorescence differential gel electrophoresis (DIGE) using CyDye™ dyes. Protein expression changes were confirmed with a volume ratio greater than 2.0 (P < 0.05). 2D-DIGE analysis revealed differential expression of three proteins for SCNT cattle (n = 4) and seven proteins for SCNT calves (n = 6) compared to controls (P < 0.05). Different protein patterning was observed among SCNT animals even if animals were generated from the same donor cell source. No differences were detected in two of the SCNT cattle. Moreover, there was no novel protein identified in any of the SCNT cattle or calves. In conclusion, variation in mitochondrial protein expression concentrations was observed in non-viable, neonatal SCNT calves and among SCNT individuals. This result implicates mitochondrial-related gene expression in early developmental loss of SCNT embryos. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.
    Molecular Reproduction and Development 04/2011; 78(4):263-73. DOI:10.1002/mrd.21298 · 2.68 Impact Factor
  • The Journal of Poultry Science 01/2011; 48(1):57-63. DOI:10.2141/jpsa.010085 · 0.79 Impact Factor
  • The Journal of Poultry Science 01/2011; 48(4):281-291. DOI:10.2141/jpsa.011045 · 0.79 Impact Factor
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    ABSTRACT: The unique accessibility of chicken primordial germ cells (PGCs) during early development provides the opportunity to combine the reproduction of live animals with genetic conservation. Male and female Gifujidori fowl (GJ) PGCs were collected from the blood of early embryos, and cryopreserved in liquid nitrogen for >6 months until transfer. Manipulated GJ embryos were cultured until hatching; fertility tests indicated that they had normal reproductive abilities. Embryos from two lines of White Leghorn (24HS, ST) were used as recipients for chimera production following blood removal. The concentration of PGCs in the early embryonic blood of 24HS was significantly higher than in ST (P < 0.05). Frozen-thawed GJ PGCs were microinjected into the bloodstream of same-sex recipients. Offspring originating from GJ PGCs in ST recipients were obtained with a higher efficiency than those originating from GJ PGCs in 24HS recipients (23.3% v. 3.1%). Additionally, GJ progeny were successfully regenerated by crossing germline chimeras of the ST group. In conclusion, the cryogenic preservation of PGCs from early chicken embryos was combined with the conservation of live animals.
    Reproduction Fertility and Development 10/2010; 22(8):1237-46. DOI:10.1071/RD10056 · 2.58 Impact Factor
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    ABSTRACT: 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.
    Biology of Reproduction 03/2010; 83(1):130-7. DOI:10.1095/biolreprod.110.083923 · 3.45 Impact Factor
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    The Journal of Poultry Science 01/2010; 47(1):53-56. DOI:10.2141/jpsa.009052 · 0.79 Impact Factor
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    ABSTRACT: Individual differentiated somatic cells and undifferentiated stem cells have common genome, although their functions or morphological characters are very different. These differences are derived from difference of gene expression pattern. DNA methylation is generally key factor of Suppression of gene and its level is globally change during mammalian early development. But, in birds, whether genome-wide changes in DNA methylation occur during embryonic development is still unknown. Here, we show that genome-wide DNA methylation to assess occurrence during early chick embryonic development. We found that the methylation status at stage 1 was approximately 57%, after which it gradually decreases, reaching a minimum at stage 10 (33%). After stage 10, DNA methylation gradually increased. These results should contribute to clarify the epigenetic mechanisms in birds.
    The Journal of Poultry Science 10/2009; 46(4):286-290. DOI:10.2141/jpsa.46.286 · 0.79 Impact Factor
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    The Journal of Poultry Science 01/2009; 46(2):136-143. DOI:10.2141/jpsa.46.136 · 0.79 Impact Factor
  • The Journal of Poultry Science 01/2009; 46(2):127-135. DOI:10.2141/jpsa.46.127 · 0.79 Impact Factor
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    ABSTRACT: Recently, many results have been reported regarding the pluripotency of bone marrow cells (BMCs) with the aim of benefiting regenerative medicine for humans. Particularly, vessel formation by hematopoietic stem cells or vascular endothelial stem cells which were derived from bone marrow has received considerable interest, since the mechanism of vessel formation has been found to be involved in neoangiogenesis of serious diseases such as cancer. Most work on neoangiogenesis and regeneration has involved mammalian experimental systems, however the avian model is useful since the process of neoangiogenesis and regeneration of vessels can be observed with the whole embryo culture system. We have established a novel system using early chick embryos, where a portion of blood vessels are degenerated by UV irradiation, and vessel regeneration is then studied. Incubated embryos were partially covered with aluminum foil, from the embryonic body to the dorsal marginal vein, and irradiated with UV for 1 min. Donor BMCs were obtained from the femurs and tibias of chicks aged 10 days, fluorescently labeled with PKH26 and injected into the anterior vitelline vein of the recipients. In BMC-treated embryos the donor BMCs were observed around the UV-degenerated vessels, and regeneration of blood vessels occurred, in contrast to the untreated embryos. These results indicate that avian BMCs have the ability to participate in vessel regeneration, and the avian model used here may be a useful tool for studies of vessel neoangiogenesis and repair.
    Cells Tissues Organs 11/2008; 189(5):348-55. DOI:10.1159/000162494 · 2.14 Impact Factor
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    ABSTRACT: The aim of the present study was to improve the efficiency of endogenous primordial germ cell (PGC) depletion and to increase the ratio of donor PGCs in the gonads of recipient chicken embryos. A sustained-release emulsion was prepared by emulsifying equal amounts of Ca(2+)- and Mg(2+)-free phosphate-buffered saline containing 10% busulfan solubilised in N,N-dimethylformamide and sesame oil, using a filter. Then, 75 microg per 50 microL busulfan sustained-release emulsion was injected into the yolk. To determine the depletion and repopulation of PGCs in the gonads after 6 days incubation, whole-mount immunostaining was performed. The busulfan sustained-release emulsion significantly reduced the number of endogenous PGCs compared with control (P < 0.05). Moreover, the busulfan sustained-release emulsion significantly depleted endogenous PGCs compared with other previously reported busulfan delivery systems (P < 0.05), but with less variation, suggesting that the sustained-release emulsion delivered a consistent amount of busulfan to the developing chicken embryos. The PGC transfer study showed that the proportion of donor PGCs in the gonads of busulfan sustained-release emulsion-treated embryos after 6 days incubation increased 28-fold compared with control. In conclusion, the results demonstrate that exogenous PGCs are capable of migrating and settling in gonads from which endogenous PGCs have been removed using a busulfan sustained-release emulsion.
    Reproduction Fertility and Development 02/2008; 20(8):900-7. DOI:10.1071/RD08138 · 2.58 Impact Factor
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    ABSTRACT: A novel method was developed to isolate chick primordial germ cells (PGCs) from circulating embryonic blood. This is a very simple and rapid method for the isolation of circulating PGCs (cPGCs) using an ammonium chloride-potassium (ACK) buffer for lysis of the red blood cells. The PGCs were purified as in vitro culture proceeded. Most of the initial red blood cells were removed in the first step using the ACK lysis buffer. The purity of the cPGCs after ACK treatment was 57.1%, and the recovery rate of cPGCs from whole blood was 90.3%. The ACK process removed only red blood cells and it did not affect cPGC morphology. In the second step, the red blood cells disappeared as the culture progressed. At 7 days of in vitro culture, the purity of the PGCs was 92.9%. Most of these cells expressed germline-specific antibodies, such as those against chicken vasa homolog (CVH). The cultured PGCs expressed the Cvh and Dazl genes. Chimeric chickens were produced from these cultured PGCs, and the donor cells were detected in the gonads, suggesting that the PGCs had biological function. In conclusion, this novel isolation system for PGCs should be easier to use than previous methods. The results of the present study suggest that this novel method will become a powerful tool for germline manipulation in the chicken.
    Biology of Reproduction 08/2007; 77(1):115-9. DOI:10.1095/biolreprod.107.061200 · 3.45 Impact Factor
  • The Journal of Poultry Science 01/2007; 44(1):73-77. DOI:10.2141/jpsa.44.73 · 0.79 Impact Factor

Publication Stats

69 Citations
31.93 Total Impact Points


  • 2014
    • National Institutes Of Natural Sciences
      Edo, Tōkyō, Japan
  • 2012–2013
    • National Institute for Basic Biology
      Okazaki, Aichi, Japan
  • 2011
    • National Institute of Livestock and Grassland Science
      Ibaragi, Ōsaka, Japan
  • 2007–2010
    • Shinshu University
      • Faculty of Agriculture
      Shonai, Nagano, Japan