Research Fellow of the Japan Society for the Promotion of Science, Japan

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1 doi: +*.,+.+/jpsa.*+**2/ Copyright,*++, Japan Poultry Science Association. +,-,, Yoshiaki Nakamura, Fumitake Usui, Daichi Miyahara, Takafumi Mori, Haruhi Watanabe, Tamao Ono, Kumiko Takeda, Keijiro Nirasawa, Hiroshi Kagami and Takahiro Tagami, +,, +, + Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano -33./32, Japan National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki -*/ *3*+, Japan - Research Fellow of the Japan Society for the Promotion of Science, Japan The purpose of this study is to clarify the e# ects of freeze-thaw treatment on viability and functionality of primordial germ cells (PGCs) in chickens. PGCs were collected separately from embryonic blood of White Leghorn, Barred Plymouth Rock and Fayoumi breeds. Some PGCs were labeled with the fluorescent lipophilic carbocyanine dye to analyze functionality by transfer assay. +** PGCs were used for each experiment to ensure accuracy of the test results. In the experimental group, PGCs were slow-frozen, then stored in liquid nitrogen for + month. In the unfrozen control group, PGCs were utilized immediately. The recovery rate of PGCs after freeze-thaw was /..-. The viability of PGCs in the frozen group was significantly lower than that of the control group ( P **/. ) ( 2/1. vs. 33,. ) with no significant di# erence between the three breeds. Thirty fluorescent-labeled PGCs were randomly chosen from each set of recovered PGCs after freeze-thaw in the frozen group, and from each set of +** PGCs in the control group, for transfer into the bloodstream. Gonadal migration of transferred PGCs was observed in all embryos in both test and control groups. The number of PGCs settled in the gonads of embryos at stage,1 was /,. 2 lower in the frozen group than in the unfrozen control group ( P *.*/). In the control group, significant di# erence in establishment of PGCs in stage,1 gonads was observed between the three breeds, with White Leghorn chickens harboring the most PGCs, and Barred Plymouth Rock chickens the fewest. We conclude that freeze-thaw treatment causes a decrease in functionality of PGCs in chicken, with only an estimated.0./ of frozen PGCs being viable after thawing. Key words: chickens, cryopreservation, functionality, primordial germ cells, viability J. Poult. Sci.,.2: /1 0-,,*++ Introduction preservation of semen has been achieved (Lake and Stewart, +312 ; Hammerstedt and Graham, +33, ), but ova The collection and preservation of genetic materials are not yet available for freezing in the same way because obtained from existing animal species is urgently needed, of their large size and their yolk-laden structure. Theresince genetic materials are unrecoverable once the animals fore, cryopreservation of male gametes allows the recovhave become extinct. At present, livestock are costly to ery of only single genes from chickens at the present time. feed and livestock populations are vulnerable to disease The first identifiable precursor cells for gametes are outbreaks and environmental disasters. Cryopreservation primordial germ cells (PGCs). In chickens, approximateof cells and/or tissues obtained from remaining animals is ly -* +/* PGCs are found in the central zone of area regarded as an available means of overcoming these prob- pellucida of stage X embryos, which contain about -0 lems. Recent advances in freezing techniques enable cells. +* cells (Tsunekawa et al.,,***; Nakamura et al.,,**1; to be conserved as genetic resources semi-permanently, Eyal-Giladi and Kochav, +310). Following formation of without changing their genetic characteristics. However, the primitive streak, PGCs migrate to the anterior edge of to disseminate these genetic materials at the cellular level, the extraembryonic region, the so-called germinal crescent techniques for regenerating live animals obtained from region. As blood vessels develop in the germinal crescent conserved cells must be developed. In chickens, the cryo- region, PGCs enter the vascular network and are transported by the embryonic circulation to the future gonadal Received: September 0,,*+*, Accepted: October /,,*+* Released Online Advance Publication: November,/,,*+* region (Nakamura et al.,,**1). Recently, PGCs have Correspondence: Dr. Takahiro Tagami, National Institute of Livestock gained much attention as an available genetic resource in and Grassland Science,, Ikenodai, Tsukuba, Ibaraki -*/ *3*+, chickens. Germline chimeric chickens have been pro- Japan. ( tagami@a# rc.go.jp) duced by transferring donor PGCs from isolated sites to

2 58 Journal of Poultry Science,.2 ( + ) the same or di# erent locations in recipient embryos, where cording to the protocol of Yamamoto et al. (,**1). The they di# erentiated normally into functional gametes chicken PGCs were distinguishable from blood cells by (Petitte et al., +33* ; Tajima et al., +33- ; Vick et al., +33-; their large size and by the presence of numerous refractive Kagami et al., +331 ; Park et al.,,**-). Several techniques granules in their cytoplasm, as observed under phase for purifying PGCs obtained from embryos have been contrast microscopy (Fujimoto et al., +310). Morphoestablished: Ficoll density gradient centrifugation (Yasuda logically normal PGCs were collected through a fine glass et al., +33, ), immunomagnetic cell sorting (Ono and micropipette from the concentrated cell suspension under Machida, +333), fluorescence-activated cell sorting (Mozdziak the phase contrast microscope (IX 1+, OLYMPUS, Tokyo, et al.,,**/), ammonium chloride-potassium lysis process Japan). Sets of +** PGCs were randomly divided into the (Yamamoto et al.,,**1) and Nycodenz density gradient frozen test group and the unfrozen control group. centrifugation (in mouse: Mayanagi et al.,,**-, in chicken: Zhao and Kuwana,,**-). It has been reported that Freezing and Thawing of PGCs In the frozen group, PGCs were separately suspended in frozen-thawed PGCs give rise to functional gametes via,** ml of Cell Banker + (Juji Field, Tokyo, Japan) in a +., germline chimeric chickens (Naito et al., +33. a; Tajima et ml cryotube (Nalgene, NY, USA), and then readily froal., +332 ; Kuwana et al.,,**0; Nakamura et al.,,*+* a). zen. In the unfrozen control group, PGCs were used im- These studies support the idea that cryopreservation of mediately for viability test or transplantation. The cryo- PGCs in liquid nitrogen can preserve both female and tubes were frozen using a freezing container (Nalgene, male chicken genetic material for a relatively long time. NY, USA) in a deep freezer at 2* overnight. There- To create better conditions for enhancing PGC-me- after, the cryotubes were stored in liquid nitrogen ( +30 diated genetic resource conservation systems in chickens, ) for + month. After storage, the cryotubes were rethe utility of frozen-thawed PGCs must be examined. The moved from the liquid nitrogen and immediately placed in aim of the present study, therefore, is to clarify the e# ects water at -3 until the ice had melted. After thawing, the of freezing and thawing on viability and functionality of cell suspension was diluted in + ml of +* FBS in PBS chicken PGCs. The results obtained from this study will ( ) and centrifuged at,** g for. min to remove the yield important information regarding the conservation of cryoprotectant. The supernatant was discarded and ap- PGC-based avian genetic resources. proximately,* ml of cell suspension was placed in a plastic Materials and Methods dish. The number of recovered PGCs after freeze-thaw was counted under a microscope (Eclipse E +***, Nikon, Fertilized Eggs and Animal Care Tokyo, Japan), and expressed as recovery rate. After Fertilized eggs obtained from White Leghorn (WL), counting, the cells were utilized for viability test or trans- Barred Plymouth Rock (BPR) and Fayoumi (FA) chick- fer. ens, maintained at the National Institute of Livestock and Grassland Science (NILGS), were reproduced by artificial Viability Test of PGCs Viability assays were conducted on both the frozen insemination. All animal care and use in this study was group (,* ml cell suspension), and on the control group conducted in accordance with the animal experimentation (,* ml cell suspensions of +** PGCs in modified bu# er). guidelines of our institute (NILGS Animal Care Committee). *.. Trypan blue solution ( +* ml) was then added to each drop of PGC suspension and the mixture incubated for, Collection of Embryonic Blood and Harvest of PGCs min at room temperature. The viability of the PGCs in Freshly obtained fertilized eggs were incubated at -3.* and relative humidity /* to 0*, with tilting by both groups was determined by the Trypan blue exclusion method (Freshney, +321). 3* twice an hour, in a forced air incubator (P-**2B Transfer of PGCs Biotype; Showa Furanki, Saitama, Japan) for /* to /. h to WL embryos were prepared as recipients and then obtain embryos at stages +. to +0 (Hamburger and incubated for.2 to /, h until they reached stage +. under Hamilton, +3/+ ). The blood of the whole embryo was the conditions described above. -* PGCs labeled with collected from the dorsal aorta and terminal vein using a PKH-,0 were randomly chosen from each set of recovered fine glass micropipette under a microscope (MS/; Leica Microsystems). The collected blood was pooled in phosphate bu# ered saline without Ca and Mg (PBS ( )) separately for each chicken breed. PGCs were then con-,, centrated by Nycodenz density gradient centrifugation PGCs after freeze-thaw in the frozen group, and from each set of +** PGCs in the control group. These sets of -* PGCs were microinjected through the dorsal aorta into the bloodstream of WL embryos. Manipulated embryos were incubated for / days (up to stage,1), after (Zhao and Kuwana,,**-), with minor modifications; which whole gonads were collected. The number of PGCs namely, +* fetal bovine serum (FBS) dissolved in PBS ( ) ( +* FBS in PBS ( )) replaced the standard KAvmedium (Kuwana et al., +330 ) as a bu# er. To assay the + migration capability of PGCs to the gonads, some concentrated cells were labeled with the fluorescent lipophilic carbocyanine dye PKH-,0 (Zynaxis, Malvern, PA) ac- labeled with PKH-,0 settled in the gonads was counted under fluorescence microscopy (DFC.2*-Note OY, Leica Microsystems). Statistical Analysis Data were presented as the least squares mean (LSM) SEM. Di# erences in viability and in number of PGCs

3 Nakamura et al. : Cryopreservation of PGCs in Chickens 59 labeled with PKH-,0 were compared between frozen and breed ( P *.*/) (Figure; Table -). In the unfrozen control groups using two-way ANOVA. Recovery rate, group, moreover, the number of PGCs settled in stage,1 viability and the number of PGCs labeled with PKH-,0 gonads was dependent on breed ( P *.*/), with average between each of the three breeds was also analyzed by PGC count highest for WL embryos, and lowest for BPR two-way ANOVA. If model e# ect was found to be sig- embryos (Table -). Although the same trend was obnificant, di# erences between mean values for each treat- served in the frozen group, the di# erences were not ment were evaluated using Bonferroni s test. Significance statistically significant. was set at P **/.. Discussion Results It has been reported that chicken PGCs obtained from Recovery Rate and Viability of PGCs after Freezing and embryonic blood and gonads can be preserved in liquid Thawing nitrogen using freezing medium containing dimethyl The number of recovered and viable PGCs after freez- sulfoxide (DMSO), and that these give rise to functional ing and thawing is shown in Table +. No significant dif- gametes via germline chimeras (Naito et al., +33. a; Tajima ferences in the number of recovered and viable PGCs after et al., +332 ; Nakamura et al.,,*+* a). Recently, we have freeze-thaw was observed between the three breeds. The demonstrated that any of the commercially available average rate of viable PGCs, as measured by the Trypan DMSO-based cryoprotectants can be used to preserve blue exclusion method, was significantly lower for the chicken PGCs (Setioko et al.,,**1). Additionally, viable frozen group than for the unfrozen control group ( 2/. 1 versus 33., ; P *.*/) (Table,). o# spring have originated from transferred PGCs which had been cryopreserved using the commercial DMSObased Migration Capability of PGCs to the Gonads after Freezing cryoprotectant, Cell Banker +, as a freezing medium and Thawing (Nakamura et al.,,*+* a). In this study, therefore, Cell To analyze the functionality of PGCs after freeze-thaw Banker + was used in the freezing process. The average treatment, sets of -* fluorescent-labeled cells were trans- rate of recovered PGCs after freeze-thaw in the present ferred into the bloodstreams of recipient WL chicken study was greater than /..-, comparable to the result of embryos. Gonadal migration of transferred PGCs from our previous study (Setioko et al.,,**1). We suspected both frozen and control groups was observed in all that some of the PGCs were broken by cryogenic damage manipulated embryos. However, the number of PGCs in and hence washed out during centrifugation. To recover stage,1 gonads transferred from the frozen group was the PGCs at a higher rate after freeze/thaw, it is imporsignificantly lower than the control group, regardless of tant to reduce intracellular ice crystal formation and Table +. The number of recovered and viable primordial germ cells after freeze-thaw in each chicken breed Replication White Leghorn Barred Plymouth Rock Fayoumi Recovered Viable Recovered Viable Recovered Viable + /, , /.,.- -0./.* /* *.1./ // , -0 0, /. / /3 /* /-.3 0* /, //.0 / * 1* // 2 /-., / / /*., *.1.* /+ ++ /1 /,.3.*./ -2 +,.* -. /2.3 /../ +- /..1 /0 /* /1 /*.2.- /+.0 +/./ -3 0* / * / // // / // 01 /0 0* /* * //.2,* 0/ /. 0+ /..0.* LSM SE / / / / +40 /04*

4 60 Journal of Poultry Science,.2 ( + ) Table,. Percentage of viable primordial germ cells before and after freeze-thaw in each chicken breed Replication White Leghorn Barred Plymouth Rock Fayoumi Unfrozen ( ) Frozen ( ) Unfrozen ( ) Frozen ( ) Unfrozen ( ) Frozen ( ) + 314* 224/ +**4* * 214+, 334* * * 204* - 324* **4* 3/41 324* 2*4*. +**4* 2,40 +**4* 2/41 +**4* 214+ / 334* * 3,4/ 334* **4* 2/4, +**4* **4* 2/ * 2/4. +**4* * **4* 134, +**4* **4* 2/4+ 3 +**4* 2-4* 334* 2.4* +**4* 2*4- +* +**4* 2/4+ +**4* 214, +**4* **4* 3+4, +**4* **4* , +**4* 2/4* +**4* 2.4/ 3/4* * 214* 334* * 2/ **4* **4* * 3*4, +/ +**4* **4* **4* **4* 3*43 +**4* 3*40 +**4* **4* **4* * * * **4* * 224, 324* * 214-,* +**4* **4* 224/ +**4* 214* LSM SE 334. *40 2/41 * * * * *40 Means with di# erent superscript are significantly di# erent ( P *.*/) a b a b a b Table -. The number of primordial germ cells settled to the gonads of embryos at stage,1 befor and after freeze-thaw in each chicken breed Replication White Leghorn Barred Plymouth Rock Fayoumi Unfrozen Frozen Unfrozen Frozen Unfrozen Frozen ,/ +/ -/ +-, 0* +1,* +2.0,+ -.,,/ ,+..+,0,*,* /* +/ / -2,/ -1 +* ,1,, //,2,. +/ -. +/ 2-0,1,2, /*,., *.1,, -, +,, *.* +. -1,. +,.+ +2,, +- -, +* +- /*, *, /, / -2, ,.,* +0., +3,1 +3 -/,, +1./,*,0 +, ,/ 2, , ,/,* /+,.,3 +1-1,+ LSM SE , , * , +4- Means with di# erent superscript are significantly di# erent ( P *.*/) a cd c ef b de

5 Nakamura et al. : Cryopreservation of PGCs in Chickens 61 Fig. +. Distribution of freshly collected and frozen-thawed primordial germ cells (PGCs) in the gonads of embryos at stage,1. Localization of transferred PGCs from White Leghorns in the embryonic gonads in unfrozen control group (A-A ) and in frozen group (B-B ). A and B: Bright field images of whole mounts of gonads. Gonadal migration of donor PGCs was tracked by staining with the fluorescent lipophilic carbocyanine dye PKH-,0 (A, B ). A, B : Merged image of A and B, and A and B. Bars +** mm. cryogenic damage due to the high-concentration solutes the di# erence in viability between our current study and that form when intracellular water freezes. This may be them. achieved using hydrophilic cryoprotectants to sequester Our PGC migration assays indicate that PGCs cryowater, and by freezing rapidly to minimize ice crystal preserved in commercial cryoprotectant are biologically growth (Freshney,,**/). In this study, sets of +** PGCs functional. The number of PGCs settled in the gonads of were cryopreserved with,** ml freezing bu# er to obtain embryos at stage,1 decreased to /,. 2 (relative to the accurate recovery and viability data after the freeze-thaw control group) after freezing and thawing treatment. treatment. Viability of frozen-thawed PGCs has been Even allowing for the di# erence in viability of transferred reported to be as high as 3..,, considerably higher than PGCs between the frozen and control groups, frozenthat of our present study (Naito et al., +33. a). The report thawed PGCs exhibit a decline in their functionality. of them was conducted using much higher cell concentra- Freeze-thaw treatment is considered to lower the migrations than those present in our study (about /*** cells tion capability and/or proliferating potential of PGCs. from early embryonic blood containing -*** PGCs, This should lead to a depression of germline transmission cryopreserved in.* ml freezing bu# er containing DMSO). e$ ciency in chimeric chickens. Recently, we have de- This di# erence in cell concentration is su$ cient to explain veloped an e$ cient method for delivering busulfan ( +,.-

6 62 Journal of Poultry Science,.2 ( + ) butanediol dimethanesulfonate), an alkylating agent with freezing condition are required to improve the e$ ciency of cytotoxic e# ect on chicken germ cells, to the early chicken PGC transfer after freeze-thaw treatment. embryo (Nakamura et al.,,**2). This method allows removal of the endogenous PGCs and repopulation with exogenous PGCs in chicken embryos at early develop- Acknowledgments This work was funded by a Research Fellowship for mental stages (Nakamura et al.,,**3). Subsequently, we Young Scientists from the Japan Society for the Promohave exploited these advances for production of chimeric tion of Sciences. The authors wish to thank the sta# of the chickens in which recipient germlines were replaced by Poultry Management Section of the NILGS for bird maindonor cells with 33./ transfer success (Nakamura et al., tenance and for providing the fertilized eggs. We are,*+* b). Meanwhile, the germline transmission rate is grateful to Dr. K. Hamano of the Faculty of Agriculture, reportedly improved by increasing the number of donor Shinshu University, for his helpful advice. The present PGCs transferred into recipient embryos (Naito et al., study was supported by all members of the Animal Breed- +333). Therefore, removing endogenous PGCs of recipi- ing and Reproduction Research Team, NILGS and all ent embryos and/or increasing the number of transferring colleagues at the Laboratory of Animal Developmental PGCs appears to enhance the e$ ciency of germline trans- Genetics, Faculty of Agriculture, Shinshu University. mission of chimeras generated from frozen-thawed PGCs. In this study, three chicken breeds, WL, BPR and FA, References were used as conservation models. No significant di# erence Eyal-Giladi H and Kochav S. From cleavage to primitive streak between the three breeds was observed in the recov- formation: a complementary normal table and a new look at ery and viability rates of PGCs following freezing and the first stages of the development of the chick. I. General morphology. Developmental Biology,.3: -, thawing. Interestingly, in the control group only, the nd Freshney RI. Culture of Animal Cells., ed. pp.,./,/0. Alan number of settled PGCs in the gonads of embryos after / R Liss, New York days incubation varied significantly between the breeds. Fujimoto T, Ukeshima A and Kiyofuji R. The origin, migration The average numbers of PGCs settled in stage,1 gonads and morphology of primordial germ cells in the chicken were highest for the WL breed, and lowest for the BPR embryo. Anatomical Record, +2/ : / breed. Although the same trend was observed in the Hamburger V and Hamilton HL. A series of normal stages in the frozen group, the di# erences were below the level of development of the chick embryo. Journal of Morphology, significance, presumably because overall numbers of 22 :.3 3,. +3/+. PGCs used for statistical analysis were low in this group. Hammerstedt RH and Graham JK. Cryopreservation of poultry These results strongly indicate that transmute ability of sperm: the enigma of glycerol. Cryobiology,,3:,0-2. PGCs to migrate into the gonads and the proliferative +33,. Kagami H, Tagami T, Matsubara Y, Harumi T, Hanada H, ability of PGCs after settlement in the gonads are di# erent Maruyama K, Sakurai M, Kuwana T and Naito M. The between breeds. Our most recent study showed that the developmental origin of primordial germ cells and the trans- mission of the donor-derived gametes in mixed-sex germline e$ ciency of generating viable o# spring derived from donor PGCs is markedly enhanced when embryos with a chimeras to the o# spring in the chicken. Molecular Reproduction and Development,.2: /*+ /+* low number of endogenous PGCs are used as recipients (Nakamura et al.,,*+* a). On the other hand, Naito et al. ( +33. b) reported a more than -./ times increase in frequency of viable donor-derived progenies after transfer of WL PGCs into BPR recipients than after transfer of vice Kuwana T, Hashimoto K, Nakanishi A, Yasuda Y, Tajima A and Naito M. Long-term culture of avian embryonic cells in vitro. International Journal of Developmental Biology,.*: +*0+ +* versa, though the number of endogenous PGCs at the time Kuwana T, Kawashima T, Naito M, Yamashita H, Matsuzaki M and Takano T. Conservation of a threatened indigenous of donor PGCs transfer was about +1. times higher in fowl (Kureko Dori) using the germline chimeras transplanted from primordial germ cells. Journal of Poultry BPR than in WL (Naito et al., +33. b). These authors suggested that removal of blood containing PGCs from Science,.-: 0* 00.,**0. recipients prior to transfer of donor PGCs might be more Lake PE and Stewart JM. Preservation of fowl semen in liquid e# ective when the number of endogenous PGCs is high. nitrogen - an improved method. British Poultry Science, +3: Therefore, the results and conclusions of Naito et al. are consistent with those of our own studies. Viability tests of recovered PGCs revealed that.0./ of the total PGCs which had been frozen by the method of Mayanagi T, Kurosawa R, Ohnuma K, Ueyama A, Ito K and Takahashi J. Purification of mouse primordial germ cells by Nycodenz. Reproduction, +,/ : /.,**-. slow-freezing were available after thawing. Frozenthawed PGCs are biologically functional enough to mi- Mozdziak PE, Angerman-Stewart J, Rushton B, Pardue SL and Petitte JN. Isolation of chicken primordial germ cells using fluorescence-activated cell sorting. Poultry Science, 2. : /3. grate and settle in the gonads after transfer, however, 0**.,**/. migration capability and/or proliferation capacity of these Naito M, Matsubara Y, Harumi T, Tagami T, Kagami H, cells are reduced by cryogenic damage. Furthermore, Sakurai M and Kuwana T. Di# erentiation of donor primordial germ cells into functional gametes in the gonads of PGC functionality di# ers between chicken breeds. Further studies on cryoprotectant use and optimization of

7 Nakamura et al. : Cryopreservation of PGCs in Chickens 63 mixed-sex germline chimaeric chickens produced by transfer Park TS, Jeong DK, Kim JN, Song KH, Hong YH, Lim JM and of primordial germ cells isolated from embryonic blood. Han JY. Improved germline transmission in chicken chime- Journal of Reproduction and Fertility, ++1 :,3+, ras produced by transplantation of gonadal primordial germ Naito M, Tajima A, Tagami T, Yasuda Y and Kuwana T. cells into recipient embryos. Biology of Reproduction, 02: Preservation of chick primordial germ cells in liquid nitro- +0/1 +00,.,**-. gen and subsequent production of viable o# spring. Journal Petitte JN, Clark ME, Liu G, Verrinder Gibbins AM and Etches of Reproduction and Fertility, +*, : -,+ -,/ a. RJ. Production of somatic and germline chimeras in the Naito M, Tajima A, Yasuda Y and Kuwana T. Production of chicken by transfer of early blastodermal cells. Developgermline chimeric chickens, with high transmission rate of ment, +*2 : +2/ *. donor-derived gametes, produced by transfer of primordial Setioko AR, Tagami T, Tase H, Nakamura Y, Takeda K, and germ cells. Molecular Reproduction and Development, -3: Nirasawa K. Cryopreservation of primordial germ cells +/ b. (PGCs) from White Leghorn embryos using commercial Nakamura Y, Usui F, Atsumi Y, Otomo A, Teshima A, Ono T, cryoprotectants. Journal of Poultry Science,..: Takeda K, Nirasawa K, Kagami H and Tagami T. E# ects of,**1. busulfan sustained-release emulsion on depletion and Tajima A, Naito M, Yasuda Y and Kuwana T. Production of repopulation of primordial germ Cells in early chicken germ line chimera by transfer of primordial germ cells in the embryos. Journal of Poultry Science,.0: +,1 +-/.,**3. domestic chicken ( Gallus domesticus). Theriogenology,.*: Nakamura Y, Usui F, Miyahara D, Mori T, Ono T, Takeda K, Nirasawa K, Kagami H and Tagami T. E$ cient system for /*3 / Tajima A, Naito M, Yasuda Y and Kuwana T. Production of preservation and regeneration of genetic resources in chick- germ-line chimeras by transfer of cryopreserved gonadal en: concurrent storage of primordial germ cells and live primordial germ cells (gpgcs) in chicken. Journal of Exanimals from early embryos of a rare indigenous fowl perimental Zoology,,2*:,0/, (Gifujidori). Reproduction, Fertility and Development,,,: Tsunekawa N, Naito M, Sakai Y, Nishida T and Noce T. +,-1 +,.0.,*+* a. Isolation of chicken vasa homolog gene and tracing the Nakamura Y, Usui F, Ono T, Takeda K, Nirasawa K, Kagami H origin of primordial germ cells. Development, +,1 :,1.+ and Tagami T. Germline replacement by transfer of primor-,1/*.,***. dial germ cells into partially sterilized embryos in the chick- Vick L, Luke G and Simkiss K. Germ-line chimaeras can proen. Biology of Reproduction, 2- : +-* +-1.,*+* b. duce both strains of fowl with high e$ ciency after partial Nakamura Y, Yamamoto Y, Usui F, Atsumi Y, Ito Y, Ono T, sterilization. Journal of Reproduction and Fertility, 32: 0-1 Takeda K, Nirasawa K, Kagami H and Tagami T Increased proportion of donor primordial germ cells in Yamamoto Y, Usui F, Nakamura Y, Ito Y, Tagami T, Nirasawa chimeric gonads by sterilisation of recipient embryos using K, Matsubara Y, Ono T and Kagami H. A novel method to busulfan sustained-release emulsion in chicken. Reproduc- isolate primordial germ cells and its use for the generation of tion, Fertility and Development,,*: 3** 3*1.,**2. germline chimeras in chicken. Biology of Reproduction, 11: Nakamura Y, Yamamoto Y, Usui F, Mushika T, Ono T, Setioko ++/ ++3.,**1. AR, Takeda K, Nirasawa K, Kagami H and Tagami T. Yasuda Y, Tajima A, Fujimoto T and Kuwana T. A method to Migration and proliferation of primordial germ cells in the obtain avian germ-line chimeras using isolated primordial early chicken embryo. Poultry Science, 20 :,+2,,+3-. germ cells. Journal of Reproduction and Fertility, 30 : /,+,**1. /,2. +33,. Ono T and Machida Y. Immunomagnetic purification of viable Zhao DF and Kuwana T. Purification of avian circulating priprimordial germ cells of Japanese quail ( Coturnix japonica). mordial germ cells by nycodenz density gradient centrifuga- Comparative Biochemistry and Physiology (Part A), +,, : tion. British Poultry Science,..: -* -/.,**-.,//,/