Yusaku Kohara, Yukio Kanai and Atsushi Tajima

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1 The Journal of Poultry Science,./: /1 0+,,**2 Copy right,**2, Japan Poultry Science Association. doi: +*.,+.+/jpsa.././1 Yusaku Kohara, Yukio Kanai and Atsushi Tajima Graduate School of Life and Environmental Sciences, University of Tsukuba, Ten-noh Dai, Tsukuba, Ibaraki -*/ 2/1,, Japan We cryopreserved gonadal germ cells (GGCs) collected from 1-day-old chick embryos using the vitrification method. Gonadal germ cells were suspended in vitrification medium and plunged directly into liquid nitrogen. The recovery rates of GGCs after freezing via vitrification and slow-freezing methods were /. and /01. -*., respectively. The survival rates of GGCs in the unfrozen control, vitrified and slow-frozen cells were 32.* *.,, 2/. 2 +., and 3+.,,. 2, respectively. The recovery and survival rates were significantly lower for vitrified GGCs than for slow-frozen GGCs ( p **/. ). To evaluate the migratory ability of cryopreserved GGCs, frozen/thawed GGCs were labeled with PKH, fluorescent dye, and,* GGCs were transferred into,-day-old chick embryos. After incubating for three days, the embryonic gonads were collected and the number of PKH, -labeled GGCs per chick was counted. These values for the control, vitrified and slow-frozen cells were 30./,-. 3, +*,.* -3.* and 3*.* -.., cells, respectively. No significant di# erences were observed among treatments ( p **/. ). These results demonstrate that it is possible to cryopreserve GGCs using vitrification, although the recovery and survival rates are lower than those observed using the slow-freezing method. Key words: chicken, cryopreservation, gonadal germ cells, migratory ability, vitrification J. Poult. Sci.,./: /1 0+,,**2 Introduction the testis or into oogonia in the ovary. PGCs originate from the epiblast (Eyal-Giladi et al., +32+ ) and appear in As the number of endangered avian species increases in the area pellucida (Swift, +3+. ). After the development of both wild (IUCN,,**0) and domesticated populations the vascular system, PGCs circulate along with embryonic (Hodges,,**0), it is increasingly important to develop blood cells and finally migrate into the embryonic gonads methods to conserve avian genetic resources. In addition, (Ukeshima and Fujimoto, +32. ; Kuwana, +33-; Kuwana avian genetic resources developed for various research and Rogulska, +333). After migration, PGCs are called purposes need to be conserved for our future use. Fur- gonadal germ cells (GGCs). Tajima et al. ( +33-) produced thermore, the genetic material of commercial parent stocks germline chimeras by transferring PGCs from needs to be preserved to reduce the risk of losses as a result White Leghorn into Rhode Island Red chickens, and vice of unexpected disease outbreaks and accidents. versa. Naito et al. ( +33. ) produced germline chimeras by In avian species, the cryopreservation of semen has transplanting cryopreserved PGCs. Tajima et al. ( +332) already been achieved in the chicken (Polge, +3/+ ), turkey also produced germline chimeras using cryopreserved (Bakst and Sexton, +313 ), duck (Maeda et al., +32. ) and GGCs collected from /-day-old embryos. All of these some non-domesticated avian species such as the Sandhill studies used the slow-freezing method and the Bicell bio- Crane (Gee et al., +32/ ). In contrast, an e# ective method freezing vessel to cryopreserve germ cells. To optimize the for the cryopreservation of avian oocytes and embryos has freezing rate in this method, the Bicell container needs to not yet been developed. Because of the unique physiolog- be placed in a deep-freeze maintained at 2*. ical and anatomical characteristics of the ovum, a method Rall and Fahy ( +32/ ) demonstrated the cryopreservaother than freezing is required for the preservation of tion of mouse embryos via vitrification. Vitrification is an oocytes. ultra-rapid cryopreservation technique that uses high concentrations Primordial germ cells (PGCs) are undi# erentiated germ of cryoprotectants that inhibit the formation cells that subsequently di# erentiate into spermatogonia in of ice crystals. Nakao et al. ( +331) developed a simplified vitrification method that uses a modified freezing solution; this method successfully produced young mice Received: April,0,,**1, Accepted: August +*,,**1 Correspondence: A. Tajima, Graduate School of Life and Environmental from vitrified embryos. However, the vitrification of chick Sciences, University of Tsukuba, Tsukuba, Ibaraki -*/ 2/1,, germ cells has not been reported until now. Therefore, we Japan. ( tajima@sakura.cc.tsukuba.ac.jp) attempted to cryopreserve avian GGCs using the vitrific-

2 58 J. Poult. Sci.,./ ( + ) ation method. ml centrifuge tube and centrifuged at 2** g for / minutes Materials and Methods at The supernatant was removed, and /* ml of PB + medium containing *.,/ M sucrose was added. Experiment + : Vitrification of GGCs b) Slow-freezing Method All equipment that came into direct contact with germ Slow-freezing was conducted according to the method cells was coated with silicone (Sigma Coat SL-,; Sigma, described by Tajima et al. ( +332), with some modific- St. Louis, MO, USA). Fertilized Y- -strain White Leg- ations. The sample was centrifuged at /** g at. for / horn (WL) eggs were incubated at for 1 days. This minutes. The supernatant was removed, and,* ml of strain was obtained from the Takikawa Animal Experi- MEM supplemented +* DMSO (MEM-D) was added. ment Station (Takikawa, Hokkaido, Japan) and main- This step was repeated twice. tained at the Agricultural and Forestry Research Center The cell suspension was incubated in ice water for / at the University of Tsukuba, Japan. After incubation, the minutes, after which,* ml of the cell suspension was embryos were dissected o# the egg yolks and washed with placed into a sterile cryogenic vial. The vial was placed, phosphate-bu# ered saline that did not contain Mg and into a biofreezing vessel (Bicell; Nihon Freezer Co., To-, Ca (PBS( ), */3+-; Nissui Pharmaceutical Co., To- kyo, Japan) and then into a deep-freezer (ULT +12/ -/- kyo, Japan). The head and tail of each embryo were fixed with needles, and the left embryonic gonad was collected D +, ; Revco, Inc., NC, USA) at 2* for at least, h. After freezing, the cryogenic vial was removed from the under an Olympus dissecting microscope (SZH; Tokyo, freezing vessel, placed in liquid nitrogen ( +30 ) and Japan). We used five embryos. stored for +- weeks. The sample was thawed at room The collected gonads were sliced using the tip of a -* G temperature and diluted stepwise by adding,*,.* and 2* dental needle (DN--*,+ K; Terumo, Tokyo, Japan) in -* ml of MEM. The diluted samples were pipetted into +./ mlof PBS ( ). The sliced gonads were placed into +./-ml ml centrifuge tubes and centrifuged at 2** g at for centrifuge tubes containing *.*/ trypsin (,*-- ++-*, ; / minutes. After centrifugation, the supernatant was re- Wako, Osaka, Japan). The tubes were incubated at placed with,** ml of MEM. for,* minutes. c) Control After the trypsin treatment, an equal volume of bovine In the control, the samples were centrifuged at 2** g at serum (B-*11+; Sigma, St. Louis, MO, USA) was added. for / minutes, after which the supernatant was reto the centrifuge tubes and agitated to form a cell suspen- placed with,* ml of MEM. This step was repeated twice. sion containing GGCs. To remove the large cell clusters A +* ml aliquot of sample was then diluted in 3* ml of and undigested tissue fragments, the cell suspension was MEM. filtered through a,*-mm nylon mesh filter (CMN-,*-D; Observation Small Parts Inc, Florida, USA) and centrifuged at,** g The viability of GGCs was evaluated three times for for +* minutes. each sample using trypan blue staining. The recovery rate After centrifugation, the supernatant was removed, and (the number of GGCs after freezing divided by number of an equal volume of minimum essential medium (MEM, GGCs before freezing) was calculated in triplicate for M *103; GIBCO BRL, New York, USA) containing +* each sample. Gonadal germ cells were identified by the bovine serum was added. The cell suspension was allo- same morphological criteria as circulating PGCs, i.e., cated into the one of the three experimental treatments: large granulated cells with large nuclei (Minematsu et al., vitrification, slow-freezing, or unfrozen control.,**.). a) Vitrification method Statistical Analysies Vitrification was performed according to the method The data were analyzed using one-way analysis of varidescribed by Nakao et al. ( +331), with some modific- ance (ANOVA) followed by Duncan s multiple range ations. The germ cell suspension was centrifuged at /** g tests. at. for / minutes, and the supernatant was replaced Experiment,: The Migratory ability of vitrified GGCs with,* ml of PB+ medium (Nakao et al., +331) containing Approximately.* frozen/thawed GGCs were collected +* DMSO. This step was repeated twice. The sample using a fine glass pipette and incubated in medium containing was then incubated in ice water for / minutes, after which PKH, fluorescent dye (ZPKH,-GL; Zynaxis, PA, +* ml of the cell suspension was placed into a sterile USA) at for / minutes. After staining,,* GGCs cryogenic vial (/***-**+,; Nalgene Co., NY, USA), followed were collected and injected through a window into the by the slow addition of 3* ml of DAP,+- medium dorsal aorta of stage embryos (Hamburger and (Nakao et al., +331). The vial was incubated in ice water Hamilton, +3/+ ). Eggshell windows were prepared +, for / minutes and then plunged into liquid nitrogen hours prior to germ cell injection; during this time, approximately ( +30 ). After storing the sample for + - weeks, the,/.. ml of blood was removed from the sample was thawed by adding /** ml of PB+ medium embryo using a fine glass pipette. containing *,/. M sucrose and warmed to -1. The frozen sample was then incubated in a water bath After germ cell injection, the eggshell window was sealed with plastic tape, and the embryo was allowed to maintained at -1, after which it was transferred to a +./ develop in an incubator for three days. The collection of

Kohara et al. : Vitrification of GGCs in Domestic Chicken 59 GGCs from embryonic gonads was conducted according secting microscope and placed into -* ml of PBS ( ).

3 Kohara et al. : Vitrification of GGCs in Domestic Chicken 59 GGCs from embryonic gonads was conducted according secting microscope and placed into -* ml of PBS ( ). to the method described by Minematsu et al. (,**.), with After slicing the gonads, an equal volume of *.*/ trypsome modifications. After incubation, the gonads were sin was added into the PBS ( ) containing gonadal collected from the GGC recipient embryos under a dis- suspension, which was then incubated at for +* minutes. The suspension was diluted and mixed with an equal volume of bovine serum. The suspension was placed on the HTCS and observed under a fluorescence microscope (IMT, RFC; Olympus, Tokyo, Japan) using a.3*-nm excitation filter to count the number of fluorescent GGCs. The migratory ability of frozen/thawed GGCs was determined by counting the total number of fluorescent GGCs in the recipient gonads. The data were analyzed, using Chi-square ( c ) tests. The proliferation rate of GGCs was estimated by calculating the ratio of the number of fluorescent GGCs to the number of transferred GGCs. Results Experiment + As shown in Figure +, the morphology of GGCs was similar to circulating PGCs. The survival of cryopreserved GGCs was determined using trypan blue staining (Fig. + ) and the survival rates of GGCs in the control, vitrified and slow frozen cells were 32*. *,., 2/2. +,. and 3+.,,. 2, respectively (Table + ); there was no significant di# erence among treatments ( p *.*/). The recovery rates of vitrified and slow-frozen GGCs were / and / *, respectively. The recovery rate of vitrified GGCs was significantly lower than that of the slow-frozen cells ( p **/. ). Experiment, For the migratory ability of transferred GGCs (Table,), the proportion of recipient embryos with fluorescent GGCs from the control, vitrified and slow-frozen samples were 2*.*, 2*.* and 0*.*, respectively. The number of fluorescent GGCs per embryo from the control, vitrified and slow-frozen samples were 30./,-. 3, +*,.* -3.* and 3*.* -.., cells, respectively ( p **/. ). No significant di# erence in proliferation rate was observed among the treatment groups; the values for the control, vitrified and slow-frozen GGCs were.2., /+. and./. times, respectively. Fig. +. Morphological characteristics of intact GGCs and trypan blue stained GGCs after cryopreservation. A: PGCs collected from dorsal aorta of,-day-old embryos. B: GGCs collected from gonad of 1-day-old embryo. C: Frozen-thawed GGCs with Trypan blue staining. Black arrow: trypan blue unstained live GGC: White arrow: trypan blue stained dead GGC. Black arrowhead: somatic cell. PGCs: Primordial Germ Cells, GGCs: Gonadal Germ Cells. Table +. Survival and recovery rates for GGCs after cryopreservation Control Vitrification Slow-freezing Survival rate ( ) Recovery rate ( ) a 324* *4, c 2/42 +4, / b 3+4,,42 /041-4* Recovery rate; (the rate of frozen GGCs/unfrozen GGCs) +**. ac : Values with di# erent letters within columns are significantly di# erent ( P *.*/). GGCs: Gonadal germ cells. b a

4 60 J. Poult. Sci.,./ ( + ) Table,. Migratory ability of transferred GGCs Recipient embryo with Number of fluorescent Proliferation rate fluorescent GGCs GGCs per embryo + ( ) Control 2* (.//) 304/, Vitrification 2* (.//) +*, 4* -34* /4+ Slow- freezing 0* (-//) 3*4* -.4,.4/ Proliferation rate; (the ratio of the number of fluorescent GGCs to the number of transferred GGCs) +**. GGCs: Gonadal germ cells. +, No significant di# erence among groups was detected using a c test ( P *.*/). Discussion sumed were the injected GGCs, were observed in all treatments. No significant di# erences in migratory ability Even though the survival rate of vitrified GGCs was were observed among treatments. In addition, the number significantly lower than that of slow-frozen cells ( p of fluorescent GGCs in all treatments was approximately **/. ), it was still possible to retrieve viable GGCs that 3* +** cells, which is./. / times the number of injected could successfully migrate to the embryonic gonad after cells. These results suggest that vitrified GGCs have northey were transferred into the blood stream of,-day-old mal migratory ability, as well as the ability to proliferate in embryos. the embryonic gonads. We encountered a significant disadvantage of vitrific- In conclusion, vitrification is a simple and inexpensive ation: the recovery rate of vitrified cells was significantly method that can be used to cryopreserve avian genetic lower than that of slow-frozen cells. However, because material in the field. Future studies are required to the method that we used was optimized for the vitrific- determine whether progeny can be produced from vitrified ation of mouse embryos, it may be possible to improve the GGCs. recovery rate of avian germ cells through a similar optimization process. Acknowledgments Several cryoprotectants have been used in freezing We thank the sta# at the Animal Science Division of the mammalian germ cells, e.g., ethylene glycol (EG) and Agricultural and Forestry Research Center, University of polyvinyl-pyrrolidone for mouse oocytes and embryos Tsukuba, for technical assistance. This study was sup- (Leibo and Oda, +33-). EG, Ficoll and sucrose have been ported in part by a Grant-in-aid awarded to AT from the used to cryopreserve mouse embryos (Kasai et al., +33* ) Ministry of Education, Culture, Sports, Science and Techand bovine embryos (Tachikawa et al., +33- ). A combi- nology, Japan ( +0-2*+2. ). nation of EG and DMSO has been used to cryopreserve bovine oocytes (Vajta et al., +332). DMSO has also been References used in combination with acetamide and propylene glycol Bakst MR and Sexton TJ. Fertilizing capacity and ultrastructure to cryopreserve mouse embryos (Nakao et al., +331). An of fowl and turkey spermatozoa before and after freezing. extensive study into the selection and concentrations of Journal of Reproduction and Fertility, //: Chen SU, Lien YR, Cheng YY, Chen HF, Ho HN and Yang YS. cryoprotectants is required to optimize the vitrification Vitrification of mouse oocytes using closed pulled straws methods for avian germ cells. (CPS) achieves a high survival and preserves good patterns The superficial dimensions of the freezing vessel strongly a# ect the cooling rate, making this an important factor open pulled straws (OPS) and grids. Human Reproduction, of meiotic spindles, compared with conventional straws, in the optimization of vitrification. Recent studies have +0 :,-/*,-/0.,**+. investigated the e# ects of several freezing vessels in working Eyal-Giladi H, Ginsburg M and Farbarov A. Avian primordial with mouse oocytes and embryos, including cryotubes germ cells are of epiblastic origin. Journal of Embryology (Nakao et al., +331), glass micropipettes (Kong et al., and Experimental Morphology, 0/ : ,***), the open pulled straw (Kong et al.,,***) and the Gee GF, Bakst MR and Sexton TJ. Cryogenic preservation of closed pulled straw (Chen et al.,,**+). The low recovery semen from the greater sandhill crane. Journal of Wildlife rates that we observed after vitrification could be improved by experimenting with di# erent freezing vessels. Management,.3:.2* /. Hamburger V and Hamilton HL. A series of normal stage in the development of the chick embryo. Journal of Morphology, Shaw et al. ( +33, ) studied the e# ect of exposure time to 2 :.3 3,. +3/+. cryoprotective agents prior to vitrification on post-thaw Hodges J. Conservation of genes and culture: historical and survival and developmental ability in mouse oocytes. contemporary issues. Poultry Science 2/ :,**,*3.,**0. They found that shorter exposure times before vitrification IUCN Red List (,**0) corresponded to increased survival rates and developmental Kasai M, Komi JH, Takakamo A, Tsunoda H, Sakurai T and ability. Machida T. A simple method for mouse embryo cryo- In Experiment,, PKH, -positive GGCs, which we aspreservation in a low-toxicity vitrification solution, without

5 Kohara et al. : Vitrification of GGCs in Domestic Chicken 61 appreciable loss of viability. Journal of Reproduction and Rall and Fahy. Ice-free cryopreservation of mouse embryos at Fertility, 23: *. +30 by vitrification. Nature, -+-: /1- /1/. +32/. Kong LK, Lee SI, Cho SG, Cho SK and Park CS. Comparison of Shaw PW, Bernard AG, Fuller BJ, Hunter JH and Shaw RW. open pulled straw (OPS) vs glass micropipette (GMP) vit- Vitrification of mouse oocytes using short cryoprotectant rification in mouse blastocysts. Theriogenology, /-: +2+1 exposure: e# ects of varying exposure time in survival. Mo- +2,0.,***. Kuwana T. Migration of avian primordial germ cells toward the lecular Reproduction and Development, --:,+*, ,. Swift CH. Origin and early history of the primordial germ cells in gonadal anlage. Development, Growth and Di# erentiation, the chick. American Journal of Anatomy, +-/ : /+ 1* /:,-1, Kuwana T and Rogulska T. Migratory mechanisms of chick Tachikawa S, Otoi T, Kondo S, Matida T and Kasai M. Successful vitrification of bovine blastocysts derived by in vitro primordial germ cells toward gonadal anlage. Cellular and maturation and fertilization. Molecular Reproduction and Molecular Biology,./: 1,/ Development, -.:,00, Leibo SP and Oda K. High survival of mouse zygotes and em- Tajima A, Naito M, Yasuda Y and Kuwana T. Production of bryos cooled rapidly or slowly in ethylene glycol plus poly- germ line chimera by transfer of primordial germ cells in the vinylpyrrolidone. Cryo-Letters, +. : domestic chicken ( Gallus domesticus). Theriogenology,.*: Maeda T, Terada T and Tsutsumi Y. Morphological observations /*3 / on frozen and thawed muscovy spermatozoa. British Poul- Tajima A, Naito M, Yasuda Y and Kuwana T. Production of try Science,,/:.* germ-line chimeras by transfer of cryopreserved gonadal Minematsu T, Tajima A and Kanai Y. The migratory ability of primordial germ cells (gpgcs) in chicken. Journal of Exgonadal germ cells in the domestic chicken. Journal of perimental Zoology,,0*:,0/, Poultry Science,.+: /.,**.. Ukeshima A and Fujimoto T. Ultrastructure of primordial germ Naito M, Tajima A, Tagami T, Yasuda Y and Kuwana T. Pre- cells in the early chick embryo. In: Ultrastructure of Reproservation of chick primordial germ cells in liquid nitrogen duction: Gametogenesis, Fertilization, and Embryogenesis and subsequent production of viable o# spring. Journal of (Van Blerkom J and Motta PM Eds). pp. +, +2. Martinus Reproduction and Fertility, +*, : -,+ -,/ Nakao K, Nakagata N and Katauki M. Simple and e$ cient Nijho# Publishers, Boston Vajta G, Holm P, Kawayama M, Greve T and Callesen H. Open vitrification procedure for cryopreservation of mouse em- pulled straw (OPS) vitrification: a new way to reduce bryos. Experimental Animals,.0:,-+, cryoinjuries of bovine ova and embryos. Molecular Repro- Polge C. Functional survival of fowl spermatozoa after freezing duction and Development, /+: /- / at 13. Nature, +01: 3.3 3/*. +3/+.

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

Research Fellow of the Japan Society for the Promotion of Science, Japan http://www.jstage.jst.go.jp/browse/jpsa doi: +*.,+.+/jpsa.*+**2/ Copyright,*++, Japan Poultry Science Association. +,-,, + + + + Yoshiaki Nakamura, Fumitake Usui, Daichi Miyahara, Takafumi Mori, Haruhi