GENETICS The potential for archiving and reconstituting valuable strains of turkey (Meleagris gallopavo) using primordial germ cells Alexander J. Wade,*1 Nick A. French,† and Grenham W. Ireland‡ *EW Group, Platts Lane, Old Moss, Stapleford, Tarvin, Chester, CH3 8HR, United Kingdom; †Aviagen, 11 Lochend Rd., Edinburgh, Midlothian, EH28 8SZ, United Kingdom; and ‡University of Manchester, Faculty of Life Sciences, Oxford Rd., M13 9PT, United Kingdom 5.4 PGC recovered, respectively. Primordial germ cells were frozen using Dulbecco’s modified Eagle medium, 20% fetal calf serum, and 10% dimethylsulfoxide and demonstrated 90 ± 1.7% viability after 3 mo frozen in liquid nitrogen. Freshly isolated and frozen thawed DiI- and Q-Tracker-labeled PGC repopulated stage 30 gonads after vascular transfer into ex ovo cultured embryos. The DiI-labeled cells repopulated gonads less frequently, with 36 ± 13.2% of gonads containing the DiI-labeled PGC, and 7 ± 3.8% of reinjected PGC reaching the gonads of positive embryos. The Q-tracker-labeled cells were detected more frequently in embryos, with 67 ± 21.1% having positive signals, and 44 ± 4.9% of reinjected Q-tracker-labeled PGC colonized the gonads of positive embryos. This study demonstrated the feasibility of using turkey PGC to archive turkey germplasm from different strains because frozen PGC reintroduced into host embryos can colonize the host gonads, suggesting the possibility of producing turkey germline chimeras.

Key words: primordial germ cell, turkey, germline chimera, cryopreservation, genetic conservation 2014 Poultry Science 93:799–809 http://dx.doi.org/10.3382/ps.2013-03629

INTRODUCTION

loss of different strains of poultry, and as a consequence a vast reduction of valuable/rare genetic material from the gene pool. Given such a scenario, it is imperative that poultry germplasm be preserved to maintain its current genetic variation (Moore et al., 2006). Primordial germ cells (PGC) are stem cell precursors to the gametes. Unlike somatic cells PGC contain all the genetic information that is capable of being transferred to the next generation upon their maturation into mature gametes (D’Costa et al., 2001). Primordial germ cells demonstrate a complex migratory pathway throughout the developing avian embryo. Initially PGC are identified on the ventral surface of the stage X– XIV chicken or equivalent stage VII–XI turkey epiblast [Roman numerals refer to the staging systems of EyalGiladi and Kochav (1976) for chicken, and Gupta and Bakst (1993) and Bakst et al. (1997) for turkey em-

Highly selected, genetically improved, populations of birds have been a major contribution to the success of the modern poultry industry (McKay, 2009). However, there are also many rare and specialized lines of poultry that are threatened with extinction primarily due to economic reasons, thereby reducing the biodiversity of poultry species (Fulton and Delany, 2003). Poultry stocks are at risk from potential disease epidemics (Blackburn, 2006). Highly virulent strains of avian influenza, if established, would inevitably result in the ©2014 Poultry Science Association Inc. Received September 17, 2013. Accepted December 3, 2013. 1 Corresponding author: [email protected]

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ABSTRACT Diseases such as avian influenza can destroy turkey flocks, potentially resulting in the loss of valuable or rare genetic material. Consequently, there is an urgent need to develop a means to archive such germplasm. Germline chimeras produced by intravascular transfer of primordial germ cells (PGC) have been reported in other avian species but not turkeys. This study examined the feasibility of both establishing an archive of frozen PGC, and producing germline chimeras by injecting the thawed PGC into host embryos. To meet these aims, the following experiments were performed: (1) PGC identification within turkey embryos; (2) development of an efficient method for isolation of turkey PGC; (3) demonstration that PGC can be cryopreserved, recovered, and retain viability; (4) reinjection into embryos and detection of injected PGC. Primordial germ cells were identified using periodic acid-Schiff reagent and the immunological marker OLP-1. Bloodstream PGC were isolated using Ficoll density gradient centrifugation with PGC recovery peaking at stages 13, 14, and 15 with 32 ± 4.9, 33 ± 6.4, and 26 ±

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for germline chimera production through the detection of reinjected PGC in the gonads of host embryos.

MATERIALS AND METHODS Sources of Eggs and Egg Incubation Turkey (Meleagris gallopavo) eggs of strain BUT BIG 5FLX were obtained from British United Turkeys Ltd. (Chester, UK). Eggs were stored at room temperature, with 55 to 60% RH. All eggs were used within 7 d to ensure the best embryonic viability. Eggs were incubated in forced-draft incubators (Brinsea Multihatch, Mark II, Sandford, UK) at 38°C with 65% RH. Eggs were automatically rotated throughout incubation by a tray sliding mechanism at 1-h intervals.

Collection of Embryo Stages 12 to 18 (2.5–3.5 d) Eggs were secured in a 50-mL glass beaker and forceps were used to open the blunt end of the egg, exposing the underlying air space. Any remaining eggshell or shell membrane was removed, exposing the blastoderm, located on the surface of the yolk, and recognized as a pale to red rounded patch, depending upon the stage of development. Embryos (stages 12–18) were removed from the eggs by placing filter paper disks (2.3 cm, Whatman, Fisher Scientific, Loughborough, UK) with a 1-cm hole cut from the center, over the embryo. Dissection scissors were used to cut around the circumference of the disk through the perivitelline layer. The disk with embryo attached was removed and inverted using forceps, and cleaned in warm PBS (pH 7.4) to remove any adherent yolk.

Collection of Embryo Stages 29 to 30 (8–8.5 d) Eggs were opened as for stages 12 to 18. A pair of curved forceps was used to locate the embryo, which was hooked under the neck and lifted from the eggshell. The adherent yolk sac was removed and the embryos rinsed using PBS and placed under a dissection microscope (WILD M3Z, Heerbrugg, Switzerland) in a Petri dish containing black dissection wax and PBS.

Embryonic Blood Collection Embryos were removed from the yolk as described previously for stages 12 to 18. The embryos were placed into Petri dishes and observed using a stereodissection microscope. Blood was collected from the embryos by using a glass microinjection needle (Borosilicate glass, 15 cm length: 1 mm outside diameter, 0.75 mm inside diameter), pulled by a Flaming Brown horizontal-P-97, Sutter, Novato, CA) puller. The needle was inserted

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bryos]. The PGC then gradually translocate into the underlying hypoblast (Karagenç et al., 1996; D’Costa and Petitte, 1999) and enter the germinal crescent phase of their migration during the gastrulation process (Ginsburg and Eyal-Giladi, 1987; Wentworth and Wentworth, 2000). During this period the hypoblast is displaced anteriorly, and the PGC is caught and carried along with it to the anterior region of the embryo. From the germinal crescent, the PGC enter the embryonic circulation associated with the forming blood vascular system at approximately stages 8 to 10 (Arabic numerals refer to the staging system of Hamburger and Hamilton, 1951), and enter the vascular phase of their migratory process. By stage 12, the extra- and intraembryonic blood system has been fully established and PGC can be found circulating throughout this system (Fujimoto et al., 1975, 1976). Chick PGC appear in the blood in increasing numbers from stage 12 up to stage 16 where numbers peak and then begin to decline as PGC gradually extravasate toward the gonadal anlagen (Nakamura et al., 2007), where, based on the sex of the embryo, they will either populate the ovary or testes as oogonia or spermatogonia at the time of hatch. These unique features of avian PGC migration have helped facilitate their subsequent isolation and transfer (Nakamura et al., 2010). The potential for using avian PGC as a means of preserving and reconstituting avian genetic material has been extensively demonstrated within chickens via the formation of germline chimeras. This process utilizes the potential for PGC transfer between individual embryos, and the ability of the donor cells to contribute to the germline of the recipient. Germline chimeric birds have been produced previously by various means; using blastodermal cells (Petitte et al., 1990), germinal crescent PGC (Vick et al., 1993), bloodstream PGC (Tajima et al., 1993; Naito et al., 1994a; van de Lavoir et al., 2006; Tagami et al., 2007; Yamamoto et al., 2007), and frozen thawed bloodstream PGC (Naito et al., 1994b; Tajima et al., 2003; Kuwana et al., 2006), and gonadal PGC (Chang et al., 1995b, 1997; Park et al., 2003; Mozdziak et al., 2006). This previous research in chickens provides evidence that PGC can be harvested, stored, and reintroduced into the germline of recipient embryos. However, there is relatively little information in the published literature, if any, about such methods to produce germline chimeras in turkeys. Early research by Reynaud (1969, 1976) demonstrated the incorporation of turkey PGC into the gonads of chicken embryos via intravascular germinal crescent PGC transfer. However, these methods only produced interspecific embryonic chimeras, which demonstrated no potential for producing donor cell-derived offspring. Therefore, the aims of this study were to investigate the feasibility of establishing a method for identifying and isolating PGC from turkey lines for the purpose of producing a cryopreserved archive of valuable genetic material, and to demonstrate the potential

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into the embryonic dorsal aorta, and gentle suction applied to facilitate blood collection. Collected blood (1 to 5 µL) was dispensed into individual wells of a 96-well plate (Corning Inc., London, UK). Each well contained a 100 μL of Medium 199 (M199) supplemented with 10% fetal bovine serum (FBS; M199-FBS).

Embryonic Gonad Isolation

Isolation of Bloodstream PGC Ficoll density gradient centrifugation was performed immediately after blood collection. Blood samples were collected into a single 1.5-mL Eppendorf tube. No more than 10 to 20 embryos worth of blood was placed into each Eppendorf tube. Collected blood was pelleted in a cooled centrifuge spun at 20°C (Sigma laboratory centrifuges, Philip Harris, model 3K18C, rotor 12154) for 4 min at 400 × g. Both 16 and 6.3% Ficoll 400 (Sigma-Aldrich) solutions were prepared and used for the isolation procedure as per the method of Yasuda et al. (1992). Briefly, the blood pellet was resuspended in 90 μL of M199-FBS. The resuspended pellet was dispersed into 900 μL of the 16% Ficoll and mixed thoroughly. Two hundred microliters of the 6.3% Ficoll was overlaid on top of the 16% Ficoll solution, and an interface line became visible between the 2 concentrations and was marked. The tube was spun for a further 30 min at 800 × g. After this spin, 200 μL was removed from the interface. The removed solution was dispersed in M199-FCS (900 μL) and centrifuged again 3 times for 4 min at 400 × g to remove residual Ficoll.

Periodic Acid-Schiff Staining Isolated cells were fixed in 4% paraformaldehyde (PFA) for 10 min. The cells were then immersed in 0.1% periodic acid (PA) solution (Sigma-Aldrich) for 5 min and subsequently treated with Schiff’s reagent (Sigma-Aldrich) for 15 min. Cells were washed in PBS by centrifugation at 400 × g for 3 min at 20°C after fix-

ation and after treatment with PA and Schiff’s reagent. All procedures were performed at room temperature, and the stained PGC were observed under an inverted microscope (TE2000-U, Nikon, Tokyo, Japan).

Immunological Staining of Isolated PGC Anti-OLP-1 (1B3, IgM; supernatant) antibody was obtained from Willi Halfter (University of Pittsburgh, Pittsburgh, PA). Both anti-EMA-1 (IgM; supernatant) and anti-SSEA-1 (MC-480, IgM; ascites) were purchased from the Developmental Studies Hybridoma Bank (Iowa City, IA). Putative PGC were isolated from embryonic blood as described previously. Primordial germ cells were first fixed in 4% PFA for 15 min and washed in PBS before use. Primordial germ cells were incubated in an appropriate dilution of primary antibody (OLP-1, EMA-1; neat; SSEA-1; 1/100) for 1 h at 4°C. Primordial germ cells were then washed 3 times in PBS. Primordial germ cells were transferred into a 1/20 dilution of goat anti-mouse IgM FITC-conjugated secondary antibody (F9259, Sigma-Aldrich). The cells were then incubated at 4°C overnight. After incubation the cells were washed 3 times in PBS. Cells were placed into bisbenzimide (Hoechst 33342, Sigma-Aldrich), 1/100 dilution, for nuclear counterstaining. A final wash in PBS was performed, and the cells transferred to glass slides for observation under a fluorescence microscope (Olympus Vanox-AHBS3, Olympus, Southend-on-Sea, UK).

Staining of Gonad Tissue Sections Embryonic gonads were isolated as described above, and frozen in OCT embedding medium (Tissue-Tek, Raymond A Lamb, Eastbourne, UK). Frozen sections were obtained by cutting using a cryostat (Kryostat 1720, Leica, Milton Keynes, UK) at a thickness of 7 to 10 μm, and collecting onto poly-l-lysine-coated slides. A 1/10 dilution of normal goat serum/PBS with 5% BSA (Sigma-Aldrich) blocking solution was applied at 50 μL per sample. Samples were placed in a humidified box for 1 h and incubated at room temperature. Slides were washed 3 times in PBS on an orbital shaker (Stuart Scientific, Stone, UK) at 5-min intervals. The PBS was removed and primary antibody added (50 μL) per section at the concentrations described above. Samples were incubated at room temperature for 1 h or 4°C overnight, in a humidified chamber. The samples were again washed 3 times with PBS. Secondary antibody was added (50 μL), and incubated in the humidified chamber at room temperature for 1 h. Secondary antibody was removed, and samples washed as above, then incubated with 1/1,000 bisbenzimide/PBS for 15 min. After 3 washes in PBS, samples were mounted in Gelvatol (pH 7.4). Slides were placed into slide wallets, wrapped in aluminum foil, left to dry for 24 h, and stored at –20°C until analysis.

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Embryos at stages 29 to 30 were obtained using the method described above and placed in PBS. The embryos were manipulated under a dissecting microscope using epi-illumination. Each embryo was positioned ventral side upwards, and extremely fine dissection forceps (Dumont No 5, Sigma-Aldrich, Gillingham, UK) were used to open the abdomen along the ventral midline. Visceral organs were removed revealing the embryonic mesenephroi and gonads. Each gonad was identifiable as an associated piece of whitish tissue attached to the lateral edges of each mesenephros. Gonads were removed from the mesenephroi using the forceps and transferred to a 1.5-mL Eppendorf tube (TreffLab, Degersheim, Switzerland) containing 500 μL of M199-FBS for storage before fixation for further analysis.

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Detection of Reinjected PGC

The PGC were isolated from embryos between stages 12 to 18 of development. Isolated cells were placed into 1 mL of freezing mix [Dulbecco’s modified Eagle medium (DMEM)] supplemented with 20% FBS and 10% dimethlysulfoxide contained within a cryotube (NUNC Cryotube Vials, Hvidovre, Denmark). Cryotubes were placed in a freezing vessel (Nalgene cryo 1°C, Nalgene Nunc International, Loughborough, UK) supplemented with 250 mL of isopropyl alcohol (Sigma-Aldrich) providing a controlled rate of cooling at −1°C/min. The freezing vessel was placed at –80°C and left overnight. The cryotubes were removed from the freezing vessels, placed into liquid nitrogen at –196°C within a liquid nitrogen cell storage unit (34HC, Taylor-Wharton, Theodore, AL), and stored for 3 mo. To recover the frozen PGC, the cells were removed from liquid nitrogen and placed into water at 37°C for 5 min. After thawing, the cell suspension was immediately diluted in 10 mL of DMEM and centrifuged at 200 × g at 20°C for 15 min. The supernatant was removed and cells resuspended in 100 µL of M199. The viability of the frozen thawed PGC was determined by Trypan blue (T8154, SigmaAldrich) dye exclusion. An equal volume of cell suspension (5 μL) was mixed with an equal volume of Trypan blue and left to incubate at room temperature for 1 to 2 min.

Gonads were removed from stage 29 to 30 embryos as described above. Whole mounts of gonadal tissue were produced by fixing in 4% PFA for 10 min at 4°C. After fixation, the tissue was washed in PBS for 1 h. Gonads were placed into 1 mL of bisbenzimide diluted 1/100 in 1% Triton X-100 (Sigma-Aldrich) diluted in PBS for 2 h at room temperature. The tissue was washed in PBS for 1 h at room temperature. Gonads were dehydrated through a series of graded alcohol solutions −50, 70, 90%, and a 50/50 alcohol glycerol solution over a 1-h period. Gonads were placed into a 100% glycerol solution (BDH Lab Supplies, UK) and allowed to clear overnight and stored at 4°C. The whole mount gonads and number of remigrated PGC were analyzed using a Nikon C1 Eclipse 90 upright confocal microscope. The nuclear counterstain bisbenzimide was excited using a blue 408 nm laser, and the emitted fluorescent light was collected using a 450/35 emission filter. Vybrant CM-DiI- and Q-tracker-labeled samples were detected using a red 568 nm laser and emitted fluorescence was collected using a 600/30 emission filter. Analysis of gonads in the z-axis was performed by taking a z-series of individual optical image slices through the tissue at different focal levels, between 100 and 150 steps at 2 to 5 µm per step. Projections of the z-series images were produced using ImageJ v 1.34 image analysis software (National Institutes of Health, Bethesda, MD). The numbers of remigrated PGC were assessed from the stacked projections by extracting individual optical slices from the different focal planes that contained labeled PGC. The numbers of cells were then counted in these associated regions.

Preparation of Recipient (Ex Ovo) Embryos Turkey embryos were incubated, as above, until they reached stage 15. The eggs were then broken into a 100 × 20 mm tissue culture dish so that the embryo was oriented on the upper surface of the yolk once broken into the dish. The embryonic stage was then assessed and the plates placed into an incubator (LEEC, Nottingham, UK) at 38°C until they were ready for PGC injection.

Injection of Frozen-Thawed DiI/Q-TrackerLabeled PGC The PGC were isolated as described previously, and fluorescently labeled using either Vybrant CM-DiI (V22888, Life Technologies, Molecular Probes, Paisley, UK) or Q-tracker 605 cell labeling kit, components A and B (Q25001, Life Technologies, Molecular Probes) as per the manufacturer’s instructions. Labeled PGC were picked up into a custom-designed microinjection capillary (Origio Ltd., Reigate, UK) produced to the following specifications: borosilicate glass, 15 cm length: 1 mm outside diameter, 0.75 mm inside diameter. The bore of the needle was produced at 40 µm and a 20° bevel was applied. The labeled PGC were then introduced into the heart of stage 15 embryos cultured ex ovo. The embryos were then placed into an incubator (LEEC) at 38°C to incubate for the desired period of time.

Statistical Analysis Statistical analysis of data was carried out using GraphPad Prism (version 3.02, GraphPad Software Inc., La Jolla, CA). This program helps organize, analyze, and graph repeated experiments and apply appropriate statistical tests. To determine if there was one or more significant differences located within a data set (when comparing 2 or more groups) the 1-way ANOVA test was used. If ANOVA analysis indicated significant differences existed between the means of the groups within the data, post hoc test analysis was applied. Post hoc multiple comparison test analysis (Tukey’s honestly significant difference) identified exactly where the significant differences lay within the data. This test was considered the most appropriate as it was considered to be the most powerful test when analyzing all possible pair-wise comparisons. In cases where only 2 columns of data were to be compared, the t-test is the corresponding ANOVA test. The unpaired t-test was used to analyze data to determine if a difference in the mean values existed when a particular variable was being compared between 2 groups. Differences were

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PGC Cryopreservation and Recovery

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regarded as significant at P < 0.05 unless otherwise stated.

RESULTS PGC Identification: Stages 12 to 18

PGC Identification in Gonad Serial cryosections of 8.5-d gonad tissue were labeled with the antibodies anti-OLP-1, anti-SSEA-1, and anti-EMA1. Results indicated that anti-EMA-1 and anti-SSEA-1 failed to label gonadal PGC (results not shown). However, the anti-OLP-1 antibody strongly labeled PGC within the gonad tissue (Figure 1C and 1D). The cells were identified as PGC on the basis of their large size (15–20 µm) and positive labeling. The majority of the staining was restricted to the cell surface, with some cytoplasmic labeling evident (Figure 1C and 1D).

PGC Recovered off Ficoll at Stages 12 to 18 After Ficoll centrifugation, a drop of media containing both blood cells and PGC was placed in the center of a tissue culture plate (100 × 20 mm). The PGC were separated from the blood cells using a micropipette under a dissection microscope and counted. The PGC numbers per embryo were increased significantly (P < 0.05) from stage 12 to stage 13. However the greatest numbers of PGC per embryo were obtained from stages 13 to 15, with 32 ± 4.9, 33 ± 6.4, and 26 ± 5.4 (mean ± SEM) PGC recovered respectively (Figure 2A). However, no significant differences were observed between these groups (P > 0.05). The highest concentration of PGC on a per microliter of blood/embryo basis was obtained from stage 13 embryos (Figure 2B). This number was highly significantly different from the numbers of PGC recovered from stage 12 embryos (P < 0.001) and stage 14 and 15 embryos (P < 0.01). After

stage 15, the numbers of PGC/embryo that could be recovered off the Ficoll system dropped significantly (P < 0.05) between stages 15 and 16. By stage 18, very few PGC could be recovered at all. This trend was also observed when results for the PGC/embryo data were being expressed as PGC recovered per microliter of blood/embryo (Figure 2B).

Viability of Frozen-Thawed PGC The PGC were removed from liquid nitrogen and thawed in a water bath. The recovered PGC were identified on the basis of their morphological criteria, and viability was found to be 90 ± 2% (mean ± SEM; Figure 3) as assessed by Trypan blue dye exclusion after 3 mo stored in liquid nitrogen. The viability of thawed PGC was less than freshly isolated PGC whose viability was 96 ± 3% (mean ± SEM; Figure 3). However, the freezing treatment was determined not to have had a significant (P = 0.18) effect on cell viability when compared with the freshly isolated cells. This result would suggest that turkey PGC are capable of being cryopreserved for prolonged periods in liquid nitrogen.

PGC Colonization of Host Gonad Tissue Detection of Frozen-Thawed DiI-Labeled PGC. Host embryos were injected with 30 to 400 DiI-labeled PGC at stage 15 and had a 74% (n = 19) survival rate 6 d postinjection when cultured ex ovo. Uninjected control embryos had an 80% survival rate (n = 15). The removed gonads were examined by confocal microscopy. Optical sectioning of whole mount gonads revealed DiIlabeled PGC (Figure 4A, 4B). However, the numbers of PGC that were detected in the gonads of these stage 30 embryos was very low. The frequency of detection of the PGC was also low with 36 ± 13.2% (mean ± SEM) of embryonic gonads containing DiI-labeled PGC (Figure 5A). The colonization of the gonads with DiIlabeled PGC was also very low with 7 ± 3.8% (mean ± SEM) of reinjected PGC reaching the gonadal tissue of positive embryos (Figure 5B). It was also found that 74% of PGC detected were located in the left gonad, with the remaining 26% located within the right gonad (data not shown).

Detection of Frozen-Thawed Q-TrackerLabeled PGC The PGC labeled with Q-tracker nanocrystals were injected into the heart of stage 15 embryos, and were left to incubate until they reached stage 29/30. These injected embryos had a survival rate of 47% (n = 17). The gonads were removed and analyzed using confocal microscopy. Optical sectioning revealed Q-tracker-labeled PGC in the gonads (Figure 4C, 4D). The frequency of detection of PGC within the surviving embryos was 67 ± 21.1% (mean ± SEM; Figure 5A), which, although

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The periodic acid-Schiff (PAS) reagent positively identified the PGC, differentiating them from blood cells (Figure 1A). The PGC because of their characteristic high glycogen content stained a darker purple/ magenta color than the surrounding smaller blood cells. These stained a lighter shade of pink because they contain smaller amounts of endogenous glycogen. Of the antibodies tested (anti-OLP-1, anti-SSEA-1, and antiEMA-1) only anti-OLP-1 antibody showed any strong positive staining for the isolated PGC (Figure 1B). Positive cells possessed characteristics of PGC that included; larger sized cells (15–20 µm), a granular appearance, and numerous highly refractive lipid droplets (Figure 1E, 1F). The anti-OLP-1 antibody showed predominantly strong staining on the cell surface (Figures 1B). Both the anti-EMA-1 and anti-SSEA-1 antibodies showed very little to no staining at all on the isolated cells (results not shown).

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higher than that of the DiI-labeled PGC (36 ± 13.2%; mean ± SEM), was not deemed significantly different (P = 0.25). The colonization of gonads with Q-trackerlabeled PGC was greater than DiI-labeled PGC, with 44 ± 4.9% (mean ± SEM) of reinjected PGC colonizing the embryonic gonads in positive embryos (Figure 5B). This was significantly different (P = 0.001) from that of DiI-labeled PGC, where 7 ± 3.8% (mean ± SEM) PGC colonized the gonads (Figure 5B). Of the PGC detected, 70% were located in the left gonad and 30% in the right gonad (data not shown).

DISCUSSION In the present experiment, PGC were efficiently isolated, cryopreserved, and reinjected/detected in the gonads of recipient turkey embryos. These results indicate that PGC could be used to preserve turkey germplasm, and potentially to produce germline chimeras. Such a method would provide a valuable strategy for recovering any turkey stocks that could inevitably be lost as a result of serious disease outbreaks. The principal aim of this study was to determine if PGC could be used

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Figure 1. Putative turkey primordial germ cells (PGC) were identified in isolation by using the histochemical periodic acid-Schiff (PAS) reagent (A) and the immunological marker anti-OLP-1 (B). Primordial germ cells were also identified within the gonads of stage 30 embryos by using anti-OLP-1 antibody (C and D). Characteristic morphology of PGC isolated from blood of stage 12 to 18 embryos (E and F). Arrows indicate PGC; B = blood cells. Bars = 20 µm (E), 50 µm (A, B, D, F), 100 µm (C).

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Figure 2. Indicates the numbers of primordial germ cells (PGC) isolated from the blood of stage 12 to 18 embryos by using Ficoll density gradient centrifugation. The data were expressed as PGC isolated on a per embryo basis (A), and per microliter of embryonic blood (B). Values are the means ± SEM. H&H = Hamburger and Hamilton.

to preserve turkey germplasm, and to demonstrate via their intravascular transfer to host embryos, the potential to produce germline chimeras. Such a method has yet to be demonstrated for turkeys, and if feasible, could provide a means to capture the entire genetics of the stock and recover a given line within only a few generations (Moore et al., 2006). In this study the immunological marker anti-OLP-1 identified isolated putative turkey PGC, both in isolation and within the gonads of turkey embryos. This is the first report of the use of this marker to help characterize turkey PGC. The histochemical marker PAS also identified isolated putative turkey PGC within the blood of embryos. These markers provided a means of identifying PGC in turkey embryos within both the blood and gonads at particular time points. The observed PAS positive staining of PGC supports previous observations by Meyer (1960) for chicken PGC, and D’Costa and Petitte (1999) for circulating turkey PGC. The OLP-1 antibody was first demonstrated to be an

efficient marker of chicken and rat PGC, where OLP was shown to label the surface of migrating chick PGC from the embryonic circulation through to their colonization of the gonads (Halfter et al., 1996). Ovomucinlike protein (OLP) is defined as a subclass of glycoproteins with multiple O-linked carbohydrate side chains on a protein core with tandem repeat peptide units (Strous and Dekker, 1992). Ovomucin-like protein is a major constituent of the oocyte perivitelline membrane and has been suggested to possess an antiadhesive property (Halfter et al., 1996). It is thought that the OLP confers this property to the surface of the PGC (Halfter et al., 1996). Therefore, OLP may facilitate the migration of the PGC to the gonads by preventing a precocious adhesion of the cells to blood vessel walls and to the mesenchyme. Two other immunological markers were tested, antiEMA-1 and anti-SSEA-1. Both of these markers have conventionally been used to identify chicken PGC (Urven et al., 1988; Jung et al., 2005). Anti-SSEA-1 has also been used to identify PGC from the germinal crescent and blood of stage 4 to 18 turkey embryos (D’Costa and Petitte, 1999). However, these markers did not result in any positive staining of the putative turkey PGC in our study. D’Costa and Petitte (1999) reported that turkey gonadal PGC lose the SSEA1 marker at approximately stage 20. This finding is contradictory to observations made by Halfter et al. (1996), who demonstrated the presence of the SSEA1 epitope on the surface of chick gonadal PGC. The former observation is consistent with the findings in our study, although the observation in this report that the anti-SSEA-1 antibody does not bind to putative PGC derived from the blood is in contradiction with the previous study by D’Costa and Petitte (1999). The failure of anti-EMA-1 to identify turkey circulating or gonadal PGC also contradicts the previous study by

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Figure 3. Indicates the percentage of viable primordial germ cells (PGC) after a prolonged period (3 mo) stored in liquid nitrogen, compared with PGC freshly isolated after Ficoll density gradient centrifugation. Viability determined by Trypan blue dye exclusion. Values are the means ± SEM (n = 4).

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Urven et al. (1988). The reasons behind the negative results obtained for both the anti-SSEA-1 and antiEMA-1 identification of turkey PGC are unclear. Because both EMA-1 and SSEA-1 antibodies recognize different parts of the same epitope, this could indicate the possibility that the epitope has been lost or masked in some way. It may also suggest that chicken PGC have different characteristics than turkey PGC. Putative PGC were identified after Ficoll isolation on the basis of their large size and characteristic morphology in comparison with blood cells. All of the features of the positively labeled cells were characteristic of avian PGC, along with the anatomical locations in which they were identified. Therefore, it was concluded that the anti-OLP-1 and PAS-positive cells were PGC. This study reports on the use of Ficoll density gradient centrifugation to purify the PGC from the blood of stage 12 to 18 turkey embryos a technique previously used for chicken PGC (Chang et al., 1992; Yasuda et al., 1992; Tajima et al., 1993; Naito et al., 1994a,b, 1998; Liu et al., 2007; Tagami et al., 2007). There is no previous report on the use of this method to isolate turkey PGC. Circulating PGC from embryonic blood

were the focus of the isolation study, due to the relative ease of access to the blood, fewer contaminating cell types (in comparison with gonadal tissue), and knowledge that the PGC were in an active state of migration. The technique was quick to perform and yielded many viable PGC. It was demonstrated that PGC numbers peaked within embryonic turkey blood at stages 13 to 15. It was found that fewer PGC could be isolated from turkey embryos than chick using Ficoll. The highest concentration of turkey PGC within this study was at stage 13. These results are similar to those found by Tajima et al. (1999) for chick embryos, where the peak concentration of PGC was at stage 14. Comparisons of PGC obtained from turkey with that of chicken indicated that turkey embryos contain fewer PGC. Within chicken embryos PGC numbers rose to almost 70 PGC/ µL of blood (Tajima et al., 1999), whereas in quail numbers peaked at around 100 PGC/µL of blood (Ono and Machida, 1999). It was demonstrated that turkey PGC could be frozen and thawed after 3 mo with high viability. Previous work on cryopreserved chick PGC found them to have similar viability (94.2%) after thawing (Naito et

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Figure 4. Donor primordial germ cells (PGC) labeled with Vybrant CM-DiI and Q-tracker nanocrystals were detected in chimeric stage 30 turkey gonads after injection into the hearts of stage 15 embryos. Confocal projections revealed Q-tracker-labeled PGC within gonads of stage 30 turkey embryos (A and B). Single confocal image slices show DiI-labeled PGC in gonads of stage 30 turkey embryos (C and D). Arrows indicate PGC, Go = gonads. Bars = 100 µm (A, D), 50 µm (B, C).

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al., 1994a). Studies which used a range of commercial cryoprotectants to freeze chick PGC found PGC to be up to 86% viable after thawing (Setioko et al., 2007). A similar study in chick (Chen et al., 2007) used 3 different cryoprotectants and protocols for freezing chick PGC, and the viability was found to be 85.9%, which is comparable to the study by Setioko et al. (2007). For turkey PGC the viability was higher than previous studies using chick PGC (Chen et al., 2007; Setioko et al., 2007), but not as high as that found by Naito et al. (1994a). This study suggests that it is feasible to produce a frozen archive of turkey PGC, thus making it possible to conserve rare genetic resources from turkey species. The reinjection of frozen-thawed fluorescently labeled turkey PGC indicated that these cells retained the potential to repopulate the host gonads. Initial experiments using CM-DiI-labeled PGC revealed donor PGC in host gonads by confocal microscopy. However, the number and proportion of reinjected turkey PGC detected was low. The CM-DiI was initially chosen in

this study because, unlike PKH26, CM-DiI is retained in cells throughout fixation and permeabilization procedures, thus making it more suitable for examining the fixed/permeabilized whole mount gonad tissue, which was analyzed in this study by confocal microscopy. The incorporation of chicken donor PGC into recipient gonads has been demonstrated previously by labeling with the fluorescent dye PKH26 (Chang et al., 1995b; Park et al., 2003). The number of PKH26-labeled PGC detected in chicken gonads ranged from 487 to 1,160 after 150 were initially injected (Chang et al., 1995b), and the frequency of detection indicated 88.9% of surviving embryos had positive signals (Park et al., 2003). These numbers were higher than what was detected in turkey gonads when CM-DiI-labeled PGC were reinjected. It was noted that the majority of reinjected PGC were detected in the left gonad of turkey embryos, a finding that was corroborated in chick embryos, where 79.6 and 20.3% of reinjected PGC were found within the left and right gonads, respectively (Chang et al., 1995b). The poor detection of CM-DiI-labeled PGC could be attributable to several factors. Studies using DiI-labeled mesenchymal stem cells indicated that labeled cells may be rendered undetectable after several mitotic divisions as the fluorescence intensity of these cells decreased exponentially by 50% after each cell division (Ferrari et al., 2001). The CM-DiI label could also influence PGC survivability. In a previous study where CM-DiI was tested for labeling human preadipocytes, it was demonstrated that CM-DiI-labeled cells were up to 55 ± 21% viable 24 h after labeling, thus demonstrating substantial toxicity (Hemmrich et al., 2006). It was due to this poor detection of CM-DiI-labeled PGC, that a relatively new cell labeling technology (Q-tracker nanocrystals) was used to better assess donor PGC incorporation into host gonads. This method of fluorescently labeling avian PGC has not previously been reported. Encouragingly Q-tracker-labeled PGC were detected in the gonads of stage 30 embryos, with a higher frequency of detection and in higher numbers than CMDiI-labeled PGC. This result suggests that Q-trackerlabeled cells offer a more efficient means of assessing donor PGC incorporation into host turkey gonad tissue than CM-DiI-labeled cells. It is likely that PGC remigration using Q-tracker was more effective because Q-tracker nanocrystals are thought to offer a greater photostability (Alvisatos, 2004), making them resistant to photobleaching, thus enabling long-term imaging experiments to be performed under conditions that may lead to the photoinduced deterioration of other more commonly used fluorophores such as CM-DiI. Overall the results from this study have helped elucidate some important questions with regards to the archiving of turkey PGC and the potential for germline chimera production in this species. Although no live chimeras or donor-derived offspring were generated from this study, these were important first steps to be addressed. It was demonstrated that it is possible to identify, isolate, cryopreserve, and recover turkey PGC

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Figure 5. A comparison between CM-DiI- and Q-tracker-labeled primordial germ cells (PGC) detected within the gonads of host stage 30 turkey embryos. Gonads removed from stage 30 embryos that had received injections of CM-DiI and Q-tracker PGC at stage 15 were analyzed. The percentage number of surviving injected embryonic gonads containing fluorescently labeled PGC (CM-DiI and Q-tracker) was calculated and compared (A). The percentage number of labeled PGC colonizing the gonads compared with the numbers originally injected was also determined for both PGC labeling methods (B). N values for (A) represent total numbers of embryos analyzed for presence of injected PGC; (B) represents total number of embryos with positive signals in which PGC were counted. Values are the means ± SEM. Significance is denoted by **P < 0.01.

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with a high viability, demonstrating that it is possible to produce a frozen genomic archive of turkey genetic material. The reinjection and detection of frozenthawed fluorescently labeled PGC in host gonads using confocal microscopy demonstrates the potential for establishing donor embryo genetics into the host germline.

ACKNOWLEDGMENTS

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The potential for archiving and reconstituting valuable strains of turkey (Meleagris gallopavo) using primordial germ cells.

Diseases such as avian influenza can destroy turkey flocks, potentially resulting in the loss of valuable or rare genetic material. Consequently, there ...
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