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Faculty of Agriculture,3 Shinshu University, Nagano 399-4598, Japan
Animal Breeding and Reproduction Research Team,4 National Institute of Livestock and Grassland Science (NILGS), Ibaraki 305-0901, Japan
Transgenic Animal Research Center,5 National Institution of Agrobiological Sciences (NIAS), Ibaraki 305-0901, Japan
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.
developmental biology, early development, gamete biology, gene regulation
Chick primordial germ cells (PGCs) circulate for a short period of time in the bloodstream, and leave the capillary vessels close to the germinal epithelium. The chick PGCs migrate into the germinal ridge [1–4], where they differentiate into either spermatogonia in the testis or oogonia in the ovary. The unique migratory pathway of avian PGCs facilitates their isolation and transfer, especially from the blood vessels.
Experiments to generate germline chimeras and donor-derived offspring by the transfer of circulating PGCs (cPGCs) have succeeded [5–8]. However, an efficient isolation method for cPGCs has not been established to date. The main methods used to purify avian cPGCs are Ficoll density gradient centrifugation [5], Nycodenz density gradient centrifugation [8], immunomagnetic cell sorting (MACS) [9], and fluorescence-activated cell sorting (FACS) [10]. However, these techniques require special training and are quite labor-intensive. In MACS and FACS, expensive antibodies that recognize cPGCs and cell sorting facilities are indispensable. The antibody against stage-specific embryonic antigen-1 (SSEA-1) [11] used in both MACS and FACS is not specific for PGCs. The cPGC recovery rates for the MACS and FACS methods are low. Many fresh cPGCs are necessary for the efficient production of germline chimeras. To date, an ideal method to isolate pure cPGCs has not been developed.
In mammals, bone marrow cells can be isolated from whole blood using the ACK lysis buffer, which disrupts red blood cells [12–14]. Therefore, the ACK process was applied to develop a more convenient and simple method to isolate PGCs from embryonic blood for the subsequent generation of germline chimeric chickens. Whole blood, which included cPGCs, was collected at stages 13, 14, and 15 [15]. The red blood cells were removed by the ACK lysis buffer, and the PGCs were purified by in vitro culture.
In the present study, a novel and simple system was developed for the isolation of chick cPGCs for subsequent use in the generation of germline chimeric chickens (Fig. 1).
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All of the procedures used in the present study were reviewed and approved by the Animal Care and Use Committee of Shinshu University, and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.
Fertilized eggs from White Leghorn chickens were cultured to stages 13–16 [15]. Approximately 5 µl of whole blood were collected from the dorsal aorta of an embryo using a fine glass micropipette under a stereomicroscope. The blood was suspended in 100 µl of 3.8% (w/v) sodium citrate in a 1.5-ml centrifuge tube and used for ACK processing.
Composition of ACK Lysis Buffer and cPGC Purification
ACK lysis buffer consisted of 150 mM NH4Cl, 1 mM KHCO3, and 0.001 mM EDTA. The buffer was filter-sterilized through a 0.2-µm filter, and the prepared blood sample was added to 900 µl of ACK lysis buffer. With occasional gentle shaking, the samples were incubated on ice for 30 min. The samples were centrifuged at 2000 rpm for 10 min at 4°C, and the pellets were resuspended in 1000 µl of ACK lysis buffer and incubated on ice for an additional 15 min. The incubated samples were centrifuged at 2000 rpm for 10 min at 4°C and the pellets were washed twice in PBS. The cPGCs were distinguished from blood cells by their large size and the presence of refractive granules in the cytoplasm when viewed under the IX70 phase-contrast microscope (Olympus, Tokyo, Japan). The percentage of cPGCs was determined for each sample.
Using a 24-well culture plate, the collected cPGCs were cultured in Kav-1 medium [17] that contained FBS, chick serum, and mLIF (500 U/ml) (Chemicon, Pittsburgh, PA) at 37°C in 5% CO2. The cells were uniformly seeded on gelatin-coated dishes at 2x105 cells/dish. For passage, the cells and medium were transferred to a centrifuge tube, pelleted by centrifugation at 2000 rpm for 5 min, and resuspended in fresh medium. PGCs were cultured for 7 days. The cells were then subjected to immunohistochemistry with anti-CVH antibodies [18] for the detection of PGCs.
Isolation of Total RNA and Reverse Transcriptase PCR (RT-PCR)
Total RNA was isolated from the cultured PGCs using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's directions. Oligt(dT) primer first-strand cDNA was prepared using Superscript II. The First-Strand Synthesis System (Invitrogen) was used for RT-PCR with 2.5 µg of total RNA. The following primer pairs were used: for chicken vasa homologue (Cvh), forward 5'-GCTCGATATGGGTTTTGGAT-3' and reverse 5'-TTCTCTTGGGTTCCATTCTGC-3'; for deleted in azoospermia-like (Dazl), forward 5'-GCTTGCATGCTTTTCCTGCT-3' and reverse 5'-TGCGTCACAAAGTTAGGCA-3'; and for actin, forward 5'-AACACCCCAGCCATGTATGTA-3' and reverse 5'-TTTCATTGTGCTAGGTGCCA-3' [19]. PCR was performed for 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min. The amplified products were verified by 1.5% agarose (Nacalai Tesque, Kyoto, Japan) gel electrophoresis.
Immunostaining of Cultured cPGCs
Some of the 7-day-cultured PGCs were fixed with 4% paraformaldehyde (PFA) for 3 h at 4°C. After washing in PBS, blocking was performed in PBS with 2.5% goat serum and 2.5% donkey serum. Samples were then incubated for 1 day with anti-CVH antibodies [18] in PBS (1:10 000) that contained 0.25% goat serum and 0.25% donkey serum. After washing three times in PBS that contained 0.25% goat serum and 0.25% donkey serum, the samples were incubated overnight in Alexa 488-conjugated goat anti-rat IgG (Invitrogen) in PBS (1:200). After washing three times in PBS, the cells were observed under a fluorescence microscope (Olympus IX70) equipped with an ORCA-ER CCD camera system (Hamamatsu Photonics, Hamamatsu, Japan). The digital images were analyzed using the AquaCosmos software (Hamamatsu Photonics).
To trace the donor cells in the chimeric embryos, the cultured PGCs were labeled with the fluorescent lipophilic carbocyanine dye PKH26-GL (Sigma Chemical Co., St. Louis, MO), according to the manufacturer's instructions. The stock solution of PKH26-GL was diluted to the appropriate concentration and the donor cells were transferred into this solution. After a 5-min exposure to the staining solution, the reaction was stopped by the addition of 200 µl of Dulbecco modified Eagle medium (DMEM) that contained 10% chicken serum. The marked donor cells were washed with 1.5 ml DMEM. The fluorescent staining reaction was assessed by fluorescence microscopy.
Production of Chimeric Embryos
Chimeric chickens were generated using 7-day-cultured PGCs stained with PKH26 as the donor. Approximately 300 labeled cPGCs were microinjected into the dorsal aorta of stage 14–15 recipient embryos. The manipulated embryos were then transferred to a host eggshell that was prepared by cutting off the blunt end of a freshly laid double-yolk egg. The manipulated embryos were cultured in system III [16] at 38°C in a light-shielded incubator for 4 days.
The whole gonads with adrenal glands from embryos at stage 29 were sampled. Each sample was fixed in 4% PFA for 1 day at 4°C. Immunostaining was performed under the conditions described above, except that the samples were incubated with the primary antibody for 3 days. The injected PGCs were monitored to detect gonadal migration using fluorescence microscopy. Immunostained samples were embedded in Tissue-Tek OCT Compound (Miles, Elkhart, IN) and frozen in liquid nitrogen. Sections (25-µm thickness) were sliced on a cryostat, and mounted on a 3-aminopropyl-triethoxysilane (APS)-coated glass slide.
Isolation and Culture of cPGCs
When the whole blood from stage 13–16 embryos was collected and centrifuged, a red pellet was observed at the base of the tube (Fig. 2A). When the whole blood was treated with ACK lysis buffer, the pellet became white and reduced in size (Fig. 2B). In the whole blood (Fig. 2A) after the first ACK treatment, the supernatants showed a weak red color, whereas after the second ACK treatment, the color of the supernatant was more clear (Fig. 2B). Many red blood cells were present in the supernatants of the ACK-processed samples. The surfaces of these cells were strained, black, and the size was almost uniform (Fig. 2C). These strained cells were rarely observed without ACK processing. When the cell samples were processed twice with ACK buffer, many of the cells were shown to have the morphological characteristics of cPGCs. These cPGCs (Fig. 2D; yellow arrows) were larger that the red blood cells (Fig. 2D; black arrows) and brighter. The percentage of cPGCs in whole blood was 0.0194 ± 0.0013% (mean ± SEM). After ACK processing, 57.1 ± 3.7% of the cells were cPGCs and the recovery rate was 90.3 ± 2.1%. However, many cells that did not resemble cPGCs were also detected. These cells showed features that were similar to those of the red blood cells observed in the supernatants (Fig. 2D). It was possible to distinguish between the small straining cells (red blood cells) and the largely rounded cells (cPGCs). Even with multiple repetitions of the ACK treatment, these cells were not removed completely (data not shown).
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Van de Lavoir et al. [19] developed a method to select PGCs from other cells using in vitro culturing. Thus, after processing (mixture of cPGCs and red blood cells) twice with ACK, the cells were cultured in vitro for 7 days. The strained cells were aggregated and their number was decreased in the culture at 3 days. On the other hand, the cells with cPGC characteristics increased in number (Fig. 3A), and cell division (Fig. 3A; red arrow) was observed. The percentages of PGCs increased to 61.2 ± 6.1%, 73.4 ± 5.2%, 86.0 ± 3.4%, and 92.9 ± 1.4% at 1, 3, 5, and 7 days of culture, respectively (Fig. 3, A–D).
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To determine the developmental potential of the cultured PGCs as germ cells, their immunoreactivities to the anti-CVH antibodies and the expression of Dazl and Cvh were analyzed. The cells cultured for 7 days had refractive granules in the cytoplasm (Fig. 4A). Many of the cells were CVH-positive (Fig. 4, B–D). RT-PCR analysis of the PGCs after 7 days of culture revealed expression of Dazl and Cvh (Fig. 5). However, the expression of these genes was not detected in whole blood (Fig. 5). The percentage of cPGCs in the whole blood should be very low. Therefore, the expression of these genes could not be detected in the RNA samples derived from whole blood. These results suggest that the enrichment of PGCs is accomplished by this method.
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Biological Functions of Cultured PGCs
To investigate whether the cultured PGCs were capable of migrating and settling in the gonads, they were tracked by the production of chimeric embryos (Fig. 6). The donor PGCs cultured for 7 days were detected in the right (Fig. 6, A' and B') and left (Fig. 6, C' and D') gonads at stage 29 (Fig. 6A'–D'). The donor cells in both the right (Fig. 6, A''' and B''') and left (Fig. 6, C''' and D''') gonads were CVH-positive. The number of donor CVH-positive cells that settled in the gonads was low compared to the recipient (Fig. 6, A'''–D'''). It was possible to distinguish the cell population of the donor and the recipient-derived PGCs in the gonads of the chimeric embryo.
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A novel and efficient method to isolate and culture cPGCs for the production of germline chimeras was established (Fig. 1). The cultured PGCs expressed germ cell markers [18] (Figs. 4 and 5), and the biological functions of the cells in terms of migration and settlement in the gonads was confirmed (Fig. 6).
The established methods for the isolation of cPGCs entail technical difficulties, mainly due to sample contamination by red blood cells, which reduces the likelihood of obtaining pure cPGCs. In the present study, the ACK lysis buffer, which removes red blood cells, was used for cPGC isolation. The unique migratory pathway of avian PGCs, which circulate in the bloodstream, was advantageous for this novel method. The purification of PGCs was achieved by removing the red blood cells from the whole blood at stages 13–16. Although there are few reports regarding the use of ACK lysis buffer to isolate chick cPGCs, we show in the present study that ACK lysis buffer can remove the red blood cells and facilitate the isolation of highly enriched cPGCs. The only experimental procedures needed for ACK processing are addition of the ACK lysis buffer and centrifugation. Therefore, the elapsed time from collection of the whole blood to the isolation of enriched cPGCs is less than 2 h. Furthermore, the frequency of opening and closing the tube is very low compared to the other methods, so the risk of contamination is reduced. In addition, all of the previous isolation methods for PGCs require serum, whereas the present method does not require serum.
The purity of the obtained PGCs was increased when lysis was combined with in vitro culture. The purity of the PGCs reached 92.9% after 7 days of culture (Fig. 3). This level of PGC purity is equal to those achieved with the FACS [4] and Nycodenz [8] methods. The red blood cells disappeared and the PGCs appeared when isolated whole blood was cultured for 1–2 weeks [19]. Similarly, with the present method, the blood cells disappeared during in vitro culture, although this phenomenon was observed within 2 days of culture (Fig. 3B). It seems that the blood cells are damaged by ACK processing, while the PGCs proliferate as the cell culture progresses. Thus, the ACK lysis buffer is suitable for the selection of cPGCs in culture. The ACK lysis buffer is used for the purification of bone marrow cells before transplantation [12, 14]. In previous studies, PGCs have been identified based mainly on morphology, PAS, and anti-SSEA-1 and anti-EMA-1 antibody staining [8, 10, 20]. However, these evaluations are not sufficient for the identification of PGCs in the chicken. In the present experiments, the cultured PGCs were evaluated using anti-CVH antibodies and most of the cells obtained were CVH-positive (Fig. 4). In addition, there was strong expression of the PGC marker genes, Dazl and Cvh (Fig. 5). Therefore, it was determined that most of the cells remaining after 7 days in culture were PGCs.
Following the injection of labeled donor cells, the populations of donor PGCs (Fig. 6, A'''–D'''; yellow arrows) and recipient PGCs (Fig. 6, A'''–D'''; green) could be distinguished in the gonads by the fluorescence of the donor and recipient PGCs. Recipient PGCs were mainly detected in the chimeric gonads, since the endogenous PGCs of the recipient embryo [21–23] were not removed (Fig. 6, A'''–D'''). However, many donor cells settled in the gonads, which suggests that cultured PGCs have high-level biological activity.
In conclusion, this novel method for PGC isolation is more rapid and easy than previous methods. The purified PGCs showed biological functionality, which may be useful for the generation of germline chimera. This novel method has the potential to be a powerful tool for germline development research and for PGC manipulation in the chicken.
FOOTNOTES
1Supported in part by Grants-in-aid from the Ministry of Education, Science, Sports and Culture of the Japanese Government (nos. 15380190, 15658081, and 18380165, to H.K.). 
Correspondence: 2Hiroshi Kagami, Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4598, Japan. FAX: 81 265 77 1419; e-mail: kagami{at}shinshu-u.ac.jp
Received: 1 March 2007.
First decision: 20 March 2007.
Accepted: 10 April 2007.
REFERENCES
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