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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY |
* Department of Food Production Science, Faculty of Agriculture, Shinshu University, Minamiminowa, Nagano 399-4598, Japan; Department of Bioscience and Food Production, Interdisciplinary Graduate School of Science and Technology, Shinshu University, Minamiminowa, Nagano 399-4598, Japan; Department of Poultry and Minor Livestock Program, Indonesian Research Institute for Animal Production (IRIAP), PO Box 221, Bogor 16002, Indonesia; Reproductive Biology and Technology Research Team, National Institute of Livestock and Grassland Science (NILGS), Ikenodai 2, Tsukuba, Ibaraki 305-0901, Japan; and # Animal Breeding and Reproduction Research Team, National Institute of Livestock and Grassland Science (NILGS), Ikenodai 2, Tsukuba, Ibaraki 305-0901, Japan
1 Corresponding author: tagami{at}affrc.go.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: primordial germ cell • vasa • early embryo • immunostaining • chicken
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chicken PGC were originally identified using morphological criteria, such as a remarkably large size, large spherical nuclei, and the presence of refractive lipids in the cytoplasm (Zhao and Kuwana, 2003), coupled with either a histochemical marker such as periodic acid-Schiff (PAS), which stains for glycogen (Meyer, 1960), or antibodies such as stage-specific embryonic antigen-1 (antiSSEA-1) and embryonic mouse antigen-1 (antiEMA-1), which recognize cell-surface carbohydrate antigens.
However, PAS staining efficiently detects PGC only after stage 4 (Arabic numerals refer to the staging system of Hamburger and Hamilton, 1951). Moreover, the SSEA-1 epitope, galactose-N-acetylglucosamine-fucose (Gooi et al., 1981), is expressed on inner cell mass, epiblastic cells, and migratory PGC in mice, suggesting that the antigen is not germline-specific. AntiEMA-1 recognizes fucosylated polylactosamine carbohydrate groups and was originally raised against mouse embryonic carcinoma cells (Hahnel and Eddy, 1986). AntiEMA-1 labels not only mouse PGC, but also chicken PGC. However, only one-third to one-fifth of PAS-positive cells are labeled by antiEMA-1 in the chicken (Urven et al., 1988), indicating that antiEMA-1 is not suitable to define germ cell lineage. Consequently, morphology, PAS staining and immunohistochemical staining, such as with antiSSEA-1 or antiEMA-1, are not specific enough to allow a direct investigation of germline segregation in the chicken.
Recently, the gene vasa has received considerable attentions as a reliable molecular marker to trace the origin of the germline. The vasa gene was originally discovered in Drosophila (Lasko and Ashburner, 1988), and genes homologous to vasa have now been identified in various species, including Caenorhabditis elegans, Xenopus laevis, zebrafish, mice, humans, trout, and rat (Roussell and Bennett, 1993; Fujiwara et al., 1994; Komiya et al., 1994; Olsen et al., 1997; Yoon et al., 1997; Castrillon et al., 2000; Yoshizaki et al., 2000). Tsunekawa et al. (2000) isolated the chicken vasa homolog (CVH) gene and demonstrated germline-specific expression of CVH protein, mainly in sections of embryos. Their research showed that CVH could be used as one of the reliable molecular markers for investigating avian germ cell lineage.
Although many studies have investigated the behavior of chicken PGC, no studies have ever investigated the proliferation of chicken PGC and their migration from the area pellucida at stage X (newly laid egg) to the future gonadal region, using eggs that were obtained from the same population. Thus, the details of this migration and proliferation in early chick embryos remain unclear. Previous studies showed that PGC use the vascular system as a vehicle to transport them to the future gonadal region. But there has been no detailed analysis showing when and where PGC move from extraembryonic region to the vascular network or from the bloodstream to the future gonadal region.
Therefore, to elucidate the details of the migration and proliferation of PGC in the early chicken embryos, whole embryos from stages X and 2 to 17, or embryonic blood taken during stages 12 to 17, were immunohistochemically stained using an antiCVH antibody.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of Embryo Samples (Stages X and 2 to 17)
Fertilized eggs of RIR chickens were freshly obtained from the farm. Whole embryos were separated from the egg yolk using a filter paper ring (Advantec, Toyo Roshi Kaisha, Tokyo, Japan) and washed with phosphate buffered saline without Ca2+ or Mg2+ [PBS(–)]. For experiments on stage X embryos, treatments were conducted immediately after the eggs were laid. For experiments on embryos at stages 2 to 17, the manipulations were carried out after incubation in conditions of 39.0°C and relative humidity of 50 to 60%, with tilting at a 90° angle twice an hour, in a forced air incubator (P-008B Biotype; Showa Furanki, Saitama, Japan).
Antibody Production and Western Blot Analysis
The production of antibody to CVH was previously described (Tsunekawa et al., 2000). Polyclonal antibody raised against CVH protein was generated in rabbits by their method. Several tissues of newly hatched chickens were separately homogenized in SDS sample buffer and centrifuged at 15,000 x g for 5 min. The supernatant was collected, and the protein content was determined using Quick Start Bradford Dye Reagent (BioRad Laboratories, Hercules, CA). Tissue extracts (15 µg) were boiled for 5 min in the sample buffer and were separated by SDS-PAGE under nonreducing conditions according to Laemmli (1970) with minor modification. Then SDS-polyacrylamide 10% gel (Atto Corp., Tokyo, Japan) were electro-transferred onto Immobilon polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). The transferred membranes were incubated an hour with antiCVH antibody (1:10,000), followed by incubation with alkaline phosphatase conjugated goat antirabbit IgG (1:1,000) for 30 min. After washing, the signals were visualized using Western Breeze Chemiluminescent Immunodetection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Immunohistochemical Staining of Whole-mount Embryos
Embryos were fixed for 3 h in 4% paraformaldehyde (PFA) in PBS(–). After 3 washes in Tris buffered saline (TBS), embryos were dehydrated in 100% methanol for 2 h. After rehydration in TBS containing 0.1% Triton (TBS-T) for 1 h, embryos were incubated in TBS containing 2% H2O2 for 45 min to inactivate endogenous peroxidase activity; embryos were then washed 3 times in TBS-T, for 10 min each. Staining was carried out using the biotin/avidin-conjugated-horseradish peroxidase system (Vectorstain Elite ABC Rabbit IgG Kit; Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions with some modifications. Briefly, the embryos were incubated for 2 h in blocking serum, in which the concentration of goat normal serum was 4.5% to decrease background staining. AntiCVH antibody was diluted 1:5,000 with 1.5% goat normal serum in TBS-T solution and applied to embryos overnight. Embryos were washed 6 times for 30 min in TBS-T and incubated overnight in biotinylated secondary antibody that had been diluted with 1.5% goat normal serum in TBS-T solution (1:200). Thereafter, embryos were washed 9 times for 30 min in TBS-T and incubated with Vectastain Elite ABC Reagent for 45 min; they were then washed 3 times in PBS(–) for 10 min each. Cells expressing the antigen were detected by NovaRED (Nova RED substrate kit for peroxidase, Vector Laboratories, Burlingame, CA) solution according to the manufacturer’s instructions. All of the steps were performed at 4°C. Stained embryos were placed on 1.5% agarose gels, and the labeled cells were counted under a microscope (DFC480-Note OY, Leica Microsystems, Tokyo, Japan).
Collection of Blood Samples (Stages 12 to 17)
Fertilized RIR eggs were cultured in a forced air incubator to reach stages 12 to 17 under the same conditions as described above. Whole blood was collected from the dorsal aorta of embryos using a fine glass micropipette under a stereomicroscope (MS5; Leica Microsystems), and suspended in 100 µL of PBS(–) in a 0.2 mL tube.
Immunohistochemical Staining of Blood Samples
Blood samples were fixed by adding 100 µL of 8% PFA in PBS(–) for 15 min, dropped onto Teflon Printed Glass slides, ADCELL (Erie Scientific Company, Portsmouth, NH), and dried. After incubation in TBS containing 0.3% H2O2 for 30 min, blood samples were washed in TBS-T for 30 min. Samples were incubated with a 1:5,000 dilution of antiCVH antibody for 5 h and washed 3 times for 30 min in TBS-T. After incubation with a 1:200 dilution of biotinylated secondary antibody for 2 h, samples were washed in TBS-T for 30 min. The samples were incubated with Vectastain Elite ABC Reagent for 15 min and washed in PBS(–) for 10 min. Cells expressing the antigen were detected using NovaRED solution according to the manufacturer’s instructions. Labeled cells were observed under a microscope (Eclipse E1000, Nikon, Tokyo, Japan).
Sexing
After observation, DNA extraction from the tissue of the embryos during stages 14 to 17 was performed according to the method of Tagami et al. (2007). Molecular sexing was conducted by amplifying conserved regions of the CHD-W and CHD-Z genes using primers 2550 F and 2718 R, following the protocol of Fridolfsson and Ellegren (1999).
Statistical Analysis
Experiments were carried out on at least 5 different samples at each developmental stage. The number of PGC at each stage is presented as the mean ± standard deviation recorded from these samples. Data for the number of PGC within the intermediate mesoderm between sexes and between left and right sides were analyzed by t-test and matched pairs t-test, respectively. Differences were regarded as significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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. The number of PGC per embryo increased gradually as development progressed to stage 10. The stage 10 embryos have more than 3 times the number of PGC as stage X embryos.
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). The majority of PGC were localized on the ventral side of the epiblast layer, with only a few PGC observed on the dorsal side of stage X blastoderms. The distribution pattern of PGC at stages 2 (after 6 h of incubation) and 3 (after 12 h of incubation) differed from that at stage X. A total of 171.4 ± 19.8 PGC were detected in the anterior region of the central zone at stage 2; PGC then gradually moved anteriorly to produce an arc-like distribution pattern by stage 3 (Figure 3C). In addition, some PGC were found on and along the primitive streak. The number of PGC per embryo at this stage was 200.4 ± 19.8. At stage 4 (after 18 h of incubation), a total of 255.8 ± 35.8 PGC were found around the anterior edge of the extraembryonic region that is called the germinal crescent region (Figure 3D). The distribution pattern of PGC at stage 5 (after 20 h of incubation) was very similar to that at stage 4, but the number of PGC per embryo was 279.8 ± 24.4.
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). The number of PGC per embryo at stages 7, 8, and 9 were 312.8 ± 55.8, 347.0 ± 77.1, and 416.7 ± 57.8, respectively. Moreover, the region containing PGC expanded on dorsal and ventral sides according to the growth of the embryos. Many PGC could be recognized at the amniocardiac vesicle at stage 9 (Figure 5B). Additionally, no PGC were seen in the head region up to and including stage 9 (Figure 6A).
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). On the ventral side, PGC could be recognized along the anterior border of the extraembryonic region (Figure 5D). In the head region, PGC could be detected from this stage (Figure 6B to 6E). The number of PGC per embryo at stage 10 (after 35 h of incubation) was 439.3 ± 93.6.
At the beginning of stage 11 (after 40 h of incubation), most PGC were present at the region anterior to the head (Figures 7A to 7C). A total of 129.8 ± 42.5 PGC were observed in this region, at this stage. At this time, PGC (194.0 ± 41.6) began to appear in the blood vessels. In the latter half of stage 11 (after 44 h of incubation), the number of PGC at the region anterior to the head decreased to 46.7 ± 4.2 (Figures 7D, 7E). By contrast, the population of PGC located inside the vascular system (Figure 7F) increased to 285.0 ± 7.5, suggesting that PGC enter the blood vessels from this anterior region. Indeed, we observed some PGC as they were invading the newly formed blood vascular system at this region (Figure 7G).
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).
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). The PGC were first observed in the intermediate mesoderm at stage 15 (Figures 9B, 9E), and they were more abundant there by stage 17 (Figures 9C, 9F). In the intermediate mesoderm, some PGC produced a small pseudopodium or looked like in cell division (Figures 9G, 9H). The total number of PGC located in the intermediate mesoderm at stages 15, 16, and 17 was 73.3 ± 16.5, 188.9 ± 45.3, and 415.0 ± 54.4 in females, and 67.0 ± 18.1, 155.4 ± 25.7, and 293.0 ± 32.3 in males, respectively (Figure 10). No significant difference was observed in the total number of PGC within the intermediate mesoderm between sexes at stages 15 and 16, whereas the number of PGC in the same region of stage 17 embryos was significantly higher in female embryos than in male embryos (P < 0.05). Interestingly, the number of PGC in the intermediate mesoderm was consistently and significantly different (P < 0.05) between the left and right sides of this region, being more abundant on the left side during stages 15 to 17 in females and males. The number of PGC in the intermediate mesoderm during stages 14 to 17 in females and males is shown in Tables 1 and 2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Embryo samples were collected from the fertilized eggs of RIR chickens during stages X and 2 to 17. All eggs used were obtained from the same flock of chickens, incubated for the same length of time and under the same conditions to avoid environmental effects. The distribution pattern of PGC in stages X and 2 to 17 embryos was roughly the same in each embryo of a given stage; however, there were some individual differences in the number of PGC at each stage.
The PGC were seen to be scattered in the area pellucida at stage X. This result is consistent with former studies (Kagami et al., 1995, 1997; Karagenç et al., 1996; Tsunekawa et al., 2000). Our study showed that PGC are especially concentrated in the central zone of the stage X blastoderm and that they are located not only on the ventral surface of the epiblast, but also, in the case of a small number of PGC, on the dorsal surface of the epiblast. The average number of PGC at stage X was more than 100 in our study. Approximately 20 SSEA-1 or EMA-1 positive cells were previously observed on the ventral surface of the stage X epiblast, in the area pellucida (Karagenç et al., 1996). Tsunekawa et al. (2000) found about 30 CVH- positive cells scattered in the central zone of the 1-cell-thick area pellucida at stage X in White Leghorn chickens from the Livestock Industry Research Institute (Kanagawa, Japan). Our results differed considerably from those of previous reports. It is possible that the size of the PGC population varies widely between RIR chickens and other strains or between the RIR population at NILGS and other populations. The distribution pattern of PGC during early stages was roughly consistent with the results of PAS staining (Ginsburg and Eyal-Giladi, 1986) and CVH-immunostaining (Tsunekawa et al., 2000). In addition, the present study shows that the size of the PGC population increased gradually during stages X and 2 to 10. This finding showed that chicken PGC continue to proliferate throughout early development. Further analysis of the developmental origin of PGC would provide an important insight into the proliferation of PGC during early development in avian species.
Details of the migration of PGC from the extraembryonic region to the blood vascular system and from the bloodstream to the future gonadal region were little known. In this study, we have clarified the entrance point of PGC from the extraembryonic region to the vascular system. The PGC were scattered widely in the anterior part of the extraembryonic region at stage 9. A number of them began to accumulate at the region anterior to the head at stage 10, where they increased in number and became more densely packed by the beginning of stage 11. It could be considered that the aggregation of PGC to one specific region is caused by passive movement during primitive streak development or active movement, or both, due to an attraction to some unknown molecule(s). Previous studies showed that the appearance and distribution of extracellular matrix molecules such as laminin, fibronectin, chondroitin sulfate, collagen type IV (Urven et al., 1989), and tenascin-C (Anstrom and Tucker, 1996), along the migratory pathways of chicken PGC. These molecules are clearly temporally and spatially correlated with the migration of PGC from the germinal crescent to the germinal ridges; however, these molecules cannot guide PGC to the germinal ridges. Recent work showed that the chemokine stromal cell-derived factor 1 (SDF-1), the ligand of the receptor CXCR4 (Doitsidou et al., 2002; Knaut et al., 2003), provides zebra-fish PGC with directional cues in the course of their migration toward the gonads. The colonization of the gonads by germ cells is impaired in mice lacking functional SDF-1 or CXCR4 (Ara et al., 2003; Molyneaux et al., 2003). However, Stebler et al. (2004) revealed that SDF-1 is not involved during the first phase of PGC migration from the extraembryonic region to the vascular system in chickens. It is possible that other high-potency chemokines attract PGC during this phase. It is important to research what kinds of molecules guide chicken PGC to the region anterior to the head. In electron microscopy studies, PGC were located inside the blood vessels forming in the germinal crescent at stage 10 or 11 and began to circulate in the blood, throughout the embryonic disk, until stage 11 (Kuwana and Fujimoto, 1984). The present study showed that the population of PGC that gathers at the region anterior to the head decreases, whereas the number of PGC localized in the blood vessels increases, during the beginning to the latter half of stage 11. Interestingly, the decrease in the number of PGC at this region is nearly equal to the increase in the number of PGC in the blood vessels. Moreover, PGC could be observed invading the vascular network during the latter half of stage 11. There are 2 possibilities, related ways by which PGC might enter into the blood vascular system. First, PGC might be passively ingested by capillaries that surrounding the PGC at the anterior part of the extraembryonic region during stage 10. Second, PGC that concentrated at the region anterior to the head might be actively invaded the peripheral vein. More detailed analysis will be needed to determine the number of PGC in stages 10 and 11.
In earlier studies, cPGC have been shown to exit blood vessels to migrate along the dorsal mesentery and collect at the germinal ridges (Fujimoto et al., 1976). Our observations showed that PGC could be recognized within the intermediate mesoderm from stage 15 and that the mean numbers of PGC increased as development progressed to stage 17. The concentration of cPGC reaches a peak in the bloodstream at stage 14 (Tajima et al., 1999). Taking our results into account, cPGC might be guided by some factor released from the intermediate mesoderm, and the attractive effect might be much stronger at stage 14 than during later stages. Stebler et al. (2004) suggested that SDF-1 might guide chicken PGC as they leave blood vessels on their way to the gonad region. However, there is still no direct evidence for its existence. Therefore, it is important to determine the molecular mechanisms governing PGC migration. Additionally, no significant difference was observed in the number of PGC within the intermediate mesoderm between sexes at stages 15 and 16, whereas the number of PGC in the same region at stage 17 was significantly higher in female embryos than in male embryos. This may imply that the proliferation rate of PGC in the intermediate mesoderm after stage 16 is higher in females than in males, the number of cPGC is higher in females than males, or both.
It has been reported that greater asymmetry in the number of germ cells within gonadal primordial started from 3 d of incubation in females and from 5 d of incubation in males, in light microscope study of histological sections (Zaccanti et al., 1990). However, the PGC observed in this study were distributed unevenly earlier than the report of Zaccanti et al. (1990). We have shown that more PGC were located on the left side of the intermediate mesoderm from stage 15 in females and males, suggesting this unevenly distribution of PGC contralateral intermediate mesoderms is independent of sexual difference. Little is known about the mechanisms of the unequal distribution of PGC in the intermediate mesoderm. The asymmetrical distribution of PGC between the left and right sides in the intermediate mesoderm might be controlled by some unknown factor expressed asymmetrically between the left and right sides.
Although previous studies have found the localization of PGC in the head region during stages 14 to 24 (Nakamura et al., 1988, 1991), it was unclear that the localization of PGC was within the head region in the earlier stages. We clarified the presence of PGC in the head region before stage 14. This study showed that PGC inside the head region appeared from stage 10.
In conclusion, immunohistochemical analysis using antiCVH antibody clarified chicken PGC continue to proliferate throughout early development, many PGC invaded into the vascular system from the region anterior to the head in stage 11, and PGC actively leave the blood vessels and migrate to the intermediate mesoderm from stage 15.
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ACKNOWLEDGMENTS |
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Received for publication December 12, 2006. Accepted for publication May 11, 2007.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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