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RESEARCH |
1 Transgenic Animal Research Center and 2 Animal Genome Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan and 3 Laboratory of Intellectual Fundamentals for Environmental Studies, National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8506, Japan
Correspondence should be addressed to M Naito; Email: mnaito{at}affrc.go.jp
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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PGCs circulating in the bloodstream have the ability to migrate to the germinal ridges of embryos, which makes it possible to transfer PGCs between embryos and produce germline chimaeric chickens (Tajima 2002, Naito 2003a, 2003b, 2003c). Interestingly, PGCs isolated from embryonic blood can migrate to the germinal ridges after transfer into the stage X blastoderm (Naito et al. 2004). Furthermore, PGCs isolated from the gonads of 5-day incubated embryos (stage 27) can migrate to the germinal ridges after transfer into the bloodstream of recipient embryos and enter the germline (Chang et al. 1997, Tajima et al. 1998). In general, it is thought that male PGCs retain the ability to migrate to the germinal ridges up to 12–13 days of incubation (stages 38 and 39) and that female PGCs retain the migratory ability up to 7 days of incubation (Howarth 1995). Recently, Minematsu et al.(2004) reported the presence of gonocytes retaining the migratory ability in the gonads of embryos up to 20.5 days of incubation (stage 45) and also germ cells (GCs) in the testes or ovaries of sexually matured chickens. Differentiation into the functional gametes of these migrated PGCs, gonocytes or GCs in the recipient gonads, however, has not been examined. Long-term tracing of the donor cells in recipient embryos has now become possible by detecting the single nucleotide polymorphism (SNP) using compatible populations of donors and recipients (Harumi et al. 2004). Germline chimaeras can thus be identified by analysing the DNA extracted from the gonads of developing embryos or from the sperm samples of adult chickens before test cross.
The spermatogonia population in the testes contains stem cells (Kanatsu-Shinohara et al. 2003). Culture of these stem cells provides a novel method for manipulating chicken germline in vitro. To manipulate these germline cells, one must assess the migratory ability and subsequently differentiate germline cells into functional gametes after transfer into the bloodstream of recipient embryos. The present study was carried out to examine whether testicular and ovarian gonocytes obtained from 20-day incubated embryos (stage 45) have the ability to migrate to the germinal ridges and contribute to the germline lineage after transfer into the bloodstream of recipient embryos.
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Results |
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and 2A and B) of 20-day cultured embryos. After dissociation of gonadal cells, GFP-positive cells were identified as gonocytes in testicular and ovarian cells (Figs 1D–F and 2C–E), and they were recognised to be relatively large cells compared with other somatic cells. Testicular and ovarian gonocytes obtained from 20-day incubated embryos were also identified by immunostaining and recognised as chicken vasa homologue (CVH)-positive cells (Fig. 3A and B). The proportion of gonocytes in the male and female gonadal cell populations were 0.94% (60/6400) and 0.75% (55/7320) respectively.
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) or ovarian gonocytes (Fig. 4D–F) had been transferred. The GFP gene was mainly expressed in the left gonads of both chimaeric embryos. The rates of GFP gene expressing embryos in the gonads were 66.7% (10/15) in embryos with transfected testicular gonocytes and 72.7% (16/22) in embryos with transfected ovarian gonocytes.
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). DNA samples from the gonads and blood (as a control) were analysed for the presence of the donor-derived D-loop region of the mitochondrial DNA by PCR. Donor-derived DNA was detected in all the DNA samples from the gonads of embryos in both the same-and mixed-sex chimaeric embryos, whereas the donor-derived DNA were detected in none of the DNA samples from the blood (Fig. 5, Table 1).
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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The results of the gonadal cell transfer experiment show that testicular and ovarian gonocytes obtained from 20-day incubated embryos have the ability to migrate to the germinal ridges of recipient embryos, enter the gonads and contribute to the germline lineage after transfer into the bloodstream of recipient embryos. The introduction of testicular and ovarian gonocytes into the recipient gonads and germline was confirmed by detecting the expression of introduced GFP gene and the presence of donor-derived mitochondrial DNA in the gonads of developing recipient embryos. The contribution of the donor cells to the recipient germline lineage was also confirmed by detecting the donor-derived mitochondrial DNA in the sperm samples of the putative chimaeric chickens. It could not be confirmed, however, that the transferred testicular and ovarian gonocytes differentiated into functional gametes, because no donor-derived offspring have so far been obtained from the putative chimaeric chickens in males and females.
Mixed-sex germline chimaeric chickens produced by the transfer of different-sex PGCs isolated from the blood of 2.5-day incubated embryos can hardly generate donor-derived offspring (Naito et al. 1999). In the present study, ovarian gonocytes were transferred into male recipient embryos, and these donor cells entering the recipient gonads were also incorporated into recipient germline. This is, to our knowledge, the first observation of ovarian gonocytes that could be incorporated into male germline. When female PGCs isolated from the blood of 2.5-day incubated embryos were transferred into male recipient embryos, the female PGCs entered the recipient germline and differentiated normally up to the round spermatid stage through the first and second meiotic divisions (Tagami et al. 2007). Further differentiation of the W-chromosome-bearing spermatids into spermatozoa (spermiogenesis) was hardly completed (Naito et al. 1999, 2001, Tagami et al. 2007), but the Z-chromosome-bearing spermatids completed spermiogenesis. The transferred ovarian gonocytes in the present study probably differentiated normally into spermatids, but only Z-chromosome-bearing spermatozoa were produced and detected as donor-derived mitochondrial DNA in the sperm samples of the mixed-sex germline chimaeric chickens.
Spermatogonia transplanted into the seminiferous tubules of recipient testes differentiated normally and reportedly gave rise to viable progenies in mice (Brinster & Zimmemann 1994). Also in chickens, testicular cells (spermatogonia and differentiated spermatogenic cells) transplanted into recipient testes of juvenile or adult chickens were incorporated into the germline and produced viable offspring (Lee et al. 2006). The frequency of donor-derived offspring from the chimaeric chickens was, however, very low (0.4–0.9%), suggesting that few, if any, testicular cells participate in the spermatogenesis in recipient testes. When gonadal cells of embryos incubated for 5–7 days (stages 27–31) were transferred into the bloodstream of recipient embryos, germline transmission rates of donor-derived gametes were only up to 27.6% (Chang et al. 1997, Tajima et al. 1998, Park et al. 2003) when compared with up to 97.6% for PGCs isolated from the blood of 2.5-day incubated embryos (Naito et al. 1994a, 1994b, 1998a, 1998b, 1999). PGCs that entered the gonads gradually lost their ability to migrate to the germinal ridges, and their incorporation rate in the recipient germline decreased when they were transferred to recipient embryos. This evidence notwithstanding, the presence of migratory GCs in the testes or ovary of adult chickens was reported (Minematsu et al. 2004). The characteristics of these migratory cells in adult testes or ovary should be examined in future.
It is confirmed that germline stem cells are present in the population of spermatogonia in the testes of adult mice, and the genetic manipulation of germline cells via cultured germline stem cells is now being explored (Kanatsu-Shinohara et al. 2003, 2006). In chickens, germline stem cell-like cells are present in a population of spermatogonia (Jung et al. 2007). The population of testicular gonocytes manipulated in the present study would, thus, contain the germline stem cells. If these germline stem cells could be isolated and cultured in vitro, a novel method for germline manipulation could be devised in chickens. A technique must be developed to produce viable offspring efficiently from the germline stem cells when they are established.
It is concluded that testicular and ovarian gonocytes obtained from 20-day incubated embryos have the ability to migrate to the germinal ridges after transfer into the bloodstream of recipient embryos, and they could enter the gonads and contribute to the germline lineage of chimaeric embryos and chickens.
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Materials and Methods |
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Preparation of PGCs
Fertilised eggs of BPR were incubated at 38 °C for about 53 h in a forced-air incubator (P-008B Bio-type; Showa Furanki, Saitama, Japan). Blood was collected from the dorsal aorta of embryos at stages 13–15 using a fine glass micropipette. PGCs were concentrated by the Nycodenz density gradient centrifugation method (Zhao & Kuwana 2003, Naito et al. 2004). Briefly, the collected blood was pooled, washed and dispersed in a 400 µl KAv-1 medium (Kuwana et al. 1996). Five millilitres of 11% Nycodenz solution was placed in a 50 ml tube (Cat. No. 2070, Becton Dickerson, Franklin Lakes, NJ, USA), and 5 ml of 5.5% Nycodenz solution and subsequently 400 µl blood solution were overlaid. The tube was centrifuged at 400 g for 15 min and 10 ml of the PGC-rich solution was recovered from the top and washed with KAv-1 medium. Then, a second purification was carried out in the same manner.
Transfection of PGCs
Transfection of PGCs was carried out by lipofection using cationic lipids (Cat. No. 11668-027, Lipofectamine2000; Invitrogen). Six microlitres of Lipofectamine2000 solution were first diluted with 50 µl Opti-MEM I reduced-serum medium (Cat. No. 31985-062; Invitrogen) and incubated for 5 min at room temperature (25 °C) in a 5 ml polystyrene culture tube (Cat. No. 2003; Becton Dickerson). Two micrograms (2 µl) of plasmid DNA (pbAEGFP; GFP gene under the control of chicken ß-actin gene promoter) were added in the tube, mixed gently and incubated for 20 min at room temperature. The PGCs (about 4000 cells) dispersed in a 400 µl KAv-1 medium were added in the tube, mixed gently and incubated for 5 h at 37 °C. The transfection efficiency by this method is usually about 30% (Naito et al. 2007). The transfected PGCs were then washed twice with KAv-1 medium, dispersed in a 60 µl fresh KAv-1 medium and placed in a plastic dish.
Preparation of recipient embryos and transfer of donor cells
Recipient embryos of WL were cultured in host eggshells at 38 °C for about 53 h as described by Naito et al.(1990). When the embryos reached stages 14 and 15, 500 donor PGCs were picked up by a fine glass micropipette and injected into the bloodstream of recipient embryos. The manipulated embryos were cultured in host eggshells at 37.8 °C for an additional 4–18 days (Perry 1988, Naito et al. 1990).
Identification of testicular and ovarian gonocytes
Testes or left ovary were removed from the embryos in which transfected PGCs had been transferred. They were washed with Dulbecco’s PBS without Ca2+ and Mg2+ (DPBS(–), Cat. No. 28-103-05 FN; Dainippon Pharmaceutical, Osaka, Japan), cut into small pieces and treated with trypsin for 20 min at 37 °C. Then, the cells were dissociated by pipetting, washed with KAv-1 medium and dispersed in 200 µl KAv-1 medium. The dispersed cells were observed under an inverted fluorescence microscope (DMIRE2; Leica Microsystems, Tokyo, Japan) and testicular and ovarian gonocytes were identified as GFP-positive cells.
Testicular and ovarian gonocytes were also identified by immunostaining. The dissociated testicular and ovarian cells were fixed by 4% paraformaldehyde (163-20145; Wako Pure Chem., Osaka, Japan) for 1 h. After washing with DPBS(–), blocking was carried out with Blocking One (03953-95; Nakalai Tesque, Kyoto, Japan) for 1 h. The cells were then incubated with CVH antibodies (Tsunekawa et al. 2000) for 1 h. After washing with DPBS(–), the cells were incubated with alkaline phosphatase-labelled goat anti-rabbit immunoglobulin (SAB1005; Open Biosystems, Huntsville, AL, USA) for 30 min. The cells were then washed with DPBS(–), incubated with 5-bromo-4-chloro-3-indoxyl phosphate/nitro blue tetrazolium chloride substrate (K0598, Dako Cytomation, Glostrup, Denmark) for 2–3 min and washed with distilled water. The treated cells were observed using an inverted microscope.
Preparation of gonadal cell populations and chimaera production
Left testis or left ovary was removed from 20-day incubated embryos. They were washed with DPBS(–), cut into small pieces and treated with trypsin containing 200 U DNase I (2210A, Takara Bio Inc., Shiga, Japan) for 20 min at 37 °C. The collected gonadal cells were then washed with KAv-1 medium and dispersed in 400 µl KAv-1 medium. The gonadal cell populations (500–2000 cells) containing testicular and ovarian gonocytes, with or without transfection of GFP gene, were picked up by a fine glass micropipette and transferred to the bloodstream of recipient embryos. The manipulated embryos were cultured in host eggshells for 4–15 days or until hatching.
Detection of GFP gene expression in gonads of embryos
Embryos cultured for 4 days (stage 30) after the injection of transfected gonadal cell populations containing testicular and ovarian gonocytes were removed from the yolk, washed with DPBS(–) and the gonads were exposed. Expression of the GFP gene was detected under a fluorescence microscope (MZFL-III, Leica Microsystems).
Detection of donor-derived D-loop mitochondrial DNA in gonads of recipient embryos
In order to detect the donor-derived cells in the gonads of recipient embryos, the recipient gonads and also the blood (as a control) were analysed for the presence of the donor-derived D-loop region of the mitochondrial DNA.
Blood (about 100 µl) was collected from the embryos cultured for 15 days (stage 43) after the injection of gonadal cells, then gonads were collected and washed with DPBS(–). DNA was extracted from the blood and gonads using a DNA extraction kit (SepaGene, Sanko Junyaku, Tokyo, Japan) according to the manufacturer’s instructions. The extracted DNA was dissolved in distilled water at a concentration of 100 ng/µl, and PCR analysis was then carried out on 200 ng DNA samples to detect the presence of the donor-derived D-loop region of the mitochondrial DNA.
In the DNA sequences of chicken mitochondrial DNA of the D-loop region (DNA database accession number: AB091008 [GenBank] ), the 686th base is fixed as ‘A’ in the WL and as ‘G’ in the BPR used in this study (Harumi et al. 2004). In order to detect this SNP by PCR, mismatch-containing primers were designed. The sequences of the primers were 5-2C: 5'-TGG GGC TTC TTC ACA GGT CA-3' and DS7: 5'-CGA CAA GCATTC ACTAAATAG CAC C-3' for detecting the WL; and 5–3C: 5'-CCG CAC CCG CAC TGT GAA GGC C-3' and DS3: 5'-CCA TTT GGT TAT GCTCGC CGT ATC-3' for detecting the BPR (Harumi et al. 2004). PCR mixture was prepared using Takara Ex Taq kit (PR001, Takara Bio Inc.) and the reaction was carried out using GenAmp PCR system 9700 (Applied Biosystems Japan, Tokyo, Japan). After an initial denaturation step of 94 °C for 2 min, 40 cycles were carried out; DNA was denatured at 94 °C for 30 s, annealed at 62 °C for 30 s and extended at 72 °C for 1 min. The reactions were then incubated at 72 °C for 5 min. After amplification, 5 µl of the PCR products were separated on a 2% agarose gel, and the bands (WL: 224 bp, BPR: 334 bp) were visualised under u.v. light after ethidium bromide staining.
Test mating
Chicks hatched were raised until sexual maturity in which gonadal cell populations containing testicular or ovarian gonocytes had been transferred at the embryonic stage. Semen and blood were collected from the male putative chimaeric chickens, DNA was extracted from the collected sperms and blood cells and analysed for the presence of the donor-derived D-loop region of the mitochondrial DNA.
Both male and female mature putative chimaeric chickens were mated with BPR using artificial insemination, and the feather colour of their offspring was recorded. BPR are homozygous recessive (i/i) at the autosomal pigment inhibitor gene and the chick feathers are black, while WL are homozygous dominant (I/I) and their feathers are white. Black offspring (i/i) indicate that the chicks were derived from the donor cells (BPR), while white offspring (I/i) show their derivation from the recipient cells (WL).
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Acknowledgements |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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