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Department of Marine Biosciences,3 Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan
PRESTO,4 Japan Science and Technology Agency, Saitama 332-0012, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, early development, embryo, gametogenesis, gene regulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These techniques open a new approach to preserving the genetic resources of fish. Although fish sperm have been successfully stored by cryopreservation, this has not been possible so far for fish eggs or embryos. Therefore, the only available method for fully preserving the genetic resources of fish is to maintain and breed live individuals. Since PGCs can differentiate into either egg or sperm, cryopreservation of fish PGCs is an ideal alternative method. Indeed, a technique to cryopreserve trout PGCs has been previously established in our laboratory [8]. Furthermore, if xenotransplantation of cryopreserved PGCs into a closely related species was also possible, gametes produced by surrogate parents could be used to reestablish fish populations, even if the target species were extinct in the wild. This technique would be particularly useful for preserving the genetic material of endangered species. Another application could be in what is termed surrogate brood stock technology, which is used in marine ranching. Seed production from large fish with long generation times is expensive, because it requires extensive rearing space and is labor intensive. If the PGCs of such target species could be transplanted into closely related fish, which are smaller and have shorter generation times, such surrogate parent fish could support the proliferation of donor PGCs and the production of mature eggs and sperm. However, PGCs isolated from transgenic fish that contain the Gfp gene would not be appropriate for applications where fish would be released into the natural environment, such as the conservation of endangered species and surrogate brood stock technology. The establishment of transgenic strains for most large fish species would also be time, labor, and space intensive. A method for labeling fish PGCs without introducing a transgene would therefore be useful.
Recently, it has been reported that the 3'-UTR of vasa RNA plays a critical role in the stabilization of mRNA in zebrafish (Danio rerio), specifically in PGCs [9]. In this study, we investigated this phenomenon in various fish species to see if this mechanism for stabilizing Gfp RNA specifically in PGCs is widespread and could be used to label PGCs with GFP in Salmonidae and other fish species by a nontransgenic method. We isolated vasa 3'-UTR RNAs from rainbow trout (O. mykiss), Nibe croaker (Nibea mitsukurii), and zebrafish (D. rerio) and attached them to the 3'-end of Gfp RNA and introduced these chimeric RNAs into various fish embryos. If these vasa 3'-UTRs stabilized Gfp RNA specifically in PGCs, we expected GFP to survive for longer in PGCs than in somatic cells, where the RNA constructs would be gradually degraded. Further, we examined the possibility of interspecies and intergenus PGC transplantation using the GFP-labeled PGCs that contained chimeric RNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, vasa 3'-UTRs were obtained from rainbow trout (O. mykiss), zebrafish (D. rerio), and Nibe croaker (N. mitsukurii), which belong to Salmonidae, Cyprinidae, and Scianidae, respectively. The trout vasa 3'-UTR (622 base pairs [bp]) was amplified by polymerase chain reaction (PCR) using rainbow trout (O. mykiss) vasa cDNA previously cloned in our laboratory [10] as the template with a forward primer designed around the termination codon (5'-TGGGAG
TGATGCACATCTACATA-3'; underline represents the termination codon) and a reverse primer flanking the poly A tail (5'-TGGGACTTCATCCATATTTCAAATGTCT-3'). The reaction was performed with LA Taq (Takara Shuzo, Kyoto, Japan) according to the manufacturer's protocol. Thirty-five reaction cycles were performed, with each cycle consisting of 30 sec at 94°C, 30 sec at 58°C, and 40 sec at 72°C. For the cloning of the zebrafish (D. rerio) vasa 3'-UTR (652 bp), total RNA was extracted from adult ovaries. Zebrafish (D. rerio) were held in 60-L fish tanks at the Laboratory of Fish Culture, Tokyo University of Marine Science and Technology (Tokyo, Japan), under established conditions [11]. For ovary isolation, fish were deeply anesthetized and killed by decapitation. Immediately after dissection, the ovaries were homogenized and used for total RNA extraction using Isogen (Nippon Gene, Tokyo, Japan). The total RNA was used as the template for cDNA synthesis with Ready-To-Go You-Prime First-Strand Beads (Amersham Biosciences, Piscataway, NJ). Forward and reverse primers were designed around the termination codon (5'-TGGGAA
CTGGCCTCACACCTGTT-3'; underline represents the termination codon) and the sequence flanking the poly A tail (5'-TCACCAGTATCCGTCTTTATTTTGA-3'), respectively, from the zebrafish (D. rerio) vasa cDNA sequence [12]. Thirty-five reaction cycles were performed, with each cycle consisting of 30 sec at 94°C, 30 sec at 59°C, and 60 sec at 72°C. To obtain vasa 3'-UTR from Nibe croaker (N. mitsukurii), we first amplified a partial cDNA fragment by degenerate reverse transcriptase (RT)-PCR. Nibe (N. mitsukurii) were caught in the vicinity of Kujukuri-hama, Chiba Prefecture, Japan, by hook and line and transferred to the Laboratory of Fish Culture, Tokyo University of Marine Science and Technology. Ovarian tissues were obtained by the method described above. Template cDNA was synthesized from total RNA extracted from postvitellogenic ovaries as described above. RT-PCR, cloning of the amplified fragment, and DNA sequencing were performed using the methods described for rainbow trout (O. mykiss) vasa cDNA cloning [10].
After sequencing the DNA, 3'-rapid amplification of cDNA ends (RACE) PCR was performed using two vasa gene-specific primers (for the initial PCR, 5'-TCGACGTACAGCACGCTAGTGAACTT-3'; for nested PCR, 5'-TCCGTGTCCTGGTGGCGACCTCCGTA-3') by a previously described method [10]. The RACE product was cloned and sequenced and a forward primer designed around the termination codon (5'-TGACTGGGAG
AGGGGATATGAA-3'; underline represents the termination codon) to amplify Nibe croaker (N. mitsukurii) vasa 3'-UTR (accession No. AB181294). The reverse primer used for 3'-RACE was also used in this reaction, and the 3'-RACE product was used as the template. Thirty-five reaction cycles were performed, with each cycle consisting of 30 sec at 94°C, 30 sec at 60°C, and 60 sec at 72°C. The vasa 3'-UTR fragments amplified from the three species were electrophoresed on agarose gels, and the DNA fragments with the predicted molecular weights were isolated using UltraClean 15 DNA Purification Kit (Mo Bio Laboratories Inc., Carlsbad, CA). The purified DNA fragments were cloned into plasmid vector pGEM-T Easy (Promega Corp., Madison, WI) and used for construction of template plasmids for in vitro transcription. All procedures described herein were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction.
Preparation of Gfp-vasa RNA
Chimeric RNAs that contained vasa 3'-UTRs immediately downstream of hrGfp (Stratagene, La Jolla, CA) or DsRed2-1 (Clontech, Palo Alto, CA) coding regions were synthesized by in vitro transcription. The template plasmids for in vitro transcription were constructed in the following manner. AatII and SacII sites were introduced at the 5'- and 3'-ends of the hrGfp coding region by a PCR reaction with forward (5'-TACAGT
CACAACCATGGTGA-3'; underline represents AatII site) and reverse (5'-TACAGT
CTATTACACCCACT-3'; underline represents SacII site) primers. After restriction enzyme cleavages, the hrGfp fragment was inserted between AatII and SacII sites of the pGEM-T Easy plasmid that contained vasa 3'-UTRs from the three different fish species. The resultant plasmid was linearized using NdeI (Takara Shuzo) and used for in vitro transcription using Message Machine T7 Kit (Ambion Inc., Austin, TX). The synthesized chimeric RNAs were extracted with phenol/ chloroform, precipitated with ethanol, and dissolved in diethylpyrocarbonate-treated water at a final concentration of 200 µg/ml. These chimeric RNAs that contained vasa-3'-UTRs of rainbow trout (O. mykiss), zebrafish (D. rerio), and Nibe croaker (N. mitsukurii) were designated as Gfp-rt-vasa, Gfp-zf-vasa, and Gfp-nc-vasa, respectively. Chimeric RNA that contained DsRed2-1 and rainbow trout (O. mykiss) vasa 3'-UTR, named DsRed-rt-vasa, was prepared as the same manner. To introduce AatII and SacII sites at the 5'- and 3'-ends of DsRed2-1 coding region, forward (5'-TATATTCGCCACCATGGCCT-3'; underline represents AatII site) and reverse (5'-TATATTCTACAGGAACAGGT-3; underline represents SacII site) primers were used.
Microinjection and Embryo Observations
The Gfp-vasa RNA injection was performed using four Salmonidae species, including rainbow trout (O. mykiss), masu salmon (Oncorhynchus masou), brown trout (Salmo trutta), and brook trout (Salvelinus fontinalis), as well as zebrafish (D. rerio). Gamete collection and insemination of all Salmonidae species were performed as previously described [13]. Fertilized eggs were activated in 1 mM reduced glutathione solution (pH 8.0) to prevent hardening of the chorion [14]. A total of 2 nl of the RNA solutions was microinjected into the blastodisc of 1-cell-stage embryos between 3 and 7 h after fertilization by the method described by Yoshizaki et al. [15]. For each species, 100 eggs were microinjected and at least 10 randomly selected embryos were used for each fluorescent observation described below. To confirm that the chimeric RNAs that contained vasa 3'-UTRs specifically labeled PGCs, DsRed-rt-vasa RNA was microinjected into transgenic embryos whose PGCs expressed GFP under the control of rainbow trout (O. mykiss) vasa regulatory regions [4, 5]. The treated embryos were reared at 10°C. All zebrafish (D. rerio) experiments, including gamete collection, egg preparation, and microinjection, were performed as described by Meng et al. [16]. The embryos were observed at various developmental stages by fluorescent microscopy (BX-50 with a BX-FLA attachment and filter sets, U-MWIB2 for GFP and U-MWIG2 for DsRed; Olympus, Tokyo, Japan).
PGC Transplantation
Intraspecies and interspecies transplantation of PGCs was performed using masu salmon (O. masou) and brown trout (S. trutta) as donors and rainbow trout (O. mykiss) as recipients. Donor PGCs were labeled with GFP by microinjecting Gfp-rt-vasa RNA into fertilized eggs. Genital ridges that contained GFP-labeled PGCs were excised from newly hatched embryos (32 days post fertilization ["f] for rainbow trout and 40 dpf for the other species) using watchmaker's forceps under a fluorescent dissecting microscope (SZX-12 with an SZX-RFL attachment and a GFP filter set; Olympus). A total of 20–30 pairs of genital ridges were treated with 0.5% trypsin solution (pH 8.2; Worthington Biochemical, Lakewood, NJ) for 2 h at 20°C followed by DNase (100 U/ml; Worthington Biochemical) treatment to reduce the viscosity of the cell suspension. After physical cell dissociation by gentle pipetting, the medium was substituted for MEM medium (Nissui Pharmaceutical, Tokyo, Japan) that contained 5% of fetal calf serum. Cell transplantation was performed by the method described previously [7]. Briefly, between 5 and 10 PGCs were microinjected into the peritoneal cavity of newly hatched rainbow trout (O. mykiss) embryos using a glass micropipette attached to a microinjector (IM-9; Narishige, Tokyo, Japan). The transplantation experiments were repeated three times using at least 20 embryos for each donor species. The recipient embryos were reared at 10°C and observed periodically under a fluorescent microscope to trace the transplanted donor PGCs.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The temporal and spatial expression patterns of Gfp-rt-vasa were examined in rainbow trout (O. mykiss) embryos. The GFP expression was first detected in most blastomeres in 3-day-old embryos (blastula stage) (Fig. 1A). Although weak green fluorescence was observed throughout embryos at 15 dpf, a cell population located in two lines between the digestive tract and dorsal side of the peritoneal cavity, where the PGCs are located (Fig. 1B), was much more strongly fluorescent. At 20 dpf the green fluorescence over the body of the embryos became weaker, whereas that in the cells aligned at the dorsal side of the peritoneal cavity remained strong (Fig. 1C) and continued to be so until at least 50 dpf (Fig. 1D). To confirm the precise location of GFP-positive cells, the newly hatched embryos were dissected and excised genital ridges were examined for fluorescence. This confirmed that the GFP-positive cells were within the excised genital ridges (Fig. 1F). Furthermore, fluorescent analysis of enzymatically dissociated genital ridges revealed that the GFP-positive cells were approximately 20 µm in a diameter and possessed large nuclei with highly granular cytoplasm, which are typical morphological characteristics of PGCs (Fig. 1G and H). It was noteworthy that all the embryos that received Gfp-rt-vasa showed the above-mentioned GFP expression pattern.
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DsRed-rt-vasa RNA Expression in vasa-Gfp Transgenic Embryos
To further confirm the identity of the GFP-positive cells, DsRed-rt-vasa RNA was injected into vasa-Gfp transgenic rainbow trout (O. mykiss) embryos whose PGCs stably expressed GFP. Our previous immunohistochemical studies with specific antibody against GFP clearly showed that this transgenic strain expresses the Gfp gene specifically in PGCs [4, 5]. In the transgenic embryos that received DsRed-rt-vasa RNA, all PGCs labeled by red fluorescence also showed green fluorescence (Fig. 1I and K), demonstrating that the cells visualized by the chimeric RNAs attached to vasa 3'-UTRs were indeed PGCs.
Gfp-vasa RNA Expression in Heterologous Host Species
We examined the visualization of PGCs in embryos of various Salmonidae species and zebrafish (D. rerio) after injection with chimeric Gfp-vasa RNAs that contained vasa 3'-UTRs derived from rainbow trout (O. mykiss), zebrafish (D. rerio), and Nibe croaker (N. mitsukurii). Embryos of all Salmonidae species, including masu salmon (O. masou) (Fig. 2A), brook trout (S. fontinalis) (Fig. 2B), and brown trout (S. trutta) (Fig. 2C), that received Gfp-rt-vasa RNA showed clear green fluorescence specifically in PGCs at 20 dpf. Furthermore, this PGC-specific green fluorescence was observed at least up to hatching in all species. Notably, PGCs in masu salmon (O. masou) fry at 55 dpf were still identifiable by the specific green fluorescence (data not shown). In addition, PGCs in 20-dpf rainbow trout (O. mykiss) embryos and 30 hours post fertilization (hpf) zebrafish (D. rerio) embryos were successfully visualized by injecting both Gfp-zf-vasa (Fig. 2D) and Gfp-nc-vasa RNAs (Fig. 2E and F).
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Interspecies and Intergenus Primordial Germ Cell Transplantation in Salmonidae
The PGCs from rainbow trout (O. mykiss), masu salmon (O. masou), and brown trout (S. trutta) were labeled with Gfp-rt-vasa RNA in these cell transplantation experiments. The PGCs dissociated by enzymatic treatment could be identified easily by their green fluorescence (data not shown) and were used in cell transplantation procedures using a fluorescent dissection microscope. Observation of the recipient embryos at 5 days post transplantation revealed that the donor PGCs were attached to the peritoneal wall or the dorsal mesentery; however, they were not found in the genital ridges of the recipients (Fig. 3A). At 10 days post transplantation, 12.3% of recipient rainbow trout (O. mykiss) carried allogeneic donor rainbow trout (O. mykiss) PGCs in its genital ridges (Table 1). Similar incorporation was observed in rainbow trout (O. mykiss) that received brown trout (S. trutta) PGCs (Fig. 3C, Table 1). Incorporation rates of masu salmon (O. masou) PGCs into rainbow trout (O. mykiss) genital ridges were even higher than those of intraspecies transplantation (Fig. 3B, Table 1). The incorporation of donor PGCs was confirmed by examining excised genital ridges from the recipients by fluorescent microscopy (Fig. 3D). The number of GFP-labeled cells observed in the genital ridges from each recipient ranged from 1 to 3. Following the donor-derived PGCs in later embryos was difficult, because the green fluorescence due to the Gfp-rt-vasa RNA declined.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Interestingly, PGCs in rainbow trout (O. mykiss), which belong to the family Salmonidae, could be visualized with chimeric Gfp RNA that contains vasa 3'-UTRs from zebrafish (D. rerio), which belong to the Cyprinid family. Even more strikingly, PGCs in zebrafish (D. rerio) and rainbow trout (O. mykiss) were visualized by Gfp RNA that contained vasa 3'-UTRs from the Nibe croaker (N. mitsukurii), which is a marine fish that belongs to a different order, Perciformes. This suggests that fish PGCs can be visualized using chimeric RNA that contains vasa 3'-UTR sequences derived even from highly diverged taxonomic groups. We have demonstrated the compatibility of vasa 3'-UTRs derived from a salmonid fish (rainbow trout), a cyprinid fish (zebrafish), and a scianid fish (Nibe croaker) in these PGC visualization experiments. Therefore, it is likely that by using one of these three chimeric RNAs, the PGCs of most teleostean species could be labeled with the method established in this study. To date, most studies of fish PGC development have been performed in zebrafish (D. rerio), in which several molecular markers are available for identifying PGCs [17]; however, the ability to label PGCs with chimeric RNAs will open the way for studying the development of PGCs in a wide range of fish species.
Previously, we established a transgenic rainbow trout (O. mykiss) strain that carries the Gfp gene driven by the vasa gene regulatory regions [4, 5]. Interestingly, a construct that contains the SV40 polyadenylation signal, instead of the 3'-UTR of the vasa gene, failed to label PGCs with GFP, suggesting that this region was essential for visualizing PGCs in the transgenic trout (unpublished results). This observation agrees with the results obtained in this study. There are two major potential mechanisms for the PGC-specific expression of GFP seen in trout that received the chimeric RNAs. First, the vasa 3'-UTR may specifically stabilize Gfp RNA in PGCs but not in somatic cells. The second possibility is that the vasa 3'-UTR enhances translation of Gfp RNA specifically in PGCs. Although we cannot rule out the second possibility from this study, the half-life of GFP is known to be approximately 24 h [18], and the enhanced translation of Gfp RNA alone would be difficult to support the visualization of PGCs seen in 50 days. A third possible explanation is the specific localization of Gfp RNA to germ plasm. However, the trout vasa 3'-UTR has been shown not to be responsible for the localization of vasa RNA in zebrafish (D. rerio) blastula embryos and Xenopus oocytes [19]. In addition, RNA localization to the germ plasm occurs during oogenesis but not during embryogenesis, which we examined in this study. This finding would suggest that specific stabilization of Gfp RNA in PGCs by vasa 3'-UTRs is at least partially responsible for the phenomenon we observed.
GFP labeling with chimeric RNAs is a powerful tool for PGC transplantation in Salmonidae. GFP-labeled PGCs could be easily selected from suspensions of genital ridge cells and transplanted under a fluorescent microscope. Furthermore, it was useful for following the initial stages of donor cell development in the recipient embryos. It was noteworthy that the donor PGCs were incorporated into genital ridges of the recipient embryos even when transplanted between different species. Although the number of donor-derived PGCs incorporated in the xenogeneic recipient genital ridges was small (between 1 and 3), in earlier studies, we found that allogeneic and xenogeneic PGCs proliferated rapidly and produced a large number of gametes within the recipient genital ridges [7, 20]. Therefore, the number of xenogeneic PGCs incorporated in the recipient genital ridges in this study would also have a good chance of producing a significant number of gametes.
In this study and our preceding studies, we have established techniques for labeling PGCs not only by transgenic technique [4, 5] but also by chimeric RNA injection (this study). It is important to choose the appropriate method for the visualization of PGCs, which depends on the experimental objectives. Although injection of chimeric RNA labels PGCs only transiently, this nontransgenic technique is especially useful for seed production using surrogate brood stock and the cryopreservation of PGCs for the conservation of genetic resources in wild fish populations. On the other hands, PGC visualization with transgenic technology has immense benefits for basic studies, allowing fluorescence-activated cell sorting, long-term tracing of donor PGCs in recipient individuals, and PGC culture studies to be performed.
To date, cell-mediated gene transfer in fish has not been possible, because fully germ line competent embryonic stem cells are not available. The transplantation of genetically modified PGCs could be an alternative approach to generating transgenic fish, particularly if in vitro culture methods for PGCs can be developed. In combination with a GFP-dependent PGC purification system [5, 6], the PGC-labeling method described in this study will make it much easier to obtain pure PGC populations as starting material for developing PGC culture methods in various fish species.
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FOOTNOTES |
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2 Correspondence. FAX: 81 3 5463 0558; goro{at}s.kaiyodai.ac.jp
Received: 20 July 2004.
First decision: 6 August 2004.
Accepted: 24 February 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-globin gene in rainbow trout. Nippon Suisan Gakkaishi 1991 57:819-824
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