| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nanae Fresh Water Laboratory,3 Field Science Center for Northern Biosphere, Hokkaido University, Hokkaido 041-1105, Japan
Laboratory of Aquaculture, Genetics and Genomics,4 Faculty of Fisheries Sciences, Hokkaido University, Hokkaido 041-8611, Japan
ABSTRACT
Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. In many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species.
developmental biology, early development, embryo, gametogenesis, primordial germ cells
Primordial germ cells (PGCs) are the founder cells of the germ-line. In fish, the large numbers of germ cells that constitute the gonad originate from only a few dozen PGCs [1]. Recently, PGCs have received considerable attention as a potentially valuable resource for genetic conservation and for the production of individuals from gametes of germ-line chimeras [2–4]. Takeuchi et al. [5, 6] achieved successful production of offspring [5] and sperm [6] from salmonid germ-line chimeras. In their studies, gonadal cells, including PGCs, were harvested from the genital ridges of hatching larvae and transplanted into the peritoneal cavities of recipient larvae by microinjection. Interspecific transplantation of gonadal cells can produce sperm derived from donor PGCs [6]. However, it is quite likely that the method established in salmonid fish cannot be applied to all other fish species, especially those that have small eggs and larvae compared with salmonids. This obstacle arises because it is extremely difficult to surgically isolate genital ridges from hatched small larvae and to transplant PGCs. Indeed, the peritoneal cavities of some species do not have sufficient space to receive the transplanted cells. Most fish species, including model species for biological research such as zebrafish and medaka, produce small eggs that are unsuitable for PGC transplantation by the method developed for salmonid fish. Clearly, it is essential that a new method be developed that overcomes this limitation and can be widely applied to fish species.
In a few fish species, germ-line chimeras have been generated through the transplantation of blastula-stage cells, including PGCs [2, 7–11]. Interspecific transplantation of blastomeres has also resulted in the production of sperm derived from donor PGCs [12]. However, to date, production of eggs by interspecific chimeras has not been reported. It is also important to note that the methods employed in all studies mentioned above could not exclude the possibility of contamination by donor somatic cells. To advance our understanding of the biology of PGCs and germ cells in fish, it will be important to overcome these limitations. What is more, it is not known how many PGCs are required for gonadal development and gametogenesis in vertebrates.
In this study, we describe a new method that ensures complete germ cell replacement in a host embryo following transplantation of a single donor PGC. We have applied the technique to a range of species and demonstrated the feasibility of using a wide range of species for xenogeneic spermatogenesis in male fish. Initially, we used the pearl danio as the PGC donor species and the zebrafish as the host species (Fig. 1, A and B). We subsequently extended this study to include a more divergent array of fish species from different genera and families, namely the goldfish (Carassius auratus, family Cyprinidae, order Cypriniformes) and the loach (Misgurnus anguillicaudatus, family Cobitidae).
|
This study was carried out in accordance with Guide for the Care and Use of Laboratory Animals in Hokkaido University and Field Science Center for Northern Biosphere, Hokkaido University.
Zebrafish were kept in the Nanae Fresh Water Laboratory, Hokkaido University. Parent fish were maintained at 26–28°C under a 16L:8D photoperiod. Fertilized eggs were obtained during the light period through natural mating: one female and two males were placed together in a10-L fish tank at 28.5°C. Embryos were dechorionated with 0.1% trypsin (Difco), 0.002% actinase E (Kaken), and 0.4% urea in Ringer culture solution (128 mM NaCl, 2.8 mM KCl, and 1.8 mM CaCl2). Dechorionated embryos were cultured in Ringer culture solution containing 0.01% penicillin, 0.01% streptomycin, and 1.6% albumen. Dechorionated embryos were cultured at 28.5°C in 24-well plates (Greiner) individually filled with Ringer culture solution for 24 h and afterward in another separate well filled with culture solution (1.8 mM CaCl2 and 1.8 mM MgCl2) containing the same antibiotics as above. The stage of embryonic development was identified according to Kimmel et al. [13].
Pearl danio were kept at the Nanae Fresh-Water Laboratory, and embryos were obtained as for zebrafish. Dechorionation and culture methods were also identical to those of zebrafish. The developmental stages of the pearl danio have not yet been described. Therefore, staging of the embryos was accomplished by observation of the numbers of blastomeres and somites and the proportion of epiboly.
The loaches used in this study were obtained from the Loach Farming Cooperation of Kitamura during the spawning season from June to July. Fertilization, dechorionation, and culture of loach embryos were carried out as described by Fujimoto et al. [14]. The stage of development was identified according to Fujimoto et al. [15].
Goldfish were kept in the Nanae Fresh-Water Laboratory, Hokkaido University. Embryos were obtained by the method of Yamaha and Yamazaki [16]. Goldfish developmental stages were determined by the criteria suggested by Yamaha et al. [17] and Kajishima [18]. Dechorionation and culture methods were as described by Yamaha et al. [19] and Otani et al. [20], respectively.
Single PGC Transplantation Method
The PGCs of pearl danio, goldfish, and loach were visualized by injection of green fluorescent protein (GFP)-nos1 3'UTR mRNA and were identified as described in Saito et al. [1]. Donor embryos were also colabeled with 5% lysine-fixable biotin-dextran (molecular weight = 10 000; Sigma) for histological observation. On average, pearl danio, goldfish, and loach embryos had 24.8, 43.2, and 16.1 GFP-tagged PGCs, respectively, at the somitogenesis stage. Embryos at the 10- to 15-somite stage were digested into single cells by 0.1% collagenase (Wako) or 0.1% trypsin (Difco) and 1% sodium citrate in Ringer solution. During the transplantation procedures, which took between 1 and 2 h, the cells were held in this enzyme solution. Isolated PGCs were collected with a glass microneedle under a stereomicroscope. One PGC was transplanted into the marginal region of the blastodisc of each blastula-stage, zebrafish embryo. Development of the host (zebrafish) PGCs was blocked by injection of a dead end (dnd) antisense morpholino oligonucleotide (MO) as described in Ciruna et al. [2]. Chimeric embryos were observed and photographed with a fluorescence stereomicroscope (Olympus SZX-12) equipped with a digital camera (Olympus Camedia).
Rearing Conditions for Chimeric Fish
Chimeric fish were reared at 26–28.5°C in 10-L fish tanks at a density of 5–10 fish per tank. Some chimeric fish carrying transplanted pearl danio or goldfish PGCs were artificially stimulated at 20 days postfertilization to undergo female development; these individuals were kept at 28.5°C for 1 mo in water containing 17-β estradiol (E2) at 100 ng/L.
Chimeric males produced by the single PGC transplantation (SPT) method of goldfish or loach PGCs were killed at 10 to 12 mo after fertilization. The testis of each fish was minced in artificial physiological saline: for goldfish, 96 mM NaCl, 70.2 mM KCl, 2.1 mM CaCl2, and 1.1 mM MgCl2, pH 7.8; for loach, 128 mM NaCl, 2.7 mM KCl, 3.6 mM CaCl2, and 2.1 mM MgCl2, pH 7.8. Diluted testis solution was mixed with mature eggs on a polypropylene sheet. Inseminated eggs were transferred to plastic dishes filled with tap water.
We extracted DNA from tissues or whole embryos and performed PCR with species-specific primers: pearl danio, 5'-GCAGACGATGAAGAATGGGAATACTGCT-3' and 5'-AGTTAGTGAGAAAGCACCACTCCGC-3'; and zebrafish, 5'-GAGGAATGGGAATAACTGGC-3' and 5'-ACTGCCAAAAGGTATTAGTC-3'. These primers were designed to amplify the 3'UTR of the vasa gene; they produce a 632-bp product from the pearl danio and a 207-bp product from the zebrafish. The 3'UTR of the vasa gene contains approximately 12% sequence differences between pearl danio and zebrafish. PCR amplification was performed with TaKaRa rTaq DNA polymerase. After the initial denaturation step, the PCR consisted of 30 cycles at 95°C for 30 sec, 60°C for 30 sec, and 72°C for 1 min.
RT-PCR Analysis of Gonadal RNA
Total RNA was isolated from the gonads of individual fish with the TRIzol reagent (Gibco BRL). Total RNA was treated with TURBO DNase (Ambion) for 15 min at 37°C. For the RT-PCR analysis, total RNAs from the gonads of pearl danio-to-zebrafish chimeras, zebrafish, pearl danio, and dnd antisense MO-injected fish were used as templates for the first-strand cDNA synthesis. The cDNA synthesis was carried out with PowerScript Reverse Transcriptase (Clontech) as described in the manufacturer's protocol. PCR was performed with the species-specific primer pairs described above.
Transplanted cells were labeled with biotin-dextran to allow immunocytological detection and to determine their positions on histological sections. Chimeric embryos at 6 days' postfertilization were fixed with Bouin fixative for 2 h and embedded in paraffin. Serial 8-µm sections were cut and attached to glass microscope slides with 0.005% poly-L-lysine (Wako). Biotin-labeled transplanted PGCs were stained with diaminobenzidine with Histofine SAB-PO (M) kit (Nichirei), as described in the manufacturer's protocol, and counterstained with eosin.
For histological analysis, gonads were fixed with Bouin fixative for 4–12 h and embedded in resin according to the manufacturer's recommended protocol (Technovit 7100: Kulzer). Serial 2-µm sections were cut and stained with hematoxylin and eosin.
Statistical analysis was performed by an unpaired t-test. Differences were considered significant at P < 0.05.
Our first step in establishing a viable PGC transplantation method was to visualize PGCs with GFP [1, 21]. GFP-labeled PGCs were isolated from 10- to 15-somite-stage embryos of the pearl danio, and a single PGC was transplanted heterochronically into the marginal region of each blastula-stage, zebrafish embryo (Fig. 1, C–E). The donor embryos were at the 10- to 15-somite stage, and the isolated PGCs were at the migration period. At this developmental stage, the PGCs were migrating along the lateral side of the somites toward the region where the gonads eventually develop. Development of the PGCs of the host embryos was blocked by injecting a dnd antisense MO [2, 22, 23], effectively sterilizing the host embryos (Fig. 2, B and E). The donor PGC migrated from its original position in the host embryo toward the genital anlage (Fig. 1, F–I). In most embryos, the PGCs migrated without undergoing cell division; in the remainder, they divided once (Fig. 1, F–I). In normal embryos, PGCs increase in number more than fivefold during the same stages. In approximately half of the embryos, the transplanted PGC was located at the genital ridge region, in the upper part of the body cavity, at 6 days' postfertilization (Fig. 1, J and K); in the remainder, the PGCs were located ectopically or could not be detected in the embryo (Table 1).
|
|
To determine whether a transplanted pearl danio PGC followed the normal migration route for PGC migration in zebrafish embryos, we labeled host and donor PGCs with either DsRed or GFP. Transplanted pearl danio PGCs tagged with DsRed showed similar migratory behavior to host PGCs. Thus, at early somitogenesis, they clustered in most embryos with the host PGCs at the side of the first somite region. They then migrated with the host PGCs toward the genital anlage (Fig. 1, L and M).
In contrast to these results, PGCs at later developmental stages—for example, 25-somite or pigmentation stages—did not migrate to the gonadal region of host embryos, even when isolated and transplanted in the same manner. A few PGCs from 25-somite-stage donor embryos did localize at the migration route, e.g., lateral side of first to third somite region (data not shown).
MO-treated zebrafish embryos, each transplanted with a single pearl danio PGC, developed into zebrafish with normal morphology as reported earlier [23]. All of these chimeric fish were phenotypically male. This was also the case when zebrafish PGCs were transplanted into MO-treated zebrafish embryos (Table 2). To obtain female chimeras to enable reproduction by natural mating, we treated the chimeras with E2 for 1 mo, starting at 20 days postfertilization. As a result, three of the four E2-treated individuals developed as phenotypical females with the characteristic swollen abdomen and distinctive blue coloration (Table 2).
|
Gonadal Development in Chimeras
Gonadal development was examined histologically and morphologically in chimeras and their controls at 100–200 days postfertilization. Control individuals had normally shaped, paired gonads in their abdomen, and oogenesis or spermatogenesis was in progress (Fig. 2A). In contrast, MO-treated individuals had threadlike gonads (Fig. 2B). Chimeras produced by the transplantation of a single PGC had one normally sized gonad. In addition, they had a threadlike, second gonadal structure similar to that seen in MO-treated individuals (Fig. 2C). The normally sized gonad of chimeras contained a considerable number of spermatozoa in the lobule lumen similar to those observed in the normal testis (Fig. 2, D–F). Our results suggested that the one normally sized gonad in chimeric fish was induced by the transplanted pearl danio PGC.
PCR Genotyping of Tissues in Chimeras
To determine whether the gonad and somatic tissues of chimeric fish contained donor cells, a PCR-based DNA analysis was performed with species-specific primers designed for the vasa 3'UTR. DNA was extracted from five tissues: brain, liver, muscle, fin, and normally sized gonad. The somatic tissue samples appeared to contain DNA from only the zebrafish genome. The normally sized gonad contained DNA from both pearl danio and zebrafish genomes. Moreover, the pearl danio PCR product stained with significantly greater intensity than that of zebrafish (Fig. 2G). Overall, our results strongly suggest that germ cells derived from donor PGCs predominate in the gonad of chimeric individuals in which formation of the host germ cells has been blocked.
To determine whether the germ cells in the gonad of chimeric fish were derived solely from the donor cells, an RT-PCR analysis was also performed with the vasa 3'UTR primers. It has been already shown that the vasa gene is exclusively expressed in the germ cell line in both male and female fish. Thus, if zebrafish germ cells are present in the gonads of chimeras, zebrafish-specific PCR products will be synthesized by RT-PCR. We extracted total RNA from the gonad of one male and one female chimera and analyzed the products of RT-PCR. We did not detect the zebrafish-specific PCR band in the cDNA of the chimeric fish, although the pearl danio specific band was detected (Fig. 2H).
These results demonstrate that the transplanted single PGCs were the only contributors to gonadal development in the chimeric fish and that the germ cells of the host species were completely replaced by those of the donor, despite being from a different species.
Offspring Were Obtained from Natural Spawning of Chimeras Between Two Related Teleost Species
To determine whether the gametes of chimeric zebrafish were functional, a chimeric male was placed with a chimeric female in a spawning tank. Following light stimulation, mating behavior was initiated, and a large number of fertilized eggs were obtained. The size of these eggs was similar to that of the zebrafish host and not of the pearl danio (Fig. 3, A–C, and Table 3). However, the coloration pattern of the larvae was similar to that of pearl danio, that is, they had fewer melanophore-producing cells than normally present in zebrafish (Fig. 3, A–C). The offspring developed, hatched out, swam, and fed normally. Externally, the fish looked like normal pearl danio (Fig. 3D). PCR analysis of 50 embryos showed that they all carried pearl danio genomic DNA (Fig. 3E). Ninety-four percent of the chimeric males (15 of 16) and 66% of the females (2 of 3) were confirmed in spawning tests to be fertile, and they produced gametes derived from donor PGCs. In the two chimeras that did not show fertility, one had bilateral, threadlike gonads similar to those seen in MO-treated individuals (male); the other had a large, partially developed gonad in which the posterior part was threadlike (female). Fertile chimeras produced numerous offspring over at least 10 cycles of mating. The offspring of the chimeric fish proved to be fertile, and we obtained subsequent generations.
|
|
Reciprocal crosses of pearl danio-to-zebrafish chimeras and normal zebrafish were set up in spawning tanks. A number of fertilized eggs were obtained, and the resulting offspring developed normally, hatched out, fed, and swam. These fish had the characteristic phenotype of hybrids between pearl danio and zebrafish [24]. PCR analysis confirmed that these fish were hybrids between pearl danio and zebrafish. The fish were healthy and showed no serious deformation during development. However, histological observation of the gonads suggested that the hybrid fish did not produce gametes (total no. of observations = five).
Extension of SPT Between Teleost Species That Are Highly Divergent in Evolution
To investigate the extent of phylogenetic divergence that is compatible with production of functional germ-line chimeras, we used the same method as above to perform transplantations with PGCs from goldfish (a species from a genus different from zebrafish) and loach (a species from a family different from zebrafish) into the blastoderm of zebrafish embryos. Morphological and histological analyses confirmed that the transplanted PGCs formed the germ-line and could differentiate into spermatozoa in the zebrafish hosts (Fig. 4A; total number of observations = eight and five, respectively). However, although a lamella structure characteristic of the ovary was observed in E2-treated fish, no mature germ cells were present (Fig. 4B). Therefore, we were unable to obtain mature eggs in this experiment. To examine whether the spermatozoa in the lobule lumen of the testis of each of the interspecies chimeras were functional, insemination tests were carried out with eggs from the respective donor species. Viable offspring were obtained when sperm that differentiated in zebrafish hosts were used to artificially inseminate eggs of the original, donor species (Tables 4 and 5). The fertilized eggs developed, hatched out, and grew normally and showed the typical donor phenotype. In contrast, hybrid fish between goldfish and zebrafish and between loach and zebrafish did not develop normally (Tables 4 and 5). Deformities were observed in embryos from epiboly to at least the somitogenesis period (data not shown). Goldfish and loach eggs activated with water showed no development in the absence of spermatozoa.
|
|
|
In this study, we described a new method that ensures complete germ cell replacement in a host embryo following transplantation of a single donor PGC.
Transplanted PGCs from different species showed migratory behavior similar to host PGCs toward the genital anlage. This result strongly suggests that an alien PGC can recognize and respond to the guide signals that direct the host PGCs and can migrate under the control of the host species developmental environment. In the experiment with loach PGCs, the donor and host species differed at the family level of taxonomic classification. Moreover, the experiment demonstrated that PGCs isolated from later stages of embryogenesis have the ability to recognize and respond to the signaling environment present in early-stage embryos, suggesting that migration of PGCs is controlled by consistent mechanisms during embryonic development. However, our transplantation experiments also suggested that the ability of PGCs to migrate toward the gonadal region declined as development progressed. In contrast, Takeuchi et al. [6] showed that PGCs isolated from the gonads of masu salmon (Salmo [Oncorhynchus] masou macrostomus) can be incorporated into the gonads of hatched rainbow trout (Salmo [Oncorhynchus] mykiss); in this case, the donor and host differ at the species level. Moreover, this group reported that spermatogonia from the testes of adult trout can be incorporated into the gonads of hatched donor larvae [25]. These results suggest that germ cells from fish that have developed beyond the embryonic stage retain the ability to recognize and enter the gonads of hatched larvae, even of a different species. It is reasonable to suppose that PGC migration during embryonic stages (observed in our experiments) and PGC/spermatogonial incorporation into the gonad during the hatching stage are controlled by separate mechanisms. In most embryos, transplanted PGCs migrated without undergoing cell division. This result suggests that the proliferation of PGCs during the migrating period is restricted by factors intrinsic to PGCs but not by external signals. It will be important to identify the factors that halt PGC migration and proliferation during embryonic development and to determine the developmental changes that occur in PGCs.
MO-treated zebrafish embryos, each transplanted with a single PGC, developed into zebrafish with a normal morphology and were phenotypically male. Previous studies have shown that sterilized embryos, that is, those without intrinsic PGCs, develop as phenotypic males [23]. It is possible that the numbers of germ cells or PGCs are more important for sex differentiation than simply whether they are present or absent. Alternatively, the presence of only one rather than two gonads in the chimeras during germ-line development might affect sex determination. It will be of interest to examine the relationship between sex determination and the number of PGCs in zebrafish as well as the role of interactions between germ cells and somatic cells. The sex determination system in zebrafish has not yet been clarified because of the lack of sex-linked marker genes. Our PGC transplantation system may provide a means to obtain a greater insight into the sex determination system.
Chimeras had one normally sized gonad and a threadlike second gonadal structure. Our RT-PCR analysis clearly showed that the gonad contained only germ cells derived from the transplanted PGC and no host PGCs. The results showed that a single PGC was sufficient to allow normal development of one of the paired gonads. We have also shown that even heterospecific transplantation of a donor PGC recovered gonadal development in a sterilized host and resulted in germ cell proliferation within the gonad. Our results demonstrated that successful replacement of zebrafish germ cells with those of donor species could be achieved without any contamination by somatic cells from the donor. Fertile chimeras, each transplanted with a single pearl danio PGC, produced numerous offspring in a spawning tank. The offspring from the chimeras were genetically pearl danio but will have been exposed to maternal factors, such as the chorion and vitellogenin supplied by the zebrafish host. The success of this method suggests that it can be of use for effective fish production, such as from endangered species. Although Takeuchi et al. [6] reported that gonadal cell transplantation at the hatched larva stage resulted in functional germ-line chimeras between rainbow trout and masu salmon, they did not elaborate on the numbers of PGCs transplanted, nor did they describe egg production by donor-derived PGCs [6]. In our system, it is possible to exclude the influence of germ cells derived from the host and of cotransplanted somatic cells. Our study clearly demonstrates that numerous offspring can be obtained from interspecific germ-line chimeras produced by transplanting single PGCs into sterilized embryos. Moreover, the technique has a high frequency of success (40.8% of manipulated embryos). By this method, it is possible to judge whether developing embryos are germ-line chimeric, and embryos in which the PGCs are located ectopically can be identified and discarded.
To date, the phylogenetic relationship between pearl danio and zebrafish has not been the subject of a molecular clock analysis. However, the two species can successfully hybridize [24]. In the present study, we produced hybrids between these two species in reciprocal crosses and confirmed that the hybrid fish developed normally, although their gonads were small, and they did not produce gametes. Consequently, it is reasonable to suppose that physiologically, pearl danio and zebrafish are closely related. We also investigated whether the SPT method could work with more distantly related species. Thus, we performed transplantations with PGCs from goldfish and loach into the blastoderm of zebrafish embryos. As mentioned above, conventional crosses between either of these species and zebrafish yield hybrid embryos that do not hatch out. In the SPT experiments, we were unable to obtain mature eggs from the chimeras, but functional sperm of the donor species were obtained. Our results clearly showed that gonadal formation and spermatogenesis occurred following transplantation of a single PGC into zebrafish embryos without any support of donor-derived gonadal somatic cells. A detailed study of phylogenetic relationships in the Cypriniformes [26] and molecular clock analyses have provided estimates for the time of divergence of the Cobitidae (loach) and the Cyprinidae (goldfish and zebrafish) of 133.4 [27], 154.7 [27], 144 ± 4 [28], or 183 [29] million yr ago (Mya), depending on the data set. The Rasborinae (zebrafish) and Cyprininae (goldfish) are estimated to have diverged 50 Mya [28]. With regard to the relationship between zebrafish and loach, it is clear that, no matter which estimate is most accurate, zebrafish and loach separated at least 133.4 Mya. Thus, loach-to-zebrafish transplantation proved feasible despite their long evolutionary divergence. This suggests that similarly successful results will be possible with domestic fish and with transplantation of PGCs from wild or endangered fish species to sterilized hosts. One disappointing outcome was the failure of the loach-to-zebrafish and goldfish-to-zebrafish chimeras to develop ovaries under our rearing conditions. Possibly, differentiation of ovaries and eggs in chimeras between species that are highly divergent in evolution requires a highly elaborate relationship between the germ-line and soma, such as signaling and transfer of egg-forming materials.
In the gonads, PGCs finally differentiate into highly specialized cells, sperm in males and eggs in females. However, the process of germ cell differentiation is still obscure because germ cells are closely supported by the somatic cells during the whole of development; thus, they are difficult to access for experimental approaches in vivo. The methodology we have developed, the SPT method, produces an unusual germ cell environment, as the germ cells originate from a single PGC of one species, and the supporting somatic tissues are from a different species. By modifying the function of the donor PGC, we should be to manipulate the interaction between the germ-line and somatic cells. Our method therefore offers a new means to study germ cell biology in teleosts, especially with regard to the interaction of germ cells and somatic cells during the whole process of gonadal formation and gametogenesis.
ACKNOWLEDGMENTS
We thank Mr. S. Kimura, Ms. M. Takagi, and the members of the Nanae Fresh-Water Laboratory, Field Science Center for Northern Biosphere, Hokkaido University, for advice and help with breeding of the fish. We also thank Dr. T. Fujimoto and Mr. H. Yoshikawa, Laboratory of Breeding Science, Graduate School of Fisheries Sciences, Hokkaido University, and Dr. S. Kusuda, Hokkaido Fish Hatchery, for technical advice.
FOOTNOTES
1Supported by the 21st Century COE program for Graduate School of Fisheries Sciences of Hokkaido University from MEXT of the Japanese government and a grant-in-aid for Young Scientists (B) from JSPS to T.S. (19780412) and R.G.-K. (18780140). 
Correspondence: 2Taiju Saito, Nanae Fresh Water Laboratory, Field Science Center for Northern Biosphere, Hokkaido University, Sakura, Nanae, Kameda, Hokkaido 041-1105, Japan. FAX: 81 138 65 2239; e-mail: taiju{at}fish.hokudai.ac.jp
Received: 8 January 2007.
First decision: 13 March 2007.
Accepted: 14 September 2007.
REFERENCES
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
