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Production of Trout Offspring from Triploid Salmon Parents

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Science  14 Sep 2007:
Vol. 317, Issue 5844, pp. 1517
DOI: 10.1126/science.1145626

Abstract

Many salmonids have become at risk of extinction. For teleosts whose eggs cannot be cryopreserved, developing techniques other than egg cryopreservation to save genetic resources is imperative. In this study, spermatogonia from rainbow trout were intraperitoneally transplanted into newly hatched sterile triploid masu salmon. Transplanted trout spermatogonia underwent spermatogenesis and oogenesis in male and female recipients, respectively. At 2 years after transplantation, triploid salmon recipients only produced trout sperm and eggs. With use of these salmon as parents, we successfully produced only donor-derived trout offspring. Thus, by transplanting cryopreserved spermatogonia into sterile xenogeneic recipients, we can generate individuals of a threatened species.

In recent decades, the number of salmonid species has declined markedly, and several species have become extinct or endangered. Because cryopreservation of fish eggs is difficult due to their large size and high fat content, we investigated the potential of surrogate broodstock technologies as a new method of genetic resource preservation for fish. Surrogate broodstock technologies involve the transplantation of primordial germ cells (PGCs) (1) or spermatogonia (2) from a target fish species into a related species for which rearing techniques are well developed. In doing so, the recipient species can produce sperm and eggs of the target species (3). Furthermore, because PGCs and spermatogonia are sufficiently small for cryopreservation, animals can be generated via the transplantation of thawed PGCs or spermatogonia into recipients, even if the target species becomes extinct. In prior work, we demonstrated that most spermatozoa produced by xenogeneic recipients are of recipient origin; few donor-derived spermatozoa are produced (4). In addition, the production of viable donor-derived eggs in xenogeneic recipients has not yet been observed in any animal species to date. The present study therefore attempted to produce only donor-derived sperm and eggs by transplanting spermatogonia into sterile xenogeneic recipients.

In this study, spermatogonia of pvasa-Gfp (where Gfp represents green fluorescent protein) hemizygous (pvasa-Gfp/–) and dominant orange-colored mutant heterozygous [OR/wild type (WT)] adult rainbow trout (Oncorhynchus mykiss) were intraperitoneally microinjected into newly hatched embryos of triploid sterile masu salmon (O. masou). Hybrids of these two species do not survive. Histological examination showed that, whereas the testes of 2-year-old triploid salmon in the control group (no transplantation) were immature and contained mostly spermatogonia, testes of recipients appeared normal (Fig. 1A). Ten of the 29 male triploid salmon recipients produced milt. Offspring produced with milt from these 10 recipients and wild-type trout eggs developed normally (fig. S1 and table S1). Five F1 progeny were collected from each of the 10 recipients (n = 50) for species determination using random amplified polymorphic DNA (RAPD) analysis. All 50 specimens exhibited the same DNA fingerprint patterns as rainbow trout (fig. S2), indicating that male triploid salmon recipients produced only donor-derived trout.

Fig. 1.

Development of donor-derived germ cells and F1 offspring generated from surrogate parents. (A) Hematoxylin and eosin (H&E)–stained section of testes from an intact triploid salmon (top) and a triploid salmon recipient that received spermatogonial transplantation (bottom). Scale bars indicate 20 μm. (B) Oocyte colony derived from donor trout spermatogonia in the ovary of triploid salmon recipient at 17 months after transplantation (bottom) and ovaries of intact triploid salmon (top) at the same age as the recipient. (Insets) Fluorescent views. Scale bars, 5 mm. (C) Lateral view of orange-colored offspring (inset), with a highly magnified image of a frame. Gfp was expressed in PGCs (asterisk). (D) Trout juveniles at 6 months old generated from surrogate triploid salmon parents.

The ovaries of four of the eight female recipients contained vitellogenic oocytes at 17 months post transplantation (Fig. 1B). All vitellogenic oocytes exhibited donor-specific green fluorescence. Ovaries of intact triploid salmon of the same age contained no vitellogenic oocytes (Fig. 1B). When recipients reached 2 to 3 years of age, 5 of the 50 female triploid salmon recipients ovulated eggs (table S2) that were then fertilized with milt harvested from the male triploid salmon recipients. Although developmental rates of the offspring varied from one female broodstock to the next, the hatching rate reached 89.5% (table S2). The ratios of orange-colored trout to wild-type trout and of pvasa-Gfp(+) to pvasa-Gfp(–) were both about 3:1 in the F1 generation (Fig. 1C and table S3). These findings show Mendelian inheritance of OR/WT and pvasa-Gfp/–, implying that the F1 generation was produced from donor-derived sperm and eggs. Resulting fry also developed normally (Fig. 1D). Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA revealed that all F1 fish specimens examined (n = 18) carried trout mitochondria (fig. S3). Thus, female triploid salmon recipients that received trout spermatogonia produced only donor-derived trout eggs. In addition, RAPD analysis of total DNA showed that the DNA fingerprinting pattern of the F1 generation was the same as that of trout (fig. S3). Further, the F1 generation was fertile and could produce normal F2 trout. We therefore established a surrogate broodstock technique for salmonids in which spermatogonia can be transplanted into sterile triploid xenogeneic recipients to produce a next generation consisting entirely of donor-derived fish. We also confirmed that trout spermatogonia frozen in a cryomedium had a high associated survival rate (45.4%). Thus, by transplanting cryopreserved spermatogonia into sterile xenogeneic recipients, it is possible to generate individuals of an endangered, and perhaps extinct, species.

Supporting Online Material

www.sciencemag.org/cgi/content/full/317/5844/1517/DC1

Materials and Methods

Figs. S1 to S3

Tables S1 to S3

References

References

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