Production of Trout Offspring from
Triploid Salmon Parents
Tomoyuki Okutsu,1 Shinya Shikina,1 Megumi Kanno,1 Yutaka Takeuchi,1 Goro Yoshizaki1,2*
n 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 donorderived 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 trans-
I
planting spermatogonia into sterile xenogeneic
recipients.
In this study, spermatogonia of pvasa-Gfp
(where Gfp represents green fluorescent protein)
hemizygous (pvasa-Gfp/–) and dominant orangecolored 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.
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.
References and Note
1. Y. Takeuchi, G. Yoshizaki, T. Takeuchi, Biol. Reprod. 69,
1142 (2003).
2. T. Okutsu, K. Suzuki, Y. Takeuchi, T. Takeuchi, G. Yoshizaki,
Proc. Natl. Acad. Sci. U.S.A. 103, 2725 (2006).
3. T. Okutsu et al., J. Reprod. Dev. 52, 685 (2006).
4. Y. Takeuchi, G. Yoshizaki, T. Takeuchi, Nature 430, 629
(2004).
5. This study was supported by the Industrial Technology
Research Grant Program from the New Energy and
Industrial Technology Development Organization (NEDO).
Downloaded from www.sciencemag.org on October 3, 2007
BREVIA
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
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
mm. (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.
www.sciencemag.org
SCIENCE
VOL 317
24 May 2007; accepted 26 July 2007
10.1126/science.1145626
1
Department of Marine Biosciences, Tokyo University of Marine
Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 1088477, Japan. 2Solution-Oriented Research for Science and
Technology (SORST), Japan Science and Technology Agency, 5
Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
*To whom correspondence should be addressed. E-mail:
[email protected]
14 SEPTEMBER 2007
1517