Research Note Chimeric plumage coloration produced by ovarian transplantation in chickens1 J. Liu,*† M. C. Robertson,* K. M. Cheng,† and F. G. Silversides*2 *Agassiz Research Center, Agriculture and Agri-Food Canada, Agassiz, British Columbia, Canada V0M 1A0; and †Faculty of Land and Food Systems, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4 ABSTRACT Ovaries from Rhode Island Red donors were transplanted orthotopically into White Leghorn recipients. At maturation, recipients were mated with Rhode Island Red roosters to test the origin of their ovaries, using plumage coloration as a marker. A chick with chimeric plumage coloration was produced, in-
dicating mechanisms that produce follicles with both donor and recipient ovarian contents. This study suggests that ovarian transplantation could be useful for in vivo studies of cytological and molecular mechanisms involved in avian folliculogenesis.
Key words: chicken, ovarian transplantation, chimera, plumage coloration, folliculogenesis 2013 Poultry Science 92:1073–1076 http://dx.doi.org/10.3382/ps.2012-02843
INTRODUCTION Orthotopic transplantation has been used to recover reproductive potential of ovarian tissue in several avian species, using fresh (Song and Silversides, 2007, 2008; Song et al., 2012) or cryopreserved (Liu et al., 2010) ovarian tissue from donor birds surgically implanted to the normal location of the left ovary in ovariectomized recipients. Incomplete ovariectomy has been common in previous studies, which permits the residual ovarian tissue of the recipients to reach maturity and produce recipient-derived offspring. Plumage coloration has been used as a marker to distinguish donor-derived offspring from recipient-derived offspring (Song and Silversides, 2007, 2008; Liu et al., 2010). In chickens, plumage color is determined by the distribution of melanins including eumelanin and pheomelanin as a result of interactions among a series of gene loci (Smyth, 1990). The distribution of eumelanin is the first genetic decision, which is primarily controlled by the E locus. At this locus, the E allele that causes extended black is dominant to other alleles including wild type (e+). The noneumelanic feather may be pigmented by pheomelanin, which is subjected to modifications by other genes such as the sex-linked silver gene at the S locus. In White Leghorn (WL) chickens, which were used as recipients in a previous study (Song and Silversides, 2007), the extended black allele (E) at the ©2013 Poultry Science Association Inc. Received October 16, 2012. Accepted December 31, 2012. 1 Agassiz Research Center Contribution Number 811. 2 Corresponding author:
[email protected]
E locus is suppressed by the presence of a dominant white allele (I) at the I locus and pheomelanin pigmentation is eliminated by a dominant silver allele (S) at the S locus, resulting in white plumage (Smyth, 1990). The dominance of the I allele to its recessive allele i+ is incomplete and the plumage of extended black individuals that are heterozygous at this locus (I/i+) show occasional black spots on a white background. When the ovarian tissue from a donor that is colored at the I locus (i+/i+) is transplanted into a WL recipient (I/I), the origin of the progeny can be tested by crossing recipients with males from the donor (colored) line. Resulting chicks with colored down (i+/i+) are derived from the transplanted ovarian tissue, whereas chicks that are white with black spots (I/i+) are derived from the regenerated recipient ovary. In this study, birds from a line with colored plumage (i+/i+ e+/e+) were used as ovarian donors, and ovarian recipients were from a WL line (I/I E/E). After sexual maturation, a progeny test was conducted by inseminating the recipients with semen collected from colored roosters (i+/i+ e+/e+), using plumage color as a marker. This research note reports a chick with chimeric plumage coloration that appears to be derived from both donor and recipient ovarian tissue.
MATERIALS AND METHODS Newly hatched Rhode Island Red (RIR) chicks carrying the rc mutation (Cheng et al., 1980) and WL chicks from a line maintained at the Agassiz Research Center (Silversides, 2010) were used as donors and recipients, respectively. The research protocol was ap-
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proved by the Animal Care Committee of the Agassiz Research Centre following principles described by the Canadian Council on Animal Care (2009). All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada) unless otherwise indicated. Ovaries from newly hatched RIR chicks were isolated immediately after euthanasia by cervical dislocation and immersed in a handling medium that consisted of Dulbecco’s PBS with 20% fetal bovine serum on ice. The isolated ovaries were kept in handling medium on ice before further treatment within 2 h. The cryopreservation and warming procedures described by Liu et al. (2010) were used in the current study. The surgical transplantation described previously (Song and Silversides, 2007) was improved and used in this study. Newly hatched WL chicks were anesthetized by administration of isoflurane. The chick was placed on its back on a heated surgical surface. The feathers were removed from the left abdominal area, where a 2-cm vertical incision was made approximately 1 cm left of the median plane. The yolk sac was removed after tying the yolk stalk with surgical suture. The abdominal organs were pushed to the right with a laboratory spatula to expose the ovary, which was illuminated with a fiber light source, and removed in small pieces using a pair of fine forceps. A donor ovary was then placed in the position of ovariectomy of the recipient, and the incision was closed. The recipient chicks were administered an immunosuppressant, mycophenolate mofetil (CellCept, Hoffmann-LaRoche Ltd., Mississauga, Ontario, Canada), orally at 100 mg/ kg per day for 2 wk, followed by once a week until the age of 2 mo. Recipient chicks were kept in a brooder for 2 wk with a temperature of 33°C and then in pens with a temperature of 25°C and a day length of 12 h. At 15 wk of age, the pullets were caged individually with the same temperature and day length. At the onset of egg laying,
the recipient hens (I/I E/E) with RIR ovarian transplants (i+/i+ e+/e+) were inseminated with pooled semen from RIR rosters (i+/i+ e+/e+) to test the genetic origin of their offspring. Brown chicks (i+/i+ e+/e+) produced from this cross were derived from the RIR ovarian transplants, whereas white chicks (I/i+ E/e+) with randomly distributed black spots originated from regenerated recipient WL ovarian tissue.
RESULTS AND DISCUSSION Donor- and recipient-derived chicks from the transplantations performed are shown in Figure 1A. One hen from this series of transplantations produced 25 white chicks with randomly distributed black spots, indicating that they were derived from the ovary of the recipient. This hen also produced one chick that had white plumage with black spots over most of her body along with randomly distributed brown patches (Figure 1B) over large areas. The white areas of this chick are clearly derived from the recipient ovary as confirmed by the presence of black spots, indicating that some cells have the E allele at the E locus (E+/e+), which is incompletely suppressed by the heterozygous I locus (I/i+). However, the brown areas clearly originate from the donor tissue because they do not carry either I or E. This chick must therefore be a chimera composed of tissue from both donor and recipient ovaries. Two other hens produced similar chimeric chicks. One of these hens also produced 48 recipient-derived chicks and 3 light-colored chicks with no black spots, which may have been donor-derived, and the other hen produced 34 recipient-derived chicks with no donor-derived chicks. Chimerism in plumage coloration cannot occur if the oocyte that contributes to the zygote is completely donor- (i+/_ e+/_) or recipient- (I/_ E/_) derived unless the composition of the oocyte is altered during follicu-
Figure 1. Chicks produced from progeny test. A: A donor-derived chick (left) was uniformly brown, and a recipient-derived chick (right) was white with randomly distributed black spots. B: A chick with chimeric plumage had areas of white with randomly distributed black spots and areas of brown coloration. Color version available in the online PDF.
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Figure 2. Possible mechanisms for chimeric folliculogenesis. A: Closely positioned donor and recipient oocytes may communicate (indicated by the double arrow) and form chimeric follicles. B: Donor and recipient oocytes may fuse to form a chimeric polyovular follicle.
logenesis. In the present study, the surgeons conducting ovariectomy were inexperienced and the production of the recipient-derived chicks demonstrated that ovariectomy of the recipients was incomplete. The production of the chimeric chicks showed that donor tissue survived cryopreservation and transplantation, making it possible for donor and residual recipient ovarian tissue to fuse. Before the formation of follicular epithelium, which starts 4 to 5 d after hatch (Greenfield, 1966), closely positioned donor and recipient primary oocytes could exchange their contents through intercellular bridges (Skalko et al., 1972) or interruptions of their plasma membranes (Chin et al., 1979), resulting in 2 individual follicles containing oocytes with chimeric compositions at a later stage (Figure 2A). Avian oogenesis is characterized by the accumulation of a large amount of maternal RNA (Olszanska and Stepinska, 2008), some of which may be involved in the signaling pathways of melanogenesis (Kerje et al., 2004; Yoshihara et al., 2012) as regulatory elements. These regulatory elements could be exchanged between donor and recipient oocytes and be unevenly distributed in the early embryonic cells after fertilization and cleavage to modify the melanogenic pathways of the cells in which they reside through epigenetic regulatory mechanisms (Rassoulzadegan et al., 2006). As an alternative, donor and recipient oocytes could fuse to form a single follicle with chimeric oocytes (Figure 2B) through a process similar to the formation of polyovular follicles that are commonly observed in avian folliculogenesis (Amer and Shahin, 1975; Wakuri and Mutoh, 1986). Polyspermy during fertilization is normal in birds, but sperm that enter the egg through regions other than the germinal vesicle are subjected to high levels of DNase I and II (Olszanska and Stepinska, 2008). The chimeric polyovular follicle provides 2 germi-
nal vesicle regions that could allow fertilization of both ova and thereby generate the chimera. On the other hand, after fertilization, at least one of the ova in the chimeric polyovular follicle could develop parthenogenically with or without sperm activation (Sarvella, 1973; Rougier and Werb, 2001). These hypotheses can be further tested by transplantation of ovaries into recipients that are incompletely ovariectomised and subsequent histological and molecular assays. Orthotopic ovarian transplantation in avian species has been attempted previously with limited success (Guthrie, 1908; Davenport, 1911; Grossman and Siegel, 1966), but Song and Silversides (2007) improved it and Liu et al. (2010) used it for functional recovery of cryopreserved avian ovarian tissue. The present study suggests that ovarian transplantation can also be used as a useful in vivo approach for investigating folliculogenesis, ovarian development, and ovarian functioning in avian species.
ACKNOWLEDGMENTS This research was supported by a grant from Poultry Industry Council (Guelph, Canada), Canadian Poultry Research Council (Ottawa, Canada), and Egg Farmers of Canada (Ottawa). The authors thank the poultry crew at the Agassiz Research Centre for taking care of the experimental birds.
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