Wednesday, August 4, 2004
10:30 AM–12:30 PM
(730) PREPARATION OF RECIPIENT GOATS FOR GERM CELL TRANSPLANTATION.
Behboodi, Esmail1, Ayres, Sandy2, Azuma, Chieko3, Blash, Steven 1, Echelard, Yann1, Honaramooz, Ali4, Dobrinski, Ina 4, 1 GTC Biotherapeutics, Inc., Framingham, MA2 Tufts University School of Veterinary Medicine, North Grafton, MA3 Tufts University School of Veterinary Medicine, North Grafton, MA4 University of Pennsylvania School of Veterinary Medicine, Kennett Square, PA
ABSTRACT- Transgenic goats are important for the production of biopharmaceuticals in their milk. Because methods currently available to generate transgenic goats are inefficient and costly, transgene introduction through the male germ line is a promising alternative. We demonstrated previously that transplantation of germ cells from transgenic donor goats to pre-pubertal wild-type recipients resulted in transmission of the donor haplotype to the offspring of the recipient. To allow donor germ cells more efficient access to the stem cell niche, endogenous germ cells in the recipient testis should be depleted. The objective of this study was to develop a testicular irradiation protocol to deplete germ cells in immature goats. Groups of 3 Alpine and Nubian goats at 1, 5 and 9.5 weeks of age received fractionated irradiation of the testes with 3 doses of 2 Gy on 3 consecutive days under short-acting anesthesia. Goats were castrated at 2, 3, or 6 months after irradiation and testis tissue was processed for histology. At least 150 tubule cross sections per testis in treated and age-matched untreated control animals were scored for presence and stage of spermatogenesis. At 2 months after irradiation, <5% of tubule cross sections contained germ cells developed to pachytene spermatocytes in goats irradiated at 1 or 5 weeks of age, compared to >50% in age-matched controls. In goats irradiated at 9.5 weeks of age, 10% of tubules contained spermatocytes 2 months after irradiation, whereas controls showed complete spermatogenesis in all tubules. At 3 months after irradiation, sperm were apparent in 18-25% of tubule cross sections in all groups, and in all tubules in control goats. By 6 months after irradiation, spermatogenesis had recovered in 60% of tubules in goats treated at 5 or 9.5 weeks of age but in only 29% of tubules after treatment at 1 week of age. These results indicate that fractionated testicular irradiation with 6 Gy results in depletion of germ cells in goats at all treated ages. Formation of a tubular lumen occurs in treated and untreated goats between 3 and 4 months of age, and will facilitate the introduction of donor cells. Therefore, testicular irradiation at 1 week of age followed by germ cell transplantation at 15 weeks of age should result in an optimized environment for donor cell colonization. Supported by NIH/NICHD 1R41 HD044780-01
KEY WORDS: Irradiation, Testis