Irradiation Enhanced Efficiency of Testicular Germ Cell Transplantation in Sheep.

Abstract

The application of germ cell transplantation technology in livestock to produce individuals containing donor-origin sperm has great potential to replace traditional artificial insemination in animal breeding systems. However, current obstacles to this strategy are the low success rate of transplantation with a corresponding small proportion of donor spermatozoa in the recipient's semen. In rodents, the deletion of endogenous testicular stem cells by irradiation or chemotherapy drugs is a prerequisite for successful transplantation. However, until now there has been little information in the literature with regard to the beneficial effects of the preparation of recipients in germ cell transplantation in livestock animals. In the present study, we have investigated the effect of different doses of irradiation on the outcome of germ cell transplantation in sheep. Prepubertal Merino ram lambs with a scrotal circumference (SC) ranging from 21-28cm were irradiated with 9 and 15 Grays (Gy). The size of the SC decreased rapidly following irradiation in both groups with a greater reduction with 15 Gy. Donor testicular cells were enzymatically isolated from 5 cross-bred and 1 Border Leicester ram with SC from 18.5-25cm and injected into both testes of 13 recipient rams (n=3, non-irradiated control) at 6 weeks of post irradiation. In order to investigate the functionality of the irradiated recipient testes, semen was collected from all recipients following 3 months of post transplantation over a period of 10 months. The percentages of donor DNA were tested by microsatellite markers. One out of 3 (33%), 2/5 (40%) and 5/5 (100%) recipients tested positive for donor DNA in the control, 9 Gy and 15 Gy groups, respectively, indicating that irradiation of recipient testis with a single dose of 15 Gy significantly increased the success rate of germ cell transplantation. Interestingly, one ram in the control group tested positive for donor DNA at 18 weeks post transplantation but from 26 weeks onwards this ram tested negative. The highest percentage (30%) of donor origin sperm was identified in a 15Gy treated ram. One positive ram was obtained from the cross breed transplantation, where 10% of Border Leicester DNA was found in the semen of a Merino recipient from the 15 Gy group. Our results demonstrated that 1) the preparation of recipient's testis with irradiation not only enhances the efficiency of germ cell transplantation but also increase the proportion of donor sperm in recipient semen 2) homologous testicular germ cell transplantation in sheep is feasible and the resultant recipients are capable of producing sperm of donor origin.