De Novo Morphogenesis of Testis Tissue in Sheep

Recovery of germ cells is an important tool to preserve the genetic resources of endangered animals. In adult males, sperm cannot always be collected and cryopreserved and testis tissue xenografting has shown little success. Formation of functional testis tissue after xenografting of dissociated testis cells has been reported for pigs and rodents and could be an alternative to the conservation of germ cells from adults. To develop this approach with bovid testis tissue, de novo morphogenesis of testis tissue was characterised in sheep testes. Preliminary work showed that isolated sheep testis cells are able to form testicular tissue de novo but germ cell differentiation had not occurred at 16 weeks postgrafting. Therefore, the objective of this study was to investigate if germ cell differentiation could be supported in testis tissue formed de novo by increasing time to tissue recovery or by increasing testosterone levels by co-grafting with intact testis tissue. Testes from 14 day old Suffolk lambs were processed in two ways: Part of every testis was cut into small tissue fragments and from the remaining testis tissue a single cell suspension was prepared by two-step enzymatic digestion. Eight pellets of 50x106 cells were transplanted under the back skin of 4 NCR-nude mice and 4 cell pellets and 4 pieces of testis tissue were grafted into another 4 NCR-nude mice. At the time of transplantation the cell pellets contained 1.27 ± 0.50 % germ cells, 55.47 ± 5.19 % Sertoli cells, 6.13 ± 0.92 % myoid cells and 4.60 ± 1.39 % Leydig cells. Histological examination was performed at 35 and 40 weeks after grafting (n=4 mice per time point). At 35 weeks postgrafting, no differentiated germ cells were found in the isolated cells grafted alone but single cell aggregates co-grafted with tissue contained pachytene spermatocytes. The weight of the seminal vesicles, an indicator of circulating levels of testosterone, was significantly higher (291.7 ± 81.7 mg) in mice carrying cell and tissue co-grafts, than in mice carrying only cell grafts (83.2 ± 69.9 mg). At 40 weeks after grafting, the seminal vesicle weights had increased 2 fold in both groups. Elongated spermatids were present in grafts from cells transplanted alone while germ cells in grafts of cells co-grafted with tissue showed no differentiation. These results show that isolated sheep testis cells are able to reorganize and form functional testis tissue. More time is required to complete germ cell differentiation than in intact animals and the efficiency of spermatogenesis is low, possibly due to the low number of spermatogonia harvested after enzymatic digestion. Increased levels of testosterone accelerated the onset of spermatogenesis in single-cell grafts. De novo formation of testis tissue might provide a novel alternative for germ line preservation in bovid species.Supported by NIH/NCRR RR17359-05, USDA/CSREES/NRICGP 03-35203-13486 and Spanish Ministry of Education and Science (BES-2004-4112).