The study and application of testis tissue xenografting
Testis tissue xenografting (TTX) provides a novel in vivo model for the study of testis function, and a previously-unavailable opportunity to produce spermatozoa in the grafts from immature donors of diverse species. The overall objectives of this thesis were to examine a number of factors that potentially affect the outcome of TTX, and to apply TTX using immature bison and deer donors as models for endangered ungulates. The objective of the first experiment was to examine the effects of recipient mouse strain, gender and gonadal status on the outcome of TTX. Eight small fragments of neonatal porcine testis tissue (~5 mg each) were grafted under the back skin of immunodeficient mice of different strains (SCID vs. nude), gender (male vs. female), and gonadal status (intact vs. gonadectomised), using a 2×2×2 factorial design (8 groups, n = 7 mice/group). The xenografts were recovered at 8 mo post-grafting and evaluated for gross and histological attributes. Gonadectomy of the recipients did not affect any of the measured outcomes of TTX (P > 0.05), and data were pooled into four groups based on recipient strain and gender. Overall, male recipient mice had grafts with higher mean (+SEM) recovery rate (97 ± 2.3% vs. 88 ± 2.4%, P = 0.004), weight (348 ± 26.3 vs. 104 ± 27.0 mg, P < 0.001), seminiferous tubular diameter (150 ± 3.3 vs. 108 ± 5.3 mg, P < 0.001), percentage of tubules containing spermatozoa (32 ± 3.2 vs. 6 ± 1.8%, P < 0.001), elongated spermatids (13 ± 1.4% vs. 4 ± 0.8%, P < 0.001), and round spermatids (10 ± 1.2% vs. 6 ± 1.1%, P = 0.006) than female mice. Overall, SCID mice had grafts with higher recovery rate (98 ± 2.4% vs. 87 ± 2.3%, P = 0.001), average weight (292 ± 27.0 vs. 160 ± 26.3 mg, P = 0.001), tubular density (44 ± 3.3 vs. 33 ± 2.1, P = 0.02), percentage of tubular cross-sections containing spermatocytes (27 ± 3.7% vs. 13 ± 2.3%, P = 0.003) than nude mice. Among the four groups of recipients, the grafts from male SCID mice had the highest weight (P < 0.05) and percentage of tubules containing spermatozoa (P < 0.05). The objective of the second experiment was to evaluate the effect of using different numbers of donor testis tissue fragments on the outcome of TTX. Fragments of donor piglet testis tissue were grafted subcutaneously under the back skin of four groups of castrated male nude mice (n = 10/group). Each group of recipient mice received 2, 4, 8, or 16 fragments per mouse. Mice were sacrificed at 8 mo post-grafting, and xenografts were evaluated for physical growth and histological development. The relative weight of the vesicular gland (index) was also determined as a measure of bioactive androgen production by grafts in castrated recipient mice. The overall graft recovery rate was ~94% (range 86-98%) which did not differ among the groups (P > 0.05). The group of mice that received 16 testis tissue fragments had higher mean (+ SEM) graft weights (278 ± 39.4 vs. 106 ± 38.0, P = 0.02), total graft weight (2,443 ± 338.8 vs. 192 ± 76.2, P < 0.001), vesicular gland index (0.5 ± 0.06 vs. 0.1 ± 0.06, P = 0.007), and percentage of seminiferous tubules with round spermatids (11 ± 1.5 vs. 3 ± 1.3, P = 0.03) than the group of mice that received two testis tissue fragments. The objective of the third experiment was to assess the use to salvage testis tissue from neonatal/immature bison or deer donors using TTX into immunodeficient recipient mice as models for closely-related rare or endangered ungulates. Donor testis tissue fragments from two newborn bison calves (Bison bison bison) and a 2-mo-old white-tailed deer fawn (Odocoileus virginianus) were grafted under the back skin of gonadectomised nude mice (n = 15 and n = 7 for bison and deer groups, respectively, 8 testis fragments/mouse). To examine the potential effect of individual donors, we grafted four testis tissue fragments from one bison calf on one side of the recipient and four fragments from the second bison calf on the other side. Single grafts were surgically removed from representative recipient mice every 2 mo for up to 16- and 14 mo post-grafting, for bison and deer groups, respectively. The overall graft recovery rates were 69% and 63% for bison and deer groups, respectively. For bison grafts, a donor effect on efficiency of spermatogenesis was also observed. The weight of bison testis tissue xenografts increased (P < 0.02) ~4-fold by 2 mo and ~10-fold by 16 mo post-grafting, and gradual maturational changes were evident in the form of seminiferous tubule expansion starting at 2 mo, first appearance of spermatocytes at 6 mo, round spermatids at 12 mo, and elongated spermatids at 16 mo post-grafting. Testis tissue xenografts from donor white-tailed deer also showed a gradual development starting with tubular expansion by 2 mo and presence of spermatocytes by 6 mo post-grafting, round and elongated spermatids by 8 mo, followed by fully-formed spermatozoa by 12 mo post-grafting. The timing of complete spermatogenesis roughly corresponded to the reported timing of sexual maturation in these species. Taken together, the findings in this thesis suggest that male SCID mice provide a more suitable recipient model for TTX with neonatal porcine testis tissue; recipient mice can be grafted with as many as 16 testis tissue fragments for optimal results; and that TTX is a feasible strategy for salvaging genetic materials from immature males of rare or endangered ungulates that die prematurely.
DegreeMaster of Science (M.Sc.)
DepartmentVeterinary Biomedical Sciences
ProgramVeterinary Biomedical Sciences
CommitteeMapletoft, Reuben; Adams, Gregg; Muir, Gillian; Barth, Albert; Baerwald, Angella
Copyright DateJune 2010