Cryopreservation and xenografting of testis tissue
The objective of this thesis was to investigate and expand the use of testis tissue xenografting as means of maintaining the developmental potential of donor testis tissue. The objective of the first study was to investigate the effect of donor age on spermatogenesis in canine testis tissue after xenografting into immunodeficient recipient mice. Fragments of testis tissue from 12 dogs of different ages were xenografted under the back skin of mice. Donors were categorized based on testis developmental status at the time of grafting into: less than four months (immature), four to six months (young), and greater than six months of age (adult). The grafts were evaluated at four, six or eight months post-grafting. At four months post-grafting, immature and young groups had higher graft recovery rates (92 ± 5.8 and 88 ± 4.4% versus 69 ± 3.5%; P = 0.001 and P = 0.001), graft weights (34 ± 8.1 and 32 ± 11.0 mg versus 7 ± 2.6 mg; P = 0.001 and P = 0.02), vesicular gland indices (1.1 ± 0.20 and 0.6 ± 0.18% versus 0.1 ± 0.03%; P < 0.0001 and P = 0.02), seminiferous tubule numbers (517 ± 114.8 and 364 ± 161.0 versus 10 ± 5.1; P < 0.0001 and P = 0.03), and larger seminiferous tubular diameters (140 ± 17.8 and 130 ± 3.4 µm versus 55 ± 21.9 µm; P = 0.003 and P = 0.001), compared to adult donor xenografts. Xenografts from immature donors maintained the growth and development for eight months, as exhibited by greater graft weights (17 ± 4.6 mg, P = 0.002), seminiferous tubule numbers (547 ± 210.3, P < 0.01) and tubular diameters (93 ± 15.9 µm, P < 0.0001), and induced greater vesicular gland indices (1.5 ± 0.46%, P = 0.0005), compared to adult donor xenografts. The growth and development of testis tissue xenografts from immature and young donors were not different after eight months (P > 0.05). Young donor xenografts had greater seminiferous tubule number and diameter compared to adult donor xenografts (P = 0.009 and P = 0.004, respectively) at eight months post-grafting. Elongated spermatids were the most advanced germ cell type present at four and eight months post-grafting in the testis grafts of immature and young age groups. The objective of the second study was to evaluate three different strategies to preserve/cryopreserve immature porcine testis tissue. Immature porcine testes were cooled at 4 °C for 24, 48 or 72 hours, and testis tissue fragments were cryopreserved using programmed slow freezing with dimethyl sulfoxide (DMSO), glycerol, or ethylene glycol, or vitrified using DMSO or glycerol at 5, 15 or 30 min exposure time. In vitro cell viability was determined by trypan blue exclusion, and in vivo developmental potential was evaluated by xenografting into immunodeficient mice. Compared to fresh tissue, short-term cooling of porcine testis tissue resulted in similar in vitro cell survival rates (93 ± 2.2% for fresh versus 95 ± 0.3, 93 ± 1.7 and 87 ± 4.3% after 24, 48 and 72 hours at 4 °C, respectively; P = 0.74) and in vivo development, with generation of elongated spermatids and sperm after four months of grafting. Cryopreservation of testis tissue with programmed slow freezing using glycerol and vitrification with DMSO (5 min equilibration) or glycerol (5 or 15 min equilibration) did not compromise the developmental competence of xenografts when compared to fresh tissue (control), characterized by the formation of elongated spermatids and sperm. These findings suggest that canine testis tissue from immature donors and cooling of immature porcine testis tissue to refrigerator temperature for up to 72 hours or cryopreservation with slow controlled freezing or vitrification could be suitable methods to restore male fertility following xenografting.
DegreeMaster of Science (M.Sc.)
DepartmentVeterinary Biomedical Sciences
ProgramVeterinary Biomedical Sciences
CommitteeMapletoft, Reuben J.; Lessard, Carl; Anzar, Muhammad; Singh, Baljit