Comparison of spermatogenesis and steroidogenesis of cryopreserved neonatal mouse testicular tissue – vitrification vs. controlled slow-rate freezing

Abstract

Ectopic allo- or xeno-grafting of frozen testicular tissue is a promising new approach that can be used to preserve testicular function in mutant animals and cancer patients for future fertility. Rescue of genetic resources and valuable mutant strain associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The aim of present study was to improve new mouse testicular tissue cryopreservation protocols by comparing spermatogenesis and testosterone levels of grafted neonatal testicular tissue after exposed to different vitrification protocols and slow-rate freezing. Experimental basic animal study. Neonatal mouse testes were frozen using 0.7 M DMSO + 5% FBS with a computer controlled slow rate freezer. Three modified vitrification protocols were used: (1) DAP 213 (2 M DMSO + 1 M Acetamid + 3 M Propylene glycol (PG)), (2) 40% Ethylene glycol (EG) + 18% Ficoll + 0.35 M Sucrose, and (3) 15% EG + 15% PG + 0.5 M Sucrose solutions. Frozen-thawed testicular tissues were grafted onto castrated immunodeficient Ncr-nude mice. Fresh neonatal testes were grafted to serve as the positive control. Animals were sacrificed, and histological analyses of grafted tissue and serum hormonal assays were performed 12 weeks post-transplantation. Survival of testicular grafts is superior in the control and computer controlled slow rate freezing groups comparing to the other 3 vitrification groups (P<0.05). Spermatogenesis from the slow rate freezing groups is also comparable to the controls. Testosterone production from the slow rate freezing group is significantly higher than the vitrification protocols tested (P<0.01). There were no significant differences between controlled slow-rate freezing technique and freshly grafted control. Our data shows a clear advantage of the computer controlled slow-rate freezing protocol over the standard vitrification techniques for neonatal mouse testis cryopreservation. Further modifications and improvement in the vitrification process is required before it can be applied to testicular tissue.