Effect of different cryoprotectant agents on spermatogenesis efficiency in cryopreserved and grafted neonatal mouse testicular tissue

Abstract

Restoration of male fertility associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The purpose of this study was to test the effects of four different cryoprotectant agents (CPA) on spermatogenesis and steroidogenesis in cryopreserved and allotransplanted neonatal mouse testicular tissue. Hank's balanced salt solution (HBSS) with 5% fetal bovine serum including either 0.7 M dimethyl sulfoxide (DMSO), 0.7 M propylene glycol (PrOH), 0.7 M ethylene glycol (EG), or glycerol was used as the cryoprotectant solution. Donor testes were collected and dissected from neonatal pups of CD-1 mice (one day old). Freezing and seeding of the testicular whole tissues was performed using an automated controlled-rate freezer. Four fresh (non-frozen) or frozen–thawed pieces of testes were subcutaneously grafted onto the hind flank of each castrated male NCr nude recipient mouse and harvested after 3 months. Fresh neonatal testes grafts recovered from transplant sites had the most advanced rate of spermatogenesis with elongated spermatid and spermatozoa in 46.6% of seminiferous tubules and had higher levels of serum testosterone compared to all other frozen–thawed-graft groups (p < 0.05). Fresh grafts and frozen–thawed grafts in the DMSO group had the highest rate of tissue survival compared to PrOH, EG, and glycerol after harvesting (p > 0.05). The most effective CPA for the freezing and thawing of neonatal mouse testes was DMSO in comparison with EG (p < 0.05) in both pre-grafted and post-grafted tissues based on histopathological evaluation. Likewise, the highest level of serum testosterone was obtained from the DMSO CPA group compared to all other cryoprotectants evaluated (p < 0.05). The typical damage observed in the frozen–thawed grafts included disruption of the interstitial stroma, intercellular connection ruptures, and detachment of spermatogonia from the basement membrane. These findings indicate that neonatal mouse testes were most effectively preserved when frozen with HBSS medium with DMSO and that the type of CPA is a significant factor to obtain the most advanced stages of spermatogenesis and steroidogenesis after cryopreservation, thawing, and transplantation of neonatal mouse testes.

Effects of different cryoprotectants on the cryopreservation of cattle testicular tissue

“…Travers et al (2011) reported that using 1.5 M DMSO as CPA had high cell viability in the cryopreservation of immature mouse testis tissue. Yildiz et al (2013) reported that DMSO could effectively prevent the formation of ice crystals with a tetrahedral structure and provided a greater protection against freezing with regard to testicular survival rates in the grafted samples compared to PrOH, EG, and glycerol. In this study, the optimal concentrations of DMSO, PrOH, glycerol, and sucrose were 10, 10, 7.5, and 10 % (P <0.05), respectively.…”

“…After the cryopreservation of testicular tissue, detecting the content of testosterone can be used to evaluate of the survival of the tissue (Keros et al, 2007;Curaba et al, 2011;Gouk et al, 2011). Yildiz et al (2013) reported that the DMSO group had similar serum testosterone levels to fresh grafted controls, while serum testosterone levels were significantly lower in the PrOH, EG, and glycerol groups. The results of previous studies, using DMSO for in vitro endocrine and partial exocrine functions in both human (Keros et al, 2005(Keros et al, , 2007 and mouse immature testes (Milazzo et al, 2008), were similar to Yildiz et al (2013).…”

“…DMSO has been found to be a more suitable CPA than ethylene glycol (EG) for immature mouse and rat testis tissue (Goossens et al, 2008;Jezek et al, 2001). Yildiz et al (2013) indicated that neonatal mouse testes in DMSO had a higher rate of tissue survival than in PrOH and glycerol after graft.…”

Cryopreservation of testicular tissue: an alternative to maintain the reproductive capacity in different animal species

“…There was no difference between the CPAs in terms of DNA fragmentation. YILDIZ et al (2013); however, observed higher cell viability and preservation of tissue structure with DMSO when comparing these four CPAs in the slow freezing of testicular fragments from mice.…”

“…The protocol by YILDIZ et al (2013) involved the use of a programmable freezer to subject the testicular fragments of mice to slow freezing. After the fragments passed through the equilibrium solution at 4°C, they were subjected to a temperature reduction of -1°C minute -1 until the temperature reached 0°C, and this temperature was maintained for 5 minutes.…”