Vitrification is a method extensively used for preserving oocytes and embryos and is also gaining acceptance for preserving gonadal tissue. Cryopreservation of spermatogonial stem cells is an applicable method for young males seeking fertility preservation before starting a treatment or can be a tool for genetic preservation of rare or high-value animals. The aim of this work was to evaluate the cryopreservation of testicular tissue from young lambs by vitrification using a new device named E.Vit (FertileSafe, Ness Ziona, Israel) that permits all cryopreservation procedures to be performed in straw. The new device consists of a 0.3-mL straw (Cryo Bio System, IMV, L’Aigle, France) with a capsule containing 50-µm pores inserted at one end. Testicular tissue extracts were prepared from testes of slaughtered lambs (n=10, 40 days old), opened by sagittal sectioning with a microblade and collecting small pieces of testicular tissue (1mm3) from the middle part of the rete testis. Three pieces of gonadal tissue were inserted into each E.Vit device. Each straw was sequentially loaded vertically in two 1.5-mL microtubes, which contained the following solutions: first, the equilibrating solution (7.5% dimethyl sulfoxide+7.5% ethylene glycol+20% FCS in TCM-199) for 6min, followed by 90min in the vitrification solution (18% dimethyl sulfoxide+18% ethylene glycol+0.5M Trehalose+BSA in TCM-199). After exposure to the equilibrating solution and vitrification solution, the solutions were removed and the straws were directly loaded into LN2. The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, and 25% warming solution (1M sucrose in TCM-199+20% FCS) at 38.6°C for 5min each before arrival into the holding medium. Samples were recovered from the straws incubated at 38.6°C in 5% CO2 in air in TCM 199+5% FCS and evaluated at 0 and 2h post-warming for viability using trypan blue staining. Expression of a panel of specific genes (SOD2, HSP90b, BAX, POUF5/OCT4, TERT, CIRBP, KIF11, AR, FSHR) was analysed by real-time PCR in cryopreserved tissue in vitro cultured for 2h post-warming (2hV), in fresh controls immediately after tissue dissection (0hF), and after 2h of in vitro culture (2hF). The majority of cells survived after vitrification, although viability immediately after warming (0hV: 56%±1.45) or after 2h of in vitro culture (IVC) (2hV: 54±7%) was significantly lower compared with non-cryopreserved fresh controls (0hF: 89%±1.45; ANOVA P<0.05). Expression analysis showed specific patterns for the different genes. Notably, BAX transcript abundance was not affected by vitrification or IVC, indicating an acceptable level of stress for the cells. The genes HSP90b and CIRBP were down-regulated in 2hF but increased in 2hV, as expected. Expression of SOD1 and OCT4 was altered by vitrification but not by IVC. Conversely, expression of TERT, KIF11, and AR was affected by both IVC and cryopreservation (ANOVA P<0.05). This novel protocol for testicular tissue cryopreservation of prepubertal animals may be a promising strategy for fertility preservation and can contribute as a new approach in the development of large-scale biodiversity programs.
Development of Spermatogenesis In Vitro in Three-Dimensional Culture from Spermatogonial Cells of Busulfan-Treated Immature Mice
