Effect of vitrification on in vitro developmental competence of rat testicular tissue.

Abstract

Cryopreservation of testicular tissue has been proposed as a potential technique for preserving fertility in pre-pubertal boys with various malignancies. The present study aimed to compare the effects of two vitrification techniques-solid surface vitrification (SSV) and needle-immersed vitrification (NIV)-on the integrity, development, cell viability, and apoptosis of rat testicular tissue. Testes from 4-week-old Wistar rats underwent a two-step vitrification process. Tissue pieces were allocated to either the SSV or NIV group. Equilibration involved a solution containing 7.5% dimethyl sulfoxide (DMSO) and 7.5% ethylene glycol (EG), followed by a vitrification solution with 0.07 mol/L sucrose, 15% DMSO, and 15% EG. The optimal protocol was determined after vitrification using either the NIV or SSV technique. Samples from the control and selected vitrification (SSV) groups were cultured for 3 weeks. Tissue integrity, cell viability, apoptosis, and gene expression were evaluated using hematoxylin and eosin staining, trypan blue staining, annexin V-PI staining, and real-time PCR. Morphological changes were more pronounced in the NIV group compared to the SSV group (P < 0.05). Although the percentage of viable cells did not significantly differ between the NIV and SSV groups, it was slightly higher in the SSV group. Thus, SSV was identified as the optimum vitrification method. Real-time PCR analysis revealed altered gene expression: spermatogonial-related genes (Lrp4, Egr3, Nanos, Gfra1, C-kit, and Sohlh1) were significantly decreased in the SSV group, while somatic-cells-related genes (Gdnf, Csf1, and Fgf2) were higher. Overall, SSV appears suitable for rat testis tissue vitrification, although it induces some molecular changes. Optimization of the culture medium is essential to support successful spermatogenesis.