Comparison of apoptosis pathway following the use of two protocols for vitrification of immature mouse testicular tissue

Abstract

Our objective was to evaluate the apoptosis incidence in immature mouse testicular tissue after two different protocols of vitrification and short-term culture. Testes of 7-day-old Naval Medical Research Institute mice were isolated and distributed into control and vitrification groups. In vitrification 1 group, testes were vitrified using a combination of ethylene glycol and DMSO in three steps, and in vitrification 2 group, testes were vitrified using a combination of ethylene glycol and sucrose in five steps. Then, fresh and vitrified-warmed testis fragments were cultured for 20 hours. Morphology, cell viability, apoptosis incidence, and apoptosis gene expression (BAX, BCL2, Caspase 3, Fas, Fas ligand, p53) were evaluated at 0, 3, and 20 hours of culture by light microscopy, flow cytometry, and real-time polymerase chain reaction, respectively. Significant decrease of early apoptosis (annexin V+/PI− cells in vitrification 1 and 2 groups at 0 hours of culture, 37.34 ± 0.91 and 30.72 ± 2.2, and at 20 hours of culture, 1.46 ± 0.28 and 0.76 ± 0.11, respectively), increase of late apoptosis (annexin V+/PI+ cells in vitrification 1 group at 0 hours of culture, 14.46 ± 0.86, and at 20 hours of culture, 37.18 ± 2.34), and BAX/BCL-2 ratio (in vitrification 1 and 2 groups at 0 hours of culture, 7.31 ± 0.31 and 6.83 ± 1.38, and at 20 hours of culture, 24.08 ± 4.32 and 9.35 ± 1.91, respectively) were observed in vitrification groups during culture period. Caspase 3 expression was significantly decreased in all groups after 3 hours of culture (in control, vitrification 1, and vitrification 2 groups at 0 hours of culture, 1.00 ± 0.0, 1.56 ± 0.09, and 0.79 ± 0.06, and at 20 hours of culture, 0.37 ± 0.0, 0.96 ± 0.10, and 0.12 ± 0.03, respectively). Expression of p53 was significantly lower in vitrification 1 (0.32 ± 0.02) and control (0.50 ± 0.03) groups in 20 hours of culture as compared with vitrification 2 (0.88 ± 0.14) group. Fas (in vitrification 1 and 2 groups at 0 hours of culture, 2.29 ± 0.23 and 1.14 ± 0.15, and at 20 hours of culture, 12.43 ± 0.46 and 6.7 ± 0.48, respectively) and Fas Ligand (in vitrification 1 and 2 groups at 0 hours of culture, 1.2 ± 0.28 and 5.24 ± 0.32, and at 20 hours of culture, 21.75 ± 2.00 and 25.82 ± 2.15, respectively) expressions significantly increased in vitrification groups after 20 hours of culture. Although both vitrification protocols cause cell death via apoptotic and necrotic pathway, it seems that vitrification 1 protocol induces cell death more via apoptotic pathway than via necrosis. The apoptosis incidence after vitrification may have occurred independent of p53.