Objective: To explore and compare the current mainstream testicular tissue freezing methods, namely vitrification and controlled slow freezing, and optimize the best testicular tissue freezing methods.
Methods: Testicular tissues of 3-week-old mice and <2-year old prepubertal cynomophage monkeys were collected and cut to 6-26 mm3, and divided into three groups: Fresh group, vitrification group and controlled slow freezing group were resuscitated after 5-7 days of freezing. HE staining, immunofluorescence staining, TUNEL staining and Western blot were used to evaluate the frozen-thawed testicular tissue.
Results: 1. In the testes of C57BL6/J mice, the expression level of spermatogonial stem cell marker UCHL1 in the controlled slow freezing group was higher than that in the vitrification group, and the content of apoptotic cells (TUNEL+ cells) was lower than that in the vitrification group. 2. In the testicular tissue of cynomolgus monkeys, the expression levels of spermatogonial stem cell markers UCHL1 and cell proliferation marker PCNA in the CSF group were higher than those in the vitrification group.
Conclusion: Both vitrification and CSF can successfully preserve the testes of immature mice and cynomolgus monkeys before puberty. However, in the vitrification, there are more frozen damaged areas in the testicular tissue with the frozen volume of 6-26mm3, which may affect the cryopreservation of spermatogonial stem cells in the testicular tissue.
Keywords: vitrification; controlled slow freezing; male fertility preservation; spermatogonial stem cell.