In Vitro Culture of Vitrified Immature Mouse Testicular Tissue in The Presence of N-acetylcysteine Antioxidant

Abstract

Background: Cryopreservation of immature testicular tissue is a suitable method for spermatogonial stem cell (SSC) preservation in prepubertal boys, who are at risk of infertility due to cancer treatments. Viable spermatozoa can be obtained by transplantation or in vitro culture of cryopreserved testicular tissue. Optimizing the culture conditions is essential for reducing tissue damage caused by oxidative stress produced during cryopreservation and culture. Our ob­jective was to improve the culture conditions of vitrified immature mouse testicular tissue by using N-acetylcysteine (NAC) antioxidant.Materials and Methods: In this experimental study, testicular tissues of 6-day-old immature NMRI mice were iso­lated, vitrified, and distributed into three groups: control, culture I (cultured without NAC), and culture II (cultured in the presence of 125 mM NAC). After seven days of culture, histological analysis, cell viability, apoptotic-related gene expression, promyelocytic leukaemia zinc finger (Plzf) gene expression, and Caspase-3 protein expression were assessed. Moreover, the malondialdehyde (MDA) level was measured in the culture media.Results: Tissue integrity and higher viability level were observed in the culture II group compared to the other two groups. Furthermore, the Bax/Bcl-2 ratio and MDA level were decreased significantly in the culture ӀӀ group, whereas Caspase-3 and Plzf gene expression were significantly increased.Conclusion: Our data revealed that the presence of 125 mM NAC improves the developmental process of vitrified-warmed immature mouse testicular fragments during in vitro culture, thus it may have potential implications for in vitro culturing of human prepubertal testicular tissues.

Keywords

• acetylcysteine
• antioxidant
• culture techniques
• testis
• vitrification