Assessment of autophage, apoptosis and dna damage/repair process in cryopreserved ovarian cortex

OBJECTIVE: The genomic integrity of oocytes subjected to ovarian tissue cryopreservation has not been thoroughly inverstigated. To understand potential causes and mechanisms of somatic and germ cell damage due to ovarian tissue cryopreservation, biomarker expression for autophagy, apoptosis and DNA damage/repair was evaluated in fresh, slow frozen-thawed, and vitrified-warmed bovine cortical sections. DESIGN: Experimental laboratory study. MATERIALS AND METHODS: Fresh ovaries were processed into thin cortical sections (5x5x1mm). These prepared cortical sections were divided into three groups (fresh, slow freezing, vitrification). For slow freezing, 1.5M DMSO based cryoprotectant was used. For vitrification, 20% ethylene glycol plus 20% DMSO based vitrification solution with a two step equilibration method was used. After thawing tissue was process for immunohistochemical localization of DNA damage (H2AX) and repair (RAD 51) markers. In parallel, samples were lysed and analyzed by Western blot for the expression of apoptosis (PARP) and autophage (LC3B) markers. RESULTS: In the fresh control group, oocytes within primordial and primary follicles exhibited nuclear staining for H2AX and cytoplasmic staining for RAD 51. However, H2AX staining in the nucleus disappeared and nuclear localization for RAD 51 increased in both vitrified and slow frozen groups. By Western blot analysis, slow frozen tissue showed activation of autophage as indicated by cleavage of LC3B. In contrast, vitrified tissue exhibited acute activation of apoptosis as evidenced by PARP cleavage. CONCLUSION: These findings indicate that oocytes within primordial and primary follicles generate an acute DNA repair process in response to DNA damage induced by tissue cryopreservation. Slow freezing/thawing appears to elicit autophage (cell survival pathway) initially followed by apoptosis, whereas the pathway to autophage is bypassed upon warming of vitrified tissue possibly due to the effects of high concentration of cryoprotectants.