Oocytes recovered after ovarian tissue slow freezing have impaired H2AX phosphorylation and functional competence

Abstract

It has been shown that oocytes isolated from ovarian tissue cryopreservation acquire DNA damage during the process of freeze-thawing. Using a mouse model, here we have investigated the functional competence and phosphorylation of H2AX (γ-H2AX) in germinal vesicle (GV) and parthenogenetically activated oocytes derived from conventional ovarian tissue slow freezing and vitrification techniques. The number of GV-stage oocytes with γ-H2AX foci was not significantly different between the slow-freezing and vitrification groups. Although the in vitro maturation (IVM) potential of GV oocytes in the slow-freezing group showed a significant delay (P<0.0001) in the process of germinal vesicle breakdown, no difference in the maturation rate was observed between the two protocols. Nevertheless, parthenogenetic activation of IVM oocytes using strontium chloride showed a significantly lower activation rate in the slow-freezing group compared with the vitrification (P<0.05) and control (P<0.01) groups. Importantly, H2AX phosphorylation was significantly perturbed in the slow-freezing group in comparison to the control (P<0.05). Therefore, we conclude that impaired sensing of DNA strand breaks and repair processes are associated with the reduced functional competence of the oocytes recovered from the slow-freezing group, which may have a significant impact on the reproductive outcome.

Analysis of nuclear maturation, DNA damage and repair gene expression of bovine oocyte and cumulus cells submitted to ionizing radiation

“…One of the earliest responses to DNA damage is the phosphorylation of histone variant H2AX, which forms foci around the DSB sites ( Firsanov et al, 2011 ; Rogakou et al, 1999 , 1998 ). The γH2AX foci play an important role in identifying DNA damage and promoting repair by allowing sensor and repair proteins to access the damaged sites ( Sudhakaran et al, 2015 ). In an experiment with human lymphocytes submitted to different radiation doses, a maximum response of γH2AX levels was observed at 1 or 2h, which was followed by a gradual loss of γH2AX over the next 6h, and a slower decline until 24h toward background levels ( Ghardi et al, 2012 ).…”

In Vitro Matured Oocytes Are More Susceptible than In Vivo Matured Oocytes to Mock ICSI Induced Functional and Genetic Changes

“…Immunofluorescence detection of γ-H2AX was performed according to the previously described method [ 31 ] with minor modifications. Oocytes were washed with phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 30 min and then permeabilized for 30 min at room temperature with PBS containing 0.1% (V/V) Triton X-100 and 0.5% bovine serum albumin.…”