Effect of vitrification at the germinal vesicle stage on the global methylation status in mouse oocytes subsequently matured in vitro

摘要

Background It is still unclear whether the vitrification procedure itself is associated with the incidence of abnormal DNA methylation during oocytes vitrification.The purpose of this study was to evaluate the epigenetic profile of mouse oocytes,which went through vitrification either at a mature stage or at an immature stage following in vitro maturation (IVM) by analyzing the global DNA methylation.Methods Metaphase Ⅱ (M Ⅱ) stage and germinal vesicle (GV) stage oocytes were collected from adult female mice and were vitrified respectively.The M Ⅱ oocytes were assessed for cryo-survival and global DNA methylation.The GV oocytes were assessed for cryo-survival and only the surviving GV oocytes were cultured in vitro for subsequent assessment of global DNA methylation in mature oocytes.In vivo matured fresh M Ⅱ oocytes without undergoing vitrification were used as control.The level of global DNA methylation in the M Ⅱ oocytes was then examined by immunofluorescence using an anti-5-methylcytosine (anti-5-MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG under a laser scanning confocal microscope.Results In terms of the effect of vitrification on global DNA methylation status in matured oocytes,in the M Ⅱ-v group,all the examined oocytes (90/90) were found with hypermethylation,including 63.3％ (57/90) of them displaying DNA methylation of a very high level,25.6％ (23/90) with a high level,and 11.1％ (10/90) with an intermediate level,whereas in the GV-v group,all the matured oocytes (129/129) were also examined with hypermethylation,including 67.4％ (87/129) of them displaying DNA methylation of a very high level,23.3％ (30/129) with a high level,and 9.3％ (12/129) with an intermediate level.Statistically,it was similar between both groups,which were similar to the control:68.6％ (83/121) of fresh M Ⅱ oocytes displayed DNA methylation of a very high level,21.5％ (26/121) with a high level,and 9.9％(12/121) with an intermediate level (P ＞0.05).In terms of the effect of IVM on global DNA methylation status in matured oocytes,in the in vivo matured oocytes group,all oocytes examined (94/94) were found with hypermethylation,including 80.9％ (76/94) displaying DNA methylation of a very high level and 19.1％ (18/94) with a high level,whereas in the in vitro matured oocytes group,all oocytes examined (69/69) were also found with hypermethylation:85.2％ (56/69) of them displayed with DNA methylation of very high level,11.9％ (11/69) with high level,and 2％ (2/69) with intermediate level.This result was similar to that in in vivo matured fresh M Ⅱ oocytes (P ＞0.05).Conclusion The vitrification procedure at GV stage does not induce widespread alteration of global DNA methylation status of mouse oocytes subsequently matured in vitro.