Female mammalian meiosis consists of a protracted G2-phase arrest followed by two consecutive meiotic M-phases (meiosis I and meiosis II) culminating in a second arrest at metaphase II. This unique sequence is underpinned by precisely regulated proteolysis, which to-date, is understood to be orchestrated exclusively by the anaphase-promoting complex (APC). The APC ubiquitinates key cell-cycle proteins such as cyclin B1 and securin, thereby earmarking them for destruction by the 26S proteasome.
SIRT2, one member of the family of NAD+-dependent sirtuin deacetylases, is emerging as a pivotal regulator of protein stability in somatic cells. Very little is known regarding the involvement of deacetylation in post-translational control in oocytes. Here we studied SIRT2 in mouse oocytes using highly specific small molecule inhibitors, which enabled temporally controlled SIRT2 disruption during meiosis.
We found that disrupting SIRT2 in G2-arrested oocytes using either of two inhibitors or by morpholino-induced knockdown severely impaired the G2-M transition marked by reduced germinal vesicle breakdown (GVBD). Using Western blotting and time-lapse fluorescence imaging, we established that suppressed GVBD was due to excessive proteasome-mediated destruction of cyclin B1 and securin that was acetylation-dependent. Excessive proteolysis also ensued when SIRT2 inhibition was selectively imposed during meiosis I or after metaphase II arrest. Remarkably, exaggerated proteolysis during any of these meiotic stages proceeded even when the APC was disabled by depleting its co-activators, Cdh1 and Cdc20, showing that APC was not responsible for protein destruction. Significantly, unrestrained proteolysis during either meiosis I or meiosis II led to spindle collapse and to exit into an interphase-like state.
These findings reveal a novel mechanism involving SIRT2-mediated deacetylation that is critical for fine-tuning proteolysis required for meiotic maturation. Since levels of SIRT2’s essential co-factor, NAD+, decline with ageing, these data uncover a direct link between metabolic health during ageing and meiotic cell-cycle control.