Functional assessment of progesterone receptor membrane component 1 (PGRMC1) action during bovine oocyte meiosis by means of PGRMC1 inhibitor and small-interfering RNA mediated gene silencing

Previous studies suggest that Progesterone Receptor Membrane Component 1 (PGRMC1) plays an essential role during bovine oocyte meiosis, since it 1) localizes to the centromeres at metaphase-I and II and 2) concentrates between the separating chromosomes at ana/telophase-I. Moreover, injection of an antibody to PGRMC1 significantly impairs completion of meiosis. The aim of the present study is to expand these findings by using a PGRMC1 inhibitor (AG205) and small-interfering RNA (RNAi) mediated gene silencing. In a first set of experiments, bovine Cumulus-Oocytes Complexes (COC) were collected from 2-6 mm antral follicles. COC or denuded oocytes (DO) were in vitro matured (IVM) for 24h in the presence of 0, 10, 20 or 40 μM of AG 205. After IVM, the oocyte capability to extrude the first polar body (PBI) and the effect on the meiotic progression were assessed. Data were analyzed by two-way ANOVA followed by Tukey’s Post hoc test. Treatment with AG205 affected oocyte meiosis of both COC and DO in a dose dependent manner by: 1) decreasing the % of oocytes that extruded the PBI (p<0.05); 2) affecting nuclear meiotic progression by increasing the % of MI stage arrested oocyte, decreasing the % of oocytes that reached MII stage and increasing the % of oocytes showing aberrant meiotic figures (p<0.05) and 3) increasing the % of oocytes with clumps of scattered DNA within the cytoplasm (p<0.05). Importantly these effects were more severe in DO, indicating that AG205 directly acts on oocytes and cumulus cells do not mediate its negative effect. In order to finally confirm PGRMC1 function during bovine oocyte meiosis, a second set of experiment were conducted, in which COC were microinjected to deliver PGRMC1 or CTRL-RNAi into the oocytes cytoplasm, kept in meiotic arrest for 18h with 10μM cilostamide and then in vitro-matured for 24h. After IVM, efficacy in depleting PGRMC1 expression was assessed by quantitative RT-PCR and western blotting. As above described, the oocyte capability to undergo meiotic maturation and to extrude the PBI were considered as biological end points. Data were analyzed by t-test. PGRMC1 expression following PGRMC1-RNAi treatment was significantly reduced by 30% (p<0.05). This was accompanied by a 22% reduction of the oocytes that extruded the PBI (p<0.05). PGRMC1-RNAi treatment induced a significant reduction of PGRMC1 mRNA and protein expression. Overall, PGRMC1 down-regulation mirrored AG205 effects, by 1) reducing the % of oocytes that extruded the PBI (p<0.05); 2) increasing the % of oocytes showing aberrant meiotic figures (p<0.05) and 3) increasing the % of oocytes with clumps of scattered DNA. Surprisingly, although we observed a decrease of the % of MII stage oocytes in PGRMC1 RNAi treated group, this effect was not significant. Therefore, we further assessed the morphology of the MII plates. Data indicated that PGRMC1 down regulation induced a decrease of the % of MII plates with aligned chromosomes together with an increase of the % of oocytes with misaligned chromosomes. The present findings are consistent with PGRMC1 localization at the centromeres and at the midbody and with a putative role in both chromosomes separation and cytokinesis. We hypothesize that lower PGRMC1 expression impairs the process of chromosome separation and/or PBI formation. As a consequence, DNA that should be extruded with the PBI forms aberrant meiotic figures or is degraded. Funding: FP7-PEOPLE-2011-CIG, contract n.:303640-Pro-Ovum.