WCRB2014 POSTER PRESENTATIONS (1) (335 abstracts)
Insight into progesterone receptor membrane component 1 action during bovine oocyte meiosis by means of siRNA-mediated gene silencing
Valentina Lodde 1 , Irene Tessaro 1 , Franca Raucci 1 , Federica Franciosi 2 , Federica Marchese 1 , Silvia Modina 1 , John J. Peluso 3 & Alberto M Luciano 1
1University of Milan, Milan, Italy; 2University of California, San Francisco, San Francisco, California, USA; 3University of Connecticut Health Center, Farmington, Connecticut, USA.
Introduction: Previous studies suggest that progesterone receptor membrane component 1 (PGRMC1) plays an essential role during bovine oocyte meiosis, since it i) localizes to the centromeres at metaphases I and II and ii) 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 siRNA (RNAi)-mediated gene silencing.
Methods: Cumulus–oocytes complexs were microinjected to deliver PGRMC1 or CTRL-RNAi into the oocytes cytoplasm, kept in meiotic arrest for 18 h with 10 μM cilostamide and then in vitro-matured (IVM) for 24 h. After IVM, efficacy in depleting PGRMC1 expression was assessed by quantitative RT-PCR and western blotting. Finally, the oocyte capability to extrude the first polar body (PBI) and the morphology of the MII plates were assessed.
Results and discussion: PGRMC1 expression following PGRMC1-RNAi treatment was significantly reduced by 30%. This was accompanied by a 22% reduction of the oocytes that extruded the PBI (P<0.05). Surprisingly, PGRMC1-RNAi treatment did not affect MII plate formation or morphology. However, a significantly higher proportion of PGRMC1-RNAi injected oocytes possessed clumps of DNA scattered throughout the ooplasm in addition to the MII plate (P<0.05). This is consistent with PGRMC1 localization at the midbody and with a putative role in cytokinesis. We hypothesize that lower PGRMC1 expression impairs the process of PBI formation. As a consequence, DNA that should be extruded with the PBI is retained in the cytoplasm and degraded.
Funding: FP7-PEOPLE-2011-CIG, contract no.: 303640-Pro-Ovum.
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