Role of progesterone receptor membrane component-1 in regulating bovine granulosa cells mitosis : a preliminary study

Progesterone Receptor Membrane Component-1 (PGRMC1) is expressed in bovine granulosa cells (bGC) during all folliculogenesis stages. Many experimental evidences in rat and human ovarian cell lines indicate that the mechanisms of action through which PGRMC1 regulates cell proliferation include a direct function during the mitotic (M) phase of the cell cycle. Therefore, the present studies were designed to assess PGRMC1’s role in regulating mitosis in bGC. First, PGRMC1’s cellular localization was evaluated by immunofluorescence at each stage of the cell cycle in bGC collected from antral follicles and cultured in vitro in the presence of serum added to stimulate mitotic activity. These studies revealed that PGRMC1 is localized in both nuclear and cytoplasmic compartments during interphase, while it concentrates in the area of the spindle apparatus during M-phase, showing a specific localization in the midzone and the midbody during Ana/Telophase and Cytokinesis, respectively. In order to assess the effect of perturbing PGRMC1 function on bGC cell proliferation, bovine PGRMC1 expression was silenced using small interfering RNA (RNAi). Cells were transfected with PGRMC1 or CTRL RNAi using lipofectamine and cultured for 24, 48 or 72h. Transfection efficiency was tested using fluorescent-labeled RNAi and was estimated to be 80%. Quantitative RT-PCR and western blotting analysis confirmed that RNAi treatment significantly reduced PGRMC1 expression up to 72 h, when compared to the CTRL- RNAi treated group (t-test, P<0.05). Importantly, cell counting at each time point revealed that PGRMC1 depletion significantly reduced cell proliferation by 30.5 and 41.7% after 48 and 72 h of treatment respectively (t-test, P<0.05). Furthermore, flow cytometry showed that the lower growth rate of PGRMC1 depleted cells associated to a higher percentage of cells arrested at G2/M phase compared to CTRL RNAi treated group at 72h (2.3±0.9% and 9.7±1.2%, t-test, P<0.05). Altogether, these data suggest that PGRMC1 depletion delays mitotic progression in bGC, confirming previous studies in rat granulosa cells. Ongoing live imaging experiments will clarify the precise stage(s) of the M-phase that is mostly affected by PGRMC1 depletion. Finally, in order to explore possible PGRMC1’s mechanisms of action in mitotic cells, we have started to hypothesize that PGRMC1 could play a role in vesicular trafficking during M- phase, which in turn is emerging as a fundamental process that ensures proper cell division, since PGRMC1 is a membrane protein found associated to cellular vesicles. Therefore, we conducted immunofluorescence and in situ- Proximity Ligation Assay studies that indicated that PGRMC1 co- localizes and directly binds to clathrin during interphase and all stages of the M-phase. This is important in that clathrin not only participates in endosomes formation but also has an important function during cell division; during mitosis, indeed, clathrin concentrates at the spindle apparatus, where it stabilizes fibers of the mitotic spindle aiding chromosomes congression (Royle et al, Nature 2005). Moreover, clathrin mediated endocytosis is important for cytokinesis and final abscission (Warner et al, Traffic 2006). Taken together, our study set the stage for further investigations that will elucidate the role of PGRMC1 in granulosa cell mitosis. Funding: ‘FP7-PEOPLE-2011-CIG, 303640, Pro-Ovum’ and ‘FONDO di RICERCA GIOVANI RICERCATORI (15-6-3027000-54) University of Milano.