Assessment of Progesterone Receptor Membrane Component 1 (PGRMC1) role in bovine granulosa cells mitosis

Progesterone Receptor Membrane Component-1 (PGRMC1) is expressed in bovine granulosa cells (bGC) during all stages of folliculogenesis. The aim of the present studies is to assess PGRMC1`s role in regulating bGC mitosis since studies in rat and human ovarian cell lines and bovine oocyte indicate its direct involvement during chromosome segregation and cytokinesis. First, PGRMC1`s sub-cellular localization was evaluated by immunofluorescence in bGCcollected from antral follicles andcultured in vitro. These studies revealed that in mitotic cells, PGRMC1 concentrates in the area of the spindle apparatus, localizing in the midzone and the midbody during Ana/Telophase and Cytokinesis, respectively. Then, in order to assess the effect of disrupting PGRMC1 function on bGC cell mitosis, bovine PGRMC1 expression was down-regulated using small interfering RNA (RNAi). Cells were transfected with PGRMC1 or CTRL RNAi and cultured for up to 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 reduced PGRMC1 expression, when compared to the CTRL-RNAi treated group (t-test, P<0.05). The effect of PGRMC1 depletion on cell proliferation was assessed by cell counting and flow cytometry which, respectively, revealed a lower growth rate by 41.7% at 72 h (P<0.05) associated to a higher % of cells arrested at G2/M phase compared to CTRL RNAi treated group at 72h (2.3±0.9% and 9.7±1.2% P<0.05). Furthermore, live imaging studies confirmed that PGRMC1 silencing directly affects mitotic division. In fact, we observed an increased % of abnormal and/or incomplete mitosis in PGRMC1 RNAi treated cells, when compared to control cells (Fisher exact test, P<0.05). Finally, to investigate PGRMC1 putative mechanism of action in mitotic cells we conducted immunofluorescence and in situ-Proximity Ligation Assay studies, which is a new immuno-based technique that allows localized and quantifiable detection of protein-protein interactions directly on fixed cells. Specifically, we considered a possible interaction between PGRMC1 and clathrin. In fact, clathrin acts during cell division concentrating at the spindle apparatus, where it stabilizes fibers of the mitotic spindle aiding chromosomes separation. The results show that PGRMC1 colocalizes and directly binds to clathrin during interphase and all stages of the M-phase. Our data confirm a direct involvement of PGRMC1 during granulosa cell mitosis and set the stage for further mechanistic studies. Funding: FP7-PEOPLE-2011-CIG, 303640, Pro-Ovum and FONDO di RICERCA GIOVANI RICERCATORI (15-6-3027000-54) University of Milan