In all experiments, the zona pellucida was removed from porcine oocytes and embryos by a brief pronase (Protease; Sigma) treatment and then processed for immunofluorescence as described previously. All animal handling procedures in the present study were approved by the Institutional Animal Care and Use Committee in accordance with the guidelines of the National Institutes of Health and the U.S. Department of Agriculture.
Western Blot Analysis
Granulosa cell extracts containing 50 |xg of protein from different experimental groups were subjected to both one- and two-dimensional gel electrophoresis as described previously. In brief, proteins separated by 12% SDS-PAGE were transferred onto 0.2-^m nitrocellulose membranes (Sigma) using the Royal Genie electrophoretic blotter (Idea Scientific, Minneapolis, MN) at 350 mA for 5 h. Blots were incubated for 1 h in Tris-buffered saline containing 0.05% Tween-20 and 5% nonfat dried milk and subsequently (overnight at 4°C) with polyclonal antiprohibitin antibody (1:2000; Neomarkers). Membranes were incubated with the appropriate secondary antibody for 2 h at room temperature (RT), and antibody binding was detected by chemiluminescence (Amersham).