Figure 5.

miR-21 is induced in bovine COCs over 24 hours in culture with or without serum and hormone supplementation but is sensitive to inhibition of protein synthesis. (A) qRT-PCR of miR-21 in cumulus cells of COCs at 0 hr or 24 hr IVM culture in serum free conditions with no growth or hormone supplementation (no supp), LIF alone, FSH alone, or FSH + LIF, as compared to standard IVM media (std IVM) which is further supplemented with LH, estradiol and FBS. Gene expression is shown relative to the 0 hour time point. Error bars represent SEM. N = 3 biological replicates. Different letters indicate statistically significant differences between groups. (B) Light micrographs of immature COCs (0 hr) and COCs matured in vitro under the same conditions described in (A). Images obtained with EVOS FL Cell Imaging System using 4x objective. (C,D) qRT-PCR quantification of pri-miR-21 transcripts and mature miR-21 in (C) oocytes and (D) cumulus cells after 24 hr culture in standard IVM media containing ethanol vehicle (VEH) or cycloheximide (CHX). Gene expression is shown relative to VEH. Error bars represent SEM. N = 3 biological replicates. Different letters indicate statistically significant differences between groups. (E) Representative Western blot of STAT3 phosphorylation (Y705) in cumulus cells at 7 hr IVM, in serum free, non-supplemented media containing ethanol vehicle (VEH), cycloheximide (CHX) or CHX + LIF. Shown with total STAT3. Beta-actin is used as a loading control. Cropped images of a single blot are shown. Full-length blots are presented in Supplemental Fig. 3. Three independent experiments were performed with similar results. (F) qRT-PCR quantification of pri-miR-21 transcripts in cumulus cells after 24 hr culture in serum free, non-supplemented media as described in (E). Gene expression is shown relative to VEH. Error bars represent SEM. N = 3 biological replicates. Different letters indicate statistically significant differences between groups.