Sam68 is required for Spag16 and Spdya mRNA translation during the meiotic divisions. (A) m7GTP-Sepharose bead (M7GTP-sepharose) pull-down assays using extracts from pachytene spermatocytes (Ctrl) or spermatocytes treated with 0.5 µM OA (OA). Control pull-down assays were performed using Sepharose beads (sepharose). Western blot analysis of the Sam68, eIF4G, and eIF4E proteins bound in the pull-down assays are shown as indicated. Bottom panels show the densitometric analysis of the pull-down assays. Data are represented as ratios between Sam68 and eIF4E or eIF4G and eIF4E band intensities. (B) Western blot analysis of the expression levels of SPAG16 and SPDYA proteins in germ cells at different stages of differentiation. ERK2 and tubulin staining were also performed as a control of loading to rule out a general increase in proteins. (C and D) Western blot (C) or RT-PCR (D) analyses of extracts from Sam68^+/+ or Sam68^−/− pachytene spermatocytes before (0) or after treatment with 0.5 µM OA for 2, 4, or 6 h. (C) The top panel shows the levels of Sam68 and its shift in electrophoretic mobility caused by phosphorylation (p-Sam68) after OA treatment. The middle panels show the accumulation of SPAG16 and SPDYA in Sam68^+/+ spermatocytes, and the bottom panel shows tubulin as loading control of the experiment. The bar graphs show the densitometry of SPAG16 and SPDYA protein expression expressed as a ratio with the intensity of tubulin in each lane analyzed. (D) The bar graphs show the densitometry of Spag16 and Spdya mRNA levels expressed as a ratio with the intensity of Gapdh in each lane analyzed.