Expression and immunolocalization of the calpain-calpastatin system in the human oocyte  Irit Ben-Aharon, M.D., Ph.D., Dalit Ben-Yosef, Ph.D., Ami Amit, M.D., Ruth Shalgi, Ph.D.  Fertility and Sterility  Volume 83, Issue 6, Pages 1807-1813 (June 2005) DOI: 10.1016/j.fertnstert.2004.12.049 Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Expression of calpain in the human oocyte by Western immunoblotting. (a) A lysate of 40 oocytes was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with polyclonal anti-calpain antibody (1:500). (b) Lysate of 200 rat MII eggs served as a control. A secondary antibody, donkey antirabbit conjugated to HRP, was used in a 1:10,000 dilution, followed by an enhanced chemiluminescence detection system. The arrow points to the calpain band at 80 kd, as calculated from the migration of known protein standards. Ben-Aharon. Calpain-calpastatin system in the human oocyte. Fertil Steril 2005. Fertility and Sterility 2005 83, 1807-1813DOI: (10.1016/j.fertnstert.2004.12.049) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Localization of calpain and calpastatin in human oocytes. Human oocytes were labeled by either (a) anti-μ calpain antibody (1:75), (b) anti-m calpain antibody (1:75), or (c) anti-calpastatin (1:75). Primary antibodies were detected by a fluorescent labeled Cy secondary antibody (1:500). Localization of calpain isoenzymes and of calpastatin was visualized by laser-scanning confocal microscopy. Bar = 25 μm. Ben-Aharon. Calpain-calpastatin system in the human oocyte. Fertil Steril 2005. Fertility and Sterility 2005 83, 1807-1813DOI: (10.1016/j.fertnstert.2004.12.049) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Expression of calpastatin in human oocyte. A lysate of 70 oocytes was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with monoclonal anti-calpastatin antibody (1:750). A secondary antibody, goat antimouse conjugated to HRP, was used in a 1:5,000 dilution, followed by an enhanced chemiluminescence detection system. The arrow points to the calpastatin band at 110 kd, as calculated from the migration of known protein standards. Ben-Aharon. Calpain-calpastatin system in the human oocyte. Fertil Steril 2005. Fertility and Sterility 2005 83, 1807-1813DOI: (10.1016/j.fertnstert.2004.12.049) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Visualization of cortical reaction and cell cycle status. Fluorescence micrographs of eggs contained for both cortical granules’ exudate (a-c) and cell cycle stage (d-f). Oocytes were double-labeled with biotinylated Lens culinaris aectin and Texas-red-streptavidin (a-c). Cortical reaction was evaluated according to its intensity: (a) none, (b) weak, and (c) strong. The DNA-specific fluorochrome Hoechst 33342 served as a marker for chromatin stage: (d) metaphase II oocyte, (e) condensed chromatin, and (f) scattered chromosomes (karyomeres). Stained oocytes were visualized by laser-scanning confocal microscopy. Original magnification, ×220 in a-c, ×350 in d-f. Ben-Aharon. Calpain-calpastatin system in the human oocyte. Fertil Steril 2005. Fertility and Sterility 2005 83, 1807-1813DOI: (10.1016/j.fertnstert.2004.12.049) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Correlation between chromatin status and cortical reaction in 48-hour and 72-hour unfertilized human oocytes. Ben-Aharon. Calpain-calpastatin system in the human oocyte. Fertil Steril 2005. Fertility and Sterility 2005 83, 1807-1813DOI: (10.1016/j.fertnstert.2004.12.049) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions