Involvement of the inflammasome in abnormal semen quality of men with spinal cord injury  Xianyang Zhang, Ph.D., Emad Ibrahim, M.D., Juan Pablo de Rivero Vaccari, Ph.D., George Lotocki, Ph.D., Teodoro C. Aballa, M.S., W. Dalton Dietrich, Ph.D., Robert W. Keane, Ph.D., Charles M. Lynne, M.D., Nancy L. Brackett, Ph.D., H.C.L.D.  Fertility and Sterility  Volume 99, Issue 1, Pages 118-124.e2 (January 2013) DOI: 10.1016/j.fertnstert.2012.09.004 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Measurement by ELISA of the inflammasome proteins caspase-1 in the seminal plasma of control subjects and spinal cord injury (SCI) subjects. (B) Detection and quantification of apoptosis associated speck-like protein containing a caspase activation and recruitment domain (ASC) in the seminal plasma by Western blot analysis: Top: a representative blot showing the levels of ASC (24 kD) in the seminal plasma from six control subjects and seven SCI subjects (S = an ASC-positive sperm cell extract; M = protein size standards); Bottom: quantification of ASC (relative intensity) in the seminal plasma is shown; each point represents the average relative intensity from duplicated specimens of 28 control subjects and 28 SCI subjects (P<.0001). Horizontal lines in graphs represent mean ± SEM. Fertility and Sterility 2013 99, 118-124.e2DOI: (10.1016/j.fertnstert.2012.09.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Detection by Western blot analysis of procaspase-1 and caspase-1 in the cell extracts of sperm fractions. Representative blots are shown. Upper left panel: sperm F1 fractions from five control subjects and five spinal cord injury (SCI) subjects. Top right: sperm F2 fractions from four control subjects and eight SCI subjects. The relative intensities of procaspase-1 (45 kD) in the F1WBC− fractions is shown in the bottom left graph (P<.001). The bottom center graph shows the relative intensities of procaspase-1 in F2 fractions (P<.0001). The bottom right graph shows the relative intensities of caspase-1 in F2 fractions (P<.0001). Fertility and Sterility 2013 99, 118-124.e2DOI: (10.1016/j.fertnstert.2012.09.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Detection of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) in the cell extracts by Western blot analysis. Representative blot including cell extracts prepared from F1 and F2 fractions of two control (Ctrl) subjects and two spinal cord injury (SCI) subjects. Graphs show the comparison of the relative intensities of ASC (24 kD) in the in the F1WBC− and F2 fractions (both P<.0001). Fertility and Sterility 2013 99, 118-124.e2DOI: (10.1016/j.fertnstert.2012.09.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Localization of caspase-1 (top left) and ASC (top right and bottom left and right) in the purified sperm cells of SCI subjects were analyzed by immunocytochemical staining; red = caspase-1 or ASC. Sperm were also stained with Mitotracker (green) and DAPI (blue) to verify integrity. Fertility and Sterility 2013 99, 118-124.e2DOI: (10.1016/j.fertnstert.2012.09.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Detection by ELISA of inflammatory cytokines (A) interleukin (IL) 1β (P<.001) and (B) IL-18 (P<.01) in the seminal plasma of control subjects and spinal cord injury (SCI) subjects. Fertility and Sterility 2013 99, 118-124.e2DOI: (10.1016/j.fertnstert.2012.09.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 (A) Linear regression analysis of the levels of apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC; relative intensity by Western blot analysis) and the levels of caspase-1 (pg/mL by ELISA) in the seminal plasma of 28 spinal cord injury (SCI) subjects and 28 control subjects (P<.0001). (B) Correlation between the levels of ASC and procaspase-1 (relative intensity by Western blot analysis) in the leukocyte-free sperm extracts (F1WBC−) of 15 SCI subjects and 27 control subjects (P<.0001). (C) Correlation between the levels of ASC and procaspase-1 (relative intensity by Western blot analysis) in the F2 sperm extracts in 28 SCI subjects and 28 control subjects (P<.0001). (D) Correlation between ASC levels in the seminal plasma and sperm motility of 28 SCI subjects and 28 control subjects (P<.0001). Fertility and Sterility 2013 99, 118-124.e2DOI: (10.1016/j.fertnstert.2012.09.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions