Identification of human spermatogenesis-related proteins by comparative proteomic analysis: a preliminary study  Ran Huo, Ph.D., Ying He, B.M., Chun Zhao, Ph.D., Xue-jiang Guo, Ph.D., Min Lin, B.M., Jia-hao Sha, Ph.D.  Fertility and Sterility  Volume 90, Issue 4, Pages 1109-1118 (October 2008) DOI: 10.1016/j.fertnstert.2007.07.1342 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Morphologies of histologically normal and pathological human testes on hematoxylin/eosin staining. (A) In the normal human testis, spermatogenesis proceeds in an orderly manner; the mean (±SEM) Johnsen score is 9.93 ± 0.03. (B) In the pathological testis, the organization of spermatogenic epithelium is abnormal, many germ cells are shed into the seminiferous tubule, and condensed spermatids are barely visible; the mean Johnsen score is 7.80 ± 0.27. Bar = 200 μm. Fertility and Sterility 2008 90, 1109-1118DOI: (10.1016/j.fertnstert.2007.07.1342) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 (A) Two-dimensional electrophoresis protein patterns of a normal human testis (n) and a pathological testis obtained from an azoospermic patient (p). Red arrows indicate 10 differentially expressed protein spots, assigned letters a–j. Blue arrows indicate two protein spots with unaltered expression levels; the identification result of spot i′ is the same as spot i, and spot j′ corresponds to the same protein as spot j. (B) Magnified specific areas showing the expression level of 12 protein spots in normal human testes and pathological testes. Corresponding protein spots are indicated with white crosses. Bar graphs at the right show the results of the statistical analyses of these proteins. The y-coordinate indicates the relative volume (% Vol), which normalized the spot volume as a percentage of the total volume of all the spots present in a gel. ∗P<.05. SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fertility and Sterility 2008 90, 1109-1118DOI: (10.1016/j.fertnstert.2007.07.1342) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Regulation map of differentially expressed proteins generated using Pathway Studio software. (A) Cellular processes regulated by the differentially expressed proteins; proteins are shown as red ovals, and regulated processes are represented by yellow squares. Regulation events are displayed with arrows and documented by literature citations. (B) Four proteins had the ability to regulate the activity of the NF-κB complex. Fertility and Sterility 2008 90, 1109-1118DOI: (10.1016/j.fertnstert.2007.07.1342) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Immunohistochemical analysis of cathepsin D (A), Prx4 (B), and Hsp27 (C) in normal (n) and pathological (p) human testes. In the normal testis, cathepsin D is strongly stained in Sertoli cells; Prx4 immunolabeling is visible in spermatids and is strongest in elongating spermatids and residual bodies; Hsp27 protein is expressed from the cytoplasm of spermatocytes and abundantly expressed in spermatids. The pathological testis showed the same staining pattern (positive cells marked with arrowheads), but the positive signal decreased dramatically or disappeared in some cells, as indicated by arrows. St = Sertoli cell nuclei; Sc = spermatocyte; Sd = spermatid. Bar = 80 μm. Fertility and Sterility 2008 90, 1109-1118DOI: (10.1016/j.fertnstert.2007.07.1342) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions