Immunologic behavior of human cysteine-rich secretory protein 1 (hCRISP1) in primates: prospects for immunocontraception  Diego A. Ellerman, Ph.D., Débora J. Cohen, Ph.D., Mariana Weigel Muñoz, M.Sc., Vanina G. Da Ros, Ph.D., Juan I. Ernesto, M.Sc., Ted L. Tollner, Ph.D., Patricia S. Cuasnicu, Ph.D.  Fertility and Sterility  Volume 93, Issue 8, Pages 2551-2556 (May 2010) DOI: 10.1016/j.fertnstert.2010.01.075 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Specificity of the immune response. Sera from animals immunized with hCRISP1 (1), mkCRISP1 (2), or MBP (3) as well as preimmune sera (4) were used as primary antibodies in Western blots against protein extracts from (A) monkey or (B) human ejaculated sperm. Human sperm extracts were also probed with either anti-hCRISP1 sera preincubated with an excess of hCRISP1 (5), anti-CRISP2 (6), or anti-CRISP3 (7). Fertility and Sterility 2010 93, 2551-2556DOI: (10.1016/j.fertnstert.2010.01.075) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of immunization on semen parameters. Semen samples obtained before (pre) or after (post) immunization with MBP or hCRISP1 were analyzed for (A) the number of sperm per ejaculate, (B) sperm morphology, (C) and sperm motility. Each value represents the mean ± standard error of mean of three different ejaculates obtained from each male. Differences that were not statistically significant were observed between samples obtained before and after immunization (Student's t-test). cd = cytoplasmic droplet; lf = looped flagellum. Fertility and Sterility 2010 93, 2551-2556DOI: (10.1016/j.fertnstert.2010.01.075) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Presence of antibodies in seminal plasma and their interaction with sperm in vivo. (A) Seminal plasma recovered from MBP- and hCRISP1-immunized animals were used as primary antibodies in ELISA against MBP (red boxes) or mkCRISP1 (green boxes). The corresponding sera titers evaluated by ELISA against MBP or mkCRISP1 are indicated between brackets. nd = not detectable. (B). Ejaculated sperm from MBP- (left) or hCRISP1- (right) immunized monkeys were fixed and directly incubated with FITC-conjugated anti-monkey IgG secondary antibody. Note the patchy fluorescent labeling on the sperm head. (Magnification, ×400.) Fertility and Sterility 2010 93, 2551-2556DOI: (10.1016/j.fertnstert.2010.01.075) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions