In vitro effects of cationic compounds on functional human sperm parameters  Marta Baptista, M.Sc., Stephen J. Publicover, Ph.D., João Ramalho-Santos, Ph.D.  Fertility and Sterility  Volume 99, Issue 3, Pages 705-712 (March 2013) DOI: 10.1016/j.fertnstert.2012.11.008 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Representative images of the different assays carried out in this work. (A) Mitochondrial membrane potential (MMP) monitored with the JC-1 probe, showing a midpiece stained orange (accompanied with green), indicating high MMP. (B) Sperm viability monitored with the Live/Dead Sperm Viability Kit: DNA of live sperm stained green and DNA from dead sperm stained red. Viability and MMP were monitored simultaneously; images are shown separately for clarity. (C) Acrosomal status monitored with PSA-FITC, intact acrosomes are shown as a green cap, which is lacking in sperm that have lost their acrosome (middle cell); DAPI (blue) was used as a nuclear stain. (D) Capacitation monitored by the detection of phosphotyrosines (red) in capacitated (right) but not uncapacitated (left) sperm, DAPI (blue) was used as a nuclear stain. (E) Intracellular calcium monitored with the use of Oregon Green Bapta: Sperm with increased calcium concentration stain brighter than the background fluorescence signal (arrowheads). Magnifications are ×1,000 (A–D) and × 400 (E). See Materials and Methods for details and abbreviations. Fertility and Sterility 2013 99, 705-712DOI: (10.1016/j.fertnstert.2012.11.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of different cationic compounds on the survival and motility (percentage of progressive motile sperm) of human sperm, after in vitro exposure for 60 and 180 minutes. Sperm viability with: (A) C12Bzk; (B) C12Pyr; and (C) C12TAB. Sperm motility with: (D) C12Bzk; (E) C12Pyr; and (F) C12TAB. Data are expressed as mean ± SEM of the percentages of viable or motile sperm. A total of five samples were used for each compound. Asterisks denote significant differences from control (P<.05). Fertility and Sterility 2013 99, 705-712DOI: (10.1016/j.fertnstert.2012.11.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of different cationic compounds after in vitro exposure for 60 and 180 minutes on: (A) sperm mitochondrial membrane potential (MMP); (B) sperm capacitation (protein tyrosine phosphorylation); and (C) the percentage of intact acrosomes. (D) Effect of different cationic compounds on intracellular sperm calcium concentration. Data are expressed as mean ± SEM of the percentages of functional mitochondrial, capacitated, acrosome-intact, and increased intracellular calcium sperm. A total of five samples were used for each compound. Asterisks denote significant differences from control (P<.05); and the cardinal sign denotes difference between experimental groups (P<.05). Fertility and Sterility 2013 99, 705-712DOI: (10.1016/j.fertnstert.2012.11.008) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions