Direct effect of macrophage migration inhibitory factor on sperm function: possible involvement in endometriosis-associated infertility  Cédric Carli, M.Sc., Pierre Leclerc, Ph.D., Christine N. Metz, Ph.D., Ali Akoum, Ph.D.  Fertility and Sterility  Volume 88, Issue 4, Pages 1240-1247 (October 2007) DOI: 10.1016/j.fertnstert.2007.04.002 Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

Figure 1 MIF effects on tyrosine phosphorylation of sperm proteins. After Percoll washing, spermatozoa were incubated with indicated MIF concentrations or with 10% FCS for 4 hours at 37°C in Ham's F10 medium. SDS-PAGE and Western blotting were performed as described in the Materials and Methods section. Membranes were reblotted with an anti-α-tubulin antibody to ensure equal protein loading. (A) Representative Western blot of tyrosine-phosphorylated proteins and α-tubulin; (B) normalized signal of the phosphotyrosine-containing protein p105 to the tubulin content measured in each treatment by densitometric analysis and expressed as a percent of control (sperm incubated with Ham's F10 medium alone without MIF); (C) normalized signal for the p81 phosphotyrosine-containing protein expressed as in (B). Data are mean ± standard error of the mean from four different donors. ∗Significantly different from control (sperm incubated with Ham's F10 medium alone without MIF) (P<.05). Carli. MIF affects sperm functions. Fertil Steril 2007. Fertility and Sterility 2007 88, 1240-1247DOI: (10.1016/j.fertnstert.2007.04.002) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effect of MIF on sperm acrosome reaction. Percoll-washed spermatozoa were incubated in Ham's F10 medium with the indicated MIF concentrations or with 10% FCS for 4 hours at 37°C. Spermatozoa were then incubated for 1 hour at 37°C with or without 5 μM A23187 to determine induced and spontaneous acrosome reactions, respectively. Acrosome is labeled using PSA-FITC and observed using fluorescence microscopy at ×100. (A) Photomicrograph of a reacted acrosome; (B) photomicrograph of an intact acrosome; (C) percentage of spontaneous (open bars) and A23187-induced (closed bars) acrosome reactions. Data are mean ± standard error of the mean from five different donors. ∗Significantly different from the induced acrosome reaction at 0 ng/mL MIF (P<.05); +Significantly different from the spontaneous acrosome reaction at the same MIF concentration (P<.05). ++Significantly different from the spontaneous acrosome reaction at the same MIF concentration (P<.01). Carli. MIF affects sperm functions. Fertil Steril 2007. Fertility and Sterility 2007 88, 1240-1247DOI: (10.1016/j.fertnstert.2007.04.002) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of MIF on sperm motility. Percoll-washed spermatozoa were incubated with the indicated concentrations of MIF in Ham's F10 medium for 4 hours at 37°C. Sperm motility parameters were determined using the Hamilton-Thorne computer-assisted sperm analysis system as described in the Materials and Methods section. Motility is expressed as a percentage of control (sperm incubated with Ham's F10 medium alone without MIF). Data are mean ± standard error of the mean from four different donors. ∗Significantly different from control (sperm incubated with Ham's F10 medium alone without MIF) (P<.05). Carli. MIF affects sperm functions. Fertil Steril 2007. Fertility and Sterility 2007 88, 1240-1247DOI: (10.1016/j.fertnstert.2007.04.002) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Western blot analysis of CD74 expression in human spermatozoa. Washed sperm proteins from three different volunteers were solubilized and separated by SDS-PAGE. Proteins were electrotransferred onto nitrocellulose, and the presence of CD74 was assessed by Western blotting using a polyclonal goat anti-hCD74 antibody. Membranes were incubated with anti-hCD74 primary antibody preabsorbed or not with a specific blocking peptide (A) or with the same concentration of goat IgGs (B). Molecular weight markers (kDa) are indicated on the left. Lines 1 and 2: THP-1 protein extracts (20 and 40 μg, respectively); line 3: sperm protein extracted from 4 ×106 spermatozoa; lines 4 and 5: THP-1 protein extracts (20 and 40 μg, respectively); line 6: sperm protein extracted from 4 ×106 spermatozoa. Membranes were reblotted with an anti-α-tubulin antibody for protein loading control. Carli. MIF affects sperm functions. Fertil Steril 2007. Fertility and Sterility 2007 88, 1240-1247DOI: (10.1016/j.fertnstert.2007.04.002) Copyright © 2007 American Society for Reproductive Medicine Terms and Conditions