Nerea Subirán, J. D. , Ekaitz Agirregoitia, J. D. , Asier Valdivia, Ph

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Expression of enkephalin-degrading enzymes in human semen and implications for sperm motility Nerea Subirán, J.D., Ekaitz Agirregoitia, J.D., Asier Valdivia, Ph.D., Carmen Ochoa, Ph.D., M.D., Luis Casis, Ph.D., Jon Irazusta, Ph.D. Fertility and Sterility Volume 89, Issue 5, Pages 1571-1577 (May 2008) DOI: 10.1016/j.fertnstert.2007.06.056 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Western blot analysis of aminopeptidase N (A) and neutral endopeptidase 24.11 (B) in spermatozoa (Sp), prostasome (P), seminal fluid (SF), and human kidney particulate fractions (K). Molecular mass is indicated on the left side of each panel. Representative Western blots obtained with 9 normozoospermic donors are shown. Fertility and Sterility 2008 89, 1571-1577DOI: (10.1016/j.fertnstert.2007.06.056) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Immunofluorescence analysis of aminopeptidase N (A, E) and neutral endopeptidase 24:11 (B, F) in human sperm cells under non-permeabilized (A–D) and permeabilized (E–H) conditions. Negative controls were treated with preimmune rabbit serum (C, G) and secondary antibody alone (D, H). The DNA of controls was stained with Hoechst 33342. Representative photomicrographs are shown; n = 5. Arrows, variability of staining in the sample. Scale bar, 10 μm. Fertility and Sterility 2008 89, 1571-1577DOI: (10.1016/j.fertnstert.2007.06.056) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Amplification cycles (Cycle threshold [Ct] ± SEM) for aminopeptidase N (APN), neutral endopeptidase 24:11 (NEP), and ribosomal 18S (18S), assessed by real-time quantitative PCR using specific primers (n = 4). Fertility and Sterility 2008 89, 1571-1577DOI: (10.1016/j.fertnstert.2007.06.056) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Effect of enkephalin-degrading enzyme inhibition on sperm motility at 0, 2, and 4 hours. (A) Percentage of grade a cells in the control (white bars), leuhistin (100 μM) treatment (black bars), and leuhistin (100 μM) plus naloxone (10 μM) treatment (gray bars) groups during the time of observation; n = 10. (B) Percentage of grade a cells in the control (white bars), thiorphan (150 nM) treatment (black bars), and thiorphan (150 nM) plus naloxone (10 μM) treatment (gray bars) groups during the time of observation; n = 10. (C) Percentage of grade a cells in the control (white bars) and naloxone (10 μM) treatment (black bars) groups during the time of observation; n = 5. The results are expressed as mean ± SEM. aP<.01 vs. control. bP<.01 vs. inhibitor treatment. Fertility and Sterility 2008 89, 1571-1577DOI: (10.1016/j.fertnstert.2007.06.056) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions