Expression of dysadherin in the human male reproductive tract and in spermatozoa  Nieves María Gabrielli, M.Sc., María Florencia Veiga, Ph.D., María Laura Matos, M.Sc., Silvina Quintana, M.Sc., Héctor Chemes, M.D., Ph.D., Gustavo Blanco, M.D., Ph.D., Mónica Hebe Vazquez-Levin, Ph.D.  Fertility and Sterility  Volume 96, Issue 3, Pages 554-561.e2 (September 2011) DOI: 10.1016/j.fertnstert.2011.06.053 Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Immunodetection of dysadherin in human ejaculated spermatozoa. (A) Indirect immunofluorescence of selected motile human spermatozoa recovered from the ejaculate, using NCC-M53 anti-dysadherin antibody (dysadherin: a–c) or mouse IgG (control: g–i). Corresponding brightfield photomicrographs (d–f and j–l, respectively) are also shown. Cells were treated without (a, d, g, j) and with methanol (b, c, e, f, h, i, k, l). Bar = 10 μm in d, e, j, and k, 5 μm in f and l. (B) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western immunoblotting of protein extracts from spermatozoa developed using the NCC-M53 antibody. Protein extracts from HUVEC and MDA-MB-231 somatic cells are included for comparison. The estimated molecular weight of dysadherin forms is indicated. Fertility and Sterility 2011 96, 554-561.e2DOI: (10.1016/j.fertnstert.2011.06.053) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Immunodetection of dysadherin after sperm capacitation and acrosomal exocytosis. (A) Indirect immunofluorescence analysis of 18h-Cap spermatozoa (a, b, e, f, i, and j) and of acrosome-reacted cells after calcium ionophore A23187 treatment (c, d, g, h, k, l) using NCC-M53 anti-dysadherin antibody (a–d). The corresponding FITC-PSA (e–h) and brightfield (i–l) images for each cell are also shown. Bar = 10 μm in i and k and 5 μm in j and l. “Acrosome-intact” and “acrosome-reacted” spermatozoa were classified with the following criteria: presence of a bright staining over the acrosomal cap (“intact”) and cell labelling in the equatorial segment or showing no label in the acrosome (“acrosome-reacted”). (B) Dysadherin immunostaining pattern in intact and in acrosome-reacted human spermatozoa. Cells were stained with NCC-M53 anti-dysadherin antibody, followed by incubation with FITC-PSA to assess the acrosomal status. A representative image is displayed, showing the signal for dysadherin (left, dysadherin), FITC-PSA (middle, PSA) staining, and the composed image (right, merge). Bar = 7.5 μm. (C) Distribution of dysadherin immunostaining patterns for the sperm acrosomal cap (left) and flagellum (right). Values for populations of noncapacitated (NC), 18h-Cap (C), and acrosome-reacted (AR) spermatozoa. Cells were stained with NCC-M53 anti-dysadherin antibody, followed by incubation with FITC-PSA to assess the acrosomal status on the cells. Results are expressed as mean ± SD, n = 3. ∗P<.001 (analysis of variance). Fertility and Sterility 2011 96, 554-561.e2DOI: (10.1016/j.fertnstert.2011.06.053) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Expression of dysadherin in human male reproductive tissues. (A) Reverse transcription–polymerase chain reaction analysis of dysadherin RNA of human testis, caput, corpus, and cauda epididymis. MDA-MB-231 cells were used as a positive control. Negative controls for RT and PCR procedures were included. Experiments were repeated three times with similar results; a representative experiment is shown. (B) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western immunoblot analysis of dysadherin in protein extracts of spermatozoa, testis, epididymis, and HUVEC cells using NCC-M53 anti-dysadherin antibody. The estimated molecular weight of dysadherin forms is indicated. (C) Immunohistochemical localization of dysadherin in human testis (a, b) and epididymis (d, e) using NCC-M53 anti-dysadherin antibody. (c, f) Controls. Bar = 10 μm in a and c, 25 μm in b, 50 μm in d–f. (D) Immunofluorescence analysis of testicular spermatozoa using NCC-M53 anti-dysadherin antibody (dysadherin) or mouse IgG (control). The corresponding brightfield photomicrograph (brightfield) for each cell is shown at right. Bar = 5 μm. (E) Reverse transcription–polymerase chain reaction analysis of dysadherin RNA in human ejaculated spermatozoa. Negative controls for RT and PCR procedures are also shown. Experiments were repeated three times with similar results. An image of the agarose gel from a typical experiment is shown. Fertility and Sterility 2011 96, 554-561.e2DOI: (10.1016/j.fertnstert.2011.06.053) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Co-immunolocalization analysis of dysadherin with E-cadherin and Na+,K+-ATPase. (A) Immunolocalization analysis of dysadherin (NCC-M53 anti-dysadherin antibody, left) and E-cadherin (H-108 anti-E-cadherin antibody, middle) on motile human spermatozoa recovered from the ejaculate. Merge images (right) revealed an overlapped signal for both proteins in the proximal acrosomal region (yellow). Bar = 10 μm for upper panel, 5 μm for lower panels. The secondary antibody used for the images shown in Figure 1A, b and Figure 4 are different: whereas a Cy3-anti mouse IgG was used in Figure 1A, b, a FITC-anti-mouse IgG was used in Figure 4, for the colocalization studies. Cy3 (indocarbocyanine) is a brighter, more photostable fluorophore that gives a stronger signal and less background than most other fluorophores, such as FITC. (B) Immunolocalization analysis of dysadherin (NCC-M53 anti-dysadherin antibody, left) and Na+,K+-ATPase (anti-Na+,K+-ATPase α4 subunit, middle) on motile human spermatozoa recovered from the ejaculate. Merge images (right) show an overlapped signal for both proteins in the flagellum (yellow). Bar = 5 μm. Fertility and Sterility 2011 96, 554-561.e2DOI: (10.1016/j.fertnstert.2011.06.053) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Nucleotide and protein sequence analysis of dysadherin from human testis. (A) Nucleotide sequence comparison between the sequence of human testicular dysadherin and the reported NM_144779.2. (B) Protein sequence comparison between the amino acidic sequences deduced from the nucleotide sequences of human testicular dysadherin and the reported NM_144779.2. Fertility and Sterility 2011 96, 554-561.e2DOI: (10.1016/j.fertnstert.2011.06.053) Copyright © 2011 American Society for Reproductive Medicine Terms and Conditions