Comparison of enzymatic digestion and mechanical dissociation of human testicular tissues  Florian Schneider, M.D., Klaus Redmann, Ph.D., Joachim Wistuba, Ph.D., Stefan Schlatt, Ph.D., Sabine Kliesch, M.D., Nina Neuhaus, Ph.D.  Fertility and Sterility  Volume 104, Issue 2, Pages 302-311.e3 (August 2015) DOI: 10.1016/j.fertnstert.2015.05.001 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Immunohistochemical stainings for germ cell and somatic cell markers. Stainings were performed on testicular tissue sections obtained from transsexual men with complete spermatogenesis and meiotic arrest. Sections from nontranssexual men with complete spermatogenesis were used as positive controls. Representative images are shown for the undifferentiated germ cell markers SALL4 and UTF1 (A–F), for the spermatogonial and spermatocyte marker MAGE A4 (G–I), and for the somatic markers SMA and VIM (J–O). Corresponding immunoglobulin G antibodies were used as negative controls, and representative images are shown in panels M–O. Scale bars represent 100 μm and 10 μm in the inserts. Fertility and Sterility 2015 104, 302-311.e3DOI: (10.1016/j.fertnstert.2015.05.001) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Total cell number and the amount of living and dead cells after mechanical dissociation and enzymatic digestion. Fresh and cryopreserved testicular tissues from men with complete spermatogenesis (A, D, n = 6; B, E, n = 7) and cryopreserved tissues from patients with meiotic arrest (C, F, n = 4) were evaluated. Results are shown as mean with SEM, and significant differences are marked with *P<.05; **P<.01. Fertility and Sterility 2015 104, 302-311.e3DOI: (10.1016/j.fertnstert.2015.05.001) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Fold change expression of the germ cell–and somatic cell–specific marker genes. Panels A–L show relative expression levels of the undifferentiated spermatogonial marker genes FGFR3 (A–C) and SALL4 (D–F) as well as of the markers UTF1 and MAGE A4 (G–L), which are expressed in spermatogonia and spermatogonia and spermatocytes, respectively. Additionally, panels M–R show results for the somatic marker genes ACTA2 and VIM. Values were determined for the initial testicular tissues (black bars) as well as for the cell fractions after mechanical dissociation (white bars) and enzymatic digestion (gray bars) for the three patient groups (complete spermatogenesis fresh, n = 6; cryopreserved n = 7; and meiotic arrest, n = 4). Results are shown as RQ data using GAPDH for normalization and results from the initial tissues as calibrator. Data are shown as mean with SEM, and significant differences are marked with *P<.05; **P<.01. Fertility and Sterility 2015 104, 302-311.e3DOI: (10.1016/j.fertnstert.2015.05.001) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Histograms showing results from flow-cytometic analyses for the germ cell marker SALL4 and the somatic marker VIM. Results were obtained using fresh testicular tissue with complete spermatogenesis, and percentages of SALL4-positive (A, B) and VIM-positive cells (C, D) are shown in the individual figures. Results for the negative IgG controls are shown in black. Fertility and Sterility 2015 104, 302-311.e3DOI: (10.1016/j.fertnstert.2015.05.001) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions