Estrogenic regulation of testicular expression of stem cell factor and c-kit: implications in germ cell survival and male fertility Sara Correia, M.S.,

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Estrogenic regulation of testicular expression of stem cell factor and c-kit: implications in germ cell survival and male fertility Sara Correia, M.S., Mário R. Alves, M.S., José E. Cavaco, Ph.D., Pedro F. Oliveira, Ph.D., Sílvia Socorro, Ph.D. Fertility and Sterility Volume 102, Issue 1, Pages 299-306 (July 2014) DOI: 10.1016/j.fertnstert.2014.04.009 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Effect of 100 nM E2 on mRNA (A) and protein (B) expression of SCF and c-kit in rat SeT cultured ex vivo for 24 hours. The mRNA expression was determined by qPCR after normalization with β-actin and GAPDH housekeeping genes. Protein expression was determined by Western blot analysis after normalization with α-tubulin. Results are expressed as the fold variation relative to 0 nM E2 (dashed line). *P<.05; **P<.01. Error bars indicate mean ± standard error of the mean (n = 5). (C) Representative immunoblots. Fertility and Sterility 2014 102, 299-306DOI: (10.1016/j.fertnstert.2014.04.009) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Apoptosis in rat SeT cultured ex vivo for 48 hours in presence (E2) or absence (control) of 100 nM of E2. (A) Expression of death factors FasL and FasR (24 hours). (B) Ratio of proapoptotic (Bax) and antiapoptotic (Bcl-2) proteins. Protein expression was determined by Western blot analysis after normalization with α-tubulin. (C) Representative immunoblots. (D) Caspase-3 activity. Results are represented as mean ± standard error of the mean (n = 5). *P<.05; **P<.01. Fertility and Sterility 2014 102, 299-306DOI: (10.1016/j.fertnstert.2014.04.009) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Representative confocal microscopy images showing TUNEL (A) and c-kit (B) positive cells in rat SeT cultured ex vivo in presence (E2) or absence (control) of 100 nM of E2 at different experimental time-points (0, 24, and 48 hours). Nuclei are stained with Hoechst 33342 (blue), and fluorescence for TUNEL- and c-kit-positive cells is red and green, respectively. Negative controls for c-kit obtained by omission of the primary antibody are provided as insert panels (−). Fertility and Sterility 2014 102, 299-306DOI: (10.1016/j.fertnstert.2014.04.009) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Proliferation index in rat SeT cultured ex vivo for 48 hours in presence (E2) or absence (control) of 100 nM of E2. Proliferation was determined by Ki67 immunofluorescence analysis. (A) Percentage of Ki67-positive cells relative to the total cell number. Results are expressed as the fold variation compared with control. Error bars indicate mean ± standard error of the mean (n = 5 in each group). ***P<.001. (B) Representative images of Hoechst stained nuclei (i, iv), Ki67 immunofluorescence (ii, v), and corresponding merged images (iii, vi) in control and E2-treated groups. Negative controls for Ki67 obtained by omission of the primary antibody are provided as insert panels (−). Fertility and Sterility 2014 102, 299-306DOI: (10.1016/j.fertnstert.2014.04.009) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions