Telomere homeostasis is compromised in spermatocytes from patients with idiopathic infertility  Rita Reig-Viader, M.Sc., Laia Capilla, M.Sc., Marta Vila-Cejudo, M.Sc., Ferrán Garcia, M.D., Begoña Anguita, Ph.D., Montserrat Garcia- Caldés, Ph.D., Aurora Ruiz-Herrera, Ph.D.  Fertility and Sterility  Volume 102, Issue 3, Pages 728-738.e1 (September 2014) DOI: 10.1016/j.fertnstert.2014.06.005 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Recombination analysis in testis samples. (A) Examples of immunolocalization of meiotic recombination events in human spermatocytes at pachynema from control (top) and an infertile patient (bottom). MLH1 foci are depicted in green, centromeres in blue, and synaptonemal complexes in red. Arrowheads indicate bivalents lacking MLH1 signals. (B) Distribution of the mean numbers of MLH1 foci per cell observed in each sample. Means are indicated by a horizontal black line. (C) Percentage of pachytene spermatocytes showing one MLH1-negative bivalent. In both cases asterisks indicate the level of significance compared with control. ∗P<.05 (one-way analysis of variance [ANOVA] and t test); ∗∗P<.001 (one-way ANOVA and t test). Fertility and Sterility 2014 102, 728-738.e1DOI: (10.1016/j.fertnstert.2014.06.005) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 TERRA levels and colocalization between TERRA and TRF2. (A) Representative images of a pachytene spermatocyte and a SYCP3(-) cell showing TERRA (red foci) localization at telomeres (light blue foci), the synaptonemal complex in green and the DNA counterstained with 6-diamino-2-phenylindole (DAPI). Insets show enlarged TERRA foci colocalizing with TRF2 signals. (B) TERRA levels expressed as the percentage of spermatocytes I (sp I) and SYCP3(-) cells included in each group according to TERRA levels: less than 15 foci, between 15 and 30 or more than 30 foci. (Clover indicates retrieved from Reig-Viader et al., 2014 [44]). Asterisks indicate significance for SYCP3(-) cells, and number signs for spermatocytes I, both compared with the control sample (χ2 test and one-way analysis of variance [ANOVA]). ∗ or #P<.05; ∗∗ or ##P<.001. The table below indicates the number of cells analyzed per sample and cell type. (C) Percentage of TERRA foci localizing at telomeres (left) and percentage of telomeres showing TERRA foci associated (right) in both primary spermatocytes and SYCP3(-) cells. (the clover symbol indicates data retrieved from Reig-Viader et al., [44]). Asterisks indicate significance for SYCP3(-) cells and number signs (#) for spermatocytes I, both compared with the control sample (one-way ANOVA). ∗ or #P<.05; ∗∗ or ##P<.001. Tables below indicate the number of cells analyzed per sample and cell type. (D) Pearson correlations between the number of TERRA foci per cell and the percentage of TERRA localized at telomeres in spermatocytes I (sp) and SYCP3(-) cells for each sample. (E) Pearson correlations between TERRA foci and the proportion of telomeres showing TERRA in spermatocytes I (sp) and SYCP3(-) cells. Significance is indicated by asterisks: ∗P<.05; ∗∗P<.001. (Clover indicates retrieved from Reig-Viader et al., 2014 [44]). See text for other acronyms. Fertility and Sterility 2014 102, 728-738.e1DOI: (10.1016/j.fertnstert.2014.06.005) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Colocalization of TERT with telomeres and with TERRA. (A) Representative images of a pachytene spermatocyte and a SYCP3(-) cell where the protein component of telomerase (red), telomeres (light blue), and synaptonemal complexes (green) were immunodetected. DNA was counterstained with 6-diamino-2-phenylindole (DAPI). Insets show enlarged regions where TERT association to telomeres can be observed. (B) Percentages of telomeres showing TERT signals in primary spermatocytes (Sp I) and SYCP3(-) cells for each sample. Asterisks and number signs indicate significance for SYCP3(-) cells and spermatocytes I, respectively, compared with the control sample. One-way analysis of variance (ANOVA), ∗P<.05; ##P<.001. (C) Images showing a pachytene spermatocyte and a SYCP3(-) cell where TERRA (red) and TERT (green) have been labeled by immunofluorescence/RNA-fluorescent in situ hybridization. Synaptonemal complexes (light blue) and DNA (DAPI) are revealed. Insets show enlarged regions with TERRA foci colocalizing with TERT signals. (D) Proportion of TERRA foci colocalizing with TERT in spermatocytes I (Sp I) and SYCP3(-) cells for each sample. Asterisks indicate significance for SYCP3(-) cells compared with the control. One-way ANOVA, ∗P<.05. Tables below graphs indicate the number of analyzed cells per sample and per cell type. (the clover symbol indicates data retrieved from Reig-Viader et al., [44]). See text for other acronyms. Fertility and Sterility 2014 102, 728-738.e1DOI: (10.1016/j.fertnstert.2014.06.005) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Telomere lengths in human testis cells. (A) Images of a pachytene spermatocyte and a SYCP3(-) cell where telomeric sequences were revealed by means of a TelC PNA probe (green). Synaptonemal complexes (light blue) and DNA (6-diamino-2-phenylindole [DAPI]) are also shown. (B) Representation of the mean values ± SEM of telomere lengths measured in the different samples displayed as HeLa-fold increase telomere fluorescent units. Asterisks indicate significance for SYCP3(-) cells and number signs for spermatocytes I, both compared with the control (one-way analysis of variance [ANOVA]). ∗ or #P<.05; ∗∗ or ##P<.001. (C) Frequency distribution of measured telomeres for each telomere fluorescent unit (TFU) interval and for each sample. The table below the graph indicates the number of analyzed telomere signals per sample and per cell type. (the clover symbol indicates data retrieved from Reig-Viader et al., [44]). See text for other acronyms. Fertility and Sterility 2014 102, 728-738.e1DOI: (10.1016/j.fertnstert.2014.06.005) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions