Inhibition of mammalian target of rapamycin signaling pathway decreases retinoic acid stimulated gene 8 expression in adult mouse testis  Pinar Sahin, M.Sc., Zeliha Sahin, Ph.D., N. Ece Gungor-Ordueri, M.Sc., Baris Ozgur Donmez, Ph.D., Ciler Celik-Ozenci, D.D.S., Ph.D.  Fertility and Sterility  Volume 102, Issue 5, Pages 1482-1490.e3 (November 2014) DOI: 10.1016/j.fertnstert.2014.08.004 Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Expressions of mTOR pathway proteins in adult mouse testis. (A) mTOR expression at different seminiferous tubule stages. Negative control. Cytoplasmic mTOR expression was seen in Sertoli cells (black arrow heads), in spermatogonia (red arrow heads), and in preleptotene spermatocytes (green arrow heads; scale bar = 20 μm). (B) p-mTOR expression at different seminiferous tubule stages. Negative control. Nuclear p-mTOR expression was seen in spermatogonia (red arrow heads) and in preleptotene spermatocytes (green arrow heads; scale bar = 20 μm, 10 μm). (C) Total p70S6K expression in adult mouse testis. Total p70S6K expression was seen in spermatogonia (red arrow heads) and in preleptotene spermatocytes (green arrow heads). Additionally, total p70S6K expression was present in Sertoli cells (black arrow heads). (D) p-p70S6K expression in adult mouse testis. The p-p70S6K expression was present in different seminiferous tubule stages. Negative control. Nuclear p-p70S6K expression was seen in spermatogonia (red arrow heads), in preleptotene spermatocytes (green arrow heads), and in round spermatids (blue arrow heads; scale bar = 20 μm). (E) p-4EBP1 expression in adult mouse testis. The p-4EBP1 expression was present in various seminiferous tubule stages. Negative control. Cytoplasmic p-4EBP1 expression was seen in Sertoli cells (black arrow heads), in spermatogonia (red arrow heads), in preleptotene spermatocytes (green arrow heads), and in interstitial cells (yellow arrow heads; scale bar = 20 μm). Fertility and Sterility 2014 102, 1482-1490.e3DOI: (10.1016/j.fertnstert.2014.08.004) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Expressions of mTOR pathway proteins in ex vivo mouse seminiferous tubule cultures. Cytoplasmic mTOR expression was seen in spermatogonia (red arrow heads), in Sertoli cell (black arrow heads), and in preleptotene spermatocytes (green arrow heads) in all groups. MTOR expression was similar among all four groups. Nuclear p-mTOR expression was specifically present in spermatogonia (red arrow heads) and in preleptotene spermatocytes (green arrow heads) in all groups. No expression of p-mTOR was seen in Sertoli cells. The p-mTOR expression was similar among all four groups. Expression of total p70S6K was seen in preleptotene spermatocytes (green arrow heads) and in Sertoli cells (black arrow heads) in all groups. Total p70S6K expression was similar among all four groups. Nuclear expression of p-p70S6K was seen in spermatogonia (red arrow heads) and in preleptotene spermatocytes (green arrow heads) in all groups. The p-P70S6K expression decreased in the rapamycin-treated group when compared with the control (0 hour), 24-hour culture, and ethanol-treated groups. The p-4EBP1 expression was seen in Sertoli cells (black arrow heads) and in spermatogonia (red arrow heads) in all groups. The p-4EBP1 expression decreased in the rapamycin-treated group when compared with the control (0 hour), 24 hour-culture, and ethanol-treated groups. Fertility and Sterility 2014 102, 1482-1490.e3DOI: (10.1016/j.fertnstert.2014.08.004) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Evaluation of protein expressions of ex vivo-cultured seminiferous tubules by western blot after rapamycin treatment. (A) Evaluation of p-p70S6K protein expression of cultured seminiferous tubules. GAPDH was used as an internal standard. The p-p70S6K protein decreased in the rapamycin-treated group when compared with the control (0 hour), 24-hour culture, and ethanol-treated groups. Quantitative analysis of Western blot results (per-group n = 6). The values in the bar graphs were obtained by dividing the protein intensity value by its corresponding GAPDH intensity value. The p-p70S6K protein significantly decreased in the rapamycin-treated group when compared with the control (0 hour), 24-hour culture, and ethanol-treated groups. ∗P<.05. (B) Evaluation of p-4EBP1 protein expression of cultured seminiferous tubules. GAPDH was used as an internal standard. The p-4EBP1 protein decreased in the rapamycin-treated group when compared with the control (0 hour), 24-hour culture, and ethanol-treated groups. Quantitative analysis of Western blot results (per-group n = 6). The values in the bar graphs were obtained by dividing the protein intensity value by its corresponding GAPDH intensity value. The p-4EBP1 protein significantly decreased in the rapamycin-treated group when compared with the control (0 hour), 24-hour culture, and ethanol-treated groups. ∗P<.05. (C) Evaluation of PCNA and STRA8 protein expression of cultured seminiferous tubules. GAPDH was used as an internal standard. PCNA and STRA8 proteins decreased in the rapamycin-treated group when compared with the control (0 hour), 24-hour culture, and ethanol-treated groups. Quantitative analysis of Western blot results (per-group n = 6). The values in the bar graphs were obtained by dividing the protein intensity value by its corresponding GAPDH intensity value. PCNA and STRA8 proteins significantly decreased in the rapamycin-treated group when compared with the control (0 hour), 24-hour culture, and ethanol-treated groups. *P<.05. (D) Evaluation of VASA protein expression of cultured seminiferous tubules. GAPDH was used as an internal standard. VASA protein expression was not different between groups. Quantitative analysis of Western blot results (per-group n = 6). The values in the bar graphs were obtained by dividing the protein intensity value by its corresponding GAPDH intensity value. Fertility and Sterility 2014 102, 1482-1490.e3DOI: (10.1016/j.fertnstert.2014.08.004) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Hematoxylen and eosin stained seminiferous tubules of control (0 hour), 24-hour culture, rapamycin-treated, and ethanol treated groups. (Scale bar = 20 μm.) Fertility and Sterility 2014 102, 1482-1490.e3DOI: (10.1016/j.fertnstert.2014.08.004) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 TUNEL staining of control (0 hour), 24-hour culture, rapamycin-treated, and ethanol-treated groups. The number of apoptotic cells (black arrow heads) was similar in all four groups. Fertility and Sterility 2014 102, 1482-1490.e3DOI: (10.1016/j.fertnstert.2014.08.004) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 MTT assay of control (0 hour), 24-hour culture, rapamycin-treated, and ethanol-treated groups. Cell viability was similar in all groups. Fertility and Sterility 2014 102, 1482-1490.e3DOI: (10.1016/j.fertnstert.2014.08.004) Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions