Androgen signaling in decidualizing human endometrial stromal cells enhances resistance to oxidative stress Takeshi Kajihara, M.D., Ph.D., Hideno Tochigi,

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Androgen signaling in decidualizing human endometrial stromal cells enhances resistance to oxidative stress Takeshi Kajihara, M.D., Ph.D., Hideno Tochigi, M.D., Japarath Prechapanich, M.D., Satomi Uchino, Atsuo Itakura, M.D., Ph.D., Jan J. Brosens, M.D., Ph.D., Osamu Ishihara, M.D., Ph.D. Fertility and Sterility Volume 97, Issue 1, Pages 185-191 (January 2012) DOI: 10.1016/j.fertnstert.2011.10.017 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Prolactin production by primary HESCs in response to treatment with 8-br-cAMP + P, DHT, and an AR antagonist (flutamide). (A) Confluent HESCs were treated with 8-br-cAMP (0.5 mM), P (10−6 M), DHT (10−7 M or 10−6 M), or a combination of these, as indicated. The medium was changed every 24 hours. (B) Confluent HESCs were treated as indicated for 6 days, with the medium changed every 24 hours. Significant differences were found in terms of PRL secretion between cultures with 8-br-cAMP + P and 8-br-cAMP + P + DHT (10−6) at 3 and 6 days. The data represent the mean of PRL concentrations in the supernatant, normalized for protein content. Data shown mean ± SD. ∗∗P<.01. Fertility and Sterility 2012 97, 185-191DOI: (10.1016/j.fertnstert.2011.10.017) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Apoptosis analysis. Decidualizing HESCs were cultured without or with various concentrations of DHT and exposed to H2O2 (100 μM) for 4 hours. Decidualizing cultures with DHT were pretreated with 8-br-cAMP and P for 6 days. The level of apoptosis was analyzed, and the data shown represent mean ± SD. ∗∗P<.01. Fertility and Sterility 2012 97, 185-191DOI: (10.1016/j.fertnstert.2011.10.017) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Western blot analysis of FOXO1, SOD2, SOD1, and catalase proteins in whole-cell lysates obtained from primary HESCs cultures treated as indicated for 6 days. Alpha-tubulin expression was used as loading control. Data shown represent mean ± SD. ∗∗P<.01. Fertility and Sterility 2012 97, 185-191DOI: (10.1016/j.fertnstert.2011.10.017) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Primary HESCs were treated with 8-br-cAMP, P, DHT, and an AR antagonist (flutamide). Western blot analysis of FOXO1 proteins in whole-cell lysates obtained from primary HESCs cultures treated as indicated for 6 days. Alpha-tubulin expression was used as loading control. Fertility and Sterility 2012 97, 185-191DOI: (10.1016/j.fertnstert.2011.10.017) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions