Leptin down-regulates γ-ENaC expression: a novel mechanism involved in low endometrial receptivity  Xian-Hua Lin, M.D., Miao-E. Liu, M.B., Hai-Yan Xu, M.B., Xue-Jun Chen, M.D., Hui Wang, M.B., Shen Tian, M.D., Jian-Zhong Sheng, Ph.D., He-Feng Huang, M.D.  Fertility and Sterility  Volume 103, Issue 1, Pages 228-235.e3 (January 2015) DOI: 10.1016/j.fertnstert.2014.10.002 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Expression of epithelial Na+ channel (ENaC) subunits in the endometrium of infertile women with male factor and polycystic ovary syndrome (PCOS). (A) Serum leptin levels of 12 control and 12 PCOS women were determined with the use of ELISA: ∗∗P<.01. (B) Immunohistochemical analysis of ENaC subunits (α, β, and γ) in human endometrial tissues collected by biopsy. Endometrial explants were fixed, paraffin embedded, and stained for anti-ENaC antibodies as described in Materials and Methods. This figure shows one representative experiment among three separate experiments. (C) H-Score analysis of immunohistochemical staining of three ENaC subunits in human endometrium (n = 12 each). The values are expressed as mean ± SEM. ∗∗P<.01 (unpaired t test). (D) Messenger RNA expressions of ENaC subunits in human endometria. Total RNA was extracted from endometrium biopsies of infertile women with male factor and PCOS (n = 12 each) and then analyzed by quantitative reverse-transcription polymerase chain reaction. The values are presented as mean ± SEM. ∗∗P<.01 (unpaired t test). (E) ENaC protein expression. Tissue lysates (80 μg) were extracted from endometrial tissues of infertile women with male factor (control; n = 3) and infertile women with PCOS (PCOS; n = 3), and then subjected to Western blot analysis with the use of anti-ENaC or anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody, as described in the Materials and Methods. This figure shows one representative experiment among three separate experiments. (F) Relative protein levels of ENaC subunits normalized to GAPDH. The values are presented as the mean ± SEM. ∗P<.05 (unpaired t test). Fertility and Sterility 2015 103, 228-235.e3DOI: (10.1016/j.fertnstert.2014.10.002) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Leptin reduced the expression of γ-ENaC and up-regulated the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in Ishikawa cells, resulting in lower JAr spheroid attachment rate in vitro. (A) Messenger RNA expression of the γ-ENaC subunit in Ishikawa cells treated with leptin at different concentrations. Total RNA was extracted from Ishikawa cells treated with different concentrations of leptin (n = 4 each) and then analyzed with the use of quantitative reverse-transcription polymerase chain reaction. The values are presented as mean ± SEM. ∗∗P<.01 (unpaired t test). (B) γ-ENaC protein expression. Cell lysates (100 μg) were extracted from Ishikawa cells subjected to Western blot analysis with the use of anti–γ-ENaC or anti-GAPDH antibody. This figure shows one representative experiment among three separate experiments. (C) Effect of leptin at the concentration of 200 ng/mL on phosphorylated STAT3 protein expression in Ishikawa cells at different time points. This figure shows one representative experiment among three separate experiments. (D) JAr spheroid attachment rate in an attachment model in vitro. JAr spheroid attachment rate was examined after treatment of Ishikawa cells with different concentrations of leptin for 24 hours (n = 4 each) as described in the Materials and Methods. The values are presented as mean ± SEM. ∗P<.05; ∗∗P<.01; compared with the value of Tris-HCl treatment; unpaired t test. Abbreviations as in Figure 1. Fertility and Sterility 2015 103, 228-235.e3DOI: (10.1016/j.fertnstert.2014.10.002) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Leptin down-regulated γ-ENaC via activating the STAT3 pathway. (A) Expression levels of γ-ENaC mRNA in Ishikawa cells with or without STAT3-specific small interfering (si) RNA in the presence of different concentrations of leptin (real-time polymerase chain reaction [PCR]; n = 4). The values are presented as mean ± SEM; ∗∗P<.01, compared with control (Tris-HCl treatment). (B) Expression levels of γ-ENaC in Ishikawa cells transfected with STAT3-specific siRNA in the presence of different concentrations of leptin (Western blot). The figure shows one representative experiment among three separate experiments. (C) The effect of leptin at 200 ng/mL on the JAr spheroid attachment rate after transfection of Ishikawa cells with or without treatment of STAT3-specific siRNA (n = 4). ∗P<.05; ∗∗P<.01. (D and E) STAT3-specific siRNA knock-down efficiency detected with the use of real-time PCR and Western blot. ∗∗P<.01, compared with control (scramble siRNA treatment). Abbreviations as in Figures 1 and 2. Fertility and Sterility 2015 103, 228-235.e3DOI: (10.1016/j.fertnstert.2014.10.002) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions