Association of aberrant expression of sex-determining gene fibroblast growth factor 9 with Sertoli cell–only syndrome  Chia-Ling Chung, M.Sc., Chun-Wun Lu, Ph.D., Yu-Sheng Cheng, M.D., Ching-Yi Lin, M.Sc., H. Sunny Sun, Ph.D., Yung-Ming Lin, M.D.  Fertility and Sterility  Volume 100, Issue 6, Pages 1547-1554.e4 (December 2013) DOI: 10.1016/j.fertnstert.2013.08.004 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Expression profiles of the FGF9 protein and the correlations between their expressions and serum hormone parameters. (A) An FGF9 immunopositive reaction was detected predominately in the cytoplasm of Leydig cells. (B) Magnification of panel A. (C) A weak immunopositive reaction was detected in the cytoplasm of Sertoli cells and the cell membranes of germ cells. (D) Normal rabbit IgG was used as a primary antibody (negative control). (E) A significantly weak immunopositive reaction of FGF9 was detected in the Leydig cells and Sertoli cells of a patient with SCOS. (F) Magnification of panel E. (G) Almost no immunopositive reaction of FGF9 was detected in the Sertoli cells of a patient with SCOS. (H) Magnification of panel G. (I) The mean IOD was determined by the total IOD value measured in the interstitial areas divided by the Leydig cell number (at least 50 Leydig cells from each patient). The mean IOD represents the immunostaining intensity of FGF9 in each patient. FGF9 protein IOD levels were significantly decreased in patients with SCOS. ***P<.0001. Data are medians with the interquartile range. (J) Testicular interstitial tissue of patients with the same histology was pooled for immunoblot analysis, and the results showed that FGF9 protein expression was significantly decreased in samples with SCOS. (K, L) Significant and positive correlations between FGF9 protein IOD levels and serum T levels and T:LH levels were found; the r2-value are shown on each graph. Scale bar = 100 μm for panels A, D, E, and G and = 50 μm for panels B, C, F, and H. NS = normal spermatogenesis; PC = positive control using human endometrial tissue; NC = negative control. Fertility and Sterility 2013 100, 1547-1554.e4DOI: (10.1016/j.fertnstert.2013.08.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Effects of FGF9 on germ cell proliferation. (A, B) GC1 and GC2 cells were treated with FGF9 recombinant protein at different doses, and the cell proliferation rates were determined at different time points. (C, D) TM3 cells were treated with different doses of FGF9 (0, 2.5, and 5 ng), and the conditioned media were collected. Then GC1 cells and GC2 cells were incubated with a mixture of 20% 5-fold concentrated conditioned medium and 80% fresh medium and subjected to cell proliferation assay. Details of two-way ANOVA results are presented in the text. Fertility and Sterility 2013 100, 1547-1554.e4DOI: (10.1016/j.fertnstert.2013.08.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Promoter polymorphism on the FGF9 gene and effects of FGF9 c.-712C→T on FGF9 expression and E2F1 binding. (A) Chromatogram of the forward sequence of FGF9 gene from a normal fertile man and an infertile patient with SCOS showing c.-712C→T. The star indicates the polymorphic site. (B) A schematic diagram of luciferase reporter constructs containing the FGF9 promoter, either wild-type (-712C) or mutagenized (-712T). For the p1949 construct, a 5′-flanking sequence, from −1949 to +40 relative to the transcription start site, was amplified and subcloned into pGL3-basic vector. The p1949mT construct was generated using a Phusion site-directed mutagenesis kit. (C) The constructs were transfected into PC3, IMR32, and HEK293 cells, respectively. Twenty-four hours after the transfection, firefly and Renilla luciferase were measured using a Dual Luciferase Assay. Firefly luminescence was normalized to Renilla luminescence and reported as relative luciferase (RLU) units. All experiments were done in triplicate and independently performed at least three times. *P<.05. (D) Patients were subdivided into three groups according to testicular phenotype and the presence or absence of FGF9 c.-712C→T, and then the FGF9 transcript level was determined. Different letters indicate significant difference (P<.05) by Tukey's test. (E) The FGF9 nucleotide sequence around -712 shows putative transcription factor binding sites revealed using PROMO and TESS analysis. (F) ChIP analysis was done on IMR32 and PC3 cells (data not shown). Cross-linked, enzymatic sheared chromatin was immunoprecipitated with E2F1 or mouse IgG antibodies, and the recovered chromatin was subjected to PCR analysis. A primer pair designed to amplify the chromosome 13 fragment, 30 kb upstream of the FGF9 promoter, was used as a negative control, and a fraction (10%) of the chromatin preparation was used as an input control. Ethidium bromide-stained gel of the PCR products shows a representative of ChIP analysis from three (labeled A, B, and C) independent experiments. (G) EMSA was done using biotin-labeled oligonucleotide probes containing -712C or -712T. The nuclear extracts from IMR32 and PC3 (data not shown) cells were incubated with the biotinylated probes, in the presence of anti-E2F1 or an increasing amount of unlabeled competitors. Samples were loaded on a 10% native acrylamide gel. The arrow indicates DNA protein complex formation in the presence of cell lysate. The star indicates the position of supershift induced by anti-E2F1 antibody. NS = normal spermatogenesis. Fertility and Sterility 2013 100, 1547-1554.e4DOI: (10.1016/j.fertnstert.2013.08.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 Purity of interstitial cells extracted from testes with normal spermatogenesis and testes with SCOS. Total RNA was extracted from different testicular tissues, and the expressions of testicular cell markers were determined by RT-PCR. INSL3 = insulin-like factor 3, (Leydig cell marker); SOX9 = SRY (sex determining region Y)-box 9 (Sertoli cell marker); SYCP3 = synaptonemal complex protein 3 (germ cell marker); 18S = 18S ribosomal RNA (loading control). Lane 1 = RNA extracted from whole testes; lane 2 = RNA extracted from interstitial cells of testes with normal spermatogenesis; lane 3 = RNA extracted from interstitial cells of testes with SCOS; lane 4 = no template control. Fertility and Sterility 2013 100, 1547-1554.e4DOI: (10.1016/j.fertnstert.2013.08.004) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions