Fertility and Sterility

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Fertility and Sterility Androgenic and antiandrogenic effects and expression of androgen receptor in mouse embryonic stem cells Chih-Yang Chang, M.D., Yan-Der Hsuuw, Ph.D., Fu-Jen Huang, M.D., Chih-Rong Shyr, Ph.D., Shiuh-Young Chang, M.D., Chiung-Kuei Huang, M.Sc., Hong-Yo Kang, Ph.D., Ko-En Huang, M.D. Fertility and Sterility Volume 85, Pages 1195-1203 (April 2006) DOI: 10.1016/j.fertnstert.2005.11.031 Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Androgenic and antiandrogenic effects on embryonic stem cell (ESC) growth. (A) ES-D3 cells were incubated with various concentrations of dihydotestosterone (DHT) and nilutamide (NT) (10 μmol/L) for indicated periods of time and applied to XTT assay for measuring cell growth. (B) ES-D3 cells were incubated with various concentrations of T and NT (10 μmol/L) for indicated periods of time. Subsequently, the cells were quantified by XTT assay for measuring cell growth. (C) ES-D3 cells were incubated with various concentrations of NT for indicated periods of time and applied to XTT assay for measuring cell growth. Each data point indicates mean ± SD of at least six independent sets for each experiment. Chang. Androgen and antiandrogen in mouse ESCs. Fertil Steril 2006. Fertility and Sterility 2006 85, 1195-1203DOI: (10.1016/j.fertnstert.2005.11.031) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 The effects of androgens and antiandrogen on the expression of Akt kinase and p27(Kip1) in embryonic stem cells. ES-D3 cells were treated with 10 nmol/L dihydrotestosterone (DHT) and testosterone (T) and 10 μmol/L nilutamide (NT) for three days and lysed. Equal amounts of cell lysates were analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent immunoblotting with anti-Akt and anti-glyceraldehyde 3 phosphate dehydrogenase (GAPDH) antibodies. Representative blots are shown, and the results were verified in at least three independent experiments. Chang. Androgen and antiandrogen in mouse ESCs. Fertil Steril 2006. Fertility and Sterility 2006 85, 1195-1203DOI: (10.1016/j.fertnstert.2005.11.031) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 The immunofluorescence pattern of androgen receptor (AR) localization was detected in embryonic stem cells. Embryonic stem cells (ESCs) were cultured with ethanol treatment (A–C) and dihydrotestosterone treatment (D–F). (G–I) Embryonic stem cells immunostained with anti-IgG antibody as a negative control. (A, D, G) ESCs stained with anti-AR antibody. (B, E, H) ESCs stained with 4,6-diamidino-2-phenylindole (DAPI). (C, F, I) Merged images. Original magnification 200×. Chang. Androgen and antiandrogen in mouse ESCs. Fertil Steril 2006. Fertility and Sterility 2006 85, 1195-1203DOI: (10.1016/j.fertnstert.2005.11.031) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Quantitative evaluation of androgen receptor (AR) gene expression in the blastocyst, ES-D3 cells, and inner cell mass (ICM) by real-time reverse transcriptase polymerase chain reaction (PCR). Each bar represents mean ± SD of six independent sets for each experiment. Samples (n = 6) were significantly different from controls. Concomitant detection of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) mRNA served as a reference for relative quantification. Chang. Androgen and antiandrogen in mouse ESCs. Fertil Steril 2006. Fertility and Sterility 2006 85, 1195-1203DOI: (10.1016/j.fertnstert.2005.11.031) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Quantitative evaluation of androgen receptor (AR) gene expression in the undifferentiated embryonic stem cells (ESCs), undifferentiated embryoid body, and differentiated embryoid body in the presence or absence of dihydrotestosterone (DHT) treatment by real-time reverse transcriptase polymerase chain reaction. Each bar represents mean ± SD of six independent sets for each experiment. Samples (n = 6) were significantly different from controls. Concomitant detection of glyceraldehyde 3 phosphate dehydrogenase (GAPDH) mRNA served as a reference for relative quantification. Chang. Androgen and antiandrogen in mouse ESCs. Fertil Steril 2006. Fertility and Sterility 2006 85, 1195-1203DOI: (10.1016/j.fertnstert.2005.11.031) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions