Fertility and Sterility Regulation of cyclooxygenase activity in cultured endometrial stromal cells by granulocyte-macrophage colony-stimulating factor  Hongbo Wang, M.D., Ph.D., Yan Wen, M.D., Mary Lake Polan, M.D., Ph.D., Robert Boostanfar, M.D., Michael Feinman, M.D., Barry Behr, Ph.D.  Fertility and Sterility  Volume 85, Pages 1118-1124 (April 2006) DOI: 10.1016/j.fertnstert.2005.09.040 Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Representative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of COX-1 and COX-2 mRNA in human endometrial stromal cells. Total RNA was prepared from cultured cells and subjected to RT-PCR using primers specific to each mRNA. The size of the COX-1, COX-2, and β-actin amplifications were 303, 254, and 248 bp, respectively (A). In each case, a 100-bp ladder was used as molecular markers. The relative amount of the mRNA of COX-1 (B) and COX-2 (C) was calculated by dividing the intensity of the band for each group by the intensity of the β-actin band for the corresponding group. Semiquantitative RT-PCR analysis of COX-1 and COX-2 mRNA abundance in stromal cells: low-dose granulocyte macrophage-colony stimulating factor (GM-CSF) (0.1 ng/mL) increases COX-2 mRNA levels in stromal cell compared with the control (C), whereas high-dose GM-CSF (1–100 ng/mL) decreases COX-1 mRNA levels compared with the control (B). *P<.05, compared with the control. Data are expressed as means ± Standard error of the mean (SEM) of separate representative experiments (n = 6). Wang. Regulation of cyclooxygenase by GMCSF. Fertil Steril 2006. Fertility and Sterility 2006 85, 1118-1124DOI: (10.1016/j.fertnstert.2005.09.040) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Enzyme activity of COX in the cultured stromal cells stimulated with various amounts of granulocyte macrophage-colony stimulating factor (GM-CSF). Low concentrations of GM-CSF (0.001–0.1 ng/mL) increased total COX enzyme activity, whereas high concentrations of GM-CSF (1–100 ng/mL) inhibited COX activity (A); COX-1 enzyme predominated in control cells but was inhibited in the presence of GM-CSF (B); COX-2 activity increased in the presence of low concentrations of GM-CSF (0.001–0.1 ng/mL) but decreased at high concentrations of GM-CSF (1–100 ng/mL; C). *P<.05, compared with the control. The data are expressed as mean ± standard error of the mean (SEM) of separate representative experiments (n = 6). Wang. Regulation of cyclooxygenase by GMCSF. Fertil Steril 2006. Fertility and Sterility 2006 85, 1118-1124DOI: (10.1016/j.fertnstert.2005.09.040) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Effect of granulocyte macrophage-colony stimulating factor (GM-CSF) on the production of prostaglandin PGE2 in the supernatant of cultured stromal cells. The PGE2 levels increased by 9% from 567.7 pg/mL to 619 pg/mL in the group incubated with 0.01 ng/mL of GM-CSF. But, PGE2 levels decreased by 31% to 393.3 pg/mL (P<.05) with increasing concentration of GM-CSF from 1 ng/mL to 100 ng/mL. *P<.05, compared with the control. The data are expressed as mean ± standard error of the mean (SEM) of separate representative experiments (n = 6). Wang. Regulation of cyclooxygenase by GMCSF. Fertil Steril 2006. Fertility and Sterility 2006 85, 1118-1124DOI: (10.1016/j.fertnstert.2005.09.040) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions