Differential in vitro actions of nitric oxide on human endometrial cell survival  M.Cecilia Johnson, Ph.D., Manuel Maliqueo, M.Sc., M.Angélica Boric, B.Sc., Alejandra Villavicencio, Ph.D., David Vantman, M.D., Margarita Vega, Ph.D.  Fertility and Sterility  Volume 81, Issue 1, Pages 176-184 (January 2004) DOI: 10.1016/j.fertnstert.2003.05.018

FIGURE 1 Immunohistochemical staining for caspase-3 in explants of human endometrial tissue from throughout the menstrual cycle. Representative human endometria explants from the (A and C) proliferative phase and (B and D) secretory phase of the menstrual cycle with positive immunostaining for (A and B) procaspase-3 and (C and D) active caspase-3. Negative controls (inserts). Black arrows indicate positive staining. Cell nuclei are stained with hematoxylin. Magnification, ×400. Johnson. NO actions on human endometrium survival. Fertil Steril 2004. Fertility and Sterility 2004 81, 176-184DOI: (10.1016/j.fertnstert.2003.05.018)

FIGURE 2 Expression of Bcl-2 and Bax mRNAs expression in human endometrial cells during culture: effect of L-arginine (L-Arg). Normalized yield of Bcl-2 and Bax PCR fragments relative to β-actin PCR products of cDNAs obtained from (A) isolated stromal and epithelial cells or (B) epithelial cell incubated in the absence (basal) or in the presence of L-Arg (1 mmol/L) with or without L-NMMA (1 mmol/L). The number of cycles for amplification was 31 for Bcl-2 and Bax genes, and 23 for β-actin. A linear relationship was observed between PCR products and amplification cycle when plotted (A, insert). The expression of eNOS mRNA was observed in isolated epithelial cells, and of iNOS mRNA in stromal and epithelial cells during cultures (B, insert). Results are given as mean ± SEM of five different cell cultures obtained from five women. *P<.05 vs. basal values. Johnson. NO actions on human endometrium survival. Fertil Steril 2004. Fertility and Sterility 2004 81, 176-184DOI: (10.1016/j.fertnstert.2003.05.018)

FIGURE 3 Apoptotic cell death evidenced by annexin V–FITC bound to human endometrial cell cultures: effect of L-arginine (L-Arg). Stromal and epithelial cells cultured in Hanks' media for 4, 10, and 24 hours in the (A) absence (basal) or (B) presence of L-Arg (1 mmol/L) for 24 hours. The cells were detached, incubated with annexin V–FITC, and counterstained with propidium iodide. Results are given as mean ± SEM of the percentage of apoptotic annexin V–FITC positive cells respect to total cells counted (a mean of 1,000 cells counted per culture) of four different cell cultures performed in duplicates obtained from four different women. #P<.05 vs. stromal cell cultures. *P<.05 vs. basal values. Johnson. NO actions on human endometrium survival. Fertil Steril 2004. Fertility and Sterility 2004 81, 176-184DOI: (10.1016/j.fertnstert.2003.05.018)

FIGURE 4 Effect of L-arginine (L-Arg) on human endometrial cell proliferation during culture. Subconfluent stromal and epithelial cells were incubated in 100 μl Hanks' media for 24 hours then incubated in the presence of L-Arg (1 mmol/L: 0 to 2 mmol/L in the insert) or N-monomethyl-L-arginine (L-NMMA: 1 mmol/L, added 15 minutes before to L-Arg treatment) for another 24 hours. For proliferation assessment, cells were incubated with assay reactive for another 2 hours at 37°C and measured. Values are given as mean ± SEM of five stromal and three epithelial cell cultures performed in quadruplicates obtained from five and three women, respectively (basal optical density/105 cells: 0.9 ± 0.1 and 1.6 ± 0.2, respectively). *P<.05 vs. basal values. Johnson. NO actions on human endometrium survival. Fertil Steril 2004. Fertility and Sterility 2004 81, 176-184DOI: (10.1016/j.fertnstert.2003.05.018)