Fig. 1: GnRH suppresses multiple beneficial functions of endometrial stem cells in vitro.

The inhibition of endometrial stem cell viability by 1 µM GnRH at 72 h was determined by an MTT assay. Stem cell viability (%) was calculated as a percent of the vehicle control (a). Endometrial stem cells were treated with GnRH for 72 h, and the effect of GnRH on stem cell migration ability was then evaluated using the transwell migration assay. GnRH treatment significantly decreased stem cell migration across the membrane compared with the negative control (b). The effects of GnRH on stem cell migration were further evaluated using a scratch assay. The migration of stem cells treated with GnRH was slower than that of cells treated with vehicle. (c). The relative expression levels of key positive regulators of cell migration (MMP-2/9) were assessed using western blotting (d). GnRH-induced actin filament disorganization and morphological changes in stem cells were visualized by staining actin filaments with phalloidin (e). Elevated levels of cleaved caspase-3 following GnRH treatment were assessed by western blotting. GnRH-induced apoptotic DNA fragmentation and condensation were visualized using DAPI staining (f). Confluent stem cells were cultured in osteogenic medium with or without GnRH. The effects of GnRH on osteoblast differentiation were determined by alizarin red staining. The relative quantification of calcium mineral content was performed by measuring the absorbance at 570 nm (g). Real-time PCR results showed the changes in the expression of the stem cell markers NANOG and OCT4 after GnRH treatment for 72 h (h). DAPI staining was used to label the nuclei. β-actin was used as the internal control. The data are presented as the mean ± SD of three independent experiments