Fig 1.
A, Immunofluorescence staining of the biomarkers of fetal membrane cells and PGRMC1. Above 95% of amnion and chorion cells stained positive for their biomarker cytokeratin (green). Amnion and chorion cells appear to be epithelial cell phenotype. Above 95% of decidua cells stained positive for their biomarker vimentin (green). Decidua cells demonstrated typical stromal cell phenotype. All cells stained positive for PGRMC1 (red). PGRMC1 protein in amnion cells is mainly localized to the cell membrane and cytoplasm. In contrast, the expression of PGRMC1 protein in chorion and decidua cells is mainly localized to nuclear and perinuclear space. DAPI counterstaining was performed to visualize nuclei (blue). Images of cells were captured using digital camera interfaced with a fluorescence microscope. Magnification is 40. B, PGRMC1 mRNA and protein knock-down in amnion (A) and chorion cells (C). PsiRNA is for PGRMC1 siRNA transfected group; CsiRNA is for control siRNA transfected group. qPCR results consistently showed about 70% knockdown of PGRMC1 mRNA production (PsiRNA vs CsiRNA, P<0.0001 for amnion cells, P<0.0001 for chorion cells, n = 5). Representative Western blotting and densitometry data are presented. Western blotting consistently showed above 50% knockdown of PGRMC1 protein production (PsiRNA vs CsiRNA, P = 0.0007 for amnion cells, P = 0.01 for chorion cells, n = 5).
Fig 2.
Adherence of Ureaplasma parvum (UPA) serotype 3 to fetal membrane cells (amnion, chorion and decidua).
The percentage of adherence of UPA to each cell type increased as the MOI increased. At the maximal MOI, UPA had a significant higher adherence in primary amnion and chorion cells compared with decidua cells. (n = 5).
Fig 3.
The effects of P4 pretreatment or PGRMC1 knockdown on IL-8 mRNA and IL-8 protein secretion induced by U. parvum infection in amnion and chorion cells.
A, B, C, D, IL-8 mRNA expression and protein secretion were significantly up-regulated following U.parvum stimulation (UPA) compared to no U. parvum treatment control (N) in amnion and chorion cells (CsiRNA). A, No significant differences were observed for U. parvum induced IL-8 mRNA expression between CsiRNA and PsiRNA in amnion cells. B, but IL-8 protein secretion induced by U. parvum exposure appears decreased in PsiRNA. C, D, No significant differences were observed for U. parvum induced IL-8 mRNA expression and protein secretion when PGRMC1 was knocked down (PsiRNA) in comparison to CsiRNA in chorion cells. A, B, C, D, P4 pretreatment followed by U. parvum treatments (P4+UPA) partially inhibited the U. parvum induced up-regulation of IL-8 mRNA expression in chorion cells and had no effects on IL-8 mRNA and protein secretion compared to vehicle control group (E+UPA) in amnion cells. This P4 effect was not changed in PsiRNA cells compared with CsiRNA cells.
Fig 4.
The effects of P4 pretreatment or PGRMC1 knockdown on COX-2 mRNA expression and PGE2 protein secretion induced by U. parvum infection in amnion and chorion cells.
In amnion cells, A, B, significant up-regulation of COX-2 mRNA expression and PGE2 secretion induced by U. parvum was observed (N vs UPA). No significant differences of this up-regulation were observed between PsiRNA and CsiRNA groups. A, B, P4 pretreatment did not affect the U. parvum induced up-regulation of COX-2 mRNA level and PGE2 secretion compared to ethanol pretreatment (E+UPA vs P4+UPA). P4 effect was not different between PsiRNA and CsiRNA groups. D, similar trend was also observed for PGE2 protein secretion in chorion cells. C, the up-regulation of COX-2 mRNA expression by U. parvum exposure was significantly enhanced in PsiRNA cells in chorion cells. P4 pretreatment partially attenuated the U. parvum induced up-regulation of COX-2 mRNA expression compared to ethanol pretreatment (CsiRNA). The differences between E+UPA and P4+UPA disappeared in PsiRNA cells. The differences in the P4 effects between the CsiRNA and PsiRNA groups are statistically significant.
Fig 5.
The effects of P4 pretreatment or PGRMC1 knockdown on MMP9 mRNA level and MMP9 activity induced by U. parvum infection in amnion and chorion cells.
In amnion cells, A and B, U. parvum exposure significantly increased MMP9 mRNA expression and activity. The induction of MMP9 activity but not MMP9 mRNA was affected by PGRMC1 knock-down. The induction of MMP9 mRNA and activity by U. parvum exposure was not influenced by P4 pretreatment. In chorion cells, C, MMP9 mRNA expression was not significantly induced by U. parvum exposure in CsiRNA cells while it was significantly induced in PsiRNA cells. P4 pretreatment appeared to partially inhibit induced MMP9 mRNA expression by U. parvum exposure in CsiRNA cells. This effect of P4 was disappeared in PsiRNA cells. The differences of UPA vs N or (E+UPA) vs (P4+UPA) between CsiRNA and PsiRNA were statistically significant. D, MMP9 activity was significantly induced by U. parvum exposure in both CsiRNA and PsiRNA chorion cells, and this induction was not affected by either PGRMC1 knockdown or P4 pretreatment.