Fig 1: Effect of sperm-BOEC binding on TGFB1 production and the expression of anti- and pro-inflammatory cytokines and PTGES in BOECs.Viable sperm were co-cultured with BOEC monolayer for 3, 6, 12, or 24 h to detect the effect of sperm-BOEC binding. a) TGFB1 concentration in BOEC-conditioned media at 12 and 24 h by ELISA and its mRNA expression at different time points. b) mRNA expression of IL10, pro-inflammatory cytokines (TNF and IL1B), and PTGES in BOECs, as measured by real-time PCR. Results are presented as the mean ± SEM of five independent experiments performed in triplicate. All mRNA expression levels were normalized to ß-actin. Asterisks denote statistical differences: * P < 0.05, ** P < 0.01, *** P < 0.001, when compared to the control at the same time point and determined using a Two-Way ANOVA followed by the Bonferroni's post hoc test.
Fig 2: Immunohistochemical localization of IL10 and TGFB1 in bovine oviduct.Representative photos for immunohistochemical labelling of IL10 (A-D) and TGFB1 (E-H) in paraffin wax sections of the ampulla (A, B, E, F) and isthmus (C, D, G, H) of the bovine oviduct. Low magnification images (A, C, E, G) show a clear co-localization of both cytokines in epithelial cells (brown stain of luminal cell layer) and vessel walls of large blood vessels (arrowheads), while other tissue components are negative. High magnification images (B, D, F, H) reveal a cytoplasmic epithelial immunoreaction (ep) and staining of stromal endothelial cells (arrowheads). Besides, in the isthmus both proteins are expressed by smooth muscle cells of the surrounding prominent muscle layer (asterisks in C and G). Occasionally occurring leukocytes are positive for both cytokines (arrow in D). Insets to B and D show representative negative control sections. Scale bars represent 200 µm in A, C, E, G and 50 µm in B, D, F, H.
Fig 3: Schematic illustration to show the local oviductal immunity after sperm binding to BOECs.Based on the present results, sperm-BOEC binding could regulate the oviduct immunological environment under physiological conditions. (A) BOECs strongly express TGFB1 and IL10. Basal PGE2 levels in oviductal cells downregulate pro-inflammatory cytokines (TNF and IL1B). (B) Sperm-BOEC binding enhances the production of PGE2 and stimulates anti-inflammatory cytokines (TGFB1 and IL10), which strengthens the anti-inflammatory environment needed for sperm protection and survival. PGE2 secreted from BOECs decreases TNF expression in PMNs; furthermore, the sperm-BOEC binding induces further secretion of PGE2 in BOECs. PGE2 production results in the up-regulation of TGFB1 and IL10, which may contribute to supply a stable anti-inflammatory environment in the oviduct.
Fig 4: Changing of PMNs gene expression after culturing in BOEC-conditioned media.Cytokine gene expression (TGFB1, IL10, TNF, and PTGES) in PMNs incubated for 4 h with BOEC-conditioned media (with and without sperm co-culture). Results are presented as the mean ± SEM of three independent experiments performed in duplicate. All expression levels were normalized to ß actin. Asterisks denote statistical differences: * P < 0.05, ** P < 0.01, *** P < 0.001, when compared to the control, as determined using a One-Way ANOVA followed by Bonferroni's post hoc test.
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