Table 1.
List of primers used for real-time PCR.
Table 2.
List of antibodies used for Western blotting.
Fig 1.
Bovine endometrium expressed TLR2 and TLR4 proteins.
Representative sections for luminal epithelium and stratum compactum (A, C, E, G), as well as stratum reticulare with endometrial glands (B, D, F, H) and blood vessels (BV) are shown. TLR2 is localized in luminal (LE) and glandular epithelium (UG) as well as a few immune cells (arrows) during follicular phase (FP) and luteal phase (LP). TLR4 is found in the same cells and additionally in stromal cells of the stratum compactum (asterisks) in some areas. Scale bars, 20 μm from A to H. Insets to A and G show representative controls for TLR2 and TLR4, respectively with scale bars: 40 μm.
Fig 2.
TLR2/4 activation induced transcription of the pro-inflammatory genes in BEECs.
Sub-confluent bovine endometrial epithelial cells (BEECs) monolayers were exposed to A) TLR2 agonist (Pam; 0, 10, 100, and 1000 ng/ml) or B) TLR4 agonist (LPS; 0, 0.1, 1, and 10 ng/ml) for 0, 1, 3, or 6 h. At each time point, mRNA expressions of TNFA, IL-1B, IL-8, and PGES in BEECs were quantified by a real-time PCR. Data are presented as mean ± SEM of 4 independent experiments using epithelial cells from 4 different uteri (3 wells per treatment per experiment). Asterisks denote a significant variance [* (P < 0.05), ** (P < 0.01), *** (P < 0.001)] between the different doses of Pam/LPS when compared to the control group (0 ng/ml) at each time point. Different small letters denote a significant variance (P < 0.05) between the different time points of 100 ng/ml Pam (A) or 1 ng/ml LPS (B). Different capital letters denote a significant difference (P < 0.05) between the different time points of 1000 ng/ml Pam (A) or 10 ng/ml LPS (B).
Fig 3.
TLR2/4 blocker inhibited the inflammatory response of BEECs towards TLR2/4 agonist.
Sub-confluent BEECs monolayers were 2 h incubated with A) TLR2 antagonist (0 μM, 0.05 μM = 18.12 ng/ml, and 0.1 μM = 36.24 ng/ml, and 0.5 μM = 181.20 ng/ml) prior to stimulation with Pam (100 ng/ml) for 1 h or B) TLR4 antibody (0, 10, 100, and 1000 ng/ml) prior to stimulation with LPS (1 ng/ml) for 3 h. TNFA mRNA expression was then quantified in BEECs. Data are presented as mean ± SEM of 3 independent experiments using epithelial cells from 3 different uteri (3 wells per treatment per experiment).
Fig 4.
TLR2/4 blocker prevented the stimulatory effect of sperm on the transcription of pro-inflammatory genes in BEECs.
Sub-confluent BEECs monolayers were incubated for 2 h with A) 0.1 μM TLR2 antagonist or B) 100 ng/ml TLR4 antibody, then stimulated with 106/ml sperm for 3 h. mRNA expressions of TNFA, IL-1B, IL-8, and PGES were then quantified in BEECs. Data are presented as mean ± SEM of 4 independent experiments using epithelial cells from 4 different uteri (3 wells per treatment per experiment). Different letters denote a significant variance (P< 0.05) between the different groups.
Fig 5.
Direct effect of TLR2/4 blocker on sperm motility.
Sperm cells at 106/ml Sp-TALP were exposed to either 0.1 μM TLR2 antagonist or 100 ng/ml TLR4 antibody for 0, 30, 60, and 120 min. At each time point, progressive motility of recovered sperm from both groups was assessed and compared with those of the control group (without either TLR2 antagonist or TLR4 antibody). Data are presented as mean ± SEM of 3 independent experiments using epithelial cells from 3 different uteri (3 wells per treatment per experiment).
Fig 6.
TLR2/4 blocker inhibited TLR signal transduction pathways in BEECs in response to sperm.
Sub-confluent BEECs monolayers were incubated for 2 h with 0.1 μM TLR2 antagonist or 100 ng/ml TLR4 antibody, then stimulated with 106/ml sperm for 1 h. Western blotting was then carried out to estimate the phosphorylation levels of A) p38MAPK, B) JNK, C) ERK1/2, D) NFkB, and E) IRF3 in the different groups. Data are presented as mean ± SEM of 3 independent experiments using epithelial cells from 3 different uteri (3 wells per treatment per experiment). Asterisk (*) denotes a significant variance (P< 0.05) between TLR2/4 blocking groups when compared with sperm group.
Fig 7.
A diagrammatic illustration of the mechanism by which sperm activate TLR2/4 signaling pathway in BEECs in vitro.
The proposed working model showing that stimulation of BEECs with sperm activates MAPKs components (p38MAPK and JNK; solid lines) without any effect on other downstream targets of TLR2/4 signaling pathways (ERK1/2, NFKB or IRF3; dotted line) down to the nuclear translocation of AP-1 protein which in turn regulates the transcription of pro-inflammatory genes in BEECs. “?” refers to unknown endogenous ligand(s) which may link sperm to TLR2/4 signaling pathway in BEECs.