Abstract
We have recently shown that sperm attachment to bovine endometrial epithelial cells (BEECs) triggers uterine local innate immunity with induction of a pro-inflammatory response in vitro, however details of the mechanism remain unknown. Here, we investigated the involvement of Toll-like receptor 2/4 (TLR2/4) pathway in mediating sperm-BEECs inflammatory process. Immunohistochemistry of the uterine tissue revealed that TLR2 and TLR4 proteins were present in the luminal and glandular epithelia of bovine endometrium. Moreover, BEECs monolayers were treated with TLR2 agonist (Pam; 0, 10, 100, and 1000 ng/ml) or TLR4 agonist (LPS; 0, 0.1, 1, and 10 ng/ml) for 0, 1, 3, or 6 h, followed by evaluating mRNA expression of the pro-inflammatory genes (TNFA, IL-1B, IL-8, and PGES) in BEECs using a real-time PCR. Both Pam and LPS treatments showed a dose-dependent stimulation of mRNA expression of the pro-inflammatory genes. To elucidate the functional role of TLR2/4 in sperm-BEECs interaction, BEECs monolayers were incubated with either TLR2 antagonist or TLR4 antibody for 2 h prior to the co-culture with sperm for 3 h. Importantly, pre-incubation of BEECs with TLR2 antagonist or TLR4 antibody prevented the stimulatory effect of sperm on the transcription of pro-inflammatory genes in BEECs. Furthermore, sperm increased the phosphorylation levels of TLR2/4 downstream targets (p38MAPK and JNK) in BEECs within 1 h of the co-culture. Treatment of BEECs with TLR2 antagonist prior to sperm addition inhibited JNK phosphorylation, while TLR4 antibody inhibited the phosphorylation of both p38MAPK and JNK. In conclusion, the present in vitro findings strongly suggest that bovine endometrial epithelial cells respond to sperm via TLR2/4 signal transduction.
Highlights
Millions of sperm are deposited into the bovine uterus during artificial insemination (AI), most of these sperm are eliminated and only a few thousands reach to the oviduct where fertilization takes place [1]
Toll-like receptor 2/4 (TLR2/4) blocker inhibited the inflammatory response of bovine endometrial epithelial cells (BEECs) towards TLR2/4 agonist
We found that 0.1 μM of TLR2 antagonist or 100 ng/ml of TLR4 antibody was the minimal concentration that could inhibit the mRNA expression of TNFA in BECs in response to Pam or LPS, respectively (Fig 3A and 3B)
Summary
Millions of sperm are deposited into the bovine uterus during artificial insemination (AI), most of these sperm are eliminated and only a few thousands reach to the oviduct where fertilization takes place [1]. The deposited sperm is considered antigenic to the uterine innate immune system and so induce an inflammatory response. This inflammatory response is characterized by a rapid and transient leukocytic infiltration, mostly polymorphonuclear cells (PMNs), into the uterine lumen which removes dead or in excess sperm cells [2]. The uterus and in particular the endometrium orchestrates many pivotal functions such as the conceptus implantation, placentation, and maintenance of pregnancy until normal parturition [3]. With these reproductive functions, the uterus requires a defense system against invading pathogens as well as tolerating allogenic sperm and semi-allogenic embryo. The uterine milieu should be equipped with a well-developed and strictly controlled immune system that can respond effectively to various antigens to which it is exposed [4]
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