الفهرس 
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Abstract
Insemination induces an inflammatory reaction in the bovine uterus, but which component of semen can motivate such reaction is still questionable. Therefore, we aimed in the current study to investigate whether sperm could modulate the bovine uterine local innate immunity towards the production of a pro-inflammatory response. In addition, the mechanism whereby the endometrial cells could differentially recognize and respond to physiological vs. pathophysiological allogens occupied a great space in our objectives as follows: First: Sub-confluent BUECs monolayers were co-cultured 104, 105, or 106 sperm/ml for 0, 1, 3, and 6 h. The mRNA expressions of TNFA, IL-1B, IL-8, and PGES were then quantified using a real-time PCR to assess the extent of the inflammatory response in BUECs at each time point. Sperm at 106 /ml upregulated mRNA expression of pro-inflammatory genes in BUECs with a maximum upregulation at 3 h post-incubation. Second: Uterine tissue samples from cows in follicular and luteal phases were examined for TLR2 and TLR4 localization using immunohistochemistry. TLR2 and TLR4 proteins are constantly expressed in the luminal and glandular epithelia of bovine endometrium throughout the estrous cycle. Third: Sub-confluent BUECs monolayers were treated with TLR2 agonist (Pam; 0, 10, 100, or 1000 ng/ml) or TLR4 agonist (LPS; 0, 0.1, 1, or 10 ng/ml) for 0, 1, 3, and 6 h. The mRNA expressions of TNFA, IL-1B, IL-8, and PGES were then quantified using a real-time PCR to assess the extent of the inflammatory response in BUECs at each time point. Both Pam and LPS induced inflammatory responses in BUECs, but with different patterns. Pam-induced inflammatory response peaked at 1 h post-incubation, while LPS-induced one was a timely progressing. Fourth: Sub-confluent BUECs monolayers were pre-incubated (2 h) with TLR2 antagonist (0.05 or 0.1 µM) or TLR4 antibody (100 ng/ml), then stimulated with sperm (106/ml) for 3 h. After that, mRNA expressions of TNFA, IL-1B, IL-8, and PGES were quantified using a real-time PCR to assess the extent of the inflammatory response in BUECs. TLR2/4 blocker abrogated the stimulatory effect of sperm on the transcription of pro-inflammatory genes in BUECs. Fifth: Sub-confluent BUECs monolayers were stimulated with sperm (106/ml), Pam (100 ng/ml) or LPS (1 ng/ml) for 1 or 3 h. Afterwards, Western blotting was carried out at each time point to estimate the phosphorylation levels of p38MAPK, JNK, ERK1/2, NFkB, and IRF3 as downstream targets of TLR2/4 activation in BUECs. Sperm increased the phosphorylation levels of p38MAPK and JNK, while phospho-NFkB and phosho-ERK1/2 were enhanced by Pam and LPS, respectively. Sixth: Sub-confluent BUECs monolayers were pre-incubated (2 h) with TLR2 antagonist (0.1 µM) or TLR4 antibody (100 ng/ml), then stimulated with sperm (106/ml) for 1 h. Afterwards, Western blotting was carried out to estimate the phosphorylation levels of p38MAPK and JNK as downstream targets of TLR2/4 activation in sperm-triggered BUECs. TLR2/4 blocker inhibited TLR signal transduction pathways in BUECs in response to sperm. In conclusion, our data clearly provide the first evidence that upon the contact of semen with the uterus during AI in cows, sperm can modulate the local innate immunity towards induction of a rapid and transient pro-inflammatory reaction in the endometrial cells with a subsequent recruitment of PMNs to the uterine lumen for removal of excess/dead sperm with associated contaminants and thus preventing immunization against sperm. Furthermore, the current study opens new insights into the molecular mechanism whereby sperm can initiate a crosstalk with maternal side at the uterine level. TLRs, definitely TLR2/4, activation pathway is a key regulator of sperm-uterine interaction. Importantly, our findings detect the potential differences in the immunological responsiveness of uterine epithelial cells to physiological (sperm derived from semen during AI) vs. pathophysiological (TLR2/4 ligands derived from G+ve/-ve bacteria during infection) allogens. Altogether, the present work points out that the bovine uterus is an immunological competent organ supported with a highly specialized local immune system which has the capacity to differentially recognize and therefore efficiently respond to array of foreign entities to which it is exposed all the time.