A proinflammatory response of bovine endometrial epithelial cells to active sperm in vitro

In the cow, cryopreserved semen is inseminated into the uterus, and most of sperm are removed by backflow and phagocytes. Nevertheless, the mechanism responsible for sperm phagocytosis is unclear. Here, we used cultured bovine uterine epithelial cells (BUECs) to investigate the uterine response to sperm and the mechanism that activates polymorphonuclear neutrophils (PMNs). BUEC monolayers were co-cultured with different numbers of washed sperm obtained from cryopreserved semen (10(4), 10(5), and 10(6) sperm/ml) for 3hr. Sperm dose-dependently up-regulated IL8 (Interleukin 8). Sperm at 10(6)/ml increased mRNA expression of TNFA (Tumor necrosis factor alpha), IL1B (Interleukin 1B), NFKB2 (Nuclear factor kappa B2), and C3 (Complement factor 3), as well as PGES (Prostaglandin E synthase) expression and PGE(2) release. Live sperm, but not dead sperm, attached to BUECs, and dead sperm did not induce an acute inflammatory response. Time-dependent effects were evaluated by co-culture of 10(6)/ml washed sperm with BUECs for 0, 1, 3, and 6hr. The number of detached sperm increased gradually toward 6hr. Maximum mRNA expression of IL8, TNFA, IL1B, and NFKB2 was induced at 3hr, while C3 continued to increase toward 6hr. Sperm did not stimulate mRNA expression of anti-inflammatory cytokines TGFB1 (Transforming growth factor beta 1) or IL10 (Interleukin 10). Medium conditioned by sperm co-incubated with BUECs stimulated PMNs phagocytosis of sperm in vitro. Fresh media supplemented with low levels of IL1B, TNFA, and PGE(2) up-regulated sperm phagocytosis by PMNs as well. In conclusion, our findings strongly suggest that the active sperm attach to BUECs and trigger uterine local innate immunity with induction of a pro-inflammatory response that enhances sperm phagocytosis by PMNs.