Endothelin-1 downregulates sperm phagocytosis by neutrophils in vitro: A physiological implication in bovine oviduct immunity

Abstract

The oviduct is an active contractile tube that provides the proper environment for sperm transport, capacitation and survival. Oviductal contractions are regulated by autocrine/paracrine secretion of several factors, such as prostaglandins (PGs) and endothelin-1 (EDN-1). We have previously shown that during the preovulatory stage, sperm are exposed to polymorphonuclear neutrophils (PMNs) in the bovine oviduct, and the bovine oviduct epithelial cells (BOECs) secrete molecules including PGE2 that suppress sperm phagocytosis by PMNs in vitro. In this study, we investigated the possible effects of EDN-1 on the phagocytic activity of PMNs toward sperm. The local concentrations of EDN-1 in oviduct fluid and BOEC culture medium ranged from 10(-10) to 10(-11) M as determined by EIA. Phagocytosis and superoxide production were assayed by co-incubation of sperm pretreated to induce capacitation with PMNs exposed to EDN-1 (0, 10(-11), 10(-10), 10(-9), and 10(-8) M) for 2 h. EDN-1 suppressed dose dependently (10(-11) to 10(-8) M) the phagocytic activity for sperm and superoxide production of PMNs in response to capacitated sperm. Moreover, this suppression was eliminated by an ETB receptor antagonist (BQ-788). EDN-1 suppressed mRNA expression of EDN-1 and ETB but not ETA receptors in PMNs, suggesting the ETB receptor-mediated pathway. Scanning electron microscopic observation revealed that incubation of PMNs with EDN-1 (10(-9) M) completely suppressed the formation of DNA-based neutrophil extracellular traps for sperm entanglement. The results provide evidence indicating that EDN-1 may be involved in the protection of sperm from phagocytosis by PMNs in the bovine oviduct, supporting sperm survival until fertilization.

Fig. 1.

(a) EDN-1 concentrations (pg/ml) in oviduct fluid during estrous cycle. Pre, preovulatory phase (n = 6); post, postovulatory phase (n = 6); and mid, mid-luteal phase (n = 5). (b) EDN-1 concentrations (pg/ml) in BOEC culture stimulated with 10 ng/ml LH (LH-BOEC) or without any stimulant (BOEC). Numerical values are presented as the mean ± SEM of 4–6 experiments. The letters a and b indicate significant differences between the treatments at P < 0.05 as determined by ANOVA followed by Bonferroni’s multiple comparison test or the t-test.

Fig. 2.

(a) Dose-dependent effect EDN-1 on percentage of in vitro phagocytosis for sperm treated to induce capacitation by polymorphonuclear neutrophils (PMNs). PMNs were incubated for 2 h in a culture medium supplemented with different concentrations of EDN-1 (10^–11 M = 24.9 pg/ml, 10^–10, 10^–9 and 10^–8 M) before a phagocytosis assay (100% = 48.4 ± 3.5, means ± SEM). (b) Dose-dependent effect of EDN-1 on superoxide generation by PMNs for sperm. PMNs were incubated for 2 h in a culture medium supplemented with different concentrations of EDN-1 (10^–11 M = 24.9 pg/ml, 10^–10, 10^–9 and 10^–8 M) before a superoxide assay (100% = 56747.5 ± 5731, means ± SEM). (c) The effect of receptor antagonists for EDN-1 on PMN phagocytosis for sperm treated to induce capacitation in vitro. PMNs were incubated for 2 h with EDN-1 (10^–9 M) together with an ET_A receptor antagonist (LU135252) (10^–7 M) or ET_B receptor antagonist (BQ-788) (10^–7 M) and then phagocytosis was assayed (100% = 50.2 ± 4.1, means ± SEM). Numerical values are presented as the mean ± SEM of 4–5 experiments. The letters a, b and c, indicate significant differences between the treatments at P < 0.05 as determined by ANOVA followed by Bonferroni’s multiple comparison test.

Fig. 3.

Relative mRNA expression of EDN-1 and the ET_A and ET_B receptors in the bovine PMNs stimulated with EDN-1 (0, 10^–11, and 10^–10 M) and harvested after 2 h. Numerical values are presented as the mean ± SEM of 5 experiments. Different letters indicate significant differences between the treatments at P < 0.05 as determined by ANOVA followed by Bonferroni’s multiple comparison test.

Fig. 4.

Effect of combinations of EDN-1 and PGE2 on phagocytic activity for sperm and superoxide production by PMNs in response to sperm. PMNs were incubated for 2 h in a culture medium supplemented with EDN-1 (10^–10 M), and PGE2 (10^–7 M) at concentrations detected in BOEC culture medium and then phagocytosis (a) and superoxide generation (b) were assayed. Numerical values are presented as the mean ± SEM of 4 experiments. Different letters indicate significant differences between the treatments at P < 0.05 as determined by ANOVA followed by Bonferroni’s multiple comparison test.

Fig. 5.

Scanning electron microscopic observation for sperm phagocytosis by polymorphonuclear neutrophils (PMNs). PMNs were incubated with culture medium supplemented with EDN-1 (10^–9 M) for 2 h and then subjected to a 1 h phagocytosis assay. In the phagocytosis assay, addition of sperm to PMNs induced neutrophil extracellular trap (NET) formation (b) compared with that without sperm addition (a). Incubation of PMNs with EDN-1 (10^–9 M) prior to the phagocytosis assay resulted in complete suppression of NET formation by PMNs for sperm entanglement (c).