Deposition of sperm during artificial insemination in the bovine female reproductive tract results in early host innate immune reactions of polymorphonuclear neutrophils (PMN). Further, sperm-mediated neutrophil extracellular trap (NET) formation ("NETosis"), was recently reported to occur in different mammalian species, including humans. We here investigated interactions of bovine PMN with different semen-derived samples and analyzed in more depth molecular aspects of this effector mechanism. Overall, confrontation of PMN with sperm/cell preparation (SCP) resulted in a rapid and dose-dependent NET formation leading to effective spermatozoa entrapment. Thereby, spermatozoa induced different phenotypes of NETs. Immunostaining analyses revealed the presence of histones (H3), neutrophil elastase (NE) and pentraxin (PTX) in sperm-triggered NET structures. Fresh SCP stronger induced NETosis than frozen-thawed ones. The level of NETosis was not related to spermatozoa viability. SCP as well as purified sperm cells (SC) and supernatant (SN) induce NETosis, although the reaction in SC was lower. Enhanced levels of oxygen consumption and proton leak in PMN revealed sperm supernatants but not purified sperm cells as PMN activators. Functional inhibition experiments revealed sperm-triggered NETosis as a NADPH oxidase (NOX)- and peptidylarginine deiminase 4 (PAD4) -dependent process and proved to be dependent on intra- and extracellular Ca++ influxes whilst myeloperoxidase (MPO) activity and as ERK1/2- and PI3K-related signaling pathways did not seem to play a pivotal role in this effector mechanism. From these findings, we speculate that sperm-derived NETosis might also occur in vivo during artificial insemination and might therefore play a role related to reduced fertility.

中文翻译：

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牛精子样本在 NADPH 氧化酶、PAD4 和 Ca++ 依赖性过程中诱导不同的 NET 表型。

在牛雌性生殖道中人工授精过程中精子的沉积导致多形核中性粒细胞 (PMN) 的早期宿主先天免疫反应。此外，最近有报道称，精子介导的中性粒细胞胞外陷阱 (NET) 形成（“NETosis”）发生在不同的哺乳动物物种中，包括人类。我们在这里研究了牛 PMN 与不同精液来源样品的相互作用，并在更深入的分子方面分析了这种效应机制。总体而言，PMN 与精子/细胞制剂 (SCP) 的对抗导致快速且剂量依赖性的 NET 形成，从而导致有效的精子截留。因此，精子诱导了 NETs 的不同表型。免疫染色分析显示存在组蛋白 (H3)，中性粒细胞弹性蛋白酶 (NE) 和五聚体 (PTX) 在精子触发的 NET 结构中。新鲜的 SCP 诱导的 NETosis 比冻融的更强。NETosis 的水平与精子活力无关。SCP 以及纯化的精子细胞 (SC) 和上清液 (SN) 诱导 NETosis，尽管 SC 中的反应较低。PMN 中氧消耗和质子泄漏水平的提高揭示了精子上清液而不是纯化的精子细胞作为 PMN 激活剂。功能抑制实验显示精子触发的 NETosis 作为 NADPH 氧化酶 (NOX) 和肽基精氨酸脱亚胺酶 4 (PAD4) 依赖性过程，并证明依赖于细胞内和细胞外 Ca ++ 流入，同时髓过氧化物酶 (MPO) 活性和作为 ERK1/2-和 PI3K 相关的信号通路似乎在这种效应机制中没有发挥关键作用。从这些发现中，