Institute of Parasitology, Justus Liebig University Giessen, Giessen, Germany.
Chair for Molecular Reproductive Medicine, Clinic of Veterinary Obstetrics, Gynecology and Andrology, Justus Liebig University Giessen, Giessen, Germany.
Biol Reprod. 2020 Apr 15;102(4):902-914. doi: 10.1093/biolre/ioaa003.
Deposition of sperm during artificial insemination in the bovine female reproductive tract results in early host innate immune reactions of polymorphonuclear neutrophils (PMNs). Furthermore, sperm-mediated neutrophil extracellular trap (NET) formation (NETosis) was recently reported to occur in different mammalian species, including humans. We, here, investigated the 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 strongly induced NETosis than frozen-thawed ones. The level of NETosis was not related to spermatozoa viability. SCP as well as purified sperm cells (SCs) and supernatant (SN) induce NETosis, although the reaction in SC was lower. Enhanced levels of oxygen consumption and proton leak in PMN revealed sperm SNs but not purified SCs as PMN activators. Functional inhibition experiments revealed sperm-triggered NETosis as an NADPH oxidase- and peptidylarginine deiminase 4-dependent process and proved to be dependent on intra- and extracellular Ca++ influxes while myeloperoxidase 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.
在牛的雌性生殖道中进行人工授精时，精子的沉积会导致多形核粒细胞（PMN）发生早期宿主固有免疫反应。此外，最近有报道称，在包括人类在内的不同哺乳动物物种中，精子介导的中性粒细胞胞外诱捕网（NET）形成（NETosis）会发生。在这里，我们研究了牛 PMN 与不同精液衍生样本的相互作用，并更深入地分析了这种效应机制的分子方面。总的来说，PMN 与精子/细胞准备物（SCP）的对抗导致了快速和剂量依赖性的 NET 形成，从而有效地捕获精子。因此，精子诱导了不同表型的 NET。免疫染色分析显示，在精子触发的 NET 结构中存在组蛋白（H3）、中性粒细胞弹性蛋白酶（NE）和五聚素（PTX）。新鲜的 SCP 比冷冻解冻的 SCP 强烈诱导 NETosis。NETosis 的水平与精子活力无关。SCP 以及纯化的精子细胞（SCs）和上清液（SN）诱导 NETosis，尽管 SC 的反应较低。增强的 PMN 耗氧量和质子泄漏表明精子 SN 而不是纯化的 SC 作为 PMN 激活剂。功能抑制实验表明，精子触发的 NETosis 是 NADPH 氧化酶和肽基精氨酸脱亚氨酶 4 依赖性过程，并证明依赖于细胞内和细胞外 Ca++流入，而髓过氧化物酶活性以及 ERK1/2 和 PI3K 相关信号通路似乎在该效应机制中不起关键作用。根据这些发现，我们推测在人工授精过程中，精子衍生的 NETosis 也可能在体内发生，因此可能与降低生育能力有关。
