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.© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction.
