Sperma-induzierte Bildung neutrophiler extrazellulärer Traps (NETs) im bovinen System
Buch in deiner Nähe kaufen
Beschreibung
During artificial insemination in cattle, sperm cells as well as seminal plasma and diluent/extender are transferred into the uterus, a location where they are not found naturally. The deposition of sperm during (artificial) insemination results in an early host innate immune reaction mainly based on polymorphonuclear neutrophils (PMN). One major effector mechanism is the formation of neutrophil extracellular traps (NETs, NETosis), a spiderweb-like structure to entrap pathogens. NETosis was recently reported to be induced by spermatozoa in humans and cattle.
In this work, we investigated interactions of bovine PMN with different semen-derived samples in vitro and analyzed the molecular aspects of this effector mechanism. Confronting of PMN with sperm cell preparations resulted in a rapid and dose-dependent NET formation. Microscopic analyses revealed different phenotypes of NETs (spread, aggregated and diffuse NETs) and the presence of characteristic components of NET structures, such as histones, neutrophil elastase (NE) and pentraxin. Functional inhibition experiments revealed sperm-triggered NETosis as a NADPH oxidase (NOX)- and peptidylarginine deiminase 4 (PAD4)-dependent process and proved it to be dependent on intra- and extracellular Ca++ influxes whilst myeloperoxidase (MPO) activity and ERK1/2- and PI3K-related signaling pathways did not seem to play a pivotal role in this effector mechanism.
As an interesting finding, all sperm-derived fractions, i. e. purified sperm cells as well as sperm cell preparations or supernatant in principle, induced NETosis, although purified sperm cells had the lowest effects. Enhanced levels of oxygen consumption and proton leak in PMN revealed sperm supernatants but not purified sperm cells as PMN activators. Fresh sperm cell preparations induced NETosis stronger than frozen-thawed ones. The level of NETosis was not related to spermatozoa viability.
Furthermore, we investigated interactions between bovine PMN and different semen extenders, various seminal plasma concentrations from young and old bulls as well as sexed and non-sexed semen. We compared three different semen extenders from two companies in fresh and frozen-thawed conditions. The reaction differed between the extenders of the two companies, and egg yolk was not per se the strongest NET inducer. Sperm-PMN binding also depended on the presence of seminal plasma since seminal plasma alone, even at low concentrations of 1 %, induced NETosis. Seminal plasma from young bulls led to significantly higher NET induction than that of old ones. We observed no difference between non-sex-sorted and sex-sorted sperm and its extenders.
In summary, we demonstrated that bovine PMN confronted with sperm cells form NETs in vitro. Overall, bovine sperm-induced NETosis follows classical signaling pathways. Surprisingly, components of insemination doses other than sperm cells also triggered the formation of NETs. These findings indicate that sperm-derived NETosis might occur in vivo during artificial insemination and may therefore play a role in reduced fertility. Consequently, more effort is needed to evaluate the role of bovine sperm-induced NETosis in vivo.
Autor*in
Theresa Fichtner
Themen in »Sperma-induzierte Bildung neutrophiler extrazellulärer Traps (NETs) im bovinen System«
Doktorarbeit Genetik Tiermedizin