Adverse Effects of Single Neutrophil Extracellular Trap-Derived Components on Bovine Sperm Function

Abstract

Neutrophil extracellular traps (NETs) play a key role in fertilisation by eliminating microorganisms and entrapping spermatozoa in the female reproductive tract (FRT). The deleterious effects of NETs on spermatozoa have been previously described; however, individual exposure to NET-derived components in bull spermatozoa has not been explored. The aim of this study was to evaluate the effects of the main NET-derived proteins, histone 2A (H2A), neutrophil elastase (ELA), myeloperoxidase (MPO), pentraxin 3 (PTX), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), at concentrations of 1, 10, and 30 μg/mL, on sperm parameters. Sperm were selected and incubated with different NET-derived proteins for 4 h. Membrane and acrosome integrity, lipoperoxidation, and membrane phospholipid disorders were also evaluated. Bovine polymorphonuclear neutrophil (PMN)/sperm co-cultures were evaluated by scanning electron microscopy and immunofluorescence. All NET-derived proteins/enzymes resulted in a reduction in membrane integrity, acrosome integrity, and lipoperoxidation at a concentration of 30 μg/mL. Bovine PMN/sperm co-cultures showed marked NET formation in the second hour. In conclusion, all NET-derived proteins/enzymes exerted cytotoxic effects on bull sperm, and this effect should be considered in future investigations on the uterine microenvironment and the advancement of spermatozoa in the FRT.

Extrusion of Neutrophil Extracellular Traps (NETs) Negatively Impacts Canine Sperm Functions: Implications in Reproductive Failure

“…In this regard, very little is known about sperm-derived factors that promote NETotic cell induction and NET formation, while some studies suggest that it is the seminal plasma and not the sperm that triggers NETosis [ 31 ]. Conversely, other work suggests that it is an inherent property of sperm to induce this process, which is consistent with our observations [ 32 , 33 ]. The canine sperm in our model favors the formation of NETs, and it is still not entirely clear whether sperm motility would be a factor favoring PMN-derived NETosis.…”

“…The results of this study demonstrated the individual damage induced by various NET proteins, including MPO, which is found in high concentrations in extracellular networks [ 9 , 38 ]. This membrane damage catalyzes the formation of a significant number of oxidative species [ 39 ], which can lead to further membrane lipid peroxidation in spermatozoa [ 32 ]. Similarly, our study demonstrated that MPO caused severe damage to the canine sperm membrane, where the acrosome was also affected.…”

“…In addition, membrane damage in the presence of NET-derived MPO may be consistent with previous findings showing a progressive decrease in the motility of porcine sperm [ 19 ]. In addition, Cat G, one of the major serine proteases released by activated PMNs, is another protein associated with lipid peroxidation damage at the sperm level [ 32 ] and plays a critical role in inflammation through the hydrolysis of many other proteins [ 40 ]. Consequently, it was recently demonstrated that Cat G had detrimental effects on both sperm membrane and acrosome integrity in exposed bovine sperm [ 32 ].…”

“…In addition, Cat G, one of the major serine proteases released by activated PMNs, is another protein associated with lipid peroxidation damage at the sperm level [ 32 ] and plays a critical role in inflammation through the hydrolysis of many other proteins [ 40 ]. Consequently, it was recently demonstrated that Cat G had detrimental effects on both sperm membrane and acrosome integrity in exposed bovine sperm [ 32 ]. NE has been involved in sperm-induced NET release in livestock such as cattle and pigs, which has also been shown to be associated with decreased motility and viability [ 32 ].…”

“…Consequently, it was recently demonstrated that Cat G had detrimental effects on both sperm membrane and acrosome integrity in exposed bovine sperm [ 32 ]. NE has been involved in sperm-induced NET release in livestock such as cattle and pigs, which has also been shown to be associated with decreased motility and viability [ 32 ]. Furthermore, the presence of NE was associated with loss of sperm membrane functions, and these findings are consistent with our observations.…”