Bovine sperm samples induce different NET phenotypes in a NADPH oxidase-, PAD4-, and Ca++-dependent process†

Area of Science:

• Reproductive immunology
• Innate immunity
• Bovine reproduction

Background:

• Artificial insemination in cattle involves sperm deposition, activating innate immune responses like neutrophil recruitment.
• Sperm-mediated neutrophil extracellular trap formation (NETosis) has been observed in various mammals, but its role in bovine reproduction is unclear.

Purpose of the Study:

• To investigate the interaction between bovine polymorphonuclear neutrophils (PMNs) and semen-derived samples.
• To analyze the molecular mechanisms of sperm-induced NETosis in cattle.
• To explore the potential implications of NETosis on bovine fertility.

Main Methods:

• Confrontation assays of bovine PMNs with sperm/cell preparations (SCP), purified sperm cells (SCs), and supernatant (SN).
• Immunostaining for histones (H3), neutrophil elastase (NE), and pentraxin (PTX) in NET structures.
• Analysis of PMN oxygen consumption and proton leak.
• Functional inhibition experiments targeting NADPH oxidase, peptidylarginine deiminase 4, and calcium influx.

Main Results:

• Bovine PMNs rapidly form NETs in response to SCP, effectively trapping spermatozoa in a dose-dependent manner.
• Fresh SCP induced higher NETosis than frozen-thawed samples; NETosis levels were independent of sperm viability.
• Sperm supernatant (SN) activated PMNs more effectively than purified sperm cells (SCs).
• Sperm-triggered NETosis is dependent on NADPH oxidase, peptidylarginine deiminase 4, and Ca++ influx, but not myeloperoxidase activity or ERK1/2/PI3K pathways.

Conclusions:

• Sperm-induced NETosis occurs in cattle and leads to spermatozoa entrapment.
• The process is molecularly defined and influenced by semen handling.
• Sperm-derived NETosis may play a role in modulating fertility during artificial insemination in cattle.