Imiquimod (R837), a TLR7-Specific Agonist, Regulates Boar Sperm Motility via PI3K/GSK3α/β/Hexokinase Pathway

Mammalian sperm has a high metabolic activity and primarily relies on glycolysis and mitochondrial oxidative phosphorylation for energy supply. It has been reported that imiquimod (R837), a specific ligand of TLR7 protein, reduces the motility of sperm. However, the specific molecular mechanism by which TLR7 modulates boar sperm is unclear. In this study, the effect of R837, a Toll-like receptor 7 (TLR7) agonist, on boar sperm motility was investigated. Sperm samples were incubated with varying concentrations of R837 (0 to 0.8 µM) at different time points (30, 60, and 90 min). Findings reveal for the first time that TLR7 protein, a key component of the immune system's Toll-like receptors, is predominantly localized in the middle section of the boar sperm tail, with a smaller concentration observed in the neck. Also, immunofluorescence (IF) revealed that approximately half of the boar sperm sample expressed TLR7. Furthermore, the TLR7 agonist influenced glycolytic hexokinase activity and mitochondrial function via the PI3K-GSK3β signaling pathway. It also selectively inhibited motility in the lower-layer sperm, while motility in the upper-layer sperm remained unaffected. Additionally, this study determined that incubation conditions for boar sperm with 0.2 μM R837 at 37 °C for 60 min yielded the most pronounced inhibition of forward motility in the lower layer of sperm, without compromising the integrity of the acrosome or plasma membrane. The present study reveals the crucial role of R837 in boar sperm motility and highlights TLR7 as an important protein that regulates boar sperm energy metabolism.