Activation of sperm toll-like receptor 2 induces the mucus penetrating ability and subsequent sperm-induced uterine gland inflammatory response in cattle

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[Introduction] We previously reported that TLR2 is localized in the posterior segment of the bull sperm head and regulates sperm sensing with TLR2 in the uterine glands to trigger the immune responses in the endometrium using a sperm-endometrial ex vivo model (SRD, 2020). Now we evaluate the impact of activation of sperm TLR2 in mucus penetrating ability and subsequent immune crosstalk with the endometrium. [Materials and methods] Frozen-thawed swim-up bull sperm were treated with TLR1/2 agonist (TLR2 ago.), antagonist (TLR2 ant.), or without treatments (non.). CASA was performed to investigate the sperm motion parameters. Sperm that able to penetrate the mucus has both higher VAP and ALH (i.e., sperm moving with increased velocity and higher side-to-side head movement) (Mortimer, 2000, J. Andrology). In the present study, we characterized the sperm with both VAP ≧ 100 μm/s and ALH ≧ 3.0 μm as the sperm that able to penetrate the mucus (i.e., mucus penetrable sperm). The percentages of sperm that penetrated the model mucus (0.7% long-chain polyacrylamide) were assessed by layering sperm (10 × 10⁶/mL, 0.5 mL) over the mucus layer (1 mL) in a microcentrifuge tube. TLR2 ago. treated-sperm immune interactions with endometrium were evaluated by the means of fluorescence microscopy and mRNA expression. [Results] TLR2 ago. increased the percentages of mucus penetrable (~33% vs control) and mucus penetrated (~20% vs control) sperm, while TLR2 ant. decreased these effects (~16% vs control and ~22% vs control respectively). Meanwhile, a higher number of TLR2 ago. sperm were entered within the uterine glands than non-treated sperm (~32% vs control). TLR2 ago. sperm increased the mRNA expression of TNFA, IL1B, IL8, PGES, and TLR2 in explants similar to non-treated sperm. Together, the activation of sperm TLR2 increases the sperm velocity and side-to-side head movement that permits the sperm penetration to mucus which is a prerequisite for entering the glands to induce the uterine inflammatory response.