Sperm TLR2 regulates Ca²⁺ influx and acrosome reaction to facilitate sperm penetration to oocytes during in vitro fertilization in cows

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[Introduction] Toll–like receptors (TLRs) are innate immune cell receptors which recognize pathogens during infection. Specifically, TLR2 pathway regulates calcium mobilization through the cell. Calcium influx is essential for acquisition of sperm fertilizing competence through regulation of capacitation and acrosome reaction (AR). Human sperm express TLR2 to recognize pathogens. Recently, we reported that TLR2 is localized in the posterior segment of bull sperm head. Here, we aimed to clarify the role of sperm TLR2 on sperm–oocytes interaction during in vitro fertilization (IVF). [Materials and Methods] Frozen–thawed bull sperm were washed and treated with TLR1/2 antagonist (0, 10 and 100 µM) for 30 min to block the sperm TLR2. Then, co–cultured with intact cumulus–oocyte complexes (COCs), denuded, or zona pellucida (ZP)–free oocytes for 6 h. After 1 h and 3 h of co–culture, the number of bound or penetrated sperm to the ZP were counted. Next, AR was induced using calcium ionophore–A23187 (CaA) to evaluate the effect of sperm TLR2 on calcium influx and AR by fluorescence microscopy and flow cytometry respectively. [Results] Co–culture of TLR2 antagonist-treated sperm either with COCs or denuded oocytes, but not with ZP–free oocytes, reduced the cleavage rate and blastocyst ratio. Computer–assisted sperm analysis (CASA) analysis revealed that motility parameters were not affected in TLR2 antagonist-treated sperm. However, TLR2 antagonist reduced the ability of sperm to bind or penetrate the ZP at 1 h and 3 h. Moreover, blockage of sperm TLR2 reduced the induction of AR either in the ZP–attached sperm or in CaA–triggered AR compared to control. Notably, exposure of TLR2 antagonist sperm to CaA clearly reduced the intracellular calcium level in sperm. Overall, the results provide evidence that sperm TLR2 is involved in sperm Ca²⁺ influx to induce AR which enables sperm to penetrate and fertilize oocytes in cows.