TLR2 pathway is involved in sperm acrosome reaction for penetration through zona pellucida for fertilization

抄録

[Introduction]Cumulus cells of ovulated cumulus-oocyte complexes (COCs) express TLR2 which mediates the physiological inflammation and sperm capacitation at the interaction of sperm with COCs during fertilization. Recently, we reported that bovine sperm express TLR2. We aimed to clarify the pivotal role of TLR2 of bovine sperm in sperm-COCs interaction during in vitro fertilization (IVF). [Materials and methods]Washed frozen bull sperm were treated with TLR1/2 antagonist (0, 10, or 100 μM) for 30 min. Then, sperm were washed and co-cultured with either intact COCs, cumulus-free oocytes, or zona pellucida (ZP)-free oocytes in IVF100 medium. After 6 h of co-culture, presumptive zygotes were denuded and transferred to IVC medium. The cleavage rate and blastocyst ratio were assessed after 42 h and 7 days post-insemination respectively. The ability of TLR1/2 antagonist-treated sperm to penetrate ZP of COCs or cumulus-free oocytes was detected using Hoechst 33342 staining and fluorescence microscopy after 1 h of co-culture. TLR1/2 antagonist-treated sperm was stimulated by calcium ionophore (A23187) for 30 min and acrosome reaction (AR) was evaluated using FITC-PNA staining andquantified by flow cytometry. [Results]Co-culture of TLR1/2 antagonist-treated sperm with either COCs or cumulus-free oocytes significantly suppressed the cleavage rate and blastocyst ratio compared to control. While, co-culture with ZP-free oocytes did not affect fertilization ratio. Furthermore, pre-treatment of sperm with TLR1/2 antagonist reduced the average number of sperm penetrated through the ZP and sperm AR induced by A23187 (62 % vs. 3.6 %) which might account for the suppression of fertilization ratio. Interestingly, a pre-treatment of sperm with TLR1/2 antagonist had no effect on sperm motility parameters as analyzed by CASA System. Together, our data provides the first evidence that sperm TLR2 is involved in sperm AR and their penetration through ZP of oocytes to induce fertilization in cows.