Fig. 1. TLR2 and TLR4 expressed on cumulus cells of ovulated COCs are functional, and both receptors can recognize HA fragments. (A) Kinetic changes in the expression of TLRs and related molecules in cumulus cells of COCs during the ovulation process. Results in each panel are representative of two separate experiments. (B) TLR2/TLR4 agonists and HA fragments induce the expression of cytokine and chemokine mRNA. Ovulated COCs were cultured with 100 ng/ml LPS, 1 µg/ml Pam3Cys or 100 µg/ml HA fragments for 2 hours. The 0 hour value was set as 1, and the data are presented as fold increase. Values are mean±s.e.m. of three replicates. Significant differences were observed as compared with COCs cultured without any agonists for 2 hours (Cont; ^*P<0.01; ^**P<0.05). (C) Effects of the anti-TLR2 or anti-TLR4 neutralizing antibody on agonist-induced expression of Il6 mRNA. Ovulated COCs were pre-cultured for 30 minutes with 50 ng/ml anti-TLR4 or anti-TLR2 neutralizing antibody, and then further cultured with LPS or Pam3Cys for 2 hours. (D) HA fragment-induced Il6 mRNA was suppressed by both anti-TLR2 and anti-TLR4 neutralizing antibodies, but not by anti-CD44 antibody. Cont, COCs cultured without any neutralizing antibodies; Free, COCs cultured without HA fragments.