Fig. 4. The roles of TLR2/TLR4 in regulating the expression and secretion of
cytokines/chemokines during the in vitro fertilization process. (A)
The expression of Il6, Ccl5 and Snap25 mRNA in cumulus cells
of COCs cultured with sperm. Ovulated COCs were cultured with sperm for 2 or 4
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.
*P<0.01 (significant difference observed compared with
that at 0 hour). Some COCs were cultured with anti-TLR2 and anti-TLR4
neutralizing antibodies (TLR2+4), anti-CD44 antibody (CD44), or HA-blocking
peptide (Pep-1) for 30 minutes, and then further cultured with the sperm for 2
hours. The Cont value was set as 1, and the data are presented as fold
increase. Values are mean±s.e.m. of three replicates. Cont, ovulated
COCs cultured for 2 hours without sperm. (B) The secretion of IL6, CCL4
and CCL5 from COCs during the fertilization process. IVF, COCs cultured with
sperm for up to 4 hours; Cont, ovulated COCs cultured for up to 4 hours
without sperm. (C) Effects of the mRNA synthesis inhibitor, or
anti-TLRs neutralizing antibodies, on the secretion of cytokines and
chemokines from COCs. COCs were pre-cultured for 30 minutes with neutralizing
antibodies (+anti-TLR2+4) or 10 µg/ml of 
-amanitin (+amanitin), and
then further cultured with sperm for 1 or 2 hours. Culture with sperm (IVF)
significantly increased secreted levels as compared with those in the control
(*P<0.01; **P<0.05). Cont,
ovulated COCs cultured for 1 or 2 hours without sperm. (D) Effects of
anti-TLR2/TLR4 neutralizing antibodies or Pep-1 on fertilization. COCs were
pre-cultured for 30 minutes with Pep-1 or neutralizing antibodies (anti-TLR2
antibody, anti-TLR4 antibody, or both), and further cultured for 12 hours with
sperm. Twelve hours after insemination, oocytes were checked the formation of
pronuclei under a phase-contrast microscope. Data are presented as the
percentage of oocytes fertilized. Cont, ovulated COCs cultured with sperm for
12 hours.