ACTIVATION OF TOLL-LIKE RECEPTORS 2 AND 4 ON CUMULUS CELLS OF OVULATED CUMULUS OOCYTE COMPLEXES STIMULATES PRODUCTION OF CYTOKINES/CHEMOKINES THAT CAN INDUCE SPERM CAPACITATION LEADING TO SUCCESSFUL FERTILIZATION

Abstract

Our microarray analyses revealed that pattern recognition receptors (PRRs) of the Toll-like receptor (TLR) family and related molecules are expressed in cumulus cells of ovulated cumulus-oocyte complexes (COCs) and exhibit functions similar to those in macrophages. Specifically, we showed that when COCs were cultured with the TLR4 ligand bacterial lipopolysaccharide (LPS), the expression of Il-6, Tnfa and Ptgs2 were induced. However, the endogenous ligand for TLR family and its physiological role in ovulated COCs was not analyzed. Recently, it was reported that hyaluronan fragments, but not the high molecular weight hyaluronan polymers, activate TLR2 and TLR4. Since cumulus cells produce a hyaluronan rich matrix during the ovulation process, and because the matrix is broken-down by sperm-secreted hyaluronidase during fertilization, we hypothesized that hyaluronan fragments generated during matrix degradation might act as an endogenous ligand for TLR2 and/or TLR4 on cumulus cells and be required for successful fertilization. To examine this, mouse ovulated COCs were cultured for 2 hr with either LPS or Pam3Cys, ligands for TLR4 or TLR2, respectively. Each ligand increased mRNAs encoding specific cytokines/chemokines: Il6, Mip2, Ccl5 ∼7-fold, ∼10-fold, ∼15-fold, respectively. When ovulated COCs were cultured with hyaluronan fragments or treated with hyaluronidase, the expression of Il6, Mip2, Ccl5 mRNAs also increased with peak levels observed using hyaluronan fragments at 100 μg/ml or hyaluronidase at 1 IU/ml for 2 hr. Anti-TLR2 and anti-TLR4 monoclonal antibodies significantly suppressed hyaluronan fragment- and hyaluronidase- induced expression of these genes. Thus, TLR2/TLR4 on cumulus cells of ovulated COCs are functional and activated by hyaluronan fragments. To determine the role of activated TLR2/TLR4 on cumulus cells in fertilization process, ovulated COCs were cultured with 1x105 sperm in the presence or absence of anti-TLR2 and anti-TLR4 antibodies. Using a Bioplex Protein Array system we identified 17 cytokine and chemokine factors that were secreted by COCs. Secretion of 10 factors, CCL-5, G-CSF, IL-1a, IL-6, IL-9, IL-12 IL-17, MCP-1, MIP-1a, and TNFa was significantly up-regultaed by co-culture with sperm. The addition of anti-TLR2 and anti-TLR4 antibodies significantly suppressed the secretion of CCL-5, G-CSF, MCP-1, MIP-1a, and IL-6, concomitantly with the reduction of fertilization rate to 32 % as compared with that in oocytes without antibodies (75 %). Additionally, Ccr1, Ccr2, Ccr3 and Ccr5 mRNAs that encode CCL5, MCP-1, and MIP-1a receptors were expressed in spermatozoa. Sperm motility was enhanced by specific ligands suggesting that the chemokines secreted from cumulus cells of ovulated COCs induces sperm capacitation and subsequent oocyte penetration by mechanisms dependent on TLR2/4 activation. Supported in part by JSPS- 18688016 (MS), HD-16229, HD07459 (JSR) (platform)