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First published online 23 April 2008
doi: 10.1242/dev.020461
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1 Department of Applied Animal Science, Graduate School of Biosphere Science,
Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8528, Japan.
2 Infertility Center, Daigo-Watanabe Clinic, Fushimi-ku, Kyoto, 601-1375,
Japan.
3 Department of Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX 77030, USA.
* Author for correspondence (e-mail: mashimad{at}hiroshima-u.ac.jp)
Accepted 4 April 2008
The toll-like receptor (TLR) system is expressed in cumulus cells of
ovulated cumulus-oocyte complexes (COCs) and is activated by bacterial
lipopolysaccharides (LPS). However, the endogenous ligand(s) for the TLRs and
the physiological role(s) in ovulated COCs remain to be defined. Based on
reports that hyaluronan fragments can activate TLR2 and TLR4 in macrophages,
and that ovulated COCs are characterized by a hyaluronan-rich matrix, we
cultured ovulated mouse COCs with purified hyaluronan fragments, treated them
with purified hyaluronidase or exposed them to sperm as a physiologically
relevant source of hyaluronidase. Hyaluronan fragments or hyaluronidase
activated the NF
B pathway and induced Il6, Ccl4 and
Ccl5 mRNA expression within 2 hours. Anti-TLR2 and anti-TLR4
neutralizing antibodies significantly suppressed hyaluronan fragment- and
hyaluronidase-induced activation of the NFB pathway and the expression
of these genes. When ovulated COCs were cultured with sperm, the expression
and secretion of cytokine/chemokine family members were induced in a
time-dependent manner that could be blocked by TLR2/TLR4 antibodies or by a
hyaluronan-blocking peptide (Pep-1). The chemokines secreted from
TLR2/TLR4-stimulated COCs activated cognate chemokine receptors (CCRs)
localized on sperm and induced sperm protein tyrosine phosphorylation, which
was used as an index of capacitation. Significantly, in vitro fertilization of
COC-enclosed oocytes was reduced by the TLR2/TLR4 neutralizing antibodies or
by Pep-1. From these results, we propose that TLR2 and TLR4 present on cumulus
cells were activated by the co-culture with sperm in a hyaluronan
fragment-dependent manner, and that chemokines secreted from COCs induced
sperm capacitation and enhanced fertilization, providing evidence for a
regulatory loop between sperm and COCs during fertilization.
Key words: Toll-like receptor, Cumulus cell, Cytokine/chemokine, Fertilization, Sperm
