Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization

Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine... RESEARCH ARTICLE 2001 Development 135, 2001-2011 (2008) doi:10.1242/dev.020461 Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization 1, 1 1 1 1 2 Masayuki Shimada *, Yoshiari Yanai , Tetsuji Okazaki , Noritaka Noma , Ikkou Kawashima , Takahide Mori and JoAnne S. Richards 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 NFκB 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 NFκB 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 INTRODUCTION Tnfaip6 mRNA was significantly lower in mice null for either Ptgs2 Ovulation is essential for successful reproduction. In mammals, this (prostaglandin-endoperoxide synthase 2, also known as COX2) or process involves the release of a mature oocyte and its surrounding Ptger2 (prostaglandin E receptor 2, also known as EP2) than in their cumulus cells, the cumulus cell oocyte complex (COC). The wild-type littermates (Ochsner et al., 2003a; Ochsner et al., 2003b), extrusion of COCs is dependent on the formation and stabilization indicating that Tnfaip6 gene expression was dependent on of a matrix composed of a polymeric hyaluronan (HA) backbone prostaglandin E2 and its receptor pathway. Ovulation in both mouse and HA-binding proteins/proteoglycans (Salustri et al., 1999; models was reduced slightly, but cumulus expansion, as well as Richards et al., 2002; Richards, 2005). Some of the matrix- fertilization, was completely suppressed in these mutant mice (Lim associated genes that have been identified encode hyaluronan et al., 1997; Kennedy et al., 1999; Hizaki et al., 1999). Additionally, synthase 2 (HAS2) (Fülöp et al., 1997a), TNF-α-induced protein 6 Ptx3-null mice showed abnormal cumulus-oocyte complex (TNFAIP6, also known as TSG6) (Fülöp et al., 1997b; Fülöp et al., morphology and reduced in vivo fertilization (Varani et al., 2002; 2003) and pentraxin 3 (PTX3) (Varani et al., 2002; Salustri et al., Salustri et al., 2004). The fertilization defects of Ptgs2-, Ptger2- or 2004). Polymeric HA directly binds to serum-derived inter-α-trypsin Ptx3-null mice were related to defective matrix stability/function inhibitor (IαI) (Chen et al., 1992; Camaioni et al., 1993) and because mature oocytes retrieved from these mutant mice could be interacts with TNFAIP6. PTX3 binds directly to TNFAIP6 to fertilized in vitro by the use of capacitated spermatozoa (Matsumoto provide an additional link of stability to the matrix scaffold within et al., 2001; Salustri et al., 2004). Collectively, these reports suggest COCs (Fülöp et al., 2003; Salustri et al., 2004). that the hyaluronan-rich matrix produced by COCs is essential not The functional relevance of HA and HA-binding proteins has only for ovulation, but also for in vivo fertilization, and perhaps been documented by a wealth of biochemical data and physiological more specifically for sperm capacitation. studies using mutant mouse models. Specifically, the level of Specific biochemical roles of the HA-rich matrix have been implicated by recent studies showing that HA fragments can impact immune cell responses by binding to specific membrane receptors. Department of Applied Animal Science, Graduate School of Biosphere Science, CD44, a cell surface molecule that is generally considered to be a Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8528, Japan. Infertility primary HA receptor (Aruffo et al., 1990; Miyake et al., 1990), is Center, Daigo-Watanabe Clinic, Fushimi-ku, Kyoto, 601-1375, Japan. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA. expressed in cumulus cells of mouse, human and porcine COCs during the ovulation process (Ohta et al., 1999; Kimura et al., 2002; *Author for correspondence (e-mail: [email protected]) Hernandez-Gonzalez et al., 2006). CD44 recognizes both high molecular weight HA present within extracellular matrices and small Accepted 4 April 2008 DEVELOPMENT 2002 RESEARCH ARTICLE Development 135 (11) fragments of HA generated during tissue injury, inducing the anti-TLR4 neutralizing antibody (MAb mTLR4/MD2) were purchased from InvivoGen (San Diego, CA), anti-CD44 neutralizing antibody (A020) expression of cytokine and chemokine families (Hodge-Dufour et al., from Calbiochem, and anti-CCL5 neutralizing antibody (anti-mouse 1997; Cuff et al., 2001; Kothapalli et al., 2007). HA fragments have CCL5/RANTES antibody) from R&D Systems. Routine chemicals and also been reported to stimulate members of the pathogen recognition reagents were obtained from Nakarai Chemical Co. (Osaka, Japan) or receptor surveillance pathway, toll-like receptor 2 (TLR2) and TLR4, Sigma. which are present in macrophages (Termeer et al., 2002; Fieber et al., 2004). Interestingly, Chang et al. reported that TLR2 and TLR4 do Animals Immature female C57BL/6 mice were obtained from Clea Japan (Tokyo, not recognize the high molecular weight of HA, but that fragments Japan). At 23 days old, female mice were injected intraperitoneally (IP) with of less than 230 kDa can activate these receptors (Chang et al., 2007); 4 IU of eCG, to stimulate follicular growth, followed 48 hours later by maximal effects were observed with 30-mer fragments, suggesting a injection with 5 IU hCG to stimulate ovulation and luteinization (Robker et gradient of responses during HA degradation. Moreover, very small al., 2000). Animals were housed under a 16-hour light/8-hour dark schedule HA fragments can activate the TLR-dependent pathway but not in the Experiment Animal Center at Hiroshima University, and provided CD44 in acute lung injury (Jiang et al., 2005), providing additional food and water ad libitum. Animals were treated in accordance with the NIH evidence that HA may exert pleomorphic effects on TLR2/TLR4- Guide for the Care and Use of Laboratory Animals, as approved by the positive cells. Our recent study (Shimada et al., 2006) documented Animal Care and Use Committee at Hiroshima University. that cumulus cells of ovulated COCs express numerous immune cell- Cumulus-oocyte complex isolation and culture related genes, including members of the TLR family, TLR4 and Ovulated COCs were recovered from oviducts and 50 COCs were cultured related molecules. When the ovulated COCs were cultured with the in separate wells of a Falcon 96-well plate (Becton Dickinson, Franklin TLR4 ligand, LPS, the expression of Il6, Ptgs2 and Tnfα mRNA was Lakes, NJ) in 150 μl of defined medium (Ochsner et al., 2003a), containing increased. These results indicated that the TLR4 pathway was present 1% (v/v) FBS with LPS, Pam3Cys or HA fragment in the presence or and functional in cumulus cells of ovulated COCs. Although the absence of each specific neutralizing antibody (anti-TLR2 rat polyclonal physiological relevance of the TLR pathway in ovulation and IgG, anti-TLR4 rabbit polyclonal IgG or anti-CD44 rabbit polyclonal IgG). fertilization has not been explored, the ability of capacitated sperm After culture of COCs, the culture medium was recovered for use in the Bio-Plex Protein Array System (BioRad, Hercules, CA, USA). Total RNA to secrete hyaluronidase, leading to the modification and breakdown or protein was extracted from cumulus cells isolated from COCs (see of the COC HA-rich matrix, is well known (Srivastava et al., 1965; below). Rogers and Morton, 1973; Talbot and Franklin, 1974). These reports, and our previous study, led us to hypothesize that, during the In vitro fertilization fertilization process, sperm-secreted hyaluronidase would generate Ovulated COCs were collected from oviductal ampullae 16 hours after the small HA fragments capable of activating TLR2 and/or TLR4 on hCG injections and placed into 150 μl of human tubal fluid (HTF) medium. Spermatozoa were collected from the cauda epididymi of 4-month-old ICR cumulus cells. strain mice into 500 μl of HTF medium. After 15, 30 or 60 minutes, the To examine this hypothesis, ovulated COCs were cultured with spermatozoa were introduced into fertilization medium at a final the TLR4 ligand LPS, the TLR2 ligand Pam3Cys-Ser-(Lys)4 HCl concentration of 1000 spermatozoa/μl. Twelve hours after insemination, (Pam3Cys), or with small HA fragments. The functional oocytes were washed thoroughly five times, and then checked for the responses of cumulus cells to these ligands were analyzed by formation of pronuclei under a phase-contrast microscope. Some COCs determining ligand-mediated secretion of specific cytokines and were recovered after 2 or 4 hours of culture with sperm; cumulus cells were chemokines. Neutralizing antibodies for TLR2, TLR4 or CD44 then isolated to prepare the total RNA. were used to verify receptor activation. siRNA approaches were Synthesis of Pep-1 and control peptide used to examine the responses (or lack thereof) in a granulosa cell Pep-1 (GAHWQFNALTVR) and scrambled control peptide culture system. Hyaluronidase and in vitro fertilization of COC- (WRHGFALTAVNQ), both with an amidated GGGS linker (Mummert et enclosed ooctyes by sperm provided further evidence consistent al., 2000; Jiang et al., 2005), were synthesized by Scrum (Tokyo Japan). with a role for HA-induced TLR activation in the fertilization Peptide solutions were prepared immediately before use by dissolution in process. Finally, we show that TLR2 and TLR4 are expressed by DMSO to a concentration of 500 mg/ml. The COCs were pre-cultured with the cumulus cells of ovulated human COCs, and that a positive 0.5 mg/ml peptides (final concentration of DMSO was 0.1%) for 30 correlation occurs between the in vitro fertilization of human minutes, and then treated with hyaluronidase or cultured with sperm as oocytes and the levels of chemokine family members secreted by described above. The scrambled control peptide did not significantly affect the COC. Therefore, the TLR pathway may have physiological the gene expression in cumulus cells and sperm penetration (data not shown). relevance in human fertility as well. Sperm culture Spermatozoa were collected from the cauda epididymi of 4-month-old mice MATERIALS AND METHODS into 500 μl of HTF medium. The sperm were cultured with 100 pg/ml of Materials CCL2, CCL4 or CCL5 for 30 or 60 minutes. After culture, sperm were lysed Equine chorionic gonadotropin (eCG) and human CG (hCG) were by Laemmli sample buffer and then analyzed for tyrosine phosphorylation purchased from Asuka Seiyaku (Tokyo, Japan). DMEM:F12 medium and as described below. penicillin-streptomycin were from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from Life Technologies (Grand Island, NY). Sperm accumulation assay Oligonucleotide poly-(dT) was purchased from Amersham Pharmacia Sperm were collected from the cauda epididymi of 4-month-old mice into Biotech (Newark, NJ), and AMV reverse transcriptase and Taq polymerase 500 μl of HTF medium and placed into a m-Slide VI flow chamber for live were from Promega (Madison, WI). α-amanitin and LPS were purchased cell analysis (Ibidi GmbH, Munich, Germany). The chamber plate has two from Sigma Chemical Co. (Sigma; St Louis, MO), and Pam3Cys was from wells connected by a narrow channel. First, 100 μl of HTM medium was Calbiochem (Los Angeles, CA). Hyaluronan fragments purified from pig added to the plate and 110 sperm were placed on one side. The agonists skin were obtained from Saikagaku Kougyou (Tokyo, Japan). CCL2 were added to other side chamber, and the plate was incubated for 30 (MCP1), CCL4 (MIP1β) and CCL5 (RANTES) were from R&D Systems minutes. After culture, the number of sperm that had moved to other side (Minneapolis, MN). Anti-TLR2 neutralizing antibody (MAb mTLR2) and was counted. DEVELOPMENT The role of TLR in fertilization RESEARCH ARTICLE 2003 Table 1. List of primers employed for RT-PCR and expected product size Anneling Gene Forward primer Reverse primer Size (bp) temperature (°C) Cycle Mouse Ccl2 5-GGTCCCTGTCATGCTTCTGG-3 5-CCTTCTTGGGGTCAGCACAG-3 236 64 Ccl4 5-ACCCTCCCACTTCCTGCTGT-3 5-GGGCAGGAAATCTGAACGTG-3 301 64 Ccl5 5-ATATGGCTCGGACACCACTC-3 5-GGGAAGCGTATACAGGGTCA-3 242 66 Ccr1 5-GGGAGTTCACTCACCGTACC-3 5-GATTGTAGGGGGTCCAGAGG-3 243 62 35 Ccr2 5-GCCATGCAGGTGACAGAGAC-3 5-AGACCTCTTGCTCCCCAGTG-3 214 66 35 Ccr3 5-GAAGTCCAGGTGTGGTGCTG-3 5-GGTATGTGAGGGCCGGTAGA-3 183 66 35 Ccr5 5-CTACCACACCGGGACTGTGA-3 5-GCCTGGAACACAGAGAGCAG-3 275 64 35 Il6 5-CCGGAGAGGAGACTTCACAG-3 5-GGAAATTGGGGTAGGAAGGA-3 421 62 L19 5-CTGAAGGTCAAAGGGAATGTG-3 5-GGACACAGTCTTGATGATCTC-3 196 60 Snap25 5-GAGATGCAGAGGAGGGCTGAC-3 5-GCTGGCCACTACTCCATCCTG-3 309 62 Tlr2 5-TTGCTCCTGCGAACTCCTAT-3 5-CAATGGGAATCCTGCTCACT-3 354 60 Tlr4 5-ACCTGGCTGGTTTACACGTC-3 5-CAGGCTGTTTGTTCCCAAAT-3 455 58 Human β-actin 5-CTACAATGAGCTGCGTGTGG-3 5-TAGCTCTTCTCCAGGGAGGA-3 450 58 33 Tlr2 5-GATGCCTACTGGGTGGAGAA-3 5-GAATGAGAATGGCAGCATCA-3 256 62 37 Tlr4 5-CCATAAAAGCCGAAAGGTGA-3 5-CAGGGCTTTTCTGAGTCGTC –3 265 62 37 siRNA treatment procedure in cultured mouse granulosa cells following parameters: 10 minutes at 95°C, followed by 45 cycles each of 15 TLR2 and TLR4 Silencer Pre-designed siRNA were purchased from seconds at 95°C and 1 minute at 60, 62 or 64°C. Specific primers were Ambion (Austin, TX). The sequences were: selected and analyzed as indicated in Table 1. TLR2, GGCAUUAAGUCUCCGGAAUtt (sense) and AUUCCGGA- For Q RT-PCR analysis, specific primers pairs, dNTP (Promega), Taq GACUUAAUGCCtt (antisense); polymerase and Thermocycle buffer (Promega) were added to PCR mixture. TLR4, GCAUCUAUGAUGCAUUUGUtt (sense) and ACAAAUG- cDNA products were resolved on 2% (w/v) agarose gels. CAUCAUAGAUGCtt (antisense). Western blot analyses Scrambled siRNA duplex (Ambion) was used as a negative control. Mouse Protein samples from cumulus cells, sperm, spleen or testes were prepared granulosa cells (110 cells/well) recovered from eCG/hCG-primed mice by homogenization in whole cell extract buffer and then diluted by the same were plated in 12-well culture plates for 3 hours before transfection. volume of 2SDS sample buffer (Hernandez-Gonzalez et al., 2006). Transfection of siRNA (25 nM) was accomplished using the HVJ envelope Extracts (5 μg protein) were resolved by SDS polyacrylamide gel (10%) vector kit GenomONE neo (Ishihara Sangyo, Tokyo, Japan), according to the electrophoresis and transferred to Immobilon-P nylon membranes manufacturer’s instructions and our previous study (Shimada et al., 2007). (Millipore, Bedford, MA). Membranes were blocked in Tris-buffered saline Cells were incubated at 37°C in a CO incubator, and the culture medium was and Tween 20 [TBST: 10 mM Tris (pH 7.5), 150 mM NaCl and 0.05% replaced 5 hours after transfection. After transfection, granulosa cells were Tween 20], containing 5% (w/v) non-fat Carnation instant milk (Nestle, cultured with 100 μg/ml of HA fragment for 2 hours. After culture, total RNA Solon, OH). Blots were incubated with primary antibody as shown in Table was collected and gene expression analyzed by real-time PCR. 2 overnight at 4°C. After washing in TBST, enhanced chemiluminescence Collection of human COC from periovulatory follicles, and the (ECL) detection was performed by using ECL western blotting detection media after in vitro fertilization reagents (Amersham) and appropriate exposure of the blots to Kodak X-ray Women were stimulated with human menopausal gonadotrophin (HMG; film. Specific bands were quantified by densitometric analyses using a Gel- HMG Injection TEIZO, Teikoku-zouki, Tokyo, Japan) according to routine Pro analyzer (Media Cybernetics, Bethesda, MD). procedures. Ovarian follicle diameter was assessed by transvaginal sonography, and gonadotropins were administered daily until the second Immunofluorescence largest follicle reached a diameter of 18 mm. When the follicle grew beyond Testes were recovered from 4- to 5-month-old male mice, and were that diameter, 10000 IU HCG (Gonatropin; Teikoku-zouki) or 600 μg embedded in OCT compound (Tissue-TEK, Miles, Elkhart, IN) and stored GnRH-agonist was administered; 35 hours later, oocytes were retrieved at –80°C before the preparation of 5 μm sections, which were fixed under ultrasonographic guidance. The oocytes underwent conventional IVF overnight in PBS-buffered 4% (w/v) paraformaldehyde at 4°C. Sections for 5 hours, before being transferred to 500 μl P-1 medium (preimplantation- were then sequentially probed with primary anti-CCR3 antibody (10 μg/ml, 1 medium; Irvine Scientific) under mineral oil with 10% (v/v) human serum Anaspec, San Hose, CA) or normal rabbit IgG (10 μg/ml, Sigma) and at 37°C in a humidified atmosphere of 5% CO , 5% O and 90% N . After secondary FITC-conjugated goat anti-rabbit IgG antibodies (diluted 1:250, 2 2 2 12 hours of culture, the formation of pronuclei was observed. The medium Sigma). Slides were mounted using VectaShield with DAPI (Vector after conventional IVF was kept below –80°C before Bio-Plex protein array Laboratories, Burlingame, CA). analysis using the Human Cytokine 9-Plex Panel (BioRad), as described Bio-Plex protein array system below. All patients gave written informed consent to participate in this study. During culture, media samples were collected and cytokines present in the RT-PCR analyses media were analyzed with the Bio-Plex Protein Array System (BioRad) Total RNA was obtained from cumulus cells or granulosa cells using the using the Bio-Plex Mouse Cytokine 23-Plex Panel, including antibodies for RNAeasy Mini Kit (Qiagen Sciences, Germantown, MD), according to the interleukin (IL) family members [IL1α, IL1β, IL2, IL3, IL4, IL5, IL6, IL9, manufacturer’s instructions. Real-time or quantitative (Q) RT-PCR analyses IL10, IL12 (p40), IL12 (p70), IL13, IL17], Eotaxin (CCL11), granulocyte were performed as described previously (Shimada et al., 2007). Briefly, total colony-stimulating factor (GCSF), granulocyte-macrophage colony RNA was reverse transcribed using 500 ng poly-dT (Amersham Pharmacia stimulating factor (GMCSF), interferon γ (IFNγ), keratinocyte-derived Biotech, Newark, NJ) and 0.25 U avian myeloblastosis virus-reverse chemokine (KC), monocyte chemotactic protein 1 (MCP1, CCL2), transcriptase (Promega, Madison, WI) at 42°C for 75 minutes and 95°C for macrophage inflammatory protein 1α (MIP1α, CCL3), MIP1β (CCL4), 5 minutes. RANTES (Regulated upon Activation, Normal T-cell Expressed and For real-time PCR analysis, cDNA and primers were added to the Power Secreted; CCL5), and tumor necrosis factor α (TNFα), or the Human SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) Cytokine 9-Plex Panel, including IL1β, IL6, IL12 (p70), IL17, Eotaxin to give a total reaction volume of 15 μl. PCR reactions were then performed (CCL11), GCSF, MCP1 (CCL2), MIP1β (CCL4), and RANTES (CCL5), using the iCycler thermocycler (Bio-Rad). Conditions were set to the according to our previous report (Shimada et al., 2007). DEVELOPMENT 2004 RESEARCH ARTICLE Development 135 (11) Table 2. List of antibodies used for western blotting Antibody Company Dilution β-Actin (AC74) Sigma 1:10,000 CCR3 Anaspec, San Hose, CA 1:250 ERK 1/2 (p44/p42 MAP kinase, 9102) Cell Signaling, Boston, MA 1:1000 pERK1/2 (phospho-p44/p42 MAPK, E10, 9106) Cell Signaling 1:2000 IκB-a (L35A5, 4814) Cell Signaling 1:2000 IRF3 Cell Signaling 1:1000 MYD88 eBioscience, San Diago, CA 1:1000 NF-κB (3034) Cell Signaling 1:1000 phospho-NF-κB p65 (Ser536, 3031) Cell Signaling 1:1000 phospho-p38MAPK (28B10, 9126) Cell Signaling 1:2000 Toll-like receptor 2 (2229) Cell Signaling 1:1000 Toll-like receptor 4 (IMG578A) IMGENEX, San Diago, CA 1:500 phospho-Tyr (P-Tyr-100, 9411) Cell Signaling 1:5000 Statistics at 0 hours, except for NFκB, confirming and extending previous Statistical analyses of data from three or four replicates were carried out by studies (Shimada et al., 2006) that the TLR2/TLR4 pathway is one-way ANOVA followed by Duncan’s multiple-range test (Statview; expressed in cumulus cells of ovulated COCs. To determine if the Abacus Concepts, Berkeley, CA). TLR2/TLR4 pathways were functional, 23 different cytokines and chemokines were analyzed in the medium of COCs that had been RESULTS cultured for 24 hours with the TLR4 ligand (LPS), the TLR2 ligand TLR2 and TLR4 expressed on cumulus cells of (Pam3Cys) or the small HA fragment, using the Bio-Plex Protein ovulated COCs are functional, and both receptors Array system. are activated by HA fragments As shown in Table 3, COCs cultured with LPS or Pam3Cys To analyze the induction of TLRs and related molecules in cumulus secreted increased levels of several cytokines and chemokines cells of COCs during the ovulation process, COCs were isolated [IL1α, IL1β, IL6, IL9, IL12 (p40), IL12 (p70), GCSF, Eotaxin, from ovaries of eCG-primed mice before (0 hours) and at 8 hours CCL2, CCL3, CCL4 and CCL5] compared with COCs cultured after hCG administration, as well as from oviducts at 16 hours post- without agonists. When COCs were cultured with HA fragments, hCG administration (Fig. 1A). Western blot analyses using the levels of IL6, as well as those of IL12 (p40), CCL2, CCL4 and antibodies to TLR2, TLR4, MYD88, IRF3, and Iκβ and NFκB CCL5, were higher than those observed in controls. Although showed that the levels of these proteins were increased in COCs COCs released IL1α, IL1β, I12 (p70), GCSF and CCL2, the levels collected at 8 and 16 hours post-hCG administration compared with between control and HA fragment-treated samples were not 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. DEVELOPMENT The role of TLR in fertilization RESEARCH ARTICLE 2005 Table 3. BioPlex Protein Array analysis of COC-secreted cytokine and chemokine family members (pg/ml) secreted following stimulation with LPS, Pam3Cys or HA fragments Control LPS Pam3Cys Hyaluronan fragment IL1α 6.34±1.51 47.93±5.33 60.9±5.07 7.02±5.69 IL1β – 35.01±8.15 78.04±6.66 15.3±2.54 IL6 9.16±2.33 344.84±28.62 472.18±52.63 284.35±18.72 IL9 – 72.23±30.51 65.11±6.25 – IL12 (p40) 0.08±0.02 36.14±6.94 28.69±3.99 33.33±5.11 IL12(p70) 52.4±10.36 139.18±11.56 196.32±21.68 27.87±6.33 GCSF 17.61±5.65 173.96±35.63 265.59±32.83 26.71±3.75 MCP1 23.51±3.89 468.82±55.88 506.21±42.68 255.82±20.63 MIP1α 5.13±1.17 182.78±30.42 404.74±25.88 2.85±0.06 MIP1β 11.07±2.20 1082.73±105.36 927.3±62.16 678.56±72.39 RANTES 24.19±5.26 1368.25±256.82 474.58±48.67 674.24±51.78 Ovulated COCs (50 COCs per/well) were cultured with 100 ng/ml LPS, 1 μg/ml Pam3Cys or 100 μg/ml of HA fragments for 24 hours. Media samples were collected and cytokines present in the media were analyzed using a Bio-Plex Mouse Cytokine 23-Plex Panel. dramatically different. The secreted levels of other tested antibodies, suggesting that HA activated the TLR receptors. cytokines/chemokines were below the detection limit. The dramatic Conversely, anti-TLR2 and anti-TLR4 neutralizing antibodies increase in the secretion of specific cytokine family members in suppressed the decrease of Iκβ and the phosphorylation of NFκBat response to LPS, Pam3Cys or HA fragments confirmed the 2 hours. Anti-CD44 antibody did not affect the NFκB pathway, at concomitant upregulation of gene expression (Fig. 1B) and least not during the time interval examined. previous studies stating that cumulus cells are highly secretory (Shimada et al., 2007). To determine the mechanisms by which HA fragments stimulated gene expression in cumulus cells, we used neutralizing antibodies to TLR2, TLR4 or CD44. The anti-TLR4 neutralizing antibody significantly suppressed LPS-induced expression of Il6 mRNA but did not alter Pam3Cys-induced expression of this gene (Fig. 1C). By contrast, Pam3Cys-induced expression of Il6 mRNA was significantly suppressed by the anti-TLR2 neutralizing antibody (Fig. 1C). When ovulated COCs were cultured with HA fragments, the level of Il6 mRNA was decreased marginally by either the anti- TLR2 or anti-TLR4 neutralizing antibody, but was significantly reduced in the presence of both antibodies when compared with the control (no neutralizing antibody). The anti-CD44 neutralizing antibody did not impact Il6 mRNA expression in cumulus cells of COCs. HA fragments induced the TLR2-, TLR4- and CD44-targeted signal transduction pathways Previous studies have implicated p38MAPK phosphorylation, ERK1/2 phosphorylation and the activation of Iκβ-NFκB signaling as downstream targets of the TLR pathway (Kawai and Akira, 2005). Therefore, we investigated whether these signaling pathways were activated via TLRs in the cumulus cells of ovulated COCs cultured with HA fragments. As shown in Fig. 2, these pathways were activated by HA fragments, but the temporal pattern of MAP kinase phosphorylation was different from that of NFκB pathway. Specifically, phosphorylation of p38MAP kinase and ERK1/2 was rapidly but transiently upregulated: high levels at 15 minutes returned to basal levels after 60 minutes. By contrast, the phosphorylation of NFκB was induced progressively from 15 Fig. 2. HA fragments induced the TLR2-, TLR4- and CD44-targeted minutes to 120 minutes. At that time, degradation of Iκβ was also signal transduction pathways. (A) Time-dependent changes of the detected. activation of p38MAP kinase, ERK1/2 and the NFκB pathway in To determine if the regulation of MAP kinase family and NFκB cumulus cells of ovulated COCs cultured with 100 μg/ml of HA pathways was mediated by TLR2/TLR4, ovulated COCs were fragments. Results in each panel are representative of two separate cultured in the presence of both anti-TLR2 and anti-TLR4 experiments. (B) Ovulated COCs were pre-cultured with neutralizing neutralizing antibodies (anti-TLRs), or the anti-CD44 neutralizing antibodies (both anti-TLR2 and anti-TLR4, or anti-CD44) for 30 minutes, antibody for 15 minutes or 2 hours, respectively. The rapid and further cultured with HA fragments for 15 minutes (p38MAP phosphorylation of p38MAP kinase and ERK1/2 by HA fragments kinase and ERK1/2), or for 2 hours (Iκβ and NFκB). Results in each panel at 15 minutes was suppressed by both anti-TLRs and anti-CD44 are representative of two separate experiments. DEVELOPMENT 2006 RESEARCH ARTICLE Development 135 (11) Hyaluronidase impacts gene expression and NFκB the phosphorylation of NFκB in cumulus cells of COCs, pathway activation in cumulus cells of ovulated responses that were reversed by anti-TLR2 and anti-TLR4 COCs neutralizing antibodies (Fig. 3B). When ovulated COCs were incubated with different concentrations of hyaluronidase for 2 hours, significant increases in the expression TLRs mediate the expression and secretion of of Il6, Ccl4 and Ccl5 mRNA were detected at the 1.0 IU/ml dose, cytokine/chemokine families during in vitro and further increases in Ccl5 mRNA were detected at hyaluronidase fertilization procedures concentrations of 10 or 100 IU/ml (Fig. 3A). Hyaluronidase (10 The expression of Il6 and Ccl5 mRNA in cumulus cells was also IU/ml) also significantly stimulated Ccl2 mRNA expression (Fig. induced within 2 hours by the co-culture of ovulated COCs with 3A). sperm (Fig. 4A). The induction of these genes in cumulus cells was The addition of anti-TLR2 and anti-TLR4 neutralizing suppressed by the addition of anti-TLR2 plus anti-TLR4 antibodies to the hyaluronidase-containing medium significantly neutralizing antibodies, or by treatment with the HA-blocking decreased the expression levels of Il6, Ccl2, Ccl4 and Ccl5 peptide Pep-1, described by Mummert et al. (Mummert et al., 2000), mRNA in cumulus cells of COCs, as compared with those in whereas the anti-CD44 antibody had no significant effect (Fig. 4A). COCs treated with 10 IU/ml hyaluronidase alone (Fig. 3B). These treatments did not significantly affect the expression of However, Snap25 mRNA expression, which is involved in the Snap25 mRNA (Fig. 4A). To analyze the secretion levels of exocytosis of these secreted factors, was not impacted by cytokines/chemokines, we collected the medium 0.5, 1, 2 or 4 hours hyaluronidase treatment and by the neutralizing antibodies (Fig. after ovulated COCs were cultured with capacitated sperm. The 3B). Hyaluronidase treatment induced the degradation of Iκβ and results show that high levels of IL6, CCL4 and CCL5 were secreted within 4 hours culture. The secretion of each cytokine was rapidly and significantly induced within 1 hour, and secretion further increased in time-dependent manner (Fig. 4B). After 1 hour of culture, the release of these cytokines from COCs was independent of de novo mRNA transcription, because addition of the transcriptional inhibitor α-amanitin (10 μg/ml) did not alter the levels of CCL4, CCL5 or IL6 in the culture medium (Fig. 4C). However, α-amanitin did significantly suppress the levels of these cytokines after 2 hours culture with sperm (Fig. 4C). Moreover, when COCs were cultured with sperm in the presence of anti-TLR2 plus anti-TLR4 neutralizing antibodies, significantly lower levels of cytokines were detected at both 1 and 2 hours of culture (Fig. 4C). Lastly, pronuclear formation in oocytes was analyzed after 12 hours of in vitro fertilization with sperm. Approximately 70% of the oocytes were fertilized and contained two pronuclei (Fig. 4D), responses that were reduced by the addition of neutralizing antibodies to TLR2/TLR4 (Fig. 4D). The HA-blocking peptide Pep- 1 also significantly suppressed pronuclear formation (Fig. 4D). Sperm express chemokine receptors (CCR1, CCR2, CCR3 and CCR5) that are required for sperm capacitation during the fertilization process Cumulus cells of cultured COCs secrete various kinds of cytokines/chemokines during the sperm-mediated fertilization process (see Table S1 in the supplementary material). Of note, IL6 and CC chemokine family members (CCL2, CCL3, CCL4 and CCL5) were predominantly produced from COCs at more than 100 pg/ml. Whereas CCL2 stimulates the CCR2 receptor, CCL3 binds mainly to Fig. 3. The effects of hyaluronidase (HY) treatment on gene CCR1 and partly to CCR5, and CCL4 selectively activates CCR5. All expression and NFκB pathway activation in cumulus cells of of receptors (CCR1, CCR2, CCR3 and CCR5) were activated by ovulated COCs. (A) Dose-dependent effects of HY on the expression CCL5 (Charo and Ransohoff, 2006). The CCR receptors are members of cytokine and chemokine mRNA. Ovulated COCs were treated with 0 of the G-protein coupling receptor family that induce phospholipase to 100 mIU/ml of HY for 2 hours. The 0 hour value was set as 1, and 2+ the data are presented as fold increase. Values are mean±s.e.m. of C to increase Ca and PKC activation in cytoplasm (Meyer et al., 2+ three replicates. Significant differences were observed as compared 1996). In sperm, the Ca -PKC pathway is involved in capacitation with COCs cultured without HY for 2 hours (Cont; *P<0.01; (Rotem et al., 1992), suggesting that the COC-secreted CC chemokine **P<0.05). (B) HY-induced cytokine and chemokine mRNA expression family might play an important role in sperm capacition during the and NFκB pathway activation were suppressed by both anti-TLR2 and fertilization process. The RT-PCR analysis shown in Fig. 5A shows anti-TLR4 neutralizing antibodies (Ab). Ovulated COCs were pre- the expression of Ccr1, Ccr2, Ccr3 and Ccr5 mRNA in sperm cultured with both anti-TLR2 and anti-TLR4 antibodies for 30 minutes, collected from the cauda epididymis. The positive immunofluorescent then 10 mIU/ml of HY was added to the medium. The addition of signals observed following the use of an anti-CCR3 antibody localized neutralizing antibodies significantly suppressed Il6, Ccl2, Ccl4 and Ccl5 CCR3 to luminal region of the testicular seminiferous tubules, and, at gene expression (*P<0.01; **P<0.05). Cont, COCs cultured without HY higher magnification, showed that CCR3 was detected near the tail of for 2 hours. Results of western blotting in each panel are representative of two separate experiments. spermatozoa; however these signals were very weak and a few cells DEVELOPMENT The role of TLR in fertilization RESEARCH ARTICLE 2007 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 α-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. also stained positive with the rabbit IgG control antibody (Fig. 5B). not dramatically changed from that of the control, for each ligand, Whole-mount preparations of spermatozoa provided additional the level of phosphorylation of other bands was increased within 30 evidence that CCR3 is present preferentially in the mid-piece of sperm minutes culture; the highest intensity was detected in sperm cultured (Fig. 5C). The immunoreactive band at about 55 kDa that corresponds with CCL5 for 60 minutes (Fig. 5E). to CCR3 was present in spleen used as positive control. In the testis To determine the role of CCL5 during the fertilization process, we sample, a 55 kDa band (as well as three other minor bands) was examined whether the addition of CCL5 could overcome the detected by the anti-CCR3 antibody. Sperm contained only one inhibitory effects of TLR neutralizing antibodies on fertilization, immuno-positive band of the correct size, indicating that sperm do and, conversely, whether antibodies to CCL5 could reduce express CCR3 (Fig. 5D). fertilization. The results showed that fertilization was suppressed by Protein tyrosine phosphorylation in spermatozoa is thought to be anti-TLR2/TLR4 neutralizing antibodies and that the addition of crucial for the acquisition of a capacitated state and the hyper- CCL5 slightly, but not significantly, increased the fertilization activation of motility (Naz et al., 1991). Tyrosine phosphorylation rate (Fig. 5F). However, the anti-CCL5 neutralizing antibody in protein extracts from sperm exposed to CCL2, CCL4 or CCL5 for significantly suppressed the percentage of oocytes that completed 30 and 60 minutes was analyzed by western blotting using a pan fertilization (Fig. 5F). anti-Tyr phosphorylation antibody. Positive immunoreactive bands It is known that when the high concentrations of sperm recovered were detected at ~110 kDa, ~75 kDa, 65 kDa and 50 kDa when from the cauda epididymis are cultured for more than 60 minutes sperm were cultured with BSA for 60 minutes (data not shown). (pre-culture), capacitation is induced spontaneously (Fraser, 1977). Although the level of phosphorylation of the band at ~110 kDa was Therefore, we examined whether the prolonged pre-culture of sperm DEVELOPMENT 2008 RESEARCH ARTICLE Development 135 (11) Fig. 5. Sperm express chemokine receptors required for sperm capacitation during the fertilization process. (A) The expression of Ccr1, Ccr2, Ccr3 and Ccr5 mRNA in sperm was determined by RT-PCR. (B,C) The localization of CCR3 in testicular seminiferous tubes (B) and sperm (C). Blue, DAPI staining of nuclei; green, FITC signal localizing the anti-CCR3 antibody. Scale bar: 10 μm. As a negative control, the slides were incubated with normal rabbit IgG and then reacted with secondary antibody. (D) Western analysis of CCR3 using the same primary antibody that was used for immunofluorescence. (E) The induction of protein tyrosine phosphorylation in sperm by CCL2, CCL4 or CCL5. Sperm collected from cauda epididymi were cultured with 100 pg/ml of CCL2, CCL4 or CCL5 for 30 or 60 minutes. Tyrosine phosphorylation (P-Tyr) was detected by an anti-phospho-Tyr antibody. (F) CCL5 regulates sperm penetration to oocytes. COCs were pre-cultured for 30 minutes with anti-TLR2 + anti-TLR4 antibodies (Anti- TLR2+TLR4), or with anti-CCL5 antibody (Anti-CCL5), and further cultured for 12 hours with sperm. In some cases, CCL5 (100 pg/ml) was added to the fertilization medium (+CCL5). Data are presented as the percentage of oocytes fertilized. Cont, ovulated COCs cultured with sperm for 12 hours. (G) The effects of the pre-culture period of sperm on oocyte fertilization in vitro. COCs were pre-cultured for 30 minutes with anti-TLR2 + anti-TLR4 antibodies (Anti-TLR2+TLR4), or with anti-CCL5 antibody (Anti-CCL5), and further cultured for 12 hours with sperm. The sperm were collected from the cauda epididymis, and then cultured for 0, 15, 30 or 60 minutes. Data are presented as the percentage of oocytes fertilized. that allows capacitation would overcome the negative effects of the and fertilization, we compared the levels of nine different chemokine anti-TLR2/4 neutralizing antibodies and the anti-CCL5 neutralizing family members and the rate of oocytes fertilized. Patients were antibody on sperm penetration. Without pre-culture or antibody separated into two groups: the Y group, in which all of the oocytes exposure, fertilization was highly variable (control, Fig. 5G). The were fertilized in vitro; and the N group, in which less than half of number of oocytes fertilized increased in controls in a duration- oocytes were fertilized. We detected IL6, CCL2, CCL4 and CCL5 dependent manner, with maximal success obtained when the sperm in the in vitro fertilization medium. The levels of IL6 and CCL2 were pre-cultured for 60 minutes (i.e. complete capacitation; Fig. were almost the same between two groups; however, CCL5 was 5G). The addition of neutralizing antibodies significantly suppressed slightly higher in Y group and CCL4 was significantly elevated (Fig. the penetration of sperm that were pre-cultured for 15 or 30 minutes 6B). (Fig. 5G). However, these negative effects were not detected when sperm were pre-cultured for 60 minutes and hence were fully DISCUSSION capacitated (Fig. 5G). Our previous microarray analyses revealed that cumulus cells of ovulated COCs express a unique set of genes associated with Tlr2 and Tlr4 mRNA are expressed in human specific immune cell-like responses. The TLRs and related factors cumulus cells and cytokine/chemokine production were of particular interest because they represent part of the immune may impact human fertility cell surveillance system that monitors changes in the external Human cumulus cells obtained from patients undergoing an IVF environment (Hernandez-Gonzalez et al., 2006; Shimada et al., protocol expressed Tlr2 and Tlr4 mRNA (Fig. 6A). To determine the 2006). Specifically, we have documented that, when ovulated COCs relationship between human COC-secreted cytokines/chemokines were cultured with the TLR4 ligand bacterial lipopolysaccharide DEVELOPMENT The role of TLR in fertilization RESEARCH ARTICLE 2009 supplementary material), blocked the effects of HA. The HA-rich matrix generated during COC expansion is presumed to be of high molecular weight, as cumulus cells express primarily HAS2 (Fülöp et al., 1997a), which is known to generate HA with a broad but extremely large size (average molecular weight of more than 210 kDa) (Itano et al., 1999). Although the functions of high molecular weight HA are not entirely clear for any cell system, it can bind and activate CD44, but not TLR2 and TLR4 in macrophages (Cuff et al., 2001; Chang et al., 2007). To determine whether HA is a physiologically relevant ligand for TLRs on cumulus cells, ovulated COCs were treated with exogenous hyaluronidase or were cultured with sperm that are known to secrete hyaluronidase. The results show that exogenous hyaluronidase significantly upregulated Il6, Ccl2, Ccl4 and Ccl5 mRNA expression, and induced activation of the NFκB pathway in cumulus cells. The induction of these genes and the activation of specific signaling pathways by hyaluronidase were suppressed by anti-TLR2/TLR4 neutralizing antibodies. Moreover, the culture with sperm induced the expression of Il6 and Ccl5 mRNA through TLR2/4-dependent, but CD44-independent, mechanisms. Additionally, we used the HA blocking peptide Pep-1, which has been shown to function effectively in vitro to inhibit the binding of HA to its receptors (Mummert et al., 2000; Taylor et al., 2004). Pep-1 significantly suppressed the expression of Il6 and Ccl5 mRNA in cumulus cells and sperm penetration in in vitro Fig. 6. The expression of Tlr2 and Tlr4 mRNA and the role of fertilization assays. It has been shown that hyaluronidase cytokines/chemokines in human fertility. (A) The expression of Tlr2 depolymerizes high molecular weight HA to 10-40 kDa end and Tlr4 mRNA in cumulus cells of human COCs. The cumulus cells products (Sampson et al., 1992), suggesting that the small HA were recovered from COCs after in vitro fertilization. (B) The fragments generated by sperm-secreted hyaluronidase probably relationship between the amount of secreted CCL cytokines and activated TLR2 and TLR4 on cumulus cells, leading to cytokine and fertilization in vitro. The cytokines present in the media were analyzed chemokine expression via the NFκB pathway. by using the Bio-Plex Human Cytokine 9-Plex Panel. Y, all oocytes were The high level of cytokine secretion from cumulus cells 2-4 hours successfully fertilized; N, more than half of the oocytes were not after exposure to sperm was dependent on the increased fertilized. P values are for Y versus N. transcription of specific genes, as α-amanitin blocked this effect. By contrast, the initial rapid release of IL6, CCL4 and CCL5 by the (LPS), the expression of Il6 and Tnfα was induced. However, the TLR2/TLR4 pathway during fertilization did not require de novo endogenous ligand(s) for TLR2/TLR4 and the physiological role of mRNA synthesis. The mechanism by which the rapid release is this signaling cascade in ovulated COCs remained unclear. Because regulated could involve an exocytosis system because cumulus cells cumulus cells produce and are surrounded by a HA-rich express components of the SNARE complex [synaptosomal- extracellular matrix and because HA fragments have been shown associated protein 25 (Snap25), syntaxin 1a (Stx1a) and recently to activate TLR2 and TLR4 (Termeer et al., 2002; Jiang et synaptotagmin 1 (Syt1)] (Shimada et al., 2007) (M.S., unpublished). al., 2005), we hypothesized that HA fragments generated during In mast cells, vesicle degranulation and the release of cytokines matrix degradation might act as an endogenous ligand for TLR2 occurs in a TLR2- and TLR4-dependent manner (Supajatura et al., and/or TLR4 that are present in cumulus cells of ovulated COCs. 2002), via exocytosis involving SNAP25 localized to the secretory Herein, we provide the first evidence that TLRs can play a functional granules (Salinas et al., 2004; Stow et al., 2006). Thus, it is possible role in the, at least in vitro, fertilization process. Notably, we show that the SNAP25-associated exocytosis system present in cumulus that hyaluronidase treatment and culture with sperm (used as a cells is also activated by the small HA fragment-induced TLR biological source of hyaluronidase) activate TLR2/TLR4 on pathway during the fertilization process, although the precise cumulus cells. Activated cumulus cells, but not sperm itself, then mechanisms remain to be resolved. release specific cytokines capable of enhancing sperm capacitation The functional role of secreted CC chemokines appears to be part and fertilization. Thus, a functional regulatory loop appears to be of a regulatory loop between COCs and sperm, because operative between sperm and the ovulated COCs. chemokines can enhance fertilization by the activation of GPCRs Specifically, the results presented here document that exposure of (CCRs) and calcium release. We show here that multiple CCRs are ovulated COCs to HA fragments induced the phosphorylation of expressed in mouse sperm, as well as in human sperm (Isobe et al., p38MAP kinase, ERK1/2 and NFκB, and the expression of specific 2002; Muciaccia et al., 2005), and that CCR3 is localized to the genes (Il6, Ccl2, Ccl4 and Ccl5) in cumulus cells. This response was mid-piece of mature sperm. During capacitation, the increase of 2+ similar to that observed when ovulated COCs were exposed to LPS, Ca is observed around the mid-piece of spermatozoa (Harper et 2+ a known ligand of TLR4 (Shimada et al., 2006), indicating that HA al., 2004), and the Ca -dependent pathway evokes protein tyrosine fragments could serve as ligands for cumulus cell TLRs. The ability phosphorylation (Carrera et al., 1996), suggesting that secreted CC of HA fragments to active these signaling cascades and to induce the chemokine families are involved in sperm capacitation. When expression of specific cytokines in cumulus cells was selectively sperm collected from cauda epididymi were cultured with CCL2, mediated by TLR2 and TLR4, as TLR2 and TLR4 neutralizing CCL4 or CCL5, increased levels of immunoreactive phospho- antibodies, but not the anti-CD44 antibody (see Fig. S1 in the tyrosine were detected in extracts of the CCL5 treatment group. DEVELOPMENT 2010 RESEARCH ARTICLE Development 135 (11) CCL5 treatment also increased sperm motility in a dose-dependent receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation. J. Clin. Invest. 108, 1031-1040. manner (see Fig. S2 in the supplementary material). Moreover, Fieber, C., Baumann, P., Vallon, R., Termeer, C., Simon, J. C., Hofmann, M., using COC-conditioned medium, we showed that factors secreted Angel, P., Herrlich, P. and Sleeman, J. P. (2004). Hyaluronan-oligosaccharide- by COCs induced not only sperm motility but also capacitation, and induced transcription of metalloproteases. J. Cell. Sci. 117, 359-367. Fraser, L. R. (1977). Differing requirements for capacitation in vitro of mouse that these effects were suppressed by the addition of anti-CCL5 spermatozoa from two strains. J. Reprod. Fertil. 49, 83-87. neutralizing antibody (see Fig. S3 in the supplementary material). Fülöp, C., Salustri, A. and Hascall, V. C. (1997a). Coding sequence of a Furthermore, the fertilization of oocytes was suppressed hyaluronan synthase homologue expressed during expansion of the mouse cumulus-oocyte complex. Biochem. Biophys. 337, 261-266. significantly by either anti-TLR2/TLR4 or anti-CCL5 neutralizing Fülöp, C., Kamath, R. V., Li, Y., Otto, J. M., Salustri, A., Olsen, B. R., Glant, T. antibodies if short-term pre-cultured sperm was used for T. and Hascall, V. C. (1997b). Coding sequence, exon-intron structure and insemination. However, after prolonged pre-culture (over 60 chromosomal localization of murine TNF-stimulated gene 6 that is specifically minutes), which mediates complete sperm capacitation, fertilization expressed by expanding cumulus cell-oocyte complexes. Gene 202, 95-102. Fülöp, C., Szántó, S., Mukhopadhyay, D., Bárdos, T., Kamath, R. V., Rugg, M. was not impaired by the presence of neutralizing antibodies. Based S., Day, A. J., Salustri, A., Hascall, V. C., Glant, T. T. and Mikecz, K. (2003). on these results, we conclude that during the fertilization process, Impaired cumulus mucification and female sterility in tumor necrosis factor- TLR2 and TLR4 present on the cumulus cells are activated by co- induced protein-6 deficient mice. Development 130, 2253-2261. Harper, C. V., Barratt, C. L. and Publicover, S. J. (2004). Stimulation of human culture with sperm in a hyaluronan fragment-dependent manner, spermatozoa with progesterone gradients to simulate approach to the oocyte. leading to the secretion of CCL5 and other CC chemokine family 2+ Induction of [Ca ] oscillations and cyclical transitions in flagellar beating. J. Biol. members. These, in turn, stimulate CC receptors on sperm to Chem. 279, 46315-46325. enhance sperm motility and to induce sperm capacitation, thereby Hernandez-Gonzalez, I., Gonzalez-Robayna, I., Shimada, M., Wayne, C. M., Ochsner, S. A., White, L. and Richards, J. S. (2006). Gene expression profiles enhancing successful fertilization. Thus, there is a functional of cumulus cell oocyte complexes during ovulation reveal cumulus cells express regulatory loop between the COCs and sperm. neuronal and immune-related genes: does this expand their role in the ovulation Finally, we report that Tlr2 and Tlr4 mRNA are also expressed in process? Mol. Endocrinol. 20, 1300-1321. Hizaki, H., Segi, E., Sugimoto, Y., Hirose, M., Saji, T., Ushikubi, F., Matsuoka, human peri-ovulatory cumulus cells, and that human COCs secret T., Noda, Y., Tanaka, T., Yoshida, N. et al. (1999). Abortive expansion of the IL6, CCL2, CCL4 and CCL5 during in vitro fertilization. Therefore, cumulus and impaired fertility in mice lacking the prostaglandin E receptor it is likely that the TLR system in human cumulus cells plays a subtype EP(2). Proc. Natl. Acad. Sci. USA 96, 10501-10506. Hodge-Dufour, J., Noble, P. W., Horton, M. R., Bao, C., Wysoka, M., Burdick, similar role to in mouse cells in facilitating fertilization. Importantly, M. D., Strieter, R. M., Trinchieri, G. and Puré, E. (1997). Induction of IL-12 human oocytes collected from COCs that produced high amounts of and chemokines by hyaluronan requires adhesion-dependent priming of resident CCL4 or CCL5 showed higher fertilization rates than did oocytes but not elicited macrophages. J. Immunol. 159, 2492-2500. from COCs that produced low levels of these cytokines. On the basis Isobe, T., Minoura, H., Tanaka, K., Shibahara, T., Hayashi, N. and Toyoda, N. (2002). The effect of RANTES on human sperm chemotaxis. Hum. Reprod. 17, of these data in human and mouse COCs, the addition of CC 1441-1446. chemokines such as CCL5 might improve IVF or intracytoplasmic Itano, N., Sawai, T., Yoshida, M., Lenas, P., Yamada, Y., Imagawa, M., sperm injection (ICSI) protocols. Shinomura, T., Hamaguchi, M., Yoshida, Y., Ohnuki, Y. et al. (1999). Three isoforms of mammalian hyaluronan synthases have distinct enzymatic The authors are grateful to Dr S. Tanabe, Hiroshima University, for technical properties. J. Biol. Chem. 274, 25085-25092. advice on the use of the Bio-Plex Protein Array System; Dr Y. Yoshimura, Dr N. Jiang, D., Liang, J., Fan, J., Yu, S., Chen, S., Luo, Y., Prestwich, G. D., Mascarenhas, M. M., Garg, H. G., Quinn, D. A. et al. (2005). Regulation of Isobe and Mr. A. Ozoe for supporting the making of the frozen sections; and lung injury and repair by Toll-like receptors and hyaluronan. Nat. Med. 11, 1173- Dr K. Uematsu for technical advice on the use of microscope. We also thank Mr. K. Kinoshita, Daigo-Watanabe Clinic, for collecting IVF medium, and Ms K. Kawai, T. and Akira, S. (2005). Toll-like receptor downstream signaling. Arthritis. Nishimatsu for technical assistance. This work was supported, in part, by Res. Ther. 7, 12-19. Grant-in-Aid for Scientific Research No.18688016 from the Japan Society for Kennedy, C. R., Zhang, Y., Brandon, S., Guan, Y., Coffee, K., Funk, C. D., the Promotion of Science (JSPS) and The Kao Foundation for Arts and Sciences Magnuson, M. A., Oates, J. A., Breyer, M. D. and Breyer, R. M. (1999). Salt- (to M.S.), and the National Institutes of Health HD-16229 and HD-07495 sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 (Project III, Specialized Cooperative Program in Reproductive Research, SCPRR; receptor. Nat. Med. 5, 217-220. to J.S.R.). Kimura, N., Konno, Y., Miyoshi, K., Matsumoto, H. and Sato, E. (2002). Expression of hyaluronan synthases and CD44 messenger RNAs in porcine Supplementary material cumulus-oocyte complexes during in vitro maturation. Biol. Reprod. 66, 707- Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/135/11/2001/DC1 Kothapalli, D., Zhao, L., Hawthorne, E. A., Cheng, Y., Lee, E., Puré, E. and Assoian, R. K. (2007). Hyaluronan and CD44 antagonize mitogen-dependent References cyclin D1 expression in mesenchymal cells. J. Cell Biol. 176, 535-544. Aruffo, A., Stamenkovic, I., Melnick, M., Underhill, C. B. and Seed, B. (1990). Lim, H., Paria, B. C., Das, S. K., Dinchuk, J. E., Langenbach, R., Trzaskos, J. M. CD44 is the principal cell surface receptor for hyaluronate. Cell 61, 1303-1313. and Dey, S. K. (1997). Multiple female reproductive failures in cyclooxygenase Camaioni, A., Hascall, V. C., Yanagishita, M. and Salustri, A. (1993). Effects of 2-deficient mice. Cell 91, 197-208. exogenous hyaluronic acid and serum on matrix organization and stability in the Matsumoto, H., Ma, W., Smalley, W., Trzaskos, J., Breyer, R. M. and Dey, S. mouse cumulus cell-oocyte complex. J. Biol. Chem. 268, 20473-20481. K. (2001). Diversification of cyclooxygenase-2-derived prostaglandins in Carrera, A., Moos, J., Ning, X. P., Gerton, G. L., Tesarik, J., Kopf, G. S. and ovulation and implantation. Biol. Reprod. 64, 1557-1565. Moss, S. B. (1996). Regulation of protein tyrosine phosphorylation in human Meyer, A., Coyle, A. J., Proudfoot, A. E., Wells, T. N. and Power, C. A. (1996). sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase Cloning and characterization of a novel murine macrophage inflammatory anchor proteins as major substrates for tyrosine phosphorylation. Dev. Biol. 180, protein-1 alpha receptor. J. Biol. Chem. 271, 14445-14451. 284-296. Miyake, K., Underhill, C. B., Lesley, J. and Kincade, P. W. (1990). Hyaluronate Chang, E. J., Kim, H. J., Ha, J., Kim, H. J., Ryu, J., Park, K. H., Kim, U. H., Lee, can function as a cell adhesion molecule and CD44 participates in hyaluronate Z. H., Kim, H. M., Fisher, D. E. and Kim, H. H. (2007). Hyaluronan inhibits recognition. J. Exp. Med. 172, 69-75. osteoclast differentiation via Toll-like receptor 4. J. Cell. Sci. 120, 166-176. Muciaccia, B., Padula, F., Vicini, E., Gandini, L., Lenzi, A. and Stefanini, M. Charo, I. F. and Ransohoff, R. M. (2006). The many roles of chemokines and (2005). Beta-chemokine receptors 5 and 3 are expressed on the head region of chemokine receptors in inflammation. N. Engl. J. Med. 354, 610-621. human spermatozoon. FASEB J. 19, 2048-2050. Chen, L., Mao, S. J. and Larsen, W. J. (1992). Identification of a factor in fetal Mummert, M. E., Mohamadzadeh, M., Mummert, D. I., Mizumoto, N. and bovine serum that stabilizes the cumulus extracellular matrix. A role for a Takasima, A. (2000). Development of a peptide inhibitor of hyaluronan- member of the inter-alpha-trypsin inhibitor family. J. Biol. Chem. 267, 12380- mediated leukocyte trafficking. J. Exp. Med. 192, 769-779. 12386. Naz, R. K., Ahmad, K. and Kumar, R. (1991). Role of membrane Cuff, C. A., Kothapalli, D., Azonobi, I., Chun, S., Zhang, Y., Belkin, R., Yeh, C., phosphotyrosine proteins in human spermatozoal function. J. Cell. Sci. 99, 157- Secreto, A., Assoian, R. K., Rader, D. J. and Puré, E. (2001). The adhesion 165. DEVELOPMENT The role of TLR in fertilization RESEARCH ARTICLE 2011 Ochsner, S. A., Day, A. J., Rugg, M. S., Breyer, R. M., Gomer, R. H. and Sampson, P. M., Rochester, C. L., Freundlich, B. and Elias, J. A. (1992). Richards, J. S. (2003a). Disrupted function of tumor necrosis factor-alpha- Cytokine regulation of human lung fibroblast hyaluronan (hyaluronic acid) stimulated gene 6 blocks cumulus cell-oocyte complex expansion. Endocrinology production. Evidence for cytokine-regulated hyaluronan (hyaluronic acid) 144, 4376-4384. degradation and human lung fibroblast-derived hyaluronidase. J. Clin. Invest. 90, Ochsner, S. A., Russell, D. L., Day, A. J., Breyer, R. M. and Richards, J. S. 1492-1503. (2003b). Decreased expression of tumor necrosis factor-alpha-stimulated gene 6 Shimada, M., Hernandez-Gonzalez, I., Gonzalez-Robanya, I. and Richards, J. in cumulus cells of the cyclooxygenase-2 and EP2 null mice. Endocrinology 144, S. (2006). Induced expression of pattern recognition receptors in cumulus 1008-1019. oocyte complexes: novel evidence for innate immune-like functions during Ohta, N., Saito, H., Kuzumaki, T., Takahashi, T., Ito, M. M., Saito, T., ovulation. Mol. Endocrinol. 20, 3228-3239. Nakahara, K. and Hiroi, M. (1999). Expression of CD44 in human cumulus and Shimada, M., Yanai, Y., Okazaki, T., Yamashita, Y., Sriraman, V., Wilson, M. mural granulosa cells of individual patients in in-vitro fertilization programmes. C. and Richards, J. S. (2007). Synaptosomal-associated protein 25 gene Mol. Hum. Reprod. 5, 22-28. expression is hormonally regulated during ovulation and is involved in cytokine/ Richards, J. S. (2005). Ovulation: new factors that prepare the oocyte for chemokine exocytosis from granulosa cells. Mol. Endocrinol. 21, 2487-2502. fertilization. Mol. Cell. Endocrinol. 234, 75-79. Srivastava, P., Adams, C. E. and Hartree, E. F. (1965). Enzymatic action of Richards, J. S., Russell, D. L., Ochsner, S. and Espey, L. L. (2002). Ovulation: lipoglycoprotein preparations from sperm-acrosomes on rabbit ova. Nature 205, new dimensions and new regulators of the inflammatory-like response. Annu. 498. Rev. Physiol. 64, 69-92. Stow, J. L., Manderson, A. P. and Murray, R. Z. (2006). SNAREing immunity: the Robker, R. L., Russell, D. L., Espey, L. L., Lydon, J. P., O’Malley, B. W. and role of SNAREs in the immune system. Nat. Rev. Immunol. 6, 919-929. Richards, J. S. (2000). Progesterone-regulated genes in the ovulation process: Supajatura, V., Ushio, H., Nakao, A., Akira, S., Okumura, K., Ra, C. and ADAMTS-1 and cathespin L proteases. Proc. Natl. Acad. Sci. USA 97, 4689- Ogawa, H. (2002). Differential responses of mast cell Toll-like receptors 2 and 4 4694. in allergy and innate immunity. J. Clin. Invest. 109, 1351-1359. Rogers, B. J. and Morton, B. E. (1973). The release of hyaluronidase from Talbot, P. and Franklin, L. E. (1974). The release of hyaluronidase from guinea- capacitating hamster spermatozoa. J. Reprod. Fertil. 35, 477-487. pig spermatozoa during the course of the normal acrosome reaction in vitro. J. Rotem, R., Paz, G. F., Homonnai, Z. T., Kalina, M., Lax, J., Breitbart, H. and Reprod. Fertil. 39, 429-432. 2+ Naor, Z. (1992). Ca -independent induction of acrosome reaction by protein Taylor, K. R., Trowbridge, J. M., Rudisill, J. A., Termeer, C. C., Simon, J. C. and kinase C in human sperm. Endocrinology 131, 2235-2243. Gallo, R. L. (2004). Hyaluronan fragments stimulate endothelial recognition of Salinas, E., Ventura, J., Córdova, L. E. and Luis Quintanar, J. (2004). Presence injury through TLR4. J. Biol. Chem. 279, 17079-17084. of SNAP-25 in rat mast cells. Immunol. Lett. 95, 105-108. Termeer, C., Benedix, F., Sleeman, J., Fieber, C., Voith, U., Ahrens, T., Miyake, Salustri, A., Camaioni, A., Di Giacomo, M., Fulop, C. and Hascall, V. C. (1995). K., Freudenberg, M., Galanos, C. and Simon, J. C. (2002). Oligosaccharides Hyaluronan and proteoglycans in ovarian follicles. Hum. Reprod. Update. 5, 293- of Hyaluronan activate dendritic cells via toll-like receptor 4. J. Exp. Med. 195, 301. 99-111. Salustri, A., Garlanda, C., Hirsch, E., De Acetis, M., Maccagno, A., Bottazzi, Varani, S., Elvin, J. A., Yan, C., DeMayo, J., DeMayo, F. J., Horton, H. F., B., Doni, A., Bastone, A., Mantovani, G., Beck Peccoz, P. et al. (2004). PTX3 Byrne, M. C. and Matzuk, M. M. (2002). Knockout of pentraxin 3, a plays a key role in the organization of the cumulus oophorus extracellular matrix downstream target of growth differentiation factor-9, causes female subfertility. and in in vivo fertilization. Development 131, 1577-1586. Mol. Endocrinol. 16, 1154-1167. DEVELOPMENT http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Development The Company of Biologists

Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization

Download PDF
11 pages

Loading next page...
 
/lp/the-company-of-biologists/hyaluronan-fragments-generated-by-sperm-secreted-hyaluronidase-W3pm6o54SD

References (53)

Publisher
The Company of Biologists
Copyright
© 2021 The Company of Biologists. All rights reserved.
ISSN
0950-1991
eISSN
0950-1991
DOI
10.1242/dev.020461
Publisher site
See Article on Publisher Site

Abstract

RESEARCH ARTICLE 2001 Development 135, 2001-2011 (2008) doi:10.1242/dev.020461 Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization 1, 1 1 1 1 2 Masayuki Shimada *, Yoshiari Yanai , Tetsuji Okazaki , Noritaka Noma , Ikkou Kawashima , Takahide Mori and JoAnne S. Richards 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 NFκB 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 NFκB 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 INTRODUCTION Tnfaip6 mRNA was significantly lower in mice null for either Ptgs2 Ovulation is essential for successful reproduction. In mammals, this (prostaglandin-endoperoxide synthase 2, also known as COX2) or process involves the release of a mature oocyte and its surrounding Ptger2 (prostaglandin E receptor 2, also known as EP2) than in their cumulus cells, the cumulus cell oocyte complex (COC). The wild-type littermates (Ochsner et al., 2003a; Ochsner et al., 2003b), extrusion of COCs is dependent on the formation and stabilization indicating that Tnfaip6 gene expression was dependent on of a matrix composed of a polymeric hyaluronan (HA) backbone prostaglandin E2 and its receptor pathway. Ovulation in both mouse and HA-binding proteins/proteoglycans (Salustri et al., 1999; models was reduced slightly, but cumulus expansion, as well as Richards et al., 2002; Richards, 2005). Some of the matrix- fertilization, was completely suppressed in these mutant mice (Lim associated genes that have been identified encode hyaluronan et al., 1997; Kennedy et al., 1999; Hizaki et al., 1999). Additionally, synthase 2 (HAS2) (Fülöp et al., 1997a), TNF-α-induced protein 6 Ptx3-null mice showed abnormal cumulus-oocyte complex (TNFAIP6, also known as TSG6) (Fülöp et al., 1997b; Fülöp et al., morphology and reduced in vivo fertilization (Varani et al., 2002; 2003) and pentraxin 3 (PTX3) (Varani et al., 2002; Salustri et al., Salustri et al., 2004). The fertilization defects of Ptgs2-, Ptger2- or 2004). Polymeric HA directly binds to serum-derived inter-α-trypsin Ptx3-null mice were related to defective matrix stability/function inhibitor (IαI) (Chen et al., 1992; Camaioni et al., 1993) and because mature oocytes retrieved from these mutant mice could be interacts with TNFAIP6. PTX3 binds directly to TNFAIP6 to fertilized in vitro by the use of capacitated spermatozoa (Matsumoto provide an additional link of stability to the matrix scaffold within et al., 2001; Salustri et al., 2004). Collectively, these reports suggest COCs (Fülöp et al., 2003; Salustri et al., 2004). that the hyaluronan-rich matrix produced by COCs is essential not The functional relevance of HA and HA-binding proteins has only for ovulation, but also for in vivo fertilization, and perhaps been documented by a wealth of biochemical data and physiological more specifically for sperm capacitation. studies using mutant mouse models. Specifically, the level of Specific biochemical roles of the HA-rich matrix have been implicated by recent studies showing that HA fragments can impact immune cell responses by binding to specific membrane receptors. Department of Applied Animal Science, Graduate School of Biosphere Science, CD44, a cell surface molecule that is generally considered to be a Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8528, Japan. Infertility primary HA receptor (Aruffo et al., 1990; Miyake et al., 1990), is Center, Daigo-Watanabe Clinic, Fushimi-ku, Kyoto, 601-1375, Japan. Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA. expressed in cumulus cells of mouse, human and porcine COCs during the ovulation process (Ohta et al., 1999; Kimura et al., 2002; *Author for correspondence (e-mail: [email protected]) Hernandez-Gonzalez et al., 2006). CD44 recognizes both high molecular weight HA present within extracellular matrices and small Accepted 4 April 2008 DEVELOPMENT 2002 RESEARCH ARTICLE Development 135 (11) fragments of HA generated during tissue injury, inducing the anti-TLR4 neutralizing antibody (MAb mTLR4/MD2) were purchased from InvivoGen (San Diego, CA), anti-CD44 neutralizing antibody (A020) expression of cytokine and chemokine families (Hodge-Dufour et al., from Calbiochem, and anti-CCL5 neutralizing antibody (anti-mouse 1997; Cuff et al., 2001; Kothapalli et al., 2007). HA fragments have CCL5/RANTES antibody) from R&D Systems. Routine chemicals and also been reported to stimulate members of the pathogen recognition reagents were obtained from Nakarai Chemical Co. (Osaka, Japan) or receptor surveillance pathway, toll-like receptor 2 (TLR2) and TLR4, Sigma. which are present in macrophages (Termeer et al., 2002; Fieber et al., 2004). Interestingly, Chang et al. reported that TLR2 and TLR4 do Animals Immature female C57BL/6 mice were obtained from Clea Japan (Tokyo, not recognize the high molecular weight of HA, but that fragments Japan). At 23 days old, female mice were injected intraperitoneally (IP) with of less than 230 kDa can activate these receptors (Chang et al., 2007); 4 IU of eCG, to stimulate follicular growth, followed 48 hours later by maximal effects were observed with 30-mer fragments, suggesting a injection with 5 IU hCG to stimulate ovulation and luteinization (Robker et gradient of responses during HA degradation. Moreover, very small al., 2000). Animals were housed under a 16-hour light/8-hour dark schedule HA fragments can activate the TLR-dependent pathway but not in the Experiment Animal Center at Hiroshima University, and provided CD44 in acute lung injury (Jiang et al., 2005), providing additional food and water ad libitum. Animals were treated in accordance with the NIH evidence that HA may exert pleomorphic effects on TLR2/TLR4- Guide for the Care and Use of Laboratory Animals, as approved by the positive cells. Our recent study (Shimada et al., 2006) documented Animal Care and Use Committee at Hiroshima University. that cumulus cells of ovulated COCs express numerous immune cell- Cumulus-oocyte complex isolation and culture related genes, including members of the TLR family, TLR4 and Ovulated COCs were recovered from oviducts and 50 COCs were cultured related molecules. When the ovulated COCs were cultured with the in separate wells of a Falcon 96-well plate (Becton Dickinson, Franklin TLR4 ligand, LPS, the expression of Il6, Ptgs2 and Tnfα mRNA was Lakes, NJ) in 150 μl of defined medium (Ochsner et al., 2003a), containing increased. These results indicated that the TLR4 pathway was present 1% (v/v) FBS with LPS, Pam3Cys or HA fragment in the presence or and functional in cumulus cells of ovulated COCs. Although the absence of each specific neutralizing antibody (anti-TLR2 rat polyclonal physiological relevance of the TLR pathway in ovulation and IgG, anti-TLR4 rabbit polyclonal IgG or anti-CD44 rabbit polyclonal IgG). fertilization has not been explored, the ability of capacitated sperm After culture of COCs, the culture medium was recovered for use in the Bio-Plex Protein Array System (BioRad, Hercules, CA, USA). Total RNA to secrete hyaluronidase, leading to the modification and breakdown or protein was extracted from cumulus cells isolated from COCs (see of the COC HA-rich matrix, is well known (Srivastava et al., 1965; below). Rogers and Morton, 1973; Talbot and Franklin, 1974). These reports, and our previous study, led us to hypothesize that, during the In vitro fertilization fertilization process, sperm-secreted hyaluronidase would generate Ovulated COCs were collected from oviductal ampullae 16 hours after the small HA fragments capable of activating TLR2 and/or TLR4 on hCG injections and placed into 150 μl of human tubal fluid (HTF) medium. Spermatozoa were collected from the cauda epididymi of 4-month-old ICR cumulus cells. strain mice into 500 μl of HTF medium. After 15, 30 or 60 minutes, the To examine this hypothesis, ovulated COCs were cultured with spermatozoa were introduced into fertilization medium at a final the TLR4 ligand LPS, the TLR2 ligand Pam3Cys-Ser-(Lys)4 HCl concentration of 1000 spermatozoa/μl. Twelve hours after insemination, (Pam3Cys), or with small HA fragments. The functional oocytes were washed thoroughly five times, and then checked for the responses of cumulus cells to these ligands were analyzed by formation of pronuclei under a phase-contrast microscope. Some COCs determining ligand-mediated secretion of specific cytokines and were recovered after 2 or 4 hours of culture with sperm; cumulus cells were chemokines. Neutralizing antibodies for TLR2, TLR4 or CD44 then isolated to prepare the total RNA. were used to verify receptor activation. siRNA approaches were Synthesis of Pep-1 and control peptide used to examine the responses (or lack thereof) in a granulosa cell Pep-1 (GAHWQFNALTVR) and scrambled control peptide culture system. Hyaluronidase and in vitro fertilization of COC- (WRHGFALTAVNQ), both with an amidated GGGS linker (Mummert et enclosed ooctyes by sperm provided further evidence consistent al., 2000; Jiang et al., 2005), were synthesized by Scrum (Tokyo Japan). with a role for HA-induced TLR activation in the fertilization Peptide solutions were prepared immediately before use by dissolution in process. Finally, we show that TLR2 and TLR4 are expressed by DMSO to a concentration of 500 mg/ml. The COCs were pre-cultured with the cumulus cells of ovulated human COCs, and that a positive 0.5 mg/ml peptides (final concentration of DMSO was 0.1%) for 30 correlation occurs between the in vitro fertilization of human minutes, and then treated with hyaluronidase or cultured with sperm as oocytes and the levels of chemokine family members secreted by described above. The scrambled control peptide did not significantly affect the COC. Therefore, the TLR pathway may have physiological the gene expression in cumulus cells and sperm penetration (data not shown). relevance in human fertility as well. Sperm culture Spermatozoa were collected from the cauda epididymi of 4-month-old mice MATERIALS AND METHODS into 500 μl of HTF medium. The sperm were cultured with 100 pg/ml of Materials CCL2, CCL4 or CCL5 for 30 or 60 minutes. After culture, sperm were lysed Equine chorionic gonadotropin (eCG) and human CG (hCG) were by Laemmli sample buffer and then analyzed for tyrosine phosphorylation purchased from Asuka Seiyaku (Tokyo, Japan). DMEM:F12 medium and as described below. penicillin-streptomycin were from Invitrogen (Carlsbad, CA). Fetal bovine serum (FBS) was obtained from Life Technologies (Grand Island, NY). Sperm accumulation assay Oligonucleotide poly-(dT) was purchased from Amersham Pharmacia Sperm were collected from the cauda epididymi of 4-month-old mice into Biotech (Newark, NJ), and AMV reverse transcriptase and Taq polymerase 500 μl of HTF medium and placed into a m-Slide VI flow chamber for live were from Promega (Madison, WI). α-amanitin and LPS were purchased cell analysis (Ibidi GmbH, Munich, Germany). The chamber plate has two from Sigma Chemical Co. (Sigma; St Louis, MO), and Pam3Cys was from wells connected by a narrow channel. First, 100 μl of HTM medium was Calbiochem (Los Angeles, CA). Hyaluronan fragments purified from pig added to the plate and 110 sperm were placed on one side. The agonists skin were obtained from Saikagaku Kougyou (Tokyo, Japan). CCL2 were added to other side chamber, and the plate was incubated for 30 (MCP1), CCL4 (MIP1β) and CCL5 (RANTES) were from R&D Systems minutes. After culture, the number of sperm that had moved to other side (Minneapolis, MN). Anti-TLR2 neutralizing antibody (MAb mTLR2) and was counted. DEVELOPMENT The role of TLR in fertilization RESEARCH ARTICLE 2003 Table 1. List of primers employed for RT-PCR and expected product size Anneling Gene Forward primer Reverse primer Size (bp) temperature (°C) Cycle Mouse Ccl2 5-GGTCCCTGTCATGCTTCTGG-3 5-CCTTCTTGGGGTCAGCACAG-3 236 64 Ccl4 5-ACCCTCCCACTTCCTGCTGT-3 5-GGGCAGGAAATCTGAACGTG-3 301 64 Ccl5 5-ATATGGCTCGGACACCACTC-3 5-GGGAAGCGTATACAGGGTCA-3 242 66 Ccr1 5-GGGAGTTCACTCACCGTACC-3 5-GATTGTAGGGGGTCCAGAGG-3 243 62 35 Ccr2 5-GCCATGCAGGTGACAGAGAC-3 5-AGACCTCTTGCTCCCCAGTG-3 214 66 35 Ccr3 5-GAAGTCCAGGTGTGGTGCTG-3 5-GGTATGTGAGGGCCGGTAGA-3 183 66 35 Ccr5 5-CTACCACACCGGGACTGTGA-3 5-GCCTGGAACACAGAGAGCAG-3 275 64 35 Il6 5-CCGGAGAGGAGACTTCACAG-3 5-GGAAATTGGGGTAGGAAGGA-3 421 62 L19 5-CTGAAGGTCAAAGGGAATGTG-3 5-GGACACAGTCTTGATGATCTC-3 196 60 Snap25 5-GAGATGCAGAGGAGGGCTGAC-3 5-GCTGGCCACTACTCCATCCTG-3 309 62 Tlr2 5-TTGCTCCTGCGAACTCCTAT-3 5-CAATGGGAATCCTGCTCACT-3 354 60 Tlr4 5-ACCTGGCTGGTTTACACGTC-3 5-CAGGCTGTTTGTTCCCAAAT-3 455 58 Human β-actin 5-CTACAATGAGCTGCGTGTGG-3 5-TAGCTCTTCTCCAGGGAGGA-3 450 58 33 Tlr2 5-GATGCCTACTGGGTGGAGAA-3 5-GAATGAGAATGGCAGCATCA-3 256 62 37 Tlr4 5-CCATAAAAGCCGAAAGGTGA-3 5-CAGGGCTTTTCTGAGTCGTC –3 265 62 37 siRNA treatment procedure in cultured mouse granulosa cells following parameters: 10 minutes at 95°C, followed by 45 cycles each of 15 TLR2 and TLR4 Silencer Pre-designed siRNA were purchased from seconds at 95°C and 1 minute at 60, 62 or 64°C. Specific primers were Ambion (Austin, TX). The sequences were: selected and analyzed as indicated in Table 1. TLR2, GGCAUUAAGUCUCCGGAAUtt (sense) and AUUCCGGA- For Q RT-PCR analysis, specific primers pairs, dNTP (Promega), Taq GACUUAAUGCCtt (antisense); polymerase and Thermocycle buffer (Promega) were added to PCR mixture. TLR4, GCAUCUAUGAUGCAUUUGUtt (sense) and ACAAAUG- cDNA products were resolved on 2% (w/v) agarose gels. CAUCAUAGAUGCtt (antisense). Western blot analyses Scrambled siRNA duplex (Ambion) was used as a negative control. Mouse Protein samples from cumulus cells, sperm, spleen or testes were prepared granulosa cells (110 cells/well) recovered from eCG/hCG-primed mice by homogenization in whole cell extract buffer and then diluted by the same were plated in 12-well culture plates for 3 hours before transfection. volume of 2SDS sample buffer (Hernandez-Gonzalez et al., 2006). Transfection of siRNA (25 nM) was accomplished using the HVJ envelope Extracts (5 μg protein) were resolved by SDS polyacrylamide gel (10%) vector kit GenomONE neo (Ishihara Sangyo, Tokyo, Japan), according to the electrophoresis and transferred to Immobilon-P nylon membranes manufacturer’s instructions and our previous study (Shimada et al., 2007). (Millipore, Bedford, MA). Membranes were blocked in Tris-buffered saline Cells were incubated at 37°C in a CO incubator, and the culture medium was and Tween 20 [TBST: 10 mM Tris (pH 7.5), 150 mM NaCl and 0.05% replaced 5 hours after transfection. After transfection, granulosa cells were Tween 20], containing 5% (w/v) non-fat Carnation instant milk (Nestle, cultured with 100 μg/ml of HA fragment for 2 hours. After culture, total RNA Solon, OH). Blots were incubated with primary antibody as shown in Table was collected and gene expression analyzed by real-time PCR. 2 overnight at 4°C. After washing in TBST, enhanced chemiluminescence Collection of human COC from periovulatory follicles, and the (ECL) detection was performed by using ECL western blotting detection media after in vitro fertilization reagents (Amersham) and appropriate exposure of the blots to Kodak X-ray Women were stimulated with human menopausal gonadotrophin (HMG; film. Specific bands were quantified by densitometric analyses using a Gel- HMG Injection TEIZO, Teikoku-zouki, Tokyo, Japan) according to routine Pro analyzer (Media Cybernetics, Bethesda, MD). procedures. Ovarian follicle diameter was assessed by transvaginal sonography, and gonadotropins were administered daily until the second Immunofluorescence largest follicle reached a diameter of 18 mm. When the follicle grew beyond Testes were recovered from 4- to 5-month-old male mice, and were that diameter, 10000 IU HCG (Gonatropin; Teikoku-zouki) or 600 μg embedded in OCT compound (Tissue-TEK, Miles, Elkhart, IN) and stored GnRH-agonist was administered; 35 hours later, oocytes were retrieved at –80°C before the preparation of 5 μm sections, which were fixed under ultrasonographic guidance. The oocytes underwent conventional IVF overnight in PBS-buffered 4% (w/v) paraformaldehyde at 4°C. Sections for 5 hours, before being transferred to 500 μl P-1 medium (preimplantation- were then sequentially probed with primary anti-CCR3 antibody (10 μg/ml, 1 medium; Irvine Scientific) under mineral oil with 10% (v/v) human serum Anaspec, San Hose, CA) or normal rabbit IgG (10 μg/ml, Sigma) and at 37°C in a humidified atmosphere of 5% CO , 5% O and 90% N . After secondary FITC-conjugated goat anti-rabbit IgG antibodies (diluted 1:250, 2 2 2 12 hours of culture, the formation of pronuclei was observed. The medium Sigma). Slides were mounted using VectaShield with DAPI (Vector after conventional IVF was kept below –80°C before Bio-Plex protein array Laboratories, Burlingame, CA). analysis using the Human Cytokine 9-Plex Panel (BioRad), as described Bio-Plex protein array system below. All patients gave written informed consent to participate in this study. During culture, media samples were collected and cytokines present in the RT-PCR analyses media were analyzed with the Bio-Plex Protein Array System (BioRad) Total RNA was obtained from cumulus cells or granulosa cells using the using the Bio-Plex Mouse Cytokine 23-Plex Panel, including antibodies for RNAeasy Mini Kit (Qiagen Sciences, Germantown, MD), according to the interleukin (IL) family members [IL1α, IL1β, IL2, IL3, IL4, IL5, IL6, IL9, manufacturer’s instructions. Real-time or quantitative (Q) RT-PCR analyses IL10, IL12 (p40), IL12 (p70), IL13, IL17], Eotaxin (CCL11), granulocyte were performed as described previously (Shimada et al., 2007). Briefly, total colony-stimulating factor (GCSF), granulocyte-macrophage colony RNA was reverse transcribed using 500 ng poly-dT (Amersham Pharmacia stimulating factor (GMCSF), interferon γ (IFNγ), keratinocyte-derived Biotech, Newark, NJ) and 0.25 U avian myeloblastosis virus-reverse chemokine (KC), monocyte chemotactic protein 1 (MCP1, CCL2), transcriptase (Promega, Madison, WI) at 42°C for 75 minutes and 95°C for macrophage inflammatory protein 1α (MIP1α, CCL3), MIP1β (CCL4), 5 minutes. RANTES (Regulated upon Activation, Normal T-cell Expressed and For real-time PCR analysis, cDNA and primers were added to the Power Secreted; CCL5), and tumor necrosis factor α (TNFα), or the Human SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) Cytokine 9-Plex Panel, including IL1β, IL6, IL12 (p70), IL17, Eotaxin to give a total reaction volume of 15 μl. PCR reactions were then performed (CCL11), GCSF, MCP1 (CCL2), MIP1β (CCL4), and RANTES (CCL5), using the iCycler thermocycler (Bio-Rad). Conditions were set to the according to our previous report (Shimada et al., 2007). DEVELOPMENT 2004 RESEARCH ARTICLE Development 135 (11) Table 2. List of antibodies used for western blotting Antibody Company Dilution β-Actin (AC74) Sigma 1:10,000 CCR3 Anaspec, San Hose, CA 1:250 ERK 1/2 (p44/p42 MAP kinase, 9102) Cell Signaling, Boston, MA 1:1000 pERK1/2 (phospho-p44/p42 MAPK, E10, 9106) Cell Signaling 1:2000 IκB-a (L35A5, 4814) Cell Signaling 1:2000 IRF3 Cell Signaling 1:1000 MYD88 eBioscience, San Diago, CA 1:1000 NF-κB (3034) Cell Signaling 1:1000 phospho-NF-κB p65 (Ser536, 3031) Cell Signaling 1:1000 phospho-p38MAPK (28B10, 9126) Cell Signaling 1:2000 Toll-like receptor 2 (2229) Cell Signaling 1:1000 Toll-like receptor 4 (IMG578A) IMGENEX, San Diago, CA 1:500 phospho-Tyr (P-Tyr-100, 9411) Cell Signaling 1:5000 Statistics at 0 hours, except for NFκB, confirming and extending previous Statistical analyses of data from three or four replicates were carried out by studies (Shimada et al., 2006) that the TLR2/TLR4 pathway is one-way ANOVA followed by Duncan’s multiple-range test (Statview; expressed in cumulus cells of ovulated COCs. To determine if the Abacus Concepts, Berkeley, CA). TLR2/TLR4 pathways were functional, 23 different cytokines and chemokines were analyzed in the medium of COCs that had been RESULTS cultured for 24 hours with the TLR4 ligand (LPS), the TLR2 ligand TLR2 and TLR4 expressed on cumulus cells of (Pam3Cys) or the small HA fragment, using the Bio-Plex Protein ovulated COCs are functional, and both receptors Array system. are activated by HA fragments As shown in Table 3, COCs cultured with LPS or Pam3Cys To analyze the induction of TLRs and related molecules in cumulus secreted increased levels of several cytokines and chemokines cells of COCs during the ovulation process, COCs were isolated [IL1α, IL1β, IL6, IL9, IL12 (p40), IL12 (p70), GCSF, Eotaxin, from ovaries of eCG-primed mice before (0 hours) and at 8 hours CCL2, CCL3, CCL4 and CCL5] compared with COCs cultured after hCG administration, as well as from oviducts at 16 hours post- without agonists. When COCs were cultured with HA fragments, hCG administration (Fig. 1A). Western blot analyses using the levels of IL6, as well as those of IL12 (p40), CCL2, CCL4 and antibodies to TLR2, TLR4, MYD88, IRF3, and Iκβ and NFκB CCL5, were higher than those observed in controls. Although showed that the levels of these proteins were increased in COCs COCs released IL1α, IL1β, I12 (p70), GCSF and CCL2, the levels collected at 8 and 16 hours post-hCG administration compared with between control and HA fragment-treated samples were not 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. DEVELOPMENT The role of TLR in fertilization RESEARCH ARTICLE 2005 Table 3. BioPlex Protein Array analysis of COC-secreted cytokine and chemokine family members (pg/ml) secreted following stimulation with LPS, Pam3Cys or HA fragments Control LPS Pam3Cys Hyaluronan fragment IL1α 6.34±1.51 47.93±5.33 60.9±5.07 7.02±5.69 IL1β – 35.01±8.15 78.04±6.66 15.3±2.54 IL6 9.16±2.33 344.84±28.62 472.18±52.63 284.35±18.72 IL9 – 72.23±30.51 65.11±6.25 – IL12 (p40) 0.08±0.02 36.14±6.94 28.69±3.99 33.33±5.11 IL12(p70) 52.4±10.36 139.18±11.56 196.32±21.68 27.87±6.33 GCSF 17.61±5.65 173.96±35.63 265.59±32.83 26.71±3.75 MCP1 23.51±3.89 468.82±55.88 506.21±42.68 255.82±20.63 MIP1α 5.13±1.17 182.78±30.42 404.74±25.88 2.85±0.06 MIP1β 11.07±2.20 1082.73±105.36 927.3±62.16 678.56±72.39 RANTES 24.19±5.26 1368.25±256.82 474.58±48.67 674.24±51.78 Ovulated COCs (50 COCs per/well) were cultured with 100 ng/ml LPS, 1 μg/ml Pam3Cys or 100 μg/ml of HA fragments for 24 hours. Media samples were collected and cytokines present in the media were analyzed using a Bio-Plex Mouse Cytokine 23-Plex Panel. dramatically different. The secreted levels of other tested antibodies, suggesting that HA activated the TLR receptors. cytokines/chemokines were below the detection limit. The dramatic Conversely, anti-TLR2 and anti-TLR4 neutralizing antibodies increase in the secretion of specific cytokine family members in suppressed the decrease of Iκβ and the phosphorylation of NFκBat response to LPS, Pam3Cys or HA fragments confirmed the 2 hours. Anti-CD44 antibody did not affect the NFκB pathway, at concomitant upregulation of gene expression (Fig. 1B) and least not during the time interval examined. previous studies stating that cumulus cells are highly secretory (Shimada et al., 2007). To determine the mechanisms by which HA fragments stimulated gene expression in cumulus cells, we used neutralizing antibodies to TLR2, TLR4 or CD44. The anti-TLR4 neutralizing antibody significantly suppressed LPS-induced expression of Il6 mRNA but did not alter Pam3Cys-induced expression of this gene (Fig. 1C). By contrast, Pam3Cys-induced expression of Il6 mRNA was significantly suppressed by the anti-TLR2 neutralizing antibody (Fig. 1C). When ovulated COCs were cultured with HA fragments, the level of Il6 mRNA was decreased marginally by either the anti- TLR2 or anti-TLR4 neutralizing antibody, but was significantly reduced in the presence of both antibodies when compared with the control (no neutralizing antibody). The anti-CD44 neutralizing antibody did not impact Il6 mRNA expression in cumulus cells of COCs. HA fragments induced the TLR2-, TLR4- and CD44-targeted signal transduction pathways Previous studies have implicated p38MAPK phosphorylation, ERK1/2 phosphorylation and the activation of Iκβ-NFκB signaling as downstream targets of the TLR pathway (Kawai and Akira, 2005). Therefore, we investigated whether these signaling pathways were activated via TLRs in the cumulus cells of ovulated COCs cultured with HA fragments. As shown in Fig. 2, these pathways were activated by HA fragments, but the temporal pattern of MAP kinase phosphorylation was different from that of NFκB pathway. Specifically, phosphorylation of p38MAP kinase and ERK1/2 was rapidly but transiently upregulated: high levels at 15 minutes returned to basal levels after 60 minutes. By contrast, the phosphorylation of NFκB was induced progressively from 15 Fig. 2. HA fragments induced the TLR2-, TLR4- and CD44-targeted minutes to 120 minutes. At that time, degradation of Iκβ was also signal transduction pathways. (A) Time-dependent changes of the detected. activation of p38MAP kinase, ERK1/2 and the NFκB pathway in To determine if the regulation of MAP kinase family and NFκB cumulus cells of ovulated COCs cultured with 100 μg/ml of HA pathways was mediated by TLR2/TLR4, ovulated COCs were fragments. Results in each panel are representative of two separate cultured in the presence of both anti-TLR2 and anti-TLR4 experiments. (B) Ovulated COCs were pre-cultured with neutralizing neutralizing antibodies (anti-TLRs), or the anti-CD44 neutralizing antibodies (both anti-TLR2 and anti-TLR4, or anti-CD44) for 30 minutes, antibody for 15 minutes or 2 hours, respectively. The rapid and further cultured with HA fragments for 15 minutes (p38MAP phosphorylation of p38MAP kinase and ERK1/2 by HA fragments kinase and ERK1/2), or for 2 hours (Iκβ and NFκB). Results in each panel at 15 minutes was suppressed by both anti-TLRs and anti-CD44 are representative of two separate experiments. DEVELOPMENT 2006 RESEARCH ARTICLE Development 135 (11) Hyaluronidase impacts gene expression and NFκB the phosphorylation of NFκB in cumulus cells of COCs, pathway activation in cumulus cells of ovulated responses that were reversed by anti-TLR2 and anti-TLR4 COCs neutralizing antibodies (Fig. 3B). When ovulated COCs were incubated with different concentrations of hyaluronidase for 2 hours, significant increases in the expression TLRs mediate the expression and secretion of of Il6, Ccl4 and Ccl5 mRNA were detected at the 1.0 IU/ml dose, cytokine/chemokine families during in vitro and further increases in Ccl5 mRNA were detected at hyaluronidase fertilization procedures concentrations of 10 or 100 IU/ml (Fig. 3A). Hyaluronidase (10 The expression of Il6 and Ccl5 mRNA in cumulus cells was also IU/ml) also significantly stimulated Ccl2 mRNA expression (Fig. induced within 2 hours by the co-culture of ovulated COCs with 3A). sperm (Fig. 4A). The induction of these genes in cumulus cells was The addition of anti-TLR2 and anti-TLR4 neutralizing suppressed by the addition of anti-TLR2 plus anti-TLR4 antibodies to the hyaluronidase-containing medium significantly neutralizing antibodies, or by treatment with the HA-blocking decreased the expression levels of Il6, Ccl2, Ccl4 and Ccl5 peptide Pep-1, described by Mummert et al. (Mummert et al., 2000), mRNA in cumulus cells of COCs, as compared with those in whereas the anti-CD44 antibody had no significant effect (Fig. 4A). COCs treated with 10 IU/ml hyaluronidase alone (Fig. 3B). These treatments did not significantly affect the expression of However, Snap25 mRNA expression, which is involved in the Snap25 mRNA (Fig. 4A). To analyze the secretion levels of exocytosis of these secreted factors, was not impacted by cytokines/chemokines, we collected the medium 0.5, 1, 2 or 4 hours hyaluronidase treatment and by the neutralizing antibodies (Fig. after ovulated COCs were cultured with capacitated sperm. The 3B). Hyaluronidase treatment induced the degradation of Iκβ and results show that high levels of IL6, CCL4 and CCL5 were secreted within 4 hours culture. The secretion of each cytokine was rapidly and significantly induced within 1 hour, and secretion further increased in time-dependent manner (Fig. 4B). After 1 hour of culture, the release of these cytokines from COCs was independent of de novo mRNA transcription, because addition of the transcriptional inhibitor α-amanitin (10 μg/ml) did not alter the levels of CCL4, CCL5 or IL6 in the culture medium (Fig. 4C). However, α-amanitin did significantly suppress the levels of these cytokines after 2 hours culture with sperm (Fig. 4C). Moreover, when COCs were cultured with sperm in the presence of anti-TLR2 plus anti-TLR4 neutralizing antibodies, significantly lower levels of cytokines were detected at both 1 and 2 hours of culture (Fig. 4C). Lastly, pronuclear formation in oocytes was analyzed after 12 hours of in vitro fertilization with sperm. Approximately 70% of the oocytes were fertilized and contained two pronuclei (Fig. 4D), responses that were reduced by the addition of neutralizing antibodies to TLR2/TLR4 (Fig. 4D). The HA-blocking peptide Pep- 1 also significantly suppressed pronuclear formation (Fig. 4D). Sperm express chemokine receptors (CCR1, CCR2, CCR3 and CCR5) that are required for sperm capacitation during the fertilization process Cumulus cells of cultured COCs secrete various kinds of cytokines/chemokines during the sperm-mediated fertilization process (see Table S1 in the supplementary material). Of note, IL6 and CC chemokine family members (CCL2, CCL3, CCL4 and CCL5) were predominantly produced from COCs at more than 100 pg/ml. Whereas CCL2 stimulates the CCR2 receptor, CCL3 binds mainly to Fig. 3. The effects of hyaluronidase (HY) treatment on gene CCR1 and partly to CCR5, and CCL4 selectively activates CCR5. All expression and NFκB pathway activation in cumulus cells of of receptors (CCR1, CCR2, CCR3 and CCR5) were activated by ovulated COCs. (A) Dose-dependent effects of HY on the expression CCL5 (Charo and Ransohoff, 2006). The CCR receptors are members of cytokine and chemokine mRNA. Ovulated COCs were treated with 0 of the G-protein coupling receptor family that induce phospholipase to 100 mIU/ml of HY for 2 hours. The 0 hour value was set as 1, and 2+ the data are presented as fold increase. Values are mean±s.e.m. of C to increase Ca and PKC activation in cytoplasm (Meyer et al., 2+ three replicates. Significant differences were observed as compared 1996). In sperm, the Ca -PKC pathway is involved in capacitation with COCs cultured without HY for 2 hours (Cont; *P<0.01; (Rotem et al., 1992), suggesting that the COC-secreted CC chemokine **P<0.05). (B) HY-induced cytokine and chemokine mRNA expression family might play an important role in sperm capacition during the and NFκB pathway activation were suppressed by both anti-TLR2 and fertilization process. The RT-PCR analysis shown in Fig. 5A shows anti-TLR4 neutralizing antibodies (Ab). Ovulated COCs were pre- the expression of Ccr1, Ccr2, Ccr3 and Ccr5 mRNA in sperm cultured with both anti-TLR2 and anti-TLR4 antibodies for 30 minutes, collected from the cauda epididymis. The positive immunofluorescent then 10 mIU/ml of HY was added to the medium. The addition of signals observed following the use of an anti-CCR3 antibody localized neutralizing antibodies significantly suppressed Il6, Ccl2, Ccl4 and Ccl5 CCR3 to luminal region of the testicular seminiferous tubules, and, at gene expression (*P<0.01; **P<0.05). Cont, COCs cultured without HY higher magnification, showed that CCR3 was detected near the tail of for 2 hours. Results of western blotting in each panel are representative of two separate experiments. spermatozoa; however these signals were very weak and a few cells DEVELOPMENT The role of TLR in fertilization RESEARCH ARTICLE 2007 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 α-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. also stained positive with the rabbit IgG control antibody (Fig. 5B). not dramatically changed from that of the control, for each ligand, Whole-mount preparations of spermatozoa provided additional the level of phosphorylation of other bands was increased within 30 evidence that CCR3 is present preferentially in the mid-piece of sperm minutes culture; the highest intensity was detected in sperm cultured (Fig. 5C). The immunoreactive band at about 55 kDa that corresponds with CCL5 for 60 minutes (Fig. 5E). to CCR3 was present in spleen used as positive control. In the testis To determine the role of CCL5 during the fertilization process, we sample, a 55 kDa band (as well as three other minor bands) was examined whether the addition of CCL5 could overcome the detected by the anti-CCR3 antibody. Sperm contained only one inhibitory effects of TLR neutralizing antibodies on fertilization, immuno-positive band of the correct size, indicating that sperm do and, conversely, whether antibodies to CCL5 could reduce express CCR3 (Fig. 5D). fertilization. The results showed that fertilization was suppressed by Protein tyrosine phosphorylation in spermatozoa is thought to be anti-TLR2/TLR4 neutralizing antibodies and that the addition of crucial for the acquisition of a capacitated state and the hyper- CCL5 slightly, but not significantly, increased the fertilization activation of motility (Naz et al., 1991). Tyrosine phosphorylation rate (Fig. 5F). However, the anti-CCL5 neutralizing antibody in protein extracts from sperm exposed to CCL2, CCL4 or CCL5 for significantly suppressed the percentage of oocytes that completed 30 and 60 minutes was analyzed by western blotting using a pan fertilization (Fig. 5F). anti-Tyr phosphorylation antibody. Positive immunoreactive bands It is known that when the high concentrations of sperm recovered were detected at ~110 kDa, ~75 kDa, 65 kDa and 50 kDa when from the cauda epididymis are cultured for more than 60 minutes sperm were cultured with BSA for 60 minutes (data not shown). (pre-culture), capacitation is induced spontaneously (Fraser, 1977). Although the level of phosphorylation of the band at ~110 kDa was Therefore, we examined whether the prolonged pre-culture of sperm DEVELOPMENT 2008 RESEARCH ARTICLE Development 135 (11) Fig. 5. Sperm express chemokine receptors required for sperm capacitation during the fertilization process. (A) The expression of Ccr1, Ccr2, Ccr3 and Ccr5 mRNA in sperm was determined by RT-PCR. (B,C) The localization of CCR3 in testicular seminiferous tubes (B) and sperm (C). Blue, DAPI staining of nuclei; green, FITC signal localizing the anti-CCR3 antibody. Scale bar: 10 μm. As a negative control, the slides were incubated with normal rabbit IgG and then reacted with secondary antibody. (D) Western analysis of CCR3 using the same primary antibody that was used for immunofluorescence. (E) The induction of protein tyrosine phosphorylation in sperm by CCL2, CCL4 or CCL5. Sperm collected from cauda epididymi were cultured with 100 pg/ml of CCL2, CCL4 or CCL5 for 30 or 60 minutes. Tyrosine phosphorylation (P-Tyr) was detected by an anti-phospho-Tyr antibody. (F) CCL5 regulates sperm penetration to oocytes. COCs were pre-cultured for 30 minutes with anti-TLR2 + anti-TLR4 antibodies (Anti- TLR2+TLR4), or with anti-CCL5 antibody (Anti-CCL5), and further cultured for 12 hours with sperm. In some cases, CCL5 (100 pg/ml) was added to the fertilization medium (+CCL5). Data are presented as the percentage of oocytes fertilized. Cont, ovulated COCs cultured with sperm for 12 hours. (G) The effects of the pre-culture period of sperm on oocyte fertilization in vitro. COCs were pre-cultured for 30 minutes with anti-TLR2 + anti-TLR4 antibodies (Anti-TLR2+TLR4), or with anti-CCL5 antibody (Anti-CCL5), and further cultured for 12 hours with sperm. The sperm were collected from the cauda epididymis, and then cultured for 0, 15, 30 or 60 minutes. Data are presented as the percentage of oocytes fertilized. that allows capacitation would overcome the negative effects of the and fertilization, we compared the levels of nine different chemokine anti-TLR2/4 neutralizing antibodies and the anti-CCL5 neutralizing family members and the rate of oocytes fertilized. Patients were antibody on sperm penetration. Without pre-culture or antibody separated into two groups: the Y group, in which all of the oocytes exposure, fertilization was highly variable (control, Fig. 5G). The were fertilized in vitro; and the N group, in which less than half of number of oocytes fertilized increased in controls in a duration- oocytes were fertilized. We detected IL6, CCL2, CCL4 and CCL5 dependent manner, with maximal success obtained when the sperm in the in vitro fertilization medium. The levels of IL6 and CCL2 were pre-cultured for 60 minutes (i.e. complete capacitation; Fig. were almost the same between two groups; however, CCL5 was 5G). The addition of neutralizing antibodies significantly suppressed slightly higher in Y group and CCL4 was significantly elevated (Fig. the penetration of sperm that were pre-cultured for 15 or 30 minutes 6B). (Fig. 5G). However, these negative effects were not detected when sperm were pre-cultured for 60 minutes and hence were fully DISCUSSION capacitated (Fig. 5G). Our previous microarray analyses revealed that cumulus cells of ovulated COCs express a unique set of genes associated with Tlr2 and Tlr4 mRNA are expressed in human specific immune cell-like responses. The TLRs and related factors cumulus cells and cytokine/chemokine production were of particular interest because they represent part of the immune may impact human fertility cell surveillance system that monitors changes in the external Human cumulus cells obtained from patients undergoing an IVF environment (Hernandez-Gonzalez et al., 2006; Shimada et al., protocol expressed Tlr2 and Tlr4 mRNA (Fig. 6A). To determine the 2006). Specifically, we have documented that, when ovulated COCs relationship between human COC-secreted cytokines/chemokines were cultured with the TLR4 ligand bacterial lipopolysaccharide DEVELOPMENT The role of TLR in fertilization RESEARCH ARTICLE 2009 supplementary material), blocked the effects of HA. The HA-rich matrix generated during COC expansion is presumed to be of high molecular weight, as cumulus cells express primarily HAS2 (Fülöp et al., 1997a), which is known to generate HA with a broad but extremely large size (average molecular weight of more than 210 kDa) (Itano et al., 1999). Although the functions of high molecular weight HA are not entirely clear for any cell system, it can bind and activate CD44, but not TLR2 and TLR4 in macrophages (Cuff et al., 2001; Chang et al., 2007). To determine whether HA is a physiologically relevant ligand for TLRs on cumulus cells, ovulated COCs were treated with exogenous hyaluronidase or were cultured with sperm that are known to secrete hyaluronidase. The results show that exogenous hyaluronidase significantly upregulated Il6, Ccl2, Ccl4 and Ccl5 mRNA expression, and induced activation of the NFκB pathway in cumulus cells. The induction of these genes and the activation of specific signaling pathways by hyaluronidase were suppressed by anti-TLR2/TLR4 neutralizing antibodies. Moreover, the culture with sperm induced the expression of Il6 and Ccl5 mRNA through TLR2/4-dependent, but CD44-independent, mechanisms. Additionally, we used the HA blocking peptide Pep-1, which has been shown to function effectively in vitro to inhibit the binding of HA to its receptors (Mummert et al., 2000; Taylor et al., 2004). Pep-1 significantly suppressed the expression of Il6 and Ccl5 mRNA in cumulus cells and sperm penetration in in vitro Fig. 6. The expression of Tlr2 and Tlr4 mRNA and the role of fertilization assays. It has been shown that hyaluronidase cytokines/chemokines in human fertility. (A) The expression of Tlr2 depolymerizes high molecular weight HA to 10-40 kDa end and Tlr4 mRNA in cumulus cells of human COCs. The cumulus cells products (Sampson et al., 1992), suggesting that the small HA were recovered from COCs after in vitro fertilization. (B) The fragments generated by sperm-secreted hyaluronidase probably relationship between the amount of secreted CCL cytokines and activated TLR2 and TLR4 on cumulus cells, leading to cytokine and fertilization in vitro. The cytokines present in the media were analyzed chemokine expression via the NFκB pathway. by using the Bio-Plex Human Cytokine 9-Plex Panel. Y, all oocytes were The high level of cytokine secretion from cumulus cells 2-4 hours successfully fertilized; N, more than half of the oocytes were not after exposure to sperm was dependent on the increased fertilized. P values are for Y versus N. transcription of specific genes, as α-amanitin blocked this effect. By contrast, the initial rapid release of IL6, CCL4 and CCL5 by the (LPS), the expression of Il6 and Tnfα was induced. However, the TLR2/TLR4 pathway during fertilization did not require de novo endogenous ligand(s) for TLR2/TLR4 and the physiological role of mRNA synthesis. The mechanism by which the rapid release is this signaling cascade in ovulated COCs remained unclear. Because regulated could involve an exocytosis system because cumulus cells cumulus cells produce and are surrounded by a HA-rich express components of the SNARE complex [synaptosomal- extracellular matrix and because HA fragments have been shown associated protein 25 (Snap25), syntaxin 1a (Stx1a) and recently to activate TLR2 and TLR4 (Termeer et al., 2002; Jiang et synaptotagmin 1 (Syt1)] (Shimada et al., 2007) (M.S., unpublished). al., 2005), we hypothesized that HA fragments generated during In mast cells, vesicle degranulation and the release of cytokines matrix degradation might act as an endogenous ligand for TLR2 occurs in a TLR2- and TLR4-dependent manner (Supajatura et al., and/or TLR4 that are present in cumulus cells of ovulated COCs. 2002), via exocytosis involving SNAP25 localized to the secretory Herein, we provide the first evidence that TLRs can play a functional granules (Salinas et al., 2004; Stow et al., 2006). Thus, it is possible role in the, at least in vitro, fertilization process. Notably, we show that the SNAP25-associated exocytosis system present in cumulus that hyaluronidase treatment and culture with sperm (used as a cells is also activated by the small HA fragment-induced TLR biological source of hyaluronidase) activate TLR2/TLR4 on pathway during the fertilization process, although the precise cumulus cells. Activated cumulus cells, but not sperm itself, then mechanisms remain to be resolved. release specific cytokines capable of enhancing sperm capacitation The functional role of secreted CC chemokines appears to be part and fertilization. Thus, a functional regulatory loop appears to be of a regulatory loop between COCs and sperm, because operative between sperm and the ovulated COCs. chemokines can enhance fertilization by the activation of GPCRs Specifically, the results presented here document that exposure of (CCRs) and calcium release. We show here that multiple CCRs are ovulated COCs to HA fragments induced the phosphorylation of expressed in mouse sperm, as well as in human sperm (Isobe et al., p38MAP kinase, ERK1/2 and NFκB, and the expression of specific 2002; Muciaccia et al., 2005), and that CCR3 is localized to the genes (Il6, Ccl2, Ccl4 and Ccl5) in cumulus cells. This response was mid-piece of mature sperm. During capacitation, the increase of 2+ similar to that observed when ovulated COCs were exposed to LPS, Ca is observed around the mid-piece of spermatozoa (Harper et 2+ a known ligand of TLR4 (Shimada et al., 2006), indicating that HA al., 2004), and the Ca -dependent pathway evokes protein tyrosine fragments could serve as ligands for cumulus cell TLRs. The ability phosphorylation (Carrera et al., 1996), suggesting that secreted CC of HA fragments to active these signaling cascades and to induce the chemokine families are involved in sperm capacitation. When expression of specific cytokines in cumulus cells was selectively sperm collected from cauda epididymi were cultured with CCL2, mediated by TLR2 and TLR4, as TLR2 and TLR4 neutralizing CCL4 or CCL5, increased levels of immunoreactive phospho- antibodies, but not the anti-CD44 antibody (see Fig. S1 in the tyrosine were detected in extracts of the CCL5 treatment group. DEVELOPMENT 2010 RESEARCH ARTICLE Development 135 (11) CCL5 treatment also increased sperm motility in a dose-dependent receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation. J. Clin. Invest. 108, 1031-1040. manner (see Fig. S2 in the supplementary material). Moreover, Fieber, C., Baumann, P., Vallon, R., Termeer, C., Simon, J. C., Hofmann, M., using COC-conditioned medium, we showed that factors secreted Angel, P., Herrlich, P. and Sleeman, J. P. (2004). Hyaluronan-oligosaccharide- by COCs induced not only sperm motility but also capacitation, and induced transcription of metalloproteases. J. Cell. Sci. 117, 359-367. Fraser, L. R. (1977). Differing requirements for capacitation in vitro of mouse that these effects were suppressed by the addition of anti-CCL5 spermatozoa from two strains. J. Reprod. Fertil. 49, 83-87. neutralizing antibody (see Fig. S3 in the supplementary material). Fülöp, C., Salustri, A. and Hascall, V. C. (1997a). Coding sequence of a Furthermore, the fertilization of oocytes was suppressed hyaluronan synthase homologue expressed during expansion of the mouse cumulus-oocyte complex. Biochem. Biophys. 337, 261-266. significantly by either anti-TLR2/TLR4 or anti-CCL5 neutralizing Fülöp, C., Kamath, R. V., Li, Y., Otto, J. M., Salustri, A., Olsen, B. R., Glant, T. antibodies if short-term pre-cultured sperm was used for T. and Hascall, V. C. (1997b). Coding sequence, exon-intron structure and insemination. However, after prolonged pre-culture (over 60 chromosomal localization of murine TNF-stimulated gene 6 that is specifically minutes), which mediates complete sperm capacitation, fertilization expressed by expanding cumulus cell-oocyte complexes. Gene 202, 95-102. Fülöp, C., Szántó, S., Mukhopadhyay, D., Bárdos, T., Kamath, R. V., Rugg, M. was not impaired by the presence of neutralizing antibodies. Based S., Day, A. J., Salustri, A., Hascall, V. C., Glant, T. T. and Mikecz, K. (2003). on these results, we conclude that during the fertilization process, Impaired cumulus mucification and female sterility in tumor necrosis factor- TLR2 and TLR4 present on the cumulus cells are activated by co- induced protein-6 deficient mice. Development 130, 2253-2261. Harper, C. V., Barratt, C. L. and Publicover, S. J. (2004). Stimulation of human culture with sperm in a hyaluronan fragment-dependent manner, spermatozoa with progesterone gradients to simulate approach to the oocyte. leading to the secretion of CCL5 and other CC chemokine family 2+ Induction of [Ca ] oscillations and cyclical transitions in flagellar beating. J. Biol. members. These, in turn, stimulate CC receptors on sperm to Chem. 279, 46315-46325. enhance sperm motility and to induce sperm capacitation, thereby Hernandez-Gonzalez, I., Gonzalez-Robayna, I., Shimada, M., Wayne, C. M., Ochsner, S. A., White, L. and Richards, J. S. (2006). Gene expression profiles enhancing successful fertilization. Thus, there is a functional of cumulus cell oocyte complexes during ovulation reveal cumulus cells express regulatory loop between the COCs and sperm. neuronal and immune-related genes: does this expand their role in the ovulation Finally, we report that Tlr2 and Tlr4 mRNA are also expressed in process? Mol. Endocrinol. 20, 1300-1321. Hizaki, H., Segi, E., Sugimoto, Y., Hirose, M., Saji, T., Ushikubi, F., Matsuoka, human peri-ovulatory cumulus cells, and that human COCs secret T., Noda, Y., Tanaka, T., Yoshida, N. et al. (1999). Abortive expansion of the IL6, CCL2, CCL4 and CCL5 during in vitro fertilization. Therefore, cumulus and impaired fertility in mice lacking the prostaglandin E receptor it is likely that the TLR system in human cumulus cells plays a subtype EP(2). Proc. Natl. Acad. Sci. USA 96, 10501-10506. Hodge-Dufour, J., Noble, P. W., Horton, M. R., Bao, C., Wysoka, M., Burdick, similar role to in mouse cells in facilitating fertilization. Importantly, M. D., Strieter, R. M., Trinchieri, G. and Puré, E. (1997). Induction of IL-12 human oocytes collected from COCs that produced high amounts of and chemokines by hyaluronan requires adhesion-dependent priming of resident CCL4 or CCL5 showed higher fertilization rates than did oocytes but not elicited macrophages. J. Immunol. 159, 2492-2500. from COCs that produced low levels of these cytokines. On the basis Isobe, T., Minoura, H., Tanaka, K., Shibahara, T., Hayashi, N. and Toyoda, N. (2002). The effect of RANTES on human sperm chemotaxis. Hum. Reprod. 17, of these data in human and mouse COCs, the addition of CC 1441-1446. chemokines such as CCL5 might improve IVF or intracytoplasmic Itano, N., Sawai, T., Yoshida, M., Lenas, P., Yamada, Y., Imagawa, M., sperm injection (ICSI) protocols. Shinomura, T., Hamaguchi, M., Yoshida, Y., Ohnuki, Y. et al. (1999). Three isoforms of mammalian hyaluronan synthases have distinct enzymatic The authors are grateful to Dr S. Tanabe, Hiroshima University, for technical properties. J. Biol. Chem. 274, 25085-25092. advice on the use of the Bio-Plex Protein Array System; Dr Y. Yoshimura, Dr N. Jiang, D., Liang, J., Fan, J., Yu, S., Chen, S., Luo, Y., Prestwich, G. D., Mascarenhas, M. M., Garg, H. G., Quinn, D. A. et al. (2005). Regulation of Isobe and Mr. A. Ozoe for supporting the making of the frozen sections; and lung injury and repair by Toll-like receptors and hyaluronan. Nat. Med. 11, 1173- Dr K. Uematsu for technical advice on the use of microscope. We also thank Mr. K. Kinoshita, Daigo-Watanabe Clinic, for collecting IVF medium, and Ms K. Kawai, T. and Akira, S. (2005). Toll-like receptor downstream signaling. Arthritis. Nishimatsu for technical assistance. This work was supported, in part, by Res. Ther. 7, 12-19. Grant-in-Aid for Scientific Research No.18688016 from the Japan Society for Kennedy, C. R., Zhang, Y., Brandon, S., Guan, Y., Coffee, K., Funk, C. D., the Promotion of Science (JSPS) and The Kao Foundation for Arts and Sciences Magnuson, M. A., Oates, J. A., Breyer, M. D. and Breyer, R. M. (1999). Salt- (to M.S.), and the National Institutes of Health HD-16229 and HD-07495 sensitive hypertension and reduced fertility in mice lacking the prostaglandin EP2 (Project III, Specialized Cooperative Program in Reproductive Research, SCPRR; receptor. Nat. Med. 5, 217-220. to J.S.R.). Kimura, N., Konno, Y., Miyoshi, K., Matsumoto, H. and Sato, E. (2002). Expression of hyaluronan synthases and CD44 messenger RNAs in porcine Supplementary material cumulus-oocyte complexes during in vitro maturation. Biol. Reprod. 66, 707- Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/135/11/2001/DC1 Kothapalli, D., Zhao, L., Hawthorne, E. A., Cheng, Y., Lee, E., Puré, E. and Assoian, R. K. (2007). Hyaluronan and CD44 antagonize mitogen-dependent References cyclin D1 expression in mesenchymal cells. J. Cell Biol. 176, 535-544. Aruffo, A., Stamenkovic, I., Melnick, M., Underhill, C. B. and Seed, B. (1990). Lim, H., Paria, B. C., Das, S. K., Dinchuk, J. E., Langenbach, R., Trzaskos, J. M. CD44 is the principal cell surface receptor for hyaluronate. Cell 61, 1303-1313. and Dey, S. K. (1997). Multiple female reproductive failures in cyclooxygenase Camaioni, A., Hascall, V. C., Yanagishita, M. and Salustri, A. (1993). Effects of 2-deficient mice. Cell 91, 197-208. exogenous hyaluronic acid and serum on matrix organization and stability in the Matsumoto, H., Ma, W., Smalley, W., Trzaskos, J., Breyer, R. M. and Dey, S. mouse cumulus cell-oocyte complex. J. Biol. Chem. 268, 20473-20481. K. (2001). Diversification of cyclooxygenase-2-derived prostaglandins in Carrera, A., Moos, J., Ning, X. P., Gerton, G. L., Tesarik, J., Kopf, G. S. and ovulation and implantation. Biol. Reprod. 64, 1557-1565. Moss, S. B. (1996). Regulation of protein tyrosine phosphorylation in human Meyer, A., Coyle, A. J., Proudfoot, A. E., Wells, T. N. and Power, C. A. (1996). sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase Cloning and characterization of a novel murine macrophage inflammatory anchor proteins as major substrates for tyrosine phosphorylation. Dev. Biol. 180, protein-1 alpha receptor. J. Biol. Chem. 271, 14445-14451. 284-296. Miyake, K., Underhill, C. B., Lesley, J. and Kincade, P. W. (1990). Hyaluronate Chang, E. J., Kim, H. J., Ha, J., Kim, H. J., Ryu, J., Park, K. H., Kim, U. H., Lee, can function as a cell adhesion molecule and CD44 participates in hyaluronate Z. H., Kim, H. M., Fisher, D. E. and Kim, H. H. (2007). Hyaluronan inhibits recognition. J. Exp. Med. 172, 69-75. osteoclast differentiation via Toll-like receptor 4. J. Cell. Sci. 120, 166-176. Muciaccia, B., Padula, F., Vicini, E., Gandini, L., Lenzi, A. and Stefanini, M. Charo, I. F. and Ransohoff, R. M. (2006). The many roles of chemokines and (2005). Beta-chemokine receptors 5 and 3 are expressed on the head region of chemokine receptors in inflammation. N. Engl. J. Med. 354, 610-621. human spermatozoon. FASEB J. 19, 2048-2050. Chen, L., Mao, S. J. and Larsen, W. J. (1992). Identification of a factor in fetal Mummert, M. E., Mohamadzadeh, M., Mummert, D. I., Mizumoto, N. and bovine serum that stabilizes the cumulus extracellular matrix. A role for a Takasima, A. (2000). Development of a peptide inhibitor of hyaluronan- member of the inter-alpha-trypsin inhibitor family. J. Biol. Chem. 267, 12380- mediated leukocyte trafficking. J. Exp. Med. 192, 769-779. 12386. Naz, R. K., Ahmad, K. and Kumar, R. (1991). Role of membrane Cuff, C. A., Kothapalli, D., Azonobi, I., Chun, S., Zhang, Y., Belkin, R., Yeh, C., phosphotyrosine proteins in human spermatozoal function. J. Cell. Sci. 99, 157- Secreto, A., Assoian, R. K., Rader, D. J. and Puré, E. (2001). The adhesion 165. DEVELOPMENT The role of TLR in fertilization RESEARCH ARTICLE 2011 Ochsner, S. A., Day, A. J., Rugg, M. S., Breyer, R. M., Gomer, R. H. and Sampson, P. M., Rochester, C. L., Freundlich, B. and Elias, J. A. (1992). Richards, J. S. (2003a). Disrupted function of tumor necrosis factor-alpha- Cytokine regulation of human lung fibroblast hyaluronan (hyaluronic acid) stimulated gene 6 blocks cumulus cell-oocyte complex expansion. Endocrinology production. Evidence for cytokine-regulated hyaluronan (hyaluronic acid) 144, 4376-4384. degradation and human lung fibroblast-derived hyaluronidase. J. Clin. Invest. 90, Ochsner, S. A., Russell, D. L., Day, A. J., Breyer, R. M. and Richards, J. S. 1492-1503. (2003b). Decreased expression of tumor necrosis factor-alpha-stimulated gene 6 Shimada, M., Hernandez-Gonzalez, I., Gonzalez-Robanya, I. and Richards, J. in cumulus cells of the cyclooxygenase-2 and EP2 null mice. Endocrinology 144, S. (2006). Induced expression of pattern recognition receptors in cumulus 1008-1019. oocyte complexes: novel evidence for innate immune-like functions during Ohta, N., Saito, H., Kuzumaki, T., Takahashi, T., Ito, M. M., Saito, T., ovulation. Mol. Endocrinol. 20, 3228-3239. Nakahara, K. and Hiroi, M. (1999). Expression of CD44 in human cumulus and Shimada, M., Yanai, Y., Okazaki, T., Yamashita, Y., Sriraman, V., Wilson, M. mural granulosa cells of individual patients in in-vitro fertilization programmes. C. and Richards, J. S. (2007). Synaptosomal-associated protein 25 gene Mol. Hum. Reprod. 5, 22-28. expression is hormonally regulated during ovulation and is involved in cytokine/ Richards, J. S. (2005). Ovulation: new factors that prepare the oocyte for chemokine exocytosis from granulosa cells. Mol. Endocrinol. 21, 2487-2502. fertilization. Mol. Cell. Endocrinol. 234, 75-79. Srivastava, P., Adams, C. E. and Hartree, E. F. (1965). Enzymatic action of Richards, J. S., Russell, D. L., Ochsner, S. and Espey, L. L. (2002). Ovulation: lipoglycoprotein preparations from sperm-acrosomes on rabbit ova. Nature 205, new dimensions and new regulators of the inflammatory-like response. Annu. 498. Rev. Physiol. 64, 69-92. Stow, J. L., Manderson, A. P. and Murray, R. Z. (2006). SNAREing immunity: the Robker, R. L., Russell, D. L., Espey, L. L., Lydon, J. P., O’Malley, B. W. and role of SNAREs in the immune system. Nat. Rev. Immunol. 6, 919-929. Richards, J. S. (2000). Progesterone-regulated genes in the ovulation process: Supajatura, V., Ushio, H., Nakao, A., Akira, S., Okumura, K., Ra, C. and ADAMTS-1 and cathespin L proteases. Proc. Natl. Acad. Sci. USA 97, 4689- Ogawa, H. (2002). Differential responses of mast cell Toll-like receptors 2 and 4 4694. in allergy and innate immunity. J. Clin. Invest. 109, 1351-1359. Rogers, B. J. and Morton, B. E. (1973). The release of hyaluronidase from Talbot, P. and Franklin, L. E. (1974). The release of hyaluronidase from guinea- capacitating hamster spermatozoa. J. Reprod. Fertil. 35, 477-487. pig spermatozoa during the course of the normal acrosome reaction in vitro. J. Rotem, R., Paz, G. F., Homonnai, Z. T., Kalina, M., Lax, J., Breitbart, H. and Reprod. Fertil. 39, 429-432. 2+ Naor, Z. (1992). Ca -independent induction of acrosome reaction by protein Taylor, K. R., Trowbridge, J. M., Rudisill, J. A., Termeer, C. C., Simon, J. C. and kinase C in human sperm. Endocrinology 131, 2235-2243. Gallo, R. L. (2004). Hyaluronan fragments stimulate endothelial recognition of Salinas, E., Ventura, J., Córdova, L. E. and Luis Quintanar, J. (2004). Presence injury through TLR4. J. Biol. Chem. 279, 17079-17084. of SNAP-25 in rat mast cells. Immunol. Lett. 95, 105-108. Termeer, C., Benedix, F., Sleeman, J., Fieber, C., Voith, U., Ahrens, T., Miyake, Salustri, A., Camaioni, A., Di Giacomo, M., Fulop, C. and Hascall, V. C. (1995). K., Freudenberg, M., Galanos, C. and Simon, J. C. (2002). Oligosaccharides Hyaluronan and proteoglycans in ovarian follicles. Hum. Reprod. Update. 5, 293- of Hyaluronan activate dendritic cells via toll-like receptor 4. J. Exp. Med. 195, 301. 99-111. Salustri, A., Garlanda, C., Hirsch, E., De Acetis, M., Maccagno, A., Bottazzi, Varani, S., Elvin, J. A., Yan, C., DeMayo, J., DeMayo, F. J., Horton, H. F., B., Doni, A., Bastone, A., Mantovani, G., Beck Peccoz, P. et al. (2004). PTX3 Byrne, M. C. and Matzuk, M. M. (2002). Knockout of pentraxin 3, a plays a key role in the organization of the cumulus oophorus extracellular matrix downstream target of growth differentiation factor-9, causes female subfertility. and in in vivo fertilization. Development 131, 1577-1586. Mol. Endocrinol. 16, 1154-1167. DEVELOPMENT

Journal

Development – The Company of Biologists

Published: Jun 1, 2008

There are no references for this article.