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First published online 23 April 2008
doi: 10.1242/dev.020461

Development 135, 2001-2011 (2008)
Published by The Company of Biologists 2008
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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

Masayuki Shimada1,*, Yoshiari Yanai1, Tetsuji Okazaki1, Noritaka Noma1, Ikkou Kawashima1, Takahide Mori2 and JoAnne S. Richards3

1 Department of Applied Animal Science, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8528, Japan.
2 Infertility Center, Daigo-Watanabe Clinic, Fushimi-ku, Kyoto, 601-1375, Japan.
3 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.


Figure 1
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  		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.

 

Figure 2
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  		Fig. 2. HA fragments induced the TLR2-, TLR4- and CD44-targeted signal transduction pathways. (A) Time-dependent changes of the activation of p38MAP kinase, ERK1/2 and the NF{kappa}B pathway in cumulus cells of ovulated COCs cultured with 100 µg/ml of HA fragments. Results in each panel are representative of two separate experiments. (B) Ovulated COCs were pre-cultured with neutralizing antibodies (both anti-TLR2 and anti-TLR4, or anti-CD44) for 30 minutes, and further cultured with HA fragments for 15 minutes (p38MAP kinase and ERK1/2), or for 2 hours (I{kappa}β and NF{kappa}B). Results in each panel are representative of two separate experiments.

 

Figure 3
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  		Fig. 3. The effects of hyaluronidase (HY) treatment on gene expression and NF{kappa}B pathway activation in cumulus cells of ovulated COCs. (A) Dose-dependent effects of HY on the expression of cytokine and chemokine mRNA. Ovulated COCs were treated with 0 to 100 mIU/ml of HY 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 HY for 2 hours (Cont; *P<0.01; **P<0.05). (B) HY-induced cytokine and chemokine mRNA expression and NF{kappa}B pathway activation were suppressed by both anti-TLR2 and anti-TLR4 neutralizing antibodies (Ab). Ovulated COCs were pre-cultured with both anti-TLR2 and anti-TLR4 antibodies for 30 minutes, then 10 mIU/ml of HY was added to the medium. The addition of neutralizing antibodies significantly suppressed Il6, Ccl2, Ccl4 and Ccl5 gene expression (*P<0.01; **P<0.05). Cont, COCs cultured without HY for 2 hours. Results of western blotting in each panel are representative of two separate experiments.

 

Figure 4
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  		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 {alpha}-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.

 

Figure 5
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  		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.

 

Figure 6
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  		Fig. 6. The expression of Tlr2 and Tlr4 mRNA and the role of cytokines/chemokines in human fertility. (A) The expression of Tlr2 and Tlr4 mRNA in cumulus cells of human COCs. The cumulus cells were recovered from COCs after in vitro fertilization. (B) The relationship between the amount of secreted CCL cytokines and fertilization in vitro. The cytokines present in the media were analyzed by using the Bio-Plex Human Cytokine 9-Plex Panel. Y, all oocytes were successfully fertilized; N, more than half of the oocytes were not fertilized. P values are for Y versus N.

 




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