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.