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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.
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