Instituto de Biología y Medicina Experimental, Buenos Aires, Argentina.
Biol Reprod. 2011 Sep;85(3):503-10. doi: 10.1095/biolreprod.111.091439. Epub 2011 May 18.
Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm during maturation and participates in fertilization. Evidence indicates the existence of two populations of CRISP1 in sperm: one loosely bound and released during capacitation, and one strongly bound that remains after this process. However, the mechanisms underlying CRISP1 binding to sperm remain mostly unknown. Considering the high concentrations of Zn(2+) present in the epididymis, we investigated the potential involvement of this cation in the association of CRISP1 with sperm. Caput sperm were coincubated with epididymal fluid in the presence or absence of Zn(2+), and binding of CRISP1 to sperm was examined by Western blot analysis. An increase in CRISP1 was detected in sperm exposed to Zn(2+), but not if the cation was added with ethylenediaminetetra-acetic acid (EDTA). The same results were obtained using purified CRISP1. Association of CRISP1 with sperm was dependent on epididymal fluid and Zn(2+) concentrations and incubation time. Treatment with NaCl (0.6 M) removed the in vitro-bound CRISP1, indicating that it corresponds to the loosely bound population. Flow cytometry of caput sperm exposed to biotinylated CRISP1/avidin-fluorescein isothiocyanate revealed that only the cells incubated with Zn(2+) exhibited an increase in fluorescence. When these sperm were examined by epifluorescence microscopy, a clear staining in the tail, accompanied by a weaker labeling in the head, was observed. Detection of changes in the tryptophan fluorescence emission spectra of CRISP1 when exposed to Zn(2+) supported a direct interaction between CRISP1 and Zn(2+). Incubation of either cauda epididymal fluid or purified CRISP1 with Zn(2+), followed by native-PAGE and Western blot analysis, revealed the presence of high-molecular-weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results support the involvement of CRISP1-Zn(2+) complexes in the association of the loosely bound population of CRISP1 with sperm during epididymal maturation.
大鼠附睾蛋白 CRISP1（富含半胱氨酸的分泌蛋白 1）在精子成熟过程中与精子结合，并参与受精。有证据表明，精子中存在两种 CRISP1 群体：一种在获能过程中松散结合并释放，另一种在这个过程后仍然紧密结合。然而，CRISP1 与精子结合的机制在很大程度上仍不清楚。考虑到附睾中存在高浓度的 Zn(2+)，我们研究了这种阳离子是否参与了 CRISP1 与精子的结合。将头段精子与附睾液在有或没有 Zn(2+)的情况下共同孵育，然后通过 Western blot 分析检查 CRISP1 与精子的结合。结果显示，暴露于 Zn(2+)的精子中 CRISP1 增加，但如果加入乙二胺四乙酸（EDTA）则没有增加。使用纯化的 CRISP1 也得到了相同的结果。CRISP1 与精子的结合依赖于附睾液和 Zn(2+)浓度以及孵育时间。用 0.6 M NaCl 处理可去除体外结合的 CRISP1，表明其对应于松散结合的群体。用生物素化 CRISP1/亲和素-荧光素异硫氰酸酯处理头段精子的流式细胞术显示，只有与 Zn(2+)孵育的细胞荧光强度增加。当用荧光显微镜检查这些精子时，在尾部观察到明显的染色，头部则显示较弱的标记。当 CRISP1 暴露于 Zn(2+)时，检测到色氨酸荧光发射光谱的变化，支持 CRISP1 与 Zn(2+)之间的直接相互作用。用 Zn(2+)孵育尾段附睾液或纯化的 CRISP1，然后进行非变性聚丙烯酰胺凝胶电泳和 Western blot 分析，结果显示在未用 EDTA 处理的溶液中未检测到高分子量的 CRISP1 复合物。综上所述，这些结果支持 CRISP1-Zn(2+) 复合物参与了精子在附睾成熟过程中与松散结合的 CRISP1 群体的结合。
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