Evidence for the involvement of testicular protein CRISP2 in mouse sperm-egg fusion
Evidence for the involvement of testicular protein CRISP2 in mouse sperm-egg fusion
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DOI：
10.1095/biolreprod.106.056770
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发表时间：
2007-04-01
影响因子：
3.6
通讯作者：
Cuasnicu, Patricia S.
中科院分区：
文献类型：
--
作者：
Busso, Dolores;Goldweic, Nadia M.;Cuasnicu, Patricia S.
CRISP 2，最初称为Tpx-1，是一种富含半胱氨酸的分泌蛋白，特异性表达于雄性单倍体生殖细胞中。虽然可能参与配子相互作用，但CRISP 2在受精中的功能作用的证据仍然很差。在本研究中，我们使用小鼠模型来研究CRISP 2在精子中的亚细胞定位及其参与受精的不同阶段。间接免疫荧光和蛋白提取实验的结果表明，小鼠CRISP 2是顶体内组分，在获能和顶体反应（AR）后仍与精子相关。在体外受精试验中，使用透明质酸完整的小鼠卵子表明，针对该蛋白质的抗体显着降低穿透卵子的百分比，同时积累的卵周精子。未能抑制透明质酸穿透排除了抗体对精子运动性或AR的不利影响，支持CRISP 2在精卵融合步骤中的特异性参与。与这一证据一致，重组小鼠CRISP 2（recCRISP 2）特异性结合到小鼠卵子的融合区域，如先前报道的大鼠CRISP 1，一种参与配子融合的附睾蛋白。体外竞争研究表明，用固定浓度的recCRISP 2和增加量的大鼠CRISP 1孵育小鼠无透明带卵子会减少recCRISP 2与卵子的结合，这表明这些蛋白质与卵子表面的共同互补位点相互作用。我们的研究结果表明，睾丸CRISP 2与附睾CRISP 1一样，通过与卵子表面的互补位点结合参与精卵融合，支持同源分子之间的功能合作以确保受精成功的想法。
CRISP2, originally known as Tpx-1, is a cysteine-rich secretory protein specifically expressed in male haploid germ cells. Although likely to be involved in gamete interaction, evidence for a functional role of CRISP2 in fertilization still remains poor. In the present study, we used a mouse model to examine the subcellular localization of CRISP2 in sperm and its involvement in the different stages of fertilization. Results from indirect immunofluorescence and protein extraction experiments indicated that mouse CRISP2 is an intraacrosomal component that remains associated with sperm after capacitation and the acrosome reaction (AR). In vitro fertilization assays using zona pellucida-intact mouse eggs showed that an antibody against the protein significantly decreased the percentage of penetrated eggs, with a coincident accumulation of perivitelline sperm. The failure to inhibit zona pellucida penetration excludes a detrimental effect of the antibody on sperm motility or the AR, supporting a specific participation of CRISP2 at the sperm-egg fusion step. In agreement with this evidence, recombinant mouse CRISP2 (recCRISP2) specifically bound to the fusogenic area of mouse eggs, as previously reported for rat CRISP1, an epididymal protein involved in gamete fusion. In vitro competition investigations showed that incubation of mouse zona-free eggs with a fixed concentration of recCRISP2 and increasing amounts of rat CRISP1 reduced the binding of recCRISP2 to the egg, suggesting that the proteins interact with common complementary sites on the egg surface. Our findings indicate that testicular CRISP2, as observed for epididymal CRISP1, is involved in sperm-egg fusion through its binding to complementary sites on the egg surface, supporting the idea of functional cooperation between homologous molecules to ensure the success of fertilization.