A novel function for CRISP1 in rodent fertilization: Involvement in sperm-zona pellucida interaction
A novel function for CRISP1 in rodent fertilization: Involvement in sperm-zona pellucida interaction
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DOI：
10.1095/biolreprod.107.061788
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发表时间：
2007-11-01
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;Cohen, Debora J.;Cuasnicu, Patricia S.
附睾蛋白CRISP1通过与卵表面互补位点的相互作用参与大鼠和小鼠配子融合。基于体内观察，在本研究中，我们研究了CRISP1在配子融合之前的精子-透明带（ZP）相互作用中发挥额外作用的可能性。利用带完整的大鼠和小鼠卵子进行的体外受精实验表明，在配子共孵育期间，抗大鼠CRISP1抗体（anti-CRISP1）或大鼠原生CRISP1抗体（rCRISP1）的存在都会显著降低受精卵的百分比。然而，与参与配子融合的蛋白质不同的是，卵泡周围精子没有积累，这表明抑制发生在精子- zp相互作用水平。细菌表达的重组CRISP1 （recCRISP1）也显著抑制卵子受精。然而，在这种情况下，卵泡周围精子的数量有所增加。随后的实验评估了抗crisp1或rCRISP1对每个卵子结合的精子数量的影响，表明该蛋白参与了精子- zp结合的初始步骤。与这些功能研究相一致的是，间接免疫荧光实验显示，尽管rCRISP1能够结合ZP和ololema，但它只结合在卵子表面。研究发现，去糖基化的rCRISP1表现为未经处理的蛋白，而热变性的rCRISP1仅与肠水肿相关，这表明蛋白质的zp结合能力存在于构象中，而不是分子的糖基部分。rCRISP1和ZP之间的相互作用再现了精子与ZP结合的行为，这可以通过rCRISP1蛋白无法与受精卵的ZP相互作用来判断。总之，这些结果支持CRISP1不仅参与精子-卵子融合，还参与精子- zp相互作用的前期阶段的观点。
Epididymal protein CRISP1 participates in rat and mouse gamete fusion through its interaction with complementary sites on the egg surface. Based on in vivo observations, in the present study we investigated the possibility that CRISP1 plays an additional role in the sperm-zona pellucida (ZP) interaction that precedes gamete fusion. In vitro fertilization experiments using zona-intact rat and mouse eggs indicated that the presence of either an antibody against rat CRISP1 (anti-CRISP1) or rat native CRISP1 (rCRISP1) during gamete co-incubation produced a significant decrease in the percentage of fertilized eggs. However, differently to that expected for a protein involved in gamete fusion, no accumulation of perivitelline sperm was observed, suggesting that the inhibitions occurred at the sperm-ZP interaction level. Bacterially expressed recombinant CRISP1 (recCRISP1) also significantly inhibited egg fertilization. in this case, however, an increase in the number of perivitelline sperm was observed. Subsequent experiments evaluating the effect of anti-CRISP1 or rCRISP1 on the number of sperm bound per egg indicated that the protein is involved in the initial step of sperm-ZP binding. in agreement with these functional studies, indirect immunofluorescence experiments revealed that although rCRISP1 is capable of binding to both the ZP and the oolema, recCRISP1 only binds to the egg surface. The finding that deglycosylated rCRISP1 behaves as the untreated protein, whereas the heat-denatured rCRISP1 associated only with the oolema, indicates that the protein ZP-binding ability resides in the conformation rather than in the glycosydic portion of the molecule. The interaction between rCRISP1 and the ZP reproduces the sperm-ZP-binding behavior, as judged by the failure of the protein to interact with the ZP of fertilized eggs. Together, these results support the idea that CRISP1 participates not only in sperm-egg fusion but also in the prior stage of sperm-ZP interaction.