Da Ros Vanina G, Munuce María J, Cohen Débora J, Marín-Briggiler Clara I, Busso Dolores, Visconti Pablo E, Cuasnicú Patricia S
Instituto de Biología y Medicina Experimental, Buenos Aires 1428, Argentina.
Biol Reprod. 2004 May;70(5):1325-32. doi: 10.1095/biolreprod.103.022822. Epub 2004 Jan 7.
Numerous studies have demonstrated that sperm capacitation is a bicarbonate-dependent process. In the rat, capacitation has not been studied as much as in other species, mainly because of the difficulties in carrying out functional assays with this animal model. In the present study, we have examined the influence of bicarbonate in the overall rat sperm capacitation process by analyzing involvement of the anion in 1) protein tyrosine phosphorylation, 2) migration of epididymal protein DE (also known as CRISP-1) from the dorsal region to the equatorial segment of the sperm head that occurs during capacitation, and 3) ability of sperm to fuse with the egg. Incubation of sperm under capacitating conditions produced a time-dependent increase in protein tyrosine phosphorylation. This phosphorylation did not occur in the absence of HCO3- and rapidly increased by either exposure of sperm to HCO3- or replacement of the anion by a cAMP analog (dibutyryl-cAMP) and a phosphodiesterase inhibitor (pentoxifylline). The absence of HCO3- also produced a significant decrease in the percentage of cells showing migration of DE to the equatorial segment. This parameter was completely restored by addition of the anion, but dibutyryl-cAMP and pentoxifylline were not sufficient to overcome the decrease in DE migration. Sperm capacitated in the absence of HCO3- were unable to penetrate zona-free eggs independent of the presence of the anion during gamete coincubation. Exposure of these sperm to bicarbonate, or replacement of the anion by dibutyryl-cAMP and pentoxifylline, only partially restored the sperm fusion ability. Altogether, these results indicate that, in addition to its influence on protein tyrosine phosphorylation, bicarbonate is required to support other rat sperm capacitation- associated events, such as migration of DE to the equatorial segment, and expression of the ability of sperm to fuse with the egg.
众多研究表明，精子获能是一个依赖碳酸氢根的过程。在大鼠中，对获能的研究不如在其他物种中深入，主要是因为用这种动物模型进行功能测定存在困难。在本研究中，我们通过分析阴离子在以下方面的作用，研究了碳酸氢根对大鼠精子整体获能过程的影响：1）蛋白质酪氨酸磷酸化；2）附睾蛋白DE（也称为CRISP-1）在获能过程中从精子头部背侧区域向赤道段的迁移；3）精子与卵子融合的能力。在获能条件下孵育精子会导致蛋白质酪氨酸磷酸化随时间增加。在没有HCO3-的情况下不会发生这种磷酸化，而通过将精子暴露于HCO3-或将阴离子替换为环磷酸腺苷类似物（二丁酰环磷酸腺苷）和磷酸二酯酶抑制剂（己酮可可碱），磷酸化会迅速增加。缺乏HCO3-还会导致显示DE迁移至赤道段的细胞百分比显著下降。通过添加阴离子可完全恢复该参数，但二丁酰环磷酸腺苷和己酮可可碱不足以克服DE迁移的减少。在没有HCO3-的情况下获能的精子无法穿透去透明带的卵子，无论配子共孵育期间是否存在阴离子。将这些精子暴露于碳酸氢根，或将阴离子替换为二丁酰环磷酸腺苷和己酮可可碱，只能部分恢复精子融合能力。总之，这些结果表明，除了对蛋白质酪氨酸磷酸化的影响外，碳酸氢根对于支持其他与大鼠精子获能相关的事件也是必需的，例如DE向赤道段的迁移以及精子与卵子融合能力的表达。
