Ajmat M T, Bonilla F, Zelarayán L, Bühler M I
Departamento de Biología del Desarrollo (INSIBIO), Chacabuco 461, 4000-San Miguel de Tucumán, Argentina.
Zygote. 2011 May;19(2):171-80. doi: 10.1017/S0967199410000444. Epub 2010 Sep 30.
Calcium is considered the most important second messenger at fertilization. Transient release from intracellular stores is modulated through both agonist-gated channels, IP₃Rs and RyRs, which can be found individually or together depending on the oocyte species. Using the four commonly used compounds (thimerosal, caffeine, heparin and ruthenium red), we investigated the existence and interdependence of both IP₃Rs and RyRs in mature Bufo arenarum oocytes. We found that caffeine, a well known specific RyRs agonist, was able to trigger oocyte activation in a dose-dependent manner. Microinjection of 10 mM caffeine showed 100% of oocytes exhibiting characteristic morphological criteria of egg activation. Ruthenium red, the specific RyR blocker, was able to inhibit oocyte activation induced either by sperm or caffeine. Our present findings provide the first reported evidence of the existence of RyR in frogs. We further explored the relationship between IP₃Rs and RyRs in B. arenarum oocytes by exposing them to the agonists of one class after injecting a blocker of the other class of receptor. We found that thimerosal overcame the inhibitory effect of RyR on oocyte activation, indicating that IP₃Rs function as independent receptors. In contrast, previous injection of heparin delayed caffeine-induced calcium release, revealing a relative dependence of RyRs on functional IP₃Rs, probably through a CICR mechanism. Both receptors play a role in Ca²+ release mechanisms although their relative contribution to the activation process is unclear.
钙被认为是受精过程中最重要的第二信使。细胞内钙库的瞬时释放通过激动剂门控通道（IP₃Rs和RyRs）进行调节，根据卵母细胞的种类，这些通道可以单独存在或同时存在。我们使用四种常用化合物（硫柳汞、咖啡因、肝素和钌红），研究了成熟的阿根廷蟾蜍卵母细胞中IP₃Rs和RyRs的存在及相互依赖性。我们发现，咖啡因作为一种众所周知的特异性RyRs激动剂，能够以剂量依赖的方式触发卵母细胞激活。显微注射10 mM咖啡因后，100%的卵母细胞呈现出卵子激活的特征性形态标准。特异性RyR阻滞剂钌红能够抑制精子或咖啡因诱导的卵母细胞激活。我们目前的研究结果首次报道了青蛙中存在RyR的证据。我们通过在注射一类受体的阻滞剂后将阿根廷蟾蜍卵母细胞暴露于另一类受体的激动剂，进一步探究了IP₃Rs和RyRs之间的关系。我们发现硫柳汞克服了RyR对卵母细胞激活的抑制作用，这表明IP₃Rs作为独立受体发挥作用。相反，预先注射肝素会延迟咖啡因诱导的钙释放，这揭示了RyRs可能通过钙诱导钙释放机制对功能性IP₃Rs存在相对依赖性。尽管它们对激活过程的相对贡献尚不清楚，但两种受体在Ca²⁺释放机制中均发挥作用。
