CatSper and the Relationship of Hyperactivated Motility to Intracellular Calcium and pH Kinetics in Equine Sperm
CatSper and the Relationship of Hyperactivated Motility to Intracellular Calcium and pH Kinetics in Equine Sperm
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DOI：
10.1095/biolreprod.113.111708
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发表时间：
2013-11-01
影响因子：
3.6
通讯作者：
Hinrichs, Katrin
中科院分区：
文献类型：
--
作者：
Loux, Shavahn C.;Crawford, Kristin R.;Hinrichs, Katrin
马的体外受精并不容易发生。这可能与马精子无法启动过度激活的运动有关，因为用普鲁卡因诱导过度激活会增加受精率。在小鼠中，过度激活的运动需要精子特异性ph门控钙通道（CatSper）；因此，我们研究了马精子中的这一通道。用计算机辅助精子运动分析评估精子运动能力，用荧光探针评估细胞内pH值和钙的变化。增加细胞内pH诱导细胞内钙的增加，这被已知的CatSper阻滞剂mibefradil抑制，支持pH门控钙通道的存在，可能是CatSper。过度激活与细胞内pH值的适度升高有关，但与细胞内钙的升高呈负相关。在缺乏钙的培养基中，高ph处理导致运动能力丧失，这与钠在缺乏环境钙的情况下通过开放的CatSper通道流入一致。然而，用普鲁卡因处理过的精子在缺乏钙的培养基中既保持了运动，又发生了过度激活，这表明普鲁卡因不是通过打开CatSper通道起作用的。用PCR方法鉴定了马精子中CATSPER1 mRNA，并在免疫细胞化学上定位到CATSPER1蛋白的主要片段。对预测的马CATSPER1蛋白的分析揭示了ph传感器区域的物种特异性结构差异。我们得出的结论是，CatSper通道存在于马精子中，但过度激活的运动性与钙内流的关系很弱。普鲁卡因似乎不通过马精子中的CatSper起作用，其最初的过度激活作用不依赖于外部钙的流入。
In vitro fertilization does not occur readily in the horse. This may be related to failure of equine sperm to initiate hyperactivated motility, as treating with procaine to induce hyperactivation increases fertilization rates. In mice, hyperactivated motility requires a sperm-specific pH-gated calcium channel (CatSper); therefore, we investigated this channel in equine sperm. Motility was assessed by computer-assisted sperm motility analysis and changes in intracellular pH and calcium were assessed using fluorescent probes. Increasing intracellular pH induced a rise in intracellular calcium, which was inhibited by the known CatSper blocker mibefradil, supporting the presence of a pH-gated calcium channel, presumably CatSper. Hyperactivation was associated with moderately increased intracellular pH, but appeared inversely related to increases in intracellular calcium. In calcium-deficient medium, high-pH treatment induced motility loss, consistent with influx of sodium through open CatSper channels in the absence of environmental calcium. However, sperm treated with procaine in calcium-deficient medium both maintained motility and underwent hyperactivation, suggesting that procaine did not act via opening of the CatSper channel. CATSPER1 mRNA was identified in equine sperm by PCR, and CATSPER1 protein was localized to the principal piece on immunocytochemistry. Analysis of the predicted equine CATSPER1 protein revealed species-specific differences in structure in the pH-sensor region. We conclude that the CatSper channel is present in equine sperm but that the relationship of hyperactivated motility to calcium influx is weak. Procaine does not appear to act via CatSper in equine sperm, and its initial hyperactivating action is not dependent upon external calcium influx.