Role of μ-Calpain in Human Sperm for Fertilization

我们评估了μ-calpain在人类精子受精中的作用。在不同浓度的孕酮（P）或低氧条件（H）下，将样品进行免疫染色、SDS-PAGE和western blot分析。用荧光显微镜（ARGUS-50 CA）观察Fura 2-AM对精子内钙离子浓度的影响。用钙蛋白酶抑制剂对人精子顶体、赤道段、头段、颈段和颈尾段进行免疫染色，观察钙蛋白酶μ-calpain在精子顶体、赤道段、头段、颈段和颈尾段的免疫染色情况，并结合精子穿透试验、汉密尔顿运动分析仪、三重染色法和精子顶珠试验，推测钙蛋白酶μ-calpain在受精中的作用。抗μ-calpain原抗体染色大多为顶体型。Western blot分析表明，P或H处理可引起μ-calpain的浓度或时间依赖性激活，calpain抑制剂可显著降低精子顶体反应和穿透率，提示μ-calpain在人类精子的受精过程中起重要作用。

We evaluated the role of μ-calpain in human sperm for fertilization.Semen collected manually from healthy donors with informed consent was liquefied and following percoll gradient centrifugation. After exposure to different concentration of progesterone (P) or under hypoxic condition (H), the samples were used for immunostaining, SDS-PAGE and western blot analysis. The increase of calcium ion concentration in the sperm was observed by fluorescent microscope (ARGUS-50CA) system using Fura 2-AM. The role of calpain for fertilization was speculated from the results of Hamster penetration test, Hamilton Motility Analyzer, Triple satin method and Acrobeads test using calpain inhibitors.Immunostaining in human sperm was observed using antibodies against the μ-calpain in the types of whole acrosome, equatorial segment, head segment, neck segment or neck and tail segment. Most of these demonstrated acrosome type staining with anti-pro μ-calpain antibody. Western blot analysis revealed P or H treatment to cause a concentration or time-dependent activation of μ-calpain.In addition, calpain inhibitors significantly reduced the acrosome reaction and penetration.The results suggest that μ-calpain in human sperm plays an important role for the fertilization.