Département d'Obstétrique et de Gynécologie, Centre de recherche en biologie de la reproduction, Université Laval, Axe reproduction, santé périnatale et santé de l'enfant, Centre de recherche du CHUQ-CHUL, Québec, Canada.
J Cell Physiol. 2013 Apr;228(4):704-13. doi: 10.1002/jcp.24215.
We have recently shown that many mediators of the JAK/STAT signaling pathway are present in ejaculated human spermatozoa. Among them, STAT3 is detected mainly in membranes and flagellar cytoskeletal fractions. In order to determine the importance of STAT3-mediated signaling, sperm were incubated with Stattic V, a specific inhibitor. Effects on motility were evaluated by CASA, sperm acrosomal integrity was evaluated by FITC conjugated lectin (PSA or PNA) staining, and protein phosphotyrosine content was assessed by Western blot using a monoclonal anti-phosphotyrosine antibody. INDO1-AM and JC-1 were used to measure sperm intracellular calcium and mitochondrial membrane potential, respectively, by flow cytometry, and reactive oxygen species (ROS) production was investigated by luminol-based assay. Percentages of motility and motility parameters were significantly affected by Stattic V. This later also significantly increased intracellular Ca(2+) levels, progesterone- and calcium ionophore (A23187)-induced acrosome reaction. On the other hand, a significant decrease in ATP content was measured when sperm were treated with Stattic V, associated with depolarization of mitochondrial membrane and elevated ROS production. These results suggest that STAT3 is involved in sperm functions, at least through regulation of mitochondrial activity. This further emphasizes that STAT3 mediates cellular activities in a manner different than strictly the activation of gene transcription.
我们最近发现，许多 JAK/STAT 信号通路的介质存在于射出的人类精子中。其中，STAT3 主要存在于膜和鞭毛细胞骨架部分。为了确定 STAT3 介导的信号的重要性，精子用 Stattic V 孵育，这是一种特异性抑制剂。通过 CASA 评估运动性，通过 FITC 缀合的凝集素（PSA 或 PNA）染色评估顶体完整性，并用单克隆抗磷酸酪氨酸抗体通过 Western blot 评估蛋白磷酸酪氨酸含量。通过流式细胞术分别使用 INDO1-AM 和 JC-1 测量精子内钙和线粒体膜电位，通过发光法测量活性氧（ROS）的产生。Stattic V 显著影响运动性和运动参数的百分比。这也显著增加了细胞内 Ca(2+)水平，孕酮和钙离子载体（A23187）诱导的顶体反应。另一方面，当精子用 Stattic V 处理时，测量到 ATP 含量显著下降，伴随着线粒体膜去极化和 ROS 产生增加。这些结果表明，STAT3 参与精子功能，至少通过调节线粒体活性。这进一步强调了 STAT3 通过不同于严格激活基因转录的方式介导细胞活动。
