Lachance C, Goupil S, Tremblay R R, Leclerc P
Département d'Obstétrique, gynécologie et reproduction, Centre de recherche en biologie de la reproduction, Université Laval, Quebec, QC, Canada.
Axe reproduction, santé de la mère et de l'enfant, Centre de recherche du CHU de Québec, Québec, QC, Canada.
Andrology. 2016 Jan;4(1):133-42. doi: 10.1111/andr.12123. Epub 2015 Nov 4.
We previously showed that Stattic V (Stat3 inhibitory compound V) reduces human sperm motility and cellular ATP levels, increases intracellular Ca(2+) concentration, and promotes mitochondrial membrane depolarization resulting in increased levels of extracellular reactive oxygen species (ROS). As these alterations in cellular function are highly similar to what is observed in a cell undergoing apoptosis, our goal was to determine if the immobilizing effect of Stattic V on spermatozoa results from apoptosis or was because of an oxidative stress. To address this question, spermatozoa were incubated with Stattic V in combination with a caspase inhibitor, a proteasome inhibitor or a cell permeant ROS scavenger. Following incubation in different conditions, sperm motility was evaluated by CASA, acrosomal integrity by FITC conjugated Pisum sativum agglutinin (PSA-FITC) labeling, intracellular pH, and mitochondrial superoxide production by flow cytometry using BCECF and MitoSoxRed dye, respectively. Levels of reduced thiols were assessed by iodoacetamidofluorescein staining on total and on sperm surface proteins, and protein tyrosine phosphorylation was evaluated by western blot. The loss in sperm motility induced by Stattic V was associated with a slight intracellular acidification and an important increase in intracellular superoxide anion. Unlike caspase and proteasome inhibitors, low molecular weight thiols, such as N-acetyl-L-cysteine (NAC), prevented Stattic V-induced sperm immobilization and increase responsiveness to acrosome reaction inducers. NAC also efficiently prevented the production of superoxide anion, mitochondrial membrane depolarization, intracellular acidification and the oxidation of protein free thiols caused by Stattic V. These results show that the deleterious effects of Stattic V on sperm functions are caused directly or indirectly by excessive intracellular ROS production without causing sperm apoptosis or necrosis.
我们之前的研究表明，Stattic V（信号转导子和转录激活子3抑制化合物V）可降低人类精子活力和细胞ATP水平，增加细胞内Ca(2+)浓度，并促进线粒体膜去极化，从而导致细胞外活性氧（ROS）水平升高。由于这些细胞功能的改变与细胞凋亡时观察到的情况高度相似，我们的目标是确定Stattic V对精子的固定作用是由凋亡引起的，还是由于氧化应激。为了解决这个问题，将精子与Stattic V联合半胱天冬酶抑制剂、蛋白酶体抑制剂或细胞渗透性ROS清除剂一起孵育。在不同条件下孵育后，分别使用BCECF和MitoSoxRed染料通过计算机辅助精子分析（CASA）评估精子活力，通过异硫氰酸荧光素（FITC）偶联的豌豆凝集素（PSA-FITC）标记评估顶体完整性，通过流式细胞术评估细胞内pH值和线粒体超氧化物生成。通过对总蛋白和精子表面蛋白进行碘乙酰胺荧光素染色评估还原型硫醇水平，并通过蛋白质免疫印迹法评估蛋白质酪氨酸磷酸化。Stattic V诱导的精子活力丧失与轻微的细胞内酸化和细胞内超氧阴离子的显著增加有关。与半胱天冬酶和蛋白酶体抑制剂不同，低分子量硫醇，如N-乙酰-L-半胱氨酸（NAC），可防止Stattic V诱导的精子固定，并增加对顶体反应诱导剂 的反应性。NAC还能有效防止Stattic V引起的超氧阴离子生成、线粒体膜去极化、细胞内酸化和游离蛋白质硫醇的氧化。这些结果表明，Stattic V对精子功能的有害影响是由细胞内ROS过量产生直接或间接引起的，而不会导致精子凋亡或坏死。
