Centre of Reproductive Biotechnology (BIOREN-CEBIOR), Faculty of Medicine, University of La Frontera, Temuco, Chile.
Centre of Reproductive Biotechnology (BIOREN-CEBIOR), Faculty of Medicine, University of La Frontera, Temuco, Chile Department of Internal Medicine, Faculty of Medicine, University of La Frontera, Temuco, Chile
Hum Reprod. 2015 Apr;30(4):767-76. doi: 10.1093/humrep/dev015. Epub 2015 Feb 5.
Does mitochondrial permeability transition (MPT) induced by calcium overload cause reactive oxygen species (ROS) production and DNA fragmentation in human spermatozoa?
Studies conducted in vitro suggest that in human spermatozoa, MPT occurs in response to intracellular calcium increase and is associated with mitochondrial membrane potential (ΔΨm) dissipation, increased ROS production and DNA fragmentation.
Oxidative stress is a major cause of defective sperm function in male infertility. By opening calcium-dependent pores in the inner mitochondrial membrane (IMM), MPT causes, among other things, increased ROS production and ΔΨm dissipation in somatic cells. MPT as a mechanism for generating oxidative stress and DNA fragmentation in human spermatozoa has not been studied.
STUDY DESIGN, SIZE, DURATION: Human sperm were exposed to ionomycin for 1.5 h (n = 8) followed by analysis of sperm IMM permeability, ΔΨm, ROS production and DNA fragmentation.
PARTICIPANTS/MATERIALS, SETTING, METHODS: To evaluate the MPT in sperm cells, the calcein-AM and cobalt chloride method was used. The ΔΨm was evaluated by JC-1 staining, intracellular ROS production was evaluated with dihydroethidium and DNA fragmentation was evaluated by a modified TUNEL assay. Measurements were performed by fluorescence microscopy, confocal laser microscopy and flow cytometry.
Decreased calcein fluorescence after treatment with ionomycin (P < 0.05) suggests the opening of pores in the sperm IMM and this was accompanied by ΔΨm dissipation, increased ROS production and DNA fragmentation. ROS production occurred prior to the decrease in ΔΨm.
LIMITATIONS, REASONS FOR CAUTION: The study was carried out in vitro using motile sperm from healthy donors; tests on sperm from infertile patients were not carried out.
We propose that the MPT, due to pores opening in sperm IMM, is an important mechanism of increased ROS and DNA fragmentation. Therefore, agents that modulate the opening of these pores might contribute to the prevention of damage by oxidative stress in human spermatozoa.
STUDY FUNDING/COMPETING INTERESTS: This study was funded by grant DI12-0102 from the Universidad de La Frontera (J.V.V.) and a doctoral scholarship from CONICYT Chile (F.T.). The authors disclose no potential conflicts of interest.
钙离子过载诱导的线粒体通透性转换（MPT）是否会导致人精子中活性氧（ROS）的产生和 DNA 碎片化？
体外研究表明，在人精子中，MPT 会对内钙离子增加作出反应，并与线粒体膜电位（ΔΨm）耗散、ROS 产生增加和 DNA 碎片化有关。
氧化应激是男性不育中精子功能缺陷的主要原因。MPT 通过打开线粒体内膜（IMM）中的钙依赖性孔，除其他外，导致体细胞中 ROS 产生和 ΔΨm 耗散增加。MPT 作为人精子中产生氧化应激和 DNA 碎片化的机制尚未得到研究。
研究设计、规模、持续时间：将人精子暴露于离子霉素中 1.5 小时（n = 8），然后分析精子 IMM 通透性、ΔΨm、ROS 产生和 DNA 碎片化。
参与者/材料、设置、方法：为了评估精子细胞中的 MPT，使用钙黄绿素-AM 和氯化钴法。通过 JC-1 染色评估 ΔΨm，通过二氢乙啶评估细胞内 ROS 产生，通过改良的 TUNEL 测定评估 DNA 碎片化。通过荧光显微镜、共聚焦激光显微镜和流式细胞术进行测量。
离子霉素处理后钙黄绿素荧光降低（P < 0.05）表明精子 IMM 中的孔打开，这伴随着 ΔΨm 耗散、ROS 产生增加和 DNA 碎片化。ROS 产生发生在 ΔΨm 下降之前。
局限性、谨慎的原因：该研究是在使用来自健康供体的运动精子的体外进行的；未对不育患者的精子进行测试。
我们提出，由于精子 IMM 中的孔打开，MPT 是 ROS 和 DNA 碎片化增加的重要机制。因此，调节这些孔打开的试剂可能有助于防止人类精子中的氧化应激损伤。
研究资金/竞争利益：本研究由 Universidad de La Frontera（J.V.V.）的 DI12-0102 资助和 CONICYT Chile（F.T.）的博士奖学金资助。作者披露没有潜在的利益冲突。
