Chávez Julio C, Darszon Alberto, Treviño Claudia L, Nishigaki Takuya
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.
Front Cell Dev Biol. 2020 Jan 17;7:366. doi: 10.3389/fcell.2019.00366. eCollection 2019.
Intracellular pH (pH ) plays a crucial role in mammalian sperm physiology. However, it is a challenging task to acquire quantitative single sperm pH images due to their small size and beating flagella. In this study, we established a robust pH imaging system using the dual-emission ratiometric pH indicator, SNARF-5F. Simultaneous good signal/noise ratio fluorescence signals were obtained exciting with a green high-power LED (532 nm) and acquiring with an EM-CCD camera through an image splitter with two band-pass filters (550-600 nm, channel 1; 630-650 nm, channel 2). After calibration, we established an imaging system that allows determination of absolute pH values in spermatozoa, minimizing cell movement artifacts. Using this system, we determined that bicarbonate increases non-capacitated human pH with slower kinetics than in mouse spermatozoa. This difference suggests that distinct ionic transporters might be involved in the bicarbonate influx into human and mouse spermatozoa. Alternatively, pH regulation downstream bicarbonate influx into spermatozoa could be different between the two species.
细胞内pH值（pH ）在哺乳动物精子生理过程中起着至关重要的作用。然而，由于精子体积小且鞭毛摆动，获取定量的单个精子pH 图像是一项具有挑战性的任务。在本研究中，我们使用双发射比率型pH指示剂SNARF-5F建立了一个强大的pH 成像系统。用绿色高功率发光二极管（532 nm）激发，并通过带有两个带通滤光片（550 - 600 nm，通道1；630 - 650 nm，通道2）的图像分离器，用电子倍增电荷耦合器件（EM-CCD）相机采集，从而获得了同时具有良好信噪比的荧光信号。校准后，我们建立了一个成像系统，该系统能够确定精子中的绝对pH 值，将细胞运动伪影降至最低。使用该系统，我们确定碳酸氢盐增加未获能人类精子的pH 值，其动力学比小鼠精子慢。这种差异表明，不同的离子转运体可能参与了碳酸氢盐进入人类和小鼠精子的过程。或者，在这两个物种中，碳酸氢盐流入精子后下游的pH 调节可能有所不同。
