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Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F.


ABSTRACT: Intracellular pH (pH i ) plays a crucial role in mammalian sperm physiology. However, it is a challenging task to acquire quantitative single sperm pH i images due to their small size and beating flagella. In this study, we established a robust pH i 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 in vivo calibration, we established an imaging system that allows determination of absolute pH i values in spermatozoa, minimizing cell movement artifacts. Using this system, we determined that bicarbonate increases non-capacitated human pH i 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 i regulation downstream bicarbonate influx into spermatozoa could be different between the two species.

SUBMITTER: Chavez JC 

PROVIDER: S-EPMC6978660 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F.

Chávez Julio C JC   Darszon Alberto A   Treviño Claudia L CL   Nishigaki Takuya T  

Frontiers in cell and developmental biology 20200117


Intracellular pH (pH <sub><i>i</i></sub> ) plays a crucial role in mammalian sperm physiology. However, it is a challenging task to acquire quantitative single sperm pH <sub><i>i</i></sub> images due to their small size and beating flagella. In this study, we established a robust pH <sub><i>i</i></sub> 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  ...[more]

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