AUTHOR=Chávez Julio C. , Darszon Alberto , Treviño Claudia L. , Nishigaki Takuya 

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

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=7

YEAR=2020

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2019.00366

DOI=10.3389/fcell.2019.00366

ISSN=2296-634X

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