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.

Time-Lapse Flow Cytometry: A Robust Tool to Assess Physiological Parameters Related to the Fertilizing Capability of Human Sperm

“…Here, we report the employment of TLFC as a robust tool to measure absolute pH i values in human sperm cells. The range of the calculated resting pH i values for the human sperm samples observed here (6.4 to 7.0) was within the reported range of pH i values that have been obtained using different methods, including spectrofluorometry (6.9 to 7.1) [ 20 ], fluorescence microscopy (6.7) [ 47 ], and even stopped-flow cytometry (6.7 to 7.0) [ 28 , 63 ]. Recently, the capacitation-associated alkalinization has been investigated from a clinical standpoint, with results suggesting that sperm samples from patients with fertility issues present an impaired regulation of pH i and fail to undergo the capacitation-associated alkalinization [ 28 ].…”

“…To obtain absolute pH i values, we placed the second aliquot in a calibration medium called H + Cal (pH 6.0), which contains the K + /H + ionophore nigericin (10 µM) and a relatively high concentration of K + (120 mM). Incubation of sperm cells in H + Cal causes the collapse of the H + gradient across their PM, equalizing the pH i with the extracellular pH (pH e ) of the medium [ 47 ]. We then started recording BCECF fluorescence; after 120 s, we began sequential additions of KOH to elicit stepwise pH e increases, which resulted in equivalent increases in pH i .…”

Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization

“…Sperm alkalization controls the function of multiple proteins involved in capacitation; i.e., activation of the KSper 21 and/or CatSper ion channels 22 . During capacitation, the basal pH of 6.7 in non-capacitated mouse sperm increased approximately by 0.3 units, similar to previously reported 23,24 (Fig. 3a).…”

Starvation induces an increase in intracellular calcium and potentiates the progesterone‐induced mouse sperm acrosome reaction

“…For pH i measurements, sperm were loaded with 20 μmol/L 5-(and-6)-Carboxy SNARF 1 AM (Invitrogen) and 0.05% pluronic acid during 30 minutes in TYH basal medium as previously described. 18,19 Once loaded sperm were washed by centrifugation and immobilized in the recording chamber as was done for [Ca 2+ ] i imaging. For excitation and emission collection of SNARF 1 AM the set Wide Green 11007v2, D: 565 dcxt, Exc: 535/50, Em: 590 lpv2 (Chroma Technology Corporation) was used.…”