Excited fluorophores produce reactive oxygen species that are toxic toward many live cells (phototoxicity) and accelerate bleaching of the fluorophores during the course of extended or repeated measurements (photobleaching). We...

CatSper is a sperm-specific calcium ion (Ca2+) channel, which regulates sperm flagellar beating by tuning cytoplasmic Ca2+ concentrations. Although this Ca2+ channel is essential for mammalian fertilization, recent bioinformatics analyses have revealed that genes encoding CatSper are heterogeneously distributed throughout the eukaryotes, including vertebrates. As this channel is activated by cytoplasmic alkalization in mammals and sea urchins, it has been proposed that the sperm-specific Na+/H+ exchanger (sNHE, a product of the SLC9C gene family) positively regulates its activity. In mouse, sNHE is functionally coupled to soluble adenylyl cyclase (sAC). CatSper, sNHE, and sAC have thus been considered functionally interconnected in the control of sperm motility, at least in mouse and sea urchin. We carried out a comparative genomic analysis to explore phylogenetic relationships among CatSper, sNHE and sAC in eukaryotes. We found that sNHE occurs only in Metazoa, although sAC occurs widely across eukaryotes. In animals, we found correlated and restricted distribution patterns of the three proteins, suggesting coevolution among them in the Metazoa. Namely, nearly all species in which CatSper is conserved also preserve sNHE and sAC. In contrast, in species without sAC, neither CatSper nor sNHE is conserved. On the other hand, the distribution of another testis-specific NHE (NHA, a product of the SLC9B gene family) does not show any apparent association with that of CatSper. Our results suggest that CatSper, sNHE and sAC form prototypical machinery that functions in regulating sperm flagellar beating in Metazoa. In non-metazoan species, CatSper may be regulated by other H+ transporters, or its activity might be independent of cytoplasmic pH.

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.

: In brief: Hyperpolarization of the membrane potential is a crucial step for mammalian sperm maturation. This work demonstrates that this membrane potential change likely activates a sperm-specific sodium/proton exchanger to induce alkalization in mouse sperm flagellum. The sperm-specific sodium/proton exchanger (sNHE) is an indispensable protein for male fertility in mammals. Nevertheless, it is still unknown how mammalian sNHE is regulated. Evidence obtained from sea urchin sNHE indicates that hyperpolarization of plasma membrane potential (Vm), which is a hallmark of mammalian capacitation, positively regulates the sNHE. Therefore, we explored the activity of sNHE in mouse and human sperm by fluorescence imaging of intracellular pH (pHi) with a ratiometric dye, SNARF-5F. A valinomycin-induced Vm hyperpolarization elevated sperm flagellar pHi of WT mouse but not in sNHE-KO mouse. Moreover, this pHi increase was inhibited in a high K+ (40 mM) medium. These results support the idea that mouse sNHE is activated by Vm hyperpolarization. Interestingly, we observed different types of kinetics derived from valinomycin-induced alkalization, including some (30%) without any pHi changes. Our quantitative pHi determinations revealed that unresponsive cells had a high resting pHi (>7.5), suggesting that the activity of mouse sNHE is regulated by the resting pHi. On the other hand, valinomycin did not increase the pHi of human sperm in the head or the flagellum, regardless of their resting pHi values. Our findings suggest that the regulatory mechanisms of mammalian sNHEs are probably distinct depending on the species.