Hyperpolarization induces cytosolic alkalization of mouse sperm flagellum probably through sperm Na+/H+ exchanger

Abstract

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. Abstract 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.

The SLC9C2 Gene Product (Na+/H+ Exchanger Isoform 11; NHE11) Is a Testis-Specific Protein Localized to the Head of Mature Mammalian Sperm

“…However, we were unable to express either NHE11 isoform in cell cultures. To our knowledge, no group has been able to express an unmodified mammalian SLC9C protein in a traditional heterologous cell culture system, despite many previous attempts to express NHE10 [ 9 , 19 , 24 , 25 , 26 ]. The inability to express mammalian NHE10 or NHE11 in cultured cells suggests that these proteins contain amino acid sequences that the transfected cells are unable to process properly, resulting in degradation of the proteins or death of the expressing cells.…”

“…Although the gene expression patterns and predicted structures of NHE10 and NHE11 are similar, the distinct localizations of the two proteins to different structural compartments of the sperm suggest that these proteins perform individually specialized roles in the context of sperm physiology. NHE10 localizes to the sperm flagellum [ 13 , 14 ], allowing this NHE to facilitate pH regulation in response to various environmental stimuli to influence sperm motility [ 9 , 19 , 24 , 26 ]. The exact role that NHE11 plays in sperm is yet to be identified but because of its localization, NHE11 may be regulating pH in the sperm head to facilitate post-capacitation events such as the acrosome reaction or sperm–egg fusion.…”

“…Characterization of the suSLC9C protein transport activity revealed that it is an electroneutral Na + /H + exchanger regulated by membrane voltage and cyclic-nucleotide binding [ 19 ]. None of the mammalian NHE10 proteins have yet to be biochemically characterized, likely due to the difficulty of expressing these proteins in heterologous cell culture systems [ 9 , 19 , 24 , 25 , 26 ] and we have encountered similar difficulties in expressing NHE11 in cultured cells. However, some insight into the potential transport function of NHE11 may be gained from the comparison of the amino acid sequences of the mammalian NHE10 and NHE11 proteins to known functional NHEs such as human NHE1 (hNHE1) and the suSLC9C protein.…”

“…A recent study utilized a novel specific inhibitor of the human SLO3 K + channel, VU0546110, that prevented human sperm from hyperpolarizing and undergoing hyperactivated activity, and prevented the Ca 2+ - or progesterone-induced acrosome reaction [ 57 ]. Interestingly, a study using human sperm found no evidence of alkalization of either the sperm head or flagellum in response to valinomycin-induced hyperpolarization [ 26 ], suggesting a potential different role of NHE10 and/or NHE11 in human sperm as compared to sea urchin sperm, in which the suSLC9C protein was shown to be responsible for the hyperpolarization-evoked alkalization in response to speract stimulation [ 19 ]. However, these human sperm were not incubated under capacitating conditions and the hyperpolarization was induced by the K + ionophore valinomycin [ 26 ]; therefore, it is possible that human NHE10 and/or NHE11 are only able to respond to capacitation-induced hyperpolarization.…”

Alkaline Dilution Alters Sperm Motility in Dairy Goat by Affecting sAC/cAMP/PKA Pathway Activity

“…The ionic requirements and signaling pathways of mammalian sperm motility have been widely studied, but they are still not completely understood [ 56 , 57 ]. At the molecular level, sperm motility enhancement requires the activation of several signaling pathways, including but not limited to cAMP-dependent pathways, an increase in pHi, changes in [Ca 2+ ]i concentration, and hyperpolarization of the sperm plasma membrane potential [ 33 , 58 ].…”