Na+/H+ Exchangers Involve in Regulating the pH-Sensitive Ion Channels in Mouse Sperm

Abstract

Sperm-specific K+ ion channel (KSper) and Ca2+ ion channel (CatSper), whose elimination causes male infertility in mice, determine the membrane potential and Ca2+ influx, respectively. KSper and CatSper can be activated by cytosolic alkalization, which occurs during sperm going through the alkaline environment of the female reproductive tract. However, which intracellular pH (pHi) regulator functionally couples to the activation of KSper/CatSper remains obscure. Although Na+/H+ exchangers (NHEs) have been implicated to mediate pHi in sperm, there is a lack of direct evidence confirming the functional coupling between NHEs and KSper/CatSper. Here, 5-(N, N-dimethyl)-amiloride (DMA), an NHEs inhibitor that firstly proved not to affect KSper/CatSper directly, was chosen to examine NHEs function on KSper/CatSper in mouse sperm. The results of patch clamping recordings showed that, when extracellular pH was at the physiological level of 7.4, DMA application caused KSper inhibition and the depolarization of membrane potential when pipette solutions were not pH-buffered. In contrast, these effects were minimized when pipette solutions were pH-buffered, indicating that they solely resulted from pHi acidification caused by NHEs inhibition. Similarly, DMA treatment reduced CatSper current and intracellular Ca2+, effects also dependent on the buffer capacity of pH in pipette solutions. The impairment of sperm motility was also observed after DMA incubation. These results manifested that NHEs activity is coupled to the activation of KSper/CatSper under physiological conditions.

Blocking NHE Channels Reduces the Ability of In Vitro Capacitated Mammalian Sperm to Respond to Progesterone Stimulus

“…Few studies have analyzed the effect of NHE blockage on motility and kinematics of mammalian sperm, but none addressed its relationship to the response to progesterone stimulus. In our approach we used the inhibitor DMA, which blocks totally NHE1 and partially NHE5 isoforms of mammalian sperm [ 20 ], its effects on sNHE remaining unknown [ 4 ]. In rodents, blockage of NHE channels with DMA results in a decrease of total and progressive motility but not of kinematic parameters [ 4 ], whereas in sheep it affects total, but not progressive motility or kinematic parameters including ALH [ 16 ].…”

“…Alkalinization of inner pH (pH i ) is considered a central event for eliciting the sequence of changes related to sperm capacitation, since it is necessary for further activation of CatSper channels and Ca 2+ entrance [ 1 , 2 , 3 , 4 ]. Different ion channels and transporters are implicated in pH i regulation of sperm cells, being the Na + /H + exchangers (NHEs), HCO 3 − membrane transporters, monocarboxylate transporters (MCTs), and voltage gated H + transporters (HVCN1) the most studied in mammals [ 2 , 5 , 6 ].…”

“…NHEs are proton carriers belonging to the family of transporters of solute carrier 9 (SLC9). This family of proteins is composed by different isoforms grouped into three different subfamilies [ 4 , 11 , 12 ]: (1) NHE1 to NHE5 isoforms, which are localized in the plasma membrane (SLC9A1-5) and NHE6 to NHE9, which are intracellular isoforms (SLC9A6-9); (2) NHA1 and NHA2 isoforms (Na + /H + antiporters; SLC9AB1-2); and (3) sNHE (sNHE; SLC9C1, or SLC9A10), which is a sperm-specific plasma membrane isoform identified in mouse and humans. Each NHE isoform is encoded by a specific gene, has a particular characteristic molecular structure and distribution, and a different sensitivity to pharmacological inhibitors [ 11 ].…”

“…Whereas NHE1 is essential for pHi regulation and sNHE isoforms regulate sperm motility, the physiological role of NHE5 in mouse sperm fertility is unknown. Interestingly, previous research using patch-clamping in mouse sperm demonstrated that NHE channels are functionally coupled to the CatSper ones [ 4 , 11 ]. Furthermore, recent studies reported that sNHE in humans [ 17 ] and NHE1 in sheep [ 18 ] are implicated in the regulation of sperm motility.…”

“…Since not only does the process through which a spermatozoon acquires their capacity to fertilize an oocyte entail changes in sperm kinematics but also in plasma membrane permeability, intracellular calcium levels, mitochondrial activity, and acrosome integrity, the objective of the present study was to determine the physiological relevance of NHE channels in the sperm ability to elicit in vitro capacitation and progesterone-induced acrosome exocytosis. These assays were based on the pharmacological blockage of NHEs channels using a specific inhibitor (5-(N,N-dimethyl)-amiloride; DMA) at different concentrations [ 4 , 18 ]. DMA induces the total blockage of NHE1 and NHE2 isoforms and the partial blockage of NHE5; in contrast, NHE3 and NHE4 are amiloride-resistant isoforms (reviewed in [ 14 ]).…”