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

Abstract

Few data exist about the presence and physiological role of Na+/H+ exchangers (NHEs) in the plasma membrane of mammalian sperm. In addition, the involvement of these channels in the ability of sperm to undergo capacitation and acrosomal reaction has not been investigated in any mammalian species. In the present study, we addressed whether these channels are implicated in these two sperm events using the pig as a model. We also confirmed the presence of NHE1 channels in the plasma membrane of ejaculated sperm by immunofluorescence and immunoblotting. The function of NHE channels during in vitro capacitation was analyzed by incubating sperm samples in capacitating medium for 300 min in the absence or presence of a specific blocker (DMA; 5-(N,N-dimethyl)-amiloride) at different concentrations (1, 5, and 10 µM); acrosome exocytosis was triggered by adding progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium and reactive oxygen species (ROS) levels, and mitochondrial membrane potential (MMP) were evaluated after 0, 60, 120, 180, 240, 250, 270, and 300 min of incubation. NHE1 localized in the connecting and terminal pieces of the flagellum and in the equatorial region of the sperm head and was found to have a molecular weight of 75 kDa. During the first 240 min of incubation, i.e., before the addition of progesterone, blocked and control samples did not differ significantly in any of the parameters analyzed. However, from 250 min of incubation, samples treated with DMA showed significant alterations in total motility and the amplitude of lateral head displacement (ALH), acrosomal integrity, membrane lipid disorder, and MMP. In conclusion, while NHE channels are not involved in the sperm ability to undergo capacitation, they could be essential for triggering acrosome exocytosis and hypermotility after progesterone stimulus.

A Review on the Role of Bicarbonate and Proton Transporters during Sperm Capacitation in Mammals

“…Few studies have focused on the expression and function of NHE in sperm cells; NHE1, NHE5, and/or sNHE exchangers are reported to be expressed in some mammal sperm cells [ 23 , 25 , 114 ], whereas the presence of NHA1 and NHA2 has only been described in mice [ 120 ]. To our knowledge, no information is available on the presence, distribution, and function of intracellular NHE channels in mature sperm cells.…”

“…In agreement with this, blockage of NHE using 5-( N,N -dimethyl)-amiloride (DMA) manifests in a decrease of K + currents across SLO channels and of Ca 2+ currents across CatSper recorded using the patch-clamp technique [ 8 , 114 ]. In pigs, NHE blocking with DMA does not affect Ca 2+ influx measured by flow cytometry during in vitro capacitation, but it reduces the ability of capacitated sperm cells to respond to the progesterone stimulus; this drives impaired hypermotility and acrosomal exocytosis [ 25 ]. In humans, an NHE blockage does not affect the acrosomal reaction after progesterone addition in capacitated sperm [ 125 ].…”

“…In sperm cells, the localization, molecular weight, and physiological role of NHE1 channels differs with species. Indeed, whereas they are located in the midpiece of flagellum in mice [ 114 , 128 ], they extend along the mid- and principal pieces and the equatorial region of the head in sheep [ 23 ] and pigs [ 25 ]. The molecular weight of NHE1 in sperm is 95 kDa in rats [ 128 ], 85 kDa in sheep [ 23 ], and 75–105 kDa in pigs [ 25 , 125 ].…”

“…Yet, it should be borne in mind that intracellular HCO 3 − levels also rely upon different carbonic anhydrase isoforms present in both plasma membrane and cytoplasm [ 20 ]. On the other hand, not only does alkalinization of sperm cytosol depend on bicarbonate transport but also on the involvement of different proton (H + ) exchangers and transporters, mainly voltage-gated proton channels, such as HVCN1 [ 3 , 21 , 22 ], monocarboxylate transporters (MCTs) [ 19 ], and Na + -H + exchangers (NHEs) [ 8 , 23 , 24 , 25 ]. In fact, the increase of pH i is known to be essential for the activation of several pH-dependent channels that play a vital function for plasma membrane hyperpolarization and acrosomal reactions [ 1 ].…”

Ion Channels of Spermatozoa: Structure, Function, and Regulation Mechanisms

“…The plasma membrane of mammal sperm also contains voltage-gated proton channels (HVCN1) [ 2 ] and/or Na + -H + exchangers (NHEs) [ 7 , 12 ]. In this special issue, different complementary approaches focused on the physiological relevance of these channels in mature sperm [ 2 , 7 ].…”