Cytosolic and Acrosomal pH Regulation in Mammalian Sperm

Abstract

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca²⁺ channel CatSper and the K⁺ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.

Keywords: acrosomal pH; bicarbonate transport; cytosolic pH; mammalian sperm capacitation; proton channels and transporters.

Figure 1

Schematic representation of the location of the proteins related to pHi regulation in mammalian sperm.

Figure 2

Structure of the sperm-specific Na⁺/H⁺ exchanger, sNHE (SLC9C). (A) depicts the domain arrangement of sNHE with color-coded features representing different domains: transporter domain composed of 13 TMs (13TM-TD, red), first cytosolic helices (H1, green), voltage sensor domain (VSD, blue), second cytosolic helices (H2, yellow), cyclic nucleotide-binding domain (CNBD, pink), and the C-terminal β strand domain (β, orange). (B) illustrates the 3D structure of an inactive state of sea urchin sNHE (SpsNHE) dimer, determined by cryo-EM analysis (PDB ID: 8OTX, [76]). To highlight the interphase of the dimer in the cytosolic helix domain, one monomer is colored according to Scheme A, while the other monomer is represented in gray using the PyMOL program. Hyperpolarization (HyperP) of the membrane potential is expected to induce a downward movement of the positively charged S4 segment of the VSD, rendering the sNHE in an active state. Additionally, the binding of cAMP to the CNBD might facilitate the conformational change toward the active state.

Figure 3

pHi changes in response to a valinomycin-induced hyperpolarization in mouse and human sperm. Sperm pHi was assessed using the pH-sensitive dual-emission fluorescence probe, SNARF-5F (Excitation= 530 nm; Emission= 575/640 nm) in single cell recordings. Panel (A) illustrates pHi changes in the midpiece of mouse sperm induced by 1 µM valinomycin (Val) followed by a 20 mM NH₄Cl control addition. WTK4.7 (red trace) represents wild-type mouse sperm in a normal medium containing 4.7 mM K⁺, while KOK4.7 (black trace) indicates sperm from sNHE (SLC9C1) null mice in a normal medium. WTK40 (blue trace) indicates wild-type sperm in a medium with 40 mM K⁺. Panel (B) depicts pHi changes in the human sperm flagellar midpiece. HTF (black trace) indicates the addition of medium, serving as a negative control against the addition of 1 µM valinomycin (Val, red trace). The results are adapted from [18], with some modifications.

Figure 4

Indicator-dependent pHi changes in response to HCO₃⁻ perfusion in human sperm. HCO₃⁻ causes a pHi alkalinization in human sperm, as reported by the SNARF-5F dye, but a slight pHi acidification when pHrodo red is used. Representative pHi recordings using SNARF-5F (A,B) and pHrodo red (C,D) perfusing 15 or 30 mM HCO₃⁻ (green rectangle) in a 5% CO₂ environment. As positive controls, perfusions of 10 mM NH₄Cl (orange rectangle) and 5 mM HCl (purple rectangle) are shown in each panel. Traces in each panel show average responses from 104 cells (for SNARF-5F) and 101 cells (for pHrodo red), with S.E.M. in gray. Ratiometric SNARF-5F measurements are reported as pHi values, whereas for pHrodo red, the F/F₀ normalization is shown, and F = fluorescence intensity. ↑F/F₀ indicates pHi acidification.

Figure 5

Model of the molecular entities that regulate pHa in human sperm. Under non-capacitated conditions (NC), the pHa is acidic, due mainly to the active pumping of H⁺ mediated by the V-ATPase into the acrosomal lumen and the flow of counterions through transport such as ClC-3. As capacitation initiates, HCO₃⁻ enters the cell through different channels and transporters, and/or it is produced inside by the conversion of CO₂, H₂O, and H⁺. sAC is stimulated by HCO₃⁻, elevating cAMP levels and activating PKA, allowing the phosphorylation of several proteins, including CFTR channels, which also may allow the entry of HCO₃⁻. During capacitation, pHi also increases, favoring Ca²⁺ influx, which also enhances sAC activity. V-ATPase allows the acrosome to remain acidic during the first hours of capacitation. The continuous entry of HCO₃⁻, as well as the exit of H⁺ from the cytosol, through the Hv1 channel in the case of human sperm or through NHEs in other mammals, stabilizes the cytosolic alkalinization, dissipates the H⁺ gradient, decreases V-ATPase activity, and induces the alkalinization of the acrosome. Other mechanisms, not yet described, could also regulate the activity of the V-ATPase. The pHa increase destabilizes the acrosomal matrix, producing acrosome swelling and probably TPC1 channel activation, releasing acrosomal Ca^2+, which in turn stimulates extracellular Ca²⁺ uptake through ORAI channels (1 and 2). Both acrosome alkalinization and [Ca²⁺]i increases induce AR. Arrow indicates increase of the ion (↑, ↑↑). We place the sign (?) to highlight that some transporters or channels, although they have been detected, their exact location and identity has not been fully established (NBC), or their function in humans is unknown (TPC1, NHE11).