Dissecting the signaling pathways involved in the function of sperm flagellum

Abstract

The mammalian flagellum is a specific type of motile cilium required for sperm motility and male fertility. Effective flagellar movement is dependent on axonemal function, which in turn relies on proper ion homeostasis within the flagellar compartment. This ion homeostasis is maintained by the concerted function of ion channels and transporters that initiate signal transduction pathways resulting in motility changes. Advances in electrophysiology and super-resolution microscopy have helped to identify and characterize new regulatory modalities of the mammalian flagellum. Here, we discuss what is currently known about the regulation of flagellar ion channels and transporters that maintain sodium, potassium, calcium, and proton homeostasis. Identification of new regulatory elements and their specific roles in sperm motility is imperative for improving diagnostics of male infertility.

Keywords: Capacitation; CatSper; EFCAB9; Fertility; Flagellum; Hv1; Motility; Progesterone; Slo1; Slo3; Sperm ion channels; pH.

Figure 1

(a) Signaling pathways in the murine sperm flagellum. pH-activated CatSper opens and carries Ca²⁺ into the cell. This channel is additionally regulated by intracellular Ca²⁺ via its EFCAB9 cytoplasmic subunit. Calcium clearance mechanisms of mouse sperm is provided mainly by ATPase (ATP2B4) and by Na⁺/Ca⁺ exchanger. Intracellular alkalization can be caused by the action of NHE exchangers. Sperm capacitation is triggered by an increase in intracellular ${\mathsf{HCO}}_3^{-}$, which enters the cell in two different ways. First, CO₂ can diffuse through the membrane and then is converted by CA into ${\mathsf{HCO}}_3^{-}$. Second, extracellular ${\mathsf{HCO}}_3^{-}$ can be carried into the cell by Cl⁻/${\mathsf{HCO}}_3^{-}$ SLC cotransporter interacting with Cl⁻-permeable CFTR. Na⁺/K⁺ gradient across the plasma membrane and, hence, membrane potential are maintained by the Na⁺/K⁺-ATPase α4. This gradient could be used to power the sNHE exchanger to promote influx of Na⁺ and efflux of H⁺ and to ensure intracellular alkalization. Potassium enters the cell through the Na⁺/K⁺-ATPase α4 and can leave through the Slo3 channel, which in mouse is activated by intracellular alkalization. Efflux of K⁺ causes hyperpolarization, further inhibiting CatSper. ${\mathsf{HCO}}_3^{-}$ and intracellular Ca²⁺ can trigger activation of sAC, which produces cAMP, leading to activation of PKA and tyrosine kinases, resulting in broad tyrosine phosphorylation leading to sperm capacitation and hyperactivation. Capacitation is also associated with cholesterol removal from the plasma membrane by albumin. Solid lines represent activation, and dotted lines represent inhibition. Dotted rectangles indicate proteins with yet to be conformed functional role in male fertility. (b) Signaling pathways in human sperm flagellum. P4 released from the cumulus oophorus binds to ABHD2, which cleaves 2-AG to AA and thus removes the inhibition imposed by 2-AG on the CatSper channel. CatSper opens and carries Ca²⁺ into the cell. Warm temperature (37 °C) in the female reproductive tract activates the TRPV4 channel, allowing cations (Na⁺ and Ca²⁺) to enter the cell and to cause membrane depolarization, which further promotes CatSper opening. Besides P4 and depolarization, CatSper also requires intracellular alkalization to produce maximal current. This can be achieved by proton efflux through Hv1, which is activated by anandamide and fatty acids released from the cumulus oophorus. As mentioned in Figure 1a, ${\mathsf{HCO}}_3^{-}$ enters the cell via CO₂ diffusion and via conversion by CA into ${\mathsf{HCO}}_3^{-}$. Additionally, it can be imported by SLC cotransporter interacting with Cl⁻-permeable CFTR. As in murine sperm, ${\mathsf{HCO}}_3^{-}$ activates sAC. Flagellar alkalinity further upregulates CatSper. Potassium enters the cell through the Na⁺/K⁺-ATPase α4 and leaves through the human KSper (Slo3/Slo1) channel, which is activated by depolarization and intracellular Ca²⁺, and is inhibited by P4. Efflux of K⁺ causes hyperpolarization, thus negatively regulating CatSper and Hv1. As in murine sperm, cAMP elevation triggered by ${\mathsf{HCO}}_3^{-}$ and intracellular Ca²⁺ leads to PKA and tyrosine kinase activation, resulting in broad tyrosine phosphorylation. The latter completes sperm capacitation and ensures hyperactivation. Additionally, cholesterol removal from the plasma membrane by albumin further facilitates capacitation. Solid lines represent activation, and dotted lines represent inhibition. Dotted rectangles indicate proteins with yet to be conformed functional role in human male fertility. Abbreviations: TRPV4, transient receptor potential cation channel subfamily member 4 vanilloid 4 channel; sAC, soluble adenylyl cyclase; PKA, protein kinase A; CatSper, cation channel of sperm; Hv1, proton voltage-gated ion channel; ABHD2, α/β hydrolase domain-containing protein 2; 2-AG, 2-arachidonoylglycerol; AA, arachidonic acid; KSper/Slo1/Slo3, potassium channel of sperm/Slowpoke homolog 1/3; SLC, solute carrier family anion exchanger; CFTR, cystic fibrosis transmembrane conductance regulator; sNHE/SLC9C1, sperm-specific Na⁺/H⁺ exchanger; EFCAB9, EF-hand calcium-binding domain-containing protein 9; ATP2B4, ATPase plasma membrane Ca²⁺ transporting 4; ATP, adenosine triphosphate; ADP; adenosine diphosphate; cAMP, cyclic adenosine monophosphate; P4, progesterone; CA, carbonic anhydrases. (a) and (b) figures are created with Biorender.com.

Figure 2. Two possible flagellar arrangements of CatSper nanodomains.

(a) Reproduced from Ref. [••42]: 4Pi single-molecule switching nanoscopy images of murine CatSper1 in wild type (WT) flagella. x–y projection colors encode the relative distance from the focal plane along the z axis. Scale bar, 500 nm. Right panel: y–z cross sections (100 nm thick). Two-row structures are indicated with arrows. Scale bar, 200 nm. (b) SEM images of the principal piece of flagella from WT (top panel) and CatSper1^−/− (bottom panel) mice. As indicated by arrows, double-row lines are observed on both sides of WT flagellum within each longitudinal column. These structures are absent in CatSper1^−/− flagellum. Scale bar, 100 nm. (a) and (b) figures are from Ref. [••42]. (c) Two possible scenarios of CatSper flagellar nanodomain architecture. As shown in (a), CatSper forms two parallel rows and as suggested by Hwang et al. [••42], CatSper-zeta associates with EFCAB9 within the same CatSper complex (left panel). According to an alternative scenario (right panel), CatSper-zeta and EFCAB9 link together two neighboring channels in a zig-zag manner, in which CatSper-zeta from one CatSper complex is associated with EFCAB9 from the neighboring CatSper complex. This hypothetical arrangement would not only structurally link CatSper complexes, but also link them functionally. This linkage could permit more efficient signal propagation along the flagellar length and could be responsible for either synchronization of CatSper opening or longitudinal propagation of Ca²⁺ waves.