Evolutionary Genomics Reveals Lineage-Specific Gene Loss and Rapid Evolution of a Sperm-Specific Ion Channel Complex: CatSpers and CatSperβ

The mammalian CatSper ion channel family consists of four sperm-specific voltage-gated Ca²⁺ channels that are crucial for sperm hyperactivation and male fertility. All four CatSper subunits are believed to assemble into a heteromultimeric channel complex, together with an auxiliary subunit, CatSperβ. Here, we report a comprehensive comparative genomics study and evolutionary analysis of CatSpers and CatSperβ, with important correlation to physiological significance of molecular evolution of the CatSper channel complex. The development of the CatSper channel complex with four CatSpers and CatSperβ originated as early as primitive metazoans such as the Cnidarian Nematostella vectensis. Comparative genomics revealed extensive lineage-specific gene loss of all four CatSpers and CatSperβ through metazoan evolution, especially in vertebrates. The CatSper channel complex underwent rapid evolution and functional divergence, while distinct evolutionary constraints appear to have acted on different domains and specific sites of the four CatSper genes. These results reveal unique evolutionary characteristics of sperm-specific Ca²⁺ channels and their adaptation to sperm biology through metazoan evolution.     

CatSperbeta, a novel transmembrane protein in the CatSper channel complex

Four CatSper ion channel subunit genes (CatSpers 1-4) are required for sperm cell hyperactivation and male fertility. The four proteins assemble (presumably as a tetramer) to form a sperm-specific, alkalinization-activated Ca(2+)-selective channel. We set out to identify proteins associating with CatSper that might help explain its unique role in spermatozoa. Using a transgenic approach, a CatSper1 complex was purified from mouse testis that contained heat shock protein 70-2, a testis-specific chaperone, and CatSperbeta, a novel protein with two putative transmembrane-spanning domains. Like the CatSper ion channel subunits, CatSperbeta was restricted to testis and localized to the principal piece of the sperm tail. CatSperbeta protein is absent in CatSper1(-/-) sperm, suggesting that it is required for trafficking or formation of a stable channel complex. CatSperbeta is the first identified auxiliary protein to the CatSper channel.     

The CatSper channel: a polymodal chemosensor in human sperm

The sperm-specific CatSper channel controls the intracellular Ca(2+) concentration ([Ca(2+)](i)) and, thereby, the swimming behaviour of sperm. In humans, CatSper is directly activated by progesterone and prostaglandins-female factors that stimulate Ca(2+) influx. Other factors including neurotransmitters, chemokines, and odorants also affect sperm function by changing [Ca(2+)](i). Several ligands, notably odorants, have been proposed to control Ca(2+) entry and motility via G protein-coupled receptors (GPCRs) and cAMP-signalling pathways. Here, we show that odorants directly activate CatSper without involving GPCRs and cAMP. Moreover, membrane-permeable analogues of cyclic nucleotides that have been frequently used to study cAMP-mediated Ca(2+) signalling also activate CatSper directly via an extracellular site. Thus, CatSper or associated protein(s) harbour promiscuous binding sites that can host various ligands. These results contest current concepts of Ca(2+) signalling by GPCR and cAMP in mammalian sperm: ligands thought to activate metabotropic pathways, in fact, act via a common ionotropic mechanism. We propose that the CatSper channel complex serves as a polymodal sensor for multiple chemical cues that assist sperm during their voyage across the female genital tract.     

Association of Catsper1 or -2 with Ca(v)3.3 leads to suppression of T-type calcium channel activity

Sperm-specific CatSper1 and CatSper2 proteins are critical to sperm-hyperactivated motility and male fertility. Although architecturally resembling voltage-gated ion channels, neither CatSper1 nor CatSper2 alone forms functional ion channels in heterologous expression systems, which may be related to the absence of yet unidentified accessory subunits. Here we isolated CatSper1- and CatSper2-associated protein(s) from human sperm and analyzed their identities by a multidimensional protein identification technology approach. We identified the T-type voltage-gated calcium channel Ca(v)3.3 as binding to both CatSper1 and CatSper2. The specificity of their interactions was verified by co-immunoprecipitation in transfected mammalian cells. Electrophysiological studies revealed that the co-expression of CatSper1 or CatSper2 specifically inhibited the amplitude of Ca(v)3.3-evoked T-type calcium current without altering other biophysical properties of Ca(v)3.3. Immunostaining studies revealed co-localization of CatSper1 and Ca(v)3.3 on the principal piece of human sperm tail. Furthermore, fluorescence resonance energy transfer analysis revealed close proximity and physical association of these two proteins on the sperm tail. These studies demonstrate that CatSper1 and CatSper2 can associate with and modulate the function of the Ca(v)3.3 channel, which might be important in the regulation of sperm function.     

Pharmacological Targeting of Native CatSper Channels Reveals a Required Role in Maintenance of Sperm Hyperactivation

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca²⁺ entry evoked by alkaline depolarization. In the absence of external Ca²⁺, Na⁺ carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na⁺]_i-reporting probe SBFI in populations of intact sperm. Removal of external Ca²⁺ increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na⁺]_i is greater for sperm alkalinized with NH₄Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na⁺]_i rise is slowed by candidate CatSper blocker HC-056456 (IC₅₀ ∼3 µM). HC-056456 similarly slows the rise of [Ca²⁺]_i that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO₃ ⁻ but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca²⁺ entry through the CatSper channel is terminated by removal of external Ca²⁺. Thus, open CatSper channels and entry of external Ca²⁺ through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.     

Identification of human and mouse CatSper3 and CatSper4 genes: Characterisation of a common interaction domain and evidence for expression in testis

Background  

CatSper1 and CatSper2 are two recently identified channel-like proteins, which show sperm specific expression patterns. Through targeted mutagenesis in the mouse, CatSper1 has been shown to be required for fertility, sperm motility and for cAMP induced Ca²⁺ current in sperm. Both channels resemble a single pore forming repeat from a four repeat voltage dependent Ca²⁺ /Na⁺ channel. However, neither CatSper1 or CatSper2 have been shown to function as cation channels when transfected into cells, singly or in conjunction. As the pore forming units of voltage gated cation channels form a tetramer it has been suggested that the known CatSper proteins require additional subunits and/or interaction partners to function.     

A Model of CatSper Channel Mediated Calcium Dynamics in Mammalian Spermatozoa

CatSpers are calcium (Ca²⁺) channels that are located along the principal piece of mammalian sperm flagella and are directly linked to sperm motility and hyperactivation. It has been observed that Ca²⁺ entry through CatSper channels triggers a tail to head Ca²⁺ propagation in mouse sperm, as well as a sustained increase of Ca²⁺ in the head. Here, we develop a mathematical model to investigate this propagation and sustained increase in the head. A 1-d reaction-diffusion model tracking intracellular Ca²⁺ with flux terms for the CatSper channels, a leak flux, and plasma membrane Ca²⁺ clearance mechanism is studied. Results of this simple model exhibit tail to head Ca²⁺ propagation, but no sustained increase in the head. Therefore, in this model, a simple plasma membrane pump-leak system with diffusion in the cytosol cannot account for these experimentally observed results. It has been proposed that Ca²⁺ influx from the CatSper channels induce additional Ca²⁺ release from an internal store. We test this hypothesis by examining the possible role of Ca²⁺ release from the redundant nuclear envelope (RNE), an inositol 1,4,5-trisphosphate (IP₃) gated Ca²⁺ store in the neck. The simple model is extended to include an equation for IP₃ synthesis, degradation, and diffusion, as well as flux terms for Ca²⁺ in the RNE. When IP₃ and the RNE are accounted for, the results of the model exhibit a tail to head Ca²⁺ propagation as well as a sustained increase of Ca²⁺ in the head.     

CATSPER channel-mediated Ca2+ entry into mouse sperm triggers a tail-to-head propagation

Many Ca(2+) channel proteins have been detected in mammalian sperm, but only the four CATSPER channels have been clearly shown to be required for male fertility. Ca(2+) entry through the principal piece-localized CATSPER channels has been implicated in the activation of hyperactivated motility. In the present study, we show that the Ca(2+) entry also triggers a tail-to-head Ca(2+) propagation in the mouse sperm. When activated with 8-Br-cAMP, 8-Br-cGMP, or alkaline depolarization, a CATSPER-dependent increase in intracellular Ca(2+) concentration starts in the principal piece, propagates through the midpiece, and reaches the head in a few seconds. The Ca(2+) propagation through the midpiece leads to a Ca(2+)-dependent increase in NADH fluorescence. In addition, CatSper1-mutant sperm have lower intracellular ATP levels than wild-type sperm. Thus, a Ca(2+) influx in the principal piece through CATSPER channels can not only initiate hyperactivated motility, but can also trigger a tail-to-head Ca(2+) propagation that leads to an increase in [NADH] and may regulate ATP homeostasis.     

SLO3 K+ Channels Control Calcium Entry through CATSPER Channels in Sperm

Here we show how a sperm-specific potassium channel (SLO3) controls Ca²⁺ entry into sperm through a sperm-specific Ca²⁺ channel, CATSPER, in a totally unanticipated manner. The genetic deletion of either of those channels confers male infertility in mice. During sperm capacitation SLO3 hyperpolarizes the sperm, whereas CATSPER allows Ca²⁺ entry. These two channels may be functionally connected, but it had not been demonstrated that SLO3-dependent hyperpolarization is required for Ca²⁺ entry through CATSPER channels, nor has a functional mechanism linking the two channels been shown. In this study we show that Ca²⁺ entry through CATSPER channels is deficient in Slo3 mutant sperm lacking hyperpolarization; we also present evidence supporting the hypothesis that SLO3 channels activate CATSPER channels indirectly by promoting a rise in intracellular pH through a voltage-dependent mechanism. This mechanism may work through a Na⁺/H⁺ exchanger (sNHE) and/or a bicarbonate transporter, which utilizes the inward driving force of the Na⁺ gradient, rendering it intrinsically voltage-dependent. In addition, the sperm-specific Na⁺/H⁺ exchanger (sNHE) possess a putative voltage sensor that might be activated by membrane hyperpolarization, thus increasing the voltage sensitivity of internal alkalization.     

Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca²⁺ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca ²⁺ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca ²⁺ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca ²⁺ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca ²⁺ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca ²⁺ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH ₄Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca ²⁺ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.     

Evolution of ionic channels of biological membranes

This paper presents a view of the evolution and phylogenetic distribution of ionic channels of biological membranes. The view is based on the assumptions that ionic channels (1) appeared very early in the history of life, (2) have evolved from a common ancestor, and (3) have been subjected to evolutionary pressure to reach precision and high speed of signaling. We propose that Ca2+ was the intracellular messenger and modulator of the most primitive biological systems, which implies that the first channel to appear may have been a calcium channel. Then, very soon the entire group of potassium channels evolved from the calcium channel to improve the shape of signals and to restore initial conditions. Sodium channels probably appeared relatively late, diversifying from calcium channels in the early metazoan groups. Mainly because Na+ ions do not interfere with cellular metabolism (thus allowing the inward current--and, consequently, the speed of conduction--to be greatly increased), sodium channels probably proved advantageous in the generation of the action potential, and selection replaced calcium channels with sodium channels in this function. Finally, with the acquisition of multicellularity, channels responsible for synaptic transmission appeared. The case of the acetylcholine receptor channel is briefly discussed.     

Functional, biochemical, and chromatographic characterization of the complete [Ca2+]i oscillation-inducing activity of porcine sperm

A cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCzeta, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1-5 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.     

Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.     

The CatSper channel controls chemosensation in sea urchin sperm

Sperm guidance is controlled by chemical and physical cues. In many species, Ca²⁺ bursts in the flagellum govern navigation to the egg. In Arbacia punctulata, a model system of sperm chemotaxis, a cGMP signaling pathway controls these Ca²⁺ bursts. The underlying Ca²⁺ channel and its mechanisms of activation are unknown. Here, we identify CatSper Ca²⁺ channels in the flagellum of A. punctulata sperm. We show that CatSper mediates the chemoattractant-evoked Ca²⁺ influx and controls chemotactic steering; a concomitant alkalization serves as a highly cooperative mechanism that enables CatSper to transduce periodic voltage changes into Ca²⁺ bursts. Our results reveal intriguing phylogenetic commonalities but also variations between marine invertebrates and mammals regarding the function and control of CatSper. The variations probably reflect functional and mechanistic adaptations that evolved during the transition from external to internal fertilization.     

PLC zeta: a sperm-specific trigger of Ca(2+) oscillations in eggs and embryo development

Upon fertilisation by sperm, mammalian eggs are activated by a series of intracellular Ca(2+) oscillations that are essential for embryo development. The mechanism by which sperm induces this complex signalling phenomenon is unknown. One proposal is that the sperm introduces an exclusive cytosolic factor into the egg that elicits serial Ca(2+) release. The 'sperm factor' hypothesis has not been ratified because a sperm-specific protein that generates repetitive Ca(2+) transients and egg activation has not been found. We identify a novel, sperm-specific phospholipase C, PLC zeta, that triggers Ca(2+) oscillations in mouse eggs indistinguishable from those at fertilisation. PLC zeta removal from sperm extracts abolishes Ca(2+) release in eggs. Moreover, the PLC zeta content of a single sperm was sufficient to produce Ca(2+) oscillations as well as normal embryo development to blastocyst. Our results are consistent with sperm PLC zeta as the molecular trigger for development of a fertilised egg into an embryo.     

Conservation of the Ca2+-permeability through the voltage sensor domain of mammalian CatSper subunit

Cation channel of Spermatozoa (CatSper) is one of the voltage-gated ion channels consisting of voltage sensor domains (VSDs) and pore-gate domains. CatSper is exclusively expressed in spermatozoa and indispensable for Ca²⁺ influx into cytosol. Recently, we have reported that the VSD of ascidian CatSper induces Ca²⁺-permeable pathways in heterologous expression systems. However, it is not known whether ion permeability through the VSD of CatSper is conserved in mammals. In the present study, electrophysiology and fluorometry in Xenopus oocytes revealed that Ca²⁺-permeable paths are also formed by expressing the VSD of murine CatSper. We also examined the permeability to monovalent cations other than Na⁺ in the VSD of ascidian CatSper.     

Intramembranal disulfide cross-linking elucidates the super-quaternary structure of mammalian CatSpers

CatSper is a voltage-dependent calcium channel located in the plasma membrane of the sperm flagellum and is responsible for triggering hyperactive motility. A homology model for the transmembrane region was built in which the arrangement of the subunits around the pseudo-four-fold symmetry axis was deduced by the pairing of conserved transmembranal cysteines across mammals. Directly emergent of the predicted quaternary structure is an architecture in which tetramers polymerize through additional, highly conserved cysteines, creating one or more double-rows channels extending the length of the principal piece of the mammalian sperm tail. The few species that are missing these cysteines are eusocial or otherwise monogamous, suggesting that sperm competition is selective for a disulfide-crosslinked macromolecular architecture. The model suggests testable hypotheses for how CatSper channel opening might behave in response to pH, 2-arachidonoylglycerol, and mechanical force. A flippase function is hypothesized, and a source of the concomitant disulfide isomerase activity is found in CatSper-associated proteins β, δ and ε.     

ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization

The sperm acrosome reaction is a Ca(2+)-dependent secretory event required for fertilization. Adhesion to the egg's zona pellucida promotes Ca2+ influx through voltage-sensitive channels, thereby initiating secretion. We used potentiometric fluorescent probes to determine the role of sperm membrane potential in regulating Ca2+ entry. ZP3, the glycoprotein agonist of the zona pellucida, depolarizes sperm membranes by activating a pertussis toxin-insensitive mechanism with the characteristics of a poorly selective cation channel. ZP3 also activates a pertussis toxin-sensitive pathway that produces a transient rise in internal pH. The concerted effects of depolarization and alkalinization open voltage-sensitive Ca2+ channels. These observations suggest that mammalian sperm utilize membrane potential-dependent signal transduction mechanisms and that a depolarization pathway is an upstream transducing element coupling adhesion to secretion during fertilization.     

Direct action of endocrine disrupting chemicals on human sperm

Synthetic endocrine disrupting chemicals (EDCs), omnipresent in food, household, and personal care products, have been implicated in adverse trends in human reproduction, including infertility and increasing demand for assisted reproduction. Here, we study the action of 96 ubiquitous EDCs on human sperm. We show that structurally diverse EDCs activate the sperm‐specific CatSper channel and, thereby, evoke an intracellular Ca²⁺ increase, a motility response, and acrosomal exocytosis. Moreover, EDCs desensitize sperm for physiological CatSper ligands and cooperate in low‐dose mixtures to elevate Ca²⁺ levels in sperm. We conclude that EDCs interfere with various sperm functions and, thereby, might impair human fertilization.