Reconstruction of the three-dimensional beat pattern underlying swimming behaviors of sperm.

 The eukaryotic flagellum propels sperm cells and simultaneously detects physical and chemical cues that modulate the waveform of the flagellar beat. Most previous studies have characterized the flagellar beat and swimming trajectories in two space dimensions (2D) at a water/glass interface. Here, using refined holographic imaging methods, we report high-quality recordings of three-dimensional (3D) flagellar bending waves. As predicted by theory, we observed that an asymmetric and planar flagellar beat results in a circular swimming path, whereas a symmetric and non-planar flagellar beat results in a twisted-ribbon swimming path. During swimming in 3D, human sperm flagella exhibit torsion waves characterized by maxima at the low curvature regions of the flagellar wave. We suggest that these torsion waves are common in nature and that they are an intrinsic property of beating axonemes. We discuss how 3D beat patterns result in twisted-ribbon swimming paths. This study provides new insight into the axoneme dynamics, the 3D flagellar beat, and the resulting swimming behavior.

Synergistic activation of CatSper Ca2+ channels in human sperm by oviductal ligands and endocrine disrupting chemicals.

STUDY QUESTION: Does the chemosensory activation of CatSper Ca2+ channels in human sperm give rise to additive, sub-additive or even synergistic actions among agonists? SUMMARY ANSWER: We show that oviductal ligands and endocrine disrupting chemicals (EDCs) activate human CatSper highly synergistically. WHAT IS KNOWN ALREADY: In human sperm, the sperm-specific CatSper channel controls the intracellular Ca2+ concentration and, thereby, several crucial stages toward fertilization. CatSper is activated by oviductal ligands and structurally diverse EDCs. The chemicals mimic the action of the physiological ligands, which might interfere with the precisely coordinated sequence of events underlying fertilization. STUDY DESIGN, SIZE, DURATION: For both oviductal ligands and EDCs, we examined in quantitative terms whether stimulation of human sperm in vitro with mixtures results in additive, sub-additive or synergistic actions. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied activation of CatSper in sperm of healthy volunteers, using kinetic Ca2+ fluorimetry and patch-clamp recordings. The combined action of progesterone and prostaglandins and of the EDCs benzylidene camphor sulfonic acid (BCSA) and α-Zearalenol was evaluated by curve-shift analysis, curvilinear isobolographic analysis and the combination-index method. MAIN RESULTS AND THE ROLE OF CHANCE: Analysis of the action of progesterone/prostaglandin and BCSA/α-Zearalenol mixtures in human sperm by fluorimetry revealed that the oviductal ligands and EDCs both evoke Ca2+ influx via CatSper in a highly synergistic fashion. Patch-clamp recordings of CatSper currents in human sperm corroborated the synergistic ligand-activation of the channel. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study. Future studies have to assess the physiological relevance in vivo. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that the fertilization process is orchestrated by multiple oviductal CatSper agonists that act in concert to control the behavior of sperm. Moreover, our results substantiate the concerns regarding the negative impact of EDCs on male reproductive health. So far, safety thresholds like the "No Observed Adverse Effect Level (NOAEL)" or "No Observed Effect Concentration (NOEC)" are set for individual EDCs. Our finding that EDCs act synergistically in human sperm challenges the validity of this procedure. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the German Research Foundation (SFB 645; CRU326), the Cells-in-Motion (CiM) Cluster of Excellence, Münster, (FF-2016-17), the 'Innovative Medical Research' of the University of Münster Medical School (BR121507), an EDMaRC research grant from the Kirsten and Freddy Johansen's Foundation, and the Innovation Fund Denmark (InnovationsFonden; 14-2013-4). The authors have no competing financial interests.

The steering gaits of sperm.

Sperm are highly specialized cells, which have been subject to substantial evolutionary pressure. Whereas some sperm features are highly conserved, others have undergone major modifications. Some of these variations are driven by adaptation to mating behaviours or fitness at the organismic level. Others represent alternative solutions to the same task. Sperm must find the egg for fertilization. During this task, sperm rely on long slender appendages termed flagella that serve as sensory antennas, propellers and steering rudders. The beat of the flagellum is periodic. The resulting travelling wave generates the necessary thrust for propulsion in the fluid. Recent studies reveal that, for steering, different species rely on different fundamental features of the beat wave. Here, we discuss some examples of unity and diversity across sperm from different species with a particular emphasis on the steering mechanisms. This article is part of the Theo Murphy meeting issue 'Unity and diversity of cilia in locomotion and transport'.

Author Correction: The solute carrier SLC9C1 is a Na+/H+-exchanger gated by an S4-type voltage-sensor and cyclic-nucleotide binding.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cyclic nucleotide-gated channels on the flagellum control Ca2+ entry into sperm.

Cyclic nucleotide-gated (CNG) channels are key elements of cGMP- and cAMP-signaling pathways in vertebrate photoreceptor cells and in olfactory sensory neurons, respectively. These channels form heterooligomeric complexes composed of at least two distinct subunits (alpha and beta). The alpha subunit of cone photoreceptors is also present in mammalian sperm. Here we identify one short and several long less abundant transcripts of beta subunits in testis. The alpha and beta subunits are expressed in a characteristic temporal and spatial pattern in sperm and precursor cells. In mature sperm, the alpha subunit is observed along the entire flagellum, whereas the short beta subunit is restricted to the principal piece of the flagellum. These findings suggest that different forms of CNG channels coexist in the flagellum. Confocal microscopy in conjunction with the Ca2+ indicator Fluo-3 shows that the CNG channels serve as a Ca2+ entry pathway that responds more sensitively to cGMP than to cAMP. Assuming that CNG channel subtypes differ in their Ca2+ permeability, dissimilar localization of alpha and beta subunits may give rise to a pattern of Ca2+ microdomains along the flagellum, thereby providing the structural basis for control of flagellar bending waves.

Cell biology. A universal bicarbonate sensor.

The life-span of sperm may be short but it is certainly busy. The three principal molecular events that prepare sperm for fertilization are all controlled by the intracellular nucleotide adenosine 3',5'-monophosphate (cAMP). One of these, capacitation, is also regulated by bicarbonate ions. The elusive connection between cAMP and bicarbonate ions now appears to be solved as Kaupp and Weyand explain in their Perspective. Bicarbonate ions enter sperm through the anion transporter in the sperm plasma membrane and activate the soluble form of adenylyl cyclase, the enzyme that synthesizes cAMP (Chen et al.)

Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels.

Sensory transduction in vertebrate photoreceptors and olfactory sensory neurons is mediated by cyclic nucleotide-gated (CNG) channels that conduct mono- and divalent cations. Ca2+ entering the cell through CNG channels intimately controls signaling pathways by regulating several key enzymes. Cloned CNG channels from photoreceptors and olfactory sensory neurons profoundly differ in their relative Ca2+ permeability, their blockage by external divalent cations, and the fraction of current carried by Ca2+. In particular, CNG channels from cone photoreceptors conduct significantly more Ca2+ than those from rod photoreceptors. Furthermore, the current through the olfactory CNG channel is entirely carried by Ca2+ at approximately 3 mM extracellular Ca2+. These results suggest that a major function of CNG channels is to provide a pathway for Ca2+ entry.

Rod and cone photoreceptor cells express distinct genes for cGMP-gated channels.

Signal transduction in vertebrate rod and cone photoreceptor cells involves ion channels that are directly gated by the internal messenger cGMP. Rods and each type of cones express genetically related yet different forms of photopigments. Enzymes that control the light-stimulated hydrolysis of cGMP in rods and cones are also the product of distinct genes. Two different cDNA clones encoding cGMP-gated channels have been characterized from the chicken retina. Expression of cDNAs in Xenopus oocytes gives rise to cGMP-stimulated channel activity. Antibodies against a synthetic peptide specific for the C-terminal amino acid sequence derived from one clone stain outer segments of cone but not rod photoreceptors. Therefore chicken rod and cone cells each express different forms of cGMP-gated channels that are genetically related to each other. Expression in COS-1 cells produces the complete form of both channel polypeptides, whereas Western blot analysis indicates that channels in outer segment membranes are present in a processed form that is significantly shorter than the full-length polypeptide.

The solute carrier SLC9C1 is a Na+/H+-exchanger gated by an S4-type voltage-sensor and cyclic-nucleotide binding.

Voltage-sensing (VSD) and cyclic nucleotide-binding domains (CNBD) gate ion channels for rapid electrical signaling. By contrast, solute carriers (SLCs) that passively redistribute substrates are gated by their substrates themselves. Here, we study the orphan sperm-specific solute carriers SLC9C1 that feature a unique tripartite structure: an exchanger domain, a VSD, and a CNBD. Voltage-clamp fluorimetry shows that SLC9C1 is a genuine Na+/H+ exchanger gated by voltage. The cellular messenger cAMP shifts the voltage range of activation. Mutations in the transport domain, the VSD, or the CNBD strongly affect Na+/H+ exchange, voltage gating, or cAMP sensitivity, respectively. Our results establish SLC9C1 as a phylogenetic chimaera that combines the ion-exchange mechanism of solute carriers with the gating mechanism of ion channels. Classic SLCs slowly readjust changes in the intra- and extracellular milieu, whereas voltage gating endows the Na+/H+ exchanger with the ability to produce a rapid pH response that enables downstream signaling events.

Signaling in Sperm: More Different than Similar.

For a given sensory cell type, signaling motifs are rather uniform across phyla. By contrast, sperm from different species use diverse repertoires of sperm-specific signaling molecules and even closely related protein isoforms feature different properties and serve different functions. This surprising diversity has consequences for strategies in fertilization research and it will take some time to get the big picture. We discuss the function of receptors, ion channels, and exchangers embedded in cellular pathways from different sperm species.

The cyclic nucleotide-gated channels of vertebrate photoreceptors and olfactory epithelium.

Cation channels that are directly gated by guanosine 3', 5'-cyclic monophosphate (cGMP) control the flow of ions across the surface membrane of vertebrate rod and cone photoreceptor cells. A similar channel, gated by adenosine 3',5'-cyclic monophosphate (cAMP), exists in vertebrate olfactory sensory neurons. The channel polypeptide of rod photoreceptors has been identified and the amino acid sequence of the channel polypeptide in rod and olfactory cells has been determined by cloning cDNA. Although the cyclic nucleotide-gated channels functionally belong to the class of ligand-gated channels, they share some structural features with voltage-gated channels.

Family of cyclic nucleotide gated ion channels.

A variety of different cyclic nucleotide gated ion channels have recently been identified using molecular cloning and electrophysiological techniques. Current research is focussed on the specific molecular determinants that endow these channels with their distinctive character of gating, selectivity and modulation. In some cases, it has been possible to identify the specific physiological roles of different cyclic nucleotide gated channels. Their interactions with Ca2+ and calmodulin are particularly important, and determine the specific functions these channels subserve in distinct cells.

Interfacial potentials at the disk membranes of isolated intact cattle rod outer segments as a function of the occupation state of the intradiskal cation-exchange binding sites.

Two different methods have been used to determine the interfacial potential at the disk membranes of intact isolated bovine rod outer segments: (1) The photolysis products of rhodopsin are known to be dependent on pH. We have used this property in order to probe the interfacial potential at disk membranes which is considered to change the surface pH at the disk membrane seen by rhodopsin. (2) The pK value of the amphiphilic pH-indicating dye neutral red (uncharged basic form) in water is 6.6, but adsorbed to disk membranes at least 7.8. This makes the distribution of neutral red between disk membranes and bulk water dependent on the interfacial potential at the disk membrane if the pH in the bulk solution is less than 7.8. Both methods yielded comparable results on the influence of ions and ion carriers on the interfacial potential at disk membranes. In particular, we have studied the effect of different occupation states on the internal binding capacity (of rod outer segments) for divalent cations. In the presence of the ionophore A23187, addition of EDTA to a suspension of intact rod outer segments removed all endogenous divalent cations (Schnetkamp, P.P.M. (1979) Biochim. Biophys. Acta 554, 441--459) and resulted in an interfacial pH at the disk membrane surface of about 6.4, whereas the bulk pH was 7.4. Subsequent addition of 2 mM Mn2+ saturated the internal binding capacity and resulted in an apparent shift towards alkaline pH of the surface pH at the disk membrane by 1.0--1.1 pH units. This could indicate a change of the interfacial potential by 60--65 mV. The same change of ionic conditions resulted in a change of the interfacial potential by 72 mV as determined from the partitioning behaviour of neutral red. These results were independent of the presence of H+ ionophores such as carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and gramicidin. We conclude that the above results can be explained by the presence of fixed net negative charges (charge density: 0.5--1.5 electronic charges/rhodopsin molecule) at the intradiskal membrane surface. That the above charge density can be attributed to the intradiskal membrane surface is inferred from the observation that the presence of A23187 was required for access of divalent cations to the membrane interface involved in both rod outer segments with an intact as well as with a leaky plasma membrane.

Light-induced calcium release in isolated intact cattle rod outer segments upon photoexcitation of rhodopsin.

By applying flash-spectrophotometry with the calcium-indicating dye arsenazo III rapid light-triggered calcium release in various cattle rod outer segment preparations was studied. It is shown that light-induced calcium signals can be unambiguously discriminated from underlying absorption changes due to photolysis of rhodopsin and apparent absorption changes resulting from lightscattering transients. The following results have been obtained: 1. Calcium-induced arsenazo III responses can be quantitatively and kinetically resolved within the time domain of the visual transduction process. 2. Photoexcitation of rhodopsin results in calcium release from intradiscal binding sites. 3. Calcium released does not appear in the cytoplasmic space unless the disc membrane is made permeable to calcium ions by an ionophore. 4. The shortest observed half-rise time of calcium release (300 ms) is possibly limited by the ionophore. 5. The stoichiometric ratio of calcium released/rhodopsin bleached is 0.5 at a free calcium concentration of 2 microM. The amount of calcium released is proportional to the precentage of rhodopsin bleaching (from 1--10%). 6. Upon disruption of the disc stack by lysis of intact rod outer segments the light-induced calcium release is greatly altered. The results are discussed in relation to previous reports on a light-induced calcium release from retinal discs and in terms of the proposed role of calcium as an intracellular transmitter in vertebrate photoreceptors.

Transduction persists in rod photoreceptors after depletion of intracellular calcium.

We have examined the role of Ca++ in phototransduction by manipulating the intracellular Ca++ concentration in physiologically active suspensions of isolated and purified rod photoreceptors (OS-IS). The results are summarized by the following. Measurement of Ca++ content using arsenazo III spectroscopy demonstrates that incubation of OS-IS in 10 nM Ca++-Ringer's solution containing the Ca++ ionophore A23187 reduces their Ca++ content by 93%, from 1.3 to 0.1 mol Ca++/mol rhodopsin. Virtually the same reduction can be accomplished in 10 nM Ca++-Ringer's without ionophore, presumably via the plasma membrane Na/Ca exchange mechanism. Hundreds of photoresponses can be obtained from the Ca++-depleted OS-IS for at least 1 h in 10 nM Ca++-Ringer's with ionophore. The kinetics and light sensitivity of the photoresponse are essentially the same in the presence or absence of the ionophore in 10 nM Ca++. The addition of A23187 in 1 mM Ca++-Ringer's results in a Ca++ influx that rapidly suppresses the dark current and the photoresponse. This indicates that there is an intracellular site at which Ca++ can modulate the light-regulated conductance. Both the current and photoresponse can be restored if intracellular Ca++ is reduced by lowering the external Ca++ to 10 nM. During the transition from high to low Ca++, the response duration becomes shorter, which suggests that it can be regulated by a Ca++-dependent mechanism. If the dark current and the photoresponse are suppressed by adding A23187 in 1 mM Ca++-Ringer's, the subsequent addition of the cyclic GMP phosphodiesterase inhibitor isobutylmethylxanthine can restore the current and photoresponse. This implies that under conditions where the rod can no longer control its intracellular Ca++, the elevation of cyclic GMP levels can restore light regulation of the channels. The persistence of normal flash responses under conditions where intracellular Ca++ levels are reduced and perturbed suggests that changes in the intracellular Ca++ concentration do not cause the closure of the light-regulated channel.

A point mutation in the pore region alters gating, Ca(2+) blockage, and permeation of olfactory cyclic nucleotide-gated channels.

Upon stimulation by odorants, Ca(2+) and Na(+) enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide-gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the alpha subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide-gated channels play a similar role. We studied the corresponding glutamate (E340) of the alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca(2+) and Mg(2+) powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca(2+) relative to Na(+) was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.

Gating by cyclic GMP and voltage in the alpha subunit of the cyclic GMP-gated channel from rod photoreceptors.

Gating by cGMP and voltage of the alpha subunit of the cGMP-gated channel from rod photoreceptor was examined with a patch-clamp technique. The channels were expressed in Xenopus oocytes. At low [cGMP] (<20 microM), the current displayed strong outward rectification. At low and high (700 microM) [cGMP], the channel activity was dominated by only one conductance level. Therefore, the outward rectification at low [cGMP] results solely from an increase in the open probability, P(o). Kinetic analysis of single-channel openings revealed two exponential distributions. At low [cGMP], the larger P(o) at positive voltages with respect to negative voltages is caused by an increased frequency of openings in both components of the open-time distribution. In macroscopic currents, depolarizing voltage steps, starting from -100 mV, generated a time-dependent current that increased with the step size (activation). At low [cGMP] (20 microM), the degree of activation was large and the time course was slow, whereas at saturating [cGMP] (7 mM) the respective changes were small and fast. The dose-response relation at -100 mV was shifted to the right and saturated at significantly lower P(o) values with respect to that at +100 mV (0.77 vs. 0.96). P(o) was determined as function of the [cGMP] (at +100 and -100 mV) and voltage (at 20, 70, and 700 microM, and 7 mM cGMP). Both relations could be fitted with an allosteric state model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric opening reaction. At saturating [cGMP] (7 mM), the activation time course was monoexponential, which allowed us to determine the individual rate constants for the allosteric reaction. For the rapid rate constants of cGMP binding and unbinding, lower limits are determined. It is concluded that an allosteric model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric reaction, describes the cGMP- and voltage-dependent gating of cGMP-gated channels adequately.

Fraction of the dark current carried by Ca(2+) through cGMP-gated ion channels of intact rod and cone photoreceptors.

The selectivity for Ca(2+) over Na(+), PCa/PNa, is higher in cGMP-gated (CNG) ion channels of retinal cone photoreceptors than in those of rods. To ascertain the physiological significance of this fact, we determined the fraction of the cyclic nucleotide-gated current specifically carried by Ca(2+) in intact rods and cones. We activated CNG channels by suddenly (<5 ms) increasing free 8Br-cGMP in the cytoplasm of rods or cones loaded with a caged ester of the cyclic nucleotide. Simultaneous with the uncaging flash, we measured the cyclic nucleotide-dependent changes in membrane current and fluorescence of the Ca(2+)-binding dye, Fura-2, also loaded into the cells. The ratio of changes in fura-2 fluorescence and the integral of the membrane current, under a restricted set of experimental conditions, is a direct measure of the fractional Ca(2+) flux. Under normal physiological salt concentrations, the fractional Ca(2+) flux is higher in CNG channels of cones than in those of rods, but it differs little among cones (or rods) of different species. Under normal physiological conditions and for membrane currents

Characterization of ether-à-go-go channels present in photoreceptors reveals similarity to IKx, a K+ current in rod inner segments.

In this study, we describe two splice variants of an ether-à-go-go (EAG) K+ channel cloned from bovine retina: bEAG1 and bEAG2. The bEAG2 polypeptide contains an additional insertion of 27 amino acids in the extracellular linker between transmembrane segments S3 and S4. The heterologously expressed splice variants differ in their activation kinetics and are differently modulated by extracellular Mg2+. Cooperativity of modulation by Mg2+ suggests that each subunit of the putative tetrameric channel binds a Mg2+ ion. The channels are neither permeable to Ca2+ ions nor modulated by cyclic nucleotides. In situ hybridization localizes channel transcripts to photoreceptors and retinal ganglion cells. Comparison of EAG currents with IKx, a noninactivating K+ current in the inner segment of rod photoreceptors, reveals an intriguing similarity, suggesting that EAG polypeptides are involved in the formation of Kx channels.

Sperm chemotaxis: a primer.

Sperm become attracted by chemical substances that are released from the outer coating of the egg, a process called chemotaxis. In this paper the cellular pathway and the motor response during chemotaxis of sperm from sea urchin and starfish are briefly outlined.