Microscopic analysis of sperm movement: links to mechanisms and protein components

Microscopic analysis of sperm movement: links to mechanisms and protein components Abstract Cilia and flagella are cell machines that power hydrodynamic forces by fast beating. They are composed of bundles of 9 + 2 microtubules that associate with several protein structures including axonemal dyneins and their regulators. Spermatozoa are single cells with in most cases a single flagellum and are good cell models to study how flagellar movements are regulated and linked to cell behavior. In addition, the assessment of sperm motility is an important diagnostic tool for evaluating male fertility in human reproductive medicine, and in livestock and fisheries sciences. Microscopic analyses of the movements of spermatozoa and their flagellar waveforms and propagation have been carried out using high-speed cameras and stroboscopic illumination. Computer-assisted sperm analysis (CASA) now comprises an automated set of methods to evaluate sperm quality for fertility. Here, we summarize the microscopy systems used for evaluating sperm motility, including CASA, and introduce updates on the molecular mechanism of flagellar movement and regulation that are linked to motility parameters. Furthermore, we introduce recent techniques employed to measure key factors controlling sperm motility. cilia, flagella, axonemal dynein, sperm motility, CASA, male fertility Introduction Profound human interest in a microscopic world was opened up by Antonie van Leeuwenhoek, who used a small hand-made microscope with a highly polished lens and observed several organisms (see [1]). In a letter written to the Royal Society in 1677, he described spermatozoa as one of the ‘animalcules’, moving in semen. After a series of works for more than a century summarized in [2], precise description of fast ciliary movements was first recorded by using photographic and stroboscopic recording [3]. Sir James Gray then used 2 ms flash photography and recorded the flagellar beating of sea urchin spermatozoa. The flagellar movements were seen ‘frozen’ at an illumination frequency of 30–50 per second [4]. Undamped oscillations of ciliary and flagellar waves suggested that the active motive elements were present from the base to tip of these motile apparatuses [5]. Several studies then followed to record the propagation of flagellar waveforms with more sophisticated systems for illumination and analysis [6,7]. These attempts, in conjugation with the structural observation of flagellar structure by thin-section electron microscopy, contributed to understanding the mechanism of sperm flagellar movement. Although cilia and flagella are discriminated by their length, numbers per cell and fashion in wave propagation, they structurally comprise the same motile machineries and the terminology is just for descriptive purpose [8–10]. Structural and functional identity is demonstrated in the simultaneous symptom of dysfunction in sperm motility and those in other ciliated organs, as seen in primary ciliary dyskinesia [11]. Understanding the structure and mechanism of cilia and flagella have been advanced in flagellated and ciliated cells and organisms, such as Chlamydomonas, ciliates and marine invertebrates (see [12]). From a series of experimental observations [13–16], it was established that ciliary and flagellar oscillatory movements are caused by the active sliding of microtubules by dyneins. Proteomics and structural description of sperm axonemes by cryoelectron tomography have revealed the detailed configuration of axonemal proteins, contributing to the understanding of molecular mechanism of ciliary and flagellar motility. In this review, we overview the microscopic recording and systems of analyzing the rapid movements in sperm flagellar motility. These systems include important methods for diagnosing human male fertility in reproductive medicine, and in livestock and fisheries science. For a comprehensive assessment of sperm motility, it would be useful to understand how the parameters of flagellar movement estimated by light microscopy link to the molecular mechanisms for dynein-mediated microtubule sliding. Mechanism of sperm motility The cytoskeletal basis of sperm flagella for motility is the axoneme, which is typically composed of a set of nine doublet and two singlet microtubules (9+2) with associated structures including outer and inner arm dynein molecules and their regulatory components (Fig. 1a). Both outer and inner dynein arms (IDA) are axonemal dyneins, comprising heavy, intermediate and light chains (LCs). The heavy chains are motor ATPases, and the intermediate/LCs are involved in the assembly and regulation of the machinery [9,17]. Axonemal dyneins are functionally and phylogenetically different from cytoplasmic dynein, which plays a role in the intracellular transport of molecules and vesicles. Although the axoneme is central to producing the basic waveform and its propagation, the structural pattern is diverse and has accessory structures in some organisms, which more or less affect their movement characteristics [18]. Biochemical and proteomic identification and structural study using cryoelectron tomography, as well as the analysis of Chlamydomonas flagella mutants have brought a great advance in understanding the molecular structure and function of each axonemal component [9,19–21] as described below. Fig. 1. View largeDownload slide Basic concepts for the analysis of sperm flagellar motility. (a) Structure of the flagellar axoneme. Doublet microtubules are composed of A- and B-tubules and numbered by their position relative to the axis of the central pair. (b) Relationship between flagellar bend angle and microtubule sliding. R, radius; D, distance between microtubules; L, length of sliding; θ, bend angle. Based on reference [22]. (c) Definitions of several parameters for sperm flagellar motility, in particular those described for flagellar asymmetry. P, principal bend; R, reverse bend; θp, bend angle of the principal bend; θr, bend angle of the reverse bend; rp, radius of inscribed circle at the principal bend; rr, radius of inscribed circle at the reverse bend; σ, shear angle at a certain point on a flagellum. The bend angle is defined as the angle between the tangents in a bend region. The shear angle is defined as the angle between the reference line at the base of flagellum (parallel to the longitudinal axis of the basal body; in practice, the sperm head axis is used). Asymmetry is expressed as θp–θr. Flagellar curvature is defined as the reciprocal of the radius of the inscribed circle. The asymmetric index (ratio of P- and R-bend maximum curvatures) is also used to evaluate wave asymmetry (see [23]). Fig. 1. View largeDownload slide Basic concepts for the analysis of sperm flagellar motility. (a) Structure of the flagellar axoneme. Doublet microtubules are composed of A- and B-tubules and numbered by their position relative to the axis of the central pair. (b) Relationship between flagellar bend angle and microtubule sliding. R, radius; D, distance between microtubules; L, length of sliding; θ, bend angle. Based on reference [22]. (c) Definitions of several parameters for sperm flagellar motility, in particular those described for flagellar asymmetry. P, principal bend; R, reverse bend; θp, bend angle of the principal bend; θr, bend angle of the reverse bend; rp, radius of inscribed circle at the principal bend; rr, radius of inscribed circle at the reverse bend; σ, shear angle at a certain point on a flagellum. The bend angle is defined as the angle between the tangents in a bend region. The shear angle is defined as the angle between the reference line at the base of flagellum (parallel to the longitudinal axis of the basal body; in practice, the sperm head axis is used). Asymmetry is expressed as θp–θr. Flagellar curvature is defined as the reciprocal of the radius of the inscribed circle. The asymmetric index (ratio of P- and R-bend maximum curvatures) is also used to evaluate wave asymmetry (see [23]). The outer dynein arm This contains three heavy chains in Chlamydomonas and ciliates [24,25] and two heavy chains in animals [9,26,27]. One dynein species is arranged at 24 nm periodicity on each doublet microtubule, with the aid of an anchoring structure called a dynein docking complex [28]. LCs contain a subunit (LC2) phosphorylated by a cAMP-dependent protein kinase [29,30]. A Ca2+-binding protein, calaxin, is associated with the outer dynein arm (ODA) in a Ca2+-dependent manner. Inhibition of calaxin results in the suppression of propagation of asymmetric flagellar bends [23]. Extraction of ODAs causes a decrease of beat frequency to half of that of intact axonemes in the demembranated sea urchin spermatozoon [31]. Thus, ODAs are involved both in the increase of flagellar beat frequency and in the propagation of asymmetric flagellar bends. The inner dynein arm In Chlamydomonas flagella, there are at least seven IDAs with different subunit compositions [21,32]. They are arranged on the doublet microtubules at 96 nm periodicity. The f (I1) dynein has two dynein heavy chains and the other six inner arm dyneins each has a single heavy chain [32,33]. The f dynein contains an intermediate chain that is dephosphorylated upon activation of motility [34,35]. Some IDAs contain actin or a Ca2+-binding protein, centrin [21]. IDAs are responsible for the basic mechanism of flagellar bend formation and propagation [21]. Radial spokes and the central pair apparatus (CP) Both RSs and central pair apparatus (CPs) (RS/CS) are involved in the regulation of dyneins, playing important roles in the production of planar flagellar waveforms [20]. Signal transmission between the CPs and RSs is mediated by a simple mechanical interaction that switches the regulation of dynein activity [36]. Biochemical and proteomic studies have shown that the subunits of RS/CP complexes contain several components for protein–protein interactions and cAMP/Ca2+-mediated cell signaling and are considered to regulate dyneins through Ca2+ and protein phosphorylation [37–42]. In fact, spermatozoa from some organisms, such as eels [43,44] and the Asian horseshoe crab [45,46], lack a CP and show propagation of not planar but helical waveforms. However, spermatozoa with CP/RS complexes do not always show planar waveforms. The conversion between planar and helical waveforms has been proposed to occur by regulation of mechanical constrains through Ca2+ and cAMP signals [47,48]. Nexin–dynein regulatory complex This structure is bound to the base of each IDA and radial spokes (RSs) and extends to the adjacent B-tubule of the doublet microtubule, formerly called a nexin link or interdoublet link [49,50]. Now, the terminology is Nexin–dynein regulatory complex (‘N–DRC’) [51]. It extends to the adjacent B-tubule, makes an elastic structure to bundle the peripheral doublet microtubules and plays a role in the regulation of dynein. Sperm flagellar movement is based on the sliding of peripheral doublet microtubules by axonemal dyneins. This sliding is converted into the bending of the axonemes if structural or mechanical resistance is present [16] (Fig. 1b). The flagellar base apparently acts as the key resistance. Another resistance for bend formation along a flagellum during wave oscillation is proposed to be given by dyneins, possibly from mechanical feedback [52]. From the recorded images of flagellar movement, several wave parameters can be obtained, including bend angle, flagellar curvature and shear angle (Fig. 1c; see section Microscopic description of sperm flagellar movement in detail). These parameters are useful to understand the nature of microtubule sliding in beating flagella and to estimate flagellar asymmetry. Flagellar beating occurs vertically to the plane of the CP. Planar waves are thought to be formed by switching of dynein activation at alternate doublet microtubules via a signal from the RS/CP apparatus. In the absence of a CP, flagella show helical movements. Exogenous increases in mechanical loading such as fluid viscosity converts the flagellar waveform from planar to helical, suggesting that the RS/CP might act as a mechanical constraint for the alternative sliding direction [47]. Ca2+-binding and phosphorylation of dynein subunits are known to modulate the sliding, resulting in an increase or decrease in beating frequency or in changes to flagellar waveforms. The regulations of dyneins by Ca2+ and protein phosphorylation are seen in response to changes in extracellular conditions, such as ions, chemoattractants from eggs, and other factors from the male or female reproductive tracts [27]. Microscopic description of sperm flagellar movement The beating frequency of sperm flagella is high but diverse among organisms: 30–40 Hz in sea urchins and starfish [4,7]; ~40 Hz in teleosts [53]; 90 Hz in eels [45]; 9–12 Hz in humans [54]; 1.5–11 Hz in the mouse [55] and 20 Hz in bovines [56]. To analyze the flagellar waveform, beat frequency, and propagation of waves, it is necessary to capture the flagellar waveform (Fig. 2). To achieve this, several devices have been developed: cinematography [4]; dark-field microscopy and videography [57]; high-speed videography and stroboscopy [4,7,58]; stroboscopic illumination with a very short flash duration [6,59–61]; multiple-flash photography on a moving film [62] and video recording by a charge-coupled device (CCD) with a rapid shutter using a videocassette recorder [63,64]. The sensitivity to light of cameras was improved by developing image sensors, such as a silicon intensifier target (SIT) or a complementary metal-oxide semiconductor (CMOS). High-speed cameras are also technically advanced with high specifications for shutter speed, frame rate and sensitivity [65,66]. For measuring flagellar beat frequency, dark-field or phase-contrast microscopy with stroboscopic illumination with short light flashes have mostly been used to ‘freeze’ the wave images [67,68] (Fig. 2). The flashing frequency of illumination to freeze the waveform has to be equal to the beat frequency. Alternatively, doubling the illumination frequency also freezes the waves, where two waves with mutual reverse phase appear overlapped, which makes it easier to determine the beat frequency as half of the illumination frequency. The frequency to trigger pulses from a power supply can be recorded in a frequency output counter. Flagellar beating at low frequency can be determined from the period required for one complete propagation from images of waveform propagation. Fig. 2. View largeDownload slide Phase-contrast microscopic images of sea urchin spermatozoa under normal xenon light source. (a) and stroboscopic LED illumination (b). Note that the flagellar waveform is ‘frozen’ and thus clearly observed in b. Scale bar = 20 μm. Fig. 2. View largeDownload slide Phase-contrast microscopic images of sea urchin spermatozoa under normal xenon light source. (a) and stroboscopic LED illumination (b). Note that the flagellar waveform is ‘frozen’ and thus clearly observed in b. Scale bar = 20 μm. A planar flagellar wave can be approximately sinusoidal [4,69], or in circular arcs and straight lines (arc-line wave) [6] or in a meandering pattern [70]. These patterns are thought to be generated with similar forces but the mechanisms for bend regulation appear different [70]. Given that sliding of microtubules by dynein is converted into bending, the analysis of flagellar bends provides information on the activity and regulation of dynein [22]. In spermatozoa showing planar beating of the flagellum, the bend angle at a given point is proportional to the extent of microtubule sliding (Fig. 1c). Sliding velocity is proportional to the product of the bend angle and flagellar beat frequency. In a more detailed analysis, the measurement of shear angle would be useful to quantify microtubule sliding along a flagellum [71,72] (Fig. 1c). Shear angle is also a parameter to understand microtubule sliding in beating flagella. A plot of shear angle against the distance from the base of flagellum is called the ‘shear curve’. Curved and straight regions in a shear curve correspond to straight and bending portions in the waveform, respectively. The amount of microtubule sliding is estimated from the change in the shear angle between two shear curves (for an example of a shear curve, see Fig. 4b). The flagellar waveform is composed of a bend with a larger angle (principal bend or P-bend) and a bend with a smaller angle (reverse bend or R-bend) [72]. This asymmetry is described by the difference between these two bend conformations (Fig. 1c). P- and R-bends are positioned at the side of doublets 1 and 5–6, respectively (Fig. 3a). The trajectories of spermatozoa showing planar flagellar waveforms are directly related to wave asymmetry: thus, those with symmetric waveforms swim straight, whereas those with asymmetric waveforms swim in circles. Transient changes in flagellar waveform asymmetry are known to be essential for sperm chemotaxis toward the egg before fertilization [73]. In rodent spermatozoa, flagellar bends are defined as pro-hook and anti-hook bends from their position relative to the hooked head [74]. Pro-hook and anti-hook bends are positioned at the sides of doublets 5–6 and 1, respectively (Fig. 3b). The asymmetry of the flagellar waveform is evaluated by several parameters, including the asymmetry index [75]. Flagellar asymmetry is also estimated from the shear curve of one beating cycle. The shear curve becomes diagonal in the case of asymmetric flagellar waveforms. Fig. 3. View largeDownload slide Definition and structural orientation of flagellar bends in sea urchin and other marine invertebrate spermatozoa showing simple planar waveforms (a). In rodent spermatozoa, flagellar bends are defined according to their position relative to the hook in the head (b). Axonemes are viewed from the base to tip of the flagellum. Fig. 3. View largeDownload slide Definition and structural orientation of flagellar bends in sea urchin and other marine invertebrate spermatozoa showing simple planar waveforms (a). In rodent spermatozoa, flagellar bends are defined according to their position relative to the hook in the head (b). Axonemes are viewed from the base to tip of the flagellum. Computer-assisted systems for analysis of sperm motility CASA – computer-aided sperm analysis or computer-assisted sperm analysis – appeared in the late 1980s and was developed mainly for the clinical analysis of human semen [76–79]. This automated system for evaluating sperm motility minimizes the errors conducted manually by different researchers or doctors. Thus, the information obtained by CASA is compatible with the quality assessment of human semen in the World Health Organization (WHO) guidelines, which is one of the reasons why this methodology has spread to basic research in sperm motility. The first commercial CASA system was the CellSoft Automated Semen Analyzer, released by CRYO Resources Ltd. (New York, NY, USA) in 1985, which was equipped with a monochrome video camera connected to a phase-contrast microscope. Because the principles of CASA enabled researchers to evaluate the motility characteristics of spermatozoa rapidly, the technique spread not only to human infertility clinics but also to animal husbandry and fisheries sciences. The analysis basically depends on frame-by-frame detection of sperm heads on video clips and the building of tracks of head trajectories between frames. Movie files or those processed using ImageJ (https://imagej.nih.gov/ij/download.html) can be incorporated for analysis. Devices for capturing images, sensitivity of CCD cameras and software for sperm detection and motion analysis have changed and become more sophisticated, although the basic concept of analysis has not changed (See [79]). CASA systems depend on tracking the sperm head but do not perform direct measurements of flagellar motility (Fig. 4a and b). In a conventional chamber for measurement, the systems cannot distinguish between helical and planar waveforms. However, parameters calculated by CASA could be partly interpreted in terms of the mechanism of axonemal activity for sperm motility (Fig. 4c), although in several cases sperm motility patterns cannot be attributed to simple dynein–microtubule interactions. The possible interpretations to be considered first for parameters obtained by CASA are described below, although these are not definitive because several unknown factors would affect each motility parameter. Fig. 4. View largeDownload slide Definition of motility parameters in CASA (a). The purple line with circles represents the trajectory of the sperm head. (b, c) An example of motility analysis using a SMAS CASA system (Ditect Co. Ltd, Tokyo, Japan). Automatically traced trajectories for 1 s of sperm movements in Sillago japonica. Sperm trajectories are shown for 1 s at 15, 60 and 180 s after the initiation of motility by sea water. Scale bar = 100 μm (b). Several parameters calculated automatically using the SMAS system. Note that velocity and linearity of sperm movements decrease with time after activation (c). Fig. 4. View largeDownload slide Definition of motility parameters in CASA (a). The purple line with circles represents the trajectory of the sperm head. (b, c) An example of motility analysis using a SMAS CASA system (Ditect Co. Ltd, Tokyo, Japan). Automatically traced trajectories for 1 s of sperm movements in Sillago japonica. Sperm trajectories are shown for 1 s at 15, 60 and 180 s after the initiation of motility by sea water. Scale bar = 100 μm (b). Several parameters calculated automatically using the SMAS system. Note that velocity and linearity of sperm movements decrease with time after activation (c). Percent motility This parameter is used for a simple evaluation of sperm motility. The threshold of swimming velocity can be set to evaluate the percentage of spermatozoa showing progressive swimming. Sperm are immotile or show low motility in male reproductive organs. For animals with external fertilization, sperm initiate motility upon spawning. In mammals, spermatozoa undergo maturation during passage through the epididymis and show capacitation and hyperactivation in the female reproductive tract [27]. Spermatozoa from mammals with defects in spermatogenesis usually show very low or no motility. Deficiencies or any abnormalities in the components of the axoneme or signaling to activate sperm motility result in reduced values for percent motility. CASA velocity parameters The curvilinear velocity (VCL) parameter represents velocity along the actual trajectory (Fig. 4a). The velocity along the trajectory of the computed average path is expressed as the average path velocity (VAP). The straight-line velocity (VSL) measure represents velocity along a straight-line path from the start to the end point of any given path analysis (Fig. 4a). These three values indicate the activity of dyneins and axonemes, because sperm velocity is proportional to the beat frequency, according to the resistive force theory [69]. These values are strongly associated with flagellar kinetics or propulsive sperm movement, expressed as the power output, which is proportional to beat frequency, flagellar amplitude and flagellar length [47,80]. Axonemal motility depends on the dynein-mediated sliding of microtubules, but there are several factors to regulate it; not only second messengers like Ca2+ or cAMP that act directly on axonemes but also membrane-mediated signal transduction mechanisms upstream of Ca2+/cAMP intracellular signals, which could be affected by several factors, including receptors of activators, ion channels, transmembrane or membrane-bound enzymes, and membrane fluidity [27,81,82]. Linearity (LIN) is calculated from the VSL/VAP ratio, representing sperm path LIN. The swimming direction of a spermatozoon changes in response to the conditions outside the cell, which are important for sperm to perform directional movements toward the egg for successful fertilization. Proper waveform asymmetry determines the swimming direction of the spermatozoon. In the case of spermatozoa with a planar waveform, alternate bend formation is caused by switching the active dynein across the CP. If dyneins at both P- and R-bends are equally active, symmetric bends are formed, while if their activities are different, asymmetric waveforms are generated. Asymmetric waveforms are thought to be made by inhibition of the dynein-mediated microtubule sliding in the reverse bend [10,23,72]. Therefore, the difference in LIN is considered due to the basic force-generation activity of dyneins or most likely to the mechanism of Ca2+-dependent regulation of waveform formation and propagation. Amplitude of lateral head displacement (ALH) represents the deviation of the head trajectory from the average path. For example, hyperactivation of mammalian spermatozoa is characterized by large bending of the flagellum caused by a more flexible midpiece and large lateral movements of the sperm head [83–86], although beat frequency is decreased [74]. Both wobble (WOB, calculated as VAP/VCL) and ALH estimate the head movements in hyperactivated spermatozoa. Because the parameters measured by CASA in observation chambers do not always reflect the real or proper movements of spermatozoa in the female reproductive tract, several improvements have been carried out [79,87]. CASA is based on the movement of sperm heads. Therefore, no information about the flagellar waveform is available. However, computer-assisted systems are also powerful in the analysis of flagellar waveforms [75]. In many of our recent studies, we used a sophisticated computer-assisted system for sperm flagellar motility, called Bohboh (Bohboh soft, Tokyo, Japan) (Fig. 5a), which is software for the analysis of cell motility, specialized for spermatozoa, flagella and cilia. Bohboh was developed by Dr Shoji Baba on a Microsoft Visual Studio platform, allowing the software to run on a personal computer (PC) with Microsoft Windows software. By anchoring a few points along the flagellum, the flagellar waveform is tracked automatically. The coordinate data from the trace are used to calculate the flagellar curvature and shear angle (Fig. 5b), with a variety of formats, including a spatiotemporal matrix for 3D maps [88]. This software also automatically tracks the sperm head and analyzes sperm trajectories and several parameters for sperm movement, velocity, trajectory of the sperm head, path curvature and flagellar beat frequency, just as in other commercial CASA systems (Fig. 5a). Fig. 5. View largeDownload slide (a) A screen capture image using the Bohboh system (Bohboh soft, Tokyo, Japan). The pink line and orange dots indicate the sperm trajectory and flagellar waveform, respectively. Both are obtained by auto-tracking. Such sequential image tracking makes it possible to analyze the waveform and its propagation, in relation to each spermatozoon’s moving trajectory. (b) An example of the analysis of flagellar waveforms in sea urchin sperm. Sequential flagellar waveforms (left), flagellar curvature along the flagellum (middle) and shear curve (right) are shown. Sperm movements were observed under a phase-contrast microscope and recorded with a high-speed CCD camera (HAS-220; Ditect Co. Ltd). Images were recorded with a frame rate of 200 fps. The flagellar wave was tracked and analyzed using the Bohboh system. Sequential images taken at 5 ms intervals are shown. Note that a high intracellular Ca2+ concentration (artificial sea water, ASW + calcium ionophore A23187) induces a more asymmetric waveform than low Ca2+ conditions (calcium-free sea water, CFSW). Data from 20 waveforms are superimposed. P, principal bend; R, reverse bend. Scale bar = 20 μm. Fig. 5. View largeDownload slide (a) A screen capture image using the Bohboh system (Bohboh soft, Tokyo, Japan). The pink line and orange dots indicate the sperm trajectory and flagellar waveform, respectively. Both are obtained by auto-tracking. Such sequential image tracking makes it possible to analyze the waveform and its propagation, in relation to each spermatozoon’s moving trajectory. (b) An example of the analysis of flagellar waveforms in sea urchin sperm. Sequential flagellar waveforms (left), flagellar curvature along the flagellum (middle) and shear curve (right) are shown. Sperm movements were observed under a phase-contrast microscope and recorded with a high-speed CCD camera (HAS-220; Ditect Co. Ltd). Images were recorded with a frame rate of 200 fps. The flagellar wave was tracked and analyzed using the Bohboh system. Sequential images taken at 5 ms intervals are shown. Note that a high intracellular Ca2+ concentration (artificial sea water, ASW + calcium ionophore A23187) induces a more asymmetric waveform than low Ca2+ conditions (calcium-free sea water, CFSW). Data from 20 waveforms are superimposed. P, principal bend; R, reverse bend. Scale bar = 20 μm. Other microscopic systems for studying the mechanisms of sperm motility The 9+2 microtubule structure, the bases of the axoneme, is the central motile machinery of the sperm flagella. The ‘Triton model’ is prepared by demembranation of sperm cells with a non-ionic detergent Triton X-100. It was first developed using sea urchin spermatozoa [89]. Oscillatory flagellar bending and propagation are reproduced in the Triton model by reactivation with ATP. The effects of several chemicals or factors on axonemal movement can be examined directly using this system. If spermatozoa show no motility but the Triton models are properly activated, it is interpreted that some membrane-related or intracellular signaling molecules cannot function normally such cases. It is worth noting that ATP acts evenly on such demembranated sperm flagella. It has long been a question on how ATP is supplied throughout the flagellum. The creatine shuttle was proposed to explain the nonattenuated waveform from the base to tip of the flagellum in sea urchin and trout sperm models [90,91]. In mammalian spermatozoa, glycolysis by enzymes localized in the fibrous sheath plays an important role in providing ATP throughout the flagellum [92,93]. Efficient ATP supply throughout a flagellum appears indispensable, in particular in spermatozoa with an unusually long flagellum, such as in the quail. Quail spermatozoa develop meandering flagellar waveforms spontaneously from the proximal region. The waves initiate from a double bend, which is oscillated by an alternating direction of propulsive force [94]. Observation of ‘sliding disintegration’ of axonemal microtubules is another direct way to understand the activity of dyneins [14,95]. After demembranation with Triton X-100 and treatment with proteases, such as trypsin and elastase, followed by addition of ATP, doublet microtubules slide out from the axonemes. Although it should be kept in mind that proteases can disrupt some dynein–microtubule interactions, sliding disintegration has been used as a model system to examine the microtubule-sliding activity of dyneins directly. More direct measurement of dynein-driven microtubule sliding is the ‘in vitro motility assay’, in which a simply reconstituted system from purified dyneins and microtubules is used, thus making it possible to determine dynein behavior without consideration of the other axonemal components [23,96–98]. In addition, to understand the fundamental processes of sperm flagellar movement, other micromanipulations using dynein-mediated microtubule sliding have been carried out (for details, see [22]). Several extracellular and intracellular signaling pathways are indispensable for the normal activation of sperm motility. Attempts have been done to visualize the behavior of spermatozoa and their internal molecules. The analysis of tactic behaviors, including thermotaxis, rheotaxis and chemotaxis, needs a proper device with specific assay chambers to record the pathways of sperm movement [99–103]. Microfluidics has been developed in assisted reproduction and applied for analysis of sperm movements [104–106]. To describe the directed free swimming of sperm, discrimination of the contact with the surface of glass slides or chambers should be taken into consideration. Most analyses of sperm swimming behaviors have been done in shallow 2D fields, which do not always reflect the real behavior of spermatozoa before fertilization. Recently, a combination of holographic microscopy and optochemical techniques has enabled the tracking of 3D movements of sea urchin spermatozoa during chemotaxis [107]. The spatiotemporal dynamics of signaling molecules are keys to understanding the molecular mechanisms involved in the regulation of sperm motility. These signaling pathways are triggered by binding of specific ligands to their receptors and ion exchange through several types of channels [108]. Only a few compounds and chemical factors, including ATP [109] and pH [110,111], have been imaged microscopically. It is well known that Ca2+ and cAMP play crucial roles in the initiation, activation and regulation of the waveform of sperm flagella [27]. Using a fluorescent indicator, several groups reported on the oscillatory behavior of intracellular Ca2+ [73,103,112,113]. We employ intracellular Ca2+ imaging in spermatozoa using stroboscopic illumination using a high-power light-emitting diode (LED) [73] (Fig. 6a). The sperm flagellum oscillates too rapidly to capture its activity using a weak fluorescent light in a microscope with continuous light source. A short pulse (~200 μs) synchronized with the camera exposure signal makes the sperm flagellar waveform ‘freeze’ on one frame. The combination of a high-intensity LED with stroboscopic flash and a high sensitivity electron-multiplying (EM) CCD camera made it possible to capture the changes of weak fluorescence from the sperm flagellum. LED lighting is controlled by low voltage and has long life and low cost. The use of an LED strobe minimizes electrical noise, which was a problem in electrophysiological measurements using Xenon-based stroboscopic illumination. Moreover, stroboscopic illumination using an LED reduces phototoxicity and photobleaching [114]. Real-time Ca2+ imaging of ascidian spermatozoa loaded with a Ca2+ indicator, Fluo8H-AM (AAT Bioquest, Sunnyvale, CA, USA), revealed transient Ca2+ increases in the flagella during sperm chemotaxis in vitro (Fig. 6b) [23]. Fig. 6. View largeDownload slide (a) A schematic setup of a fluorescence microscope system for real-time imaging of intracellular Ca2+. (b) Sequential images of ascidian spermatozoa during chemotaxis at 20 ms intervals, showing transient increase in intracellular Ca2+ concentration. Spermatozoa loaded with a fluorescent dye (Fluo8H-AM) were suspended in ASW. A capillary filled with chemoattractant was inserted into ASW. The spermatozoa showed chemotactic behavior around the tip of the capillary with transient fluorescence changes. Fluorescence was visualized by excitation with a Power LED (Luxeon III Star Lambertian Cyan LED part no. LXHL-LE3C; Luxeon Star LEDs) driven by a laboratory-made stroboscopic controller. The output was synchronized with the exposure-out signal of a high sensitivity imaging camera (ImagEM, Hamamatsu Photonics KK, Hamamatsu City, Japan). Images are represented in pseudo color with an look-up-table scale for fluorescence signals. Scale bar = 20 μm. Fig. 6. View largeDownload slide (a) A schematic setup of a fluorescence microscope system for real-time imaging of intracellular Ca2+. (b) Sequential images of ascidian spermatozoa during chemotaxis at 20 ms intervals, showing transient increase in intracellular Ca2+ concentration. Spermatozoa loaded with a fluorescent dye (Fluo8H-AM) were suspended in ASW. A capillary filled with chemoattractant was inserted into ASW. The spermatozoa showed chemotactic behavior around the tip of the capillary with transient fluorescence changes. Fluorescence was visualized by excitation with a Power LED (Luxeon III Star Lambertian Cyan LED part no. LXHL-LE3C; Luxeon Star LEDs) driven by a laboratory-made stroboscopic controller. The output was synchronized with the exposure-out signal of a high sensitivity imaging camera (ImagEM, Hamamatsu Photonics KK, Hamamatsu City, Japan). Images are represented in pseudo color with an look-up-table scale for fluorescence signals. Scale bar = 20 μm. Prospects Sperm motility is based on the interaction between dyneins and microtubules in the flagellar axoneme. Therefore, any deficiency in axonemal formation during spermiogenesis results in reduced or absent motility. Although the mechanism of flagellar motility and its regulation are not yet fully understood, recent advances in cryoelectron microscopy and molecular imaging, as well as several molecular tools, ensure better technical strategies to reveal the cause of changes in sperm motility. Detailed observations of flagellar movements and analyses of several parameters by light microscopy would not only provide us with important information on dynein behavior and microtubule sliding but also give a clue to understand the causes of abnormal motility. Motility analysis software, such as CASA, made motility analysis widespread for practical uses, including human clinical diagnosis, livestock and fisheries science, and evaluation of the efficacy of sperm cryopreservation. Development of microscopic analysis systems for sperm motility promises to play major roles to improve the function of spermatozoa from infertile men and male animals and advance the techniques of sperm preservation for reproducible insemination. Acknowledgements We thank Dr Makoto Okuno and Dr Shoji Baba for their critical reading of the manuscript. We thank James Cummins, PhD, from EDANZ Group (www.edanzediting.com/ac) for editing a draft of this manuscript. Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology and the Japan Society for the Promotion of Science (JP16H06280 ‘Advanced Bioimaging Support’ and 17H01440). Conflict of interest The author declares no competing financial interests. References 1 Porter J R ( 1976 ) Antony van Leeuwenhoek: tercentenary of his discovery of bacteria . Bacteriol. Rev. 40 : 260 – 269 . Google Scholar PubMed 2 Gray J ( 1928 ) Ciliary Movement , ( Macmillan , New York ). 3 Gray J ( 1930 ) The mechanism of ciliary movement. VI. Photographic and stroboscopic analysis of ciliary movement . Proc. R. Soc. London Ser. B 107 : 313 – 332 . Google Scholar CrossRef Search ADS 4 Gray J ( 1955 ) The movement of sea-urchin spermatozoa . J. Exp. Biol. 32 : 775 – 800 . 5 Machin K E ( 1958 ) Wave propagation along flagella . J. Exp. Biol. 35 : 796 – 806 . 6 Brokaw C J ( 1965 ) Non-sinusoidal bending waves of sperm flagella . J. Exp. Biol. 43 : 155 – 169 . Google Scholar PubMed 7 Hiramoto Y , and Baba S A ( 1978 ) A quantitative analysis of flagellar movement in echinoderm spermatozoa . J. Exp. Biol. 76 : 85 – 104 . 8 Sleigh M A ( 1962 ) The biology of cilia and flagella , ( Macmillan , New York ). Google Scholar CrossRef Search ADS 9 Inaba K ( 2011 ) Sperm flagella: comparative and phylogenetic perspectives of protein components . Mol. Hum. Reprod. 17 : 524 – 538 . Google Scholar CrossRef Search ADS PubMed 10 Inaba K ( 2015 ) Calcium sensors of ciliary outer arm dynein: functions and phylogenetic consideration for eukaryotic evolution . Cilia 4 : 6 . Google Scholar CrossRef Search ADS PubMed 11 Inaba K , and Mizuno K ( 2016 ) Sperm dysfunction and ciliopathy . Reprod. Med. Biol. 15 : 77 – 94 . Google Scholar CrossRef Search ADS PubMed 12 Dentler W , and Witman G ( 1995 ) Cilia and flagella . Methods Cell Biol. 47 : 603 . 13 Satir P ( 1968 ) Studies on cilia. 3. Further studies on the cilium tip and a ‘sliding filament’ model of ciliary motility . J. Cell Biol. 39 : 77 – 94 . Google Scholar CrossRef Search ADS PubMed 14 Summers K E , and Gibbons I R ( 1971 ) Adenosine triphosphate-induced sliding of tubules in trypsin-treated flagella of sea-urchin sperm . Proc. Natl. Acad. Sci. USA 68 : 3092 – 3096 . Google Scholar CrossRef Search ADS 15 Brokaw C J ( 1972 ) Flagellar movement: a sliding filament model . Science 178 : 455 – 462 . Google Scholar CrossRef Search ADS PubMed 16 Shingyoji C , Murakami A , and Takahashi K ( 1977 ) Local reactivation of Triton-extracted flagella by iontophoretic application of ATP . Nature 265 : 269 – 270 . Google Scholar CrossRef Search ADS PubMed 17 King S M ( 2000 ) AAA domains and organization of the dynein motor unit . J. Cell Sci. 113 : 2521 – 2526 . Google Scholar PubMed 18 Inaba K ( 2007 ) Molecular basis of sperm flagellar axonemes: structural and evolutionary aspects . Ann. NY Acad. Sci. 1101 : 506 – 526 . Google Scholar CrossRef Search ADS 19 Porter M E , and Sale W S ( 2000 ) The 9 + 2 axoneme anchors multiple inner arm dyneins and a network of kinases and phosphatases that control motility . J. Cell Biol. 151 : F37 – F42 . Google Scholar CrossRef Search ADS PubMed 20 Smith E F , and Yang P ( 2004 ) The radial spokes and central apparatus: mechano-chemical transducers that regulate flagellar motility . Cell Motil. Cytoskeleton 57 : 8 – 17 . Google Scholar CrossRef Search ADS PubMed 21 Kamiya R ( 2002 ) Functional diversity of axonemal dyneins as studied in Chlamydomonas mutants . Int. Rev. Cytol. 219 : 115 – 155 . Google Scholar CrossRef Search ADS PubMed 22 Shingyoji C ( 2013 ) Measuring the regulation of dynein activity during flagellar motility . Methods Enzymol. 524 : 147 – 169 . Google Scholar CrossRef Search ADS PubMed 23 Mizuno K , Shiba K , Okai M , Takahashi Y , Shitaka Y , Oiwa K , Tanokura M , and Inaba K ( 2012 ) Calaxin drives sperm chemotaxis by Ca2+-mediated direct modulation of a dynein motor . Proc. Nat. Acad. Sci. USA 109 : 20497 – 20502 . Google Scholar CrossRef Search ADS 24 King S M ( 2000 ) The dynein microtubule motor . Biochim. Biophys. Acta 1496 : 60 – 75 . Google Scholar CrossRef Search ADS PubMed 25 DiBella L M , Gorbatyuk O , Sakato M , Wakabayashi K , Patel-King R S , Pazour G J , Witman G B , and King S M ( 2005 ) Differential light chain assembly influences outer arm dynein motor function . Mol. Biol. Cell 16 : 5661 – 5674 . Google Scholar CrossRef Search ADS PubMed 26 Gibbons I R ( 1981 ) Cilia and flagella of eukaryotes . J. Cell Biol. 91 : 107s – 124s . Google Scholar CrossRef Search ADS PubMed 27 Inaba K ( 2003 ) Molecular architecture of the sperm flagella: molecules for motility and signaling . Zool. Sci. 20 : 1043 – 1056 . Google Scholar CrossRef Search ADS PubMed 28 Takada S , and Kamiya R ( 1994 ) Functional reconstitution of Chlamydomonas outer dynein arms from alpha-beta and gamma subunits: requirement of a third factor . J. Cell Biol. 126 : 737 – 745 . Google Scholar CrossRef Search ADS PubMed 29 Inaba K , Morisawa S , and Morisawa M ( 1998 ) Proteasomes regulate the motility of salmonid fish sperm through modulation of cAMP-dependent phosphorylation of an outer arm dynein light chain . J. Cell Sci. 111 : 1105 – 1115 . Google Scholar PubMed 30 Inaba K , Kagami O , and Ogawa K ( 1999 ) Tctex2-related outer arm dynein light chain is phosphorylated at activation of sperm motility . Biochem. Biophys. Res. Commun. 256 : 177 – 183 . Google Scholar CrossRef Search ADS PubMed 31 Gibbons B H , and Gibbons I R ( 1973 ) The effect of partial extraction of dynein arms on the movement of reactivated sea-urchin sperm . J. Cell Sci. 13 : 337 – 357 . Google Scholar PubMed 32 Piperno G , Ramanis Z , Smith E F , and Sale W S ( 1990 ) Three distinct inner dynein arms in Chlamydomonas flagella: molecular composition and location in the axoneme . J. Cell Biol. 110 : 379 – 389 . Google Scholar CrossRef Search ADS PubMed 33 Kagami O , Takada S , and Kamiya R ( 1990 ) Microtubule translocation caused by three subspecies of inner-arm dynein from Chlamydomonas flagella . FEBS Lett. 264 : 179 – 182 . Google Scholar CrossRef Search ADS PubMed 34 Habermacher G , and Sale W S ( 1997 ) Regulation of flagellar dynein by phosphorylation of a 138-kD inner arm dynein intermediate chain . J. Cell Biol. 136 : 167 – 176 . Google Scholar CrossRef Search ADS PubMed 35 King S J , and Dutcher S K ( 1997 ) Phosphoregulation of an inner dynein arm complex in Chlamydomonas reinhardtii is altered in phototactic mutant strains . J. Cell Biol. 136 : 177 – 191 . Google Scholar CrossRef Search ADS PubMed 36 Oda Y , Yanagisawa H , Yagi Y , and Kikkawa M ( 2014 ) Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity . J. Cell Biol. 204 : 807 – 819 . Google Scholar CrossRef Search ADS PubMed 37 Yang P , Diener D R , Yang C , Kohno T , Pazour G J , Dienes J M , Agrin N S , King S M , Sale W S , Kamiya R , Rosenbaum J L , and Witman G B ( 2006 ) Radial spoke proteins of Chlamydomonas flagella . J. Cell Sci. 119 : 1165 – 1174 . Google Scholar CrossRef Search ADS PubMed 38 Wargo M J , Dymek E E , and Smith E F ( 2005 ) Calmodulin and PF6 are components of a complex that localizes to the C1 microtubule of the flagellar central apparatus . J. Cell Sci. 118 : 4655 – 4665 . Google Scholar CrossRef Search ADS PubMed 39 Dymek E E , Goduti D , Kramer T , and Smith E F ( 2006 ) A kinesin-like calmodulin-binding protein in Chlamydomonas: evidence for a role in cell division and flagellar functions . J. Cell Sci. 119 : 3107 – 3116 . Google Scholar CrossRef Search ADS PubMed 40 Padma P , Satouh Y , Wakabayashi K , Hozumi A , Ushimaru Y , Kamiya R , and Inaba K ( 2003 ) Identification of a novel leucine-rich repeat protein as a component of flagellar radial spoke in the ascidian Ciona intestinalis . Mol. Biol. Cell 14 : 774 – 785 . Google Scholar CrossRef Search ADS PubMed 41 Satouh Y , and Inaba K ( 2009 ) Proteomic characterization of sperm radial spokes identifies a novel spoke protein with an ubiquitin domain . FEBS Lett. 583 : 2201 – 2207 . Google Scholar CrossRef Search ADS PubMed 42 Satouh Y , Padma P , Toda T , Satoh N , Ide H , and Inaba K ( 2005 ) Molecular characterization of radial spoke subcomplex containing radial spoke protein 3 and heat shock protein 40 in sperm flagella of the ascidian Ciona intestinalis . Mol. Biol. Cell 16 : 626 – 636 . Google Scholar CrossRef Search ADS PubMed 43 Baccetti B , Burrini A G , Dallai R , and Pallini V ( 1979 ) The dynein electrophoretic bands in axonemes naturally lacking the inner or the outer arm . J. Cell Biol. 80 : 334 – 340 . Google Scholar CrossRef Search ADS PubMed 44 Woolley D M ( 1998 ) Studies on the eel sperm flagellum. 2. The kinematics of normal motility . Cell Motil. Cytoskeleton 39 : 233 – 245 . Google Scholar CrossRef Search ADS PubMed 45 Gibbons B H , Gibbons I R , and Baccetti B ( 1983 ) Structure and motility of the 9 + 0 flagellum of eel spermatozoa . J. Submicrosc. Cytol. 15 : 15 – 20 . Google Scholar PubMed 46 Ishijima S , Sekiguchi K , and Hiramoto Y ( 1988 ) Comparative study of the beat patterns of American and Asian horseshoe crab sperm: evidence for a role of the central pair complex in forming planar waveforms in flagella . Cell Motil. Cytoskeleton 9 : 264 – 270 . Google Scholar CrossRef Search ADS 47 Ishijima S ( 2012 ) Mechanical constraint converts planar waves into helices on tunicate and sea urchin sperm flagella . Cell Struct. Funct. 37 : 13 – 19 . Google Scholar CrossRef Search ADS PubMed 48 Ishijima S ( 2013 ) Regulations of microtubule sliding by Ca2+ and cAMP and their roles in forming flagellar waveforms . Cell Struct. Funct. 38 : 89 – 95 . Google Scholar CrossRef Search ADS PubMed 49 Huang B , Ramanis Z , and Luck D J L ( 1982 ) Suppressor mutations in Chlamydomonas reveal a regulatory mechanism for flagellar function . Cell 28 : 115 – 124 . Google Scholar CrossRef Search ADS PubMed 50 Piperno G , Mead K , LeDizet M , and Moscatelli A ( 1994 ) Mutations in the ‘dynein regulatory complex’ alter the ATP-insensitive binding sites for inner arm dyneins in Chlamydomonas axonemes . J. Cell Biol. 125 : 1109 – 1117 . Google Scholar CrossRef Search ADS PubMed 51 Heuser T , Raytchev M , Krell J , Porter M E , and Nicastro D ( 2009 ) The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella . J. Cell Biol. 187 : 921 – 933 . Google Scholar CrossRef Search ADS PubMed 52 Shingyoji C , Nakano I , Inoue Y , and Higuchi H ( 2015 ) Dynein arms are strain-dependent direction-switching force generators . Cytoskeleton 72 : 388 – 401 . Google Scholar CrossRef Search ADS PubMed 53 Ishijima S ( 2012 ) Comparative analysis of movement characteristics of lancelet and fish spermatozoa having different morphologies . Biol. Bull. 222 : 214 – 221 . Google Scholar CrossRef Search ADS PubMed 54 Burkman L J ( 1984 ) Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm populations . Arch Androl 13 : 153 – 165 . Google Scholar CrossRef Search ADS PubMed 55 Si Y , and Okuno M ( 1993 ) Multiple activation of mouse sperm motility . Mol. Reprod. Dev. 36 : 89 – 95 . Google Scholar CrossRef Search ADS PubMed 56 Ishijima S , and Witman G B ( 1987 ) Flagellar movement of intact and demembranated, reactivated ram spermatozoa . Cell Motil. Cytoskeleton 8 : 375 – 391 . Google Scholar CrossRef Search ADS PubMed 57 Rothschild L , and Swann M M ( 1949 ) The fertilization reaction in the sea-urchin egg; a propagated response to sperm attachment . J. Exp. Biol. 26 : 164 – 176 . Google Scholar PubMed 58 Rikmenspoel R , and Van Herpen G ( 1957 ) Photoelectric and cinematographic measurements of the motility of bull sperm cells . Phys. Med. Biol. 2 : 54 – 63 . Google Scholar CrossRef Search ADS PubMed 59 Brokaw C J ( 1970 ) Bending moments in free-swimming flagella . J. Exp. Biol. 53 : 445 – 464 . Google Scholar PubMed 60 Okuno M , and Brokaw C J ( 1979 ) Inhibition of movement of trition-demembranated sea-urchin sperm flagella by Mg2+, ATP4-, ADP and Pi . J. Cell Sci. 38 : 105 – 123 . Google Scholar PubMed 61 Inaba K , Okuno M , and Mohri H ( 1989 ) Anthraniloyl ATP, a fluorescent analog of ATP, as a substrate for dynein ATPase and flagellar motility . Arch. Biochem. Biophys. 274 : 209 – 215 . Google Scholar CrossRef Search ADS PubMed 62 Brokaw C J ( 1986 ) Sperm motility . Methods Cell Biol. 27 : 41 – 56 . Google Scholar CrossRef Search ADS PubMed 63 Suarez S S , Dai X B , DeMott R P , Redfern K , and Mirando M A ( 1992 ) Movement characteristics of boar sperm obtained from the oviduct or hyperactivated in vitro . J Androl 13 : 75 – 80 . Google Scholar PubMed 64 Ishijima S ( 1995 ) High-speed video microscopy of flagella and cilia . Methods Cell Biol. 47 : 239 – 243 . Google Scholar CrossRef Search ADS PubMed 65 Miyashiro D , Shiba K , Miyashita T , Baba S A , Yoshida M , and Kamimura S ( 2015 ) Chemotactic response with a constant delay-time mechanism in Ciona spermatozoa revealed by a high time resolution analysis of flagellar motility . Biol. Open 4 : 109 – 118 . Google Scholar CrossRef Search ADS PubMed 66 Lenaghan S C , Davis C A , Henson W R , Zhang Z , and Zhang M ( 2011 ) High-speed microscopic imaging of flagella motility and swimming in Giardia lamblia trophozoites . Proc. Natl. Acad. Sci. USA 108 : E550 – E558 . Google Scholar CrossRef Search ADS 67 Okuno M ( 1980 ) Inhibition and relaxation of sea urchin sperm flagella by vanadate . J. Cell Biol. 85 : 712 – 725 . Google Scholar CrossRef Search ADS PubMed 68 Okuno M , Asai D J , Ogawa K , and Brokaw C J ( 1981 ) Effects of antibodies against dynein and tubulin on the stiffness of flagellar axonemes . J. Cell Biol. 91 : 689 – 694 . Google Scholar CrossRef Search ADS PubMed 69 Gray J , and Hancock G J ( 1955 ) The propulsion of sea-urchin spermatozoa . J. Exp. Biol. 32 : 802 – 814 . 70 Holwill M E ( 1977 ) Some biophysical aspects of ciliary and flagellar motility . Adv. Microb. Physiol. 16 : 1 – 48 . Google Scholar CrossRef Search ADS PubMed 71 Brokaw C J ( 1991 ) Calcium sensors in sea urchin sperm flagella . Cell Motil. Cytoskeleton 18 : 123 – 130 . Google Scholar CrossRef Search ADS PubMed 72 Brokaw C J ( 1979 ) Calcium-induced asymmetrical beating of triton-demembranated sea urchin sperm flagella . J. Cell Biol. 82 : 401 – 411 . Google Scholar CrossRef Search ADS PubMed 73 Shiba K , Baba S A , Inoue T , and Yoshida M ( 2008 ) Ca2+ bursts occur around a local minimal concentration of attractant and trigger sperm chemotactic response . Proc. Natl. Acad. Sci. USA 105 : 19312 – 19317 . Google Scholar CrossRef Search ADS 74 Ishijima S , Baba S A , Mohri H , and Suarez S S ( 2002 ) Quantitative analysis of flagellar movement in hyperactivated and acrosome-reacted golden hamster spermatozoa . Mol. Reprod. Dev. 61 : 376 – 384 . Google Scholar CrossRef Search ADS PubMed 75 Brokaw C J ( 1990 ) Computerized analysis of flagellar motility by digitization and fitting of film images with straight segments of equal length . Cell Motil. Cytoskeleton 17 : 309 – 316 . Google Scholar CrossRef Search ADS PubMed 76 David G , Serres C , and Jouannet P ( 1981 ) Kinematics of human spermatozoa . Gamete Res. 4 : 83 – 95 . Google Scholar CrossRef Search ADS 77 Boyers S P , Davis R O , and Katz D F ( 1989 ) Automated semen analysis . Curr. Prob. Obstet. Gynecol. Fertil. XII : 167 – 200 . 78 Mortimer S T , Swan M A , and Mortimer D ( 1998 ) Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa . Hum. Reprod. 13 : 2139 – 2146 . Google Scholar CrossRef Search ADS PubMed 79 Mortimer S T , van der Horst G , and Mortimer D ( 2015 ) The future of computer-aided sperm analysis . Asian J. Androl. 17 : 545 – 553 . Google Scholar CrossRef Search ADS PubMed 80 Rikmenspoel R ( 1984 ) Movements and active moments of bull sperm flagella as a function of temperature and viscosity . J. Exp. Biol. 108 : 205 – 230 . Google Scholar PubMed 81 Cosson J , Prokopchuk G , and Inaba K ( 2015 ) The flagellar mechanics of spermatozoa and its regulation. In: Cosson J (ed.) Flagellar Mechanics and Sperm Guidance , pp 3 – 134 (Bentham Science Publisher Ltd, Sharjah, UAE). Google Scholar CrossRef Search ADS 82 Freitas M J , Vijayaraghavan S , and Fardilha M ( 2017 ) Signaling mechanisms in mammalian sperm motility . Biol. Reprod. 96 : 2 – 12 . Google Scholar PubMed 83 Katz D F , Overstreet J W , Samuels S J , Niswander P W , Bloom T D , and Lewis E L ( 1986 ) Morphometric analysis of spermatozoa in the assessment of human male fertility . J. Androl. 7 : 203 – 210 . Google Scholar CrossRef Search ADS PubMed 84 Chang H , and Suarez S S ( 2010 ) Rethinking the relationship between hyperactivation and chemotaxis in mammalian sperm . Biol. Reprod. 83 : 507 – 513 . Google Scholar CrossRef Search ADS PubMed 85 Mohri H , Inaba K , Ishijima S , and Baba S A ( 2012 ) Tubulin–dynein system in flagellar and ciliary movement . Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 88 : 397 – 415 . Google Scholar CrossRef Search ADS PubMed 86 Yanagimachi R ( 1969 ) In vitro capacitation of hamster spermatozoa by follicular fluid . J. Reprod. Fertil. 18 : 275 – 286 . Google Scholar CrossRef Search ADS PubMed 87 Goodson S G , White S , Stevans A M , Bhat S , Kao C Y , Jaworski S , Marlowe T R , Kohlmeier M , McMillan L , Zeisel S H , and O’Brien D A ( 2017 ) CASAnova: a multiclass support vector machine model for the classification of human sperm motility patterns . Biol. Reprod. 97 : 698 – 708 . Google Scholar CrossRef Search ADS PubMed 88 Iida T , Iwata Y , Mohri T , Baba S A , and Hirohashi N ( 2017 ) A coordinated sequence of distinct flagellar waveforms enables a sharp flagellar turn mediated by squid sperm pH taxis . Sci. Rep. 7 : 12938 . Google Scholar CrossRef Search ADS PubMed 89 Gibbons B H , and Gibbons I R ( 1972 ) Flagellar movement and adenosine triphosphatase activity in sea urchin sperm extracted with triton X-100 . J. Cell Biol. 54 : 75 – 97 . Google Scholar CrossRef Search ADS PubMed 90 Tombes R M , and Shapiro B M ( 1985 ) Metabolite channeling: a phosphorylcreatine shuttle to mediate high-energy phosphate-transport between sperm mitochondrion and tail . Cell 41 : 325 – 334 . Google Scholar CrossRef Search ADS PubMed 91 Saudrais C , Fierville F , Loir M , Le Rumeur E , Cibert C , and Cosson J ( 1998 ) The use of phosphocreatine plus ADP as energy source for motility of membrane-deprived trout spermatozoa . Cell Motil. Cytoskeleton 41 : 91 – 106 . Google Scholar CrossRef Search ADS PubMed 92 Mukai C , and Okuno M ( 2004 ) Glycolysis plays a major role for adenosine triphosphate supplementation in mouse sperm flagellar movement . Biol. Reprod. 71 : 540 – 547 . Google Scholar CrossRef Search ADS PubMed 93 Miki K , Qu W , Goulding E H , Willis W D , Bunch D O , Strader L F , Perreault S D , Eddy E M , and O’Brien D A ( 2004 ) Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility . Proc. Natl. Acad. Sci. USA 101 : 16501 – 16506 . Google Scholar CrossRef Search ADS 94 Woolley D M ( 2007 ) A novel motility pattern in quail spermatozoa with implications for the mechanism of flagellar beating . Biol. Cell. 99 : 663 – 675 . Google Scholar CrossRef Search ADS PubMed 95 Nakano I , Kobayashi T , Yoshimura M , and Shingyoji C ( 2003 ) Central-pair-linked regulation of microtubule sliding by calcium in flagellar axonemes . J. Cell Sci. 116 : 1627 – 1636 . Google Scholar CrossRef Search ADS PubMed 96 Paschal B M , King S M , Moss A G , Collins C A , Vallee R B , and Witman G B ( 1987 ) Isolated flagellar outer arm dynein translocates brain microtubules in vitro . Nature 330 : 672 – 674 . Google Scholar CrossRef Search ADS PubMed 97 Moss A G , Sale W S , Fox L A , and Witman G B ( 1992 ) The alpha subunit of sea urchin sperm outer arm dynein mediates structural and rigor binding to microtubules . J. Cell Biol. 118 : 1189 – 1200 . Google Scholar CrossRef Search ADS PubMed 98 Inoue Y , and Shingyoji C ( 2007 ) The roles of noncatalytic ATP binding and ADP binding in the regulation of dynein motile activity in flagella . Cell Motil. Cytoskeleton 64 : 690 – 704 . Google Scholar CrossRef Search ADS PubMed 99 Bahat A , Tur-Kaspa I , Gakamsky A , Giojalas L C , Breitbart H , and Eisenbach M ( 2003 ) Thermotaxis of mammalian sperm cells: a potential navigation mechanism in the female genital tract . Nat. Med. 9 : 149 – 150 . Google Scholar CrossRef Search ADS PubMed 100 Miki K , and Clapham D E ( 2013 ) Rheotaxis guides mammalian sperm . Curr. Biol. 23 : 443 – 452 . Google Scholar CrossRef Search ADS PubMed 101 Yoshida M , Inaba K , and Morisawa M ( 1993 ) Sperm chemotaxis during the process of fertilization in the ascidians Ciona savignyi and Ciona intestinalis . Dev. Biol. 157 : 497 – 506 . Google Scholar CrossRef Search ADS PubMed 102 Kaupp U B , Solzin J , Hildebrand E , Brown J E , Helbig A , Hagen V , Beyermann M , Pampaloni F , and Weyand I ( 2003 ) The signal flow and motor response controling chemotaxis of sea urchin sperm . Nat. Cell Biol. 5 : 109 – 117 . Google Scholar CrossRef Search ADS PubMed 103 Wood C D , Nishigaki T , Tatsu Y , Yumoto N , Baba S A , Whitaker M , and Darszon A ( 2007 ) Altering the speract-induced ion permeability changes that generate flagellar Ca2+ spikes regulates their kinetics and sea urchin sperm motility . Dev. Biol. 306 : 525 – 537 . Google Scholar CrossRef Search ADS PubMed 104 Kricka L J , Nozaki O , Heyner S , Garside W T , and Wilding P ( 1993 ) Applications of a microfabricated device for evaluating sperm function . Clin. Chem. 39 : 1944 – 1947 . Google Scholar PubMed 105 Koyama S , Amarie D , Soini H A , Novotny M V , and Jacobson S C ( 2006 ) Chemotaxis assays of mouse sperm on microfluidic devices . Anal. Chem. 78 : 3354 – 3359 . Google Scholar CrossRef Search ADS PubMed 106 Chang H , Kim B J , Kim Y S , Suarez S S , and Wu M ( 2013 ) Different migration patterns of sea urchin and mouse sperm revealed by a microfluidic chemotaxis device . PLoS One 8 : e60587 . Google Scholar CrossRef Search ADS PubMed 107 Jikeli J F , Alvarez L , Friedrich B M , Wilson L G , Pascal R , Colin R , Pichlo M , Rennhack A , Brenker C , and Kaupp U B ( 2015 ) Sperm navigation along helical paths in 3D chemoattractant landscapes . Nat. Commun. 6 : 7985 . Google Scholar CrossRef Search ADS PubMed 108 Darszon A , Guerrero A , Galindo B E , Nishigaki T , and Wood C D ( 2008 ) Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility . Int. J. Dev. Biol. 52 : 595 – 606 . Google Scholar CrossRef Search ADS PubMed 109 Chen D T , Heymann M , Fraden S , Nicastro D , and Dogic Z ( 2015 ) ATP Consumption of eukaryotic flagella measured at a single cell level . Biophys. J. 109 : 2562 – 2573 . Google Scholar CrossRef Search ADS PubMed 110 Nakajima A , Morita M , Takemura A , Kamimura S , and Okuno M ( 2005 ) Increase in intracellular pH induces phosphorylation of axonemal proteins for activation of flagellar motility in starfish sperm . J. Exp. Biol. 208 : 4411 – 4418 . Google Scholar CrossRef Search ADS PubMed 111 Gonzalez-Cota A L , Silva P A , Carneiro J , and Darszon A ( 2015 ) Single cell imaging reveals that the motility regulator speract induces a flagellar alkalinization that precedes and is independent of Ca2+ influx in sea urchin spermatozoa . FEBS Lett. 589 : 2146 – 2154 . Google Scholar CrossRef Search ADS PubMed 112 Fukami K , Yoshida M , Inoue T , Kurokawa M , Fissore R A , Yoshida N , Mikoshiba K , and Takenawa T ( 2003 ) Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm . J. Cell Biol. 161 : 79 – 88 . Google Scholar CrossRef Search ADS PubMed 113 Kaupp U B , Kashikar N D , and Weyand I ( 2008 ) Mechanisms of sperm chemotaxis . Annu. Rev. Physiol. 70 : 93 – 117 . Google Scholar CrossRef Search ADS PubMed 114 Nishigaki T , Wood C D , Shiba K , Baba S A , and Darszon A ( 2006 ) Stroboscopic illumination using light-emitting diodes reduces phototoxicity in fluorescence cell imaging . Biotechniques 41 : 191 – 197 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Microscopy Oxford University Press

Microscopic analysis of sperm movement: links to mechanisms and protein components

Microscopy , Volume Advance Article (3) – May 8, 2018

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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0022-0744
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Abstract

Abstract Cilia and flagella are cell machines that power hydrodynamic forces by fast beating. They are composed of bundles of 9 + 2 microtubules that associate with several protein structures including axonemal dyneins and their regulators. Spermatozoa are single cells with in most cases a single flagellum and are good cell models to study how flagellar movements are regulated and linked to cell behavior. In addition, the assessment of sperm motility is an important diagnostic tool for evaluating male fertility in human reproductive medicine, and in livestock and fisheries sciences. Microscopic analyses of the movements of spermatozoa and their flagellar waveforms and propagation have been carried out using high-speed cameras and stroboscopic illumination. Computer-assisted sperm analysis (CASA) now comprises an automated set of methods to evaluate sperm quality for fertility. Here, we summarize the microscopy systems used for evaluating sperm motility, including CASA, and introduce updates on the molecular mechanism of flagellar movement and regulation that are linked to motility parameters. Furthermore, we introduce recent techniques employed to measure key factors controlling sperm motility. cilia, flagella, axonemal dynein, sperm motility, CASA, male fertility Introduction Profound human interest in a microscopic world was opened up by Antonie van Leeuwenhoek, who used a small hand-made microscope with a highly polished lens and observed several organisms (see [1]). In a letter written to the Royal Society in 1677, he described spermatozoa as one of the ‘animalcules’, moving in semen. After a series of works for more than a century summarized in [2], precise description of fast ciliary movements was first recorded by using photographic and stroboscopic recording [3]. Sir James Gray then used 2 ms flash photography and recorded the flagellar beating of sea urchin spermatozoa. The flagellar movements were seen ‘frozen’ at an illumination frequency of 30–50 per second [4]. Undamped oscillations of ciliary and flagellar waves suggested that the active motive elements were present from the base to tip of these motile apparatuses [5]. Several studies then followed to record the propagation of flagellar waveforms with more sophisticated systems for illumination and analysis [6,7]. These attempts, in conjugation with the structural observation of flagellar structure by thin-section electron microscopy, contributed to understanding the mechanism of sperm flagellar movement. Although cilia and flagella are discriminated by their length, numbers per cell and fashion in wave propagation, they structurally comprise the same motile machineries and the terminology is just for descriptive purpose [8–10]. Structural and functional identity is demonstrated in the simultaneous symptom of dysfunction in sperm motility and those in other ciliated organs, as seen in primary ciliary dyskinesia [11]. Understanding the structure and mechanism of cilia and flagella have been advanced in flagellated and ciliated cells and organisms, such as Chlamydomonas, ciliates and marine invertebrates (see [12]). From a series of experimental observations [13–16], it was established that ciliary and flagellar oscillatory movements are caused by the active sliding of microtubules by dyneins. Proteomics and structural description of sperm axonemes by cryoelectron tomography have revealed the detailed configuration of axonemal proteins, contributing to the understanding of molecular mechanism of ciliary and flagellar motility. In this review, we overview the microscopic recording and systems of analyzing the rapid movements in sperm flagellar motility. These systems include important methods for diagnosing human male fertility in reproductive medicine, and in livestock and fisheries science. For a comprehensive assessment of sperm motility, it would be useful to understand how the parameters of flagellar movement estimated by light microscopy link to the molecular mechanisms for dynein-mediated microtubule sliding. Mechanism of sperm motility The cytoskeletal basis of sperm flagella for motility is the axoneme, which is typically composed of a set of nine doublet and two singlet microtubules (9+2) with associated structures including outer and inner arm dynein molecules and their regulatory components (Fig. 1a). Both outer and inner dynein arms (IDA) are axonemal dyneins, comprising heavy, intermediate and light chains (LCs). The heavy chains are motor ATPases, and the intermediate/LCs are involved in the assembly and regulation of the machinery [9,17]. Axonemal dyneins are functionally and phylogenetically different from cytoplasmic dynein, which plays a role in the intracellular transport of molecules and vesicles. Although the axoneme is central to producing the basic waveform and its propagation, the structural pattern is diverse and has accessory structures in some organisms, which more or less affect their movement characteristics [18]. Biochemical and proteomic identification and structural study using cryoelectron tomography, as well as the analysis of Chlamydomonas flagella mutants have brought a great advance in understanding the molecular structure and function of each axonemal component [9,19–21] as described below. Fig. 1. View largeDownload slide Basic concepts for the analysis of sperm flagellar motility. (a) Structure of the flagellar axoneme. Doublet microtubules are composed of A- and B-tubules and numbered by their position relative to the axis of the central pair. (b) Relationship between flagellar bend angle and microtubule sliding. R, radius; D, distance between microtubules; L, length of sliding; θ, bend angle. Based on reference [22]. (c) Definitions of several parameters for sperm flagellar motility, in particular those described for flagellar asymmetry. P, principal bend; R, reverse bend; θp, bend angle of the principal bend; θr, bend angle of the reverse bend; rp, radius of inscribed circle at the principal bend; rr, radius of inscribed circle at the reverse bend; σ, shear angle at a certain point on a flagellum. The bend angle is defined as the angle between the tangents in a bend region. The shear angle is defined as the angle between the reference line at the base of flagellum (parallel to the longitudinal axis of the basal body; in practice, the sperm head axis is used). Asymmetry is expressed as θp–θr. Flagellar curvature is defined as the reciprocal of the radius of the inscribed circle. The asymmetric index (ratio of P- and R-bend maximum curvatures) is also used to evaluate wave asymmetry (see [23]). Fig. 1. View largeDownload slide Basic concepts for the analysis of sperm flagellar motility. (a) Structure of the flagellar axoneme. Doublet microtubules are composed of A- and B-tubules and numbered by their position relative to the axis of the central pair. (b) Relationship between flagellar bend angle and microtubule sliding. R, radius; D, distance between microtubules; L, length of sliding; θ, bend angle. Based on reference [22]. (c) Definitions of several parameters for sperm flagellar motility, in particular those described for flagellar asymmetry. P, principal bend; R, reverse bend; θp, bend angle of the principal bend; θr, bend angle of the reverse bend; rp, radius of inscribed circle at the principal bend; rr, radius of inscribed circle at the reverse bend; σ, shear angle at a certain point on a flagellum. The bend angle is defined as the angle between the tangents in a bend region. The shear angle is defined as the angle between the reference line at the base of flagellum (parallel to the longitudinal axis of the basal body; in practice, the sperm head axis is used). Asymmetry is expressed as θp–θr. Flagellar curvature is defined as the reciprocal of the radius of the inscribed circle. The asymmetric index (ratio of P- and R-bend maximum curvatures) is also used to evaluate wave asymmetry (see [23]). The outer dynein arm This contains three heavy chains in Chlamydomonas and ciliates [24,25] and two heavy chains in animals [9,26,27]. One dynein species is arranged at 24 nm periodicity on each doublet microtubule, with the aid of an anchoring structure called a dynein docking complex [28]. LCs contain a subunit (LC2) phosphorylated by a cAMP-dependent protein kinase [29,30]. A Ca2+-binding protein, calaxin, is associated with the outer dynein arm (ODA) in a Ca2+-dependent manner. Inhibition of calaxin results in the suppression of propagation of asymmetric flagellar bends [23]. Extraction of ODAs causes a decrease of beat frequency to half of that of intact axonemes in the demembranated sea urchin spermatozoon [31]. Thus, ODAs are involved both in the increase of flagellar beat frequency and in the propagation of asymmetric flagellar bends. The inner dynein arm In Chlamydomonas flagella, there are at least seven IDAs with different subunit compositions [21,32]. They are arranged on the doublet microtubules at 96 nm periodicity. The f (I1) dynein has two dynein heavy chains and the other six inner arm dyneins each has a single heavy chain [32,33]. The f dynein contains an intermediate chain that is dephosphorylated upon activation of motility [34,35]. Some IDAs contain actin or a Ca2+-binding protein, centrin [21]. IDAs are responsible for the basic mechanism of flagellar bend formation and propagation [21]. Radial spokes and the central pair apparatus (CP) Both RSs and central pair apparatus (CPs) (RS/CS) are involved in the regulation of dyneins, playing important roles in the production of planar flagellar waveforms [20]. Signal transmission between the CPs and RSs is mediated by a simple mechanical interaction that switches the regulation of dynein activity [36]. Biochemical and proteomic studies have shown that the subunits of RS/CP complexes contain several components for protein–protein interactions and cAMP/Ca2+-mediated cell signaling and are considered to regulate dyneins through Ca2+ and protein phosphorylation [37–42]. In fact, spermatozoa from some organisms, such as eels [43,44] and the Asian horseshoe crab [45,46], lack a CP and show propagation of not planar but helical waveforms. However, spermatozoa with CP/RS complexes do not always show planar waveforms. The conversion between planar and helical waveforms has been proposed to occur by regulation of mechanical constrains through Ca2+ and cAMP signals [47,48]. Nexin–dynein regulatory complex This structure is bound to the base of each IDA and radial spokes (RSs) and extends to the adjacent B-tubule of the doublet microtubule, formerly called a nexin link or interdoublet link [49,50]. Now, the terminology is Nexin–dynein regulatory complex (‘N–DRC’) [51]. It extends to the adjacent B-tubule, makes an elastic structure to bundle the peripheral doublet microtubules and plays a role in the regulation of dynein. Sperm flagellar movement is based on the sliding of peripheral doublet microtubules by axonemal dyneins. This sliding is converted into the bending of the axonemes if structural or mechanical resistance is present [16] (Fig. 1b). The flagellar base apparently acts as the key resistance. Another resistance for bend formation along a flagellum during wave oscillation is proposed to be given by dyneins, possibly from mechanical feedback [52]. From the recorded images of flagellar movement, several wave parameters can be obtained, including bend angle, flagellar curvature and shear angle (Fig. 1c; see section Microscopic description of sperm flagellar movement in detail). These parameters are useful to understand the nature of microtubule sliding in beating flagella and to estimate flagellar asymmetry. Flagellar beating occurs vertically to the plane of the CP. Planar waves are thought to be formed by switching of dynein activation at alternate doublet microtubules via a signal from the RS/CP apparatus. In the absence of a CP, flagella show helical movements. Exogenous increases in mechanical loading such as fluid viscosity converts the flagellar waveform from planar to helical, suggesting that the RS/CP might act as a mechanical constraint for the alternative sliding direction [47]. Ca2+-binding and phosphorylation of dynein subunits are known to modulate the sliding, resulting in an increase or decrease in beating frequency or in changes to flagellar waveforms. The regulations of dyneins by Ca2+ and protein phosphorylation are seen in response to changes in extracellular conditions, such as ions, chemoattractants from eggs, and other factors from the male or female reproductive tracts [27]. Microscopic description of sperm flagellar movement The beating frequency of sperm flagella is high but diverse among organisms: 30–40 Hz in sea urchins and starfish [4,7]; ~40 Hz in teleosts [53]; 90 Hz in eels [45]; 9–12 Hz in humans [54]; 1.5–11 Hz in the mouse [55] and 20 Hz in bovines [56]. To analyze the flagellar waveform, beat frequency, and propagation of waves, it is necessary to capture the flagellar waveform (Fig. 2). To achieve this, several devices have been developed: cinematography [4]; dark-field microscopy and videography [57]; high-speed videography and stroboscopy [4,7,58]; stroboscopic illumination with a very short flash duration [6,59–61]; multiple-flash photography on a moving film [62] and video recording by a charge-coupled device (CCD) with a rapid shutter using a videocassette recorder [63,64]. The sensitivity to light of cameras was improved by developing image sensors, such as a silicon intensifier target (SIT) or a complementary metal-oxide semiconductor (CMOS). High-speed cameras are also technically advanced with high specifications for shutter speed, frame rate and sensitivity [65,66]. For measuring flagellar beat frequency, dark-field or phase-contrast microscopy with stroboscopic illumination with short light flashes have mostly been used to ‘freeze’ the wave images [67,68] (Fig. 2). The flashing frequency of illumination to freeze the waveform has to be equal to the beat frequency. Alternatively, doubling the illumination frequency also freezes the waves, where two waves with mutual reverse phase appear overlapped, which makes it easier to determine the beat frequency as half of the illumination frequency. The frequency to trigger pulses from a power supply can be recorded in a frequency output counter. Flagellar beating at low frequency can be determined from the period required for one complete propagation from images of waveform propagation. Fig. 2. View largeDownload slide Phase-contrast microscopic images of sea urchin spermatozoa under normal xenon light source. (a) and stroboscopic LED illumination (b). Note that the flagellar waveform is ‘frozen’ and thus clearly observed in b. Scale bar = 20 μm. Fig. 2. View largeDownload slide Phase-contrast microscopic images of sea urchin spermatozoa under normal xenon light source. (a) and stroboscopic LED illumination (b). Note that the flagellar waveform is ‘frozen’ and thus clearly observed in b. Scale bar = 20 μm. A planar flagellar wave can be approximately sinusoidal [4,69], or in circular arcs and straight lines (arc-line wave) [6] or in a meandering pattern [70]. These patterns are thought to be generated with similar forces but the mechanisms for bend regulation appear different [70]. Given that sliding of microtubules by dynein is converted into bending, the analysis of flagellar bends provides information on the activity and regulation of dynein [22]. In spermatozoa showing planar beating of the flagellum, the bend angle at a given point is proportional to the extent of microtubule sliding (Fig. 1c). Sliding velocity is proportional to the product of the bend angle and flagellar beat frequency. In a more detailed analysis, the measurement of shear angle would be useful to quantify microtubule sliding along a flagellum [71,72] (Fig. 1c). Shear angle is also a parameter to understand microtubule sliding in beating flagella. A plot of shear angle against the distance from the base of flagellum is called the ‘shear curve’. Curved and straight regions in a shear curve correspond to straight and bending portions in the waveform, respectively. The amount of microtubule sliding is estimated from the change in the shear angle between two shear curves (for an example of a shear curve, see Fig. 4b). The flagellar waveform is composed of a bend with a larger angle (principal bend or P-bend) and a bend with a smaller angle (reverse bend or R-bend) [72]. This asymmetry is described by the difference between these two bend conformations (Fig. 1c). P- and R-bends are positioned at the side of doublets 1 and 5–6, respectively (Fig. 3a). The trajectories of spermatozoa showing planar flagellar waveforms are directly related to wave asymmetry: thus, those with symmetric waveforms swim straight, whereas those with asymmetric waveforms swim in circles. Transient changes in flagellar waveform asymmetry are known to be essential for sperm chemotaxis toward the egg before fertilization [73]. In rodent spermatozoa, flagellar bends are defined as pro-hook and anti-hook bends from their position relative to the hooked head [74]. Pro-hook and anti-hook bends are positioned at the sides of doublets 5–6 and 1, respectively (Fig. 3b). The asymmetry of the flagellar waveform is evaluated by several parameters, including the asymmetry index [75]. Flagellar asymmetry is also estimated from the shear curve of one beating cycle. The shear curve becomes diagonal in the case of asymmetric flagellar waveforms. Fig. 3. View largeDownload slide Definition and structural orientation of flagellar bends in sea urchin and other marine invertebrate spermatozoa showing simple planar waveforms (a). In rodent spermatozoa, flagellar bends are defined according to their position relative to the hook in the head (b). Axonemes are viewed from the base to tip of the flagellum. Fig. 3. View largeDownload slide Definition and structural orientation of flagellar bends in sea urchin and other marine invertebrate spermatozoa showing simple planar waveforms (a). In rodent spermatozoa, flagellar bends are defined according to their position relative to the hook in the head (b). Axonemes are viewed from the base to tip of the flagellum. Computer-assisted systems for analysis of sperm motility CASA – computer-aided sperm analysis or computer-assisted sperm analysis – appeared in the late 1980s and was developed mainly for the clinical analysis of human semen [76–79]. This automated system for evaluating sperm motility minimizes the errors conducted manually by different researchers or doctors. Thus, the information obtained by CASA is compatible with the quality assessment of human semen in the World Health Organization (WHO) guidelines, which is one of the reasons why this methodology has spread to basic research in sperm motility. The first commercial CASA system was the CellSoft Automated Semen Analyzer, released by CRYO Resources Ltd. (New York, NY, USA) in 1985, which was equipped with a monochrome video camera connected to a phase-contrast microscope. Because the principles of CASA enabled researchers to evaluate the motility characteristics of spermatozoa rapidly, the technique spread not only to human infertility clinics but also to animal husbandry and fisheries sciences. The analysis basically depends on frame-by-frame detection of sperm heads on video clips and the building of tracks of head trajectories between frames. Movie files or those processed using ImageJ (https://imagej.nih.gov/ij/download.html) can be incorporated for analysis. Devices for capturing images, sensitivity of CCD cameras and software for sperm detection and motion analysis have changed and become more sophisticated, although the basic concept of analysis has not changed (See [79]). CASA systems depend on tracking the sperm head but do not perform direct measurements of flagellar motility (Fig. 4a and b). In a conventional chamber for measurement, the systems cannot distinguish between helical and planar waveforms. However, parameters calculated by CASA could be partly interpreted in terms of the mechanism of axonemal activity for sperm motility (Fig. 4c), although in several cases sperm motility patterns cannot be attributed to simple dynein–microtubule interactions. The possible interpretations to be considered first for parameters obtained by CASA are described below, although these are not definitive because several unknown factors would affect each motility parameter. Fig. 4. View largeDownload slide Definition of motility parameters in CASA (a). The purple line with circles represents the trajectory of the sperm head. (b, c) An example of motility analysis using a SMAS CASA system (Ditect Co. Ltd, Tokyo, Japan). Automatically traced trajectories for 1 s of sperm movements in Sillago japonica. Sperm trajectories are shown for 1 s at 15, 60 and 180 s after the initiation of motility by sea water. Scale bar = 100 μm (b). Several parameters calculated automatically using the SMAS system. Note that velocity and linearity of sperm movements decrease with time after activation (c). Fig. 4. View largeDownload slide Definition of motility parameters in CASA (a). The purple line with circles represents the trajectory of the sperm head. (b, c) An example of motility analysis using a SMAS CASA system (Ditect Co. Ltd, Tokyo, Japan). Automatically traced trajectories for 1 s of sperm movements in Sillago japonica. Sperm trajectories are shown for 1 s at 15, 60 and 180 s after the initiation of motility by sea water. Scale bar = 100 μm (b). Several parameters calculated automatically using the SMAS system. Note that velocity and linearity of sperm movements decrease with time after activation (c). Percent motility This parameter is used for a simple evaluation of sperm motility. The threshold of swimming velocity can be set to evaluate the percentage of spermatozoa showing progressive swimming. Sperm are immotile or show low motility in male reproductive organs. For animals with external fertilization, sperm initiate motility upon spawning. In mammals, spermatozoa undergo maturation during passage through the epididymis and show capacitation and hyperactivation in the female reproductive tract [27]. Spermatozoa from mammals with defects in spermatogenesis usually show very low or no motility. Deficiencies or any abnormalities in the components of the axoneme or signaling to activate sperm motility result in reduced values for percent motility. CASA velocity parameters The curvilinear velocity (VCL) parameter represents velocity along the actual trajectory (Fig. 4a). The velocity along the trajectory of the computed average path is expressed as the average path velocity (VAP). The straight-line velocity (VSL) measure represents velocity along a straight-line path from the start to the end point of any given path analysis (Fig. 4a). These three values indicate the activity of dyneins and axonemes, because sperm velocity is proportional to the beat frequency, according to the resistive force theory [69]. These values are strongly associated with flagellar kinetics or propulsive sperm movement, expressed as the power output, which is proportional to beat frequency, flagellar amplitude and flagellar length [47,80]. Axonemal motility depends on the dynein-mediated sliding of microtubules, but there are several factors to regulate it; not only second messengers like Ca2+ or cAMP that act directly on axonemes but also membrane-mediated signal transduction mechanisms upstream of Ca2+/cAMP intracellular signals, which could be affected by several factors, including receptors of activators, ion channels, transmembrane or membrane-bound enzymes, and membrane fluidity [27,81,82]. Linearity (LIN) is calculated from the VSL/VAP ratio, representing sperm path LIN. The swimming direction of a spermatozoon changes in response to the conditions outside the cell, which are important for sperm to perform directional movements toward the egg for successful fertilization. Proper waveform asymmetry determines the swimming direction of the spermatozoon. In the case of spermatozoa with a planar waveform, alternate bend formation is caused by switching the active dynein across the CP. If dyneins at both P- and R-bends are equally active, symmetric bends are formed, while if their activities are different, asymmetric waveforms are generated. Asymmetric waveforms are thought to be made by inhibition of the dynein-mediated microtubule sliding in the reverse bend [10,23,72]. Therefore, the difference in LIN is considered due to the basic force-generation activity of dyneins or most likely to the mechanism of Ca2+-dependent regulation of waveform formation and propagation. Amplitude of lateral head displacement (ALH) represents the deviation of the head trajectory from the average path. For example, hyperactivation of mammalian spermatozoa is characterized by large bending of the flagellum caused by a more flexible midpiece and large lateral movements of the sperm head [83–86], although beat frequency is decreased [74]. Both wobble (WOB, calculated as VAP/VCL) and ALH estimate the head movements in hyperactivated spermatozoa. Because the parameters measured by CASA in observation chambers do not always reflect the real or proper movements of spermatozoa in the female reproductive tract, several improvements have been carried out [79,87]. CASA is based on the movement of sperm heads. Therefore, no information about the flagellar waveform is available. However, computer-assisted systems are also powerful in the analysis of flagellar waveforms [75]. In many of our recent studies, we used a sophisticated computer-assisted system for sperm flagellar motility, called Bohboh (Bohboh soft, Tokyo, Japan) (Fig. 5a), which is software for the analysis of cell motility, specialized for spermatozoa, flagella and cilia. Bohboh was developed by Dr Shoji Baba on a Microsoft Visual Studio platform, allowing the software to run on a personal computer (PC) with Microsoft Windows software. By anchoring a few points along the flagellum, the flagellar waveform is tracked automatically. The coordinate data from the trace are used to calculate the flagellar curvature and shear angle (Fig. 5b), with a variety of formats, including a spatiotemporal matrix for 3D maps [88]. This software also automatically tracks the sperm head and analyzes sperm trajectories and several parameters for sperm movement, velocity, trajectory of the sperm head, path curvature and flagellar beat frequency, just as in other commercial CASA systems (Fig. 5a). Fig. 5. View largeDownload slide (a) A screen capture image using the Bohboh system (Bohboh soft, Tokyo, Japan). The pink line and orange dots indicate the sperm trajectory and flagellar waveform, respectively. Both are obtained by auto-tracking. Such sequential image tracking makes it possible to analyze the waveform and its propagation, in relation to each spermatozoon’s moving trajectory. (b) An example of the analysis of flagellar waveforms in sea urchin sperm. Sequential flagellar waveforms (left), flagellar curvature along the flagellum (middle) and shear curve (right) are shown. Sperm movements were observed under a phase-contrast microscope and recorded with a high-speed CCD camera (HAS-220; Ditect Co. Ltd). Images were recorded with a frame rate of 200 fps. The flagellar wave was tracked and analyzed using the Bohboh system. Sequential images taken at 5 ms intervals are shown. Note that a high intracellular Ca2+ concentration (artificial sea water, ASW + calcium ionophore A23187) induces a more asymmetric waveform than low Ca2+ conditions (calcium-free sea water, CFSW). Data from 20 waveforms are superimposed. P, principal bend; R, reverse bend. Scale bar = 20 μm. Fig. 5. View largeDownload slide (a) A screen capture image using the Bohboh system (Bohboh soft, Tokyo, Japan). The pink line and orange dots indicate the sperm trajectory and flagellar waveform, respectively. Both are obtained by auto-tracking. Such sequential image tracking makes it possible to analyze the waveform and its propagation, in relation to each spermatozoon’s moving trajectory. (b) An example of the analysis of flagellar waveforms in sea urchin sperm. Sequential flagellar waveforms (left), flagellar curvature along the flagellum (middle) and shear curve (right) are shown. Sperm movements were observed under a phase-contrast microscope and recorded with a high-speed CCD camera (HAS-220; Ditect Co. Ltd). Images were recorded with a frame rate of 200 fps. The flagellar wave was tracked and analyzed using the Bohboh system. Sequential images taken at 5 ms intervals are shown. Note that a high intracellular Ca2+ concentration (artificial sea water, ASW + calcium ionophore A23187) induces a more asymmetric waveform than low Ca2+ conditions (calcium-free sea water, CFSW). Data from 20 waveforms are superimposed. P, principal bend; R, reverse bend. Scale bar = 20 μm. Other microscopic systems for studying the mechanisms of sperm motility The 9+2 microtubule structure, the bases of the axoneme, is the central motile machinery of the sperm flagella. The ‘Triton model’ is prepared by demembranation of sperm cells with a non-ionic detergent Triton X-100. It was first developed using sea urchin spermatozoa [89]. Oscillatory flagellar bending and propagation are reproduced in the Triton model by reactivation with ATP. The effects of several chemicals or factors on axonemal movement can be examined directly using this system. If spermatozoa show no motility but the Triton models are properly activated, it is interpreted that some membrane-related or intracellular signaling molecules cannot function normally such cases. It is worth noting that ATP acts evenly on such demembranated sperm flagella. It has long been a question on how ATP is supplied throughout the flagellum. The creatine shuttle was proposed to explain the nonattenuated waveform from the base to tip of the flagellum in sea urchin and trout sperm models [90,91]. In mammalian spermatozoa, glycolysis by enzymes localized in the fibrous sheath plays an important role in providing ATP throughout the flagellum [92,93]. Efficient ATP supply throughout a flagellum appears indispensable, in particular in spermatozoa with an unusually long flagellum, such as in the quail. Quail spermatozoa develop meandering flagellar waveforms spontaneously from the proximal region. The waves initiate from a double bend, which is oscillated by an alternating direction of propulsive force [94]. Observation of ‘sliding disintegration’ of axonemal microtubules is another direct way to understand the activity of dyneins [14,95]. After demembranation with Triton X-100 and treatment with proteases, such as trypsin and elastase, followed by addition of ATP, doublet microtubules slide out from the axonemes. Although it should be kept in mind that proteases can disrupt some dynein–microtubule interactions, sliding disintegration has been used as a model system to examine the microtubule-sliding activity of dyneins directly. More direct measurement of dynein-driven microtubule sliding is the ‘in vitro motility assay’, in which a simply reconstituted system from purified dyneins and microtubules is used, thus making it possible to determine dynein behavior without consideration of the other axonemal components [23,96–98]. In addition, to understand the fundamental processes of sperm flagellar movement, other micromanipulations using dynein-mediated microtubule sliding have been carried out (for details, see [22]). Several extracellular and intracellular signaling pathways are indispensable for the normal activation of sperm motility. Attempts have been done to visualize the behavior of spermatozoa and their internal molecules. The analysis of tactic behaviors, including thermotaxis, rheotaxis and chemotaxis, needs a proper device with specific assay chambers to record the pathways of sperm movement [99–103]. Microfluidics has been developed in assisted reproduction and applied for analysis of sperm movements [104–106]. To describe the directed free swimming of sperm, discrimination of the contact with the surface of glass slides or chambers should be taken into consideration. Most analyses of sperm swimming behaviors have been done in shallow 2D fields, which do not always reflect the real behavior of spermatozoa before fertilization. Recently, a combination of holographic microscopy and optochemical techniques has enabled the tracking of 3D movements of sea urchin spermatozoa during chemotaxis [107]. The spatiotemporal dynamics of signaling molecules are keys to understanding the molecular mechanisms involved in the regulation of sperm motility. These signaling pathways are triggered by binding of specific ligands to their receptors and ion exchange through several types of channels [108]. Only a few compounds and chemical factors, including ATP [109] and pH [110,111], have been imaged microscopically. It is well known that Ca2+ and cAMP play crucial roles in the initiation, activation and regulation of the waveform of sperm flagella [27]. Using a fluorescent indicator, several groups reported on the oscillatory behavior of intracellular Ca2+ [73,103,112,113]. We employ intracellular Ca2+ imaging in spermatozoa using stroboscopic illumination using a high-power light-emitting diode (LED) [73] (Fig. 6a). The sperm flagellum oscillates too rapidly to capture its activity using a weak fluorescent light in a microscope with continuous light source. A short pulse (~200 μs) synchronized with the camera exposure signal makes the sperm flagellar waveform ‘freeze’ on one frame. The combination of a high-intensity LED with stroboscopic flash and a high sensitivity electron-multiplying (EM) CCD camera made it possible to capture the changes of weak fluorescence from the sperm flagellum. LED lighting is controlled by low voltage and has long life and low cost. The use of an LED strobe minimizes electrical noise, which was a problem in electrophysiological measurements using Xenon-based stroboscopic illumination. Moreover, stroboscopic illumination using an LED reduces phototoxicity and photobleaching [114]. Real-time Ca2+ imaging of ascidian spermatozoa loaded with a Ca2+ indicator, Fluo8H-AM (AAT Bioquest, Sunnyvale, CA, USA), revealed transient Ca2+ increases in the flagella during sperm chemotaxis in vitro (Fig. 6b) [23]. Fig. 6. View largeDownload slide (a) A schematic setup of a fluorescence microscope system for real-time imaging of intracellular Ca2+. (b) Sequential images of ascidian spermatozoa during chemotaxis at 20 ms intervals, showing transient increase in intracellular Ca2+ concentration. Spermatozoa loaded with a fluorescent dye (Fluo8H-AM) were suspended in ASW. A capillary filled with chemoattractant was inserted into ASW. The spermatozoa showed chemotactic behavior around the tip of the capillary with transient fluorescence changes. Fluorescence was visualized by excitation with a Power LED (Luxeon III Star Lambertian Cyan LED part no. LXHL-LE3C; Luxeon Star LEDs) driven by a laboratory-made stroboscopic controller. The output was synchronized with the exposure-out signal of a high sensitivity imaging camera (ImagEM, Hamamatsu Photonics KK, Hamamatsu City, Japan). Images are represented in pseudo color with an look-up-table scale for fluorescence signals. Scale bar = 20 μm. Fig. 6. View largeDownload slide (a) A schematic setup of a fluorescence microscope system for real-time imaging of intracellular Ca2+. (b) Sequential images of ascidian spermatozoa during chemotaxis at 20 ms intervals, showing transient increase in intracellular Ca2+ concentration. Spermatozoa loaded with a fluorescent dye (Fluo8H-AM) were suspended in ASW. A capillary filled with chemoattractant was inserted into ASW. The spermatozoa showed chemotactic behavior around the tip of the capillary with transient fluorescence changes. Fluorescence was visualized by excitation with a Power LED (Luxeon III Star Lambertian Cyan LED part no. LXHL-LE3C; Luxeon Star LEDs) driven by a laboratory-made stroboscopic controller. The output was synchronized with the exposure-out signal of a high sensitivity imaging camera (ImagEM, Hamamatsu Photonics KK, Hamamatsu City, Japan). Images are represented in pseudo color with an look-up-table scale for fluorescence signals. Scale bar = 20 μm. Prospects Sperm motility is based on the interaction between dyneins and microtubules in the flagellar axoneme. Therefore, any deficiency in axonemal formation during spermiogenesis results in reduced or absent motility. Although the mechanism of flagellar motility and its regulation are not yet fully understood, recent advances in cryoelectron microscopy and molecular imaging, as well as several molecular tools, ensure better technical strategies to reveal the cause of changes in sperm motility. Detailed observations of flagellar movements and analyses of several parameters by light microscopy would not only provide us with important information on dynein behavior and microtubule sliding but also give a clue to understand the causes of abnormal motility. Motility analysis software, such as CASA, made motility analysis widespread for practical uses, including human clinical diagnosis, livestock and fisheries science, and evaluation of the efficacy of sperm cryopreservation. Development of microscopic analysis systems for sperm motility promises to play major roles to improve the function of spermatozoa from infertile men and male animals and advance the techniques of sperm preservation for reproducible insemination. Acknowledgements We thank Dr Makoto Okuno and Dr Shoji Baba for their critical reading of the manuscript. We thank James Cummins, PhD, from EDANZ Group (www.edanzediting.com/ac) for editing a draft of this manuscript. Funding This work was supported by the Ministry of Education, Culture, Sports, Science and Technology and the Japan Society for the Promotion of Science (JP16H06280 ‘Advanced Bioimaging Support’ and 17H01440). Conflict of interest The author declares no competing financial interests. References 1 Porter J R ( 1976 ) Antony van Leeuwenhoek: tercentenary of his discovery of bacteria . Bacteriol. Rev. 40 : 260 – 269 . Google Scholar PubMed 2 Gray J ( 1928 ) Ciliary Movement , ( Macmillan , New York ). 3 Gray J ( 1930 ) The mechanism of ciliary movement. VI. Photographic and stroboscopic analysis of ciliary movement . Proc. R. Soc. London Ser. B 107 : 313 – 332 . Google Scholar CrossRef Search ADS 4 Gray J ( 1955 ) The movement of sea-urchin spermatozoa . J. Exp. Biol. 32 : 775 – 800 . 5 Machin K E ( 1958 ) Wave propagation along flagella . J. Exp. Biol. 35 : 796 – 806 . 6 Brokaw C J ( 1965 ) Non-sinusoidal bending waves of sperm flagella . J. Exp. Biol. 43 : 155 – 169 . Google Scholar PubMed 7 Hiramoto Y , and Baba S A ( 1978 ) A quantitative analysis of flagellar movement in echinoderm spermatozoa . J. Exp. Biol. 76 : 85 – 104 . 8 Sleigh M A ( 1962 ) The biology of cilia and flagella , ( Macmillan , New York ). Google Scholar CrossRef Search ADS 9 Inaba K ( 2011 ) Sperm flagella: comparative and phylogenetic perspectives of protein components . Mol. Hum. Reprod. 17 : 524 – 538 . Google Scholar CrossRef Search ADS PubMed 10 Inaba K ( 2015 ) Calcium sensors of ciliary outer arm dynein: functions and phylogenetic consideration for eukaryotic evolution . Cilia 4 : 6 . Google Scholar CrossRef Search ADS PubMed 11 Inaba K , and Mizuno K ( 2016 ) Sperm dysfunction and ciliopathy . Reprod. Med. Biol. 15 : 77 – 94 . Google Scholar CrossRef Search ADS PubMed 12 Dentler W , and Witman G ( 1995 ) Cilia and flagella . Methods Cell Biol. 47 : 603 . 13 Satir P ( 1968 ) Studies on cilia. 3. Further studies on the cilium tip and a ‘sliding filament’ model of ciliary motility . J. Cell Biol. 39 : 77 – 94 . Google Scholar CrossRef Search ADS PubMed 14 Summers K E , and Gibbons I R ( 1971 ) Adenosine triphosphate-induced sliding of tubules in trypsin-treated flagella of sea-urchin sperm . Proc. Natl. Acad. Sci. USA 68 : 3092 – 3096 . Google Scholar CrossRef Search ADS 15 Brokaw C J ( 1972 ) Flagellar movement: a sliding filament model . Science 178 : 455 – 462 . Google Scholar CrossRef Search ADS PubMed 16 Shingyoji C , Murakami A , and Takahashi K ( 1977 ) Local reactivation of Triton-extracted flagella by iontophoretic application of ATP . Nature 265 : 269 – 270 . Google Scholar CrossRef Search ADS PubMed 17 King S M ( 2000 ) AAA domains and organization of the dynein motor unit . J. Cell Sci. 113 : 2521 – 2526 . Google Scholar PubMed 18 Inaba K ( 2007 ) Molecular basis of sperm flagellar axonemes: structural and evolutionary aspects . Ann. NY Acad. Sci. 1101 : 506 – 526 . Google Scholar CrossRef Search ADS 19 Porter M E , and Sale W S ( 2000 ) The 9 + 2 axoneme anchors multiple inner arm dyneins and a network of kinases and phosphatases that control motility . J. Cell Biol. 151 : F37 – F42 . Google Scholar CrossRef Search ADS PubMed 20 Smith E F , and Yang P ( 2004 ) The radial spokes and central apparatus: mechano-chemical transducers that regulate flagellar motility . Cell Motil. Cytoskeleton 57 : 8 – 17 . Google Scholar CrossRef Search ADS PubMed 21 Kamiya R ( 2002 ) Functional diversity of axonemal dyneins as studied in Chlamydomonas mutants . Int. Rev. Cytol. 219 : 115 – 155 . Google Scholar CrossRef Search ADS PubMed 22 Shingyoji C ( 2013 ) Measuring the regulation of dynein activity during flagellar motility . Methods Enzymol. 524 : 147 – 169 . Google Scholar CrossRef Search ADS PubMed 23 Mizuno K , Shiba K , Okai M , Takahashi Y , Shitaka Y , Oiwa K , Tanokura M , and Inaba K ( 2012 ) Calaxin drives sperm chemotaxis by Ca2+-mediated direct modulation of a dynein motor . Proc. Nat. Acad. Sci. USA 109 : 20497 – 20502 . Google Scholar CrossRef Search ADS 24 King S M ( 2000 ) The dynein microtubule motor . Biochim. Biophys. Acta 1496 : 60 – 75 . Google Scholar CrossRef Search ADS PubMed 25 DiBella L M , Gorbatyuk O , Sakato M , Wakabayashi K , Patel-King R S , Pazour G J , Witman G B , and King S M ( 2005 ) Differential light chain assembly influences outer arm dynein motor function . Mol. Biol. Cell 16 : 5661 – 5674 . Google Scholar CrossRef Search ADS PubMed 26 Gibbons I R ( 1981 ) Cilia and flagella of eukaryotes . J. Cell Biol. 91 : 107s – 124s . Google Scholar CrossRef Search ADS PubMed 27 Inaba K ( 2003 ) Molecular architecture of the sperm flagella: molecules for motility and signaling . Zool. Sci. 20 : 1043 – 1056 . Google Scholar CrossRef Search ADS PubMed 28 Takada S , and Kamiya R ( 1994 ) Functional reconstitution of Chlamydomonas outer dynein arms from alpha-beta and gamma subunits: requirement of a third factor . J. Cell Biol. 126 : 737 – 745 . Google Scholar CrossRef Search ADS PubMed 29 Inaba K , Morisawa S , and Morisawa M ( 1998 ) Proteasomes regulate the motility of salmonid fish sperm through modulation of cAMP-dependent phosphorylation of an outer arm dynein light chain . J. Cell Sci. 111 : 1105 – 1115 . Google Scholar PubMed 30 Inaba K , Kagami O , and Ogawa K ( 1999 ) Tctex2-related outer arm dynein light chain is phosphorylated at activation of sperm motility . Biochem. Biophys. Res. Commun. 256 : 177 – 183 . Google Scholar CrossRef Search ADS PubMed 31 Gibbons B H , and Gibbons I R ( 1973 ) The effect of partial extraction of dynein arms on the movement of reactivated sea-urchin sperm . J. Cell Sci. 13 : 337 – 357 . Google Scholar PubMed 32 Piperno G , Ramanis Z , Smith E F , and Sale W S ( 1990 ) Three distinct inner dynein arms in Chlamydomonas flagella: molecular composition and location in the axoneme . J. Cell Biol. 110 : 379 – 389 . Google Scholar CrossRef Search ADS PubMed 33 Kagami O , Takada S , and Kamiya R ( 1990 ) Microtubule translocation caused by three subspecies of inner-arm dynein from Chlamydomonas flagella . FEBS Lett. 264 : 179 – 182 . Google Scholar CrossRef Search ADS PubMed 34 Habermacher G , and Sale W S ( 1997 ) Regulation of flagellar dynein by phosphorylation of a 138-kD inner arm dynein intermediate chain . J. Cell Biol. 136 : 167 – 176 . Google Scholar CrossRef Search ADS PubMed 35 King S J , and Dutcher S K ( 1997 ) Phosphoregulation of an inner dynein arm complex in Chlamydomonas reinhardtii is altered in phototactic mutant strains . J. Cell Biol. 136 : 177 – 191 . Google Scholar CrossRef Search ADS PubMed 36 Oda Y , Yanagisawa H , Yagi Y , and Kikkawa M ( 2014 ) Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity . J. Cell Biol. 204 : 807 – 819 . Google Scholar CrossRef Search ADS PubMed 37 Yang P , Diener D R , Yang C , Kohno T , Pazour G J , Dienes J M , Agrin N S , King S M , Sale W S , Kamiya R , Rosenbaum J L , and Witman G B ( 2006 ) Radial spoke proteins of Chlamydomonas flagella . J. Cell Sci. 119 : 1165 – 1174 . Google Scholar CrossRef Search ADS PubMed 38 Wargo M J , Dymek E E , and Smith E F ( 2005 ) Calmodulin and PF6 are components of a complex that localizes to the C1 microtubule of the flagellar central apparatus . J. Cell Sci. 118 : 4655 – 4665 . Google Scholar CrossRef Search ADS PubMed 39 Dymek E E , Goduti D , Kramer T , and Smith E F ( 2006 ) A kinesin-like calmodulin-binding protein in Chlamydomonas: evidence for a role in cell division and flagellar functions . J. Cell Sci. 119 : 3107 – 3116 . Google Scholar CrossRef Search ADS PubMed 40 Padma P , Satouh Y , Wakabayashi K , Hozumi A , Ushimaru Y , Kamiya R , and Inaba K ( 2003 ) Identification of a novel leucine-rich repeat protein as a component of flagellar radial spoke in the ascidian Ciona intestinalis . Mol. Biol. Cell 14 : 774 – 785 . Google Scholar CrossRef Search ADS PubMed 41 Satouh Y , and Inaba K ( 2009 ) Proteomic characterization of sperm radial spokes identifies a novel spoke protein with an ubiquitin domain . FEBS Lett. 583 : 2201 – 2207 . Google Scholar CrossRef Search ADS PubMed 42 Satouh Y , Padma P , Toda T , Satoh N , Ide H , and Inaba K ( 2005 ) Molecular characterization of radial spoke subcomplex containing radial spoke protein 3 and heat shock protein 40 in sperm flagella of the ascidian Ciona intestinalis . Mol. Biol. Cell 16 : 626 – 636 . Google Scholar CrossRef Search ADS PubMed 43 Baccetti B , Burrini A G , Dallai R , and Pallini V ( 1979 ) The dynein electrophoretic bands in axonemes naturally lacking the inner or the outer arm . J. Cell Biol. 80 : 334 – 340 . Google Scholar CrossRef Search ADS PubMed 44 Woolley D M ( 1998 ) Studies on the eel sperm flagellum. 2. The kinematics of normal motility . Cell Motil. Cytoskeleton 39 : 233 – 245 . Google Scholar CrossRef Search ADS PubMed 45 Gibbons B H , Gibbons I R , and Baccetti B ( 1983 ) Structure and motility of the 9 + 0 flagellum of eel spermatozoa . J. Submicrosc. Cytol. 15 : 15 – 20 . Google Scholar PubMed 46 Ishijima S , Sekiguchi K , and Hiramoto Y ( 1988 ) Comparative study of the beat patterns of American and Asian horseshoe crab sperm: evidence for a role of the central pair complex in forming planar waveforms in flagella . Cell Motil. Cytoskeleton 9 : 264 – 270 . Google Scholar CrossRef Search ADS 47 Ishijima S ( 2012 ) Mechanical constraint converts planar waves into helices on tunicate and sea urchin sperm flagella . Cell Struct. Funct. 37 : 13 – 19 . Google Scholar CrossRef Search ADS PubMed 48 Ishijima S ( 2013 ) Regulations of microtubule sliding by Ca2+ and cAMP and their roles in forming flagellar waveforms . Cell Struct. Funct. 38 : 89 – 95 . Google Scholar CrossRef Search ADS PubMed 49 Huang B , Ramanis Z , and Luck D J L ( 1982 ) Suppressor mutations in Chlamydomonas reveal a regulatory mechanism for flagellar function . Cell 28 : 115 – 124 . Google Scholar CrossRef Search ADS PubMed 50 Piperno G , Mead K , LeDizet M , and Moscatelli A ( 1994 ) Mutations in the ‘dynein regulatory complex’ alter the ATP-insensitive binding sites for inner arm dyneins in Chlamydomonas axonemes . J. Cell Biol. 125 : 1109 – 1117 . Google Scholar CrossRef Search ADS PubMed 51 Heuser T , Raytchev M , Krell J , Porter M E , and Nicastro D ( 2009 ) The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella . J. Cell Biol. 187 : 921 – 933 . Google Scholar CrossRef Search ADS PubMed 52 Shingyoji C , Nakano I , Inoue Y , and Higuchi H ( 2015 ) Dynein arms are strain-dependent direction-switching force generators . Cytoskeleton 72 : 388 – 401 . Google Scholar CrossRef Search ADS PubMed 53 Ishijima S ( 2012 ) Comparative analysis of movement characteristics of lancelet and fish spermatozoa having different morphologies . Biol. Bull. 222 : 214 – 221 . Google Scholar CrossRef Search ADS PubMed 54 Burkman L J ( 1984 ) Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm populations . Arch Androl 13 : 153 – 165 . Google Scholar CrossRef Search ADS PubMed 55 Si Y , and Okuno M ( 1993 ) Multiple activation of mouse sperm motility . Mol. Reprod. Dev. 36 : 89 – 95 . Google Scholar CrossRef Search ADS PubMed 56 Ishijima S , and Witman G B ( 1987 ) Flagellar movement of intact and demembranated, reactivated ram spermatozoa . Cell Motil. Cytoskeleton 8 : 375 – 391 . Google Scholar CrossRef Search ADS PubMed 57 Rothschild L , and Swann M M ( 1949 ) The fertilization reaction in the sea-urchin egg; a propagated response to sperm attachment . J. Exp. Biol. 26 : 164 – 176 . Google Scholar PubMed 58 Rikmenspoel R , and Van Herpen G ( 1957 ) Photoelectric and cinematographic measurements of the motility of bull sperm cells . Phys. Med. Biol. 2 : 54 – 63 . Google Scholar CrossRef Search ADS PubMed 59 Brokaw C J ( 1970 ) Bending moments in free-swimming flagella . J. Exp. Biol. 53 : 445 – 464 . Google Scholar PubMed 60 Okuno M , and Brokaw C J ( 1979 ) Inhibition of movement of trition-demembranated sea-urchin sperm flagella by Mg2+, ATP4-, ADP and Pi . J. Cell Sci. 38 : 105 – 123 . Google Scholar PubMed 61 Inaba K , Okuno M , and Mohri H ( 1989 ) Anthraniloyl ATP, a fluorescent analog of ATP, as a substrate for dynein ATPase and flagellar motility . Arch. Biochem. Biophys. 274 : 209 – 215 . Google Scholar CrossRef Search ADS PubMed 62 Brokaw C J ( 1986 ) Sperm motility . Methods Cell Biol. 27 : 41 – 56 . Google Scholar CrossRef Search ADS PubMed 63 Suarez S S , Dai X B , DeMott R P , Redfern K , and Mirando M A ( 1992 ) Movement characteristics of boar sperm obtained from the oviduct or hyperactivated in vitro . J Androl 13 : 75 – 80 . Google Scholar PubMed 64 Ishijima S ( 1995 ) High-speed video microscopy of flagella and cilia . Methods Cell Biol. 47 : 239 – 243 . Google Scholar CrossRef Search ADS PubMed 65 Miyashiro D , Shiba K , Miyashita T , Baba S A , Yoshida M , and Kamimura S ( 2015 ) Chemotactic response with a constant delay-time mechanism in Ciona spermatozoa revealed by a high time resolution analysis of flagellar motility . Biol. Open 4 : 109 – 118 . Google Scholar CrossRef Search ADS PubMed 66 Lenaghan S C , Davis C A , Henson W R , Zhang Z , and Zhang M ( 2011 ) High-speed microscopic imaging of flagella motility and swimming in Giardia lamblia trophozoites . Proc. Natl. Acad. Sci. USA 108 : E550 – E558 . Google Scholar CrossRef Search ADS 67 Okuno M ( 1980 ) Inhibition and relaxation of sea urchin sperm flagella by vanadate . J. Cell Biol. 85 : 712 – 725 . Google Scholar CrossRef Search ADS PubMed 68 Okuno M , Asai D J , Ogawa K , and Brokaw C J ( 1981 ) Effects of antibodies against dynein and tubulin on the stiffness of flagellar axonemes . J. Cell Biol. 91 : 689 – 694 . Google Scholar CrossRef Search ADS PubMed 69 Gray J , and Hancock G J ( 1955 ) The propulsion of sea-urchin spermatozoa . J. Exp. Biol. 32 : 802 – 814 . 70 Holwill M E ( 1977 ) Some biophysical aspects of ciliary and flagellar motility . Adv. Microb. Physiol. 16 : 1 – 48 . Google Scholar CrossRef Search ADS PubMed 71 Brokaw C J ( 1991 ) Calcium sensors in sea urchin sperm flagella . Cell Motil. Cytoskeleton 18 : 123 – 130 . Google Scholar CrossRef Search ADS PubMed 72 Brokaw C J ( 1979 ) Calcium-induced asymmetrical beating of triton-demembranated sea urchin sperm flagella . J. Cell Biol. 82 : 401 – 411 . Google Scholar CrossRef Search ADS PubMed 73 Shiba K , Baba S A , Inoue T , and Yoshida M ( 2008 ) Ca2+ bursts occur around a local minimal concentration of attractant and trigger sperm chemotactic response . Proc. Natl. Acad. Sci. USA 105 : 19312 – 19317 . Google Scholar CrossRef Search ADS 74 Ishijima S , Baba S A , Mohri H , and Suarez S S ( 2002 ) Quantitative analysis of flagellar movement in hyperactivated and acrosome-reacted golden hamster spermatozoa . Mol. Reprod. Dev. 61 : 376 – 384 . Google Scholar CrossRef Search ADS PubMed 75 Brokaw C J ( 1990 ) Computerized analysis of flagellar motility by digitization and fitting of film images with straight segments of equal length . Cell Motil. Cytoskeleton 17 : 309 – 316 . Google Scholar CrossRef Search ADS PubMed 76 David G , Serres C , and Jouannet P ( 1981 ) Kinematics of human spermatozoa . Gamete Res. 4 : 83 – 95 . Google Scholar CrossRef Search ADS 77 Boyers S P , Davis R O , and Katz D F ( 1989 ) Automated semen analysis . Curr. Prob. Obstet. Gynecol. Fertil. XII : 167 – 200 . 78 Mortimer S T , Swan M A , and Mortimer D ( 1998 ) Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa . Hum. Reprod. 13 : 2139 – 2146 . Google Scholar CrossRef Search ADS PubMed 79 Mortimer S T , van der Horst G , and Mortimer D ( 2015 ) The future of computer-aided sperm analysis . Asian J. Androl. 17 : 545 – 553 . Google Scholar CrossRef Search ADS PubMed 80 Rikmenspoel R ( 1984 ) Movements and active moments of bull sperm flagella as a function of temperature and viscosity . J. Exp. Biol. 108 : 205 – 230 . Google Scholar PubMed 81 Cosson J , Prokopchuk G , and Inaba K ( 2015 ) The flagellar mechanics of spermatozoa and its regulation. In: Cosson J (ed.) Flagellar Mechanics and Sperm Guidance , pp 3 – 134 (Bentham Science Publisher Ltd, Sharjah, UAE). Google Scholar CrossRef Search ADS 82 Freitas M J , Vijayaraghavan S , and Fardilha M ( 2017 ) Signaling mechanisms in mammalian sperm motility . Biol. Reprod. 96 : 2 – 12 . Google Scholar PubMed 83 Katz D F , Overstreet J W , Samuels S J , Niswander P W , Bloom T D , and Lewis E L ( 1986 ) Morphometric analysis of spermatozoa in the assessment of human male fertility . J. Androl. 7 : 203 – 210 . Google Scholar CrossRef Search ADS PubMed 84 Chang H , and Suarez S S ( 2010 ) Rethinking the relationship between hyperactivation and chemotaxis in mammalian sperm . Biol. Reprod. 83 : 507 – 513 . Google Scholar CrossRef Search ADS PubMed 85 Mohri H , Inaba K , Ishijima S , and Baba S A ( 2012 ) Tubulin–dynein system in flagellar and ciliary movement . Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 88 : 397 – 415 . Google Scholar CrossRef Search ADS PubMed 86 Yanagimachi R ( 1969 ) In vitro capacitation of hamster spermatozoa by follicular fluid . J. Reprod. Fertil. 18 : 275 – 286 . Google Scholar CrossRef Search ADS PubMed 87 Goodson S G , White S , Stevans A M , Bhat S , Kao C Y , Jaworski S , Marlowe T R , Kohlmeier M , McMillan L , Zeisel S H , and O’Brien D A ( 2017 ) CASAnova: a multiclass support vector machine model for the classification of human sperm motility patterns . Biol. Reprod. 97 : 698 – 708 . Google Scholar CrossRef Search ADS PubMed 88 Iida T , Iwata Y , Mohri T , Baba S A , and Hirohashi N ( 2017 ) A coordinated sequence of distinct flagellar waveforms enables a sharp flagellar turn mediated by squid sperm pH taxis . Sci. Rep. 7 : 12938 . Google Scholar CrossRef Search ADS PubMed 89 Gibbons B H , and Gibbons I R ( 1972 ) Flagellar movement and adenosine triphosphatase activity in sea urchin sperm extracted with triton X-100 . J. Cell Biol. 54 : 75 – 97 . Google Scholar CrossRef Search ADS PubMed 90 Tombes R M , and Shapiro B M ( 1985 ) Metabolite channeling: a phosphorylcreatine shuttle to mediate high-energy phosphate-transport between sperm mitochondrion and tail . Cell 41 : 325 – 334 . Google Scholar CrossRef Search ADS PubMed 91 Saudrais C , Fierville F , Loir M , Le Rumeur E , Cibert C , and Cosson J ( 1998 ) The use of phosphocreatine plus ADP as energy source for motility of membrane-deprived trout spermatozoa . Cell Motil. Cytoskeleton 41 : 91 – 106 . Google Scholar CrossRef Search ADS PubMed 92 Mukai C , and Okuno M ( 2004 ) Glycolysis plays a major role for adenosine triphosphate supplementation in mouse sperm flagellar movement . Biol. Reprod. 71 : 540 – 547 . Google Scholar CrossRef Search ADS PubMed 93 Miki K , Qu W , Goulding E H , Willis W D , Bunch D O , Strader L F , Perreault S D , Eddy E M , and O’Brien D A ( 2004 ) Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility . Proc. Natl. Acad. Sci. USA 101 : 16501 – 16506 . Google Scholar CrossRef Search ADS 94 Woolley D M ( 2007 ) A novel motility pattern in quail spermatozoa with implications for the mechanism of flagellar beating . Biol. Cell. 99 : 663 – 675 . Google Scholar CrossRef Search ADS PubMed 95 Nakano I , Kobayashi T , Yoshimura M , and Shingyoji C ( 2003 ) Central-pair-linked regulation of microtubule sliding by calcium in flagellar axonemes . J. Cell Sci. 116 : 1627 – 1636 . Google Scholar CrossRef Search ADS PubMed 96 Paschal B M , King S M , Moss A G , Collins C A , Vallee R B , and Witman G B ( 1987 ) Isolated flagellar outer arm dynein translocates brain microtubules in vitro . Nature 330 : 672 – 674 . Google Scholar CrossRef Search ADS PubMed 97 Moss A G , Sale W S , Fox L A , and Witman G B ( 1992 ) The alpha subunit of sea urchin sperm outer arm dynein mediates structural and rigor binding to microtubules . J. Cell Biol. 118 : 1189 – 1200 . Google Scholar CrossRef Search ADS PubMed 98 Inoue Y , and Shingyoji C ( 2007 ) The roles of noncatalytic ATP binding and ADP binding in the regulation of dynein motile activity in flagella . Cell Motil. Cytoskeleton 64 : 690 – 704 . Google Scholar CrossRef Search ADS PubMed 99 Bahat A , Tur-Kaspa I , Gakamsky A , Giojalas L C , Breitbart H , and Eisenbach M ( 2003 ) Thermotaxis of mammalian sperm cells: a potential navigation mechanism in the female genital tract . Nat. Med. 9 : 149 – 150 . Google Scholar CrossRef Search ADS PubMed 100 Miki K , and Clapham D E ( 2013 ) Rheotaxis guides mammalian sperm . Curr. Biol. 23 : 443 – 452 . Google Scholar CrossRef Search ADS PubMed 101 Yoshida M , Inaba K , and Morisawa M ( 1993 ) Sperm chemotaxis during the process of fertilization in the ascidians Ciona savignyi and Ciona intestinalis . Dev. Biol. 157 : 497 – 506 . Google Scholar CrossRef Search ADS PubMed 102 Kaupp U B , Solzin J , Hildebrand E , Brown J E , Helbig A , Hagen V , Beyermann M , Pampaloni F , and Weyand I ( 2003 ) The signal flow and motor response controling chemotaxis of sea urchin sperm . Nat. Cell Biol. 5 : 109 – 117 . Google Scholar CrossRef Search ADS PubMed 103 Wood C D , Nishigaki T , Tatsu Y , Yumoto N , Baba S A , Whitaker M , and Darszon A ( 2007 ) Altering the speract-induced ion permeability changes that generate flagellar Ca2+ spikes regulates their kinetics and sea urchin sperm motility . Dev. Biol. 306 : 525 – 537 . Google Scholar CrossRef Search ADS PubMed 104 Kricka L J , Nozaki O , Heyner S , Garside W T , and Wilding P ( 1993 ) Applications of a microfabricated device for evaluating sperm function . Clin. Chem. 39 : 1944 – 1947 . Google Scholar PubMed 105 Koyama S , Amarie D , Soini H A , Novotny M V , and Jacobson S C ( 2006 ) Chemotaxis assays of mouse sperm on microfluidic devices . Anal. Chem. 78 : 3354 – 3359 . Google Scholar CrossRef Search ADS PubMed 106 Chang H , Kim B J , Kim Y S , Suarez S S , and Wu M ( 2013 ) Different migration patterns of sea urchin and mouse sperm revealed by a microfluidic chemotaxis device . PLoS One 8 : e60587 . Google Scholar CrossRef Search ADS PubMed 107 Jikeli J F , Alvarez L , Friedrich B M , Wilson L G , Pascal R , Colin R , Pichlo M , Rennhack A , Brenker C , and Kaupp U B ( 2015 ) Sperm navigation along helical paths in 3D chemoattractant landscapes . Nat. Commun. 6 : 7985 . Google Scholar CrossRef Search ADS PubMed 108 Darszon A , Guerrero A , Galindo B E , Nishigaki T , and Wood C D ( 2008 ) Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility . Int. J. Dev. Biol. 52 : 595 – 606 . Google Scholar CrossRef Search ADS PubMed 109 Chen D T , Heymann M , Fraden S , Nicastro D , and Dogic Z ( 2015 ) ATP Consumption of eukaryotic flagella measured at a single cell level . Biophys. J. 109 : 2562 – 2573 . Google Scholar CrossRef Search ADS PubMed 110 Nakajima A , Morita M , Takemura A , Kamimura S , and Okuno M ( 2005 ) Increase in intracellular pH induces phosphorylation of axonemal proteins for activation of flagellar motility in starfish sperm . J. Exp. Biol. 208 : 4411 – 4418 . Google Scholar CrossRef Search ADS PubMed 111 Gonzalez-Cota A L , Silva P A , Carneiro J , and Darszon A ( 2015 ) Single cell imaging reveals that the motility regulator speract induces a flagellar alkalinization that precedes and is independent of Ca2+ influx in sea urchin spermatozoa . FEBS Lett. 589 : 2146 – 2154 . Google Scholar CrossRef Search ADS PubMed 112 Fukami K , Yoshida M , Inoue T , Kurokawa M , Fissore R A , Yoshida N , Mikoshiba K , and Takenawa T ( 2003 ) Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm . J. Cell Biol. 161 : 79 – 88 . Google Scholar CrossRef Search ADS PubMed 113 Kaupp U B , Kashikar N D , and Weyand I ( 2008 ) Mechanisms of sperm chemotaxis . Annu. Rev. Physiol. 70 : 93 – 117 . Google Scholar CrossRef Search ADS PubMed 114 Nishigaki T , Wood C D , Shiba K , Baba S A , and Darszon A ( 2006 ) Stroboscopic illumination using light-emitting diodes reduces phototoxicity in fluorescence cell imaging . Biotechniques 41 : 191 – 197 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Published: May 8, 2018

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