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 ). 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 , precise description of fast ciliary movements was first recorded by using photographic and stroboscopic recording . 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 . Undamped oscillations of ciliary and flagellar waves suggested that the active motive elements were present from the base to tip of these motile apparatuses . 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 . 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 ). 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 . 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 . (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 ). 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 . (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 ). 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 . 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 . Extraction of ODAs causes a decrease of beat frequency to half of that of intact axonemes in the demembranated sea urchin spermatozoon . 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 . IDAs are responsible for the basic mechanism of flagellar bend formation and propagation . 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 . Signal transmission between the CPs and RSs is mediated by a simple mechanical interaction that switches the regulation of dynein activity . 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’) . 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  (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 . 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 . 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 . 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 ; 90 Hz in eels ; 9–12 Hz in humans ; 1.5–11 Hz in the mouse  and 20 Hz in bovines . 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 ; dark-field microscopy and videography ; 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  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)  or in a meandering pattern . These patterns are thought to be generated with similar forces but the mechanisms for bend regulation appear different . 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 . 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) . 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 . In rodent spermatozoa, flagellar bends are defined as pro-hook and anti-hook bends from their position relative to the hooked head . 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 . 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 ). 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 ). 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 . 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 . Only a few compounds and chemical factors, including ATP  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 . 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)  (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 . 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) . 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 . 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Microscopy – Oxford University Press
Published: May 8, 2018
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