Flagellar Beat Research Articles

Magnetotactic Sperm Cells for Assisted Reproduction.

Biohybrid micromotors are active microscopic agents consisting of biological and synthetic components that are being developed as novel tools for biomedical applications. By capturing motile sperm cells within engineered microstructures, they can be controlled remotely while being propelled forward by the flagellar beat. This makes them an interesting tool for reproductive medicine that can enable minimally invasive sperm cell delivery to the oocyte in vivo, as a treatment for infertility. The generation of sperm-based micromotors in sufficiently large numbers, as they are required in biomedical applications has been challenging, either due to the employed fabrication techniques or the stability of the microstructure-sperm coupling. Here, biohybrid micromotors, which can be assembled in a fast and simple process using magnetic microparticles, are presented. These magnetotactic sperm cells show a high motility and swimming speed and can be transferred between different environments without large detrimental effects on sperm motility and membrane integrity. Furthermore, clusters of micromotors are assembled magnetically and visualized using dual ultrasound (US)/photoacoustic (PA) imaging. Finally, a protocol for the scaled-up assembly of micromotors and their purification for use in in vitro fertilization (IVF) is presented, bringing them closer to their biomedical implementation.

Human sperm rotate with a conserved direction during free swimming in four dimensions.

Head rotation in human spermatozoa is essential for different swimming modes and fertilisation, as it links the molecular workings of the flagellar beat with sperm motion in three-dimensional (3D) space over time. Determining the direction of head rotation has been hindered by the symmetry and translucent nature of the sperm head, and by the fast 3D motion driven by the helical flagellar beat. Analysis has been mostly restricted to two-dimensional (2D) single focal plane image analysis, which enables tracking of head centre position but not tracking of head rotation. Despite the conserved helical beating of the human sperm flagellum, human sperm head rotation has been reported to be uni- or bi-directional, and even to intermittently change direction in a given cell. Here, we directly measure the head rotation of freely swimming human sperm using multi-plane 4D (3D+t) microscopy and show that: (1) 2D microscopy is unable to distinguish head rotation direction in human spermatozoa; (2) head rotation direction in non-capacitating and capacitating solutions, for both aqueous and viscous media, is counterclockwise (CCW), as seen from head to tail, in all rotating spermatozoa, regardless of the experimental conditions; and (3) head rotation is suppressed in 36% of spermatozoa swimming in non-capacitating viscous medium, although CCW rotation is recovered after incubation in capacitating conditions within the same viscous medium, possibly unveiling an unexplored aspect of the essential need of capacitation for fertilisation. Our observations show that the CCW head rotation in human sperm is conserved. It constitutes a robust and persistent helical driving mechanism that influences sperm navigation in 3D space over time, and thus is of critical importance in cell motility, propulsion of flagellated microorganisms, sperm motility assessments, human reproduction research, and self-organisation of flagellar beating patterns and swimming in 3D space.

Swimming zygote formation mediated by symmetrically arranged cell fusion sites in the gametes of Chlamydomonas eugametos (Chlorophyceae, Chlorophyta)

SUMMARYThe spatial arrangement of cell fusion sites and eyespots of the gametes in vis‐à‐vis mating pairs of Chlamydomonas eugametos, corresponding to the swimming zygote of Chlamydomonas reinhardtii, were studied using field emission scanning electron microscopy and light microscopy, respectively. After the adhesion of mt+ and mt− gametes, each of which has one eyespot and two flagella elongated from the cell anterior, gamete–gamete fusion occurred at the anterior papilla on the cell apex of each gamete, leading to the formation of a vis‐à‐vis mating pair, indicating that the cell fusion site is arranged symmetrically relative to the flagellar beat plane. In vis‐à‐vis mating pairs, two eyespots could be seen on the same or on opposite sides of the cell. As only the mt+ flagella and eyespot are functional for swimming and phototaxis in the vis‐à‐vis mating pair of C. eugametos, it is suggested that coordinated alignment of the parental gamete flagella and eyespots in the swimming zygote and asymmetric positioning of the gamete mating structure/cell fusion sites, which is prevalent in C. reinhardtii and other chlorophytes, is not necessary for C. eugametos.

The Role of Soluble Adenylyl Cyclase in the Regulation of Flagellar Motility in Ascidian Sperm.

Flagellar motility in sperm is activated and regulated by factors related to the eggs at fertilization. In the ascidian Ciona intestinalis, a sulfated steroid called the SAAF (sperm activating and attracting factor) induces both sperm motility activation and chemotaxis. Cyclic AMP (cAMP) is one of the most important intracellular factors in the sperm signaling pathway. Adenylyl cyclase (AC) is the key enzyme that synthesizes cAMP at the onset of the signaling pathway in all cellular functions. We previously reported that both transmembrane AC (tmAC) and soluble AC (sAC) play important roles in sperm motility in Ciona. The tmAC plays a major role in the SAAF-induced activation of sperm motility. On the other hand, sAC is involved in the regulation of flagellar beat frequency and the Ca2+-dependent chemotactic movement of sperm. In this study, we focused on the role of sAC in the regulation of flagellar motility in Ciona sperm chemotaxis. The immunochemical analysis revealed that several isoforms of sAC protein were expressed in Ciona sperm, as reported in mammals and sea urchins. We demonstrated that sAC inhibition caused strong and transient asymmetrization during the chemotactic turn, and then sperm failed to turn toward the SAAF. In addition, real-time Ca2+ imaging in sperm flagella revealed that sAC inhibition induced an excessive and prolonged Ca2+ influx to flagella. These results indicate that sAC plays a key role in sperm chemotaxis by regulating the clearance of [Ca2+]i and by modulating Ca2+-dependent flagellar waveform conversion.

Insect Cell-Based Expression of Cytoskeletal Motor Proteins for Single-Molecule Studies.

Cytoskeletal motor proteins are essential molecular machines that hydrolyze ATP to generate force and motion along cytoskeletal filaments. Members of the dynein and kinesin superfamilies play critical roles in transporting biological payloads (such as proteins, organelles, and vesicles) along microtubule pathways, cause the beating of flagella and cilia, and act within the mitotic and meiotic spindles to segregate replicated chromosomes to progeny cells. Understanding the underlying mechanisms and behaviors of motor proteins is critical to provide better strategies for the treatment of motor protein-related diseases. Here, we provide detailed protocols for the recombinant expression of the Kinesin-1 motor KIF5C using a baculovirus/insect cell system and provide updated protocols for performing single-molecule studies using total internal reflection fluorescence microscopy and optical tweezers to study the motility and force generation of the purified motor.

The reaction-diffusion basis of animated patterns in eukaryotic flagella

The flagellar beat of bull spermatozoa and C. Reinhardtii are modelled by a minimal, geometrically exact, reaction-diffusion system. Spatio-temporal animated patterns describe flagellar waves, analogous to chemical-patterns from classical reaction-diffusion systems, with sliding-controlled molecular motor reaction-kinetics. The reaction-diffusion system is derived from first principles as a consequence of the high-internal dissipation by the flagellum relative to the external hydrodynamic dissipation. Quantitative comparison with nonlinear, large-amplitude simulations shows that animated reaction-diffusion patterns account for the experimental beating of both bull sperm and C. Reinhardtii. Our results suggest that a unified mechanism may exist for motors controlled by sliding, without requiring curvature-sensing, and uninfluenced by hydrodynamics. High-internal dissipation instigates autonomous travelling waves independently of the external fluid, enabling progressive swimming, otherwise not possible, in low viscosity environments, potentially critical for external fertilizers and aquatic microorganisms. The reaction-diffusion system may prove a powerful tool for studying pattern formation of movement on animated structures.

CCDC176 stabilizes microtubule doublets 1 and 9 to ensure proper sperm movement

The molecular mechanism underlying asymmetric axonemal complexes in sperm flagella is still largely unknown. Here, we showed that the knockout of the coiled-coil domain-containing 176 (CCDC176) in mice led to male infertility due to decreased sperm motility. Ccdc176 knockout specifically destabilized microtubule doublets (MTDs) 1 and 9 during sperm maturation in the corpus epididymis. Single-sperm immunofluorescence showed that most CCDC176 was distributed along the axoneme, and further super-resolution imaging revealed that CCDC176 is asymmetrically localized in the sperm axoneme. CCDC176 could cooperate with microtubule and radial spoke proteins to stabilize MTDs 1 and 9, and its knockout results in the destabilization of some proteins in sperm flagella. Furthermore, as predicted by the sperm multibody dynamics (MBD) model, we found that MTDs 1 and 9 jutted out from the sperm flagellum annulus region in Ccdc176-/- spermatozoa, and these flagellar defects alter sperm flagellar beat patterns and swimming paths, potentially owing to the reduction and disequilibration of bending torque on the central pair. These results demonstrate that CCDC176 specifically stabilizes MTDs 1 and 9 in the sperm flagellum to ensure proper sperm movement for fertilization.

Fast detection of slender bodies in high density microscopy data

Computer-aided analysis of biological microscopy data has seen a massive improvement with the utilization of general-purpose deep learning techniques. Yet, in microscopy studies of multi-organism systems, the problem of collision and overlap remains challenging. This is particularly true for systems composed of slender bodies such as swimming nematodes, swimming spermatozoa, or the beating of eukaryotic or prokaryotic flagella. Here, we develop a end-to-end deep learning approach to extract precise shape trajectories of generally motile and overlapping slender bodies. Our method works in low resolution settings where feature keypoints are hard to define and detect. Detection is fast and we demonstrate the ability to track thousands of overlapping organisms simultaneously. While our approach is agnostic to area of application, we present it in the setting of and exemplify its usability on dense experiments of swimming Caenorhabditis elegans. The model training is achieved purely on synthetic data, utilizing a physics-based model for nematode motility, and we demonstrate the model’s ability to generalize from simulations to experimental videos.

Versatile properties of dynein molecules underlying regulation in flagellar oscillation

Dynein is a minus-end-directed motor that generates oscillatory motion in eukaryotic flagella. Cyclic beating, which is the most significant feature of a flagellum, occurs by sliding spatiotemporal regulation by dynein along microtubules. To elucidate oscillation generated by dynein in flagellar beating, we examined its mechanochemical properties under three different axonemal dissection stages. By starting from the intact 9 + 2 structure, we reduced the number of interacting doublets and determined three parameters, namely, the duty ratio, dwell time and step size, of the generated oscillatory forces at each stage. Intact dynein molecules in the axoneme, doublet bundle and single doublet were used to measure the force with optical tweezers. The mean forces per dynein determined under three axonemal conditions were smaller than the previously reported stall forces of axonemal dynein; this phenomenon suggests that the duty ratio is lower than previously thought. This possibility was further confirmed by an in vitro motility assay with purified dynein. The dwell time and step size estimated from the measured force were similar. The similarity in these parameters suggests that the essential properties of dynein oscillation are inherent to the molecule and independent of the axonemal architecture, composing the functional basis of flagellar beating.

Radial spoke protein 9 is necessary for axoneme assembly in Plasmodium but not in trypanosomatid parasites.

Flagella are important for eukaryote cell motility, including in sperm, and are vital for life cycle progression of many unicellular eukaryotic pathogens. The "9+2" axoneme in most motile flagella comprises nine outer doublet and two central-pair singlet microtubules. T-shaped radial spokes protrude from the outer doublets towards the central pair and are necessary for effective beating. We asked if there were radial spoke adaptations associated with parasite lineage-specific properties in apicomplexans and trypanosomatids. Following an orthologue search for experimentally uncharacterised radial spoke proteins (RSPs), we identified and analysed RSP9. Trypanosoma brucei and Leishmania mexicana, have an extensive RSP complement including two divergent RSP9 orthologs, necessary for flagellar beating and swimming. Detailed structural analysis showed that neither ortholog is needed for axoneme assembly in Leishmania. In contrast, Plasmodium has a reduced set of RSPs including a single RSP9 ortholog. deletion of which in Plasmodium berghei leads to failure of axoneme formation, failed male gamete release, greatly reduced fertilisation and inefficient life cycle progression in the mosquito. This indicates contrasting selection pressures on axoneme complexity, likely linked with the different mode of assembly of trypanosomatid versus Plasmodium flagella.

Flagellum-driven cargoes: Influence of cargo size and the flagellum-cargo attachment geometry.

The beating of cilia and flagella, which relies on an efficient conversion of energy from ATP-hydrolysis into mechanical work, offers a promising way to propel synthetic cargoes. Recent experimental realizations of such micro-swimmers, in which micron-sized beads are propelled by isolated and demembranated flagella from the green algae Chlamydomonas reinhardtii (C. reinhardtii), revealed a variety of propulsion modes, depending in particular on the calcium concentration. Here, we investigate theoretically and numerically the propulsion of a bead as a function of the flagellar waveform and the attachment geometries between the bead and the flagellum. To this end, we take advantage of the low Reynolds number of the fluid flows generated by the micro-swimmer, which allows us to neglect fluid inertia. By describing the flagellar waveform as a superposition of a static component and a propagating wave, and using resistive-force theory, we show that the asymmetric sideways attachment of the flagellum to the bead makes a contribution to the rotational velocity of the micro-swimmer that is comparable to the contribution caused by the static component of the flagellar waveform. Remarkably, our analysis reveals the existence of a counter-intuitive propulsion regime in which an increase in the size of the cargo, and hence its drag, leads to an increase in some components of the velocity of the bead. Finally, we discuss the relevance of the uncovered mechanisms for the fabrication of synthetic, bio-actuated medical micro-robots for targeted drug delivery.

Axonemal regulation by curvature explains sperm flagellar waveform modulation.

Flagellar motility is critical to natural and many forms of assisted reproduction. Rhythmic beating and wave propagation by the flagellum propels sperm through fluid and enables modulation between penetrative progressive motion, activated side-to-side yaw and hyperactivated motility associated with detachment from epithelial binding. These motility changes occur in response to the properties of the surrounding fluid environment, biochemical activation state, and physiological ligands, however, a parsimonious mechanistic explanation of flagellar beat generation that can explain motility modulation is lacking. In this paper, we present the Axonemal Regulation of Curvature, Hysteretic model, a curvature control-type theory based on switching of active moment by local curvature, embedded within a geometrically nonlinear elastic model of the flagellum exhibiting planar flagellar beats, together with nonlocal viscous fluid dynamics. The biophysical system is parameterized completely by four dimensionless parameter groupings. The effect of parameter variation is explored through computational simulation, revealing beat patterns that are qualitatively representative of penetrative (straight progressive), activated (highly yawing) and hyperactivated (nonprogressive) modes. Analysis of the flagellar limit cycles and associated swimming velocity reveals a cusp catastrophe between progressive and nonprogressive modes, and hysteresis in the response to changes in critical curvature parameter. Quantitative comparison to experimental data on human sperm exhibiting typical penetrative, activated and hyperactivated beats shows a good fit to the time-average absolute curvature profile along the flagellum, providing evidence that the model is capable of providing a framework for quantitative interpretation of imaging data.

Spokes and barrels tune the asymmetric beating of mammalian sperm flagella.

Spokes and barrels tune the asymmetric beating of mammalian sperm flagella.

The Intracellular Amastigote of Trypanosoma cruzi Maintains an Actively Beating Flagellum.

Throughout its complex life cycle, the uniflagellate parasitic protist, Trypanosoma cruzi, adapts to different host environments by transitioning between elongated motile extracellular stages and a nonmotile intracellular amastigote stage that replicates in the cytoplasm of mammalian host cells. Intracellular T. cruzi amastigotes retain a short flagellum that extends beyond the opening of the flagellar pocket with access to the extracellular milieu. Contrary to the long-held view that the T. cruzi amastigote flagellum is inert, we report that this organelle is motile and displays quasiperiodic beating inside mammalian host cells. Kymograph analysis determined an average flagellar beat frequency of ~0.7 Hz for intracellular amastigotes and similar beat frequencies for extracellular amastigotes following their isolation from host cells. Inhibitor studies reveal that flagellar motility in T. cruzi amastigotes is critically dependent on parasite mitochondrial oxidative phosphorylation. These novel observations reveal that flagellar motility is an intrinsic property of T. cruzi amastigotes and suggest that this organelle may play an active role in the parasite infection process. IMPORTANCE Understanding the interplay between intracellular pathogens and their hosts is vital to the development of new treatments and preventive strategies. The intracellular "amastigote" stage of the Chagas disease parasite, Trypanosoma cruzi, is a critical but understudied parasitic life stage. Previous work established that cytosolically localized T. cruzi amastigotes engage physically and selectively with host mitochondria using their short, single flagellum. The current study was initiated to examine the dynamics of the parasite flagellum-host mitochondrial interaction through live confocal imaging and led to the unexpected discovery that the T. cruzi amastigote flagellum is motile.

Pathogenic gene variants in CCDC39, CCDC40, RSPH1, RSPH9, HYDIN, and SPEF2 cause defects of sperm flagella composition and male infertility.

Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder affecting the function of motile cilia in several organ systems. In PCD, male infertility is caused by defective sperm flagella composition or deficient motile cilia function in the efferent ducts of the male reproductive system. Different PCD-associated genes encoding axonemal components involved in the regulation of ciliary and flagellar beating are also reported to cause infertility due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we performed genetic testing by next generation sequencing techniques, PCD diagnostics including immunofluorescence-, transmission electron-, and high-speed video microscopy on sperm flagella and andrological work up including semen analyses. We identified ten infertile male individuals with pathogenic variants in CCDC39 (one) and CCDC40 (two) encoding ruler proteins, RSPH1 (two) and RSPH9 (one) encoding radial spoke head proteins, and HYDIN (two) and SPEF2 (two) encoding CP-associated proteins, respectively. We demonstrate for the first time that pathogenic variants in RSPH1 and RSPH9 cause male infertility due to sperm cell dysmotility and abnormal flagellar RSPH1 and RSPH9 composition. We also provide novel evidence for MMAF in HYDIN- and RSPH1-mutant individuals. We show absence or severe reduction of CCDC39 and SPEF2 in sperm flagella of CCDC39- and CCDC40-mutant individuals and HYDIN- and SPEF2-mutant individuals, respectively. Thereby, we reveal interactions between CCDC39 and CCDC40 as well as HYDIN and SPEF2 in sperm flagella. Our findings demonstrate that immunofluorescence microscopy in sperm cells is a valuable tool to identify flagellar defects related to the axonemal ruler, radial spoke head and the central pair apparatus, thus aiding the diagnosis of male infertility. This is of particular importance to classify the pathogenicity of genetic defects, especially in cases of missense variants of unknown significance, or to interpret HYDIN variants that are confounded by the presence of the almost identical pseudogene HYDIN2.

Hexane fraction of Prunus japonica thunb. Seed extract enhances boar sperm motility via CatSper ion channel

IntroductionMammalian sperm motility is facilitated by flagellar beating, which depends on active ion movement through ion channels and their regulation. Prunus japonica Thunb., also known as oriental bush cherry, is a widely used traditional medicinal plant. However, its significance in improving fertility and sperm quality has not been fully elucidated yet. One of our previous reports revealed that P. japonica seed extract (PJE) can improve human sperm motility through intracellular pH modulation. Aim of the studyThe present study was designed to investigate the effects of PJE on boar spermatozoa and potential underlying mechanisms. Materials and methodsSperm motility changes were examined using a computer-assisted sperm analysis (CASA) system under both capacitated and non-capacitated conditions. Intracellular calcium concentration was measured using either confocal microscopy or a fluorescent microplate reader with Fluo-4AM calcium fluorescent dye. Sperm capacitation-related proteins were analyzed using western blotting. ResultsA significant increase in rapid motility, velocity, and linear displacement of sperm was observed in PJE-treated capacitated boar sperm, whereas the effect was insignificant in the non-capacitated counterparts. Intracellular calcium levels were significantly elevated upon PJE treatment (20–100 μg/L) in a concentration-dependent manner. The increase in intracellular calcium levels was inhibited when the sperm were treated with a CatSper (cation channel of sperm) channel inhibitor, 10 μM Mibefradil, indicating the involvement of the ion channel in the PJE modulatory mechanism. In addition, western blotting revealed an increased level of protein phosphorylation (p-tyrosine and p-PKA), which is a hallmark of sperm capacitation. ConclusionsPJE treatment resulted in a combination of increased motility, intracellular calcium concentration, and capacitation, thereby indicating its potential to ameliorate sperm motility parameters and induce capacitation of boar spermatozoa as a result of intracellular calcium elevation via the CatSper channel. Our observations further elaborate ion channel-related underlying mechanisms and show putative implications of the seed extract of traditionally used P. japonica Thunb. in ameliorating sperm quality.

Action at a distance along the microtubule couples kinesin motors.

Coupling of kinesin motors drives flagellar beating and chromosome segregation, among other important biophysical processes. Current models of motor coupling invoke either direct mechanical linkage or protein crowding, which rely on short-range motor-motor interactions. In contrast, coupling mechanisms that act at longer length scales remain largely unexplored. We found that microtubules can physically couple motor movement in the absence of detectable short-range interactions. This occurs for the human kinesin-4 Kif4A, which contributes to the stability of antiparallel microtubule overlaps in the mitotic spindle. In a reconstituted system, Kif4A changes the run length and velocity of other motors on the same microtubule in the dilute binding limit, when approximately 10-nm-sized motors are much farther apart than the motor size. This effect does not depend on specific motor-motor interactions because similar changes in Kif4A motility are induced by kinesin-1 motors. A micrometer-scale attractive interaction potential between motors is sufficient to recreate the experimental results in a biophysical model. Unexpectedly, our theory suggests that long-range microtubule-mediated coupling affects not only binding kinetics but also motor mechanochemistry. Therefore, the model predicts that motors can sense and respond to motors bound several micrometers away on a microtubule. Our results are consistent with a paradigm in which long-range motor interactions along the microtubule enable additional forms of collective motor behavior, possibly due to changes in the microtubule lattice.

Phototaxis of Chlamydomonas arises from a tuned adaptive photoresponse shared with multicellular Volvocine green algae.

A fundamental issue in biology is the nature of evolutionary transitions from unicellular to multicellular organisms. Volvocine algae are models for this transition, as they span from the unicellular biflagellate Chlamydomonas to multicellular species of Volvox with up to 50,000 Chlamydomonas-like cells on the surface of a spherical extracellular matrix. The mechanism of phototaxis in these species is of particular interest since they lack a nervous system and intercellular connections; steering is a consequence of the response of individual cells to light. Studies of Volvox and Gonium, a 16-cell organism with a plate-like structure, have shown that the flagellar response to changing illumination of the cellular photosensor is adaptive, with a recovery time tuned to the rotation period of the colony around its primary axis. Here, combining high-resolution studies of the flagellar photoresponse of micropipette-held Chlamydomonas with 3D tracking of freely swimming cells, we show that such tuning also underlies its phototaxis. A mathematical model is developed based on the rotations around an axis perpendicular to the flagellar beat plane that occur through the adaptive response to oscillating light levels as the organism spins. Exploiting a separation of timescales between the flagellar photoresponse and phototurning, we develop an equationof motion that accurately describes the observed photoalignment. In showing that the adaptive timescales in Volvocine algae are tuned to the organisms' rotational periods across three orders of magnitude in cell number, our results suggest a unified picture of phototaxis in green algae in which the asymmetry in torques that produce phototurns arise from the individual flagella of Chlamydomonas, the flagellated edges of Gonium, and the flagellated hemispheres of Volvox.

Testis-specific proteins, TSNAXIP1 and 1700010I14RIK, are important for sperm motility and male fertility in mice.

TSN (translin), also called testis brain RNA-binding protein, binds to TSNAX (translin-associated factor X) and is suggested to play diverse roles, such as RNA metabolism and DNA damage response. TSNAXIP1 (Translin-associated factor X-interacting protein 1) was identified as a TSNAX-interacting protein using a yeast two-hybrid system, but its function in vivo was unknown. To reveal the function of TSNAXIP1 in vivo in mice. We generated Tsnaxip1 knockout mice using the CRISPR/Cas9 system and analyzed their fertility and sperm motility. Further, we generated 1700010I14Rik knockout mice, because 1700010I14RIK is also predominantly expressed in testes and contains the same Pfam (protein families) domain as TSNAXIP1. Reduced male fertility and impaired sperm motility with asymmetric flagellar waveforms were observed in not only Tsnaxip1 but also 1700010I14Rik knockout mice. Unlike Tsn knockout mice, no abnormalities were found in testicular sections of either Tsnaxip1 or 1700010I14Rik knockout mice. Furthermore, TSNAXIP1 was detected in the sperm tail and fractionated with axonemal proteins. Unlike the TSN-TSNAX complex, whose disruption causes abnormal vacuoles in mouse testes, TSNAXIP1 and 1700010I14RIK may play roles in regulating sperm flagellar beating patterns.

Locomotion of bovine spermatozoa during the transition from individual cells to bundles

Various locomotion strategies employed by microorganisms are observed in complex biological environments. Spermatozoa assemble into bundles to improve their swimming efficiency compared to individual cells. However, the dynamic mechanisms for the formation of sperm bundles have not been fully characterized. In this study, we numerically and experimentally investigate the locomotion of spermatozoa during the transition from individual cells to bundles of two cells. Three consecutive dynamic behaviors are found across the course of the transition: hydrodynamic attraction/repulsion, alignment, and synchronization. The hydrodynamic attraction/repulsion depends on the relative orientation and distance between spermatozoa as well as their flagellar wave patterns and phase shift. Once the heads are attached, we find a stable equilibrium of the rotational hydrodynamics resulting in the alignment of the heads. The synchronization results from the combined influence of hydrodynamic and mechanical cell-to-cell interactions. Additionally, we find that the flagellar beat is regulated by the interactions during the bundle formation, whereby spermatozoa can synchronize their beats to enhance their swimming velocity.