Rotational motion and rheotaxis of human sperm do not require functional CatSper channels and transmembrane Ca 2+ signaling
2020; Springer Nature; Volume: 39; Issue: 4 Linguagem: Inglês
10.15252/embj.2019102363
ISSN1460-2075
AutoresChristian Schiffer, Steffen Rieger, Christoph Brenker, Samuel Young, Hussein Hamzeh, Dagmar Wachten, Frank Tüttelmann, Albrecht Röpke, U. Benjamin Kaupp, Tao Wang, Alice Wagner, Claudia Krallmann, Sabine Kliesch, Carsten Fallnich, Timo Strünker,
Tópico(s)Plant Reproductive Biology
ResumoArticle19 January 2020Open Access Transparent process Rotational motion and rheotaxis of human sperm do not require functional CatSper channels and transmembrane Ca2+ signaling Christian Schiffer Christian Schiffer Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Search for more papers by this author Steffen Rieger Steffen Rieger Optical Technologies Group, Institute of Applied Physics, University of Münster, Münster, Germany Search for more papers by this author Christoph Brenker Christoph Brenker Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Search for more papers by this author Samuel Young Samuel Young Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Search for more papers by this author Hussein Hamzeh Hussein Hamzeh orcid.org/0000-0002-3056-788X Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany Search for more papers by this author Dagmar Wachten Dagmar Wachten Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany Institute of Innate Immunity, University Hospital, University of Bonn, Bonn, Germany Search for more papers by this author Frank Tüttelmann Frank Tüttelmann Institute of Human Genetics, University of Münster, Münster, Germany Search for more papers by this author Albrecht Röpke Albrecht Röpke Institute of Human Genetics, University of Münster, Münster, Germany Search for more papers by this author U Benjamin Kaupp U Benjamin Kaupp orcid.org/0000-0002-0696-6397 Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany Search for more papers by this author Tao Wang Tao Wang Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Institute of Life Science and School of Life Science, Nanchang University, Nanchang, China Search for more papers by this author Alice Wagner Alice Wagner Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Institute of Human Genetics, University of Münster, Münster, Germany Search for more papers by this author Claudia Krallmann Claudia Krallmann Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Search for more papers by this author Sabine Kliesch Sabine Kliesch Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Search for more papers by this author Carsten Fallnich Corresponding Author Carsten Fallnich [email protected] orcid.org/0000-0002-2837-0615 Optical Technologies Group, Institute of Applied Physics, University of Münster, Münster, Germany Cells-in-Motion Cluster of Excellence (EXC1003-CiM), Münster, Germany Search for more papers by this author Timo Strünker Corresponding Author Timo Strünker [email protected] orcid.org/0000-0003-0812-1547 Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Cells-in-Motion Cluster of Excellence (EXC1003-CiM), Münster, Germany Search for more papers by this author Christian Schiffer Christian Schiffer Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Search for more papers by this author Steffen Rieger Steffen Rieger Optical Technologies Group, Institute of Applied Physics, University of Münster, Münster, Germany Search for more papers by this author Christoph Brenker Christoph Brenker Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Search for more papers by this author Samuel Young Samuel Young Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Search for more papers by this author Hussein Hamzeh Hussein Hamzeh orcid.org/0000-0002-3056-788X Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany Search for more papers by this author Dagmar Wachten Dagmar Wachten Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany Institute of Innate Immunity, University Hospital, University of Bonn, Bonn, Germany Search for more papers by this author Frank Tüttelmann Frank Tüttelmann Institute of Human Genetics, University of Münster, Münster, Germany Search for more papers by this author Albrecht Röpke Albrecht Röpke Institute of Human Genetics, University of Münster, Münster, Germany Search for more papers by this author U Benjamin Kaupp U Benjamin Kaupp orcid.org/0000-0002-0696-6397 Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany Search for more papers by this author Tao Wang Tao Wang Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Institute of Life Science and School of Life Science, Nanchang University, Nanchang, China Search for more papers by this author Alice Wagner Alice Wagner Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Institute of Human Genetics, University of Münster, Münster, Germany Search for more papers by this author Claudia Krallmann Claudia Krallmann Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Search for more papers by this author Sabine Kliesch Sabine Kliesch Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Search for more papers by this author Carsten Fallnich Corresponding Author Carsten Fallnich [email protected] orcid.org/0000-0002-2837-0615 Optical Technologies Group, Institute of Applied Physics, University of Münster, Münster, Germany Cells-in-Motion Cluster of Excellence (EXC1003-CiM), Münster, Germany Search for more papers by this author Timo Strünker Corresponding Author Timo Strünker [email protected] orcid.org/0000-0003-0812-1547 Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany Cells-in-Motion Cluster of Excellence (EXC1003-CiM), Münster, Germany Search for more papers by this author Author Information Christian Schiffer1, Steffen Rieger2, Christoph Brenker1, Samuel Young1, Hussein Hamzeh3, Dagmar Wachten4,5, Frank Tüttelmann6, Albrecht Röpke6, U Benjamin Kaupp3, Tao Wang1,7, Alice Wagner1,6, Claudia Krallmann1, Sabine Kliesch1, Carsten Fallnich *,2,8 and Timo Strünker *,1,8 1Centre of Reproductive Medicine and Andrology, University Hospital Münster, University of Münster, Münster, Germany 2Optical Technologies Group, Institute of Applied Physics, University of Münster, Münster, Germany 3Molecular Sensory Systems, Center of Advanced European Studies and Research, Bonn, Germany 4Minerva Max Planck Research Group, Molecular Physiology, Center of Advanced European Studies and Research, Bonn, Germany 5Institute of Innate Immunity, University Hospital, University of Bonn, Bonn, Germany 6Institute of Human Genetics, University of Münster, Münster, Germany 7Institute of Life Science and School of Life Science, Nanchang University, Nanchang, China 8Cells-in-Motion Cluster of Excellence (EXC1003-CiM), Münster, Germany *Corresponding author. Tel: +49 251 83 36160; E-mail: [email protected] *Corresponding author. Tel: +49 251 83 58238; E-mail: [email protected] The EMBO Journal (2020)39:e102363https://doi.org/10.15252/embj.2019102363 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Navigation of sperm in fluid flow, called rheotaxis, provides long-range guidance in the mammalian oviduct. The rotation of sperm around their longitudinal axis (rolling) promotes rheotaxis. Whether sperm rolling and rheotaxis require calcium (Ca2+) influx via the sperm-specific Ca2+ channel CatSper, or rather represent passive biomechanical and hydrodynamic processes, has remained controversial. Here, we study the swimming behavior of sperm from healthy donors and from infertile patients that lack functional CatSper channels, using dark-field microscopy, optical tweezers, and microfluidics. We demonstrate that rolling and rheotaxis persist in CatSper-deficient human sperm. Furthermore, human sperm undergo rolling and rheotaxis even when Ca2+ influx is prevented. Finally, we show that rolling and rheotaxis also persist in mouse sperm deficient in both CatSper and flagellar Ca2+-signaling domains. Our results strongly support the concept that passive biomechanical and hydrodynamic processes enable sperm rolling and rheotaxis, rather than calcium signaling mediated by CatSper or other mechanisms controlling transmembrane Ca2+ flux. Synopsis Mammalian sperm navigation in the oviductal fluid is considered to depend on calcium signaling via the CatSper channel. Here, newly-developed live imaging microscopy of trapped sperm suggests that passive biomechanical processes, rather than channel-mediated ion fluxes instruct sperm motility. Rolling and rheotaxis persist in CatSper-deficient human sperm from infertile patients. Sperm display continuous full 360° rotations, rather than incomplete rotations. Calcium influx is not required for sperm movements. Rolling and rheotaxis persist in CatSper-deficient mouse sperm. Introduction In fluid flow, mammalian sperm realign their swimming path and move upstream—a mechanism called rheotaxis (Miki & Clapham, 2013; El-Sherry et al, 2014; Kantsler et al, 2014; Tung et al, 2014, 2015; Bukatin et al, 2015). In the oviduct, long-range navigation via rheotaxis directs sperm to the site of fertilization (Miki & Clapham, 2013). An important ingredient of rheotaxis is the rotation of sperm around their longitudinal axis, called rolling (e.g., Miki & Clapham, 2013; Kantsler et al, 2014; Bukatin et al, 2015), resulting in a cone-shaped beating envelope. Through this mechanism, vertical shear flow, e.g., near boundary surfaces, exerts a torque that aligns the longitudinal axis of sperm against the flow direction (Miki & Clapham, 2013; Kantsler et al, 2014; Bukatin et al, 2015). However, whether sperm rolling involves full 360° or incomplete rotations of alternating direction is debated (Miki & Clapham, 2013; Muschol et al, 2018). The intracellular Ca2+ concentration ([Ca2+]i) controls the flagellar beat and swimming behavior of sperm (Kaupp et al, 2003; Publicover et al, 2008; Fechner et al, 2015). In most sperm species, [Ca2+]i is set by the voltage- and alkaline-activated CatSper Ca2+ channel (Quill et al, 2001; Ren et al, 2001; Kirichok et al, 2006; Lishko et al, 2010; Lishko et al, 2011; Strünker et al, 2011; Loux et al, 2013; Seifert et al, 2015). Mammalian CatSper comprises four homologous pore-forming subunits (CatSper 1–4) (e.g., Navarro et al, 2008) and at least six auxiliary subunits (CatSper β, γ, δ, ɛ, ζ, and Efcab9) (Liu et al, 2007; Wang et al, 2009; Chung et al, 2011, 2017; Hwang et al, 2019). The CatSper-channel complex is organized as quadrilateral threads along the flagellum; the CatSper threads encompass several other proteins, including Ca2+-binding proteins and protein kinases, forming local Ca2+-signaling domains near the membrane surface (Chung et al, 2014, 2017). In Catsper1−/− mouse sperm, longitudinal rolling and rheotaxis were abolished (Miki & Clapham, 2013), suggesting that control of [Ca2+]i by CatSper is required for rolling and rheotaxis of mammalian sperm. For human sperm, it was specifically proposed that rolling is created by asymmetrical Ca2+ influx via CatSper channels, stimulated by local pHi signaling (Miller et al, 2018). The H+ channel Hv1 is organized along the flagellum of human sperm in two threads near two of the four CatSper threads (Miller et al, 2018). It was proposed that H+ efflux via Hv1 organizes localized Ca2+ signaling that, ultimately, creates an asymmetry in calcium-dependent inhibition of dynein-powered microtubule sliding (Miller et al, 2018). However, the concept that rolling and rheotaxis are enabled by Ca2+ influx cannot be reconciled with the finding that rolling of mouse sperm does not require extracellular Ca2+ (Babcock et al, 2014; Muschol et al, 2018), and that exposure of human sperm to gradients of flow velocities does not evoke measurable changes in [Ca2+]i (Zhang et al, 2016). Moreover, the inventory and regulation of signaling molecules are different among mammalian sperm (Kaupp & Strünker, 2017). For example, mouse sperm lack Hv1 channels (Lishko et al, 2010; Berger et al, 2017), and human CatSper is activated by nanomolar concentrations of prostaglandins and progesterone (Lishko et al, 2011; Strünker et al, 2011) that do not activate mouse CatSper (Lishko et al, 2011). Thus, if the quadrilateral arrangement of CatSper and its control by pHi were key to rolling and rheotaxis of mouse and human sperm, the underlying mechanisms ought to be vastly different. Here, we show that human sperm undergo continuous full 360° rotations rather than incomplete rotations of alternating directions. Moreover, to scrutinize the role of CatSper and Ca2+ in rolling and rheotaxis of human sperm, we studied sperm of healthy donors and patients who suffer from the deafness-infertility syndrome (DIS). DIS patients lack the CATSPER2 gene (Zhang et al, 2007; Hildebrand et al, 2010). We show by 3D-STORM that, in the absence of CatSper 2, other pore-forming CatSper subunits still assemble into quadrilateral threads of non-functional CatSper complexes. We demonstrate that rolling and rheotaxis persist in CatSper-deficient sperm from DIS patients. Furthermore, we show that rolling and rheotaxis of human sperm are preserved even when Ca2+ influx is completely abolished. Finally, we demonstrate that rolling and rheotaxis are also preserved in Catsper1−/− mouse sperm, which lack the CatSper complex and the quadrilateral threads altogether. We conclude that in mouse and human sperm, neither Ca2+ influx via CatSper nor the quadrilateral Ca2+-signaling threads organized by CatSper are required for rolling and rheotaxis. Results The expression of pore-forming CatSper subunits is not strictly interdependent We examined sperm from five infertile patients suffering from a homozygous deletion of contiguous genes on chromosome 15, including the CATSPER2 gene (Fig EV1). This deletion at 15q15.3 is the hallmark of DIS (Zhang et al, 2007; Hildebrand et al, 2010). Motile sperm isolated from patients' ejaculates by the swim-up procedure were morphologically normal, but lacked CatSper-mediated Ca2+ influx (Fig 1A) and CatSper currents (Fig 1B and C), confirming that the deletion of the CATSPER2 gene abrogates the expression of functional CatSper channels (Smith et al, 2013; Brenker et al, 2018a). Antibodies directed against CatSper 3 and CatSper 4 stained the principal piece of sperm from healthy donors and DIS patients (Fig 1D and E). 3D-STORM analysis revealed that the quadrilateral arrangement of CatSper 3 and CatSper 4 along the flagellum was preserved in DIS patients (Fig 1F and G). Thus, in the absence of CatSper 2, CatSper 3 and CatSper 4 subunits still assemble into non-functional protein complexes, whose sub-cellular arrangement is similar to that of the functional CatSper-channel complex (Chung et al, 2014, 2017). Click here to expand this figure. Figure EV1. Array comparative genomic hybridization analysis (array CGH)Array CGH analysis (e.g., Tüttelmann et al, 2011) of DNA copy number variants (CNV) in a healthy donor (WT) and the five patients (CATSPER2−/− 1–5) suffering from the deafness-infertility syndrome. The black squares represent the fluorescence intensity ratios (log2) upon cohybridization of fluorescence-labeled genomic DNA fragments of the healthy donor or patients and controls (reference). Gene dosage variations are indicated by a fluorescence ratio ≠ 0. A ratio of < −1.5 indicates a homozygous deletion. Download figure Download PowerPoint Figure 1. Characterization of CATSPER2-deficient human sperm A. Representative Ca2+ signals in sperm from a patient with deafness-infertility syndrome lacking functional CatSper channels (CATSPER2−/−; red) and a healthy donor (black), evoked by progesterone, PGE1, NH4Cl, or ionomycin. NH4Cl increases the intracellular pH. Bar graph: Amplitudes (n = 4; mean ± SD) of Ca2+ signals in CATSPER2−/− sperm. B. Representative monovalent CatSper currents in CATSPER2−/− sperm (blue, green, orange, purple, brown) and in sperm from a healthy donor (black), and corresponding current-voltage relationship (right). The membrane voltage was stepped from −100 to +100 mV in increments of 10 mV from a holding potential of −80 mV. C. Outward and inward current amplitudes (mean ± SD) at + 100 mV and -100 mV, respectively, in CATSPER2−/− sperm (color code: panel B) and sperm from healthy donors (black). D, E. Representative immunocytochemical staining of control sperm from healthy donors and CATSPER2−/− sperm from DIS patients using antibodies directed against CatSper 3 (D) or CatSper 4 (E); DNA was labeled with DAPI (blue). Scale bars represent 10 μm. F. 3D-STORM image in xy projection of sperm from a healthy donor labeled with the anti-CatSper 3 antibody (left). Axial projection of the boxed region (right). Scale bars represent 5 μm in xy projections and 200 nm in axial projections. G. 3D-STORM images in xy projection of CATSPER2−/− sperm (left) labeled with the anti-CatSper 3 (upper panel) or anti-CatSper 4 (lower panel) antibody. Axial projection of the boxed regions (right). Scale bars represent 5 μm in xy projections and 200 nm in axial projections. Download figure Download PowerPoint Human sperm do not require functional CatSper channelsfor longitudinal rolling We examined whether longitudinal rolling is impaired or even abolished in CatSper-deficient human sperm. Under dim dark-field illumination, we monitored rolling of sperm in population via periodic changes in brightness (blinking) of the sperm heads (Fig 2A–C; Movie EV1). Semi-automated analysis of blinking events revealed the rotation frequency of each sperm cell in the field of view. In non-capacitated and capacitated control sperm from healthy donors, the rotation frequency was normally distributed (Fig 2D) with a mean value of 4.8 ± 1.5 Hz (n = 1,455) and 7.0 ± 2.2 Hz (n = 1,097), respectively (Fig 2E) (Rigler & Thyberg, 1984; Aitken et al, 1985; Miller et al, 2018). Bicarbonate (25 mM) used for capacitation stimulates cAMP synthesis (Carlson et al, 2007; Tresguerres et al, 2011; Brenker et al, 2012) and, thereby, accelerates the flagellar beat (Esposito et al, 2004; Xie et al, 2006) and rotation frequency (Miki & Clapham, 2013). The rotation frequency decreased with increasing viscosity (Fig 2F), in line with previous results (e.g., Nosrati et al, 2015; Gallagher et al, 2019). To study rolling of single sperm cells with high time resolution and for long recording times, we combined bright-field microscopy with an optical tweezer (Ashkin et al, 1986) (Fig 2G). Sperm were trapped perpendicular to the optical axis (Fig 2H, Movie EV2), and the periodic intensity fluctuations of the laser light, which was back-scattered from the cell into the microscope objective, provided a measure of the rotation frequency (Fig 2I). For optically trapped control sperm from healthy donors, the rotation frequency was constant for several tens of seconds (Fig 2I). The frequency distribution and mean frequency of trapped sperm (6.0 ± 2.1 Hz, n = 32) and freely moving sperm (7.0 ± 2.2 Hz, n = 1,097) were similar (compare Fig 2J and D). Trapping of sperm parallel to the optical axis allowed a frontal view onto the tip of the sperm head; this view reveals that human sperm display continuous full 360° rotations (Fig 2K, Movie EV3), in contrast to incomplete rotations of alternating directions that have been reported for mouse sperm (Babcock et al, 2014; Muschol et al, 2018). Remarkably, also CatSper-deficient human sperm displayed longitudinal rolling (Movie EV4): In freely moving CatSper-deficient sperm incubated under non-capacitating or capacitating conditions, the rotation frequency was normally distributed around a mean value of 6.0 ± 2.6 Hz (n = 1,009) and 6.8 ± 3.1 Hz (n = 946), respectively (Fig 2L and M). The CatSper-deficient human sperm swam progressively also in highly viscous media (Movie EV5), and, like in control sperm, the rotation frequency of CatSper-deficient sperm decreased with increasing viscosity (Fig 2N). When optically trapped, the CatSper-deficient sperm clearly displayed continuous full 360° rotations (Fig 2O and P, Movie EV6), and the rotation frequency remained constant over several tens of seconds (Fig 2Q). In conclusion, human sperm do not require functional CatSper for longitudinal rolling. If anything, the longitudinal rolling of CatSper-deficient sperm might be slightly enhanced in the absence of bicarbonate. Figure 2. Analysis of longitudinal rolling of human sperm Experimental setup for population analysis by dark-field microscopy. Dark-field microscopy of a single sperm cell; shown are single frames obtained at t = 0, 48, 96, and 136 ms. Scale bar = 25 μm. Dark-field imaging of a sperm population; left: single frames at t = 185, 363, 540, and 718 ms. Sperm selected for analysis are highlighted (1–4). Right: temporal change in the brightness (blinking) of sperm heads. The blue lines correspond to the time-points of the single frames. Scale bar = 25 μm. Representative distribution of rotation frequencies of freely swimming sperm incubated under non-capacitating (0 mM bicarbonate; black; n = 218) and capacitating (25 mM bicarbonate; red; n = 232) conditions determined by dark-field imaging. Rolling frequency (mean ± SD) of sperm incubated under non-capacitating (0 mM bicarbonate, n = 1,455; three experiments) and capacitating (25 mM bicarbonate, n = 1,097, eight experiments) conditions. Rolling frequency (mean ± SD) of freely swimming sperm in 0 (n = 1,175; five experiments), 0.2 (n = 832; three experiments), and 1% (n = 599; three experiments) methyl cellulose (w/v). Experimental setup for the laser-based optical tweezer. Bright-field images of an optically trapped sperm cell obtained at t = 0, 55, 155, and 185 ms. Scale bar represents 10 μm. Representative time-course of the rotation frequency of trapped sperm (each sperm cell is represented by a different color). Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Distribution of rotation frequencies in trapped sperm (n = 32; mean frequency ± SD 6.0 ± 2.1 Hz). Image series of a sperm cell trapped parallel to the optical axis; images were obtained at t = 0, 95, 165, and 205 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 μm. Representative distribution of rotation frequencies of freely swimming CatSper-deficient sperm incubated under non-capacitating (0 mM bicarbonate; black; n = 73) and capacitating conditions (25 mM bicarbonate; red; n = 272). Rolling frequency (mean ± SD) of freely swimming CATSPER2−/− sperm incubated under non-capacitating (0 mM bicarbonate, n = 1,009; four experiments) and capacitating (25 mM bicarbonate, n = 946; seven experiments) conditions. Rolling frequency (mean ± SD) of freely swimming CATSPER2−/− sperm in 0 (n = 457; four experiments), 0.2 (n = 389; two experiments), and 1% (n = 187; two experiments) methyl cellulose (w/v). Image series of a CATSPER2−/− sperm cell optically trapped perpendicular to the optical axis; images were obtained at t = 0, 75, 150, and 225 ms. Scale bar represents 10 μm. Image series of a CATSPER2−/− sperm cell optically trapped parallel to the optical axis; images were obtained at t = 0, 60, 195, and 320 ms. The red bar indicates the 360° rotation of the tip of the head. Scale bar represents 10 μm. Representative time courses of the rotation frequencies of optically trapped CATSPER2−/− sperm (each sperm is represented in a different color). Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Download figure Download PowerPoint Longitudinal rolling of human sperm does not require an influx of Ca2+ We further examined whether Ca2+ is required for rolling of human sperm, using both dark-field microscopy of sperm populations and optical trapping of single sperm cells. Control sperm from healthy donors held by the optical tweezer were dragged between parallel laminar flows of three different solutions (Figs 3A and EV2). This setup allows monitoring of the rotation frequency upon rapid switching of solutions. A stimulus buffer (stimulus stream) and sperm in control buffer (control stream) were separated by a barrier stream containing fluorescein in control buffer; the buffers were fed into a capillary via three inlets. The transfer from one to the other stream was monitored by changes in the fluorescence of fluorescein: When entering the barrier stream, the fluorescence rose and resumed basal values when sperm reached the stimulus stream (Fig 3B). Dragging of control sperm from healthy donors across the barrier stream was completed within ≤ 10 s (Movie EV7). Dragging itself did not affect rolling (Fig 3B and C): The mean rotation frequency before and after dragging between control buffers was 6.7 ± 2.8 Hz and 6.5 ± 2.8 Hz (n = 14), respectively. After dragging from bicarbonate-free to bicarbonate-containing buffer, the rotation frequency increased from 6.6 ± 2.9 to 11.3 ± 2.5 Hz (Fig 3D and E, n = 5). Next, the rotation frequency of trapped sperm cells before and after transition from 2 mM to ˂ 20 nM extracellular Ca2+ was studied. The rotation frequency was similar in the absence and presence of Ca2+ (5.5 ± 3.6 Hz versus 5.9 ± 3.1 Hz, n = 5; Fig 3F and G). Although in dark-field microscopy of sperm populations, the fraction of motile sperm decreased in Ca2+-free buffer with a time constant (τ) of 5.3 min (Fig 3H) (Aaberg et al, 1989; Jin et al, 2007; Torres-Flores et al, 2011), at any time-point during the decay, motile sperm were also rolling (Movie EV8). The mean rotation frequency and the rotation frequency-histogram (determined at ≤ 5 min in Ca2+-free buffer) were similar to those under control conditions (6.3 ± 1.9 Hz, n = 224; Fig 3I). These results show that Ca2+ influx is not required for rolling of human sperm. Figure 3. The action of bicarbonate and Ca2+ on longitudinal rolling of human sperm Experimental setup to subject optically trapped sperm to different conditions in a three-channel microfluidic capillary. Rotation frequency of a trapped sperm cell before and after dragging across the barrier stream. The green trace indicates the fluorescence of fluorescein included in the barrier stream. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm cells before and after dragging across the barrier stream. Rotation frequency of a trapped sperm cell before and after dragging from the control stream containing 0 mM bicarbonate into the stimulus stream containing 25 mM bicarbonate. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies of individual sperm at 0 and 25 mM bicarbonate. Rotation frequency of a trapped sperm cell in the presence and absence of extracellular Ca2+. Error bars indicate the full width at half prominence of the frequency peaks determined by the fast Fourier analysis. Paired plot of rotation frequencies in the presence and absence of extracellular Ca2+. Fraction of motile sperm (mean ± SD) in a sperm population incubated in the presence (black) and absence (at t = 0) of extracellular Ca2+ (red; n ≥ 5). Distribution of rotation frequencies in populations of freely swimming sperm in the presence (black, n = 335) and absence (red, n = 224) of extracellular Ca2+. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Optical trapping and microfluidics setup to study longitudinal rotation in human sperm A detailed description is provided in the materials and methods section. BS, beam splitter. DM, dichroic mirror. L, lens. LP, long-pass filter. M, mirror. PMT, photomultiplier tube. R, reflectivity. SP, short-pass filter. Scale bar represents 10 μm. Visualization of the laminar flow profile inside the microfluidics capillary. The center ("barrier") stream was supported with blue ink for the ease of illustration. Download figure Download PowerPoint CatSper-deficient human sperm display rheotaxis Next, we studied the swimming behavior of human sperm in a glass capillary with and without fluid flow. Sperm were tracked in the field of view, and the starting point of each track was shifted to the origin of a coordinate system (Fig 4A, C, E, G, I, K). To quantify the rheotactic behavior, we determined the angular swimming directions and plotted the mean relative frequency of sperm swimming with angular directions of 45°–135°, 135°–225°, 225°–315°, and 315°–45° in a spider plot. Under no-flow conditions, control sperm swam randomly without any
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