Two Distinct Ca²⁺ Signaling Pathways Modulate Sperm Flagellar Beating Patterns in Mice

Abstract

Hyperactivation, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization. It is characterized by asymmetrical flagellar beating and an increase of cytoplasmic Ca²⁺. We observed that some mouse sperm swimming in the oviduct produce high-amplitude pro-hook bends (bends in the direction of the hook on the head), whereas other sperm produce high-amplitude anti-hook bends. Switching direction of the major bends could serve to redirect sperm toward oocytes. We hypothesized that different Ca²⁺ signaling pathways produce high-amplitude pro-hook and anti-hook bends. In vitro, sperm that hyperactivated during capacitation (because of activation of CATSPER plasma membrane Ca²⁺ channels) developed high-amplitude pro-hook bends. The CATSPER activators procaine and 4-aminopyridine (4-AP) also induced high-amplitude pro-hook bends. Thimerosal, which triggers a Ca²⁺ release from internal stores, induced high-amplitude anti-hook bends. Activation of CATSPER channels is facilitated by a pH rise, so both Ca²⁺ and pH responses to treatments with 4-AP and thimerosal were monitored. Thimerosal triggered a Ca²⁺ increase that initiated at the base of the flagellum, whereas 4-AP initiated a rise in the proximal principal piece. Only 4-AP triggered a flagellar pH rise. Proteins were extracted from sperm for examination of phosphorylation patterns induced by Ca²⁺ signaling. Procaine and 4-AP induced phosphorylation of proteins on threonine and serine, whereas thimerosal primarily induced dephosphorylation of proteins. Tyrosine phosphorylation was unaffected. We concluded that hyperactivation, which is associated with capacitation, can be modulated by release of Ca²⁺ from intracellular stores to reverse the direction of the dominant flagellar bend and, thus, redirect sperm.

Two distinct Ca²⁺ signaling pathways alter the symmetry of mouse sperm flagellar beating.

INTRODUCTION

In the oviduct, mammalian sperm enter a state of vigorous flagellar beating known as hyperactivation. Hyperactivation is a swimming pattern characterized by high-amplitude, asymmetrical flagellar beating, resulting in circular or helical swimming trajectories when sperm are placed on glass slides [1]. This contrasts with the activated motility of sperm in the ejaculate, which is characterized by low-amplitude, symmetrical flagellar beating, resulting in a progressive and linear swimming pattern. Hyperactivation is generally considered to be a part of capacitation, which is a network of processes that sperm must undergo to be able to fertilize oocytes (for review, see [2]).

Evidence exists that hyperactivation plays various roles in the oviduct. First, sperm use hyperactivation to detach from the oviductal epithelium to escape from the oviductal storage reservoir (for review, see [3]; see also [4–7]). Second, hyperactivation provides sperm with a greater thrusting force to swim through oviductal mucus and to penetrate the viscoelastic cumulus matrix surrounding the oocyte [8–10]. Third, hyperactivation is required by sperm to penetrate the zona pellucida of the oocyte to reach and fuse with the oocyte plasma membrane [11–13].

The critical importance of hyperactivation to fertilization was confirmed with the discovery of CATSPER channels. CATSPER channels are pH-sensitive, voltage-dependent Ca²⁺ channels specific to male germ cells. CATSPER channels are located in the plasma membrane of the principal piece of the flagellum [12, 13]. CATSPER-null male mice are infertile, primarily because their sperm are unable to hyperactivate (for reviews, see [14–16]).

Even though hyperactivation assists sperm in reaching oocytes, the circumstances under which fertilization occurs suggest that hyperactivation must be modulated to enable successful fertilization. In rabbits and mice, hyperactivation begins in the lower oviduct [5, 17], and this enables mouse sperm to escape from the sperm storage reservoir [5, 7]. This region is quite far from the site of fertilization; in mice, a long length of coiled oviduct separates the reservoir from the ampulla where fertilization occurs. Furthermore, mouse oocytes are buried deeply within a large cumulus oophorus. It seems unlikely that a hyperactivated sperm could reach an oocyte without adjusting its trajectory periodically. Therefore, the flagellar beating of hyperactivated sperm very likely is modulated, at least intermittently, to redirect the sperm toward the oocyte.

Mouse sperm provide an excellent model for understanding the modulation of flagellar bending during hyperactivation, because the hook-shaped head of the sperm enables one to determine the direction of the flagellar bend. In reviewing videotapes of sperm swimming in the oviduct, we observed that some sperm produced high-amplitude pro-hook bends, whereas others produced high-amplitude anti-hook bends.

The Ca²⁺ signaling plays an important role in determining the pattern of the flagellar beat, especially during hyperactivation. Although the factors that initiate hyperactivation remain elusive, strong evidence indicates that a rise in flagellar Ca²⁺ is necessary for hyperactivation (for review, see [16]). The predominant source of Ca²⁺ is extracellular Ca²⁺, which enters the flagellum primarily through CATSPER channels (for review, see [14–16]; see also [18]). In addition, Ca²⁺ may be provided by a Ca²⁺ store in the redundant nuclear envelope (RNE) that lies at the base of the flagellum. Treatment of sperm with thimerosal [19] or thapsigargin [20], which stimulate release of Ca²⁺ from stores, raises flagellar Ca²⁺ and produces asymmetrical flagellar beating resembling hyperactivation [21]; however, preliminary observations of mouse sperm indicate that the response is actually the reverse of hyperactivation—that is, the high-amplitude bends form in the anti-hook direction [22]. We recently proposed that switching the dominant flagellar bend from the pro-hook to the anti-hook side modulates hyperactivation and redirects the path of the sperm [23]. Therefore, in the present study, we tested the hypothesis that different Ca²⁺ signaling pathways induce dominant pro-hook and dominant anti-hook bending. To do this, we compared the patterns of Ca²⁺ rise, pH rise, and protein phosphorylation in sperm treated to activate plasma membrane CATSPER channels with those of sperm treated to release Ca²⁺ from internal stores.

MATERIALS AND METHODS

Media and Chemicals

All routine chemicals and compounds were purchased from Sigma-Aldrich Co. with the exceptions noted below. A mouse sperm capacitating medium [4] was used for incubating and washing sperm. The medium consisted of 110 mM NaCl, 2.68 mM KCl, 0.36 mM NaH₂PO₄, 25 mM NaHCO₃, 25 mM Hepes (EMD Chemicals), 5.56 mM glucose, 1.0 mM pyruvic acid, 0.006% penicillin G (Na), 2.4 mM CaCl₂, and 0.49 mM MgCl₂. It also included 10 mg/ml of bovine serum albumin (BSA; EMD Chemicals) except when otherwise specified. The medium was adjusted to pH 7.6 and 290–310 mOsm/kg.

Animals

A hybrid cross (C57BL/6J × BALB/cByJ) of wild-type F₁ males aged 3–6 mo were obtained from Dr. John Schimenti (Cornell University, Department of Biomedical Sciences). The F₁ hybrids produced sperm of consistently high quality in terms of morphology and fertility, more so than outbred strains such as CD1. Mice were euthanized by CO₂ inhalation. All procedures were approved by the Institutional Animal Care and Use Committee at Cornell University.

Sperm Preparation and Hyperactivation

Sperm were obtained from freshly dissected epididymides as follows: A 100-μl droplet of medium was covered by mineral oil in a 35- × 10-mm Petri dish (Falcon), which was equilibrated in an incubator at 37°C and 5% CO₂ before use. Caudal epididymides were cleaned of fat, and then blood was gently pushed out of surface vessels. The caudal epididymides were placed under the mineral oil in the Petri dish. Several cuts were made in the coiled tubules near the vas deferens, and the emerging thick fluid containing sperm was gently pulled out of each cut using no. 5 tweezers and then transferred under the oil to the droplet of medium. Sperm were allowed to disperse for 10 min in the incubator, and samples were counted using a hemocytometer. Then, sperm were diluted with medium to 5 × 10⁶ sperm/ml and used immediately.

Immediately after dilution in medium, sperm were treated with 5 mM procaine for 4 min or with 4 mM 4-aminopyridine (4-AP; Tocris Bioscience) for 1 min [22, 24, 25] to activate CATSPER channels or with the inositol trisphosphate (IP₃) receptor agonist thimerosal (100 μM) for 30 sec [19] to stimulate release of Ca²⁺ from stores. As a negative control, medium alone was added to sperm, which were then incubated for 4 min. In addition, 500-μl aliquots of sperm were incubated in medium at 37°C under 5% CO₂ for 2 h to promote capacitation.

To examine the combined effects of the pharmacological agents, sperm that achieved maximal responses when treated with procaine or 4-AP, or when incubated under capacitating conditions for 2 h, were treated with 100 μM thimerosal for 30 sec. Sperm were also treated with thimerosal to achieve maximal response first, then treated with procaine or 4-AP as described above.

Analysis of Sperm Motility

Samples of sperm were placed on slides on a 37°C stage of a Zeiss Axiovert 35 microscope and videotaped using bright-field microscopy with a 20× objective and a Dage CCD 72 video camera (Dage-MTI, Inc.) connected to a Panasonic AG-1970 Super VHS videocassette recorder (Panasonic Industrial Co.). Playback of the videotapes was used to determine the percentages of motile sperm (i.e., sperm showing any type of flagellar activity) and of sperm swimming in various patterns. For each sample, 100–120 motile sperm were analyzed, and for each experiment, replicate tests were performed using samples from three males.

Intracellular Ca²⁺ and pH Detection

Sperm (5 × 10⁶ sperm/ml) were loaded by incubation with the Ca²⁺ indicator Fluo-4 AM (10 μM; Invitrogen) or the acetoxymethyl (AM) ester derivative of the pH-sensitive dye 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF-AM; 5 μM; Invitrogen) for 30 min in BSA-free medium. Extracellular dye was removed by centrifugation at 170 × g for 5 min. For the 4-AP treatment, Fluo-4 AM-loaded sperm were resuspended in BSA-free medium and incubated for an additional 30 min to allow de-esterification of the dye into its membrane-impermeable, Ca²⁺-sensitive form. For thimerosal treatment, dye-loaded sperm were resuspended in BSA- and Ca²⁺-free medium to produce a final concentration of 30 μM Ca²⁺ and incubated for 30 min to allow de-esterification [21]. Sperm loaded with BCECF-AM were resuspended in BSA-free medium to de-esterify for 30 min into its membrane-impermeable, pH-sensitive form. All incubations were performed in an incubator at 37°C under 5% CO₂ in humidified air.

To examine fluorescence patterns, aliquots of sperm suspension were transferred to glass-bottom wells in a 96-well plate (In Vitro Scientific). The glass bottoms had been coated with Cell-Tak (BD Biosciences), which caused sperm to adhere to the glass bottom. The fluorescence of individual sperm was monitored before and after application of treatments. The temperature was maintained at 37°C throughout each experiment. Fluorescence intensity was detected using a 480- ± 40-nm excitation filter, a 535- ± 50-nm emission filter, and a 505-nm long-pass dichroic mirror (Chroma Technology Corp.) with an oil-immersion, 100× Fluar objective (N.A. 1.3; Carl Zeiss, Inc.). Stroboscopic illumination was provided by a 75-W xenon arc flash lamp (Chadwick-Helmuth Co.). Images were captured with a sensicam em high-performance camera (The Cooke Corporation) at 4 frames/sec controlled by IPLab Spectrum software (Version 4.0; Signal Analytics). Data for each treatment were collected from 15 to 20 sperm/mouse, and the experiments were replicated three times, each time with sperm from a different mouse.

The fluorescence of pH in sperm populations was also monitored before and after treatments using a Bio-Tek Synergy 2 kinetic microplate reader. Sperm loaded with BCECF-AM were placed in wells of a prewarmed, 96-well, black-bottom plate (Thermo Fisher Scientific). Each well contained 200 000 sperm. Samples were analyzed immediately on the plate reader at an excitation wavelength of 490 nm and an emission wavelength of 535 nm. The fluorescence dynamics were recorded every 3 sec. Readings for each time point were normalized, averaged, and expressed as relative fluorescence units. The procedure was repeated using sperm from a total of three mice.

Acrosome Reaction Analysis

Coomassie blue staining [26] was used to determine if the procaine, 4-AP, or thimerosal treatments induced acrosome reactions. As a positive control, the Ca²⁺ ionophore A23187 (20 μM) was used to treat sperm for 15 min to induce acrosome reactions. Treated sperm were fixed in 4% paraformaldehyde for 10 min and washed with PBS by centrifuging at 700 × g for 3 min. The sperm pellet was resuspended in 0.1 M ammonium acetate (pH 9) and dried on microscope slides. The sperm were stained with 0.22% Brilliant Blue G for 2 min. Excess stain was rinsed off with PBS. Slides were mounted using Permount (Fisher Scientific) and examined using bright-field optics at 400×. At least 100 sperm were examined for acrosomal status for each treatment. Samples from three mice were evaluated.

One-Dimensional Gel Electrophoresis and Western Blot Analysis

Aliquots of 1 × 10⁶ sperm were treated with procaine, 4-AP, thimerosal, or medium control or were incubated under capacitating conditions as described above, and then protease (Complete, EDTA-free; Roche) and phosphatase (PhosStop; Roche) inhibitors were added and the sperm collected by centrifugation at 1900 × g for 10 min at 4°C. The sperm pellet was solubilized in an SDS lysis buffer with 5% 2-mercaptoethanol and boiled for 5 min. Lysates were centrifuged to remove insoluble material and resolved by SDS–PAGE. The resolved proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore), and then the membranes were blocked for 1 h in Tris-buffered saline containing 0.05% Tween 20 (TTBS) and 5% gelatin from cold-water fish skin (G7765; Sigma). Tyrosine-phosphorylated proteins were detected by incubating overnight at 4°C with 0.1 μg/ml of monoclonal antiphosphotyrosine antibody (05321; Millipore). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (1:10 000; Sigma) and enhanced chemiluminescence (SuperSignal West Pico; Pierce) were used to visualize antibody-labeled proteins.

Two-Dimensional Gel Electrophoresis and Western Blot Analysis

Aliquots of 5 × 10⁶ sperm were treated with procaine, 4-AP, thimerosal, or medium control or sperm were incubated under capacitating conditions as described above, and then protease and phosphatase inhibitors were added and the sperm collected by centrifugation at 1900 × g for 10 min at 4°C. The sperm pellet was solubilized in a lysis buffer containing 7 M urea (EMD Chemicals), 2 M thiourea, 1% 3-(4-Heptyl)phenyl-3-hydroxypropyl)dimethylammoniopropanesulfonate (C7BzO; Sigma), 50 mM dithiothreitol (DTT; Pierce) and 0.2% Ampholine (pH 3–10; Fluka) at room temperature for 1 h. Lysates were centrifuged to remove insoluble material.

For the first dimension of two-dimensional gel electrophoresis, IPG strips (7 cm, pH 3–10 nonlinear; Bio-Rad) were rehydrated with 125 μl of protein lysate for 14 h at 50 V and then focused at 4000 V for 2.5 h. After equilibration of the IPG strips in 125 mM Tris, 6 M urea, 10% glycerol, 2% SDS, and 4% DTT (pH 6.8), second-dimension SDS-PAGE gels were run and then transferred to PVDF membranes. Membranes were blocked for 1 h with 5% gelatin from cold-water fish skin in TTBS. Phosphoserine- and phosphothreonine-containing proteins were detected by incubating overnight at 4°C with 0.25 μg/ml (1:1000) of antiphosphoserine (AB1603; Millipore) or antiphosphothreonine (AB1607; Millipore) rabbit polyclonal antibodies diluted in TTBS containing 2% normal goat serum (EMD Chemicals). HRP-conjugated secondary antibodies (1:10 000; Sigma) and enhanced chemiluminescence were used to visualize target proteins. Blots were stripped by the Restore Western Blot stripping buffer (Thermo Scientific) and then reprobed with the next antibody. Equal protein loading was verified with MemCode Reversible Protein Stain kit (Thermo Scientific) after the Western blot analysis.

Immunocytochemistry

Sperm treated with procaine, 4-AP, thimerosal, or medium control were fixed by 1% paraformaldehyde in PBS with gentle agitation for 15 min at room temperature. Fixed sperm were collected by centrifugation at 700 × g for 3 min and then resuspended in 0.2 M glycine in PBS to quench residual fixative and agitated for 15 min at room temperature. Sperm were then either concentrated by centrifugation or diluted with PBS to give a final concentration of 5 × 10⁶ sperm/ml. Samples of 10 μl were spotted onto ethanol-cleaned microscope slides and allowed to dry at room temperature overnight. Sperm smears were permeabilized with cold methanol for 10 min and then rinsed with PBS (three times for 5 min each time). Then, sperm were blocked with 3% BSA in PBS for 1 h. Antiphosphoserine (0.5 μg/ml, 1:500) or antiphosphothreonine (0.5 μg/ml, 1:500) antibodies were added, and the slides were incubated for 1 h at room temperature. The secondary antibody, goat-anti-rabbit fluorescein isothiocyanate (F9887; Sigma), was used at a dilution of 1:200.

Statistical Analysis

In motility studies, the percentages of sperm showing dominant pro-hook and anti-hook beating patterns were normalized using arcsin square root transformation and were compared using the paired Student t-test with Bonferroni corrections made for multiple comparisons. For assessing acrosome reactions, the percentage data were normalized using arcsin square root transformation and were analyzed using one-way ANOVA to test for statistically significant differences among treatments, followed by a Tukey honestly significant difference pairwise comparison test using VassarStats online software (http://faculty.vassar.edu/lowry/VassarStats.html).

RESULTS

Pharmacological Agents Induced High-Amplitude Pro-Hook or Anti-Hook Flagellar Bending

Analysis of video-recorded images of free-swimming sperm revealed that after 2 h of incubation under capacitating conditions, sperm predominantly showed high-amplitude pro-hook flagellar beating—that is, hyperactivation (Figs. 1, A and F, and 2; see also Supplemental Movie A; all Supplemental Data are available online at www.biolreprod.org). Treatment of fresh, uncapacitated sperm with procaine (Fig. 1, B and G) or 4-AP (Fig. 1, C and H) also induced high-amplitude pro-hook beating (see also Supplemental Movies B and C). The maximal response (>90% sperm) was achieved 4 min after adding 5 mM procaine and 1 min after adding 4 mM 4-AP (Fig. 2). The response triggered by each agent lasted more than 15 min.

FIG. 1.

Flagellar beating patterns in mouse sperm. Images are individual frames taken from the Supplemental Movies A–E. The upper row of images (A–E) shows the maximal pro-hook flagellar bends after various treatments, whereas the lower row of images (F–J) shows the maximal anti-hook flagellar bends. A and F) Sperm hyperactivated under capacitating conditions. B and G) Uncapacitated sperm treated with 5 mM procaine. C and H) Uncapacitated sperm treated with 4 mM 4-AP. D and I) Uncapacitated sperm treated with 100 μM thimerosal. E and J) Control (uncapacitated sperm treated with medium).

FIG. 2.

Percentage of motile sperm swimming in dominant pro- and anti-hook beating patterns after each treatment. Data are from three males and are presented as the mean ± SEM. For all treatments, an asterisk (*) indicates a significant difference (P < 0.01) between the percentage of dominant pro-hook and anti-hook patterns for each treatment. Cap, capacitated sperm; Pro, 5 mM procaine; 4-AP, 4 mM 4-AP; Thi, 100 μM thimerosal; Con, control sperm.

In contrast to the response to procaine and 4-AP, treatment with 100 μM thimerosal induced high-amplitude anti-hook flagellar beating in 30 sec (Figs. 1, D and I, and 2; see also Supplemental Movie D). The response lasted up to 3 min, and then sperm gradually became sluggish and immotile. Control sperm treated with medium retained nearly symmetrical, low-amplitude flagellar beating, commonly referred to as activated or progressive motility (Fig. 1, E and J; see also Supplemental Movie E).

Effect of Thimerosal Was Dominant

Thimerosal treatment of sperm that were hyperactivated by incubation under capacitating conditions reversed the direction of the high-amplitude bend in the midpiece region to the anti-hook side of the flagellum. Thimerosal produced a similar reversal in uncapacitated sperm treated with procaine or 4-AP (Fig. 3A). When uncapacitated sperm were treated first with thimerosal and then with procaine or 4-AP, the dominant beat remained oriented in the anti-hook direction (Fig. 3B).

FIG. 3.

Percentage of motile sperm swimming in dominant pro- and anti-hook beating patterns after treatment with a combination of pharmacological agents. A) Sperm were treated with 100 μM thimerosal after achieving maximal response to 5 mM procaine or 4 mM 4-AP or after incubation under capacitating conditions for 2 h. B) Sperm were treated with 5 mM procaine or 4 mM 4-AP after achieving maximal response by 100 μM thimerosal. Data are from three males and are presented as the mean ± SEM. An asterisk (*) indicates a significant difference (P < 0.01) between the percentage of dominant pro-hook and anti-hook patterns for each treatment.

Patterns of Ca²⁺ Increase Differed Between Dominant Pro-Hook and Anti-Hook Beating Patterns

To compare Ca²⁺ increase patterns, sperm were loaded with the fluorescent Ca²⁺ indicator Fluo-4 AM and then adhered to the glass bottom of wells. The fluorescence dynamics were monitored before and after treatments.

After treatment with 4-AP to enhance pro-hook flagellar beating, a Ca²⁺ rise initiated at the midpiece-principal piece junction (Fig. 4Aa; see also Supplemental Movie F) in 81% (47/58) of sperm and spread to both midpiece and principal piece, whereas during treatment with thimerosal to enhance anti-hook flagellar beating, a Ca²⁺ rise initiated at the base of the flagellum (Fig. 4Ac; see also Supplemental Movie H) in 53% (27/51) of sperm. The initiation at the midpiece-principal piece junction was best seen when cytoplasmic droplets were present, because the droplets helped to tether sperm flagellum firmly to the glass bottom of the well. Medium control with 2 mM Ca²⁺ elicited a Ca²⁺ rise in only 4% (2/57) and 5% (3/55) of sperm when no Ca²⁺ was added in the medium (Fig. 4, Ab and Ad; see also Supplemental Movies G and I). Procaine suppressed the fluorescent signal of the Fluo-4 AM and could not be used for these experiments.

FIG. 4.

A) Pseudocolor images of fluorescence of the Ca²⁺ indicator Fluo-4 in sperm treated with 4 mM 4-AP in the presence of 2 mM Ca²⁺ (a) or sperm treated with medium as a control (b), or with 100 μM thimerosal in the presence of 30 μM Ca²⁺ (c) or sperm treated with medium as a control (d). Images are colorized individual frames taken from Supplemental Movies F, G, H, and I, respectively. Elapsed time is indicated above the images, and arrows indicate time of addition. The base of the flagellum is indicated by triangles, and the cytoplasmic droplet is indicated by arrowheads. Warmer colors indicate higher Ca²⁺. B) Pseudocolor images of fluorescence of pH indicator BCECF in sperm treated with 4 mM 4-AP (a), 100 μM thimerosal (b), or medium control (c). Images are colorized individual frames taken from Supplemental Movies J, K, and L, respectively. Elapsed time is indicated above the images, and arrows indicate time of addition. In a, the arrowheads indicate the increased signal in the principal piece; in b and c, the arrowheads indicate the proximal principal piece. Warmer colors indicate higher pH.

During Enhancement of Pro-Hook Bending by 4-AP, Rise in Flagellar pH Accompanied Ca²⁺ Rise

To investigate whether pH change was involved in increasing either pro- or anti-hook flagellar bends, sperm were loaded with the fluorescent pH indicator BCECF-AM to monitor pH change before and after various treatments. Imaging of individual sperm revealed that 4-AP application produced a pH rise in the principal piece of the flagellum within 1 sec in 85% (47/55) of sperm examined (Fig. 4Ba; see also Supplemental Movie J). Neither thimerosal nor medium control triggered a detectable pH change (Fig. 4, Bb and Bc; see also Supplemental Movies K and L). When the fluorescence of populations of sperm in multiwell plates was monitored, only 4-AP triggered a steady increase in fluorescence (Fig. 5).

FIG. 5.

The fluorescence of the pH indicator BCECF in sperm populations monitored using a microplate reader. Arrow indicates time of addition. Data are from three males and are presented as the mean ± SEM.

As was the case with our attempts at detecting fluorescence of the Ca²⁺ indicator, we were unable to test the effect of procaine on pH dynamics, because procaine interfered with the fluorescent signals, which was verified using the Bio-Tek plate reader (data not shown).

Pharmacological Treatments Did Not Induce Acrosome Reactions

Increases of Ca²⁺ in the head can induce acrosome reactions, which could complicate analysis. The Coomassie blue staining showed that none of the pharmacological treatments, which were performed on uncapacitated sperm, induced acrosome reactions except for the positive-control treatment with the Ca²⁺ ionophore A23187 (Fig. 6).

FIG. 6.

Percentage of acrosome-reacted sperm in response to treatments with procaine, 4-AP, thimerosal, and medium control. The positive control is A23187, a Ca²⁺ ionophore. Data are from three males and are presented as the mean ± SEM. An asterisk (*) indicates a significant difference (P < 0.01) from the medium (negative) control. Con, control sperm; Thi, 100 μM thimerosal; Pro, 5 mM procaine; A23, 20 μM A23187; 4-AP, 4 mM 4-AP.

Treatment by Pharmacological Agents Did Not Phosphorylate Proteins on Tyrosine

One-dimensional SDS-PAGE electrophoresis followed by Western blot using antiphosphotyrosine antibody revealed that neither procaine, 4-AP, nor thimerosal stimulated detectable increases in tyrosine phosphorylation, despite producing motility responses in nearly 90% of the sperm. Only incubation of sperm under capacitating conditions promoted tyrosine phosphorylation (Fig. 7).

FIG. 7.

One-dimensional electrophoresis followed by Western blot detection of proteins phosphorylated on tyrosine after various treatments. Con, control sperm; Thi, 100 μM thimerosal; Pro, 5 mM procaine; 4-AP, 4 mM 4-AP; Cap, capacitated sperm.

Enhancement of Pro- and Anti-Hook Flagellar Bending Produced Different Threonine and Serine Phosphorylation Patterns

Treatment with procaine, 4-AP, or thimerosal resulted in the phosphorylation of so many proteins on threonine and serine that two-dimensional electrophoresis and Western blot analysis were required to distinguish phosphorylation patterns. Incubation under capacitating conditions or treatment with procaine or 4-AP produced phosphorylation of several proteins on threonine and serine relative to the control (Fig. 8), in particular proteins of approximately 75 kDa and pH 4–6 and proteins of 25–30 kDa and pH 4.5–9.3 (Fig. 8, A–C). Differences were seen in phosphorylation patterns among sperm treated by capacitation, procaine, and 4-AP. For example, capacitation produced more phosphorylation than the other two treatments in proteins with a mass of approximately 60 kDa and pH 6.0–9.3 (Fig. 8A). Also, the blots from sperm treated with procaine did not show phosphoserine-containing proteins at 50–75 kDa and pH 5.8–6.5. Enhancement of anti-hook bending by thimerosal dephosphorylated some proteins, such as those of approximately 60 kDa and pH 4–4.5 (Fig. 8D).

FIG. 8.

Two-dimensional electrophoresis followed by Western blot detection of proteins phosphorylated on serine and threonine after various treatments. A) Capacitated sperm. B) 5 mM procaine. C) 4 mM 4-AP. D) 100 μM thimerosal (circles indicate major reductions in phosphorylation compared with E). E) Control sperm.

Immunocytochemistry was used to locate the threonine- or serine-phosphorylated proteins involved in dominant pro- and anti-hook bending. Antiphosphothreonine and antiphosphoserine antibody labeling showed that procaine and 4-AP generally increased phosphorylation (Fig. 9, A and B), whereas thimerosal decreased phosphorylation (Fig. 9C). Serine phosphorylation by procaine and 4-AP was concentrated at the base of the flagellum, whereas threonine phosphorylation was concentrated in the head and principal piece of the flagellum (Fig. 9, A and B).

FIG. 9.

Immunofluorescent localization of proteins phosphorylated on serine and threonine after various treatments. Immunofluorescence images are shown on the left and corresponding bright-field images on the right. For phosphothreonine images, the principal piece of sperm is indicated by triangles, and the head is indicated by arrowheads. For phosphoserine images, the base of the flagellum is indicated by triangles. A) 5 mM Procaine. B) 4 mM 4-AP. C) 100 μM thimerosal. D) Medium control. Bar = 10 μm.

DISCUSSION

In the present study, we characterized the dynamics of Ca²⁺, pH, and protein phosphorylation patterns associated with dominant pro- and anti-hook flagellar bending in mouse sperm. Because capacitation [27, 28], procaine [22, 24], and 4-AP [25] have been shown to activate CATSPER channels, our data indicate that activation of CATSPER channels produces dominant pro-hook beating. In contrast, stimulation of Ca²⁺ release from stores by thimerosal produced dominant anti-hook beating. Only dominant pro-hook beating was accompanied by a rise in pH and was associated with increased serine/threonine phosphorylation, whereas dominant anti-hook beating was associated with dephosphorylation of some proteins. Tyrosine phosphorylation was unaffected by any of the pharmacological agents. Altogether, our results demonstrate that two distinct Ca²⁺ signaling pathways induce these two types of asymmetrical beating.

Although the asymmetry of the flagellar bending is integral to the definition of hyperactivation, little attention has been given to characterizing this asymmetry with reference to the orientation of sperm heads. This is because the sperm heads of many species, including humans and cattle, are symmetrical, making it impossible to discern the direction of the bend. By using the asymmetrical hook on the head of mouse sperm as a reference for the direction of the flagellar bend, we were able to identify two distinct kinds of asymmetrical beating. Procaine [24, 29–32], 4-AP [33, 34], and thimerosal [21] are reported to trigger hyperactivation in the sperm of humans, guinea pigs, and cattle, but the response to thimerosal might actually be the reverse of the response to procaine and 4-AP.

In our experiments, 4-AP increased Ca²⁺ and pH in the principal piece, indicating the activation of CATSPER channels. We found that this 4-AP-triggered Ca²⁺ rise began at the midpiece-principal piece junction of the flagellum of sperm and spread not only distally to the principal piece, where CATSPER channels are located [12, 13], but also proximally to the midpiece and head (Fig. 4A). This observation is similar to that reported by Xia et al. [35], who observed that the Ca²⁺ entry mediated by CATSPER channels starts at the principal piece before propagating to the head. It has been reported that 4-AP enhances the CATSPER channel current [25], and it has been suggested that 4-AP induces intracellular alkalinization [36]. In sperm, alkalinization activates CATSPER channels [13, 25, 27, 28, 35, 37, 38] (for review, see [14]). Our results show that only 4-AP triggered a pH increase in the principal piece of the flagellum. The pH increase in the principal piece upon 4-AP treatment occurred within 1 sec (Fig. 4Ba), which was before the initiation of the Ca²⁺ increase (Fig. 4Aa). This is consistent with previous findings that intracellular alkalization occurs before increase of Ca²⁺ in hyperactivated bovine sperm [39]. Thus, we conclude that 4-AP indirectly activates CATSPER channels to produce pro-hook bending.

Thimerosal triggers asymmetrical beating by releasing Ca²⁺ from the RNE store. In our experiments with mouse sperm, thimerosal triggered a Ca²⁺ release at the neck of the flagellum that spread into the postacrosomal region of the head and the midpiece. Thimerosal has been found to trigger Ca²⁺ release from both IP₃- and ryanodine-sensitive calcium stores in many cell types (for review, see [40]). The presence of RNE at the base of the flagellum has been observed in mature sperm of several species [41–44]. In bovine sperm, labeling with antibody to IP₃ receptor demonstrated the presence of the receptor in the RNE stores. Also, treatment with thimerosal and thapsigargin, even in the absence of available extracellular Ca²⁺, triggered asymmetrical flagellar beating in bull sperm [21].

Despite inducing a rise in Ca²⁺ in the sperm head, neither 4-AP, procaine, nor thimerosal treatments elicited acrosome reactions in these uncapacitated sperm, which would have confounded interpretation of the effects of these agents on flagellar beating patterns. In other work, procaine and thimerosal also did not induce acrosome reactions in equine sperm [32] and in bull sperm [21].

In many types of cells, Ca²⁺ signaling is often accompanied by protein phosphorylation [45]. Our results show that procaine and 4-AP triggered phosphorylation on serine and threonine sites of several proteins, whereas thimerosal treatment dephosphorylated some proteins. Serine phosphorylation induced by procaine and 4-AP were generally located in the head and the entire flagellum but most prominently at the base, whereas threonine phosphorylated proteins were most prominent in the head and principal piece of the flagellum. In all labeled regions, increased Ca²⁺ was detected in response to 4-AP. In contrast to 4-AP, thimerosal generally reduced phosphorylation, suggesting that the downstream proteins of Ca²⁺ mobilization were quite different from those involved in pro-hook bending triggered by 4-AP. We concluded that the Ca²⁺ signaling pathways were different for dominant pro- and anti-hook bending, which might be referred to as hyperactivation and reverse hyperactivation.

Different patterns of protein phosphorylation/dephosphorylation could occur if the initial targets of the increased Ca²⁺ were different during amplification of pro-hook and anti-hook bending. Calmodulin is known to play a role in Ca²⁺-dependent modulation of mammalian sperm flagellar beating by directly affecting the axoneme [46]. Evidence indicates additional Ca²⁺-binding proteins in sperm flagella, such as CABYR in mammals [47, 48] and calaxin, in the ascidian Ciona intestinalis [49]. Also, in Chlamydomonas sp., the dynein light chain protein LC4 has been localized in the flagella and shown to undergo a conformation change in response to Ca²⁺ [50]. Calaxin and LC4, as well as additional Ca²⁺-binding axonemal proteins, may yet be identified in mammalian sperm. The downstream targets of these proteins would likely differ from those of calmodulin. Also, it would be expected that the different Ca²⁺-binding proteins responsible for amplifying pro-hook and anti-hook flagellar beating would show different ultrastructural distributions in the axoneme, particularly with respect to the side of the axoneme on which they are located.

To our knowledge, the present publication is the first report of the occurrence of serine/threonine phosphorylation during mouse sperm hyperactivation. Serine and threonine phosphorylation in general is not well understood because of a historical lack of antibodies with high specificity. To date, most serine/threonine phosphorylation studies in sperm have focused on activation, capacitation, and the acrosome reaction. In those studies, both phosphorylation and dephosphorylation have been implicated in those processes [51–55]. Several kinases [46, 56–58] and phosphatases [59–63] have been implicated in the regulation of sperm motility patterns.

Tyrosine phosphorylation on a series of proteins is an established hallmark of capacitation [64]. Hyperactivation is generally considered to be a part of capacitation. However, hyperactivation has been observed to occur independently of other aspects of capacitation [31]. Neither 4-AP, procaine, nor thimerosal induced detectable tyrosine phosphorylation in our experiments; instead, the tyrosine phosphorylation was affected only by capacitating conditions. It was reported that procaine does not affect tyrosine phosphorylation in bovine and equine sperm [31, 32]. This means proteins that are tyrosine phosphorylated during capacitation do not play a direct role in hyperactivation downstream of the action of procaine, 4-AP, or thimerosal, although tyrosine phosphorylation may operate upstream of the effects of these agents in the activation of Ca²⁺ influx or release from stores.

In our experiments, we found it was possible to reverse capacitation-associated hyperactivation or agent-amplified pro-hook beating using thimerosal, indicating that the effect of thimerosal is dominant. Thimerosal (100 μM) arrested sperm in approximately 3 min. The arrest of motility could be the result of deleterious effects of prolonged elevation of cytosolic Ca²⁺.

In other cell types, release of Ca²⁺ from stores briefly produces micromolar levels of Ca²⁺ in the immediate vicinity of the store [45, 65]. In vivo, the short burst of Ca²⁺ from the RNE triggered by physiological factor(s) could induce a brief reversal of the dominant flagellar bend from pro- to anti-hook, and then Ca²⁺ could be rapidly removed by the mitochondria near the RNE and also pumped back into the RNE stores or out through the plasma membrane by Ca²⁺ ATPases [65, 66]. The momentary large anti-hook bend could produce a course correction in sperm, perhaps reorienting them toward the oocyte.

Sperm hyperactivated during capacitation exhibited a small anti-hook bend very close to the head during the reverse phase of the beat, similar to that seen in activated sperm. Surprisingly, this small anti-hook bend was not seen in sperm hyperactivated by procaine or 4-AP (Fig. 1; see also Supplemental Movies A–C). Carlson et al. [24] also reported that capacitated sperm showed different midpiece curvature in the pro- and anti-hook directions than procaine-treated sperm. The small anti-hook bend could result from a small release of Ca²⁺ from stores that is not triggered by procaine or 4-AP or other ionic or molecular differences in the cytoplasm at the base of the flagellum.

In a variety of marine invertebrate species, including the sea urchin Arbacia punctulata, sperm respond to chemotactic signals from the oocytes through Ca²⁺-mediated changes in flagellar beat asymmetry (for review, see [67]). Specifically, the flagellar beating patterns alternate between asymmetrical and nearly symmetrical patterns, resulting in a swimming trajectory that alternates between high and low curvature. This alternation forms a “turn and run” pattern, which produces a loosely helical configuration with an axis that is directed toward the source of the attractant (for review, see [68]). Some evidence exists for sperm chemotaxis in mammals as well (for review, see [23]). Nevertheless, the details of the flagellar response of mammalian sperm to chemoattractants are not well elucidated and may be different from those of invertebrate sperm. In mammals, chemoattractant(s) from the oocytes could modulate the flagellar beating of hyperactivated sperm by releasing Ca²⁺ from the RNE store. This is supported by our observation that thimerosal could reverse the bend pattern of sperm that were hyperactivated by incubation under capacitating conditions.

We have proposed that hyperactivated sperm require intermittent course corrections to reach the oocyte, because sperm begin to hyperactivate in the oviduct far from the site of fertilization [4]. We have now provided evidence for a mechanism to make course corrections. Here, we have shown that the dominant bend of hyperactivated sperm can be switched to the anti-hook direction by release of Ca²⁺ from intracellular Ca²⁺ stores via a signal transduction pathway that is distinct from the Ca²⁺ pathway that triggers hyperactivation through activation of CATSPER channels. Much remains to be learned about the how hyperactivation is modulated to bring sperm to the oocyte. Hopefully, the clues offered by the hook-shaped heads of rodent sperm will continue to offer insight regarding these mechanisms.