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BOR - Papers in Press, published online ahead of print December 20, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.055038
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BIOLOGY OF REPRODUCTION 76, 660–665 (2007)
DOI: 10.1095/biolreprod.106.055038
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Bovine Sperm Hyperactivation Is Promoted by Alkaline-Stimulated Ca2+ Influx1

Becky Marquez , and Susan S. Suarez 2 

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

ABSTRACT

Sperm hyperactivated motility is characterized by high flagellar bend amplitude and asymmetrical beating, which are detected by computer-assisted sperm motility analysis as increased curvilinear velocity and lateral head movement. It is required for sperm penetration of the oocyte zona pellucida during fertilization and is induced by an increase in flagellar Ca2+. Our objective was to determine whether pH plays a role in promoting Ca signaling of hyperactivated motility. The cell-permeant weak base NH4Cl increased curvilinear velocity and amplitude of lateral head movement of bovine sperm, indicative of hyperactivation. Fluorometric recordings of sperm loaded with BCECF-AM or fluo3-AM, revealed that NH4Cl evoked elevations of intracellular pH and Ca2+, respectively, with the rise in pH occurring more rapidly than that of Ca2+. Single-cell image analysis showed increased Ca2+ levels in the flagellum in response to NH4Cl. When extracellular Ca2+ was lowered with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) prior to treatment with NH4Cl, intracellular pH was increased, but elevation of Ca2+ and hyperactivation were diminished. This suggests that the rise in intracellular pH precedes an influx of Ca2+. The Ca2+ channel blocker Ni2+ also diminished NH4Cl stimulation of hyperactivation, demonstrating that Ca2+ entry is required for maximal expression of hyperactivation. Ca2+ ionophore produced an increase in Ca2+ that was 3-fold greater than that produced by NH4Cl; however, it produced a weaker hyperactivation response. These results indicate that a rise in pH increases intracellular Ca2+and promotes hyperactivation primarily by stimulating Ca2+ influx, but also by other mechanisms.

calcium, gamete biology, sperm, sperm motility and transport

INTRODUCTION

Increased pH is a key element in the signaling pathways that control Ca2+-dependent responses critical for fertilization in marine invertebrate and mammalian sperm. Chemotaxis in invertebrate sperm involves such a response, whereby egg peptides evoke a pH-mediated Ca2+ influx that modulates flagellar beat asymmetry to direct sperm toward the egg [1–3]. In mammals, hyperactivated motility of sperm resembles chemotactic turning of invertebrate sperm, suggesting the existence of a mechanism in which an increase in internal pH alters flagellar bending patterns by affecting Ca2+ levels.

Hyperactivated motility is characterized by asymmetrical flagellar beating derived from an increase in the amplitude of the principal flagellar bend. Hyperactivation is displayed by sperm at the site of fertilization [4–6] and can also be exhibited by sperm undergoing capacitation in vitro [7]. Capacitation is the process in which sperm acquire the ability to undergo the acrosome reaction and fertilize an oocyte [8]. The signaling pathways that capacitate sperm are poorly understood, but evidence to date indicates that pH [9], Ca2+ [10, 11], and cAMP [12, 13] are important regulatory components. Although the pathway that supports hyperactivation has not been completely elucidated, Ca2+, through calmodulin, has been shown to signal the transition from symmetrical to asymmetrical flagellar beating at the axoneme [14, 15].

Extracellular Ca2+ is required to maintain hyperactivation [16], and Ca2+ is increased in the flagella of hyperactivated sperm [17, 18]. The mechanism(s) that trigger Ca2+ influx to support hyperactivation are not well characterized, but Ca2+ channels have been localized to the flagellum. These channels include transient receptor potential channels [19–21], cyclic-nucleotide gated Ca2+ channels [22], and voltage-dependent Ca2+ channels [23, 24], including CATSPER1 [25, 26] and CATSPER2 [27, 28]. Male mice lacking functional CATSPER1 or CATSPER2 genes are sterile. Their sperm fail to develop hyperactivation during capacitation in vitro and, unlike wild-type sperm, do not show increased intracellular Ca2+ in response to treatment with high K+, high pH medium [26, 29, 30]. Interestingly, the amino terminus of CATSPER1 contains an abundance of histidine residues [25], and patch-clamp experiments indicate that alkalinization of intracellular pH potentiates an inward Ca2+ current through CATSPER1-associated channels [26].

During capacitation, pH elevation may contribute to activation of channels permeable to Ca2+, such as CATSPER1, because capacitation is inhibited when intracellular alkalinization is prevented [9]. Maintenance of precapacitation levels of internal pH could be a means to regulate Ca2+ channels to prevent premature Ca2+ influx and Ca2+-dependent events such as hyperactivation. Weak bases, such as procaine, that require extracellular Ca2+ to induce hyperactivation [31, 32] have further implicated pH as a component in the signaling pathway that controls Ca2+ entry and hyperactivation.

Our objective was to determine whether pH plays a role in promoting Ca2+ signaling of hyperactivated motility.

MATERIALS AND METHODS

Chemicals and Media

All chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO), with the following exceptions: HEPES, BSA, and ionomycin were obtained from Calbiochem Corp. (La Jolla, CA). Molecular Probes (Eugene, OR) provided fluo3-AM, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluor-escein (BCECF-AM), and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA).

A modified Tyrode balanced salt solution (TALP) [33] was used for washing and incubating sperm. The medium consisted of 3.1 mM KCl, 99 mM NaCl, 25 mM NaHCO3, 0.4 mM NaH2PO4, 1.1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 1 mM pyruvate, 25.4 mM lactate, 50 µg/ml gentamycin, and 6 mg/ml BSA (pH 7.6, 290–300 mOsm/kg). A modified alkaline, K+-substituted medium was used to raise intracellular pH [34, 35]. This medium resembled TALP with the following exceptions: 99 mM KCl, 3.1 mM NaCl, and 10 mM TAPS (N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid) for optimal buffering at pH 8.6 (osmolality maintained at 290–300 mOsm/kg) [24].

Sperm Preparation and Hyperactivation

Semen was collected from bulls of proven fertility and generously donated by the bovine artificial insemination service Genex Cooperative Inc. (Ithaca, NY). Semen was diluted 1:5 in TALP immediately after collection and transported to the laboratory within 60 min in a 37°C water jacket. Sperm were washed free of seminal plasma by twice-repeated dilution and centrifugation (170 x g, 10 min) in TALP. Washed sperm were incubated at 39°C (bovine core body temperature) under 5% CO2 for the experiments described below.

Motility of sperm (10 x 106/ml) was analyzed after 1 min of incubation in TALP alone or TALP containing 25 mM ammonium chloride (NH4Cl) or 3 µM ionomycin. To test the effects of extracellular Ca2+ on induction of hyperactivation, sperm were preincubated in 100 µM nickel chloride (Ni2+) for 1 min before addition of treatments. To prevent precipitation, nickel chloride was prepared in TALP lacking NaH2PO4. BAPTA (10 mM) was used to buffer extracellular Ca2+ to approximately 25 nM (calculated using MaxChelator: www.stanford.edu/~cpatton/maxc.html), which is at or below the intracellular Ca2+ level of nonhyperactivated sperm with normal progressive motility [14, 17].

Analysis of Sperm Motility

Treated sperm were placed on slides on a 39°C stage of a Zeiss Axiovert 35 microscope and were videotaped using 400x differential interference microscopy (Carl Zeiss Inc., Thornbrook, NY) with stroboscopic illumination at 60 Hz provided by a 75-W xenon flash tube (Chadwick-Helmuth Co., El Monte, CA). Videotaping was conducted using a black-and-white Dage CCD 72 video camera (Dage-MTI Inc., Michigan City, IN) connected to a Panasonic AG-7300 Super VHS videocassette recorder (Panasonic Industrial Co., Secaucus, NJ). Videotapes were used to determine percent motility and hyperactivation of at least 200 sperm per treatment [18].

Sperm motility also was evaluated using computer-assisted motion analysis. Sperm movement was imaged using a 10x Olympus negative phase objective (Hi-Tech Instruments, Philadelphia, PA) and recorded using a Panasonic AG-7300 Super VHS video recorder. The video images were digitized (30 frames at 60 Hz) and analyzed using HTM-IVOS (version 10; Hamilton Thorne Biosciences, Beverly, MA). Motion parameters measured were curvilinear velocity (VCL, the rate of travel of the sperm head), linearity (LIN, straightness of trajectory, measured as the ratio of straight line velocity divided by the curvilinear velocity), and amplitude of lateral head displacement (ALH, degree of side-to-side head movement measured as the mean width of head oscillations). Because movement of the sperm head depends on flagellar bend and beat patterns, increased VCL and ALH as well as decreased LIN are indicative of hyperactivation [36].

To assess the degree of flagellar bend amplitude, the flagellar curvature ratio (FCR) was determined as the straight-line distance from the head-midpiece junction to the first inflection point in the flagellar bend divided by the curvilinear distance along the flagellum between the two points [14]. This measurement had been developed because the asymmetry of flagellar waves precludes direct measurement of wave amplitude. A lower FCR denotes a higher amplitude bend.

Data Analysis

Motility data were analyzed using Minitab statistical software (Minitab Inc., State College, PA). Treatment effects were detected using analysis of variance followed by the Tukey test for individual posthoc comparisons and were considered statistically significant at P < 0.05. For each set of experiments, one ejaculate each from three bulls was tested.

Intracellular pH and Ca2+ Determinations

Sperm (5 x 106/ml) were loaded with 5 µM of the pH-sensitive dye BCECF-AM or the Ca2+-sensitive dye fluo3-AM (in 0.5% dimethyl sulfoxide) for 40 min at 39°C. Extracellular dyes were removed by centrifugation at 170 x g for 5 min. Sperm were resuspended in medium and incubated for an additional 20 min to allow de-esterification of the dyes.

For fluorescence measurements, cells were excited at 490 nm, and fluorescence emission was recorded at 530 nm using a Perkin-Elmer LS-5 fluorescence spectrophotometer (Oak Brook, IL). At the end of each experiment, the fluo3 fluorescence signal was calibrated after lysis of sperm with 0.1% Triton X-100 for dye saturation with excess (2 mM) Ca2+ to acquire maximal fluorescence (Fmax), and then addition of 8 mM EGTA to obtain minimal fluorescence (Fmin) [37]. Extracellular dye fluorescence of nonlysed sperm suspensions was quenched using 4 mM MnCl2, and values were subtracted from both Fmax and observed fluorescence. The BCECF fluorescence signal was calibrated after sperm lysis with 0.1% Triton X-100 followed by serial additions of HCl to modify pH [37].

For single-cell Ca2+ imaging, fluorescence intensity was detected with an epifluorescence microscope using a 480 ± 40 nm excitation filter, 535 ± 50 nm emission filter, and a 505 nm long-pass dichroic mirror (Chroma Technology Corp., Rockingham, VT) with an oil immersion 40x Fluar objective (numerical aperture 1.3; Carl Zeiss Inc.). Stroboscopic illumination was provided by a 75-W xenon arc flash lamp. The fluorescence of individual motile sperm was monitored before and after application of treatments while in an open microchamber at 39°C (PDMI-2 Micro-Incubator; Medical Systems Corp., Greenvale, NY). Sperm were tethered by the head to the glass coverslip by adding them to the chamber in medium lacking BSA, then flooding the chamber with complete medium. Images were captured with a Sensicam High-Performance camera (The Cooke Corp., Auburn Hills, MI) and digitized at two images per second controlled by IPLab Spectrum software (Signal Analytics, Vienna, VA).

RESULTS

Hyperactivation Elicited by Weak Base NH4Cl Involves Influx of Ca2+

Cell-permeant NH4Cl hyperactivated sperm within 2 min of addition (Fig. 1), which was detected by computer-assisted motion analysis as increased mean VCL and ALH and decreased LIN (Table 1). Preliminary studies had indicated that the effect of NH4Cl on hyperactivation was dose dependent, and 25 mM produced the maximum effect. Motility declined immediately after additions of more than 25 mM NH4Cl. The Ca2+ ionophore ionomycin also stimulated hyperactivation, with a maximum effect observed at 3 µM. As measured by spectrofluorimetry, both NH4Cl and ionomycin stimulated a rise in intracellular Ca2+; however, the Ca2+ rise stimulated by ionomycin was 3.2 ± 0.59 times (mean ± SD for samples from five bulls) the rise stimulated by NH4Cl at 2 min after addition, and yet the hyperactivation response stimulated by ionomycin was actually lower (Table 1). Simultaneous addition of NH4Cl and ionomycin did not further enhance VCL or ALH. Sperm motility declined after 5 min of incubation in NH4Cl and/or ionomycin.


Figure 01
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  		FIG. 1. Hyperactivation induced by NH4Cl and Ca2+. Representative motion tracks of control sperm (A, B) and sperm treated with NH4Cl (C, D) or ionomycin (E, F) in TALP medium without (A, C, E) or with (B, D, F) 10 mM BAPTA. Points on the track represent positions of the head in 30 successive video frames in 0.5 sec.


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  		TABLE 1. CASA measurements of the effects of alkalization and Ca2+ on sperm motility.a

When BAPTA was used to lower available extracellular Ca2+, hyperactivation was reduced in response to NH4Cl or ionomycin (Table 1). Computer-assisted tracking of movement revealed that flagellar beat asymmetry decreased and the swimming path straightened (Fig. 1). However, VCL, ALH, and LIN of NH4Cl-treated sperm were not at the levels of controls, but rather were intermediate between controls and the responses elicited by NH4Cl in the presence of 2 mM Ca (Table 1).

Similar to the effects of lowering Ca2+ with BAPTA, addition of Ni2+ decreased hyperactivation induced by NH4Cl (Table 2). The amplitude of the flagellar bend dampened and the swimming trajectory of sperm straightened.


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  		TABLE 2. Effects of blocking Ca2+ channels on sperm motility.a

Intracellular Alkalization Precedes Ca2+ Influx

NH4Cl treatment of sperm rapidly evoked elevations of both intracellular pH and Ca2+ that persisted as long as NH4Cl was present (Fig. 2). The rise in pH occurred at a faster rate than that of Ca2+, reaching a peak in about 30 sec, whereas the Ca2+ rose more slowly. Lowering extracellular Ca2+ with BAPTA diminished the elevation of intracellular Ca2+ without affecting intracellular alkalization (Fig. 2). Increased Ca2+ (Fig. 3) could be seen in both the head and flagellum of sperm within 30 sec of addition of NH4Cl.


Figure 02
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  		FIG. 2. Fluorometric traces of the effects of NH4Cl on internal pH (A) and Ca2+ (B) in the presence of TALP with or without 10 mM BAPTA. The traces are representative of three replicate experiments, each using sperm from a different bull. No Ca2+ response was detected after addition of TALP alone (C).


Figure 03
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  		FIG. 3. Ca2+ images of fluo3-loaded sperm. Images of the same field were captured before (A, C) and 30 sec after (B, D) initial addition of TALP (B) or NH4Cl in TALP (D). Warmer colors (green and yellow) indicate higher fluorescent intensities and increased intracellular Ca2+. Bar = 10 µm.

Flagellar Beat Asymmetry is Stimulated by Activation of pH-Sensitive Channels

Alkaline K+ medium, which was used to raise intracellular pH, elicited asymmetrical flagellar beating that generated a circular swimming trajectory, which was detected by computer-assisted motion analysis as a decrease in LIN (Table 3). The effect was evident in 96.7% ± 1.8% (1.7% ± 0.3% for control) of motile sperm within 15 sec. After approximately 2 min, motility progressively declined until flagella arrested in a curved position and vibrated (Fig. 4). Removal of alkaline K+ medium by 10-fold dilution with TALP caused immediate resumption of flagellar beating (81%–90% motile), with all motile sperm swimming in circles at first, but 95%–98% of motile sperm assuming linear trajectories within 10 min (n = 3). Thus, the effects of the alkaline K+ medium could be reversed. Chelating extracellular Ca2+ with BAPTA down to 40–50 nM briefly delayed (30–60 sec) but did not prevent the effects of alkaline K+ medium.


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  		TABLE 3. Effects of alkaline K+ medium on path linearity and flagellar curvature.a


Figure 04
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  		FIG. 4. Alkaline K+ medium increased flagellar beat asymmetry and curvature. A) Sperm in TALP medium with FCR 0.84 exhibiting low amplitude and symmetrical flagellar beating. B) Sperm with FCR 0.67 after alkaline K+ medium induced the amplitude of the principal bend to increase, thereby increasing the asymmetry of the flagellar beat. C) Arrested sperm with curved flagellum caused by prolonged exposure to alkaline K+ medium. In A and B, the image represents two flashes of a stroboscope spaced 1/60 sec apart, allowing visualization of the same sperm in sequential frames. Bar = 10 µm.

To further manipulate intracellular pH, sperm suspensions in TALP (containing 2 mM CaCl2) were supplemented with HCl or Tris-base to produce acidic or alkaline media, respectively (Table 4). At pH 6.6, sperm developed lower flagellar bend amplitude and more symmetrical flagellar beating compared with control (pH 7.6). In contrast, at pH 8.6, sperm vigorously hyperactivated (86.3% ± 2.3% vs. 3.0% ± 0.6% for control) and resembled NH4Cl-treated sperm. Unlike treatment with NH4Cl, sperm were slower to respond to Tris-base by about 5 min, but motility and hyperactivation were maintained for several hours, whereas motility declined in NH4Cl-treated sperm in 5 min. The effects of acidic or alkaline media were reversible, as sperm regained motility resembling that of controls when extracellular pH was restored to 7.6.


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  		TABLE 4. Effects of pH on sperm motility.a

DISCUSSION

Our data demonstrate the involvement of a pH-stimulated intracellular Ca2+ increase in triggering hyperactivated motility in sperm. Sperm were capable of responding quickly to changes in pH and transduced such changes into modulation of flagellar bend amplitude and beat asymmetry. In these experiments, alkaline pHi primarily acted upstream of the Ca2+ signaling pathway by promoting a rapid increase of Ca2+ in the flagellum to hyperactivate sperm.

The role of pH in mediating signaling important for flagellar activity also is evident during sperm activation, where a rise in intracellular pH is associated with acquisition of progressive motility when sperm are released from the cauda epididymidis [35, 38–40]. Interestingly, a sperm-specific Na+/H+ exchanger located at the principal piece of the flagellum has been found to be required for motility and fertility [41].

In our experiments, the rise in intracellular pH induced by NH4Cl occurred more rapidly than the rise in Ca2+. Lowering of extracellular Ca2+ by BAPTA diminished the elevation of Ca2+ and hyperactivation but did not prevent the pHi increase. These results indicate that Ca2+ entry is required to produce hyperactivation and that increased pHi precedes the influx of Ca2+. Consistent with its ability to reduce Ca2+ responses elicited by NH4Cl, the Ca2+ channel blocker Ni2+ [42] impaired the effectiveness of alkaline pHi to stimulate hyperactivation, confirming that increased pHi induces hyperactivation primarily by stimulating Ca2+ influx.

Increased asymmetry of flagellar beating elicited by alkaline K+ medium provides further evidence for the involvement of pHi-modulated Ca2+ entry in promoting hyperactivation. Alkaline K+ medium was previously shown to increase pH and Ca2+ in sperm [34], although effects on hyperactivation were not examined. In our experiments, sperm treated with alkaline K+ medium eventually arrested with curved flagella, which is indicative of calcium overload [14] and demonstrates that alkaline K+ medium increased beat asymmetry by elevating flagellar Ca2+.

The existence of sperm Ca2+ channels modulated by internal pH had been suggested by various experimental approaches [1, 34, 37, 42] and recently verified by patch-clamp experiments that identified CATSPER1 as the key component of flagellar channels [26]. Sperm from CATSPER1 null mice lack pH-stimulated calcium current [26] and cannot hyperactivate [30].

The localization of CATSPER1 [29] and an Na+/H+ exchanger [41] to the principal piece in mice can explain the development of hyperactivation in that region of the flagellum. Nevertheless, because each flagellar beat arises in the flagellar midpiece before propagating down to the principal piece, additional mechanisms may be required for raising Ca2+ in the midpiece.

Reducing extracellular Ca2+ delayed but failed to prevent the onset of asymmetrical beating stimulated by alkaline K+ medium, suggesting that this medium could also have stimulated release of Ca2+ from an internal store to initiate asymmetrical flagellar beating. The role of an internal store in providing Ca2+ to increase flagellar beat amplitude and asymmetry was further implicated when, in the absence of available external Ca2+, NH4Cl evoked a small increase in intracellular Ca2+ and mild hyperactivation. Intracellular alkalization may have promoted release of Ca2+ from an internal store sufficient to support only low-intensity hyperactivation. There is evidence that an IP3R-gated Ca2+ store in the base of the flagellum provides Ca2+ to initiate hyperactivation [18, 43]. Because binding of IP3 to its receptor increases with increasing pHi, alkalization could have enhanced IP3-induced Ca2+ release from an internal store [44, 45].

In addition, in the absence of available extracellular Ca2+, increased pH could have directly affected the axoneme to some extent, accounting for the mild form of hyperactivation observed. When sperm membranes are permeabilized by detergent and then motility is reactivated by addition of ATP, addition of 400-1000 nM Ca2+ hyperactivates the permeabilized sperm only in pH 7.9–8.5 [14]. This indicates that both Ca2+ and alkaline pH can have direct effects on the flagellar axoneme. High pH may affect the phosphorylation state of axonemal proteins [38, 46] or optimize the activity of dynein ATPase [47]. In our experiments, it is possible that NH4Cl-imposed alkalization enhanced flagellar bending.

Ionomycin, which would have bypassed NH4Cl stimulation of Ca2+ influx, induced hyperactivation; however, NH4Cl stimulated more intense hyperactivation than ionomycin, despite eliciting only a third of the Ca2+ response evoked by ionomycin, suggesting that although increased pHi was not required downstream of Ca2+ entry, it enhanced hyperactivation by elevating axonemal pH.

In conclusion, our results indicate that fully developed hyperactivation depends on intracellular alkalinization that stimulates Ca2+ influx. A large part of this response may be attributed to CATSPER channels in the principal piece, whereas the remainder may be due to release of Ca2+ from a midpiece store and alkaline-increased sensitivity of the axoneme to existent available Ca2+.

ACKNOWLEDGMENTS

The authors thank Dr. Clare Fewtrell of Cornell University for her guidance regarding the use of the spectrofluorimeter.

FOOTNOTES

1Supported by National Science Foundation grant MCB-0421855 to S.S.S. and National Institutes of Health grant F31 HD 43693 to B.M. Back

Correspondence: 2Susan S. Suarez, Department of Biomedical Sciences, T5-006 Veterinary Research Tower, Cornell University, Ithaca, NY 14853. FAX: 607 253 3541; e-mail: sss7{at}cornell.edu

Received: 25 June 2006.

First decision: 27 August 2006.

Accepted: 18 December 2006.

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