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BOR - Papers in Press, published online ahead of print May 4, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.040733
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BIOLOGY OF REPRODUCTION 73, 519–526 (2005)
DOI: 10.1095/biolreprod.105.040733
© 2005 by the Society for the Study of Reproduction, Inc.

Calcium/Calmodulin and Calmodulin Kinase II Stimulate Hyperactivation in Demembranated Bovine Sperm1

George G. Ignotz , and Susan S. Suarez 2 

Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperactivated motility is observed among sperm in the mammalian oviduct near the time of ovulation. It is characterized by high-amplitude, asymmetrical flagellar beating and assists sperm in penetrating the cumulus oophorus and zona pellucida. Elevated intracellular Ca2+ is required for the initiation of hyperactivated motility, suggesting that calmodulin (CALM) and Ca2+/CALM-stimulated pathways are involved. A demembranated sperm model was used to investigate the role of CALM in promoting hyperactivation. Ejaculated bovine sperm were demembranated and immobilized by brief exposure to Triton X-100. Motility was restored by addition of reactivation medium containing MgATP and Ca2+, and hyperactivation was observed as free Ca2+ was increased from 50 nM to 1 µM. However, when 2.5 mM Ca2+ was added to the demembranation medium to extract flagellar CALM, motility was not reactivated unless exogenous CALM was readded. The inclusion of anti-CALM IgG in the reactivation medium reduced the proportion hyperactivated in 1 µM Ca2+ to 5%. Neither control IgG, the CALM antagonist W-7, nor a peptide directed against the CALM-binding domain of myosin light chain kinase (MYLK2) inhibited hyperactivation. However, when sperm were reactivated in the presence of CALM kinase II (CAMK2) inhibiting peptides, hyperactivation was reduced by 75%. Furthermore, an inhibitor of CAMK2, KN-93, inhibited hyperactivation without impairing normal motility of intact sperm. CALM and CAMK2 were immunolocalized to the acrosomal region and flagellum. These results indicate that hyperactivation is stimulated by a Ca2+/CALM pathway involving CAMK2.

calcium, gamete biology, male reproductive tract, sperm motility and transport


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Sperm acquisition of hyperactivated motility is critical for transport through the female tract and for fertilization [1]. While the motility of sperm in the ejaculate is characterized by symmetrical flagellar bending and linear trajectories, hyperactivated motility is characterized by a high-amplitude, asymmetrical flagellar waveform. Hyperactivation can be observed in situ in transilluminated murine oviducts around the time of ovulation and near the site of fertilization [2, 3]. Sperm eluted from the ampullae of oviducts of mated rabbits [4] and ewes [5] also exhibit hyperactivated motility.

Hyperactivation evidently serves key functions in vivo. Because hyperactivated sperm are more effective at penetrating highly visco-elastic media, it is believed that hyperactivation enables sperm to pass efficiently through mucus in the oviduct to arrive at the site of fertilization and then to pass through the matrix of the cumulus oophorus [6, 7]. Hyperactivation also assists sperm penetration of the zona pellucida surrounding the oocyte [8, 9].

In addition to hyperactivation, mammalian sperm must undergo capacitation to fertilize. Capacitation is a sequence of events required for initiation of the acrosome reaction and completion of fertilization. Capacitation is incompletely understood but is known to involve loss of extrinsic proteins and cholesterol from the plasma membrane, followed by increased protein tyrosine phosphorylation [10]. Because sperm incubated in vitro under capacitating conditions have been observed to hyperactivate, hyperactivation has been considered a part of capacitation. Nevertheless, hyperactivation and acrosomal responsiveness are separate processes that have been shown to occur independently of each other [11, 12].

Although the physiological roles of hyperactivation have been established and its importance to fertilization recognized, biochemical signaling events that initiate and maintain hyperactivated motility remain largely unknown. It has been demonstrated that Ca2+ signaling is critical for hyperactivation. Extracellular Ca2+ is required to maintain hyperactivation in hamster sperm [13], and cytoplasmic concentrations of free Ca2+ are increased in flagella of hyperactivated sperm [14]. Bovine sperm can be hyperactivated in vitro by stimulating Ca2+ influx with caffeine [15]. The Ca2+-ATPase inhibitor thapsigargin induces hyperactivation of bovine sperm by promoting Ca2+ release from IP3 receptor-gated internal stores and thimerosal, an IP3 receptor agonist, has been shown to increase intracellular Ca2+ levels and stimulate hyperactivation [15]. The internal Ca2+ store has been identified as a portion of the redundant nuclear envelope in the base of the flagellum [16]. Sperm from male mice that are null mutants for CatSper1 and CatSper2 genes, which encode putative plasma membrane Ca2+ channels, cannot hyperactivate [9, 17]. However, the downstream targets of Ca2+ have not been identified.

Demembranated sperm models have been used to demonstrate that Ca2+ acts directly on the flagellar axoneme to stimulate hyperactivation [18]. When sperm are treated with Triton X-100, the plasma membrane is removed and mitochondrial membranes are disrupted, leaving the axoneme intact and exposed to the environment [18]. The demembranated sperm can be reactivated in a medium that contains ATP. Models derived from detergent-demembranated sperm and Chlamydomonas flagella have been used to investigate mechanisms involved in regulating flagellar bending and waveform patterns [19–23]. Bull sperm demembranated with Triton X-100 are immotile until reactivated with ATP and 50 nM Ca2+. Increasing concentrations of Ca2+ in the reactivation medium result in a switch from activated to hyperactivated motility [18]. In this study, we employed demembranated sperm models to identify the targets of Ca2+ signaling during hyperactivation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media and Chemicals

Routine laboratory chemicals, bovine testis calmodulin (CALM), and secondary antibodies were from Sigma-Aldrich (St. Louis, MO). CAMK2 and MYLK2 inhibitory peptides, obtained from Calbiochem (San Diego, CA), were as follows: CALM-binding domain (290–309; LKKFNARRKLKGAILTTMLA), CAMK2 inhibitor (281–309; MHRQETVDCLKKFNARRKLKGAILTTMLA), CAMK2 Ntide (Myr-GGGKRPPKLGQIGRAKVVIEDDRIDDVLK), MYLK2 inhibitor (RRKTQKTQHAVRAIGRL). The inhibitors W-7, ML-7, KN-93, and KN-92 were purchased from BioMol International, LP (Plymouth Meeting, PA). Anti-CALM1 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, Ca).

A modified Tyrode balanced salt solution (TALP, [24]) was used for semen dilution, sperm transport, and washing. It consisted of 99 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.35 mM NaH2PO4, 10 mM Hepes, 2 mM CaCl2, 1.1 mM MgCl2, 21.6 mM sodium lactate, 1.1 mg/ml sodium pyruvate, 6 mg/ml BSA, and 1 µg/ml gentamycin (pH 7.4, 290 mOsm/ kg). Where indicated, sperm were washed or incubated in BSA- and Ca2+-free TALP.

Sperm Preparation

Bovine semen was provided by Genex/CRI (Ithaca, NY). Fresh semen, diluted fivefold with TALP immediately after collection, was transported within a 37°C water jacket to the laboratory shortly after collection. Sperm were recovered by centrifugation (170 x g for 10 min), washed twice in 5 ml TALP and, when required, thrice in Ca2+- and BSA-free TALP. Sperm were diluted to 100 x 106/ml in final wash medium and held at 38.5°C, 5% CO2 until used for experiments.

Sperm Demembranation and Reactivation

Demembranation and reactivation were performed as previously described [18, 25] with minor modifications. To remove the plasma membrane, a 20-µl portion of washed sperm was added to 480 µl of room-temperature extraction solution (0.2% Triton X-100, 0.2 M sucrose, 1 mM dithiothreitol [DTT], 25 mM potassium glutamate, and 40 mM Hepes, pH 7.9) in a well of a 24-well tissue culture plate. For simultaneous demembranation and CALM extraction, the extraction solution was supplemented with 2.5 mM CaCl2 [21, 23, 26]. In hyperactivation assays involving the use of CALM antagonists or inhibitors, these were included in the demembranation step at concentrations corresponding to the levels used during reactivation. The demembranated suspension was mixed by gently swirling for 30 sec, then 20 µl of the mixture were transferred to another well containing 480 µl of warmed (38°C) reactivation solution. The reactivation solution consisted of 0.2 M sucrose, 1 mM DTT, 25 mM potassium glutamate, 40 mM HEPES, 3 mM MgSO4, 3 mM disodium ATP, 0.5 mM 1,2-bis(2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid (BAPTA), with desired amounts of CaCl2, CALM, and inhibitors noted below. Final concentrations of free Ca2+ were determined in all solutions by using MaxChelator [27] at http://www.stanford.edu/~cpatton/maxc.html, taking into account temperature, pH, and concentrations of ions present, including ATP, Mg2+, and BAPTA.

To assess reactivation of demembranated sperm, 100 µl of sperm suspension from the reactivation wells were transferred to 0.7-mm-deep compartments of a 24-well tissue culture plate cover previously coated with 0.4% agarose to prevent sperm from sticking. Samples were coverslipped and placed on a 38.5°C stage of a Zeiss Axiovert microscope. Sperm were videotaped using Hoffman Modulation Contrast optics at 300x with stroboscopic illumination at 30 Hz (Strobex, Chadwick-Helmuth Co., El Monte, CA). A black-and-white video camera (Model CCD72, Dage-MTI, Inc., Michigan City, IN) was used with a Panasonic AG-7300 Super VHS videocassette recorder (Panasonic Industrial Co., Secaucus, NJ). Elapsed time in 0.01 sec was superimposed on the recorded images using a video timer (Model VTG33; For-A Co., Ltd., Newton, MA), and taping of individual reactivation assays continued for approximately 3 min.

Playback of videotapes was used to determine the extent of reactivation and hyperactivation of demembranated sperm. At least 200 motile sperm per assay were counted, and the proportion of those exhibiting hyperactivated motility was determined. Immotile sperm were not included in the final analysis.

Immunocytochemistry

Sperm (106), either intact, demembranated, or demembranated and Ca2+ extracted, were washed and suspended in 0.5 ml PBS and then fixed by adding 4.5 ml 1% paraformaldehyde in PBS, followed by gentle agitation for 15 min at room temperature. Fixed sperm were recovered by centrifugation, then suspended in 5 ml of 0.2 M glycine in PBS to quench residual fixative and agitated an additional 15 min. Sperm were then either concentrated by centrifugation or diluted with PBS to give a final concentration of 100 x 105/ml. Ten microliters were spotted onto ethanol-cleaned glass slides and allowed to dry overnight. Sperm smears were blocked with 3% BSA in PBS (3 x 20 min) before antibody addition. Intact, nondemembranated sperm were permeabilized with cold methanol for 5 min before the blocking step.

CALM was detected by indirect immunofluorescence in whole or demembranated sperm using a rabbit polyclonal antibody raised against a recombinant protein corresponding to full-length human CALM1 (sc-5537; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Sperm smears were treated with 2 µg/ml antibody in 3% BSA in PBS for 1 h at room temperature. Secondary antibody, goat-anti-rabbit FITC (Sigma #F9887), was used at a dilution of 1:200. CAMK2 was localized to sperm flagella using a mouse monoclonal antibody raised against rat CAMK2 (Clone 6G9, Chemicon International, Temecula, CA) at 10 µg/ml coupled with FITC-conjugated goat-anti-mouse IgG (Sigma #F2057) at a dilution of 1: 200. As controls, sperm were labeled with each secondary antibody alone.

Gel Electrophoresis and Western Blotting

Lysates of whole or demembranated sperm (5 x 106) were prepared by resuspending and boiling PBS-washed sperm pellets in SDS sample buffer [28]. Lysates were clarified by centrifugation and supernatants were resolved by 12% SDS-PAGE [28]. Gels were either stained with silver nitrate [29] or transferred to nitrocellulose [30]. Triton X-100 extracts of sperm were concentrated by centrifugal ultrafiltration (Nanosep, 10 000 mwco; Pall Life Sciences, Ann Arbor, MI), desalted by gel filtration (Protein Desalting Spin Columns; Pierce, Rockford, IL), and diluted with one-fifth volume 6x SDS sample buffer before reduction by boiling in the presence of 40 mM DTT. Samples were resolved by SDS-PAGE and stained or blotted as for sperm lysates.

For Western blot detection of CALM and CAMK2 in sperm lysates or extracts, nitrocellulose membrane blots were blocked overnight at 4°C with 5% BSA in Tris-buffered saline containing 0.05% Tween-20 (TTBS), then probed with either 400 ng/ml anti-CALM or 2 µg/ml anti-CAMK2, HRP-conjugated secondary antibodies, and enhanced chemiluminescence (SuperSignal West Pico; Pierce, Rockford, IL).

Statistical Analysis

Experiments were repeated at least three times and results are expressed as means ± SEM. All data were analyzed using one-way analysis of variance (ANOVA) to test for statistically significant differences among the means of percentage sperm hyperactivated (normalized by the arcsine transformation), followed by a Tukey honestly significant difference (HSD) pairwise comparison test. Dose-response relationships were tested by linear regression with P < 0.05 considered significant. VassarStats interactive web-based freeware, available at http://faculty.vassar.edu/lowry/VassarStats.html, was used for statistical analyses.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sperm Demembranation and CALM Extraction

Immunolabeling of intact bovine sperm localized CALM to the acrosomal ridge, the postacrosomal region, and the flagellum (Fig. 1A). Bovine sperm demembranated using 0.2% Triton X-100 had previously been shown to maintain intact axonemes despite disruption of the plasma membrane and acrosomal and mitochondrial membranes [18]. Consequently, demembranated sperm did not label in the acrosomal region, but retained postacrosomal and flagellar labeling (Fig. 1B). Addition of 2.5 mM Ca2+ to the extraction buffer had been shown to remove a portion of axonemal CALM from sea urchin sperm [20, 21]. When bull sperm were demembranated and extracted with 2.5 mM Ca2+, CALM was removed from flagella, as shown by Western blots (Fig. 2) and by the absence of immunolabeling of demembranated sperm flagella with anti-CALM antibodies (Fig. 1C). Secondary antibody alone produced no labeling in intact, demembranated, or extracted sperm.



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  		FIG. 1. Immunofluorescent localization of CALM in bovine sperm. Fixed permeabilized or demembranated sperm were immunolabeled with anti-CALM antibody and FITC-conjugated anti-rabbit IgG. Secondary antibody alone produced no labeling. A) Methanol-permeabilized whole sperm labeled on the acrosomal ridge, the post-acrosomal region, and the entire flagellum. B) Triton X-100 demembranated sperm. Detergent treatment removed the acrosome and hence acrosomal labeling. C) Sperm demembranated in the presence of 2.5 mM Ca2+ showed only faint head labeling



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  		FIG. 2. Western blot of sperm lysates and extracts probed with anti-CALM demonstrate CALM removal from sperm. Lane 1: purified bovine testis CALM (200 ng); lane 2: whole sperm lysate; lane 3: Triton X-100 extract of sperm; lane 4: lysate of Triton-extracted sperm; lane 5: Ca2+/ Triton extract of sperm; lane 6: lysate of Ca2+/Triton extracted sperm. Lanes 2–6 represent lysates or extracts from 5 x 106 sperm

Reactivation and Hyperactivation of Demembranated Sperm

Sperm demembranated by Triton X-100 in the absence of Ca2+ were largely immotile following transfer to reactivation solutions containing 3 mM ATP but in which free Ca2+ was calculated to be buffered below 1 nM (Fig. 3A). Motility was restored as free Ca2+ was increased to 50 nM and above. Following reactivation with 1 µM free Ca2+, 59.6% ± 3.5% of sperm regained normal activated motility. No further increase in the proportion of motile sperm was observed with the addition of CALM. CALM was also without effect when demembranated sperm were reactivated in <1 nM Ca2+.



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  		FIG. 3. Reactivation of demembranated sperm requires basal levels of Ca2+ for motility and increased Ca2+ for hyperactivation. Sperm were demembranated using 0.2% Triton X-100 then placed into reactivation solution with <1 nM free Ca2+, 50 nM free Ca2+, 1000 nM free Ca2+, 10 µg/ml CALM, or CALM plus 1000 nM Ca2+. A) Total motility (activated + hyperactivated). B) Proportion of motile sperm exhibiting hyperactivated motility upon reactivation. Different letters denote significant differences among treatment groups (P < 0.01). Means ± SEM are shown for three bulls

The extent to which reactivated motile sperm were hyperactivated is shown in Figure 3B. When sperm were demembranated in the absence of added Ca2+ and reactivated in <1 nM Ca2+, less than 20% of motile sperm were hyperactivated regardless of whether or not CALM was added. Hyperactivation occurred in nearly 60% of motile sperm when free Ca2+ was raised to 1 µM during reactivation. Here, too, exogenous CALM failed to increase the proportion of hyperactivated sperm beyond that seen with elevated Ca2+ alone.

Removal of axonemal CALM by Triton X-100 demembranation in the presence of 2.5 mM Ca2+ produced sperm that were largely refractory to reactivation unless exogenous CALM was added (Figs. 4A, 5A). Under Ca2+-free reactivation conditions, few demembranated sperm were motile, and adding either 50 nM Ca2+, 1 µM Ca2+, or 10 µg/ml CALM alone did not reactivate them. However, the addition of both CALM and Ca2+ at either 50 nM or 1 µM significantly increased motility. Both Ca2+ and exogenous CALM were also required to hyperactivate sperm under these conditions, with elevated Ca2+ (1 µM) resulting in increased hyperactivation as compared with 50 nM Ca2+ (Figs. 4B, 5B).



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  		FIG. 4. Reactivation and hyperactivation of CALM-depleted demembranated sperm. Sperm were demembranated using 0.2% Triton X-100 plus 2.5 mM CaCl2 to extract axonemal CALM then placed into reactivation solution with 50 nM free Ca2+, 10 µg/ml CALM plus 50 nM free Ca2+, 1000 nM free Ca2+, or 10 µg/ml CALM plus 1000 nM free Ca2+. A) Total motility (activated + hyperactivated). B) Proportion of motile sperm exhibiting hyperactivated motility upon reactivation. Different letters denote significant differences among treatment groups (P < 0.01). Means ± SEM are shown for four replicates using three bulls



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  		FIG. 5. Reactivation and hyperactivation of CALM-depleted demembranated sperm. Sperm were demembranated using 0.2% Triton X-100 plus 2.5 mM CaCl2 to extract axonemal CALM, then placed into reactivation solution (<1 nM Ca2+), with 10 µg/ml CALM, 1000 nM free Ca2+, or CALM plus Ca2+. A) Total motility (activated + hyperactivated). B) Proportion of motile sperm exhibiting hyperactivated motility upon reactivation. Different letters denote significant differences among treatment groups (P < 0.01). Means ± SEM are shown for four replicates using three bulls

Figure 6 shows still images from videos of intact and demembranated activated and hyperactivated sperm, which can be seen in supplemental Quicktime files (available at http://www.biolreprod.org).



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  		FIG. 6. Representative images from videos of activated and hyperactivated bovine sperm. A) Intact activated sperm, ACTIVE.mov B) Intact hyperactivated sperm, HYPER.mov C) Demembranated activated sperm, DMACTIVE.mov D) Demembranated hyperactivated sperm, DMHYPER.mov. Videos from which these still images were taken can be seen in supplemental files (available at http://www.biolreprod.org). Original magnification x300

Effect of CALM Antagonists and MYLK2 Inhibitors on Hyperactivation

Demembranated, CALM-extracted sperm were reactivated with 1 µM Ca2+ and 10 µg/ml CALM in the presence or absence of CALM antagonists and MYLK2 inhibitors. W-7, an inhibitor of Ca2+/CALM activation of MYLK2 and Ca2+/CALM-dependent phosphodiesterase activity, had no effect on CALM-induced hyperactivation (Table 1). Neither a peptide inhibitor of MYLK2 activation (MYLKp) nor the specific inhibitor of MYLK2 activity, ML-7, were able to reduce the level of hyperactivation among CALM-treated demembranated sperm. Interestingly, when the reactivation solution was supplemented with anti-CALM antibody at a twofold molar excess over added CALM, hyperactivation of reactivated sperm was significantly reduced. Nonimmune rabbit IgG at the same concentration did not inhibit CALM-induced hyperactivation.


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  		TABLE 1. Effect of CALM antagonists on reactivation and hyperactivation of demembranated sperm

CAMK2 Inhibitors Block CALM-Induced Hyperactivation of Demembranated Sperm

Three peptides that block Ca2+/CALM activation of CAMK2 and/or inhibit CAMK2 enzymatic activity were tested for their ability to block stimulation of hyperactivation in demembranated, CALM-depleted sperm. The peptides covered various regions of CAMK2 and contained the CALM-binding domain, the CALM-binding domain plus the autophosphorylation site Thr286 on CAMK2, or the inhibitory domain of CAMK2. Hyperactivation of motile sperm in control assays (no peptide) ranged from 62% to 68%. When present during reactivation, each peptide produced a dose-dependent inhibition of CALM-stimulated hyperactivation (Fig. 7). At the highest level tested (5 µM), hyperactivation was reduced to less than 20% of motile reactivated sperm. Sperm not hyperactivated exhibited normal progressive motility.



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  		FIG. 7. CAMK2 peptides inhibit hyperactivation of demembranated, CALM-extracted sperm. Demembranated, CALM-extracted sperm were reactivated in 10 µg/ml CALM and 1 µM free Ca2+ ± CAMK2 inhibitory peptides. Black squares, dotted regression line: CALM-binding domain (r = –0.96); white circles, solid regression line: Ca2+/CAMK2 inhibitor (r = –0.96); black circles, dashed regression line: CAMK2 Ntide (r = –0.98). Each inhibitory peptide produced a dose-dependent inhibition of hyperactivation (P < 0.01). Results are presented as means ± SEM of the percentage motile sperm exhibiting hyperactivated motility following reactivation (n = 3 bulls)

KN-93, an inhibitor of CAMK2 activation by Ca2+/ CALM, was also tested for its ability to prevent the CALM-induced hyperactivation of demembranated sperm. Sperm were treated with KN-93 for 10 min before and during demembranation under CALM-extracting conditions, then reactivated with 1 µM Ca2+, 10 µg/ml CALM, and KN-93. In the presence of concentrations greater than 5 µM, KN-93 inhibited CALM-induced hyperactivation (Fig. 8). At the highest level tested, 50 µM, KN-93 reduced the proportion of hyperactivated sperm by 89% without reducing the percentage of motile sperm (mean 62% ± 2.5%). The inactive analog, KN-92, had no effect on either motility or hyperactivation. In sperm demembranated with detergent alone, i.e., leaving axonemal CALM in place, KN-93 inhibited hyperactivation upon reactivation with ATP and 1 µM Ca2+. The proportion of motile sperm hyperactivated was reduced from 68% (control) to 26% (25 µM KN-93). Because KN-93 is cell permeable, its effect on intact sperm was also investigated. Sperm were treated for 10 min with 25 µM KN-93, 25 µM KN-92, or 1% DMSO (solvent control), then exposed to 10 mM caffeine to induce hyperactivation [12], and observed immediately after caffeine addition. Caffeine alone produced 96.8% ± 1.7% hyperactivation, while sperm treated with KN-93 exhibited significantly lower levels of hyperactivation (24.8% ± 2.7%, P < 0.01, n = 4 bulls). Hyperactivation in sperm treated with KN-92 was not significantly different from caffeine alone (94.5% ± 2.6%).



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  		FIG. 8. KN-93 inhibits hyperactivation of demembranated, CALM-extracted sperm. Sperm were reactivated in 10 µg/ml CALM and 1 µM free Ca2+ ± KN-93 (black circles) or KN-92 (white circles). KN-93 produced a dose-dependent inhibition of hyperactivation (P < 0.01; r = –0.97) whereas KN-92 had no effect. Results are presented as means ± SEM of the percentage motile sperm exhibiting hyperactivated motility following reactivation (n = 5, 4 bulls)

CAMK2 Localization in Demembranated Bovine Sperm

Demembranated sperm were immunolabeled with anti-CAMK2 ({alpha} subunit) and goat anti-mouse-FITC. CAMK2 was detected along the flagella of both demembranated (Fig. 9A) and CALM-depleted demembranated sperm (Fig. 9B). Extraction of CALM during demembranation had no apparent effect on CAMK2 distribution. Labeling with secondary antibody alone was negative. The presence of CAMK2 in bovine sperm was verified using Western blots of sperm lysates (Fig. 9C). A single band of approximately 50 kDa, corresponding to the {alpha} subunit of CAMK2, was detected in lysates of intact, demembranated, and CALM-extracted sperm.



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  		FIG. 9. Immunofluorescent localization of CAMK2 in bovine sperm. Demembranated sperm were fixed, then immunolabeled with anti-CAMK2 antibody and FITC-conjugated anti-mouse IgG. Secondary antibody alone did not result in labeling. A) Triton X-100 demembranated sperm labeled in the acrosomal region and the flagellum. B) Sperm demembranated in the presence of 2.5 mM Ca2+ to extract CALM showed identical labeling. C) Western blot detection of CAMK2 in bovine sperm. Lane 1: mouse brain cytosol (positive control); lane 2: whole-sperm lysate; lane 3: demembranated sperm; lane 4: CALM-extracted sperm


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results demonstrate, for the first time, that a Ca2+/ CALM CAMK2 signaling pathway initiates hyperactivated motility in sperm. Previous studies have shown that the initiation of hyperactivation is associated with an increase in free intracellular Ca2+ [14, 15] and that agents promoting Ca2+ influx or Ca2+ release from internal stores induce hyperactivation [12, 15]. Models using demembranated mammalian [18, 31–33], avian [34–36], sea urchin [19, 20, 26, 37], and ascidian sperm [38, 39], as well as flagella from the unicellular biflagellate alga Chlamydomonas [22, 23, 40] have been used to demonstrate the direct action of Ca2+ on the axoneme in regulating flagellar waveform. Calcium signaling is mediated through a number of Ca2+-binding proteins, of which CALM is the most ubiquitous. CALM is a component of both ciliary and flagellar axonemes from organisms as diverse as algae, protozoa, and mammals [41– 44].

The actions of Ca2+/CALM (Ca2+-activated CALM) are in some instances promulgated by CALM-binding proteins such as myosin light chain kinase (MYLK2) [35, 36], flagellar radial spoke proteins [45, 46], and the Ca2+/CALM-dependent kinases (CAMK1–4) [47, 48]. MYLK2 or a MYLK-like kinase has been implicated in the temperature-dependent restoration of motility of fowl sperm [35, 36]. MYLK inhibitors prevented the phosphorylation-dependent dephosphorylation of axonemal components critical for the maintenance of motility of both intact and demembranated rooster sperm [35]. The Chlamydomonas radial spoke protein RSP2 has recently been shown to be both a CALM-binding protein and to possess kinase activity [46]. It is postulated to transduce Ca2+ signals, localize CALM within the axoneme, and, through its predicted kinase activity, regulate flagellar motility and waveform patterns. Chlamydomonas pf24 mutants, which contain only trace amounts of RSP2, exhibit paralysis of their flagella [46].

The Ca2+/CALM-dependent kinase CAMK2 is a widely distributed, multifunctional serine-threonine kinase that has been implicated in the regulation of flagellar and ciliary motility [39, 40, 49]. In ascidian sperm, CAMK2 activity is required for induction of motility by the sperm activating and attracting factor (SAAF) [39]. The CALM antagonist W-7 and the CAMK2 inhibitor KN-93 prevented SAAF-induced motility as well as the attendant membrane hyperpolarization and cAMP production necessary for sperm activation [38]. CAMK2 is involved in the regulation of ciliary beat frequency [49] and also stimulates dynein activity in flagellar axonemes of Chlamydomonas [40], thereby increasing microtubule sliding velocity that produces changes in the size and shape of flagellar bends.

Demembranated sperm provide an excellent model for the investigation of sperm motility and hyperactivation by allowing direct access to the axoneme. Bovine sperm are immotile following membrane removal and disruption of the mitochondria. The addition of ATP and Ca2+ restores motility, and as free Ca2+ in the medium is increased to 400 nM or greater, hyperactivation occurs [18]. When sea urchin sperm are demembranated in the presence of millimolar Ca2+, CALM is extracted from the flagellum [26]. We have now demonstrated by Western blots and immunocytochemistry that the same approach can be used to deplete CALM from the flagella of bull sperm. Restoration of motility in these bull sperm required the addition of CALM and ≥50 nM Ca2+ to the reactivation solution. Higher levels of Ca2+ were required in addition to CALM to hyperactivate the sperm. These findings indicate that CALM plays an essential role in activating bovine sperm motility, as seen in sea urchins, fowl, and Chlamydomonas [21, 34, 40].

The roles of CALM in sperm physiology are diverse and include the support of capacitation-related changes in plasma membrane properties [50], modulation of cAMP levels [39, 51, 52], and the ability to undergo the acrosome reaction [50, 52]. CALM operates on various targets in sperm. Sperm contain the Ca2+/CALM-dependent cyclic nucleotide phosphodiesterase PDE1 [52], and CALM also regulates the activity of human sperm protein phosphatases [32] and protein phosphatase 2B (PPP3CA, calcineurin) in hamster sperm [53]. To identify the targets of Ca2+/CALM in sperm hyperactivation, we tested various CALM antagonists for their ability to block CALM-dependent hyperactivation. W-7, a CALM antagonist and inhibitor of PDE1 and MYLK2 activation, failed to block Ca2+/CALM-induced hyperactivation. Although W-7 was previously shown to reduce the proportion of hyperactivation among intact epididymal mouse sperm [54], in our study, W-7 produced no observable changes in motility of demembranated sperm (Table 1). Similarly, the motility of demembranated fowl spermatozoa was not inhibited by the addition of W-7 or trifluoperazine [34]. W-7 did not impair reactivation of demembranated carp sperm; however, motility of intact sperm was completely blocked [55]. Other inhibitors also had no effect on bull sperm hyperactivation. Neither direct inhibition of MYLK2 by ML-7 nor inhibition of Ca2+/ CALM activation of MYLK2 by a CALM antagonist peptide were able to prevent CALM-induced hyperactivation, indicating that PDE1 and MYLK2 are not involved in regulating hyperactivation at the axoneme.

Pharmacological studies have implicated CAMK2 in sperm capacitation and initiation of the acrosome reaction [50] and motility [39]. We report here, for the first time, the flagellar localization of CAMK2 in mammalian sperm and its role in hyperactivation. In contrast with the loss of CALM during demembranation in the presence of Ca2+, CAMK2 remained associated with the axoneme of demembranated sperm. The Ca2+/CALM induced hyperactivation was inhibited in a dose-dependent manner in demembranated sperm treated with KN-93, an inhibitor of CAMK2, but not by KN-92, an inert analog. KN-93 inhibited hyperactivation, but not reactivation, in sperm that had been demembranated with Triton X-100 alone, leaving axonemal CALM in place, and also inhibited caffeine-induced hyperactivation in intact sperm. In addition, three peptide inhibitors of CAMK2 produced a dose-dependent inhibition of hyperactivation without inhibiting reactivation of motility.

When antibodies to CALM were added in the reactivation solution along with CALM, thereby reducing its availability, the sperm reactivated without hyperactivating, despite the presence of sufficient Ca2+. This indicates that hyperactivation involves recruitment of additional Ca2+/ CALM to the axoneme above that required for activation of motility. This additional CALM may be the activator of CAMK2.

Increasing Ca2+ in the reactivation medium produces the asymmetrical bending characteristic of hyperactivation by increasing the amplitude of the principal flagellar bend while leaving the reverse bend unchanged [18]. This indicates that the downstream targets of the Ca2+/CALM CAMK2 pathway allow or stimulate increased microtubule sliding on one side of the axoneme. Pathway components may thus be concentrated on one side of the flagellum. Identifying these targets should elucidate how sliding is enhanced by activation of CAMK2 and subsequent phosphorylation.


   ACKNOWLEDGMENTS
 
The authors thank Bryan Krick and the staff of Genex Cooperative (Ithaca, NY) for supplying bovine semen. We would also like to thank Dr. Mark Roberson for providing anti-CALM antibody.


   FOOTNOTES
 
1 Supported by grant MCB-0421855 from the National Science Foundation. Back

2 Correspondence: Susan S. Suarez, Department of Biomedical Sciences, Cornell University, T5-002B Veterinary Research Tower, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853. FAX: 607 253 3541; sss7{at}cornell.edu Back

Received: 4 February 2005.

First decision: 7 March 2005.

Accepted: 27 April 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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