SEARCH

SEARCH BY CITATION

Keywords:

  • Motility;
  • flagellum

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

ABSTRACT: Both cyclic AMP (cAMP)/protein kinase A (PKA) and calcium (Ca2+) signaling pathways are known to be involved in the regulation of motility in mammalian sperm. Calmodulin (CaM) is a ubiquitous Ca2+ sensor that has been implicated in the acrosome reaction. In this report, we identify an insoluble pool of CaM in sperm and show that the protein, in addition to its presence in the acrosome, is found in the principal piece of the flagellum. These findings are consistent with, though not proof of, the presence of a pool of CaM in the fibrous sheath. The Ca2+/CaM-dependent protein kinase IIβ (CaMKIIβ), which is a downstream target of Ca2+/CaM, similarly localizes to the principal piece. In addition, we confirm earlier reports that a CaM inhibitor decreases sperm motility. However, we find that this inhibition can be largely reversed by stimulation of PKA if substrates for oxidative respiration are present in the medium. Our results suggest that the Ca2+/CaM/CaMKII signaling pathway in the sperm principal piece is involved in regulating sperm motility, and that this pathway functions either in parallel with or upstream of the cAMP/PKA pathway.

Sperm motility is central to male fertility in mammals. However, we do not have a full understanding of the signaling pathways that regulate flagellar function. Two pathways have emerged as key regulators of normal mammalian activated and hyperactivated motility. These are the cyclic AMP(cAMP)/protein kinase A (PKA) pathway and Ca2+ signaling (Heffner and Storey, 1981; Suarez et al, 1987; Tash and Means, 1987; Lindemann and Goltz, 1988; White and Aitken, 1989; Brokaw, 1991; Yanagimachi, 1994; Ho et al, 2002; Nolan et al, 2004; Marin-Briggiler et al, 2005).

There is evidence to suggest that the cAMP-dependent phosphorylation of flagellar proteins is involved in the initiation and maintenance of sperm motility (Tash and Means, 1982; Tash and Means, 1983; San Augustin and Witman, 1994). Since PKA is a major downstream target of cAMP in sperm, it likely that this kinase plays a central role in these phosphorylation events (Visconti et al, 1997). As further support of a role for PKA in sperm function, it has been shown that mice that lack the male germ cell-specific catalytic subunit of PKA (Cα2) are infertile due to several abnormalities, including aberrations of motility (Nolan et al, 2004).

Extracellular Ca2+ is required for motility in most epididymal sperm samples, and Ca2+ is known to regulate both activated and hyperactivated motility (Suarez et al, 1987; Tash and Means, 1987; Lindemann and Goltz, 1988; White and Aitken, 1989; Yanagimachi, 1994; Ho et al, 2002). One mechanism by which Ca2+ is directly linked to flagellar function is through its regulation of the atypical, “soluble” adenylyl cyclase, sAC, which generates cAMP and is required for sperm motility (Jaiswal and Conti, 2003; Litvin et al, 2003; Esposito et al, 2004).

Calmodulin (CaM) is a ubiquitous, highly conserved, 17-kd protein that serves as a classical intracellular Ca2+ receptor (Means et al, 1982). At least some of the effects of Ca2+ on the flagellum are likely to be achieved through CaM, since inhibition of CaM decreases sperm motility (White and Aitken, 1989; Ahmad et al, 1995; Si and Olds-Clarke, 2000). Interestingly, the effects of Ca2+ on sAC are independent of CaM (Jaiswal and Conti, 2003; Litvin et al, 2003), which suggests that Ca2+ affects motility via multiple pathways, only some of which require CaM.

Since sperm are highly compartmentalized, proteins must be targeted accurately to the appropriate region(s) of the cell. Thus, proteins involved directly in the regulation of motility typically localize to the flagellum. We use indirect immunofluorescence to show that CaM is present in the principal piece of the flagellum. In addition, we show that a portion of sperm CaM is insoluble, consistent with its localization to the cytoskeleton and similar to the extraction profile of a known fibrous sheath (FS) protein (the pro-domain of pro-AKAP4). These findings indicate that a pool of CaM localizes to the flagellum and possibly to the FS, an insoluble accessory structure that is found exclusively in the principal piece of the mammalian flagellum. We also suggest that CaM is involved in the regulation of sperm motility, since a CaM inhibitor decreased motility. This inhibition was largely reversed by stimulation of PKA, but only when lactate and pyruvate were present in the medium. Furthermore, the Ca2+/CaM-dependent protein kinase IIβ (CaMKIIβ), which is a downstream target of Ca2+/CaM, colocalized with CaM in the principal piece, which suggests that a Ca2+/CaM/CaMKII signaling pathway is present in the sperm principal piece.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Immunologic Reagents

Polyclonal rabbit antiserum against CaM (Zymed, San Francisco, Calif) was used for immunoblotting. For indirect immunofluorescence, a monoclonal mouse anti-CaM antiserum was used (Upstate, Lake Placid, NY). The monoclonal anti-CaMKIIβ antibody was obtained from Zymed. Rabbit polyclonal antiserum against the pro-domain of pro-AKAP4 (anti-DYSKIPSEN) was generously provided by Drs George L. Gerton and Stuart B. Moss of the University of Pennsylvania (Johnson et al, 1997).

The secondary antiserum used for most of the immunoblotting experiments was HRP-conjugated donkey anti-rabbit IgG (Amersham Biosciences, Piscataway, NJ). For membranes probed with the anti-CaMKIIβ antiserum, an alkaline phosphatase conjugated goat anti-mouse secondary antiserum was used (Jackson Immunoresearch Laboratories, West Grove, Pa). The secondary antiserum used for indirect immunofluorescence was Alexa Fluor 594-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, Ore).

Tissue and Cell Preparation and Protein Isolation

Sperm were collected from the cauda epididymides of retired breeder CD-1 mice (Taconic ICR) (Travis et al, 2001a). Sperm suspensions were washed in PBS that contained a protease inhibitor cocktail (Complete Tablets; Roche, Indianapolis, Ind). After the final wash, sperm pellets were mixed with SDS sample buffer that contained 40 mM DTT and boiled for 5 minutes. Samples were centrifuged and the supernatants, which contained the extracted proteins, were saved.

Mixed germ cells were prepared from decapsulated adult mouse testes by sequential dissociation with collagenase and trypsin/DNAse I, and individual germ cell types were separated by sedimentation velocity (STAPUT), as previously described (Romrell et al, 1976; Bellve et al, 1977). Protein was extracted from the resulting germ cell pellets as described above for sperm.

The amount of protein in each sample was determined using the Bradford assay with the test sample diluted such that the final DTT and SDS concentrations were within the limits specified by the manufacturer of the assay (Bio-Rad Laboratories, Hercules, Calif).

Immunoblotting

Proteins were separated under reducing conditions by SDS-PAGE on either 9% (for detection of CaMKII), 4–15% gradient precast (for detection of CaM) or 12.5% (for detection of the pro domain of pro-AKAP4) gels. After electrophoresis, proteins were electrophoretically transferred to polyvinylidene fluoride membranes (Immobilion-P Transfer Membranes, Millipore Corp, Bedford, Mass; 10 μg protein/lane unless otherwise stated). After blocking and washing, the membranes were probed for 1 hour at room temperature or overnight at 4°C with the appropriate primary antibody: anti-CaM (1:5000 [v/v]), anti-DYSKIPSEN (1:5000 [v/v]) or anti-CaMKIIβ (1:500 [v/v]). The membranes were washed and then probed with the appropriate secondary antibody diluted 1:5000 (v:v), as previously described (Carrera et al, 1994; Johnson et al, 1997; Eshete and Fields, 2001).

After additional washes, blots probed with HRP-conjugated secondary antibodies were overlaid with ECL reagents (Amersham, Piscataway, NJ) and developed before being exposed to Reflection film (New England Nuclear, Boston, Mass). Blots probed with an alkaline phosphatase-conjugated secondary antibody were developed using ECF substrate (Amersham), and fluorescence was detected using the Storm 860 system (Molecular Dynamics, Sunnyvale, Calif).

Immunoblots that were to be probed with the anti-CaM antibody were treated as previously described, to improve the retention of CaM on the membrane (Yang et al, 2001). Briefly, the polyacrylamide gels were presoaked in KP buffer (25 mM KH2/K2HPO4 [pH 7.0]) for 15 minutes prior to electrophoretic transfer to membranes. The membranes were prewet in methanol and washed for 15 minutes in the KP buffer prior to transfer. Transfer of proteins was performed in KP buffer overnight at 4°C. Following transfer, membranes were soaked in 0.2% glutaraldehyde in KP buffer, and then blocked and probed using the techniques described above.

Sperm Fractionation

Fractionation of sperm was carried out as previously described (Travis et al, 2001b). Briefly, sperm were lysed by Dounce homogenization and sonication in PBS in the presence of protease inhibitors (Complete Protease Inhibitor Cocktail; Boehringer Mannheim, Mannheim, Germany). The mixture then was centrifuged at 10 000 × g for 10 minutes at 4°C and separated into supernatant (S10) and pellet (P10) fractions. An aliquot of the S10 fraction was then centrifuged at 100 000 × g for 1 hour, yielding a pellet of membranes and associated proteins (P100) and a supernatant fraction (S100). An aliquot of the membrane pellet was suspended in PBS that contained protease inhibitors and 0.5% Triton X-100, incubated for 15 minutes at 4°C, and centrifuged again at 100 000 × g for 1 hour, yielding a detergent-soluble supernatant and a detergent-insoluble pellet. The appearance of a protein in the detergent-insoluble membrane fraction has sometimes been used to indicate localization to membrane subdomains known as lipid rafts (regions enriched in sterols and sphingolipids, as opposed to phospholipids). However, assigning this designation based solely on detergent insolubility is susceptible to artifact, due to perturbation of the membranes by the detergent from their physiologically native state (Munro, 2003). Nonetheless, the differentiation between detergent-soluble and detergent-insoluble partitioning is a useful initial biochemical screen, which can provide information on how a protein might associate with a membrane. We performed 3 separate membrane preparations, and performed immunoblot analysis on each of the 3 associated blots. Identical results were obtained each time.

Indirect Immunofluorescence of Sperm and Testis Sections

Ten-microliter aliquots of sperm diluted in PBS to 60 × 106 cells/mL were transferred onto microscope slides, smeared, and air-dried. Cells were then acetone-fixed on the slides for 15 seconds, dried, and then permeabilized in cold methanol for 15 minutes. Slides were washed with PBS and blocked for 1 hour at room temperature in 3% non-fat milk, before being washed again in PBS.

Sperm were incubated in the appropriate secondary antibody diluted to 5 μg/mL in 10% goat serum. The slides were then washed and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) before being coverslipped. Images were obtained using a fluorescent microscope (Leica Microsystems, Bannockburn, Ill) equipped with the Open Lab software (Improvision, Lexington, Mass). Paired sample and control images were exposed for equal amounts of time.

Assessment of Sperm Motility Following Addition of CaM Antagonist

N-(6-Aminohexyl)-5-chloro-1-naphthalenesufonamide hydro-chloride (W-7) is a water soluble and cell-permeable competitive antagonist of CaM (Calbiochem, San Diego, Calif). The W-7 IC50 values for CaM-dependent enzymes range from 25 μM to 50 μM (Hidaka and Kobayashi, 1992). Concentrations of up to 100 μM have been used to assess the role of CaM in sperm motility (Si and Olds-Clarke, 2000; Morita et al, 2006). A stock solution of W-7 was prepared in DMSO to a final concentration of 10 mM and stored at −20°C.

Male Swiss Webster mice (Taconic, Germantown, NY) between 10 and 12 weeks of age were killed by cervical dislocation. The cauda epididymides were removed, minced, and placed in a modified Whitten medium (22 mM HEPES [pH 7.4], 1.2 mM MgCl2, 100 mM NaCl, 4.7 mM KCl, 1 mM pyruvic acid, 4.8 mM lactic acid, 3 mM CaCl2, 5.5 mM glucose, 10 mM sodium bicarbonate) (Travis et al, 2001a) for 10 minutes to allow the sperm to swim out. The resulting liquid was filtered through a 70-μm cell strainer (BD Biosciences, San Jose, Calif) to remove tissue debris, and then placed in a water bath at 37°C. The motility of untreated sperm was determined using a computer-assisted sperm motion analysis system (IVOS Analyzer; Hamilton Thorne Research, Beverly, Mass). A progressively motile cell was defined as having Path Velocity (VAP) greater than 79 μm/s and Straightness (STR) greater than 60%. VAP is defined as the total distance a sperm moves over time, while STR is a measure of the straightness of the sperm track (defined as progressive velocity divided by VAP). The sperm solution was loaded into a 20-μm chambered slide (Leja Products, Amsterdam, The Netherlands) and viewed with the 10× objective on the IVOS Analyzer. Distal droplets were excluded from analysis using the sort option with the following parameters: head size greater than 9 pixels and beat frequency of more than 4 μm/s.

To analyze the effects of W-7 on sperm motility, 100-μL aliquots of sperm were placed in different microcentrifuge tubes and incubated with 2.5 μM to 200 μM W-7 in DMSO. Solvent controls were treated with an equivalent amount of DMSO. The resulting mixtures were incubated at 37°C and sperm motility was assessed at 0, 5, 10, 20, and 30 minutes after the addition of DMSO/W-7. The motility results are expressed as both the number of progressively motile cells divided by the number of total cells (% progressive motility) and as the number of motile cells divided by the number of total cells (% total motility).

To test whether components of the sAC/PKA pathway could restore motility to CaM-inhibited cells, sperm suspended in complete medium were treated with 1 mM dibutyryl cAMP (db-cAMP; Sigma Chemical Co, St Louis, Mo) and 100 μM 3-isobutyl-1-methylxanthine (IBMX, Sigma) or 3 mM 8-bromo cAMP (Sigma) and 100 μM IBMX, all diluted in water, 1 minute after treatment with 100 μm W-7. In a parallel assay, the same treatment was given to sperm suspended in medium with lactate and pyruvate omitted, in order to reproduce the conditions used previously (Si and Olds-Clarke, 2000). Sperm motility was recorded prior to and immediately after treatment. Control samples were treated with W-7, followed by water without cAMP/IBMX.

Statistical Analysis

The Wilcoxon signed rank test was used to determine differences between sample motilities. A P-value of < .05 was considered statistically significant.

Ethical Guidelines

The care and use of all animals used in this study were within standard ethical guidelines, as approved by the University of Pennsylvania or Cornell University Institutional Animal Care and Use Committees.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

CaM is expressed in germ cells and localizes to the acrosome and principal piece of mature sperm. To determine the expression patterns of CaM in the testes and sperm, we performed immunoblotting and indirect immunofluorescence experiments. A protein of the predicted size for CaM (Mr 16.3) was identified on immunoblots that contained sperm and mixed germ cell protein extracts (Figure 1A). Further separation of mixed germ cells into pachytene spermatocytes and round and condensing spermatids indicated that CaM was present in each examined germ cell stage (Figure 1B). Similarly, indirect immunofluorescence analysis of whole testis sections indicated that CaM was present in all germ cell stages (Figure 1C and D). Some immunofluorescence was detected within the lumen of the seminiferous tubule, suggesting that CaM was present in the flagella of testicular sperm.

image

Figure 1. . CaM is expressed in germ cells and is present in the acrosome and principal piece of mature sperm. (A) Immunoblot analysis of sperm and mixed germ cell proteins probed with an anti-CaM antibody. A band consistent with the size of CaM is present in both lanes. (B) Immunoblot analysis of separated germ cell proteins probed with the same anti-CaM antibody. Bands consistent with CaM are present in all germ cell stages examined. P, Pachytene spermatocytes; R, round spermatids; C, condensing spermatids; MGC, mixed germ cells. The numbers to the left of both blots indicate relative molecular weights in kd. (C) Indirect immunofluorescence analysis of adult mouse testis using the anti-CaM primary antibody. Immunofluorescence is identified in essentially all the germ cell stages. Some faint immunofluorescence is also present within the seminiferous tubule lumen, suggesting that CaM is also present in testicular sperm. (D) Paired bright-field image of (C). (E) Indirect immunofluorescence of cauda epididymal mouse sperm using the anti-CaM primary antibody. Immunofluorescence is present in the acrosome and in the principal piece of the flagellum (green). Sperm nuclear DNA is labeled with DAPI (blue). Incubation of sperm with nonimmune serum produced no significant signal (data not shown). (F) Paired bright-field image of (E).

Download figure to PowerPoint

To determine more definitively whether CaM was present in the sperm flagellum, we performed indirect immunofluorescence analysis of cauda epididymal sperm using the anti-CaM antibody. Fluorescence was detected in the acrosome and along the full length of the flagellar principal piece (Figure 1E and F). Since the only structure exclusive to the principal piece is the FS, this finding is consistent with the possibility that, in addition to the acrosome, CaM is present in the FS.

CaM Is Found in the Soluble and Insoluble Fractions of the Sperm Cell

To determine the solubility of CaM in sperm, we fractionated mouse sperm and performed immunoblot analysis of each fraction using the anti-CaM antibody. We identified pools of CaM in the insoluble (P10) sperm fraction, the soluble S10 sperm fraction, and some of its derivatives, including the S100, total membrane, and detergent-soluble membrane fractions (Figure 2). No CaM was found in the detergent-insoluble membrane pellet.

image

Figure 2. . Biochemical extraction of sperm components indicates that an FS-specific protein (pro-domain) and a pool of CaM are present in an insoluble fraction of the sperm cell. Immunoblots of fractionated sperm extracts probed with an antibody raised against the pro-domain of pro-AKAP4 (anti-DYSKIPSEN [top]) and anti-CaM (bottom). Lane 1, Whole sperm extract; lane 2, S10 fraction; lane 3, P10 fraction; lane 4, S100 fraction; lane 5, P100 (total membrane) fraction; lane 6, detergent-soluble membrane fraction; lane 7, detergent-insoluble membrane fraction. Essentially all the pro-domain partitions into the insoluble P10 fraction, consistent with localization to the fibrous sheath and with previous reports (Johnson et al, 1997). A proportion of CaM also partitions to the P10 fraction. However, CaM partitions in a relatively complex manner since, in addition to the insoluble P10 fraction, some CaM is found in the soluble S10 fraction and its derivatives, the S100, P100, and detergent-soluble fractions. CaM does not appear to be present in the detergent-insoluble fraction. The numbers to the left indicate relative molecular weights in kd.

Download figure to PowerPoint

We compared the fractionation pattern of CaM to that of a known FS protein, the pro-domain of pro-AKAP4. Pro-AKAP4 is the precursor of AKAP4, a major A-kinase anchor protein (AKAP) of the mammalian sperm FS (Carrera et al, 1994; Fulcher et al, 1995; Turner et al, 1998; Mandal et al, 1999; Moss et al, 1999; Jha and Shivaji, 2002). In the developing sperm flagellum, pro-AKAP4 is transported to the FS/principal piece, where most of it is processed by the proteolytic cleavage of the amino-terminal 179 amino acids to produce mature AKAP4 and the free, 179-amino acid pro-domain. Some proportion of the free pro-domain persists in cauda epididymal sperm as an insoluble component of the FS (Johnson et al, 1997). Consistent with this previous report, we identified the pro-domain only in the insoluble (P10) fraction (Figure 2).

Thus, both the pro-domain and CaM are present in an insoluble fraction of cauda epididymal sperm. While the pro-domain was exclusive to this fraction, the CaM fractionation pattern was broader. These findings are consistent with the localization of a pool of CaM to the FS, and suggest that CaM has roles both within and outside the flagellum.

Sperm Motility Is Inhibited by a CaM Antagonist and Partially Recovers After Stimulation of PKA

As CaM is present in the principal piece and since Ca2+ has long been implicated in the regulation of flagellar motility, we hypothesized that some of the effects of Ca2+ on sperm motility might be mediated by CaM. To test this hypothesis, we examined the effects of a CaM antagonist, W-7, on murine sperm motility. Cauda epididymal sperm were allowed to swim out into modified Whitten medium, and were then exposed to various concentrations of W-7. As previously reported, sperm treated with W-7 showed concentration-dependent declines in total motility and progressive motility, as compared to controls (data not shown) (Si and Olds-Clarke, 2000). The decline in progressive motility was due to declines in both VAP and STR. The W-7 concentration of 100 μM proved to be the most effective in our hands (Figure 3). Previous studies have demonstrated that this reduction in motility is not due to a toxic effect of W-7 on sperm (Si and Olds-Clarke, 2000).

image

Figure 3. . The addition of IBMX/cAMP restores motility to W-7-treated sperm only when sperm are incubated in the presence of pyruvate and lactate. Cauda epididymal sperm were incubated in the presence ([+] pyruvate/lactate) or the absence ([-] pyruvate/lactate) of the mitochondrial substrates pyruvate and lactate and exposed to either no treatment (control) or to 100 μM W-7. W-7 significantly reduces the percentages of motile sperm in both media compared to controls (P < .05). Subsequent addition of IBMX/8-bromo cAMP to W-7-treated sperm results in a significant increase in sperm motility in sperm incubated with pyruvate and lactate (P < .05). This restored motility approaches the motility levels of the untreated controls (P = .06). This increase in motility is not observed in sperm incubated in the absence of pyruvate and lactate. Comparable results are obtained for progressive motility and for sperm treated with W-7 and IBMX/db-cAMP. Bars represent the means standard deviations from 3 separate experiments. Different letters indicate significant differences.

Download figure to PowerPoint

Although a concentration of 100 μM W-7 has been used by us and others to study the role of CaM in sperm motility, we can not rule out the possibility that W-7 at this dose may affect other enzymes in addition to CaM. Therefore, some of the decreases in motility that we identified at the higher concentrations of W-7 may not be solely attributable to the inhibition of CaM.

It has been reported that the effects of Ca2+ on sAC are independent of CaM (Jaiswal and Conti, 2003; Litvin et al, 2003). If this is correct, part of the effect of Ca2+/CaM on motility must be achieved through a pathway that is separate from sAC/PKA or from an effect on that pathway either upstream or downstream of sAC. We tested whether components of the sAC/PKA pathway could restore motility to W-7-treated sperm suspended in a complete medium that contained glucose, pyruvate, and lactate. Motility was inhibited by the addition of 100 μM W-7. One minute after exposure to W-7, sperm were treated with either 1 mM db-cAMP and 100 μM of the phosphodiesterase inhibitor IBMX or 3 mM 8-bromo cAMP and 100 μM IBMX. The addition of either db-cAMP/IBMX or 8-bromo cAMP/IBMX resulted in significant increases in both total and progressive sperm motility associated with increases in both VAP and STR (P < .05) (Figure 3). This restored motility approached the motility levels of the untreated controls (P = .06).

The experiment was then repeated in the same medium, except that lactate and pyruvate were omitted (Si and Olds-Clarke, 2000). Under these conditions, there was no increase in either the total or progressive motility of sperm treated with W-7/cAMP/IBMX compared to sperm treated only with W-7 (Figure 3). These data suggest that the stimulation of PKA can partially compensate for the reduction in sperm motility seen following inhibition of CaM by W-7 but that lactate and pyruvate are required for this rescue to occur. In addition, these data confirm that the reduction in sperm motility seen following the addition of W-7 is not due to a toxic effect of W-7 on sperm.

CaMKII Is Present in Sperm and Localizes to the Principal Piece

In somatic cells, many of the actions of Ca2+ are mediated by its interaction with CaM and the subsequent activation of a variety of Ca2+/calmodulin-dependent protein kinases (CaMK). Several CaMK isoforms have been reported in the testis. These include CaMKII, CaMKIV and Pnck in the mouse, and CamKIIN, CaMK-gr, and CaMKIβI in the rat (Frangakis et al, 1991; Jones et al, 1991; Naito et al, 1997; Gardner et al, 2000; Moriya et al, 2000; Chang et al, 2001). More recently, CamKIV and CaMKIIα have been identified in the flagella of human and bovine sperm, respectively (Ignotz and Suarez, 2005; Marin-Briggiler et al, 2005). CaMKII has also been identified in ascidian sperm, where it plays a role in sperm motility (Nomura et al, 2004).

Given the reports on the presence of CaMK isoforms in testes and sperm, and since we had evidence that CaM was involved in the regulation of murine sperm motility, we searched for a CaMK isoform in murine sperm. Since CamKII had been reported in murine testes, we performed immunoblot analysis on mouse sperm protein extracts using an antibody against CaMKIIβ and identified a band of Mr 62 500 (Figure 4A). This band was identical to a control band identified in hippocampus extract (Kelly et al, 1984; Scholz et al, 1988). Note that the anti-CaMKIIβ antibody it not absolutely specific for the β-isoform. This antibody also recognizes CaMKIIγ, particularly if the γ isoform is more abundant. Therefore, our results raise the possibility that the γ isoform is also present in sperm.

image

Figure 4. . CaMKIIβ, a downstream target of CaM, is present in sperm and localizes to the principal piece. (A) Immunoblot of sperm proteins using the anti-CaMKIIβ antibody. The positive control (+) is 1 μg of hippocampus. A band identical to the hippocampal CaMKII band is present in the sperm extract lane (Sp), which contains proteins extracted from 2 million sperm equivalents. The numbers to the left of the blot indicate relative molecular weights in kd. (B) Indirect immunofluorescence of cauda epididymal sperm using the anti-CaMKIIβ antibody. Immunoreactivity is present exclusively in the principal piece of the flagellum (green). Sperm nuclear DNA is labeled with DAPI (blue). (C) Paired bright-field image of (B).

Download figure to PowerPoint

As we had evidence to suggest that a Ca2+/CaM signaling pathway is involved in mammalian sperm motility, we looked for CaMKII in the flagella of mouse sperm. Indirect immunofluorescence of cauda epididymal sperm using the same anti-CaMKII antibody revealed that, similar to CaM, CaMKII was present specifically in the flagellar principal piece (Figure 4B and C). These results reveal that the components of a putative Ca2+/CaM/CaMKII signaling pathway are present in the sperm flagellar principal piece.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

Using both indirect immunofluorescence and immunoblotting, we have shown that CaM is present in all germ cell stages, including sperm. The indirect immunofluorescence and fractionation studies reveal a complex distribution pattern of CaM in sperm. Immunofluorescence localized CaM to both the principal piece and the acrosome. The fractionation studies suggest that CaM is found in both insoluble structures (eg, cytoskeletal structures, such as the FS) and soluble structures found in the S10 fraction and its derivatives, the S100, P100, and detergent-soluble membrane fractions. CaM was not found in the detergent-insoluble membrane pellet. Consistent with this broad distribution of CaM in sperm, there is convincing evidence, including the evidence presented here, that Ca2+/CaM is involved in multiple functions in sperm, including motility, capacitation, and the acrosome reaction (Jones et al, 1980; Si and Olds-Clarke, 2000; Bendahmane et al, 2001; Lopez-Gonzalez et al, 2001). Some of the effects of Ca2+/CaM on sperm motility and capacitation may involve calcineurin (Tash et al, 1988; Tash and Bracho, 1994; Carrera et al, 1996). Other studies have identified a link between CaM and the regulation of sperm T-type Ca2+ currents (Lopez-Gonzalez et al, 2001).

Our findings of an insoluble pool of CaM in sperm and of CaM in the principal piece are consistent with, though not proof of, the existence of a pool of CaM in the FS (Tash and Means, 1987; Tash et al, 1988). It has been suggested that PKA is anchored to the FS through 1 or more AKAPs (Carrera et al, 1994; Mei et al, 1997; Miki and Eddy, 1998; Turner et al, 1998; Mandal et al, 1999; Vijayaraghavan et al, 1999). If CaM also is associated with the FS, then components of both of the major sperm motility signaling pathways (cAMP/PKA and Ca2+ signaling through CaM) find homes in this important accessory structure. Regardless of whether or not CaM is present in the FS, its presence in the flagellar principal piece provides indirect evidence for a role of CaM in the regulation of sperm motility. Inhibition of sperm motility by the addition of the CaM antagonist W-7 provides more direct evidence for this role.

It has previously been reported that 8-br-cAMP and IBMX do not restore motility to W-7-treated sperm when the sperm are incubated in a medium that contains glucose but lacks pyruvate and lactate (Si and Olds-Clarke, 2000). Our results concur with this finding. However, similar to reports on demembranated ascidian sperm (Nomura et al, 2000), we found that if lactate and pyruvate were present in the medium, then IBMX and either 8-br-cAMP or db-cAMP could largely compensate for the loss of function of CaM. Taken together, these findings provide insight into the signaling and metabolic control of mammalian sperm motility. In the mouse, it has been demonstrated that glycolysis produces ATP in the principal piece that is essential for fully normal sperm motility and for the phosphorylation events that are believed to facilitate the regulation of motility (Travis et al, 2001a; Miki et al, 2004; Mukai and Okuno, 2004). Our data show that CaM functions in the principal piece in that, when CaM is inhibited, sperm motility is significantly decreased. In addition, without substrates for oxidative respiration (lactate and pyruvate), and even in the presence of a substrate for glycolysis (glucose), increased intracellular cAMP cannot restore motility to CaM-inhibited sperm. This suggests the possibility that CaM is involved in the regulation of glycolysis or in the utilization of glycolytic ATP. There have been several previous reports linking Ca2+/CaM to the regulation of glycolytic enzymes in somatic cells (Ashkenazy-Shahar et al, 1998; Ashkenazy-Shahar and Beitner, 1999; Singh et al, 2004). Thus, it is possible that W-7, by inhibiting CaM, indirectly inhibits glycolysis.

As they are substrates for oxidative respiration, lactate and pyruvate may be able to compensate partially for the lack of glycolytic ATP by enabling the production of ATP in the midpiece. It has been shown that the production of ATP in the midpiece in the absence of glycolytic ATP can support normal motility for short periods of time (Mukai and Okuno, 2004). It is unclear whether some of this ATP is able to move to the proximal principal piece or whether it remains entirely in the midpiece. Regardless, these data show the partially compensatory abilities of the metabolic pathways that support flagellar motility, and suggest a new potential function for CaM in sperm (ie, the regulation of glycolysis in the principal piece).

Other than the addition of lactate and pyruvate to our medium, there were other more subtle differences between our experiments (in which agonists of the PKA pathway were able to restore motility to CaM-inhibited sperm) and those of Si and Olds-Clarke (in which agonists of the PKA pathway were unable to restore motility to CaM-inhibited sperm). For example, we utilized a different mouse strain in our experiments. Therefore, we can not rule out the possibility that the relationship of the CaM pathway to the PKA pathway may not be identical in all genetic backgrounds.

Although the addition of cAMP/IBMX to W-7-treated sperm significantly increased motility compared to sperm treated with W-7 alone, it should be noted that motility was still slightly but significantly less than the motility seen in the untreated control sample (P = .06 for 8-bromo cAMP/IBMX). This finding still argues strongly for our hypothesis that agonists of the PKA pathway rescue the inhibited CaM pathway. However, another possible explanation for this incomplete restoration of motility is that, at the 100 μM concentration used in our study, W-7 may have been inhibiting other enzymes in addition to CaM. If this was the case, then it is possible that cAMP/IBMX rescues these other pathways, and that the remaining sperm motility deficit that persists in the presence cAMP/IBMX is due to the still-inhibited CaM pathway.

Ca2+ also affects sperm motility through its role as a regulator of the predominant flagellar cyclase, sAC, which catalyzes the synthesis of cAMP to activate the PKA pathway. This cyclase is molecularly and biochemically distinct from the transmembrane ACs (tmACs), in part because sAC is uniquely sensitive to both bicarbonate and Ca2+ (Buck et al, 1999; Chen et al, 2000; Wuttke et al, 2001; Liguori et al, 2004). However, the effects of Ca2+ on sAC are independent of CaM (Jaiswal and Conti, 2003; Litvin et al, 2003). Therefore, although it is known that sAC is required for sperm motility (Esposito et al, 2004) and sAC is regulated by Ca2+, the effect of CaM on motility is not achieved via this cyclase.

These observations support a model in which Ca2+ affects motility at 2 different points within the sAC pathway. Alternatively, separate calcium signaling pathways may exist; one that is independent of CaM (eg, sAC/PKA) and one that is not. Since deletion of the sAC gene results in immotile sperm, it is clear that Ca2+/CaM cannot compensate for the loss of sAC function. However, our data suggest that IBMX/cAMP (ie, agonists of the PKA pathway) can restore motility when CaM is inhibited. Thus, components of the sAC/PKA pathway can compensate for a loss of function in the Ca2+/CaM component of the pathway(s), provided that the metabolic substrates pyruvate and lactate are present. The mechanism for this restoration of motility provides a novel avenue for further investigation. One possible model that is consistent with these data is that Ca2+/CaM functions upstream of sAC/PKA.

In Chlamydomonas, it has been shown that Ca2+ may act through CaM and CaMKII to control flagellar motility by regulating dynein-driven microtubule sliding (Smith, 2002). Furthermore, in ascidian sperm, it has recently been documented that CaMKII mediates sperm-activating and -attracting factor (SAAF)-induced motility activation (Nomura et al, 2004). Conflicting data exist regarding a potential role for CaMK isoforms in mammalian sperm motility. One group has reported that targeted mutagenesis of the CaMKIV isoform has no effect on male fertility (Blaeser et al, 2001), while another group has reported that loss of CaMKIV results in male sterility in association with decreased sperm motility (Wu et al, 2000). Inhibitors of CaMKIV have been reported to decrease human sperm motility (Marin-Briggiler et al, 2005) and CaMKII stimulates hyperactivation in bovine sperm (Ignotz and Suarez, 2005). Our data indicate that an isoform of CaMKII is present in the principal piece of murine sperm. This is probably CaMKIIβ but it could also be CaMKIIγ. Taken together, these and additional data (Weinman et al, 1986; Bendahmane et al, 2001) suggest that a Ca2+/CaM/CaMKII pathway in the sperm principal piece is active in mammalian sperm motility regulation.

Acknowledgement

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References

We thank Drs Stuart B. Moss, George Gerton, and Bayard Storey for their continuing support and for critical reading of this manuscript. We also thank Ms Lee Schlingmann for her assistance in preparing several of the figures.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgement
  7. References
Footnotes
  1. Supported by NIH grant HD01189–03, CONRAD Mellon Foundation grant 10100710, and the University of Pennsylvania Research Foundation (to RMT), NIH/Fogarty Training Grant 5-D43TW00671 (to MAM), and by NIH grants K01-RR00188 and R01-HD-045664 (to AJT).