Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 66, 823-829 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Surface Expression of Complement Receptor gC1q-R/p33 Is Increased on the Plasma Membrane of Human Spermatozoa after Capacitation1

Kristen S. Gracea,b, Richard A. Bronsonb, and Berhane Ghebrehiwet2,a

a Departments of Medicine b Obstetrics and Gynecology, State University of New York, Stony Brook, New York 11794-8161


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence is increasing that complement components might play a role in fertilization. C1q, the first component of the classical complement cascade, has the ability to promote sperm agglutination in a capacitation-dependent manner as well as an effect on sperm-oolemma binding and fusion. We have previously detected gC1qR, the receptor for the globular head portion of C1q, on the surface of capacitated sperm. In this study, we examined the expression of gC1qR in both fresh and capacitated human spermatozoa. We performed immunoprecipitation for gC1qR and analyzed biotinylated sperm membrane by Western blot to illustrate an increase in receptor density after overnight capacitation. These results were confirmed by flow cytometric analysis of spermatozoa using fluorescein isothiocyanate-labeled monoclonal anti-gC1qR antibody. Confocal, indirect immunofluorescence microscopy revealed an increase in receptor expression over the rostral portion of the sperm head after capacitation. In addition, the ability of live spermatozoa to bind to monoclonal anti-gC1qR antibody-coated microtiter wells was also increased after capacitation. These results suggest that gC1qR may play a role in human fertilization.

acrosome reaction, fertilization, immunology, sperm, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fertilization is a multistep process whereby each step is dependent on the previous and is controlled by a discrete and specific set of receptor-ligand interactions. Freshly ejaculated cells are unable to fertilize oocytes. Dramatic reorganization of the sperm plasma membrane must take place within the female reproductive tract to render sperm capable of acrosomal exocytosis and subsequent fertilization [1]. The various changes that occur to prime mammalian sperm for fertilization are known collectively as capacitation.

Capacitation involves biochemical modifications of membrane characteristics, enzymatic activity, and motility changes. Cholesterol efflux is a characteristic of capacitation that results in a loss of membrane rigidity [2]. An asymmetric phospholipid distribution is established, and various low-voltage ion channels are activated. In addition, sperm surface proteins are modified, added, or removed, all to ready the cell for zona pellucida binding and calcium induced-acrosomal exocytosis (for review, see [3]).

Once capacitation has occurred, the cell is able to undergo the acrosome reaction, an exocytotic event releasing hydrolytic and proteolytic enzymes as well as exposing receptors that enable the spermatozoon to penetrate the egg's vestments and to fuse with its plasma membrane [1]. On binding to ZP3, one of three zona pellucida glycoproteins that coat and protect the egg and embryo, a large calcium flux is thought to be induced, triggering a phosphorylation cascade leading to inositol triphosphate (IP3) production, protein kinase C (PKC) activation, and finally, exocytosis [3]. Whereas the exact receptors responsible for ZP3 binding and acrosomal exocytosis in vivo are not completely known, in vitro experiments show that aggregation of the ZP3 receptors is important for induction of the acrosome reaction [4].

Evidence is increasing that complement components and related proteins play a role in fertilization. At least four cell surface complement-regulatory proteins are expressed on human sperm: membrane cofactor protein (CD46) [5], vitronectin [6], decay-accelerating factor (CD55) [7], and membrane inhibitor of reactive lysis (CD59) [8]. Both CD46 [5, 9] and vitronectin [10] are exposed only after the acrosome reaction. Studies have indicated that these molecules may also participate in the sperm-oocyte interaction or in signal transduction as well as protect the sperm from complement-mediated damage in the reproductive tract [11–13]. Recently, a significant reduction in acrosome-intact sperm was shown to occur when capacitated sperm are incubated in normal serum containing activated complement, as opposed to those incubated in heat-inactivated serum [14]. C3 plays an important role in cell-cell adhesion reactions, both in natural and induced immunity, and it participates in the binding of bacteria and viruses to eukaryotic cells. Therefore, it is not surprising that C3 fragments and receptors have been shown to play a role in sperm-egg binding and fusion [11, 15]. Furthermore, C1 inhibitor-like protein is present on murine spermatozoa, and its antibody inhibits in vitro fertilization [16].

Human gC1qR was first characterized as a receptor for the globular "heads" of C1q, the first component of the classical complement pathway [17]. Recently, however, gC1qR has been redesignated as a multifunctional and multicompartmental cellular protein based on its ubiquitous expression, lack of transmembrane segment, and involvement in various ligand-mediated cellular responses [18]. The cDNA of gC1qR encodes a pre-pro-protein of 282 residues, the first 73 residues of which contain a mitochondrial targeting motif that is removed by site-specific cleavage during posttranslational processing to form the mature protein of 209 residues. Mature gC1qR is a highly acidic protein that migrates as a single chain of 33 kDa under reducing conditions, but it behaves as a trimer of 97 kDa under nonreducing conditions [17, 18]. Indeed, the recently solved crystal structure portrays a unique, doughnut-shaped homo-trimer containing a partially covered channel of approximately 10 Å [19]. Surface-expressed gC1qR is a glycosylated protein with a site for PKC phosphorylation and tyrosine kinase recognition [17, 18]. Interestingly, gC1qR contains neither a conventional transmembrane motif in its sequence nor glycosyl phosphatidylinositol anchoring, suggesting that any role of gC1qR in cellular signaling must take place in association with other transmembrane proteins such as ß1-integrins [18].

C1q is known to participate in many cellular events in addition to its role in the classical complement pathway, including IP3 production, enhancement of Fc-receptor and CR1-mediated phagocytosis, procoagulant activity on platelets, and chemotactic activity on mast cells, eosinophils, neutrophils, and fibroblasts (reviewed in [18]). We have previously demonstrated that C1q and its receptors play a role in gamete interactions leading to fertilization [20]. Exogenous C1q promotes the binding of human spermatozoa to the oolemma of zona-free hamster eggs in a concentration-dependent manner but inhibits sperm penetration of the oocytes. Addition of C1q also promotes sperm agglutination in capacitated, but not in fresh, spermatozoa. Our laboratory has also presented evidence that gC1qR and cC1qR, the receptors for the globular head and collagen-like tail, respectively, of C1q [21] are found in the lysates of human spermatozoa after Triton X-114 phase separation [22].

The present study was undertaken to examine the presence of gC1qR on the surface of human spermatozoa and to assess whether its expression on the plasma membrane was altered after capacitation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Capacitation of Human Spermatozoa

Spermatozoa were obtained from 6 known fertile donors whose semen exhibited normal parameters or from 4 men undergoing semen analysis whose semen was normal based on World Health Organization criteria [23]. A highly motile sperm population, free of round cells, was recovered by Percoll density centrifugation [24] and resuspended in Biggers-Whitten-Whittingham (BWW) medium containing 5 mg/ml of human serum albumin (HSA; Sigma, St. Louis, MO). The samples were mixed well by pipetting and separated into two aliquots: one to be utilized immediately as fresh cells, and the other to be washed once with 5% (w/v) and once with 30% (w/v) HSA in BWW and then resuspended in 30% BWW-HSA at a concentration of 10 x 106 cells/ml. These sperm were incubated overnight at 37°C in 5% CO2 in air to induce capacitation.

Chlortetracycline Staining

The capacitation status of the spermatozoa was assessed using a modification of the method described by DasGupta et al. [25]. Briefly, chlortetracycline (CTC) staining solution was prepared fresh daily by adding 750 µM CTC (Sigma) to buffer composed of 130 mM NaCl, 5 mM cysteine, and 20 mM Tris-HCl to a final pH of 7.8. Then, 100 µl of this solution were added to 100 µl of sperm suspension, mixed thoroughly, and immediately fixed by adding 16 µl of 12.5% (w/v) paraformaldehyde in 0.5 M Tris-HCl. One drop of 0.22 M diazabicyclo[2.2.2]octane (DABCO; Sigma) in glycerol-PBS (9:1 [w/v]) was placed on a prewarmed slide, and 10 µl of stained, fixed cells were added to the drop and mixed. A cover slip was applied, and the slides were gently, but firmly, compressed between two paper towels to maximize the number of sperm oriented in a flat position. The slides were sealed with clear varnish and assessed on the same or the following day. The staining patterns described by Lee et al. [26] were assessed using filter block D (ultraviolet plus violet excitation range), passing the Hg excitation beam through a 355- to 425-nm band filter. The CTC fluorescence emission was observed through an RKP 455 beam-splitting mirror (Leitz-GMBH, Wetzlar, Germany). Uncapacitated cells were scored based on uniform fluorescence over the surface of the sperm head, whereas capacitated cells exhibited a fluorescent-free band in the postacrosomal region.

Solubilization of Human Spermatozoa

Cells were washed three times in 37°C PBS and resuspended to a concentration of 25 x 106 cells/ml. Sulfo-NHS-biotin (Pierce, Rockford, IL) was added (0.5 µg/ml reaction volume) to the samples and incubated (37°C, 30 min). The excess biotin was removed by washing the cells with warm PBS, and the cells were then resuspended in 1 ml of PBS containing 100 µl of the protease-inhibitor cocktail Complete (Pierce) and immediately frozen in liquid nitrogen until solubilization.

After a quick thaw and refreeze, the cells were thawed on ice and incubated in sperm solubilization buffer containing 2% NP-40, 2% Emulphogene B720 (Sigma), 5 mM EDTA, 100 mM dithiothreitol, and 5 mM iodoacetamide in PBS. The cells were rocked overnight at 4°C. The following morning, the samples were microfuged at 12 000 rpm for 30 min (4°C). The supernatant containing the solubilized proteins was transferred to a new tube, and the pellet was discarded.

Immunoprecipitation and Western Blot Analysis

The total protein concentration of each cell lysate was determined using the detergent-compatible BCA protein assay (Pierce) as described in the manufacturer's protocol. Then, 50 µg of total cellular protein were precleared with Ultralink-BSA beads, made according to manufacturer's instructions (Pierce), overnight at 4°C with constant rocking. The beads were centrifuged for 5 min at 1000 rpm, and the pellet was discarded. The Ultralink (NIMG) beads were fully saturated with either monoclonal antibody (mAb) 60.11, directed against the N-terminus of the protein, nonimmune mouse immunoglobulin (Ig) G, or anti-ß-tubulin, and 50 µl of the beads (50% slurry) were added to the precleared lysate. The volume was brought to 400 µl with H2O, and 400 µl of immunoprecipitation (IP) buffer (50 mM Tris-HCl, 0.5 M NaCl, 1 mM CaCl, 1 mM MgCl, and 0.1% Tween 20) were added. The beads were incubated for 2 h at 4°C with constant rocking. They were then pelleted by centrifugation, washed twice with PBS containing 1 M NaCl and 3 times with IP buffer, resuspended in 30 µl of denaturing reducing loading buffer, and boiled for 5 min.

Solubilized membrane proteins were then prepared as described above. Forty microliters of protein solution were applied to each lane of a 1.5-mm-thick slab of 10% SDS-PAGE, and the proteins were separated by electrophoresis under reducing conditions using the buffer system of Laemmli [27]. The separated proteins were then electrotransferred to polyvinyl difluoride nitrocellulose membrane, and the membrane was blocked with 5% powdered nonfat milk in TBS (Tris-buffered saline) with 0.05% Tween 20, then washed 3 times before probing with Extravidin (Pierce) coupled to horse radish peroxidase. The protein bands were detected using the enzyme chemoluminescence Western blotting detecting kit (Amersham, Aylebury, U.K.).

Whole-Cell Sperm-Binding ELISA

Immulon 4 microtiter wells (Dynatech Laboratories, Inc., Chantilly, VA) were coated with increasing concentrations (0–20 µg) of either C1q, monoclonal anti-gC1qR (60.11), NIMG, or BSA. Wells were blocked overnight with TBS containing 1% BSA and washed 3 times with TBS containing 0.1% Tween 20.

On Day 1, half the plate was prepared with 1 x 106 fresh spermatozoa suspended in 100 µl of BWW-5% (v/v) HSA placed in each microtiter well, and the plate was spun at 1000 rpm for 3 min. The plates were then incubated for 1 h at 37° in 5% CO2 in air. Following incubation, the unbound cells were removed, and the wells were gently washed 3 times with warm PBS. Bound cells were fixed with 3.7% paraformaldehyde, and the plate was stored at 4°C until the following day.

On Day 2, the plate was removed and brought to 37°C. The second half of the plate was then prepared and processed in the same manner using cells from the same semen sample that had been capacitated overnight. After the paraformaldehyde had been thoroughly washed from the wells, the bound cells were determined either by blindly counting the number of cells across the widest diameter of each well or, alternatively, by calculating the total protein concentration using the BCA and read on an MR700 microplate reader (Dynatech Labs) at an optical density of 570 nm.

Flow Cytometry and Confocal Microscopy

Cells were fixed in 3.7% paraformaldehyde for 30 min at 37°C, either immediately after Percoll density centrifugation or after overnight capacitation. Cells were washed 3 times in warm Dulbecco PBS and resuspended to a final concentration of 20 x 106 cells/ml. Then, 20 µg of mAb 60.11 or NIMG were added to 500 µl of cell suspension, which was then incubated for 2 h at 37°C with occasional shaking. After washing, the cells were resuspended in 500 µl of PBS, 10 µg of Alexa 594 (rhodamine spectrum)-conjugated goat-anti-mouse (GAM) F(ab')2 antibody, and 10 µg of Alexa 488 (flourocine spectrum)-conjugated anti-CD46 (Molecular Probes, Eugene, OR). The CD46 is expressed only on the inner acrosomal membrane and is used as a marker for the acrosome reaction. This mixture was incubated (37°C, 1 h) and, after incubation, thoroughly washed in PBS. Cells were stored at 4°C until low cytometric analysis or were prepared on slides for confocal microscopy.

For confocal microscopy, a slide was prepared with one drop (~15 µl) of 0.22 M DABCO in a 1:10 (v/v) solution of PBS:glycerine. Then, 15 µl of cells, treated as described above, were gently mixed in by pipetting, and the cell mixture was covered with cover slips. To assure the sperm lay evenly flat, firm and even pressure was applied, after which the slides were sealed with clear nail polish.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Confirmation That Spermatozoa Underwent Capacitation

To confirm that overnight incubation in BWW resulted in capacitation, we utilized CTC staining to estimate the percentage of cells that either underwent spontaneous capacitation in the fresh population or were capacitated after incubation. In all men, less than 5% of fresh sperm exhibited spontaneous capacitation based on CTC-staining patterns, and only men who scored at least 70% capacitation after overnight capacitation were used in this study. The variation among donors used for all experiments ranged between 72% and 89%.

Biotinylated gC1qR Is Increased after Capacitation

We have previously shown that extracts of human spermatozoa contain both cC1qR and gC1qR [22]. To illustrate that gC1qR is, indeed, present on the cell surface and increases in concentration after capacitation, cell surface proteins were first labeled with the membrane-impermeable sulfo-NHS-LC-biotin. Because gC1qR has been shown to bind protein A [28], we utilized antibody coupled to Ultralink beads for our immunoprecipitation.

Western blot analysis revealed the presence of a 33-kDa band in both the fresh and capacitated samples. When equal amounts were loaded per lane, the capacitated sperm band was markedly darker than the fresh sperm band, as shown in Figure 1. However, no staining was observed when NIMG was used instead of mAb 60.11 to coat the beads. The increase in gC1qR expression appeared to be purely cell surface-associated, because ß-tubulin, a cytoplasmic protein, showed no indication of biotin incorporation (Fig. 1B).



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  		FIG. 1. Following immunoprecipitation, biotinylated sperm membrane extracts were subjected to SDS-PAGE and analyzed by Western blotting using horse radish peroxidase-extravidin. A) Lane 1: freshly ejaculated spermatozoa from two known fertile men; lane 2: spermatozoa from the same ejaculate, capacitated overnight in BWW supplemented with 30% HSA. Lanes 1 and 2 are both immunoprecipitated with mAb 60.11. Lanes 3 and 4 are uncapacitated and capacitated sperm, respectively, immunoprecipitated with isotype and species-matched IgG. B) Lane 1: freshly ejaculated spermatozoa; lane 2: capacitated spermatozoa. Lanes 1 and 2 are both immunoprecipitated with anti-ß-tubulin-coated beads. In all cases, 50 µg of total cellular protein were immunoprecipitated in a volume of 250 µl; 40 µl of this solution were loaded in each lane

Flow Cytometric Analysis Shows Increase Surface gC1qR after Capacitation

Flow cytometric analysis was performed to confirm the above findings in a more quantifiable manner using mAb 60.11 and Alexa-conjugated GAM. As shown in Figure 2, a specific increase in gC1qR staining was observed after overnight capacitation. The interaction between the mAb 60.11 and gC1qR was specific, because cells labeled with a primary nonimmune mouse antibody did not show any staining.



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  		FIG. 2. Flow cytometric analysis of spermatozoa. 1) Spermatozoa capacitated overnight as described in Materials and Methods, labeled with 20 µg control mouse IgG. 2) Freshly ejaculated spermatozoa. 3) Capacitated spermatozoa from the same ejaculate shown in 2 labeled with mAb 60.11. All three populations were probed with 10 µg of Alexa 594 GAM F(ab')2

Confocal Microscopy Shows Increased Cell Surface gC1qR over the Rostral Portion of the Sperm Head Plasma Membrane

The same cells that had been labeled for flow cytometric analysis were analyzed by confocal microscopy to ascertain the expression of gC1qR on the cell surface and whether this expression changed in distribution during the capacitation of spermatozoa. As predicted from the flow cytometric analysis, a substantially higher fluorescent signal was noted in the capacitated spermatozoa, localized to the plasma membrane (Fig. 3). Serial sections through the cell showed that fluorescence was restricted to the plasma membrane and not the cytoplasm (Fig. 4). A change in the distribution of gC1qR was also observed between the fresh and capacitated states. Tail and midpiece staining was evident in both populations, but these areas also showed some nonspecific staining when mAb was replaced with NIMG (data not shown). A majority of capacitated cells exhibited staining on the rostral portion of the head, whereas this pattern was not evident on those sperm that were freshly ejaculated (Fig. 3).



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  		FIG. 3. Confocal microscopy. Freshly ejaculated spermatozoa (A) or sperm from the same ejaculate capacitated overnight (B) were first incubated with mAb 60.11 and probed with 10 µg of Alexa 594 GAM as described in Materials and Methods. Control antibody (not shown) had no significant staining



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  		FIG. 4. Confocal microscopic demonstration of surface expression of gC1qR. Capacitated spermatozoa were labeled for gC1qR as in Figure 3, and a series of 1.5-µm optical sections were taken

Increased Sperm Bind Antibody-Coated Microtiter Wells after Capacitation

We have previously demonstrated that C1q can alter sperm-egg interactions, increasing the binding of spermatozoa to the oolemma while inhibiting fusion. These effects were abrogated by anti-C1q-receptor antibodies [20]. To determine whether the observed increase in the expression of C1q receptor resulted in differing sperm-binding capabilities, both fresh and capacitated live cells were incubated in microtiter wells coated with either C1q or mAb directed against gC1qR. Nonimmune mouse IgG- and BSA-coated plates were used as controls for nonspecific binding. As judged by the number of adherent cells, capacitated sperm bound to antibody-coated plates with an average of 50% greater efficiency compared with the uncapacitated population (Table 1). We further characterized the binding of spermatozoa to mAb 60.11-coated wells in a more detailed and quantifiable manner, studying the effect of increasing concentrations of antibody on cell binding by measuring total bound protein using BCA development and spectrophotometer readings. Our results showed a concentration-dependent increase in binding for both populations, but the capacitated sperm exhibited a rate of increase twice that of the fresh cells for most antibody concentrations (Fig. 5). Western blot and FACS analysis explain these results by illustrating a small surface-receptor expression before capacitation.


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  		TABLE 1. Number of spermatozoa adherent to mAb 60.11 (10 µ/ml)-coated microtiter plates before and after capacitation (mean ± SEM)



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  		FIG. 5. ELISA with intact sperm. Microtiter wells coated with increasing concentrations of either mAb 60.11 or nonimmune mouse IgG were first incubated (1 h, 37°C) with intact spermatozoa (105 cells/well), washed, and fixed with 3.7% paraformaldehyde. After incubation, the total protein concentration was determined using the BCA detection technique to assess the number of cells attached. Each data point is a mean of three separate experiments. Closed shapes represent freshly ejaculated sperm; open shapes represent capacitated sperm. Both fresh and capacitated sperm are from the same ejaculate


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Successful fertilization depends on a series of receptor-ligand interactions. Our laboratory's previous work has provided evidence that C1q may play a role in fertilization based on its ability to effect sperm-egg interaction. In the presence of exogenously added C1q, spermatozoa adherence to the oolemma was increased, whereas fusion was inhibited [20]. The observation that the addition of C1q leads to sperm agglutination only after capacitation [22] led us to speculate that C1q receptors might be up-regulated on these spermatozoa. The present study provides evidence that this is the case.

Immunoprecipitation and Western blot analysis using equal amounts of total cellular protein obtained from solubilized fresh and capacitated spermatozoa clearly demonstrate an increase of the 33-kDa gC1qR band density after capacitation. This finding is consistent with reports that other cell surface proteins such as glycine receptors, fibronectin, vitronectin, {alpha}3- and ß1-integrins, and others are either modified, up-regulated, or redistributed after capacitation [6, 29–32]. This finding is further supported by the flow cytometric analysis in which specific gC1qR staining is noted. Whereas gC1qR has been shown to be a ubiquitous protein on circulating and noncirculating cells, the observation that this multifunctional receptor is expressed on the surface of human spermatozoa only after capacitation suggests that gC1qR might play a role in those steps leading to fertilization.

Results of confocal microscopic analysis using mAb 60.11 not only confirm gC1qR localization on the cell surface but also show gC1qR expression localized to the rostral portion of the sperm head subsequent to capacitation, indicating that it may be involved with sperm-zona interaction, because this region of the sperm recognizes and binds the zona pellucida [1]. Whereas both fresh and capacitated spermatozoa exhibited staining in the tail and midpiece, these results were also seen when mAb 60.11 was replaced with NIMG (data not shown), indicating that these two regions are susceptible to nonspecific Fc binding. Small numbers of cells from fresh populations of sperm in some donors also demonstrate this rostral staining, but this is most likely due to spontaneous capacitation. In these same populations, CTC staining indicated that spontaneous capacitation occurs at a rate of 0%–5%, depending on the donor. It has been theorized that the cholesterol content of the plasma membrane determines, in part, the rate of capacitation [3]. A positive correlation has been shown between cholesterol content and the time required for capacitation among different species [1]. Similarly, the proportion of cells that exhibited CTC staining consistent with the capacitated pattern described by Lee et al. [26] varied between our donors but averaged 80%. This might explain why not all cells showed such changes in distribution.

Fertilization has been described by investigators as being quasiphagocytotic in nature [33–35]. C1q is known to enhance phagocytosis in other cell types in cooperation with antibodies and its receptors [36–38]. The receptor for the globular portion of C1q, gC1qR, has been found to play a role in the entry of certain pathogens into host cells [28, 39]. That gC1qR is present on the sperm surface as well as the fact that exogenous C1q is also able to enhance binding to the oocytes by sperm [20] support the hypothesis that the oocyte may, indeed, be a "nonprofessional" phagocyte, and that gC1qR could be involved in this process of sperm incorporation by the oocyte.

Because gC1qR is not a transmembrane protein, it has been theorized that any signaling events mediated by this receptor must result from association with other transmembrane proteins [18]. Recently, it has been shown that C1q can support the adherence and spread of endothelial cells, and that anti-gC1qR antibodies can abrogate this effect. Interestingly, the tripeptide RGD (Arg-Gly-Asp), an integrin-binding peptide sequence, also blocks this adherence [18]. The theory that gC1qR may associate with integrins to transduce cellular signals is additionally supported by a previous observation that fibroblast adherence to C1q can also be inhibited by an RGD-containing peptide [40]. Integrins are implicated in many signaling-pathway mediators, including Ca2+ influx, stimulation of inositol lipid synthesis, and protein tyrosine phosphorylation [41]. Evidence has been presented that integrins are involved in fertilization and embryo implantation [42–44]. The {alpha}5ß1 is also expressed on spermatozoa and increased after capacitation [45].

Our findings fit well into the model proposed by Benoff [46] for the action of human sperm mannose receptors. This hypothesis states that freshly ejaculated spermatozoa contain a store of mannose-specific receptors in the subplasmalemmal space that are translocated to the plasma membrane overlying the acrosome during capacitation. This action has been shown to result in an increase of sperm-mannose binding and of the mannose-induced acrosome reaction [47, 48]. On binding mannose, the most abundant carbohydrate on the surface of the human zona pellucida [49], these receptors aggregate and move to the equatorial segment of the sperm head, activating a signaling cascade that leads to the acrosome reaction.

The {alpha}-D-mannosidase activity that occurs on the sperm plasma membrane of several species, including human, seems to have a role in sperm-egg interactions. Inhibition of this enzyme activity results in a dose-dependent decrease in the number of sperm bound per egg [50–52]. Exogenous D-mannose inhibits sperm-zona interactions. Furthermore, mannose can be used to mimic zona pellucida-induced acrosome reaction [53]. Findings from our laboratory (data not shown) that gC1qR can bind mannose in a specific manner may have important implications for sperm-zona interaction and the mechanisms that induce the acrosome reaction. The cell surface association of gC1qR with cC1qR [21], a calreticulin homologue and known mannoside lectin [54], in this context is also intriguing.

Finally, the signaling cascades that lead to the acrosome reaction and the known effects of gC1q-R ligand binding have many overlapping features. Zona binding and receptor aggregation are thought to lead to tyrosine phosphorylation, induction of a membrane depolarization, and calcium influx coupled with activation of G proteins. The calcium increase activates phospholipase C, converting phosphatidylinositol-4,5-biphosphate to IP3 and diacylglycerol, which, in addition to the generated free fatty acids and lysophosphatidylcholine, activates PKC, leading finally to the fusion of the outer acrosomal membrane with the plasma membrane [3]. These same signaling pathways have also been shown to involve gC1qR in their induction, including IP3 production as well as integrin activation [55].

We have demonstrated that gC1qR, a multifunctional, multicompartmental protein, is present on the plasma membrane of human spermatozoa, and that its expression is increased after capacitation. After capacitation, gC1qR expression is increased on the plasma membrane over the rostral potion of the sperm head, indicating that this molecule may have a unique function in the process of fertilization.


   ACKNOWLEDGMENTS
 
The authors thank Susan Bronson and Lucilla Oula for their expert technical assistance as well as Dr. Rajeev Kumar for his insight and ideas.


   FOOTNOTES
 
First decision: 14 September 2001.

1 Supported in part by grants RPG-95068-03-CIM and RPG-95068-06 from the American Cancer Society, by a generous gift from Larry and Sheila Dalzell (B.G.), and by training grant GM 08444 from the National Institutes of Health. This publication represents part of K.S.G.'s work, performed in partial fulfillment of the Ph.D. Degree in Molecular and Cellular Biology. Back

2 Correspondence: Berhane Ghebrehiwet, Department of Medicine, Health Sciences Center, T-16-040, SUNY-Stony Brook, NY 11794-8161. FAX: 631 444 2493; berhane{at}mail.som.sunysb.edu Back

Accepted: October 30, 2001.

Received: August 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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