Molecular Human Reproduction, Vol. 6, No. 11, 977-982,
November 2000
© 2000 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Vitronectin is sequestered within human spermatozoa and liberated following the acrosome reaction
1 Department of Obstetrics and Gynecology 2 Department of Pathology, State University of New York at Stony Brook, New York, USA 3 Institute of Biochemistry and Justus-Leibig-Universitat, Giessen, Germany
Abstract
Vitronectin plays a role in the regulation of complement and thrombin activities and in cell surface proteolysis. Vitronectin is also an intrinsic protein of human spermatozoa. Vitronectin message has been detected in whole testis poly-A mRNA and localized by in-situ reverse transcription-polymerase chain reaction to spermatocytes. The proportion of spermatozoa that express vitronectin increases following their capacitation. In this study, spermatozoa from a man of proven fertility were probed with an anti-vitronectin monoclonal antibody (VN7) before and after their permeabilization with 0.1% Triton X-100. Of fresh spermatozoa observed by confocal microscopy, 0–8% showed vitronectin staining. However, 75% of those observed displayed vitronectin following permeabilization. Serial confocal sections through the sperm head confirmed the internal localization of vitronectin. The acrosomal status of capacitated spermatozoa that expressed vitronectin was then determined. Dual colour microscopy with rhodamine-conjugated anti-vitronectin antibody and a fluorescein-conjugated antibody directed against CD46 (a complement regulatory protein expressed on the inner acrosomal membrane) revealed that only acrosome-reacted (CD46-positive) spermatozoa displayed vitronectin. Two populations of these spermatozoa were observed. Fifty-seven of 260 (22%) were CD46-positive/vitronectin-positive and 72 of 260 (28%) were CD46-positive/vitronectin-negative. No spermatozoa were CD46-negative/vitronectin-positive. These results confirm that vitronectin is released from a sequestered location within the spermatozoon following the acrosome reaction.
acrosome reaction/capacitation/integrins/spermatozoa/vitronectin
Introduction
Integrins are a family of cell membrane receptors that play roles in cell adhesion and signal transduction, as well as in mediating the incorporation of bacteria and viruses into cells (Hynes, 1992
). Several integrins that are receptors for vitronectin have been detected on human oocytes and spermatozoa (Fusi et al., 1993, 1996a). Vitronectin is a multifunctional molecule which is present in blood and plays a role in the regulation of complement and thrombin activities as well as in cell surface proteolysis (Preissner, 1991). It is a major secretory product of the liver and exists in monomeric and multimeric forms. Vitronectin possesses both a heparin binding site as well as an RGD recognition sequence through which it binds to integrin receptors.
Evidence has accumulated that vitronectin is also an intrinsic protein of human spermatozoa. Vitronectin message has been detected by Northern analysis of whole testis poly-A mRNA (Fusi et al., 1993) and has been localized by in-situ reverse transcription-polymerase chain reaction (RT PCR) to spermatocytes within the seminiferous tubules of human testis (Nuovo et al., 1995). Vitronectin can be extracted from human spermatozoa (Fusi et al., 1992), and its expression increases during their capacitation. Vitronectin is liberated from spermatozoa in response to a calcium ionophore (Fusi et al., 1994). As exogenous vitronectin promotes the adhesion of human spermatozoa to zona-free hamster oocytes in a calcium-dependent manner, it has been suggested that multimeric vitronectin may act as a `molecular velcro', promoting gamete adherence once present on the sperm surface (Fusi et al., 1996b).
Semi-quantitative analysis of sperm lysates by densitometry of 1-D polyacrylamide gels suggests that vitronectin content of spermatozoa varies widely between different infertile men (Bronson and Preissner, 1997). The proportion of spermatozoa expressing vitronectin following capacitation also varies between different men (Fusi et al., 1994). It is not known whether spermatozoa that express vitronectin have undergone a spontaneous acrosome reaction or remain acrosome-intact. In this study, we have performed further experiments to address this question.
The vitronectin expression of fresh and capacitated spermatozoa of four men who had previously fathered children was first studied by flow cytometry, using a cyanine-conjugated monoclonal anti-vitronectin antibody (VN7-cy) that recognizes all forms of vitronectin (Stockmann et al., 1993). Spermatozoa from one of these men, who had exhibited a large increase of vitronectin expression following capacitation, were selected for further study. Fresh spermatozoa were probed with VN7-cy, before and after their permeabilization with 0.1% Triton-X, and were serially sectioned by confocal microscopy to document the internal localization of vitronectin. The acrosomal status of capacitated spermatozoa that expressed vitronectin was then determined. Spermatozoa obtained from the same semen donor were studied by dual colour confocal microscopy using rhodamine-conjugated VN7 (VN7-rhod) in conjunction with fluorescein-conjugated anti-CD46 antibody, following capacitation. CD46, the complement regulatory protein MCP (Rooney et al., 1993), was chosen as a marker of the acrosome reaction, as it is expressed on the inner acrosomal membrane, the limiting membrane of the acrosome-reacted sperm head, but not on the outer acrosomal nor plasma membranes (Anderson et al., 1989; Cervoni et al., 1993; Bronson et al., 1999a).
Materials and methods
Sperm preparation
Capacitation was performed as previously described (Bronson et al., 1981). Spermatozoa were obtained using a three-layer Isolate (Irvine Scientific, Santa Ana, CA, USA) gradient (90%, 1.5 ml; 70%, 1 ml, 40%; 1.5 ml) centrifugation. Semen (2 ml) was layered over a column of Isolate in human tubal fluid medium (HTF; Irvine Scientific) in 15 ml conical centrifuge tubes (Falcon; Becton Dickinson, Mountainview, CA, USA). Tubes were centrifuged at 200 g for 25 min, the pellet was collected and spermatozoa were washed twice by centrifugation at 300 g for 5 min using Biggers-Whitten-Whittington (BWW) medium, containing 5 mg/ml human serum albumin (HSA Fraction V, Lot. No. 126H9322; Sigma, St Louis, MO, USA). Spermatozoa (20x106 cells/ml) were resuspended in BWW-30 mg/ml HSA and capacitated by incubation overnight (18 h) at 37°C, 5% CO2.
Scoring of the expression of sperm-associated vitronectin by direct immunofluorescence and flow cytometry
Spermatozoa were processed as previously described for scoring of acrosome-reacted spermatozoa by FACScan (Becton-Dickinson. Mountainview, CA, USA) using a monoclonal anti-CD46 antibody (Bronson et al., 1999a). Briefly, fresh spermatozoa or spermatozoa capacitated as above were washed in 2 ml of phosphate-buffered saline (PBS). Cells were pelleted at 500 g for 5 min and resuspended in PBA [PBS + 0.5% bovine serum albumin (BSA)] at a concentration of 10x106/ml. In preliminary experiments, the amount of each fluorochrome-conjugated antibody required for visualization of vitronectin was determined. Spermatozoa were incubated for 30 min at room temperature in the dark with 10 µg/ml of cyanine-conjugated anti-vitronectin antibody (VN7-cy) per 1x106 cells. VN7 is a monoclonal antibody known to recognize all forms of vitronectin (Stockmann et al., 1993). Spermatozoa were then pelleted at 500 g for 5 min, the supernatant was discarded, and cells were washed twice with 2 ml PBA. Spermatozoa were resuspended in 500 µl of 1% paraformaldehyde in PBS for fixation. Tubes were stored in the dark at 4°C until analysed by flow cytometry (FACScan). When vitronectin was determined, only live cells were scored, as determined by dye exclusion of 7-aminoactinomycin D (7-AAD). 25 µl of 7-AAD (1 µg/ml) was added to 1x106 spermatozoa together with the VN7-cy antibody. The matching isotype murine myeloma protein for the anti-vitronectin monoclonal antibody MOPC21 (IgG1; Sigma) was used as a control for non-specific immunoglobulin binding. Following labelling, the specimens were gated by light scatter properties for spermatozoa and analysed. 7-AAD-stained dead cells were excluded and VN7 was detected only among living cells. Approximately 20 000 cells were scored in each experimental group.
Scoring of the acrosome reaction and vitronectin expression by dual colour confocal microscopy
Spermatozoa, fresh or capacitated, were washed with 2 ml of PBS. Cells were pelleted at 500 g for 5 min and resuspended in PBA at a concentration of 10x106/ml. As there can be a variable decrease in the affinity of antibodies for epitopes following their conjugation with fluorochromes, preliminary experiments were performed to determine the concentration of each antibody that optimized the labelling of spermatozoa. 200–300 µl of washed matched fresh or capacitated spermatozoa were incubated with rhodamine-conjugated mouse anti-human vitronectin monoclonal antibody (VN7-rhod) 300 µg/ml and 10 µl of fluorescein-conjugated anti-CD46 monoclonal antibody (Sertotec, Raleigh, NC, USA) for 1 h, at room temperature, in the dark. Cells were pelleted at 500 g for 5 min and washed once in PBA and twice in PBS. Spermatozoa were resuspended in 100 µl of 1% paraformaldehyde and placed on a slide at room temperature. After drying, slides were rinsed with equilibration buffer from the Slow Fade kit (Molecular Probes, Eugene, OR, USA) and mounted with Slow Fade to avoid bleaching.
Permeabilization of spermatozoa
Fresh, uncapacitated spermatozoa, after Isolate (Irvine Scientific) centrifugation, were washed twice with 5 mg/ml BWW/HSA. Washed spermatozoa (10x106/ml) were fixed in freshly made 3% (w/v) paraformaldehyde-0.05% (v/v) glutaraldehyde in PBS. 20x106 cells were fixed in 1 ml of this solution for 1 h at 4°C. After fixation, spermatozoa were washed with PBS twice and pelleted. The washed sperm pellet was permeabilized with 10 volumes of 0.1% Triton-X 100 in PBS for 7 min at room temperature. The conditions for permeabilization of cells were determined in preliminary experiments such that acrosomal contents were made accessible to fluoresceinated Pisum sativum agglutinin (PSA) lectin yet remained intact. Permeabilized cells were washed in PBS three times. Cells were then resuspended in PBA to reach the concentration ~10x106/ml. It was necessary to incubate spermatozoa with VN7-cy at a final concentration considerably higher (400 µg/ml) than that required for flow cytometric measurements (10 µg/ml) utilizing the same antibody, in order to achieve visualization of fluorochrome. Controls consisted of mouse IgG1-fluorescein isothiocyanate (FITC) (MPOC21; Sigma) with permeabilized cells, as well as live, fresh cells incubated with VN7-cy or MOPC21-FITC. After incubation with antibodies, for 1–2 h in the dark, at room temperature, the cells were washed with PBS three times, resuspended in 50–100 µl of PBS, placed on polylysine coated-slides, air-dried, and washed in PBS. Slides were mounted with Slow Fade in PBS, covered with cover slips, and sealed. If necessary, slides were kept until the next day in the dark, in a refrigerator, before scoring.
Results
In preliminary studies, direct observations of spermatozoa of donor 142 by confocal imaging, using VN7 conjugated with each of two different fluorochromes (cyanine versus rhodamine), both confirmed antibody activity and demonstrated an increased proportion of spermatozoa exhibiting vitronectin staining following capacitation. There was no difference in results when VN7-cy antibody was compared with VN7-rho. When tested with VN7-rho, 8% (8/101) of fresh spermatozoa enumerated exhibited vitronectin staining compared with 30% (173/590) of capacitated spermatozoa (P < 0.01, 
2-test). Similarly, 11% (3/27) of fresh spermatozoa and 30% (15/50) of capacitated spermatozoa exhibited vitronectin staining when tested with VN7-cy.
The effects of capacitation on vitronectin expression of spermatozoa were then studied, as judged by FACScan, using four men who had previously fathered children. This allowed the scoring of a larger population of spermatozoa than could be achieved by direct observations. The proportion of spermatozoa expressing vitronectin increased following their capacitation, but varied between semen donors (Table I). The increase in percentage of spermatozoa expressing vitronectin ranged from a low of 3% to a high of 29%. Spermatozoa from donor 142 were selected for further study (Figure 1)
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Following their permeabilization with 0.1% Triton X-100, ~75% of spermatozoa (30/40 cells observed) displayed vitronectin over their heads and tails, while none of the spermatozoa observed exhibited vitronectin staining before permeabilization. Vitronectin was localized within permeabilized cells, in a patchy distribution throughout the entire sperm head, with much fainter staining along the tail (Figure 2
). Serial confocal sections through the sperm head, using VoxelView software, confirmed the internal localization of vitronectin within the sperm head (Figure 3).
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The acrosomal status of capacitated spermatozoa that expressed vitronectin was then determined. In an initial experiment, different samples of the same population of spermatozoa were scored in parallel, but separately, with either VN7-cy or anti-CD4-FITC antibody. Spermatozoa were studied by indirect immunofluorescence and confocal microscopy both before and after their capacitation. The proportion of spermatozoa, whether fresh or capacitated, that had undergone an acrosome reaction was found to equal that expressing vitronectin. Of 100 spermatozoa, seven were CD46-positive (acrosome-reacted) before capacitation compared with 30 CD46-positive spermatozoa out of 100 observed following their incubation in BWW containing 30 mg/ml HSA. Of 100 spermatozoa freshly recovered from semen, eight exhibited vitronectin staining before capacitation compared with 30/100 spermatozoa that did so after capacitation
In the second experiment, simultaneous dual labelling of capacitated spermatozoa with both antibodies was performed, using rhodamine-conjugated VN7, which fluoresced in the red range, and fluorescein-conjugated CD46, which appeared green. Hence, the same spermatozoa were scored concurrently for their acrosomal status and vitronectin expression. The percentage of the total population of capacitated spermatozoa observed in bright field illumination that exhibited immunostaining for CD46 and vitronectin in matched dark fields was also determined (Table II).
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Only CD46-positive spermatozoa (acrosome-reacted) stained positive for vitronectin (Figure 4
). Vitronectin was localized to the post-acrosomal region of the acrosome-reacted sperm head (red staining-rhodamine), with CD46 staining apparent on the acrosomal region (green-staining fluorescein) over the rostral portion of the head. A yellow band of overlapping stain was occasionally noted in the equatorial region of the acrosome as well (Figure 5). Bright red vitronectin staining was also observed along the sperm tail.
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While all spermatozoa that exhibited vitronectin staining were observed to be acrosome-reacted, a population of spermatozoa was identified that were CD46-positive yet did not stain for vitronectin. Fifty-seven of 260 (22%) observed cells stained green and red (CD46-positive and vitronectin-positive), and 72 of 260 (28%) stained green only (CD46-positive and vitronectin-negative). The remainder of spermatozoa were acrosome-intact (CD46-negative), and none were observed to be stained red (vitronectin-positive) only.
Discussion
We have previously documented that the proportion of spermatozoa expressing vitronectin on their surface increases following their capacitation (Fusi et al., 1992). As shown by DasGupta et al. (1993) using a chlortetracycline (CTC) binding assay, human spermatozoa do not undergo capacitation uniformly. A sub-population of spermatozoa exhibit changes suggestive of capacitation following their incubation in capacitating medium yet remain acrosome-intact, while others undergo spontaneous acrosomal loss. Our prior studies could not determine whether spermatozoa that expressed vitronectin were capacitated and acrosome-intact or were acrosome-reacted. The present experiments were designed to address this question. The results indicate that vitronectin is released from a sequestered location within the spermatozoon during the acrosome reaction.
Vitronectin was not detected by confocal microscopy on spermatozoa freshly recovered from ejaculates, but was visualized both following their permeabilization with Triton X-100 and after capacitation. Using dual colour labelling and confocal imaging, vitronectin appeared to be localized to the post-acrosomal region of the acrosome-reacted sperm head and on the sperm tail, with CD46 staining apparent on the acrosomal region over the rostral portion of the head. Only acrosome-reacted spermatozoa expressed vitronectin. The location of CD46 is consistant with our prior observations (Bronson et al., 1999a), as well as those of Anderson et al. (1989) and Cervoni et al. (1993), who documented its presence on the inner acrosomal membrane.
In contrast, vitronectin was localized in Triton X-100-permeabilized spermatozoa in a patchy distribution throughout the entire sperm head, was not only limited to the acrosomal region, and faint staining of the sperm tail was observed. These results suggest that vitronectin is liberated from a sequestered location and redistributed on the sperm surface during the acrosome reaction. While confocal sectioning of permeabilized spermatozoa demonstrated its subsurface location within the sperm head, further ultrastructural studies are needed to clarify the exact site of vitronectin storage within spermatozoa. Whether the faint vitronectin staining observed along the tail of permeabilized cells was due to the release of vitronectin from the head region with subsequent secondary binding or whether it originated within the tail could not be determined.
Although these results were obtained observing spermatozoa from a single man, we believe that they may be illustrative of a general phenomenon. Donor 142 is proven fertile, having fathered several children, and his semen analysis is normal by World Health Organization (1992) standards. His spermatozoa have repeatedly exhibited high penetration frequencies of zona-free hamster oocytes, when used as a control in clinical sperm penetration assays, and have exhibited significant increases in the proportion of vitronectin-expressing cells, as determined by flow cytometry.
The proportion of capacitated spermatozoa expressing vitronectin varied between semen donors (Table I
), when studied using a monoclonal anti-vitronectin antibody that detected all forms of vitronectin (VN7). These observations are consistant with our prior findings using a polyclonal antibody raised in rabbits against human vitronectin (Fusi et al., 1992). In that study, a correlation was found between the proportion of capacitated spermatozoa expressing vitronectin and the subsequent ability of spermatozoa from this population to acrosome react in response to exposure to progesterone. This result suggests a different sensitivity of spermatozoa of individual men to capacitating conditions, reflected in their ability to undergo both spontaneous and induced acrosome reactions. In this prior study, in contrast to the present results, the proportion of spermatozoa expressing vitronectin following capacitation was greater than the proportion that had acrosome-reacted, as judged by staining with fluorescein-PSA lectin, rather than detection of CD46 on the inner acrosomal membrane in the present experiments. We suggest that early stages of membrane fusion and fenestration occurring during the acrosome reaction may have liberated vitronectin from its sequestered location within the spermatozoon, making it accessible to the polyclonal antibody prior to the completion of the acrosome reaction, as judged by the complete loss of PSA-stainable acrosomal contents.
In the present study, two populations of acrosome-reacted (CD46-positive) spermatozoa were observed by dual colour labelling, those that expressed vitronectin and those that did not. Whether these represent two distinct populations of spermatozoa, that contain differing amounts of vitronectin, or encompass the same population but at different stages of vitronectin release from a sequestered location cannot be addressed by the present experiments. Semi-quantitative analysis of sperm lysates by densitometry of 1-D polyacrylamide gels suggests that vitronectin content of spermatozoa varies between different infertile men (Bronson and Preissner, 1997). These results were normalized to 5x106 motile cells. Whether all spermatozoa within a population possess this mean level of vitronectin, or whether its content varies between individual spermatozoa is not known. Alternatively, the detection or absence of vitronectin could reflect differences in sperm viability.
Fourteen per cent of fresh spermatozoa from donor 142 displayed vitronectin, as judged by flow cytometric analysis, and 0–8% spermatozoa were observed by confocal microscopy to exhibit vitronectin staining. These low numbers are consistent with our results and suggest that only acrosome-reacted spermatozoa express vitronectin. In our prior studies utilizing CD46 staining and flow cytometry to score acrosomal status (Bronson et al., 1999a), we have found that a similar proportion of spermatozoa observed within a short time of their recovery from semen were acrosome-reacted.
We have previously suggested that multimeric forms of vitronectin could act as cross-linking ligands between acrosome-reacted spermatozoa and the oolemma, which has also been shown to express 
v-containing integrins (Fusi et al., 1996b). When spermatozoa were capacitated in strontium-containing, calcium-free medium, which supported their capacitation but not the acrosome reaction (Mortimer et al., 1986), addition of exogenous vitronectin did not promote the adhesion of spermatozoa to the oolemma of zona-free hamster eggs. In contrast, this effect was observed following addition of vitronectin, in the presence of Ca2+. Under these conditions, the proportion of spontaneously acrosome-reacted spermatozoa was shown to increase following the addition of calcium ions to the medium.
We have shown that sperm-associated vitronectin is displayed upon the sperm surface at this time and becomes accessible to associate with soluble, multimeric forms of vitronectin derived from follicular fluid (Stockmann et al., 1993; R.Bronson and K.Preissner, unpublished observations). Multimeric vitronectin might then bind with oolemmal integrins such as vß3, promoting the initial adherence of spermatozoa to the oocyte surface. Such adherent spermatozoa would then become liable to other ligand-receptor interactions between the closely associated gametes leading to sperm incorporation by the oocyte (Bronson, 1998; Bronson et al., 1999b). Fc
receptors (Bronson et al., 1992), integrins (Fusi et al., 1993; Tarone et al., 1993; Almeida et al., 1995; Evans et al., 1995; Chen and Sampson, 1999), and complement receptors (Anderson et al., 1993; Fenichel et al., 1994) have been detected on mammalian oocytes. In support of this hypothesis, v-containing integrins have been shown to play a role in the incorporation of viruses into cells (Nemerow et al., 1994; Roivanen et al., 1994)
An RGD-containing thioester-bridged cyclic peptide, G4120, known to block vitronectin receptor integrins (Barker et al., 1992) has recently been shown to significantly inhibit the binding of human spermatozoa to zona-free human oocytes (Bronson et al., 1999c), confirming our earlier observations of the heterologous interactions of human spermatozoa and zona-free hamster oocytes (Bronson and Fusi, 1990). This effect was apparent at concentrations as low as 10–50 µmol/l peptide concentration. These results further substantiate the hypothesis (Bronson and Fusi, 1990, 1996; Myles, 1993) that gamete interactions occur at the mammalian oocyte surface through interactions between sperm-associated ligands and as yet undefined oolemmal integrins.
Acknowledgments
We acknowledge the skilful assistance of David Colflesh, of the University Microscopy Imaging Center, Health Sciences Center SUNY, Stony Brook, in the acquisition of confocal images.
Notes
4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Health Sciences Center, State University of New York, Stony Brook, New York 11794–8091, USA. E-mail: rbronson{at}notes.cc.sunysb.edu 
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Submitted on June 27, 2000; accepted on August 16, 2000.
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 K. S. Grace, R. A. Bronson, and B. Ghebrehiwet Surface Expression of Complement Receptor gC1q-R/p33 Is Increased on the Plasma Membrane of Human Spermatozoa after Capacitation Biol Reprod, March 1, 2002; 66(3): 823 - 829. [Abstract] [Full Text] [PDF]
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