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a Department of Basic Sciences, Division of Biochemistry,
b Department of Herd Health and Reproduction and
c Department of Functional Morphology, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht,The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The membrane specificity of a number of lectin conjugates was tested with fluorescence microscopy and transmission electron microscopy. Membrane binding of these lectin conjugates was quantified with flow-cytometry and an enzyme-linked lectin binding assay. Wheat germ agglutinin was specific for the plasma membrane while peanut agglutinin was specific for the outer acrosomal membrane. The use of these lectins made it possible for the first time to discriminate between these two membranes. The isolated plasma membrane fraction was enriched more than 10-fold (17-fold after further purification by a sucrose gradient) in plasma membrane material compared to outer acrosomal membrane material.
Highly purified sperm plasma membranes should prove to be useful for research on primary sperm-zona interactions.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The purity of the plasma membrane has been expressed in terms of enrichment in relative activities of plasma membrane marker enzymes [3, 4, 6–12]. Mostly these reports also have shown decreased activities of marker enzymes from other cellular membranes (i.e., mitochondria and acrosome). The acrosomal outer membrane is the most probable contaminant in plasma membrane isolates from sperm cells because it is situated very close to the apical plasma membrane of the sperm head. However, marker enzymes for the outer acrosomal membrane have not been described, and therefore the amount of outer acrosomal membrane contamination has never been determined.
In this study, the specificity of labeled lectins and marker enzymes for various sperm membranes were used to measure the purity of the isolated plasma membranes from boar sperm cells. A novel method for measuring the purity and quantity of plasma membranes is described. With this novel assay, we were able to discriminate between the plasma membrane and the outer acrosomal membrane. The applicability of this new technique in fertilization research is discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Boar semen was obtained from the cooperative center for artificial insemination in pigs "Utrecht en de Hollanden" (Bunnik, The Netherlands). Semen was filtered through gauze to remove gelatinous material. All buffers or other solutions used were iso-osmotic (285–300 mOsm) and at room temperature.
Semen was allowed to cool to room temperature within 1.5 h after collection. Semen samples were diluted in fixative (0.5% [w:v] formaldehyde in saline), and the sperm concentration was estimated in a Bürker cell chamber under a phase contrast microscope (x200; Olympus, Tokyo, Japan). The ejaculate was diluted in Beltsville Thawing Solution (BTS: 0.2 M glucose, 20 mM sodium citrate, 15 mM NaHCO3, 3.36 mM Na2 EDTA, 10 mM KCl, 20 mM Hepes, pH 7.4) to 1.5 x 108 cells/ml, and seminal plasma was removed by washing over a Percoll (Pharmacia, Uppsala, Sweden) density gradient (35–70%). Percoll was removed by washing pelleted sperm 3 times in Tris-buffered sucrose solution (TBSS: 5 mM Tris, 0.25 M sucrose, pH 7.4).
Plasma Membrane Isolation
Plasma membranes from boar spermatozoa were isolated as described by Buhr et al. [13] with modifications. Briefly, washed sperm cells in TBSS (± 4 x 108 cells/ml) were subjected to nitrogen cavitation (10 min, 45 bar) in a cell disruption bomb (Parr Instrument Company, Moline, IL). The cavitate was slowly extruded into a clean tube, and 0.2 mM PMSF (dissolved in dimethyl sulfoxide) was added to the cavitate, which was then gently rotated for 15 min. Subsequently, the cavitate was centrifuged for 10 min at 1000 x g (room temperature). The pellet was washed with 5 ml TBSS and centrifuged for 10 min at 1000 x g (room temperature). Supernatants were combined and centrifuged for 10 min at 6000 x g (room temperature). The 6000 x g supernatant was then centrifuged for 70 min at 285 000 x g (4°C). The 285 000 x g membrane pellet was washed in Hepes-buffered saline (HBS: 5 mM Hepes, 2.7 mM KCl, 146 mM NaCl, pH 7.4) for 40 min at 285 000 x g (4°C). Samples were frozen in liquid nitrogen and kept at -20°C before examination.
Plasma membrane isolates were also layered on a discontinuous sucrose gradient and centrifuged according to Gilles et al. [5]. Fractions of 1 ml were collected, and protein content as well as lectin binding properties were determined.
Biochemical Analysis
Alkaline (EC 3.1.3.1) and acid (EC 3.1.3.2) phosphatase were assayed according to Soucek and Vary [7]. ß-N-acetylglucosaminidase (EC 3.2.1.30) activity was measured according to Khar and Anand [14]. 5'-Nucleotidase (EC 3.1.3.5) activity was determined by incubation of samples in a buffer containing 0.1 M glycine, 10 mM MgCl2, and 5 mM 5'-AMP (pH 8.5) for 30 min at 37°C. The reaction was stopped by adding phosphate reagent, and phosphate measurement was done according to Rouser et al. [15]. Acrosin (EC 3.4.21.10) activity was determined by its esterolytic activity on N-
-benzoyl-L-arginine-p-nitroanilide (L-BAPNA, 4.0 mM; Sigma, St. Louis, MO). The formation of the product p-nitroaniline was measured continuously at 410 nm (
410 = 9.9 mM-1/cm [16]) during incubation in a buffer containing 0.1 M Tris and 67 mM NaCl (pH 8.0) at 25°C. Succinate dehydrogenase (SDH, EC 1.3.99.1) was measured at 25°C according to Van Hellemond et al. [17]. Lactate dehydrogenase (EC 1.1.1.27) activity was continuously measured in a spectrophotometer at 340 nm (340 = 6.6 mM-1/cm). Samples were incubated at 25°C in 0.1 M sodium phosphate buffer (pH 7.2) containing 1.1 mM pyruvate and 0.2 mM NADH.
Protein amount was determined by the method of Lowry et al. [18] with a slight modification, using BSA as the standard. Samples were boiled for 10 min in 30 mM SDS, 160 mM Na2CO3, and 12 mM KNa-tartrate in 80 mM NaOH. Samples were incubated for 10 min in 1.5 mM CuSO4, and the color was enhanced with 0.1 M Folin-Ciocalteu's phenol reagent for 30 min.
DNA content was measured according to Young-Jo et al. [19] (DNA standard from Sigma).
Detection of Lectin Binding
Fluorescence microscopy To visualize the sperm regions to which fluorescein isothiocyanate (FITC)-lectins bind, samples were fixed in Karnovsky solution (2% paraformaldehyde, 2.5% glutaraldehyde, 80 mM sodium-cacodylate, 250 µM CaCl2, 500 µM MgCl2, pH 7.4) or in pure methanol. Before the incubations with lectins, Karnovsky-fixed samples were centrifuged (500 x g, 10 min), and the pellets were recovered in PBS containing 50 mM glycine (pH 7.4). Fixed samples were dried at room temperature on slides and then incubated in HBS containing 1 mM CaCl2 and 2 or 10 µg/ml of one of the following FITC-conjugated lectins (EY Laboratories, San Mateo, CA): PNA (Arachis hypogaea [peanut] agglutinin), LEA (Lycopersicon esculentum agglutinin), UEA-I (Ulex europeaeus agglutinin), GS-I (Griffonia simplicifolia I agglutinin), GS-II (Griffonia simplicifolia II agglutinin), WGA (Triticum vulgare [wheat germ] agglutinin), ConA (Canavalia ensiformis agglutinin), MPA (Maclura pomifera agglutinin), AIA (Artocarpus integrifolia agglutinin), DBA (Dolichos biflorus agglutinin), BPA (Bauhinia purpurea agglutinin), SBA (Glycine max [soybean] agglutinin), LPA (Limulus polyphemus agglutinin). After incubation, slides were washed with 2 ml HBS containing 1 mM CaCl2, counterstained with 2 µM ethidium homodimer (Eth D-1) and embedded in 1 drop of Slowfade (Molecular Probes, Eugene, OR). Coverslips were placed on top of the samples and were sealed with nail polish. The sperm cells were studied with a fluorescence microscope (BH-2; Olympus) equipped with differential interference contrast optics, a 100-W Hg lamp, and a filter set (470 nm; a 30-nm BP excitation filter, a 515-nm dichroic mirror, and a 530-nm LP emission filter).
Electron microscopy Binding sites for FITC-conjugated lectins were localized using immunogold-labeling combined with electron microscopy. Percoll-washed spermatozoa were centrifuged, and pelleted spermatozoa were fixed in Hepes-buffered sucrose solution (HBSS: Tris was replaced by Hepes in TBSS) containing 2% (w:v) paraformaldehyde and 0.5% (w:v) glutaraldehyde, and stored overnight at 4°C. Subsequently the sample was treated as described by Fazeli et al. [1].
FACS analysis Percoll-washed and permeabilized sperm cells were incubated for 5 min in HBSS containing 1 µg/ml of the lectin-FITC conjugate of choice. The membrane-impermeable vital stain propidium iodide (PI) was added to counterstain deteriorated cells (final concentration 10 µg/ml). The labeled sperm suspensions were analyzed in a FACS-Scan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) equipped with a 100-mW argon laser. Cell fluorescence was exited at 488 nm while the FITC- and the Eth D-1-emission intensity per cell was detected in the logarithmic mode of FL-1 (530/30-nm band pass filter) and FL-3 (620-nm long pass filter) respectively. Forward and sideward light scatter (FSC and SSC) data were collected in linear mode. At the light scatter settings for FSC (E00) and SSC (400 mV), the sperm-specific events were recognizable as a typical L-shaped scattering profile. The non-sperm-specific events (mostly small particles, < 3% of total events) were gated out for further analysis. The flow cytometric data were stored in Becton Dickinson software and analyzed on WinMDI version 2.1.4 (J. Trotter; free software, e-mail: trotter@scripps.edu). Regions were drawn on specific cell populations reflecting dead and live cells, respectively, and mean fluorescence as well as proportions of the subpopulation were calculated.
Quantification of lectin binding An ELLBA (enzyme-linked lectin binding assay) was developed to quantify lectin binding of the cavitate and the membrane samples; ELISA plates were coated with samples (500 to 4 ng protein) in 0.1 M Na2CO3 (pH 9.6) for 8 h at room temperature. Plates were blocked for 10 min with 0.3% (v:v) Tween-20 in HBS and subsequently for 1 h with 0.05% (v:v) Tween-20 and 1% (w:v) casein in HBS. Biotin-conjugated lectins (EY Laboratories; 0.5 µg/ml) were allowed to bind to the coated plates for 2 h at room temperature in incubation buffer (0.05% [v:v] Tween-20, 0.1% [w:v] casein, 1 mM CaCl2 in HBS). Free lectin was removed by washing the plates 5 times with wash buffer (10 mM Tris, 150 mM NaCl, 5 mM CaCl2, 0.05% [v:v] Tween-80, 0.0001% [w:v] benzalkonium chloride, pH 7.5). Horseradish peroxidase (HRP) conjugated to streptavidin (Sigma; 0.1 µg/ml) was allowed to bind to the biotin-lectin conjugates for 1 h in incubation buffer. Free HRP-streptavidin conjugates were removed by washing 5 times with wash buffer. Bound enzyme activity was measured according to Bos et al. [20], and absorption was measured in a plate reader at 450 nm. Calculations were made with OD450 corrected for background absorption (same procedure, but the lectin biotin conjugate was omitted). The purification rate was calculated from the linear range as the increase or decrease in the amount of bound lectin upon differential centrifugation.
Western Blotting
To visualize proteins with WGA and PNA binding properties, samples containing 10 µg protein were separated on 12% gels (SDS-PAGE) and blotted on polyvinylidene fluoride membranes (Millipore Corporation, Bedford, MO). Blots were blocked and incubated with PNA-biotin or WGA-biotin as described for the ELLBA, except that casein was replaced by BSA and the blots were washed with HBS containing 0.05% (v:v) Tween-20, 1 mM CaCl2 and 0.1% (w:v) BSA. Proteins that bound lectin were visualized with 1.7 mM diaminobenzidine in 50 mM Tris (pH 7.2) containing 1.3 µM NiCl2 and 1.8 µM H2O2 [21].
Morphological Study
Percoll-washed spermatozoa, nitrogen-cavitated spermatozoa (1000 x g and 6000 x g pellets), and the isolated plasma membrane fraction were pelleted and fixed overnight at 4°C in Karnovsky fixative. Pellets were washed with 0.1 M sodium-cacodylate (pH 7.4), post-fixed with 1% osmiumtetroxide in 0.1 M sodium-cacodylate (pH 7.4) for 1 h, washed with distilled water, and stained with 2% aqueous uranylacetate for 1 h. Samples were dehydrated in graded series of acetone, and embedded in Durcupan ACM resin (Fluka, Bachs, Switzerland). Ultrathin sections (50 nm) were cut on a Reichert Ultracuts (Leica Aktiengesellschaft, Vienna, Austria) and stained for 2 min with Reynolds' lead citrate [22]. Sections were observed and photographed in a Philips CM10 electron microscope (Philips, Eindhoven, The Netherlands) at 80 kV.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Purification of the cavitated plasma membranes by differential centrifugation steps resulted in a membrane fraction that was enriched in relative enzyme activities for alkaline phosphatase, acid phosphatase, and 5'-nucleotidase, three marker enzymes of the plasma membrane of boar spermatozoa [7]. The specific activities of these plasma membrane marker enzymes were respectively 9, 13, and 10 times higher in the membrane isolate than in the complete cavitate (Table 1). Contamination of marker enzymes from the inner acrosomal membrane (acrosin [23, 24]) and the inner mitochondrial membrane (SDH [25]) was minimal in the plasma membrane fraction (below the detection limit, see Table 1). The commonly used acrosomal marker enzyme ß-N-acetylglucosaminidase, however, had a higher specific enzyme activity in the plasma membrane fraction than in the complete cavitate (4 times, see Table 1). DNA was measured to detect nuclear contamination. Only a minor amount could be detected in the plasma membrane samples (< 0.1% of the total DNA amount in the cavitate).
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Only a trace amount of the three analyzed plasma membrane markers (< 1% of alkaline phosphatase, 11% of acid phosphatase, and < 1% of 5'-nucleotidase) were confined to the sperm cell while the rest of the enzyme activity was recovered in seminal plasma (probably soluble enzymes, Table 1). Likewise, only 3% of the total ß-N-acetylglucosaminidase activity was found in the sperm cell fraction, while 81% of this enzyme (previously designated as an acrosomal marker enzyme [6, 12, 26]) was also recovered as a soluble enzyme in the seminal plasma. In contrast to the above-described marker enzymes, only minor amounts of the enzyme activities of SDH, acrosin, and lactate dehydrogenase could be detected in seminal plasma (< 2%, 8%, and 18%, respectively, see Table 1).
Localization of Lectin Binding-Sites
Several lectins were tested on membrane specificity. Transmission electron microscopy revealed that FITC-conjugated WGA, detected by anti-FITC and protein A-gold, bound primarily to the plasma membrane (Fig. 1A). LEA bound to the plasma membrane of fixed boar spermatozoa, as is shown in Figure 1B, but also to the inner acrosomal membrane and the acrosomal content. However, LEA did not bind to the outer acrosomal membrane. The acrosomal marker lectin PNA [1] was used to detect the outer acrosomal membrane (Fig. 1C). Other tested lectins (GS-I, GS-II, AIA, DBA, ConA, UEA-I, MPA, BPA, SBA, LPA) did not show defined binding patterns.
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Fluorescence microscopy revealed that WGA-FITC (green) stained the complete surface of Percoll-washed spermatozoa fixed with Karnovsky solution (Fig. 2A). Similar staining patterns were found after methanol fixation. Some spermatozoa (approximately 10%) showed a patchy WGA distribution (not shown). Most of the green (WGA) fluorescence on the sperm head disappeared after nitrogen cavitation except for the post-equatorial subdomain (Fig. 2B). The tails and midpieces remained also fluorescent. LEA-FITC bound over the entire plasma membrane of the sperm head of Karnovsky-fixed spermatozoa (Fig. 2C). Methanol-fixed spermatozoa showed a slightly increased fluorescence at the acrosomal region (not shown). After nitrogen cavitation, most of the LEA-FITC fluorescence had disappeared (Fig. 2D) from the sperm head, but the post-equatorial region was still labeled. Like WGA, the tails and midpieces were still fluorescent after nitrogen cavitation. PNA-FITC exclusively bound to the acrosomal region in permeabilized (methanol-fixed) cells (not shown) and also to approximately 5% of the Karnovsky-fixed spermatozoa (probably cells with deteriorated acrosomes, Fig. 2E). Nitrogen cavitation resulted in staining of most acrosomes by PNA-FITC (Fig. 2F). Although Karnovsky-fixed cells were impermeable for lectin conjugates, ethidium homodimer stained the nucleus of Karnovsky-fixed as well as methanol-fixed spermatozoa.
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FACS analysis revealed that WGA-FITC was able to bind to living cells (Fig. 3A) and that permeabilization by Triton X-100 did not result in an increase of bound WGA-FITC (Fig. 3B). LEA-FITC also bound to living cells (Fig. 3C), and the intensity of labeling to permeabilized cells increased slightly (Fig. 3D). PNA-FITC bound much more intensively to dead cells (Fig. 3E) than to living cells (mean intensity for living and dead cells: 14 vs. 123). Triton X-100 permeabilization resulted in even a higher intensity of FITC-PNA (mean intensity: 201, Fig. 3F).
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Contamination by the Outer Acrosomal Membrane
Biotin-conjugated lectins were used in an ELLBA, in order to determine the contamination by outer acrosomal membranes in isolated sperm plasma membranes. Biotin-conjugated lectin was allowed to bind to ELISA plates coated with isolated sperm plasma membrane or cavitate. The amount of lectin binding was determined after coupling of HRP-streptavidin to the immobilized biotin and detection of HRP activity normalized on the basis of coated protein. WGA binding to the plasma membrane increased on a protein basis to 5-fold by purification (Fig. 4), while LEA binding increased to 2 times normalized on protein basis. PNA binding to the isolated membrane fraction decreased by a factor of 2 compared to the cavitate (Fig. 4). This implies that the membrane fraction was at least 10 times enriched in plasma membrane material compared to outer acrosomal membrane material.
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Plasma membrane isolates were also further purified by centrifugation of the isolates layered on top of a sucrose density gradient. About 60% of the material (on protein basis) was recovered on top of the 1 M sucrose layer with a density of 1.13 mg/ml, which is typical for plasma membrane material [27]). The WGA/PNA affinity ratio of this membrane subfraction was 170% higher than for the original membrane isolate placed on top of the sucrose density gradient (see Fig. 5).
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Protein samples from washed sperm cells, cell-free seminal plasma, and purified membranes were separated by SDS-PAGE, and Western blots were prepared in order to identify PNA and WGA binding sites (see Fig. 6). PNA binding sites were absent in seminal plasma and in the plasma membrane isolate; however, a 60-kDa band from washed sperm cells showed high affinity for PNA (Fig. 6). In contrast, the protein sample of washed sperm had only low affinity for WGA (plasma membrane material was highly diluted by intracellular membrane proteins within this sample) while an 18-kDa protein present in the plasma membrane but also in the seminal plasma showed high affinity for WGA (Fig. 6).
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Morphological examination of the cavitated spermatozoa by electron microscopy revealed that approximately 80–90% of the spermatozoa lost their plasma membrane at the periacrosomal region (Fig. 7A). This finding is in agreement with the fluorescence microscopical observations. The 1000 x g pellet mostly consisted of spermatozoa with released plasma membrane at the apical region (Fig. 7B) and unattached sperm heads. The 6000 x g pellet contained tails and midpieces, and some vesicles were also present (Fig. 7C). The membrane vesicles were recovered in the 285 000 x g pellet, and most of the membrane isolate was composed of unilamellar vesicles (Fig. 7D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Marker enzymes are commonly used to determine the purity of isolated plasma membranes of spermatozoa [3, 4,6–12, 29, 31], in analogy to that of somatic cells [27]. However, in contrast to somatic cells [32–36], the ultrastructural localization of most marker enzymes is poorly described for mammalian sperm cells. In this paper, we describe the presence of high activities of several putative plasma membrane (e.g., 5'-nucleotidase) and acrosomal marker enzymes in seminal plasma. This observation is supported by the literature [37, 38], and in fact a specific extracellular soluble form (isoenzyme) of 5'-nucleotidase has been described [8]. The existence of an extracellular soluble form of a marker enzyme in the seminal plasma is not crucial for a plasma membrane marker. If the enzyme adheres to the plasma membrane and will be detached during the isolation procedure, the purification of this membrane will only be underestimated. More crucial is the adherence of a marker enzyme for an intracellular membrane to the plasma membrane, like the acrosomal marker ß-N-acetylglucosaminidase [11, 12, 14, 39]. Our results showed that this acrosomal enzyme was present in our plasma membrane isolates. Interestingly, a very high activity of the enzyme was recovered in the seminal plasma, but more importantly, the majority of the enzyme activity was detected extracellularly in Percoll-washed sperm cells. We therefore believe that extracellular ß-N-acetylglucosaminidase is adsorbed to the surface of sperm cells, which already has been proposed for human spermatozoa [37]. The use of membrane marker enzymes [5–7, 29], which are abundant in seminal plasma, is unreliable for intracellular membrane structures. Therefore, membrane characterization employing membrane marker lectins is superior to the old method using marker enzymes.
In contrast to the marker enzymes described above, other marker enzymes such as SDH (inner mitochondrial membrane [25]) and acrosin (inner acrosomal membrane [23,24]) are virtually absent in seminal plasma of freshly ejaculated semen. Most likely the small amounts of enzyme activities detected in the seminal plasma are a result of enzyme leakage from deteriorated sperm cells. In fact, these enzymes are used in clinical assays as a parameter that reflects the relative integrity of sperm cells. Another intracellular marker for sperm cells that has been used in similar clinical assays is the cytosolic enzyme lactate dehydrogenase [40]. However, similar to others [41], we also found a rather high enzyme activity in the seminal plasma. Therefore, SDH is the preferred enzyme for checking sperm membrane integrity, and acrosin is a reliable enzyme for checking the integrity of the sperm acrosomes. The use of these marker enzymes shows that the isolated plasma membrane fractions are free of inner mitochondrial membrane and inner acrosomal membrane. These results are comparable to or lower than the activities reported in other studies [5, 29, 39]. DNA measurements showed that only minor amounts of the nucleus were present in the isolated plasma membrane fractions, which is in agreement with previous reported data [39]. Taken together our data demonstrate that DNA and enzymes can only be used as markers for intracellular structures in sperm cells when they are not present in seminal plasma of freshly ejaculated semen.
Lectins are widely used in biochemistry and are known to bind to carbohydrates in a very specific manner [42]. In theory, the specific carbohydrate binding properties can make lectins useful to detect specific subcellular substances. In this respect, a well-known application in spermatology is the use of fluorescent conjugates of PNA [1], ConA [43, 44], or Pisum sativum agglutinin (PSA) [45] to detect specific carbohydrates from the inner side of the acrosome. This property makes PNA, ConA, and PSA useful for the detection of acrosome-reacted spermatozoa [46, 47]. Using ultrastructural labeling techniques, Fierro et al. [48] showed that WGA bound to the surface of human spermatozoa. In this paper, the use of PNA as a marker lectin for the outer acrosomal membrane and WGA as a marker for the sperm plasma membrane is described for the first time. Another lectin with affinity for the plasma membrane (LEA) was less suitable as a marker for this membrane because it also recognized an intra-acrosomal glycoconjugate. Fluorescence microscopy showed that WGA-FITC and LEA-FITC bound to the entire surface of the sperm cell. The observation that the sperm surface was exclusively labeled with WGA-FITC was supported by FACS analysis: in labeling experiments all sperm cells showed intensive staining with WGA-FITC (also observed for LEA-FITC), while the amount of staining was unaffected in sperm samples that had been permeabilized (a slight but significant increase of LEA-FITC staining was observed due to intra-acrosomal staining). Fluorescence microscopy and FACS analysis also supported the specificity of PNA binding to the acrosomal region of sperm cells with permeable membranes. An ELLBA has been developed to quantify lectin binding sites originating from the plasma membrane and the outer acrosomal membrane in order to qualify the progress of plasma membrane purity upon our isolation procedure. With the use of PNA conjugates, we were able to detect the amount of outer acrosomal contamination in sperm plasma membrane fractions. Obviously, it is important to follow especially the outer acrosomal membrane contamination in these fractions because this membrane is positioned close to the apical sperm plasma membrane and it was found to be the primary source of membrane contamination in our plasma membrane isolates. In fact, as evidenced in this paper by ultrastructural (see also [6, 30]) and fluorescence microscopical data, only the plasma membrane overlying the acrosome is stripped off the sperm cells during the nitrogen cavitation procedure. This observation demonstrates even further the necessity of evaluating membrane isolates with a reliable marker for not only the plasma membrane but also for the outer acrosomal membrane.
We consider the isolation of sperm plasma membrane from the outer acrosomal membrane of great importance for molecular studies on 1) the adhesion of the sperm cell to the epithelial lining of the female genital tract and 2) the adhesion of the sperm cell to the zona pellucida.
1) Acrosome-intact sperm cells have intimate contact with the epithelial lining of the female genital tract [49]. During this contact, sperm cells may acquire capacitated properties [50] such as hypermotility and a higher affinity for the zona pellucida [51]. Only the interaction of intact sperm cells (i.e., by molecules on the plasma membrane) to the epithelial lining is of physiological interest since only these cells can fertilize the oocyte. The outer acrosomal membrane may contain other adhesive molecules that also recognize the female genital epithelia, but those interactions may only serve to filter deteriorated sperm cells from the fertile sperm subpopulation. Therefore it is very important to be sure about the quality of the plasma membrane isolates (i.e. the amount of acrosomal contamination).
2) A similar scenario is valid for sperm cell adhesion to the zona pellucida. Most likely this adhesion is initiated by one or more (peripheral or integral) glycoproteins of the apical sperm plasma membrane [52]. However, after this adhesion event, the sperm cell is primed to undergo the acrosome reaction. The acrosomal membranes as well as the glycoprotein matrix in the lumen of the acrosome probably contain glycoproteins that adhere to the zona pellucida [2, 53, 54]. Only glycoproteins from the plasma membrane can serve as primary zona adhesion molecules, and therefore acrosomal contamination should be eliminated from plasma membranes as far as possible in order to distinguish between primary and secondary (e.g., acrosin [55, 56], which is not present in the plasma membrane isolate) zona binding.
Interestingly, lectins can be employed for further purification of sperm membrane fractions by using immobilized marker lectins as the solid state in affinity chromatography. In fact, the plasma membrane fraction can be isolated from a crude membrane isolate from hepatocytes with the use of WGA linked to dextran [57].
The morphology of the isolated plasma membranes was detected with transmission electron microscopical techniques. The majority of the pieces of isolated plasma membrane fraction were resealed as unilamellar vesicles. Such vesicles probably have a right-side outside orientation [28], which makes these vesicles potent tools for adhesive studies. The membrane proteins are in a more physiological condition in these vesicles than after solubilization from the membrane with detergents [27]. These vesicles can be used to determine fusion properties of the sperm plasma membrane (i.e., the acrosome reaction with inside-outside vesicles, and the fertilization fusion with right side-outside vesicles).
In conclusion, with the use of WGA and PNA it is now possible to assay cavitated plasma membrane isolates from sperm cells for the amount of plasma membrane material and outer acrosomal membrane material, respectively. Further purification was obtained after centrifugation over a sucrose density gradient. In the future, better results are expected with lectin affinity chromatography methods. Highly purified plasma membrane preparations and, more specifically, preparations almost free from outer acrosomal membrane contamination, are of great importance for biochemical characterization of plasma membrane specific molecules involved in sperm adhesion processes for fertilization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Frits M. Flesch, Department of Basic Sciences, Division of Biochemistry, Faculty of Veterinary Medicine, Utrecht University, PO Box 80.176, 3508 TD Utrecht, The Netherlands. FAX: 31 30 2535492; f.flesch{at}vet.uu.nl
Accepted: August 11, 1998.
Received: February 3, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 M. Katayama, M. Koshida, and M. Miyake Fate of the acrosome in ooplasm in pigs after IVF and ICSI Hum. Reprod., October 1, 2002; 17(10): 2657 - 2664. [Abstract] [Full Text] [PDF]
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S. S. Baker, R. A. Cardullo, and C. D. Thaler Sonication of Mouse Sperm Membranes Reveals Distinct Protein Domains Biol Reprod, January 1, 2002; 66(1): 57 - 64. [Abstract] [Full Text]
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R. Rathi, B. Colenbrander, M. M. Bevers, and B. M. Gadella Evaluation of In Vitro Capacitation of Stallion Spermatozoa Biol Reprod, August 1, 2001; 65(2): 462 - 470. [Abstract] [Full Text] [PDF]
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 F. M. Flesch, J. F. H. M. Brouwers, P. F. E. M. Nievelstein, A. J. Verkleij, L. M. G. van Golde, B. Colenbrander, and B. M. Gadella Bicarbonate stimulated phospholipid scrambling induces cholesterol redistribution and enables cholesterol depletion in the sperm plasma membrane J. Cell Sci., January 10, 2001; 114(19): 3543 - 3555. [Abstract] [Full Text] [PDF]
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 B. Gadella and R. Harrison The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane Development, January 6, 2000; 127(11): 2407 - 2420. [Abstract] [PDF]
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