MOLECULAR REPRODUCTION AND DEVELOPMENT 30:258-264 (1991)

Acrosome Reaction Induced by Immunoaggregation of a Proteinase Inhibitor Bound to the Murine Sperm Head DAVID AARONS, HOLLY BOETTGER-TONG, GINGER HOLT, AND GARY R. POIRIER Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama ZP3, a glycoprotein of the murine ABSTRACT zona pellucida, functions both to bind acrosome intact sperm and to induce the acrosome reaction. Solubilized whole zonae as well as purified ZP3 are able to induce acrosome reactions in capacitated sperm. Pronase digests of whole zonae yield glycopeptides that bind to sperm but are unable to induce acrosome reactions. However, immunoaggregation of these glycopeptides results in the exocytosis of the acrosome in the majority of treated sperm. The data suggest that ZP3 triggers the acrosome reaction by the aggregation of ZP3 binding sites on the sperm head. If aggregation of ZP3 binding sites is important in the induction of the acrosome reaction, then it may be possible to induce the acrosome reaction in the absence of zona by immunoaggregation of the sites. This presentation deals with the immunoaggregation of a proteinase inhibitor of seminal vesicle origin (SVI) that binds to a site on the sperm head known to participate in zona binding. We show that capacitated murine sperm, pretreated with the SVI, will acrosome react, as determined by Coomassie brilliant blue staining,when incubated with rabbit antiinhibitor antiserum (anti-SVI).The percentage of SVI-treated sperm displaying an acrosome reaction is dependent on the concentration of the immune serum. Sperm stain positive for intact acrosomes when anti-SVI Fab fragments or normal rabbit serum is substituted for the immune serum. However, when capacitated sperm, treated with both SVI and anti-SVI Fab fragments, are incubated with goat antirabbit IgG, the majority of sperm acrosome react. The data suggest that the aggregation of SVI bound to the sperm surface,,in the absence of zona glycoproteins, is sufficient to induce the acrosome reac-

tion. Key Words: Zona binding, Fab fragments, Seminal vesicles INTRODUCTION The zona pellucida is a noncellular glycoprotein covering of the mammalian egg. In the mouse i t is composed of three components, ZP1, ZP2, and ZP3 (Bleil and Wassarman, 1980a). All three of these subunits are secreted by the growing oocyte (Bleil and Wassarman, 1980b; Greve et al., 1982) and are arranged in long, interconnecting filaments. ZP1, with a n

0 1991 WILEY-LISS, INC.

average molecular weight of 200,000 daltons, is the largest and may play a structural role by linking strands of the other two components. ZP2 has a n average molecular weight of 120,000 daltons and is thought to bind acrosome-reacted sperm to the zona through a trypsin-like site on the sperm head (Bleil et al., 1988). ZP3, with a molecular weight of approximately 83,000 daltons, binds acrosome intact sperm through its 0-linked oligosaccharides (Bleil and Wassarman, 1980c; Florman and Wassarman, 1985). However, the presence of the protein portion of the molecule is necessary for inducing the acrosome reaction (Bleil and Wassarman, 1983; Wassarman e t al., 1985). Solubilized whole zonae as well as purified ZP3 will induce the acrosome reaction in capacitated sperm. Pronase treatment of the whole zonae produces low-molecularweight glycopeptides, which bind to the sperm head but are unable to induce the acrosome reaction (Florman and Wassarman, 1985; Florman et al., 1984). However, the acrosome reaction can be induced by immunoaggregation of the bound glycopeptides (Leyton and Saling, 1989). These observations suggest that the trigger for the reaction may be the aggregation of zona binding sites on the sperm head (Leyton and Saling, 1989; Wassarman, 1990). If receptor aggregation is the key, then i t may be possible to induce the acrosome reaction by the aggregation of zona binding sites in the absence of zonae components (Saling, 1989). The purpose of this presentation is to test this hypothesis by the immunoaggregation of a proteinase inhibitor of seminal vesicle origin (SVI) bound to the acrosomal cap region of the sperm head. SVI has a molecular weight of 6.4 kDa and is heat and acid stable (Poirier and Jackson, 1981). It effectively reduces the enzymatic activity of trypsin and murine acrosin but has no effect on chymotrypsin, thrombin, plasmin, or kallikrein. The inhibitor binds the sperm head a t ejaculation (Irwin et al., 1983) and is removed during in vitro (Boettger et al., 1989) and in utero (Robinson et al., 1987) incubation. The sperm, however, will rebind SVI in the acrosomal cap region if

Received April 8, 1991; accepted June 12, 1991. Address reprint requests to Gary R. Poirier, Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294.

ACROSOME REACTION INDUCED BY IMMUNOAGGREGATION given the opportunity, suggesting that the inhibitor binding sites on the sperm head remain functional. Sperm-zona binding can be blocked by pretreating capacitated sperm with SVI (Poirier et al., 1986).The SVI binding site, the acceptor, has been purified by affinity chromatography using the inhibitor as the ligand (Aarons et al., 1984). Treating cumulus-free, zona-intact oocytes with purified acceptor reduces in a concentration-dependent manner the number of sperm able to bind to the zona (Poirier et al., 1986). These observations suggest that the acceptor, the SVI binding site, functions in zona binding. Thus immunoaggregating sperm-bound SVI should help determine if aggregation of zona binding sites, in the absence of ZP3, will induce the acrosome reaction.

MATERIALS AND METHODS Animals ICR mice 6-12 weeks of age were used throughout the study. These animals were subjected to controlled lighting (16L:BD)and constant temperature (22°C -+ 2°C) and had free access to food and water.

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SVI-specific antibodies were removed from a portion of the immune serum by absorption with purified inhibitor. Equal volumes of the immune serum and inhibitor (300 mIU/ml) in PBS were incubated overnight at 4°C. The immune complexes were removed by centrifugation (1,OOOg) for 10 min. The absorption process was repeated and the efficiency of removal of anti-SVI antibodies was verified by indirect immunofluorescence with inhibitor-treated sperm.

Fab Fragments Fab fragments prepared from rabbit anti-SVI serum were obtained by the procedure described by Mage (1980). IgG was precipitated with Na,SO, (1:0.75 ratio of serum to 36%salt). The precipitate, dialyzed against 10 mM phosphate buffer, pH 7.6, was applied to a DEAE cellulose column equilibrated with the same buffer. The IgG that did not bind to the resin was collected. The Fab fragments were obtained by proteolysis of the purified IgG. Mercuripapain was added to the purified IgG (1:lOO ratio of enzyme to protein) in 10 mM phosphate, pH 7.3, with 0.15 M NaC1,l mM EDTA, and 25 mM mercaptoethanol. The mixture was incubated for 1 h r at 37°C. Iodacetamide, final concentration 30 mM, was then added and incubated for a n additional 15 min at 37°C. Purification of the Fab fragments was done by ionexchange chromatography using CM-cellulose with a 10 mM acetate buffer, pH 5.4. Fab was eluted with the starting buffer. Digestion and purification were followed by SDS-PAGE on a 13% gel under reducing conditions. Protein concentrations were determined by the BioRad miniassay using bovine serum albumin as a standard.

SVI Isolation The inhibitor was isolated from homogenized seminal vesicle tissues as previously described (Poirier and Jackson, 1981). The procedure consisted of gel filtration (Sephadex G-75), affinity chromatography using bovine pancreatic trypsin as the ligand and ion-exchange chromatography (SP-Sephadex G-25). Once purified, the inhibitor solution was dialyzed against PBS and stored a t -20°C until used. Measures of inhibitory activity were determined against porcine pancreatic trypsin with N-benzoylDL-arginine-p-nitroanilide (BAPNA) as the substrate in Ca2+-free triethanolamine hydrochloride (TRA) Isolation of SVI-Specific IgG buffer, pH 7.8, 0.2 M (Fritz et al., 1974). Each 3.0 ml SVI was coupled to Sepharose4B (Pharmacia Fine assay contained 6.0 pg trypsin, 0.77 mM BAPNA, and various amounts of inhibitor. An inhibitor unit (IU) is Chemical, Piscataway, NJ) as described by Nicholson defined as the amount of inhibitor which reduces the e t al. (1983). The column contained 5 ml packed beads containing approximately 0.25 mg protein. The column hydrolysis of the substrate by 1 pmole/min. was washed in 0.1 M borate buffer (pH 8.0,0.5 M NaC1, Coomassie Brilliant Blue (CB) Method of 0.02% NaN,). One milliliter of anti-SVI serum was Acrosome Staining diluted 1:lO with the borate buffer, applied to the Sperm were scored for acrosomes by the CB staining column, followed by washing, with additional buffer to technique (Moller et al., 1990). Sperm were fixed with remove substance not absorbed. The column was then 5% formaldehyde in phosphate-buffered saline (PBS) washed with 0.1 M citrate phosphate buffer (pH 3.0,0.5 for 10 min, washed once with PBS at 10,000 RPM, and M NaC1, 0.02% NaN,). The fractions collected were dried onto glass slides. The cells were stained for 2 min neutralized by the dropwise addition of 2 N NaOH, in 0.04% Coomassie G-250 made with 3.5% perchloric pooled, concentrated (Amicon YM; 100,000 MW cutoff acid. The slides were rinsed with PBS and the sperm filter) and dialyzed against PBS. The final concentrate covered with PBS/glycerol (1:l by volume) and ob- was recognized by goat antirabbit IgG on a n ELISA plate a t a concentration of 4 pg/ml. served. Indirect Immunofluorescence Antiserum The indirect immunof luorescent technique was used Antiserum to the seminal vesicle inhibitor was raised in rabbits and the specificity of the antiserum to detect the presence of seminal inhibitor on sperm. All was established as reported earlier (Poirier and Jack- incubations were carried out in 1.5 ml plastic centrison, 1981; Irwin et al., 1983; Richardson et al., 1987). fuge tubes. The sperm samples were suspended in 1.0%

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Fig. 1. Estimation of the titer of the immune serum, anti-SVI serum, by ELISA methodology. Purified SVI was added to a microtiter plate and probed with dilutions of anti-SVI serum (open circles) or normal rabbit serum (solid circles). The bound immunoglobulins were detected with a peroxidase labeled antirabbit IgG. The titer was defined as the dilution that gave one-half maximum activity.

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Fig. 2. Western blot analysis of SVI a s recognized by the immune serum, its purified IgG, and the Fab fragment. The supernatant of homogenized seminal vesicle tissue (1.22 mgiml) was prepared under reducing conditions and electrophoresed in a 13%SDS polyacrylamide gel. The protein was transferred to nitrocellulose. Lane a was probed with anti-SVI (1:2,000 dilution), lane b with 5 pg/ml anti-SVI Fab fragment, lane c with 0.4 pgiml of affinity purified anti-SVI IgG, lane d with a 1:2,000 dilution ofnormal rabbit serum, and lane e with PBS. The bound immunoglobulins were localized with a peroxidase-labeled antirabbit IgG. Prestained molecular weight markers (2,512 to 20,400) purchased from Diversified Biotech (Newton Centre, MA) were used. Lane f is a silver-stained electrophorectic analysis of purified SVI (300 ng) on a 13% SDS gel.

formaldehyde in PBS for 30 min at room temperature. The sperm were washed twice in PBS, diluted to 1 x 106/ml and incubated for 30 rnin with SVI a t 30 mIU/106 cells. After a wash, Fc binding sites were blocked by a 30 min incubation in 3% normal goat serum in PBS. The cells were washed and resuspended in 200 pl of a 1/40 dilution of the immune serum for 1 hr at room temperature. The cells were washed thrice, and a 1:40 dilution of goat antirabbit IgG, fluoresceinlabeled (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) was added for 1 hr. The undiluted solution contained 1 mg/ml antibody. The excess anti- The absorbance was read at 415 nm in a microtiter body was washed away, and the cells were resuspended plate reader. in PBS. A drop of the sperm suspension was dried on a Acrosome Reaction Induction glass slide. The dried sperm were covered with PBS/ Sperm ( 2 x 106/ml), suspended in Earle’s modified glycerol and observed in a Leitz Dialux microscope medium (M199-M) containing 1.8 mM CaC1, and supequipped with epifluorescence. plemented with bovine serum albumin and pyruvate, ELISA were incubated for 1 h r at 37°C in a moist atmosphere The titer of the immune serum was determined using of 5% C 0 2 in air. These conditions are known to induce ELISA methodology. A microtiter plate was coated capacitation (Bleil and Wassarman, 1980c, 1983). Puwith 100 pl/well of purified SVI (30 mIU/ml, 10 pg/ml) rified preparations of SVI in PBS were added to 1.0 ml in coating buffer (carbonate-bicarbonate, pH 9.6). The sperm samples to give a final inhibitor concentration of plate was incubated at room temperature for 2 hr, 30 mIU/106 sperm. After a 30 min incubation a t room washed in PBS, and then blocked for 30 min a t room temperature, the sperm were then washed twice a t temperature in 3% nonfat milk. The immune serum or 5,000 rpm for 1min in M199-M in a n Eppendorf 5415C normal rabbit serum was serially diluted in PBS and minicentrifuge. Separate samples of the inhibitor100 pl of each dilution was added per well for a n treated cells were incubated for a n additional 30 min in additional 2 hr. The plates were thoroughly washed, anti-SVI serum, anti-SVI Fab fragments, normal raband 100 pl of peroxidase-labeled goat antirabbit IgG bit serum or PBS. Portions of these samples were mixed (Jackson Immunoresearch Laboratories, Inc.), diluted with equal volumes of 10% formaldehyde in PBS and 1:1500 in PBS, was added per well for a 2 h r incubation. stained with CB. The remaining portions were washed The original solution contained 1.0 mg/ml antibody. twice in PBS and incubated for 30 min in goat antirabAfter washing, 100 pI of the substrate [0.1 M of bit IgG (Jackson Immunoresearch Laboratories, Inc.). 2,2,-azinobis(3-ethylbenzthiazoline sulfonic acid)] in a The cells were then fixed with formaldehyde and citrate buffer, pH 4.2,O.l M plus 0.3% H,O, was added. stained with CB.

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Fig. 4. Coomassie blue-stained sperm. The arrow shows a sperm that has acrosome reacted. ~ 1 , 0 0 0 .

Fig. 3. Indirect immunofluorescence of cauda epididymal sperm. Anti-SVI, diluted 1:40, was used as antiserum in A, anti-SVI Fab fragments (16 Fgiml) were used in B, affinity purified anti-SVI IgG (4 pg/ml) was used in C. D is a bright field image of the sperm seen in c. x1,ooo.

Zonae Zonae were isolated from ovarian oocytes by a procedure described by Boettger et al. (1989). Zonae were solubilized just prior to use by incubation at 70°C for 30 min (Cholewa-Stewart and Massaro, 1972). The concentration of zonae used is expressed as zonae equivalents per microliter. This number is based on the addition of a minimal volume (