Journal of Reproductive Immunology, 11 (1987) 101-116 101 Elsevier Scientific Publishers Ireland Ltd.

JRI 00468

A monoclonal antibody to a zona pellucida-proteinase

inhibitor binding component on murine spermatozoa

R i c h a r d R i c h a r d s o n , T o d d B u c k i n g h a m , Hol ly B o e t t g e r and G a r y R. Po i r ie r

Department of Biology, University of Alabama at Birmingham, University Station, Birmingham, AL 35294 (U.S.A.)

(Accepted for publication 6 March 1987)

Summary

A monoclonal antibody, J-23, was produced to an epitope of a binding (acceptor) component on the plasma membrane in the acrosomal cap region of the mouse sperm head. The component binds a proteinase inhibitor of seminal vesicle origin at ejaculation and participates in the in vitro binding of capacitated sperm to the zona pellucida. The antibody, an IgM molecule, recognizes affinity purified acceptor, crude acceptor and whole sperm as determined by ELISA methodology. The antibody reacts with a 15,000 molecular weight component, the size previously determined for the acceptor, found in the supernatants of frozen- thawed cauda epididymal sperm. In addition, it binds to a 21,000 molecular weight component generated by mixing an excess of purified inhibitor (6400 daltons) with a crude acceptor preparation. J-23 binds to an epitope in the same region of the sperm head as does the inhibitor. This epitope becomes fully expressed during epididymal maturation and is found only in the lumen of epididymal tissues. Pretreating sperm with J-23 inhibits their ability to bind the inhibitor as well as the zona pellucida. Pretreating sperm with the inhibitor has little effect on the binding of J-23. These data indicate that J-23 recognizes an epitope on the acceptor but the epitope is not directly involved with inhibitor binding.

Correspondence to: G.R. Poirier.

0165-0378/87/$03.50 ~) 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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Key words: spermatozoa, sperm-zona binding, monoclonal antibody

Introduction

Recent observations suggest the presence of an acceptor type molecule on murine sperm which recognizes both the zona pellucida and a protein- ase inhibitor of seminal vesicle origin (Poirier et al., 1986). The compo- nent, isolated by affinity chromatography using the seminal inhibitor as the ligand, is heat labile and has a molecular weight of 15,000 (Aarons et al., 1984). The inhibitor is acid and heat stable with a molecular weight of 6400 (Poirier and Jackson, 1981). It is an effective inhibitor of trypsin and murine acrosin but not chymotrypsin,~ plasmin, kallikrein or thrombin. To bind to the zona peUucida, murine sperm are incubated for approximately 1 h in a medium known to induce capacitation (Poirier et al., 1986; Saling et al., 1978; Inoue and Wolf, 1975). The inhibitor will, however, bind to the plasma membrane over the acrosomal cap region of both incubated and non-incubated cauda epididymal sperm. These observations are inter- preted to mean that the site is modified by the incubation to allow zona binding. The modification has no effect on the number of sperm able to bind the inhibitor or the position on the sperm head where the inhibitor binds. The inhibitor also binds to the acrosomal cap region of the sperm head at ejaculation (Irwin et al., 1983). The percentage of sperm showing the presence of the inhibitor decreases from 90% to about 10% after 4 h in utero. However, 70-80% of these sperm show inhibitor specific fluoresc- ence in exactly the same area as on freshly ejaculated sperm if given the opportunity to rebind the inhibitor (Robinson et al., 1987). These sperm have an intact plasma membrane over the acrosome. These data suggest that the site remains intact during in utero incubation.

Substances that bind to sperm at ejaculation and come off during incubation in the female reproductive tract are considered to be decapaci- tation factors if their rebinding inhibits fertilization (Yanagimachi, 1981). Proteinase inhibitors associated with the male genital tract have been included as decapacitation factors (Bhattacharyya and' Zaneveld, 1982; Rogers and Bentwood, 1982). If the murine seminal inhibitor is consi- dered to be such a factor, then the acceptor site would participate in two important aspects of fertilization; (1) capacitation, (2) zona binding.

In order to further characterize this important site a monoclonal anti- body has been prepared to it. This presentation describes the features of the antibody as well as additional characteristics of the binding site.

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Materials and Methods

Animals ICR mice 9 to 10 weeks of age were used throughout the study. These

animals were subjected to controlled lighting (16L:8D) and constant temperature (22 ___ 2°C) and had free access to food and water.

Inhibitor and acceptor isolation The inhibitor was isolated from seminal vesicle tissue by gel filtration on

Sephadex G-75, affinity chromatography with trypsin as the ligand, and ion exchange chromatography using SP-Sephadex C-25 (Poirier and Jackson, 1981). The binding component was isolated from the supernates of frozen-thawed cauda epididymal sperm suspensions by affinity chromatography with the seminal inhibitor as the ligand (Aarons et al., 1984). Both isolation procedures resulted in single bands upon elec- trophoresis in sodium dodecyl sulfate carried out on 15% polyaerylamide gels (pH 8.3) according to the procedure of Laemmli (1970) (see Poirier and Jackson, 1981; Aarons et al., 1984).

Inhibitor and acceptor assays Measures of inhibitory activity were determined against porcine pan-

creatic trypsin using N-benzoyl-oL-arginine-p-nitroanilide (BAPNA) as the substrate in Ca 2+-free triethanolamine hydrochloride buffer (pH 7.8), 0.2M (Fritz et al., 1974). Each 3-ml assay contained 6.0 Ixg trypsin, 0.77 mM BAPNA, and various amounts of inhibitor. An inhibitor unit (IU) is defined as the amount of inhibitor that reduces the hydrolysis of BAPNA by 1 mol/min. The binding component assay is a modification of the BAPNA assay. The binding component was mixed with trypsin and then 0.1-0.3 ml of inhibitor was added. An increase in the rate of BAPNA hydrolysis over the inhibitor-treated sample was used, es an indicator of acceptor activity (Aarons et al., 1984). An acceptor unit (AU) is defined as the amount of the component needed to increase the rate of BAPNA hydrolysis over the inhibitor treated sample by 0 .10D/min.

Zona binding assay Sperm were collected by mincing portions of the cauda epididymis in an

Earle's modified medium (M199-M) containing 1.8 mM CaC12 and sup- plemented with bovine serum albumin and pyruvate (Bleil and Wassar- man, 1980). After 15 min the sperm were filtered through two layers of Kimwipes, washed and diluted to 1 to 2 x 106 cell/ml in M199-M. Sperm were then incubated under oil for 1 h at 37°C in an atmosphere of 5% CO 2 in air.

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Ovulated eggs were obtained from the oviducts of superovulated females (Rafferty, 1970). The cumulus cells were removed by treatment with 0.2 mg /ml hyaluronidase in M199-M and washed three times.

To treat the eggs, cumulus-free eggs were transferred in a minimal volume to 0.1 ml of J-23 or normal rat serum diluted 1/500 with M199-M for 30min at room temperature. The eggs were washed twice and transferred to 0.1 ml of the incubated sperm suspension under a covering of oil.

To treat the sperm, a 0.05 ml aliquot of J-23 or normal rat serum dilute 1/500 with M199-M was added to an equal volume of an incubated sperm suspension. The eggs in a minimal volume of M199-M were added to the treated sperm suspension.

The egg-sperm suspension was cultured for 45 min at 37°C under oil in a moist a tmosphere of 5% CO2 in air. The eggs were removed from the drops, washed in M199-M using mouth-opera ted micropipettes to remove excess sperm, and fixed in 2.5% glutaladehyde buffered to pH 7.2 with 0.1 M sodium cacodylate. Measurements of binding were made by count- ing the number of sperm attached to the zona under phase optics.

Indirect immunofluorescence The indirect immunofluorescent technique (Sternberger, 1979) was used

to detect both the inhibitor and the binding component on murine sperm. For the inhibitor, sperm samples were extracted from epididymal frag- ments in (pH 7.2) phosphate buffered saline (PBS). The sperm were allowed to disperse at room temperature for 15 min, filtered through two layers of Kimwipes and fixed in a 0.5% solution of formaldehyde in PBS for 30 min at 5°C. These samples were centrifuged and the pellet resuspen- ded in PBS to give a final concentrat ion of 6-10 x 1 0 6 sperm/ml . The fixed sperm sample was mixed with a purified inhibitor preparat ion to give 30mlU/106 sperm. These mixtures were incubated for 15 min at room temperature and washed twice to remove unbound inhibitor. Control sperm were t reated with PBS and washed twice.

Sperm smears were made on acetone cleaned slides. The sperm were allowed to dry, fixed in absolute ethanol for 20 min, washed twice in PBS, and treated with 3% normal goat serum for 20 min at room temperature. The smears were covered with the immune serum (rabbit anti-mouse seminal vesicle inhibitor) (Poirier and Jackson, 1981), normal rabbit serum, or immune serum previously absorbed with the seminal inhibitor. All sera were diluted 1/40 with PBS and incubated on the slide for 30 min at room temperature in a moist chamber. The slides were then washed and treated with a 1/40 dilution of goat anti-rabbit IgG conjugated with fluorescein isothiocyanate (Southern Biotechnology Associates, Inc. Bir-

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mingham, AL). After a 30-rain incubation the excess antibody was washed away and the sperm covered with PBS/glycerol (1:1, by vol.) solution and observed in a Leitz Dialux microscope equipped with epifluorescence. Photographs were taken with Kodak Tri-X Pare Film.

For localization of the acceptor, epididymal fragments (caput, corpus and cauda) as well as the ductus deferens were minced in PBS. After 15 min the sperm were filtered through Kimwipes and adjusted to 2-4 x 10 6 sperm/ml. Equal volumes (0.5 ml) of sperm and hybridoma supernat- ant (J-23), sperm and 1/500 dilution of normal rat serum or sperm and PBS were incubated for 20 min at room temperature. The sperm were washed twice and sperm smears were made on acetone cleaned slides. The sperm were allowed to dry and blocked with 3% normal goat serum and then treated for 20 min at room temperature with a 1/20 dilution of goat anti-rat IgG (whole molecule) conjugated with fluorescein isothiocyanate (United States Biochemical Corporation, Cleveland, OH).

Frozen sections (8 ~m) of mouse tissues (heart, liver, muscle, testes, seminal vesicles and the caput and cauda epididymis) were cut, transferred to clean slides, thawed and air dried. The sections were treated with J-23 for 20 min at room temperature, washed in PBS and incubated in goat anti-rat IgG (whole molecule) conjugated with ttuorescein isothiocyanate. Controls consisted of substituting normal rat serum (1/40 dilution) or PBS for J-23.

Western blots The rabbit anti-mouse seminal inhibitor antiserum has been previously

tested for specificity by double diffusion and immunoelectrophoresis (Poirier and Jackson, 1981). Its specificity has been substantiated here using the more sensitive Western-Blot technique. A purified, as well as a crude sample of the inhibitor were electrophoresed in 15% gels (pH 8.3) according to the procedure of Laemmli (1970). The transfer to nitrocellul- ose was made at 25 V for 5 h. The gel was then stained with Coomassie Blue to assay the extent of transfer. A portion of the nitrocellulose was stained with India ink (Hancock and Tsang, 1983) to determine the position of molecular weight standards. The nitrocellulose was blocked with 0.1% BSA in 20mMTris, 500mMNaCl (TBS-BSA), pH 7.5, washed in TBS and incubated overnight in 1/500 diluted rabbit anti-mouse seminal inhibitor in TBS-BSA. The nitrocellulose was then washed twice and incubated in goat anti-rabbit IgG labeled with alkaline phosphatase (Bio Rad Labs, Richmond, CA). The excess secondary antiserum was removed by washing and the substrate added. The final substrate solution was made by diluting 2ml of a stock solution to 100ml with 0.1M NaHCO 3 (pH 9.8) and 1.0 mM MgCl 2. The stock was made by adding

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Fig. 1. A Western blot of a pure seminal inhibitor preparation (a) and a crude inhibitor prepara t ion (b) probed with a polyclonal antiserum to the mouse seminal inhibitor. The numbers to the right represent the molecular weight calibrations. Five microliters of a 2 ttg protein/ml preparation of the pure inhibitor and 5 I~l of 24 txg protein/ml of the crude preparation were loaded on the gel.

equal volumes of 0.3 mg/ml of p-nitro blue tetrazolium chloride in 70% dimethyl formamide (DMF) and 0.15mg/ml of 5-bromo-4-chloro-3- indolyl phosphate toluidine salt in 100% DMF. A single component was localized in both the crude as well as the purified sample (Fig. 1).

A crude sample of the acceptor was electrophoresed in 15% gels (pH 8.3) under non-denaturing conditions and transferred to nitrocellulose. The nitrocellulose was blocked with BSA and washed in TBS. The nitrocellulose was then divided into two portions. One part was probed overnight with J-23 diluted 1/10 with TBS-BSA. The nitrocellulose was then washed twice and incubated in goat anti-rat IgG labeled with alkaline phosphatase. The other portion was incubated in a solution of purified seminal inhibitor (130 mIU/ml) for 4 h at room temperature. The excess inhibitor was removed by washing and the nitrocellulose was probed for the inhibitor using the rabbit anti-mouse seminal inhibitor serum as described above.

Monoclonal antibody A monoclonal antibody J-23, to the binding site was produced according

to the procedure described by Kearney (1984). Two female rats were

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immunized with supernatant of frozen-thawed cauda epididymal sperm. "The first injection, consisting of 1 mg of protein mixed with equal volumes of complete Freund's adjuvant, was given in 0.1 ml aliquots in the hind foot pads. At 3-day intervals for the next 18 days, the rats were injected with 500 I~g protein in each hind foot pad. One day after the last injection the inguinal lymph nodes were removed, teased into a single cell suspen- sion and fused with a mouse myeloma (S194/5XXO.BU.1) cell line using 40% polyethylene glycol. The cells were washed and transferred by pipetting into a flask containing hypoxanthine, aminopterin and thymidine (HAT) medium. Feeder (peritoneal exudate) cells were added to the fused cells at this stage. The fusion mixture was then dispensed in 1 ml aliquots into 24 well culture plates at a density of 5 x 10S/ml. On day 14 of culture, supernatants were slowly replaced with non-HAT medium during the feeding of the cultures. Clones were considered positive if they secreted an antibody reacting with an affinity purified acceptor prepara- tion, crude acceptor and cauda epididymal sperm. The positive hybridoma was recloned twice by limiting dilution in a 96-well cluster plate to insure that a given well contained the progeny of only one hybridoma cell. The immunoglobulin class was determined by ELISA methodology using class-specific goat anti-rat immunoglobulin antibodies (Southern Biotech- nology Associates, Inc.). J-23 has a titer of 1/10 when used on whole cauda epididymal sperm as determined by ELISA methodology.

ELISA Sperm used to detect the acceptor were extracted from epididymal

tissue in PBS, filtered through two layers of Kimwipes and diluted to a final concentration of 2 x 106cell/ml. If sperm were to be tested for inhibitor the filtered sperm were incubated for 15 min at room tempera- ture with a purified inhibitor preparation (15 mlU/106 sperm). The sperm were washed and the volume adjusted with PBS to give 2 x 10 6 sperm/ml. A 0.1 ml aliquot of the sperm suspension was added to the 96-well microtiter plates that were previously primed with 0.1% glutaraldehyde.

The plates were centrifuged for 5m in at 1000rev./min and 0.1ml of 0.1% glutaraldehyde in borate saline (BS) (pH 8.4) was added. The cells were fixed for 30min at room temperature and the unreactive sites blocked with 1% BSA in BS for 1 h at room temperature.

Soluble material was added directly to a microtiter plate or diluted with BS. The material was added in triplicate rows in 0.1 ml aliquots and incubated overnight at 4°C. The plates were washed and blocked with 1% BSA-BS.

The primary antibody (be it J-23 or rabbit anti-mouse seminal inhibitor) was added in 0.1 ml aliquots and incubated for 4 h at room temperature.

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The plates were then washed 5 times with BS and incubated for 4 h with 0.1 ml of goat anti-rat (whole molecule) IgG (acceptor) or goat anti-rabbit IgG (inhibitor) labeled with alkaline phosphatase and diluted 1 to 500 with 1% BSA-BS (Southern Biotechnology Associates, Inc.). After washing the plates three times with BS, 0.1 ml of the substrate ( lmg /ml of p-nitrophenylphosphate in a pH 9.8 diethanolamine buffer) was added. The absorbance was read at 405 nm in an ELISA microtiter plate reader (Flow Laboratories, Inc. McClean, VA).

Sephadex chromatography A molecular weight estimate of the component in the crude acceptor

homogenate to which the monoclonal binds was made on fractions de- veloped from a Sephadex G-75 superfine column. The column was stan- dardized by determining the elution volume (Ve) of a number of compo- nents of known molecular weight. The void volume (Vo) was determined with blue dextran 2000. The V e of the component to which J-23 binds was determined by testing each fraction of the elution profile by ELISA and immunodot methodology. To determine if the epitope recognized by J-23 is found in other parts of the body, testes, liver, heart and muscle tissue were homogenized and the supernatants chromatographed on the G-75 column. Individual fractions were dot-blotted and probed with J-23.

Immunodot Each fraction of the Sephadex separations was dotted (100 ~l/dot) to

nitrocellulose using a filtration manifold (Bio Rad Labs). The dots were then blocked with 200 ~1 of 1% BSA in TBS and washed with 400 I~1 of 0.05% Tween 20 in TBS (TFBS). The first antibody, 100 I~1 of J-23, was added to each well and incubated for 30 min. The wells were washed with 400 ixl of TFBS and 100 wl of goat anti-rat IgG (whole molecule) labeled with alkaline phosphatase, 1/3000 dilution (Bio Rad Labs), was added for 30 min. The wells were then washed twice with 400 Ixl of TI]3S. The membrane was then removed from the apparatus, the substrate added and the color allowed to develop for 15 min at room temperature.

Results

J-23 proved to be an IgM molecule. A crescent shaped fluorescence was visible on the acrosomal cap region of the sperm head when cauda epididymal sperm were treated with J-23 as the primary antibody treat- ment (Fig. 2a). This fluorescence was not seen when normal rat serum or PBS was substituted for J-23. An identical fluorescent pattern was seen when the inhibitor was localized on cauda epididymal sperm, previously

i:

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Fig. 2. Indirect immunofluorescence of cauda epididymal mouse sperm (a) treated with J-23 as the primary antibody treatment and (b) incubated in a pure seminal inhibitor preparation and then treated with rabbit anti-mouse seminal inhibitor, ×1000.

treated with a purified inhibitor preparation, using a rabbit anti-mouse seminal inhibitor antiserum (Fig. 2b). No inhibitor specific fluorescence was observed when normal rabbit serum, the immune serum absorbed with the inhibitor or PBS was substituted for the immune serum. The percentage of sperm showing the fluorescent pattern seen with J-23 treatment increased as sperm traverse the epididymis (Table 1).

Intense fluorescence was observed in the lumen of the tubules in the caput region of the epididymis when J-23 was used as the primary antibody treatment (Fig. 3a). This fluorescence was not seen when normal rat serum or PBS was substituted for J-23 (Fig. 3b). A speckled fluoresc- ence pattern was seen in the lumen of cauda epididymal tubules when J-23 was used as the primary antibody treatment (Fig. 3c). This pattern was not observed when normal rat serum or PBS was used. No fluorescence was seen in the epithelium of any region of the epididymis using J-23 as the primary treatment. Furthermore, no fluorescence was seen by exposing sections of testes, seminal vesicle, liver, heart or muscle tissue to J-23.

T A B L E 1 Percentage of sperm from different levels of the epididymis recognized by J-23 as determined by indirect immunofluorescence. Each number represents the percentage obtained from a random count of 200 cells. The experiment was done twice with two animals per trial.

Region Percentage with Fluorescing acrosomes

Caput 8, 6 Corpus 36, 18 Cauda 61, 57 Ductus deferens 86, 85

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Fig. 3. Indirect immunofluorescence of unfixed cryostat sections, (a) caput region of the epididymis treated with J-23 (b) caput region treated with normal rat serum, 1/40 dilution and (c) cauda region of the epididymis treated with J-23, x400.

Pretreating cauda epididymal sperm with J-23 decreased in a concentra- tion dependent manner the amount of seminal inhibitor able to bind to the sperm (Fig. 4). However , pre t rea tment with a 1/500 dilution of normal rat serum, or with PBS had no effect on the amount of seminal inhibitor able to bind (Fig. 4). The reverse, i.e. pretreating sperm with the seminal inhibitor had little effect on the amount of J-23 able to bind (data not

to o

1.O

o so i i

Fig. 4. ELISA data showing the effect of seminal inhibitor binding to sperm pretreated with decreasing amounts of the hybridoma supernatant (J-23), or with normal rat serum (NRtS) or PBS substituted for J-23 (0.0). The primary antiserum treatment (lst antiserum) consisted of the polyclonal rabbit anti-mouse seminal inhibitor (anti SVI), normal rabbit serum (NRbS), the immune serum absorbed with the seminal inhibitor (IA) or PBS. Each point represents the mean of triplicate values and the horizontal bars the standard deviations.

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>,ooJ .,i.) <100 Z O N 8 0 ,

60 . w

4 0

2 0 ¸

(=?, ,21,, (3;,

( 5 5 )

I S P E R M N J - 2 3 R A T N N

E G G N N N J - 2 3 R A T

Fig. 5. The effects on the number of sperm binding to zonae by treating the gametes with J-23 or normal rat serum (RAT) diluted 1/500. N refers to M199-M treatment. The number in parentheses refers to the number of eggs observed.

Fig. 6. Western blots of a crude acceptor preparation. Lane (a) was probed with J-23. Lane (c) was initially probed with the seminal inhibitor and then with the rabbit anti-mouse seminal inhibitor serum. Lane (b) was stained with India ink to demonstrate molecular weight standards.

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shown). Pretreating sperm, with J-23 reduced their ability to bind to zonae (Fig. 5). However, if normal rat serum was substituted for J-23 no decrease in the number of sperm able to bind zonae was observed. Similarly, if the eggs were pretreated with J-23 or normal rat serum no decrease in the number of sperm binding to zonae was observed.

J-23 binds to a single component of the crude acceptor preparation (Fig. 6). The component has a molecular weight of 15,000. Likewise, the seminal inhibitor also binds to 15,000 molecular component in the crude preparation.

A crude acceptor preparation was chromatographed on a Sephadex G-75 superfine column and each fraction was tested for the epitope recognized by J-23 using ELISA and immunodot methodology. Both techniques localized components in the same region of the optical density profile. The elution volume of the component suggested a molecular weight of 15,000 (Fig. 7). The epitope recognized by J-23 was not localized in chromatographed liver, heart, muscle or testicular homoge- nates.

4 0 -

2 0 "

~ O 1 0 . X

5 '

Ve Vo

Fig. 7. Molecular weight estimates of two components recognized by J-23. One of the components, with a molecular weight of 15,000, was found in the supernatant of frozen-thawed epididymal sperm (c single arrow). The other component with a molecular weight of 21,000 was formed by mixing a purified inhibitor sample with the supernatant of a frozen-thawed sperm suspension (b two arrows). The fractionations were carded out on a 2.5 x 40 cm Sephadex G-75 superfine column with a flow rate of 15 ml/h. One-milliter sample was applied and 1-ml fractions were collected. Standards include the seminal inhibitor (e = 6,400 daltons), cytochrome C (d = 12,300 daltons) and trypsin (a = 23,800 daltons).

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Fig. 8. Immunodot-blot demonstrating the ability to detect the seminal inhibitor by probing initially with a crude acceptor preparation and then J-23. Dots one (1) through four (4) represents the blotting of 100 Va of a pure seminal inhibitor solution containing 60, 30, 15 and 7.5 mlU/ml. Position 5 was dotted with pure acceptor (50 mAU/ml), 6 with a crude acceptor preparation, 7 with J-23 hybridoma supernatant, 8 with normal rat serum (1 to 500 dilution), 9 with PBS and 10 with 1% bovine serum albumin in PBS.

To determine if the seminal inhibitor bound to the acceptor in vitro a crude acceptor preparation was mixed with a purified seminal inhibitor preparation and chromatographed on the Sephadex G-75 column. When the fractions were dot-blotted to nitrocellulose and probed for the accep- tor using J-23 a single region of the profile with a molecular weight of 21,000 was identified (Fig. 7). When the fractions were probed for the seminal inhibitor two regions of the profile with molecular weights of 21,000 and 6400 were identified.

To explore additional methods to study the interactions between the binding component and the inhibitor and to help define the specificity of J-23 portions of a purified seminal inhibitor preparation were dot-blotted to nitrocellulose. The dots were initially probed with a crude acceptor preparation which was then localized with J-23. The acceptor can recog- nize the inhibitor on the nitrocellulose and the amount of binding compo- nent attached depends on the amount of inhibitor blotted (Fig. 8). Positive controls involved applying J-23 and normal rat serum directly to the nitrocellulose. This demonstrated that the secondary antiserum (goat anti-rat IgG labeled with alkaline phosphatase) wotrld, bind to the rat immunoglobulin in J-23. The negative controls consisted of substituting PBS and BSA for the inhibitor.

Discussion

The monoclonal antibody, J-23, appears to be made to an epitope of the seminal inhibitor-zona binding component. The hybridoma selected for cloning secreted an antibody able to bind to affinity purified acceptor, a crude acceptor preparation and cauda epididymal sperm as judged by the ELISA method. The seminal inhibitor binds to the apical portion of the sperm head at ejaculation and when cauda epididymal sperm are incu- bated in vitro with a purified inhibitor preparation (Irwin et al., 1983).

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Sperm removed from the uterus 4 h post coitus or from a medium known to induce capacitation will rebind or bind inhibitor such that it is imposs- ible to distinguish them from freshly ejaculated sperm (Robinson et al., 1987). This is the same region of the sperm head where the epitope, recognized by J-23, resides and is what is expected from an antibody to an exposed epitope of the acceptor.

Sperm develop the ability to bind to the seminal inhibitor (Irwin et al., 1983) and to the zona (Saling et al., 1978) during the epididymal sojourn. Furthermore, ten times more acceptor can be isolated from cauda and ductus sperm than from equal numbers of caput and corpus sperm (Robinson et al., 1987). These observations support data presented here, i.e. there is an increase in the percentage of sperm exhibiting the epitope. recognized by J-23 during epididymal maturation. The mechanism of expression of the epitope is not clear. The testes, as well as the seminal vesicles, liver, heart and muscle, show no reactive epitopes by indirect immunofluorescence of tissue sections or by the immunodot of Sephadex fractionated homogenates. It appears then that the testes are not the source of the acceptor. However, it is possible that the epitope is made in the testes but not exposed until the epididymis. Recent evidence indicates that in the hamster the caput region of the epididymis secretes a protein involved in sperm-egg binding (Robitaille et al., 1986). The intense fluorescent staining of the lumenal contents in the caput region would support such a suggestion. However, the inability to localize the acceptor in the epithelium of frozen sections in any portion of the epididymis adds little to localizing its source. One of the major problems effecting the localization and identification of antigens in unfixed cryostat sections is diffusion. If an antigen is present in limited quantity, diffusion may dilute it such that it is no longer detectable. Additionally, diffusion would give a false indication of antigen localization. However, all fixations used to date, be they on cryostat sections or for paraffin embedding, modified the epitope such that it was no longer recognized by J-23. Thus the epitope recognized by J-23 appears to be labile. However, the inhibitor binding portion of the molecule retains its activity after mild paraformaldehyde fixation (Irwin et al., 1983).

The molecular weight of the component in the frozen thawed-sperm supernatant recognized by J-23 is estimated, by two different techniques, to be 15,000. This is the same as the estimated weight of the sperm component which binds the inhibitor as well as the weight of the compo- nent seen by SDS-PAGE electrophoresis of affinity purified acceptor (Aarons et al., 1984). Recently, zona binding proteins have been demon- strated in sperm extracts from a number of mammalian species (O'Rand et al., 1985). The proteins range in size from 14,000 to 19,000 daltons and

115

there appears to be more than one zona binding component per species. In the murine system there appears to be a single component, molecular weight of 15,000, which binds a seminal inhibitor and which also partici- pates in zona binding. There may be other components in that molecular weight range which do not bind the inhibitor, are not recognized by J-23 hut do function in zona binding.

The molecular weight of the acceptor-inhibitor complex was estimated to be 21,000. Since that is the sum of the individual components it appears that, at least in vitro, only one inhibitor molecule binds per acceptor molecule.

Pretreating sperm with J-23 inhibits their ability to bind to the zona as well as the binding of seminal inhibitor. However, pretreating sperm with the seminal inhibitor has little effect on the amount of J-23 able to bind. This observation suggests that the epitope to which the antibody binds is not directly associated with the inhibitor binding site. Since the size of the antibody, an IgM molecule, is 64x larger than the acceptor an antibody on an epitope some distance from the binding site may easily block inhibitor as well as zona binding. That the epitope recognized by J-23 is not associated with the binding site is further evidenced by the fact that J-23 can recognize inhibitor-acceptor complexes.

Acknowledgements

We are indebted to Nannette Nicholson and David Mulvaney, in the laboratory of Dr. John Kearney, for their assistance in the preparation of the monoclonal antibody. We are also indebted to Dr. William Gathings of Southern Biotechnology Associates, Inc. for determining the class of the antibody. We wish to thank Jan Thibodeaux for typing the text.

References

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Irwin, M., Nicholson, N., Haywood, LT. and Poirier, G.R. (1983) Immunofluorescent localization of a murine seminal vesicle proteinase inhibitor. Biol. Reprod. 28, 1201-1206.

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