The Role of Complement Component C3b and Its Receptors in Sperm-Oocyte Interaction (Ferilzation/Inferility) DEBORAH J

Home , C3b, Complement component 3, Complement receptor, Trypsin 1

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10051-10055, November 1993 Developmental Biology The role of complement component C3b and its receptors in sperm-oocyte interaction (ferilzation/inferIlity) DEBORAH J. ANDERSON*t, AMY F. ABBOTT*, AND RICHARD M. JACKt *Fearing Research Laboratory, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital, and tDepartment of Rheumatology and Immunology, Brigham and Women's Hospital, and the Department of Medicine, Harvard Medical School, Boston, MA 02115 Communicated by K. Frank Austen, June 30, 1993

ABSTRACT Previous studies have shown that human functions other than binding and regulation of complement sperm that have undergone the acrosome reaction express a component C3b. Monoclonal antibodies (mAbs) directed unique tissue-specific variant of the complement component 3 against MCP inhibit the penetration of hamster oocytes by (C3)-binding molecule membrane cofactor protein (MCP, human sperm (2-4), suggesting that MCP and perhaps C3 also CD46) and that damaged or dead sperm activate the alternative play a role in sperm-oocyte interaction. To further define the pathway of complement and bind C3 catabolites. In this study role of C3 and its binding proteins in sperm-ovum interac- we provide evidence that MCP on sperm that have undergone tions, we assessed the expression of other C3-binding pro- the acrosome reaction specifically binds dimeric C3b and that teins and receptors on sperm and oocytes, the effects of human sperm acrosomal proteases released during the ac- sperm acrosomal enzymes on C3 cleavage to C3b, and the rosome reaction directly cleave C3, facilitating its binding to binding of C3 and C3b to gametes. We also investigated the MCP. Furthermore, human and hamster oocytes can activate effects of C3b and antibodies to C3 on sperm penetration of the alternative pathway of complement and also bind human oocytes. The data support a role for complement-binding C3 fragments. Monoclonal antibodies specific for complement proteins and C3 fragments in human gamete interactions receptors type 1 (CD35) and type 3 (CD11b/CD18) bind to the leading to fertilization. human oocyte plasma membrane, indicating that specific complement-binding molecules may play a role in the attachment of C3 catabolites to oocytes. Subsaturating concentrations of MATERIALS AND METHODS dimeric C3b (0.01-1 FM) promoted penetration of hamster Gamete Preparation. Semen donors were healthy men oocytes by human sperm, whereas saturating doses (>10 FM) between 22 and 38 yr old with normal semen parameters. inhibited this process. In addition, antibodies to both MCP and Motile sperm were isolated from fresh liquified semen by C3 signiicantly inhibited penetration of hamster oocytes by swim-up technique or centrifugation through discontinuous human sperm. These data provide evidence that regulated 47%/90%o Percoll (Pharmacia) gradients (7). AR sperm were gamete-induced generation of C3 fragments and the binding of prepared by incubation with 5 ,uM calcium ionophore A23187 these fragments by selectively expressed receptors on sperm in Ham's F-10 medium (GIBCO)/3.5% human serum albumin and oocytes may be an initial step in gamete interaction, leading (HSA; Sigma) at 37°C for 30 min (2). Human ova were to membrane fusion and fertilization. obtained from the Brigham and Women's Hospital In Vitro Fertilization Service (courtesy ofR. Clarke and K. Jackson). Mammalian fertilization entails a complex series of events Methods for ovulation induction and oocyte retrieval have including (i) attachment of sperm to the zona pellucida of the been described (8). Hamster oocytes were obtained from oocyte via receptors on the sperm plasma membrane; (ii) the sexually mature female golden hamsters, Mesocricetus au- sperm acrosome reaction (AR), exocytosis of the acrosomal ratus (Charles River Breeding Laboratories) (9). Oocytes contents from the acrosome, a specialized granule containing were washed and resuspended in sperm washing medium proteases and other degradative enzymes; (iii) penetration of (SWM; Irvine Scientific), which contains HSA at 5 mg/ml sperm through the zona pellucida, which occurs concurrently and was supplemented with 10% heat-inactivated fetal calf with the AR; (iv) attachment of sperm to the oocyte plasma serum (GIBCO). For some experiments, oocytes were transmembrane (oolema) via receptors exposed on the sperm ferred to a drop ofTyrode's salt solution (Sigma), pH 2.0, to surface after the AR; and (v) sperm-oocyte fusion. Receptors dissolve the zona pellucida (10). and ligands mediating these events are only now beginning to Immunofluorescence Staining and Microscopy. Expression be identified and characterized (1). of complement-binding proteins and receptors on gamete Recently, we and others have shown that human sperm membranes was assessed by indirect fluorescence staining. that have undergone the AR express membrane cofactor Sperm (1 x 106 in 0.1 ml of Ham's F-10 medium/0.3% HSA) protein (MCP, CD46), a complement component 3 (C3)- or ova (2 ova per 0.1 ml of F-10/HSA medium) were binding and regulatory protein, on the inner acrosomal mem- incubated with predetermined saturating doses (6-12 ug/ml) brane (2-4). Sperm MCP has cofactor activity for factor of murine anti-human monoclonal IgG specific for MCP I-mediated cleavage of C3, and one of its apparent functions (mAb H316) (2) and the complement receptors CR1 (mAb is protection of sperm from the lytic consequences of com- YZ-1) (11), CR2 (mAb HB-5) (12), or CR3 (mAb OKM1) (13) plement resulting from interactions with antisperm antibodies (5). However, sperm MCP is a tissue-specific isoform Abbreviations: C3, complement component 3; MCP, membrane characterized by differential glycosylation (5) and deletions cofactor protein; CR1, -2, and -3, complement receptor types 1, 2, resulting in a shorter transmembrane region and distinctive and 3, respectively; AR, acrosome reaction; mAb, monoclonal cytoplasmic tail (6); this suggests that sperm MCP may have antibody; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine B isothiocyanate; NHS, normal human serum; HI-NHS, heat-inactivated normal human serum; HSA, human serum albumin; The publication costs of this article were defrayed in part by page charge PSA, Pisum sativum agglutinin. payment. This article must therefore be hereby marked "advertisement" tTo whom reprint requests should be addressed at: 250 Longwood in accordance with 18 U.S.C. §1734 solely to indicate this fact. Avenue, Room 204, Boston, MA 02115. 10051 Downloaded by guest on October 2, 2021 10052 Developmental Biology: Anderson et al. Proc. Natl. Acad. Sci. USA 90 (1993) for 60 min at 4°C. mAbs were purified as described (11). nique and capacitated in SWM for 1 hr at 37°C, were Control isotype-matched mAbs included MOPC-21 (IgGl; incubated with dimeric C3b (concentrations as above) and Sigma), HLe-1 (IgGl; Becton Dickinson) and HLA-DR antibodies or with SWM medium alone for 1 hr at 37°C. (IgG2a; Becton Dickinson). Sperm were washed in F-10/ Identically treated oocytes and sperm were then combined in HSA by centrifugation at 4°C for 10 min at 400 x g and organ culture dishes (Falcon) and incubated at 37°C in 5% resuspended to 1 x 106 per ml in F-10/HSA medium. Oocytes CO2 for 3 hr. Oocytes were then washed twice in SWM were washed by transfer through three 0.1-ml drops of medium and examined under phase-contrast microscopy for F-10/HSA. Gametes were then stained with affinity-purified evidence of sperm penetration. fluorescein isothiocyanate (FITC)-coupled F(ab')2 goat anti- Acrosin C3 Cleavage Studies. An acrosin-enriched extract mouse F(ab')2 at 5 ug/ml (Jackson ImmunoResearch). To containing =100 milli-international units of acrosin per ml determine whether complement was activated by gametes was prepared from 108 fresh human sperm by the acid- and whether C3 products were deposited on their surface, extraction method (17). To study the dose dependence of sperm or ova were incubated for 30 min at 37°C with normal acrosin-mediated C3 cleavage, replicate 0.05-ml samples of human serum (NHS), NHS that had been heat-inactivated 125I-labeled C3 (0.5 mg/ml) were incubated at 37°C for 30 min (HI-NHS) at 56°C for 30 min, or NHS treated with 10 mM with an equal volume of Hanks' balanced salt solution EDTA (Sigma). Washed gametes were then incubated in 50% without cations (HBSS), with an equal volume of trypsin (1 normal goat serum for 10 min (blocking step) and assessed for mg/ml in HBSS; Worthington), with 2-50 p1 of acrosin bound C3 products by direct fluorescent staining at 4°C with extract diluted to 50 ul in HBSS, or with 50 Al of hyaluroni- FITC-coupled F(ab')2 goat anti-human C3 (25 Ag/ml; Cap- dase (2 mg/ml in HBSS). The time dependence of acrosin- pel). To detect binding of dimeric C3b, gametes were incu- mediated cleavage ofC3 was analyzed by incubating replicate bated with a 1 uM solution of dimeric C3b in F-10/HSA samples of 25 ,g of 125I-labeled C3 (0.5 mg/ml) at 37°C for up medium for 60 min at 4°C and visualized by immunofluores- to 30 min with either 0.05 ml of acrosin extract or with 10 pg cent staining by using affinity-purified FITC-coupled F(ab')2 oftrypsin (1 mg/ml) in 50-,l volumes. Samples were resolved goat anti-human C3. FITC-coupled F(ab')2 goat anti-mouse on a SDS/5-15% polyacrylamide gel and visualized by IgG was also run for each assay condition to serve as a autoradiography. negative control. Gametes were examined for fluorescence by using a Zeiss epifluorescence microscope. Photomicro- graphs were taken with a Nikon camera on Kodak Tri-X or RESULTS Ektar 400 ASA film. Immunofluorescence studies were done on sperm at various Correlation of C3 Binding with the AR. To simultaneously stages of the AR (Fig. 1). MCP (CD46) was detected on the assess the C3-binding pattern and acrosomal status of indi- surface of ionophore-treated AR sperm but not on untreated vidual sperm, sperm were incubated with sera or dimeric C3b (non-AR) replicate samples as has been shown (2-4). The and treated with FITC-conjugated anti-C3 or control anti- human complement receptors CR1 (CD35), CR2 (CD21), and body as described above, then were dried onto microscope CR3 (CDllb/CD18) were not detected on either untreated or slides, and fixed with acetone (4°C, 10 min). Subsequently, ionophore-treated AR sperm (data not shown). Dimeric C3b tetramethylrhodamine B isothiocyanate (TRITC)-conjugated bound to the acrosomal region of acrosome-reacting sperm Pisum sativum agglutinin (PSA; Sigma) was applied to iden- (speckled TRITC-PSA acrosomal binding pattern) and to the tify AR sperm. AR sperm show no binding of lectin in the equatorial region of acrosome-reacted sperm (equatorial region of the acrosomal cap but show some binding to the TRITC-PSA binding pattern) (Fig. 2A) but did not bind at all equatorial segment, whereas non-AR sperm show strong to fresh sperm that had not undergone AR (data not shown). PSA staining in the entire acrosomal-cap region (14). Sperm This pattern resembled that described for MCP (2), suggest- were individually assessed for FITC and TRITC fluorescence ing that MCP binds dimeric C3b on AR sperm. 125I-labeled patterns by using a dual-filter system on a Zeiss epifluorescence microscope. A B c Preparation of Dimeric C3b and Binding Studies. C3 was purified from fresh-frozen human plasma (American Red Cross, Dedham, MA) (15). C3b was prepared by trypsin cleavage, crosslinked with glutaraldehyde, and sized on pm. sucrose gradients (16). Dimeric C3b was labeled with 125I by oam using lodo-Gen (Pierce) to a specific activity of 2 x 106 am . I. cpm/pg. Replicate samples of untreated or AR sperm (2 x zI. 107/ml) were incubated in Ham's F-10 medium alone or in iam medium containing a 20-fold molar excess of unlabeled - es dimeric C3b for 30 min at 4°C. Replicate samples of 2 x 106 m~~ cells from the untreated (medium alone) or dimer-pretreated cell suspensions were then incubated for 60 min at 4°C with 1251-labeled C3b2 (1 pg/ml to 50 pg/ml). Binding was assessed by centrifuging samples through dibutyl/dinonyl mp phthalate oil (ICN), and the pellets and supernatants were assessed for cell-bound and free 125I-labeled ligand, respectively (11). Specific binding of dimeric ligand was calculated t- by subtracting the amount bound to cells pretreated with unlabeled excess ligand from the amount bound in the absence of unlabeled ligand. FIG. 1. Diagram of human spermatozoa undergoing the AR. (A) Hamster Egg-Penetration Test. Approximately 20 zona Normal sperm have an intact plasma membrane (pm) and an acrosome delimited by the outer (oam) and inner (iam) acrosomal pellucida-free hamster oocytes were added to 0.1 ml of SWM membranes. mp, Midpiece; t, tail. (B) Early stages of the AR are medium containing human dimeric C3b (10 nm-20 uM) or characterized by pm and oam fusion and the release of acrosomal various concentrations of anti-MCP or anti-C3 antibodies or matrix and contents (am) and expression ofMCP. (C) AR sperm bind medium alone; subsequent incubation was for 1 hr at 37°C. to the oolemma via receptors expressed on the iam, which are Simultaneously, human sperm, isolated by swim-up tech- concentrated at its equatorial segment (es). Downloaded by guest on October 2, 2021 Developmental Biology: Anderson et al. Proc. Natl. Acad. Sci. USA 90 (1993) 10053

FIG. 2. Immunofluorescence photomicrographs of C3 binding to FIG. 3. Immunofluorescence photomicrographs of human human spermatozoa. (A) Localization of dimeric C3b to the acroso- oocytes. (A) Binding of anti-CR1 mAb to the surface of a zona mal region of acrosome-reacting sperm (specked pattern; upper left peliucida-free human oocyte. (x360.) (B) Binding of anti-CR3 mAb and lower left quadrants) and equatorial segment ofAR human sperm to the surface of a zona pellucida-free human oocyte. (x360.) (C) (upper right and lower right quadrants). Brightly fluorescent spher- Lack of binding of anti-MCP antibody to viable human oocyte. ical particle in the upper left quadrant resembles a cytoplasmic (x360.) (D) Binding ofdimeric C3b to the surface ofa zona pellucida- droplet. (x900.) (B) Localization of C3 fragments from normal free human oocyte. (x360.) human serum on the surface of AR human sperm. Neck, midpiece, and tail localization was most frequently seen; binding to the sperm were detected by characteristic equatorial PSA staining (Fig. head was occasionally observed. (x900.) (C) TRITC-PSA staining of 2C). Thus, AR or nonviable sperm can activate C3 by the ionophore-treated human sperm preparation to detect individual AR sperm [lack of acrosomal fluorescence, except in equatorial region alternate pathway, but C3 deposition may also occur from (1)] and acrosome-intact sperm [solid staining of acrosome (2)]. direct activation of C3 by sperm enzymes (see below). After (x900.) (D) Same field as C, except with filter detecting FITC- exposure to NHS, but not to HI-NHS or EDTA/NHS, zona anti-C3 immunofluorescence. Photomicrograph shows localization pellucida-free human and hamster oocytes displayed heavy of C3 catabolites from HI-NHS to equatorial segment of an AR (1) deposition of C3 catabolites to the plasma membrane in a but not to an acrosome-intact (2) sperm. (x900.) pattern similar to that seen for dimeric C3b (Fig. 3D), indicating that these cells, too, can activate the alternative dimeric C3b bound in a specific and saturable manner to AR pathway of complement. sperm. The AR-sperm population specifically bound 280 To test the possibility that sperm acrosomal enzymes molecules of dimeric C3b per cell (Ka = 4 AM), whereas directly cleave C3, increased doses of sperm acrosin extract non-AR sperm bound 36 molecules per cell. The actual were added to 1251-labeled C3. The acrosin extract cleaved number of dimeric C3b molecules bound per AR sperm was the C3 a chain to the a' chain in a dose-dependent manner likely higher because only 30% of sperm in the population (Fig. 4A, lanes 3-6) but had no effect on the , chain at the underwent the AR. doses used. Hyaluronidase, another prominent sperm ac- Human oocytes were examined by indirect immunofluo- rosomal enzyme, did not cleave C3 a or ,B chains (Fig. 4A, rescence assay for the expression of complement receptors. lane 7). Trypsin completely degraded both the a and 3 chains Both zona pellucida-intact and zona pellucida-free human of C3 by 30 min (Fig. 4A, lane 2). The kinetics of acrosin- oocytes were positive for plasma-membrane expression of mediated cleavage ofC3 a chain to the a' form (Fig. 4B, lanes CR1 (Fig. 3A) and CR3 (Fig. 3B) epitopes when the YZ-1 and 1-4) was slower and less complete than that seen with OKM1 mAbs were used, respectively. Various commercially trypsin, where both C3 a and (3 chains were completely available mAbs to CR1 and CR3 (anti-CR1 and anti-CR3, cleaved (Fig. 4C, lanes 5-8). To determine whether acroso- Becton Dickinson; anti-CR1, Dako) did not bind to oocytes, mal enzymes cleave C3 during the AR, allowing C3 catabo- either due to their lower affinity and titer or different epitope lites to bind to the plasma membrane of AR sperm, we used specificity. In contrast, intact fresh human oocytes did not double-label immunofluorescence to simultaneously detect express detectable MCP (Fig. 3C) or CR2 (data not shown). AR sperm and C3 binding. AR sperm were incubated in Oocytes had little or no endogenous C3 associated with the HI-NHS and then double-labeled with TRITC-conjugated plasma membrane when examined by immunofluorescence PSA to identify AR sperm and FITC-labeled goat anti-C3 to assay immediately after harvest but did bind significant detect C3 on the sperm surface. AR (acrosome PSA negative) dimeric C3b on the plasma membrane after incubation with sperm but not acrosome-intact (acrosome PSA positive) dimeric C3b (1 ,uM in SWF) for 30 min at room temperature sperm bound C3 products in the equatorial segment adjacent (Fig. 2D). to the acrosome (Fig. 2 C and D). These data provide further Sperm and oocytes were tested for their ability to activate evidence that acrosin released during the AR directly cleaves C3. AR sperm, but not viable non-AR sperm, had C3 catab- C3, resulting in covalent attachment of C3b to the sperm olites deposited in various sites on the plasma membrane surface or C3 catabolite binding to MCP. after incubation in NHS (Fig. 2B). Different C3-staining C3 fragments and C3 receptors are involved in cell-cell patterns resulted from sperm being in various states of AR adhesion reactions in both innate and adaptive immunity (18, and viability. The amount of C3 fragments bound to the 19) and in membrane apposition before fusion and entry of surface of AR sperm was greatly diminished when EDTA- eukaryotic cells by bacteria, viruses, and protozoa (20). To treated or HI-NHS was used (data not shown). However, assess the role of C3 fragments and C3 receptors in sperm- after incubation in HI-NHS, C3 antigens were noted in the egg apposition and fusion, dimeric C3b or antibodies to C3 equatorial region of individual AR sperm (Fig. 2D), which were added to medium during the hamster egg-penetration Downloaded by guest on October 2, 2021 10054 Developmental Biology: Anderson et al. Proc. Natl. Acad. Sci. USA 90 (1993)

A ; . t- 0. Acrosin B Acrosin ry5 1s2n S s . 2 10 20 50 = 2' 5' 0O' 2(' s 2? 5' IO 20's

.. .. :. .~;:~... .:..._: a _ :: : ..kjj~: a . '...... -a FIG. 4. Autoradiographs showing 9( -4 the effects of acrosin on 125I-labeled C3. (A) Autoradiograph of cleavage ...... pattern of 1251-labeled C3 incubated -6M- -68 for 30 min at 37°C with buffer (lane 1), trypsin (lane 2), increased doses of acrosin (lanes 3-6), or hyaluronidase .43' (HU) (lane 7). (B) Autoradiograph of cleavage pattern of 125I-labeled C3 incubated at 37°C for increased times with 50 j. of acrosin (lanes 1-4) or U7 - with 10 pg of trypsin (lanes 5-8). Mr 27- standards are in lanes marked S. a, a', and ,3 chains ofC3 heterodimer are I 1 2 3 4 5 6 7 x marked.

test. As shown in Table 1, dimeric C3b at subsaturating doses deposition of C3 fragments on the surface of AR sperm in the (10 nM-1 ,uM) significantly enhanced both the percentage of equatorial segment (Fig. 1), the domain that binds to and eggs penetrated by sperm and the average number of sperm fuses with the oolema (28). that entered each ovum (sperm-penetration index) compared Complement is involved in cell-cell interactions in several with medium alone. In contrast, saturating doses of dimeric systems (18-20), and recent studies have suggested that it C3b (10-20 ,uM) significantly reduced both percentage fer- might play a role in fertilization. C3 is secreted by rat and tilization and sperm-penetration index compared with con- human uterine glandular epithelium; highest levels are pro- trols. Anti-C3 and anti-MCP antibodies also significantly duced at fertilization (29-31). Furthermore, sperm, which decreased the percentage fertilization and sperm-penetration constitutively express the host-protective complement regu- index in cultures containing medium alone. C3 was detected latory proteins decay-accelerating factor (CD55) and CD59 by immunoblot in the commercial medium (SWM) used for (32), also express the C3-binding protein MCP (CD46), but the hamster egg-penetration tests (data not shown). only after the AR, which prepares them for fertilization (2). Data from the present study showing that dimeric C3b binds to AR sperm but not to acrosome-intact sperm provide DISCUSSION further evidence that sperm MCP binds C3 catabolites im- Functional levels of C3 and other complement components mediately before fertilization. Furthermore, our data show have been found in the female reproductive tract (21-23). In that intact human oocytes express CR1 and CR3 epitopes and addition to its antipathogen effect, complement is thought to bind dimeric C3b (Fig. 3). In addition, whereas most fresh function in the normal clearance from the female reproduc- oocytes examined did not have detectable levels of C3 on tive tract of dead or dysfunctional sperm, which activate their surface, zona pellucida-free oocytes could activate the complement, whereas intact sperm do not (24). C3-derived alternative pathway and bind C3b on the plasma membrane fragments may opsonize dead sperm, whereas comple- (Fig. 3). That anti-C3 and anti-MCP antibodies inhibited ment-derived chemotactants (25) attract neutrophils to the human sperm penetration in the hamster egg test, a common insemination site. Such activation may also enhance host assay for male fertility (9), further implicates a role for defense against microbes introduced with sperm. We have complement in gamete membrane apposition (Table 1). shown that sperm also activate complement enzymatically. Hamster egg-penetration studies involving different con- Our evidence indicates that the sperm acrosomal enzyme centrations of dimeric C3b shed further light on the possible acrosin, a serine protease, can cleave directly the C3 a chain role of C3b in fertilization. At subsaturating levels ofdimeric (Fig. 4). Acrosin is the major acrosomal protease released C3b, the interaction of sperm and egg was enhanced, whereas during the sperm AR (26); hyaluronidase, the other major at saturating doses the interaction was inhibited (Table 1). acrosomal enzyme, is incapable of C3 cleavage (Fig. 4). The This result suggested that dimeric C3b at low levels could pattern of cleavage differs from that seen with other serine serve as a bridge between sperm (MCP) and oocyte (CR1, proteases that cleave C3, such as trypsin and C3 convertases CR3) complement receptors, facilitating fertilization, (27). Our data also suggest that C3 cleavage by acrosin causes whereas at high levels dimeric C3b saturated all receptor- binding sites for C3 fragments and inhibited the apposition of Table 1. Effects of antibodies or dimeric C3b in the hamster gamete membranes (Fig. 5). In this context it is of interest egg-penetration assay that the commercial medium commonly used for hamster Eggs Penetration egg-penetration assays contains a small amount ofC3, which penetrated, % index may facilitate fertilization, and that abnormally high levels of uterine C3 have been reported in women with endometriosis, Control (medium alone) 76 ± 8.9 1.07 ± 0.22 a condition associated with infertility (33, 34). Other inflam- Rab preimmune Ig (25 ug/ml) 63 ± 9.7 0.89 ± 0.21 matory conditions of the female reproductive tract (i.e., Rab anti-C3 (25 Ag/ml) 25 ± 5* 0.40 ± 0.16* subclinical infections and pelvic inflammatory disease) are H316 anti-MCP (50 pg/ml) 24.7 ± 12.5* 0.41 ± 0.05* also associated with infertility and may also produce high C3b2 (0.01-0.1 ,uM) 90 ± 9.7* 1.70 ± 0.44* levels of C3 fragments capable of blocking fertilization. C3b2 (0.1-1 ,uM) 90 ± 4.5* 2.06 ± 0.58** CR3 may also facilitate gamete membrane apposition C3b2 (10-20 ,LM) 48.3 ± 6.7*** 0.47 ± 0.17* through another mechanism. The guinea pig-sperm plasma- Data are presented as means ± SDs of three to five individual membrane protein PH-30 is an a,8 heterodimer in which the experiments. Rab, rabbit; Ig, immunoglobulin. *, P < 0.05; **, P < ,( chain contains a disintegrin motif and the a chain contains 0.001; ***, P < 0.0001 (ANOVA). a membrane-fusion domain. It has been proposed that mem- Downloaded by guest on October 2, 2021 Developmental Biology: Anderson et al. Proc. Natl. Acad. Sci. USA 90 (1993) 10055 We acknowledge the technical assistance ofHuai An Wang and the Acrosome secretarial assistance of Bernadette Aidonidis. Research was sup- reacted sperm Ovum plasma ported by Grants AI26292 (R.M.J.) and AI25305 (D.J.A.) from the membrane membrane National Institutes of Health, Bethesda, MD, and by the Fearing Laboratory Endowment. a [ 1. Saling, P. M. (1991) Biol. Reprod. 44, 246-251. 2. Anderson, D. J., Michaelson, J. S. & Johnson, P. M. (1989) Biol. Covalent Reprod. 41, 285-293. ) [ Attachment 3. Fenichel, P., Dohr, G., Grivaux, C., Cervani, F., Donseau, M. & Hsi, B. L. (1990) Mol. Reprod. Dev. 27, 173-178. c [ 4. Okabe, M., Ying, X., Nagira, M., Ikawa, M., Yauhiro, K., Mimura, T. & Tanaka, K. (1992) J. Pharmacobio-Dyn. 15, 455-459. 5. Cervoni, F., Oglesby, T. J., Adams, E. M., Milesifluet, C., Nick- (I [ ells, M., Fenichel, P., Atkinson, J. P. & Hsi, B. L. (1992) J. Immunol. 148, 1431-1437. 6. Russell, S., Sparrow, R. L., McKenzie, I. F. C. & Purcell, D. F. J. eL[ (1992) Eur. J. Immunol. 22, 1513-1518. 7. Berger, T., Marrs, R. P. & Moyer, D. L. (1985) Fertil. Steril. 43, 268-273. 8. Jackson, K. V., Nureddin, A., Clarke, R. N., Hornstein, M. D., FIG. 5. Model for role of C3 and complement-binding proteins in Rein, M. S. & Friedman, A. J. (1992) Fertil. Steril. 58, 366-372. gamete membrane apposition. (a) C3b/iC3b (C3b/bi) acting as a 9. Hill, J. A., Cohen, J. & Anderson, D. J. (1989) Am. J. Obstet. bridging ligand between sperm MCP and oocyte complement recep- Gynecol. 160, 1154-1159. tor (CR). (b) C3b/C3bi covalently attached to oocyte acting as ligand 10. Yanagimachi, R., Yanagimachi, H. & Rogers, B. J. (1976) Biol. for sperm MCP. (c) C3b/C3bi covalently attached to sperm acting as Reprod. 15, 471-476. ligand for oocyte complement receptor (CR). (d and e) Excess 11. Changelian, P. S., Jack, R. M., Collins, L. A. & Fearon, D. T. C3b/C3bi saturating all MCP/complement receptor (CR) sites and (1985) J. Immunol. 134, 1851-1858. inhibiting membrane apposition. 12. Weis, J. J., Tedder, T. F. & Fearon, D. T. (1984) Proc. Natl. Acad. Sci. USA 81, 881-885. brane apposition is mediated by the sperm disintegrin motif 13. Wright, S. D., Rao, P. E., VanVorrhis, W. C., Craigmyle, L. S., Iida, K., Talle, M. A., Westberg, E. F., Goldstein, G. & Silverstein, binding to its oocyte integrin counter-ligand (35). Our study S. C. (1983) Proc. Natl. Acad. Sci. USA 80, 5699-5703. shows that CR3, a (32 integrin (36), is expressed on oocytes 14. Fichorova, R. & Anderson, D. J. (1991) J. Reprod. Immunol. 20, and may bind the human-sperm homologue of the PH-30 1-13. disintegrin to facilitate membrane fusion by the PH-30 15. Hammer, C. H., Wirtz, G. H., Renfert, L., Gresham, H. D. & a-chain homologue. Furthermore, RGD, a tripeptide se- Tack, B. F. (1981) J. Biol. Chem. 256, 3995-4006. 16. Kalli, K. R., Hsu, P. H., Bartow, T. J., Ahearn, J. M., Matsumoto, quence in a number of ,B-integrin-binding molecules, includ- A. K., Klickstein, L. B. & Fearon, D. T. (1991) J. Exp. Med. 174, ing C3 (37), has been shown to inhibit sperm-egg interaction 1451-1460. in the hamster egg-penetration test (38). Because CR3 also 17. Goodpasture, J. C., Polakoski, K. L. &Zaneveld, L. J. D. (1980)J. binds C3b and iC3b, the inhibition of hamster-egg penetration Androl. 1, 16-27. by human sperm in the presence ofconcentrations ofdimeric 18. Ahearn, J. M. & Fearon, D. T. (1989) Adv. Immunol. 46, 183-219. 19. Springer, T. A. (1990) Nature (London) 346, 425-434. C3b be a function of C3b to thus may binding CR3, inhibiting 20. Joiner, K. A. (1988) Annu. Rev. Microbiol. 42, 201-230. CR3 binding to the sperm disintegrin. Low doses of dimeric 21. Price, R. J. & Boettcher, B. (1979) Fertil. Steril. 32, 61-66. 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