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 com- plement-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 trans- membrane (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 antibod- ies (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, tetramethyl- rhodamine 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.