Immunoinhibition of human fertilization in vitro by antibodies to the cumulus oophorus intercellular matrix J. Tesa\l=r%v\\l=i'\k Centre for Reproductive Medicine, Purkyne University Medical Faculty, nam Sov. hrdinu 11, CS-65677 Brno, Czechoslovakia Summary. Rabbit polyvalent antiserum raised against the solubilized cumulus matrix was a powerful inhibitor of human fertilization in vitro, affecting sperm\p=n-\zonapellucida interaction. Both sperm binding to, and penetration of, the zona pellucida were severely impaired by the anti\p=n-\cumulusmatrix antiserum, whereas no effects of this antiserum on cumulus matrix solubilization or penetration of zona-free human eggs were evident. Moreover, the anti-cumulus antiserum partly neutralized the acrosome reaction-inducing activity of the cumulus matrix. These results warrant further research into the functional specificity of different cumulus matrix components and into the effects of the respective antibodies on reproductive function as a possible lead to a new approach to contraceptive vaccine development. Keywords: contraceptive vaccine; cumulus oophorus antigens; sperm\p=n-\zonapellucida interaction; sperm acrosome reaction; immunoinhibition of fertilization; man Introduction There is general consensus that the contraceptive methods currently available are far from covering the increasing demand for birth control, especially in Third World countries. Accordingly, a cheap and simple method, affording prolonged yet reversible protection against conception and preferen¬ tially acting before or during the early stages of fertilization, is needed. Since the early seventies, when the concept of immunological interference with fertilization was born and seemed to comply with the above criteria (Shivers, 1974), research in this field has been focussed on both sperm and egg antigens. Ongoing studies involving sperm antigens deal with enzymes essential to sperm energetic metabolism and with numerous types of membrane antigens (for a recent review see Carrón et al, 1988). On the other hand, the parallel research dealing with egg-derived antigens is virtually limited to antigens from the zona pellucida (reviewed by Henderson et al, 1988). Even though antibodies raised against antigens of the pig zona pellucida cross-react with the human zona pellucida and produce an effective block to fertilization under conditions in vitro (Mori et al, 1985; Henderson et al, 1987), the usefulness of zona-derived antigenic determinants as a basis for a human contraceptive vaccine has been somewhat compromised by the possibility that anti-zona antibodies might also attack ovarian components other than the zona pellucida, with resulting adverse effects on ovarian function. In fact, some in-vivo studies have confirmed the binding of anti-zona antibodies not only to ovulated oocytes, but also to those still in their follicles (Sacco, 1979), resulting in serious disturbances of follicular growth with ultimate reduction of the developing oocyte pool (Gwatkin et al, 1980; Wood et al, 1981; Gulyas et al, 1983; Skinner et al, 1984). Moreover, adverse effects of anti-zona pellucida antibodies on hormonal status of actively immunized animals have been described (Skinner et al, 1984; Bamezai et al, 1986). Although many of the above side-effects may apparently be overcome by purification of the antigen, the *Present address: INSERM Unite 187, Hôpital Antoine-Béclère, 92141 Clamart, France. Downloaded from Bioscientifica.com at 09/29/2021 09:42:33PM via free access possibility of developing a contraceptive vaccine based on antigens other than those of the zona pellucida should be reconsidered, especially in the light of the recent data concerning the effects of the cumulus oophorus intercellular matrix on human sperm function (Tesafík, 1985; Tesafík et al, 1988a) and on sperm-zona pellucida interaction (Tesafík et al, 1988b). It has to be stressed that, unlike the zona pellucida, this material is produced late in the preovulatory period and the respect¬ ive antibodies can therefore be expected not to react with components of younger follicles. In the present study, I examined the effects of antiserum against the cumulus intercellular matrix on human sperm-egg interaction in vitro. Materials and Methods Source of oocytes and cumuli oophori. Oocytes (n = 45) were obtained from women treated in an in-vitro fertilization programme and came from cases in which the treatment had to be cancelled after oocyte recovery, either due to the inability of the husband to produce a semen sample or to unexpected severe asthenozoospermia. Ovarian stimulation and follicle puncture were carried out as described by Tesafík et al. (1988b). Parts of cumuli oophori were dissected from oocyte-cumulus complexes (other than those subsequently used for testing the effects of anti-cumulus matrix antisera on fertilization) just after recovery using sharp hypodermic needles. Solubilization ofcumulus matrix. Parts of cumuli oophori were washed thoroughly from follicular fluid by incubat¬ ing each cumulus fragment for 10 min in 5 ml Dulbecco's phosphate-buffered saline (DPBS; Serva, Heidelberg, FRG). After this incubation, the cumuli were transferred into a fresh washing solution and the whole procedure was repeated three times. The intercellular matrix was solubilized by incubation of cumuli with 10 units of bovine testicular hyaluronidase (Spofa, Prague, Czechoslovakia) dissolved in 1 ml DPBS at 37°C for 30 min. The basic proteinase inhibitor aprotinin (Antilysin; Spofa) and the SH proteinase inhibitor 4-aminomercury benzoate Na (Serva) were added at concentrations of 100pg/ml and 10pg/ml, respectively. The digested matrix was then separated from cumulus cells by centrifugation at 500g for 15 min. The supernatants were decanted and freeze-dried. Preparation ofzona-free human eggs. After the removal of the cumulus oophorus, some eggs were stripped of their zona pellucida by the combined action of pronase and mechanical forces (Tesafík et al, 1988b). Production ofanti-cumulus matrix antiserum. Two female rabbits were bled before immunization to obtain normal rabbit serum for control incubations. The rabbits were then injected subcutaneously with 250 pg of solubilized cumulus matrix preparations in Freund's complete adjuvant (Gibco, Grand Island, NY, USA). Three booster injec¬ tions of the same preparation emulsified with Freund's incomplete adjuvant were given at 8-week intervals. Rabbits were bled by heart puncture 4 weeks after the last injection, the blood was allowed to coagulate and serum, obtained by centrifugation, was pooled, heated at 56°C for 30 min, absorbed by normal human serum in a 1:1 ratio and stored at 20°C until used. Antibody titres were monitored by ELISA using the solubilized antigen preparation (as above). The— reactivity of the antiserum with intact cumuli oophori spread on microscope slides was checked by indirect immunofluorescence. The antiserum used in this study exhibited a titre between 1:2000 and 1:2500. The tissue speci¬ ficity of the anti-cumulus matrix antiserum was checked by indirect immunofluorescence after the reaction with cryostat sections of human ovaries. There was no specific reaction with ovarian stromal and thecal cells. A slight reaction with mural granulosa cells was occasionally observed, but only with undiluted antiserum. In-vitro insemination. Spermatozoa from normospermic donors with proven fertility were used in all experiments. Sperm preparation and in-vitro insemination were performed using standard protocols (Tesafík et al, 1988b). Fertili¬ zation outcomes were evaluated 18-20 h after in-vitro insemination by inspection for pronuclei and polar bodies in an inverted microscope equipped with differential interference-contrast optics. An egg was considered penetrated when at least 2 pronuclei were visible in the egg cytoplasm. Eggs were then fixed and processed for electron microscopy using standard procedures (Tesafík et al, 1988b). Incubations with anti-cumulus matrix antisera. For the evaluation of the effects of the anti-cumulus matrix anti¬ bodies on fertilization and sperm-zona pellucida interaction, freshly obtained or briefly cultured (1-6 h) oocyte- cumulus complexes were preincubated for 90 min at 37°C in the presence of anti-cumulus matrix antiserum, followed by thorough washing of the oocyte-cumulus complexes in medium and in-vitro insemination. Each oocyte-cumulus complex was placed in 5 ml medium and incubated for 10 min at 37°C. This step was repeated four times using fresh medium to remove all unbound antibody and so to prevent any potential direct reaction of non-specific (in relation to the cumulus) antibodies with subsequently added spermatozoa. Other oocytes inseminated under the same conditions but preincubated with preimmune rabbit serum instead of the antiserum served as controls. For testing the effects of anti-cumulus matrix antisera on the cumulus matrix-induced acrosome reaction, samples of solubilized cumulus matrix were mixed with anti-cumulus matrix antisera and incubated at 37°C for 2 h. Aliquants of this mixture (100 pi) were then added to sperm suspensions (about 1 IO7 spermatozoa in 1 ml medium) preincu¬ bated in capacitating medium for 5 or 16 h. After an additional 1 h spermatozoa were prepared for quantitation of the acrosome reaction (see below). Sperm cultures in which either the anti-cumulus matrix antiserum was replaced with Downloaded from Bioscientifica.com at 09/29/2021 09:42:33PM via free access normal rabbit serum or in which the solubilized cumulus