Complement-mediated effects of sperm head-directed human antibodies on the ability of human spermatozoa to penetrate zona-free hamster eggs

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1 FERTILITY AND STERILITY Copyright " 1983 The American Fertility Society Printed in U.BA. Complement-mediated effects of sperm head-directed human antibodies on the ability of human spermatozoa to penetrate zona-free hamster eggs Richard A. Bronson, M.D. * George W. Cooper, Ph.D. David L. Rosenfeld, M.D. Division of Human Reproduction, Department of Obstetrics and Gynecology, North Shore University Hospital, Manhasset, New York The ability of immunoglobulins of IgA, IgO, and IgM classes to mediate complement-dependent membrane damage varies. Sera containing antisperm antibodies of differing immunoglobulin classes were studied, in association with complement, for their ability to alter human sperm penetration of zona-free hamster eggs. Sera that contained immunoglobulins of IgO, IgA, or IgM classes directed primarily against the sperm head (as determined by immunobead binding) were selected from men and women judged to be at risk for immune causes of infertility. Spermatozoa were incubated in these sera in the presence and absence of complement. Following an additional incubation in a modified Biggers, Whitten and Whittingham medium, zona-free hamster eggs were inseminated with these spermatozoa. Antibodies known to fix complement ([go and IgM) diminished the percentage of eggs penetrated and the number of penetrating sperm per egg without impairing the ability of sperm to contact the egg surface, as judged by comparable numbers of spermatozoa adherent to the oolemma. IgA, which cannot fix the first component of complement, did not alter the ability of sperm to penetrate eggs. Fertil Steril40:91, 1983 Immunoglobulins of IgA, IgG, and IgM classes interact with elements of the complement system in different ways,1, 2 and their ability to mediate complement-dependent erythrocyte damage also varies. 3, 4 The effect of antisperm antibodies on complement-mediated motility loss depends on immunoglobulin class as well as regional binding specificity to the spermatozoan surface. 5 It has also been shown that autoantibodies induced in guinea pigs by immunization with purified testic- Received October 7,1982; revised and accepted March 17, *Reprint requests: Richard A. Bronson, M.D., Director, Laboratory of Human Reproduction, Department of Obstetrics and Gynecology, North Shore University Hospital, 300 Community Drive, Manhasset, New York ular antigens, in the presence of complete Freund's adjuvant, will mediate complement-dependent damage of the plasma membrane and the acrosomal membrane of epididymal sperm. 6 We studied whether the ability of human spermatozoa to penetrate eggs would be altered following their exposure to spontaneously occurring antibodies directed against the sperm head. Sera were selected that contained immunoglobulins of either IgA, IgG, or IgM classes directed primarily against the sperm head, as determined by immunobead binding. These antibodies were transferred to spermatozoa of a fertile man in the presence of guinea pig serum as a source of complement or heat-inactivated guinea pig serum. A comparison was made of the ability of these sperm to adhere to and penetrate zona-free eggs Bronson et ai. Impairment of sperm penetrating ability 91

2 Head Directed Human Antlsperm Antibody with Guinea Pig Serum Incubate for 30' or 90' at 37 C I Wash Sperm Free of Sera 1 Head Directed Human Antlsperm Antibody with Heat Inactivated Guinea Pig Serum! Incubate in Modified B.W.W. Medium! I!Insemlnate Zona-Free EggS! I 1. Determine./. Zona-Free Eggs Penetrated 2. Score No. of Sperm Adherent to Oolema and No. of Penetrating Sperm per Inseminated Egg Figure 1 The ability of antibody-bound spermatozoa preincubated in the presence or absence of complement to penetrate zona-free hamster eggs is compared. after prior exposure of spermatozoa to antibodies of each immunoglobulin class. The percentage of zona-free eggs penetrated, the number of sperm adherent to the oolemma, and the number of penetrating sperm per inseminated egg were determined. The use of sera containing primarily headdirected antibodies allowed assessment of the penetrating ability of spermatozoa while minimizing loss of motility, which in itself could impair gamete contact (Fig. 1). MATERIALS AND METHODS Sera of couples at risk for immune causes of infertility were screened for antisperm antibodies. Indications for screening consisted of (1) altered sperm motion within cervical mucus on postcoital testing despite normal semen parameters, (2) unexplained infertility, or (3) spontaneous agglutination of sperm in seminal plasma. Class-specific rabbit anti-human antibodies linked to immunobeads were used as antibody detectors on motile sperm, 7 and the regional specificity of each immunoglobulin class for the sperm surface was determined. PASSIVE ANTIBODY TRANSFER Antibody-free semen of a fertile donor diluted 1:5 with Biggers, Whitten and Whittingham (BWW) medium was centrifuged for 8 to 10 minutes at 800 x g. Five million spermatozoa, washed free of seminal plasma, in BWW medium, were added to 0.4 ml of antibody-positive human serum that had been heat-inactivated 30 minutes at 56 C and diluted 1:3 with BWW medium. Eighty to 100 I.d of male guinea pig serum as a source of complement, or heat-inactivated (30 minutes at 56 C) guinea pig serum was added to each sperm-human serum-bww mixture. Guinea pig serum had previously been absorbed with human sperm to remove heterologous antisperm antibodies,s which could impair sperm motility. Complement activity of guinea pig serum was verified by loss of sperm motility following incubation in human serum containing IgG antisperm antibodies directed against the entire principal piece of the sperm tai1. 5 Complete loss of motility of sperm was apparent within 60 to 90 minutes. Sperm motility was scored by observing 200 consecutive cells. Following incubation at 37 C for 30 or 90 minutes, spermatozoa were pelleted and washed twice by centrifugation at 800 x g in BWW medium with 6 mg/ml human serum albumin (HSA; Pentex, Miles Laboratories, Elkhart, IN). Spermatozoa were resuspended at 20 million/ ml in BWW medium containing 30 mg/ml HSA and 7 mm caffeine, for capacitation during a 2- to 4-hour preincubation period. INSEMINATION OF ZONA-FREE EGGS Mature cycling hamsters were housed in a reversed light-dark cycle with a 10-hour period of darkness centered at noon. Ovulation was induced by intraperitoneal injection of pregnant mares serum (Gestyl, Organon, West Orange, NJ), 35 IV at 4:00 P.M. to 6:00 P.M. on the evening of the postestrus vaginal discharge. Human chorionic gonadotropin (Pregnyl, Organon) (35 IV) was administered intraperitoneally 50 hours thereafter. The eggs were harvested 15 to 16 hours following administration of human chorionic gonadotropin. Animals were killed with an overdose of pentobarbital, oviducts were removed, washed free of blood with phosphate-buffered saline, and then transferred to droplets of BWW medium containing 1 mg/ml HSA. The distended ampullae were then incised for retrieval of the cumulus oophorus, which was then dispersed with hyaluronidase (Sigma Chemical Co., St. Louis, MO) (1 mg/md in BWW/HSA (1 mg/md. Cumulus-free eggs were transferred through three 50-ILl washes of BWW/HSA (1 mg/md and 92 Bronson et al. Impairment of sperm penetrating ability Fertility and Sterility

3 Table 1. Effect of Antisperm Antibodies in the Presence or Absence of Complement on Ability of Human Sperm to Penetrate Zona-Free Hamster Eggs Complement present b Heat-inactivated complement Immunoglobulin class of antisperm anti- % Eggs penetrated No. penetrating sperm Eggs penetrated No. penetrating sperm body present in (no. eggs in- per inseminated (range) per inseminated test serum a seminated) egg" egg IgM (4 experiments) 40.3 ± 1.93 (61) 0.50 ± ± 1.26 (51) 1.3 ± 0.17 IgG or IgAllgG (6 ex ± 2.88 (95) 0.89 ± ± 1.78 (97) 1.8 ± 0.67 periments) IgA (5 experiments) 93.9 ± 1.05 (80) 2.7 ± ± 1.89 (58) 2.6 ± 2.0 a Antisperm antibodies directed solely against the sperm head (postacrosomal and/or acrosomal regions) as determined by immunobead binding. bguinea pig serum from a single animal was used as a source of complement. cmean ± standard error of the mean. placed in 150-fll droplets of trypsin (Sigma) (1 mg/ml) in BWW/HSA. Zona lysis occurred within 90 to 120 seconds, at which time the zona-free eggs were promptly removed and transferred through three washes of BWW/HSA (100 fll). Those eggs exhibiting pale, granular cytoplasm of uneven distribution, or evidence of gross cellular damage were excluded from insemination and subsequent scoring. Fifteen to 20 zona-free eggs were placed in 150 fll of modified BWW medium (30 mg/ml HSA and 7 mm caffeine) and inseminated with 50 fll of capacitation medium containing 20 million spermatozoa. 9, 10 Two hours after insemination, the eggs were retrieved, washed free of excess sperm with BWW/HSA, and stained with acridine orange (1 mm acridine orange-3% dimethylsulfoxide [Sigma Grade I] diluted in phosphate-buffered saline), under direct observation at low power ( x 9.6 magnification). These stained eggs were then washed free of excess acridine solution in BWWI HSA and prepared as whole mounts. Eggs were scored by combined phase-contrast and fluorescence microscopy for numbers of penetrating and adherent sperm. RESULTS The ability of antibody-bound spermatozoa to penetrate zona-free hamster eggs in the absence of complement (heated guinea pig serum) was similar for sperm exposed to antibodies of each of the three immunoglobulin classes (Table 1). The mean number of spermatozoa adherent to the oolemma was also comparable for penetrated and nonpenetrated eggs. This would confirm the premise that all zona-free hamster eggs were fertilizable between treatment groups. A marked difference was apparent in the penetrating ability of spermatozoa exposed to IgM and IgG class antibodies in the presence of complement, when compared with IgA. Antisperm antibodies of the IgA class alone did not depress sperm penetrating ability (Table 1). The percentage of zona-free hamster eggs penetrated (93.9% and 87.6%) and the number of penetrating sperm per egg (2.7 and 2.6) were no different, whether complement was active or not. In contrast to this finding, IgG and IgM immunoglobulins, in the presence of active complement, were able to suppress the ability of antibody-bound spermatozoa Table 2. Effect of Antisperm Antibodies in the Presence or Absence of Complement on Sperm Motility and the Ability of Human Spermatozoa to Contact the Vitellus No. sperm adherent to oolemma Immunoglobulin class of per inseminated egg % Motile sperm at insemination antisperm antibodies --=-_-:-_----, ---::::_--:- --=-_--,- ---=_--:- present in test Complement b Complement' Complement Complement serum a active inactive active inactive IgM IgG + IgAllgG IgA 5.7 ± 5.5 d 4.7 ± ± ± ± ± ± ± ± ± ± ± 7.2 a Antisperm antibodies directed solely against the sperm head (postacrosomal and/or acrosomal regions) as determined by immunobead binding. bguinea pig serum from a single animal was used as a source of complement. ccomplement was inactivated by heating at 56 C for 6 minutes. dmean ± standard deviation. Bronson et ai. Impairment of sperm penetrating ability 93

4 Table 3. Statistical Comparison a Between Groups With and Without Complement Immuno- No. penetrating globulin % Eggs sperm per inclass penetrated seminated egg IgM 0.01 > P > P < IgG or 0.05 < P < < P < IgAlIgG IgA NS NS aone-way analysis of variance. bnot significant. No. sperm adherent to oolemma NS b NS to penetrate zona-free eggs with little loss of motility. The percentage of eggs penetrated (40.3 and 54.6, respectively) and the mean number of penetrating sperm per egg (0.50 and 0.89, respectively) were depressed when compared with heatinactivated controls. A greater effect was seen for IgM class antibodies than for IgG. A small loss of sperm motility was noted with IgG antibodies; that is, there was a 10% difference in motility between those spermatozoa exposed to active complement versus those exposed to heat-inactivated guinea pig serum (Table 2). A loss of up to 30% in sperm motility was noted following incubation with active complement and sera containing head-directed IgM antibodies. This loss of motility did not prevent sperm-egg contact, as noted by comparable numbers of sperm adherent to the oolemma for all treatment groups (Table 3). Hence, the loss of motility in itself could not account for the impaired ability of spermatozoa exposed to IgM class antibodies, in the presence of active complement, to penetrate eggs. In addition, when the total number of motile sperm to which zona-free eggs were exposed was held constant between treatment groups by addition of greater numbers of spermatozoa to compensate for motility loss, there was no increase in the percentage of eggs penetrated or the number of penetrating sperm per egg. DISCUSSION Auto- and isoantibodies of IgG and IgM classes directed against antigens associated with the acrosomal and postacrosomal regions of the sperm plasma membrane in association with complement impaired the ability of spermatozoa to penetrate zona-free hamster eggs. IgM antibodies, which have been shown to be looo-fold more efficient than IgG in mediating complement-dependent red blood celllysis,3 similarly were associated with the greatest loss of sperm penetrating 94 Bronson et al. Impairment of sperm penetrating ability NS ability. Although we had previously shown that IgA class antibodies impaired the ability of spermatozoa to bind to the human zona pellucida,11 these head-directed IgA antibodies failed to impair sperm adherence to the oolemma, or their subsequent penetration of zona-free eggs, irrespective of whether complement was absent or present. This finding correlates with the known inefficiency of this immunoglobulin class to promote complement-mediated red blood cell lysis4 and its inability to activate the early components of the complement cascade.12 In addition, it rules against the presence of sperm-specific receptors on the oolemma, given the inability of immunoglobulins, lectins, and proteases13 to block sperm penetration of zona-free eggs, in contrast to those findings for the zona pellucida. The mechanism by which immunoglobulins in association with complement impair the penetrating ability of human sperm is not known, although the work of LeBouteiller et al.6 in the guinea pig provides ultrastructural evidence that the plasma membrane and acrosomal membrane are damaged. Following preincubation in all the sera tested, whether in the presence or absence of active complement, there was no difference in the mean number of sperm adherent to the oolemma of the hamster egg. Talbot and Chacon 14 have recently shown that only acrosome-reacted sperm adhere to the oolemma of the zona-free hamster egg. This observation suggests that the failure of spermatozoa to penetrate was not due to an inhibition of the acrosome reaction or inability of sperm to contact the egg proper, through loss of motility. Hence, the inhibitory effect of complement in the presence of complement-fixing antibodies was at the level of sperm-egg membrane fusion and not attachment of sperm to the oolemma. These results are important in documenting the premise that antisperm antibodies are capable of playing a significant role in impairing fertility. Because seminal plasma contains complement inhibitors,15 sperm motility in the ejaculate is maintained despite the presence of antibody binding to the sperm surface. Once such antibodybound sperm enter the female reproductive tract, however, they become liable to complement-dependent membrane damage. The limiting factor in impairment of sperm function would then be the concentration of complement components at different levels of the female tract. Price and Boettcher16 have shown that an aqueous extract Fertility and Sterility

5 of human cervical mucus possesses - 10% of the complement activity of human serum, as determined by release of 51Cr-Iabeled hemoglobin from red blood cells and by sperm immobilization. The complete complement cascade may not be present, however, within oviductal secretions. Oliphant and Cabot17 have failed to find complement activity as determined by red blood cell lysis, in, 18 the oviducts of estrous rabbits. Schumacher has also been unable to detect a complete complement cascade in oviductal fluid retrieved from rhesus monkeys late in the follicular phase of ovulatory cycles induced with human menopausal gonadotropins. Complement activity within the serum of mice is suppressed by estrogens and would be expected to be at its nadir in the periovulatory period.19 The activity of complement at the site of fertilization then becomes a significant factor in determining whether antibody-bound spermatozoa retain or lose their ability to penetrate eggs. REFERENCES 1. Rapp HJ, Borsos T: Molecular Basis of Complement Action. New York, Appleton-Century-Crofts, Muller-Eberhard HJ: Chemistry and reaction mechanisms of complement. Adv Immunol 8:2, Humphrey JH, Dourmashkin RR: Electron microscope studies on immune cell lysis. In CIBA Foundation Symposium on Complement, Edited by GEW Wolstenholme, J Knight. Boston, Little, Brown and Co., 1965, p Colton HR, Borsos T, Rapp HJ: Titration of the first component of complement on a molecular basis: suitability of IgM and unsuitability of IgG hemolysins as a sensitizer. Immunochemistry 6:461, Bronson RA, Cooper GW, Rosenfeld DL: Correlation between regional specificity of antisperm antibodies to the spermatozoan surface and complement-mediated sperm immobilization. Am J Reprod Immunol 2:222, LeBouteiller P, Toullet F, Voisin GA: Ultrastructural lesions induced in vitro in guinea pig spermatozoa by a specific autoantibody (anti-tj and complement. Immunology 28:983, Bronson R, Cooper G, Rosenfeld D: Ability of antibodybound human sperm to penetrate zona-free hamster ova in vitro. Fertil Steril 36:778, Witkin SS, Brown CA, Good RA, Day NK: Sperm immobilization by sera from unimmunized guinea pigs: requirements for immunoglobulin and complement. J' Reprod Immunol 2:65, Yanagimachi R, Yanagimachi H, Rogers BJ: The use of zona-free animal ova as a test system for the assessment offertilizing capacity of human spermatozoa. BioI Reprod 15:471, Perreault SD, Rogers BJ: Stimulation of human sperm fertilizing ability by caffeine and theophylline. Presented at the Thirteenth Annual Meeting of the Society for the Study of Reproduction, August 11 to 14, 1980, Ann Arbor, Michigan 11. Bronson RA, Cooper GW, Rosenfeld DL: Sperm-specific isoantibodies and autoantibodies inhibit the binding of human sperm to the human zona pellucida. Fertil Steril 38:724, Gotze 0, Muller-Eberhard HJ: Paroxysmal nocturnal hemoglobinuria: hemolysis initiated by the C3 activator system. N Engl J Med 286:180, Yanagimachi R: Specificity of sperm-egg interaction. In Immunobiology of Gametes, Edited by M Edidin, MH Johnson. London, Cambridge University Press, 1977, p Talbot P, Chacon RS: Ultrastructural observations on binding and membrane fusion between human sperm and zona pellucida-free hamster oocytes. Fertil Steril 37:240, Petersen BH, Lammel CJ, Stites DP, Brooks GF: Human seminal plasma inhibition of complement. J Lab Clin Med 96:582, Price RJ, Boettcher B: The presence of complement in human cervical mucus and its possible relevance to infertility in women with complement-dependent sperm immobilizing antibodies. Fertil Steril 32:61, Oliphant G, Cabot C: Control of humoral immune cytotoxic activity by the oviduct. Presented at the Fourteenth Annual Meeting of the Society for the Study of Reproduction, August 10 to 13, 1981, Corvallis, Oregon. Abstract Schumacher GFB: Humoral immune factors in the female reproductive tract and their changes during the cycle. In Immunologic Aspects of Infertility and Fertility Regulation, Edited by DS Dhindsa, GFB Schumacher. New York, Elsevier-North Holland, 1980, p Weintraub RM, Churchill WH Jr, Crisler C, Rapp HJ, Borsos T: Mouse complement: influence of sex hormones on its activity. Science 152:783, 1966 Bronson et al. Impairment of sperm penetrating ability 95