FERTILITY AND STERILITY
Vol. 57, No.6, June 1992
Copyright © 1992 The American Fertility Society
Printed on acid-free paper in U.S.A
Effects of antibodies to sperm surface fertilization antigen-l on human sperm-zona pellucida interaction*
Rajesh K. Naz, Ph.D.H Charlene Brazil, B.S.§ James W. Overstreet, M.D., Ph.D.§ Albert Einstein College of Medicine, Bronx, New York, and University of California, Davis, California
Objective: To investigate the effects of antibodies to well-defined sperm surface antigens (the fertilization antigen [FA-I] and germ-cell antigen [GA-I]) and nuclear antigen (protamine) on human sperm-zona interaction. Design: Number of total and acrosome-reacted human sperm bound to the human zona pellucida and the sperm movement characteristics assessed by computer-aided sperm analysis were evaluated after incubation of sperm with the antibodies. Setting: Academic research environment approved by the Institute Review Board. Patients: Human oocytes were obtained from ovaries removed at surgery. Semen from fertile donors was used in all assays. Interventions: Human oocytes were stored in salt solution at -80°C until used. Spermatozoa were treated with the antibodies to various sperm antigens. Main Outcome Measures: Total and acrosome-reacted sperm bound to zona pellucida and sperm movement characteristics were evaluated after 3 to 5 hours of incubation of the antibodies with human sperm. Results: Anti-FA-I antibodies significantly reduced human sperm fusion with zona-free hamster oocytes and sperm binding to the human zona pellucida but did not affect binding of acrosomereacted sperm and sperm movement characteristics. Anti-GA-I and antiprotamine antibodies did not affect sperm-oocyte interaction, acrosomal reaction, or sperm motility. Conclusions: Antibodies to FA-I but not to GA-I and protamine inhibit human sperm-zona Fertil SterilI992;57:1304-10 interaction. Key Words: Sperm antigens, antisperm antibodies, fertilization, sperm-zona interaction, human oocytes
We have isolated and characterized a sperm surface glycoprotein, the fertilization antigen (FA-1) from human and murine male germ cell plasma Received July 18, 1991; revised and accepted February 7, 1992.
* Supported by grant HD 24425 from the National Institutes of Health, Bethesda, Maryland (to R.K.N.). t Department of Obstetrics and Gynecology, Albert Einstein College of Medicine. § Department of Obstetrics and Gynecology, University of California. :j: Reprint requests: Rajesh K. Naz, Ph.D., Reproductive Immunology and Molecular Biology, Ullmann Research Building, Room 123, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. 1304
N az et at.
Role of FA -1 in human fertilization
membranes (1, 2). The antibodies (monoclonal and polyclonal) to FA-1 are germ-cell specific but crossreact among various mammalian species and are involved in involuntary immunoinfertility in humans (3, 4). Anti-FA-1 antibodies inhibit binding and penetration of sperm to zona-intact (but not zonafree) oocytes in mice and also block human sperm fusion with zona-free hamster oocytes (2, 5). In the experiments described in this communication, we evaluated the effects of anti-FA-1 antibodies on human sperm -zona pellucida interaction. For comparison, we also evaluated antibodies to two other sperm antigens that are not involved with sperm binding to the zona pellucida. The second Fertility and Sterility
antibody we examined was to the germ-cell antigen (GA-I), which we have also isolated from male germcell membranes (6). Antibodies to GA-I do not inhibit fertilization but cause postfertilization reduction of fertility in rabbits and mice by inhibiting early embryonic development (7, 8). Anti-GA-I antibodies bind to human sperm but do not inhibit their fusion with zona-free hamster oocytes. The third antibody that we studied was to the germ-cell specific intrinsic antigen, protamine. Protamine is a nuclear protein, and antibodies against this antigen cross-react with sperm from various species including salmon, rabbit, and human (9). Although antibodies to protamine have been found in the sera of infertile men (10, 11), this internal sperm antigen is not relevant to fertilization. The present study was conducted to investigate whether the antibodies to these three antigens (FA1, GA-I, and protamine) have any affect: (1) on the binding of human sperm to the human zona pellucida; (2) on human sperm capacitation and the acrosome reaction as induced by zona pellucida binding; and (3) on the percentage of motile sperm and sperm movement characteristics as assessed by computer-aided sperm analysis (CASA).
Protamine
Salmon protamine (salmine, free base, grade VI, Sigma Chemical Co., St. Louis, MO) was used for immunization. Protamines are small «10,000 d), strongly basic proteins (pH ~ 12) present exclusively in the nuclei of sperm. Salmon protamine immunologically cross-reacts with human protamine (9). The commercial protamine was tested for its homogeneity by size exclusion high-performance liquid chromatography before use (11). Antibodies
Antisera were raised against the three antigens (FA-I, GA-I, and protamine) and against lithium diiodosalicylate-solubilized human sperm preparation by immunizing sexually mature virgin female rabbits of New Zealand white strain (n = 3, for each group) as described elsewhere (1,3,8, 10, 11). The antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA) (1, 2, 10), Western blot procedure involving lithium diiodosalicylate-solubilized human sperm preparation (10), indirect immunofluorescence technique (1FT), using methanolfixed human sperm (11) and swollen-sperm head (9, 11), sperm agglutination technique (12), sperm immobilization technique (SIT) (13), and the sperm penetration assay (SPA) as described below.
MATERIALS AND METHODS
Sperm Penetration Assay Antigens
Fertilization Antigen
Fertilization antigen was purified from lithium diiodosalicylate-solubilized human testes by immunoaffinity chromatography involving monoclonal antibody (mAb) that inhibits fertilization (1, 2). Each batch of FA-I was tested for its homogeneity. Only those batches that showed specific bands of 23,000 d and/or 47,000 to 50,000 d in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) were used in the present study. Fertilization antigen showed its dimeric form in SDS-PAGE. Germ-Cell Antigen
Germ-cell antigen was purified from lithium diiodosalicylate-solubilized murine testes by immunoaffinity chromatography involving mAb that causes postfertilization inhibition of fertility (6, 7). Only those batches that showed a specific band in 63,000 d region in nonreduced SDS-PAGE were used. Vol. 57, No.6, June 1992
The effects of sera on fertilizing capacity of human sperm was assessed by the zona-free hamster egg human SPA as described elsewhere (10). Briefly, sperm were capacitated in TEST-yolk buffer for 42 to 48 hours at 4°C, washed three times, and resuspended in Biggers, Whitten, and Wittingham (BWW) medium containing 1% bovine serum albumin (BSA). The sperm suspension was adjusted to 5 X 106 sperm/mL, a 50-~L drop containing 7 ~L of the test antibody solution was mixed with 10 to 20 zona-free hamster eggs obtained from superovulated golden hamsters and incubated together for 2 to 3 hours at 37°C under oil. After incubation, the ova were washed, stained with acridine orange (0.2%), and penetration was determined by the presence of swollen sperm head in the cytoplasm of the ovum. The assay was repeated two to three times using three fertile donors, and each serum was tested with 20 to 50 oocytes. Preparation of Human Sperm and Oocytes for Sperm-Zona Pellucida Interaction
Spermatozoa from fertile donors were prepared by the swim-up technique in modified BWW meN az et al.
Role of FA -1 in human fertilization
1305
dium as described elsewhere (14), and after washing two times were resuspended in BWW containing human serum albumin (HSA) (35 mg/mL) at a concentration of 2 X 106 sperm/mL. The sperm suspension was divided into four aliquots of 380 ML, and each aliquot was incubated with 20 ML of the antibody (5% final concentration of anti-FA-1, antiGA-1, or anti-protamine) or with 20 ML of 3.5% HSA in BWW as a control. Sperm were incubated (37°C, in 5% CO 2 :95% air mixture) with the antibodies for 3 hours, and a 150 ML-drop of antibody-sperm suspension was transferred to a petri dish and covered with mineral oil for coincubation with oocytes. Human oocytes were obtained as previously described (14) from ovaries removed at surgery and were stored at -80°C until used. Such oocytes are not viable, but human sperm can bind to and penetrate the zona pellucida. The human oocytes were thawed, washed five times in BWW and suspended in 3.5% HSA-BWW. The oocytes were then added to 150 ML-sperm drops (4 to 5 oocytes per treatment) and coincubated for 25 to 30 minutes. The oocytes were washed three times in 3.5% HSA-BWW and passed repeatedly through a pipette to dislodge the more loosely adherent sperm. The oocytes were then fixed in cold 100% ethanol in small well dishes. The experiment was repeated three times on different days using sperm from different donors. Assessment of Sperm-Zona Binding and Acrosome Reaction
Fluorescence microscopy was used to count the number of sperm bound to the zona pellucida and to assess the acrosomal status of bound sperm. All sperm on the zonae were labeled using the stain Hoechst 33258 (H-258; Sigma Chemical Co., St. Louis, MO) that bound to the deoxyribonucleic acid of the ethanol-permeablized sperm. Sperm acrosomes were evaluated by 1FT after treatment with a polyclonal antisperm antiserum (15, 16). After removal from ethanol, the oocytes were transferred to a droplet of rabbit antisperm antiserum (15) for 60 minutes at room temperature, rinsed three times in Dulbecco's phosphate-buffered saline (DPBS; GIBCO, Grand Island, NY) containing 10 mg/mL BSA, incubated 30 minutes in 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG (Sigma Chemical Co.) and rinsed in DPBS. The oocytes were then transferred to H258 (0.5 mg/mL in DPBS) for 5 minutes and, after rinsing, they were mounted on slides in the glycerolmounting medium. The glycerol-mounting medium (pH 8.5 to 9.0) had 90% glycerol in DPBS containing 1306
N az et al.
Role of FA -1 in human fertilization
sodium azide (1 mg/mL) and 1,4-diazabicyclo (2,2,2) octane (100 mg/mL) (Sigma Chemical Co.) to reduce the photobleaching of the fluorescent probe during observation (15). Fluorescein isothiocyanate fluorescence was observed using an FITC excitation filter and a DM500 + 0515 dichroic mirror, with a 0530 barrier filter. A UG 1 excitation filter was used to visualize H-258. The number of bound sperm was first counted using the H258 label, and then the acrosomal status of the bound sperm was evaluated. Because of the difficulty in visualizing overlapping sperm, some bound sperm were not analyzed for their acrosomal status. Sperm with heavily labeled anterior heads were considered acrosome intact, and those with faintly labeled heads and/or an equatorial band were classified as acrosome reacted (see Fig. 1 of Cross et al. [16]). Sperm Motility Analysis
Sperm motility was evaluated in all sperm antibody suspensions and in controls immediately after preparation (0 hour) and after 5 hours of incubation at 37°C in 5% CO 2:95% air. The sperm that were assessed for motility were taken from the same suspensions that were used for coincubation with 00cytes. At each time point, an 8-ML aliquot of sperm suspension was observed at 37°C using a 20 Mmdeep microcell slide. The percentage motility was determined at each time by visually scoring 100 sperm as motile or immotile using a mechanical counter. At the 5 hours time point, >50 microscopic fields with one to five sperm/field were video recorded with a Cohn 48102 CCR video camera (Cohn Inc., San Diego, CA) and a Panasonic AG6300 VHS recorder (Panasonic, Secaucus, NJ) using an Olympus BH-2S microscope (Olympus, Tokyo, Japan) with lOX negative phase-contrast optics. Video tapes were analyzed by CellTrak System (Motion Analysis, Santa Rosa, CA). The parameter settings used for CASA analysis are given in Table 1. The following parameters of sperm motion were measured: straight line velocity (VSL), curvilinear velocity (VCL), linearity (LIN), amplitude of lateral head displacement (ALH), and mean angular deviation (MAD). The definitions of these parameters have been discussed previously (17). Statistical Analysis
Significance of differences between antibodytreated and controls was based on unpaired Student's t-test. Fertility and Sterility
Table 1 Parameter Settings Used for CASA Analysis of Sperm Motility Using Cell Trak Parameter
Setting
Frame rate (frames/s) Duration of data capture (frames) Minimum path length (frames) Minimum motile speed (!Lm/s) Maximum burst speed (!Lm/s) Centroid X search neighborhood (pixels) Centroid Y search neighborhood (pixels) Centroid cell size minimum Centroid cell size maximum Path maximum interpolation (frames) Path prediction (%) No. of edges
60 60 60 10 500 2 2
5 35 2
o 4
RESULTS
Anti-FA-1 antibodies having an ELISA titer of >1:4096 against FA-1 recognized a single band of 51,000 ± 3,000 d on Western blot of lithium diiodosalicylate-solubilized human sperm preparation (Table 2). Anti-FA-1 antibodies did not show any sperm agglutinating and immobilizing activity but reacted with postacrosome, midpiece, and tail regions of majority (60% to 80%) of methanol-fixed human sperm, and the remaining (20% to 40%) demonstrated binding with acrosomal, midpiece, and tail regions in 1FT. Anti-FA-1 antibodies inhibited
Table 2
human sperm penetration of zona-free hamster eggs (Table 2). Anti-GA-1 antibodies having an ELISA titer of >1:4096 against GA-1 recognized two bands in 63,000 ± 4,000 d region on Western blot of lithium diiodosalicylate-solubilized human sperm (Table 2). Anti-GA-1 antibodies did not have any sperm agglutinating and immobilizing activity but reacted with the acrosomal regions of methanol-fixed human sperm in 1FT. Anti-GA-1 antibodies did not inhibit human sperm penetration of zona -free hamster eggs (Table 2). Antiprotamine antibodies having ELISA titer of > 1:8192 against protamine did not react with any specific band corresponding to protamine on lithium diiodosalicylate-solubilized human sperm penetration (Table 2). Antiprotamine antibodies did not have sperm agglutinating or immobilizing activity and did not react with any region of methanol-fixed human sperm in 1FT. However, these antibodies reacted strongly with swollen human sperm heads in 1FT. These antibodies also did not inhibit human sperm penetration of zona-free hamster eggs (Table 2). When anti-FA-1, anti-GA-1, and antiprotamine antibodies were investigated for their effects on human sperm-human zona interaction, only the antiFA-1 antibodies significantly (P < 0.04) reduced the
Analysis of Antisperm Antibodies by Various Techniques SPA
Antibodies
ELISA titer
1. Anti-FA-1
>1:4096
2. Anti-GA-1
>1:4096
3. Anti-protamine
>1:8192
4. Negative control (HSA) 5. Positive control (antilithium diiodosalicylate sperm antibodies)1f
0 >1:8192
Western blot procedure
SATt
SIT:j:
Ova penetration
60% to 80% with post0 0 3.60 ± 2.20§ acrosome, midpiece, and tail regions; 20% to 40% with acrosome, midpiece, and tail regions 2 bands in 63,000 90% with acrosomal 0 0 13.80 ± 4.10 ± 4,000 d regions Bound only to swollen None 0 0 18.20 ± 4.90 sperm headll Negative None 0 0 19.30 ± 5.00 8 bands between Acrosome, midpiece, >1:2048 >1:128 2.38 ± 1.48§ 14,000 to and tail regions 92,000 d 1 band of 51,000 ± 3,000 d
• Indirect immunofluorescence technique was performed on methanol-fixed sperm from three different fertile donors. t Expressed as dilution titer in sperm agglutination technique (SAT). :j: Expressed as dilution titer in sperm immobilization technique (SIT). Vol. 57, No.6, June 1992
1FT·
Sperm penetration per egg
68§
100 100 100 47§
§ Values are means ± so. P < 0.01 to 0.001 versus negative control; others were insignificant versus negative control. II Antiprotamine antibodies did not bind to methanol-fixed sperm but bound to swollen sperm head in 1FT. 1f Positive control serum was raised against lithium diiodosalicylate-solubilized human sperm preparation.
Naz et aI. Role of FA-l in human fertilization
1307
Table 3 Effects of Antibodies on Human Sperm-Human Zona Interaction
Antibodies
Total sperm bound/egg
Acrosome-reacted sperm on zona surface %
1. 2. 3. 4.
Anti-FA-l Anti-GA-l Anti-protamine Control (HSA)
42.00 63.83 122.55 119.75
± ± ± ±
58.84* 79.51 95.62 92.45
26.0 21.3 16.3 20.5
± ± ± ±
7.0 11.7 10.8 5.0
* Values are means ± SD. P < 0.04 versus control; others were insignificant (P > 0.05) versus control.
number of sperm bound per oocyte (Table 3). The other antibodies did not significantly decrease the number of sperm bound to zona pellucida. None of the three antibodies demonstrated any significant effect on the percentages of acrosome-reacted human sperm bound to the zona pellucida (Table 3). These three antibodies were also studied for their effects on percent sperm motility and sperm movement characteristics. None of the antibodies showed any significant effect on sperm motility either initially or after 5 hours of incubation in vitro (Fig. 1). DISCUSSION
The FA-1 was immunogenic in rabbits and formed high titer antibodies that were monospecific, recognizing only FA-Ion the Western blot. These antibodies did not agglutinate or immobilize the sperm cells. These results are in agreement with the previous findings demonstrating the strong immunogenicity of the antigenic molecule (4, 5). Fertilization antigen is a sperm surface glycoprotein, and antibodies to it inhibit human sperm fusion with zona-free hamster oocytes and reduce binding and penetration of zona pellucida by murine sperm (1-3). In the murine in vitro fertilization assay, FA-l antibodies reduced sperm penetration of zona-encased but not zona-free oocytes. It was further found that the human spermatozoal FA -1 binds to purified zona pellucida antigen ZP3 and neutralizes its sperm ligand activity in sperm-zona binding assay in porcine system (18). These results suggested that FA-l may have zona ligand/receptor activity. The present findings in humans support the previous data. Fertilization antigen antibodies inhibited spermzona interaction in humans without affecting the motility characteristics of human sperm. These antibodies did not apparently affect the acrosome reaction of human sperm in the present study. However, they did reduce human sperm fusion with zona1308
Naz et al. Role of FA-l in human fertilization
free hamster oocytes. These findings are consistent with the effect of the antibody on the spontaneous acrosome reaction rather than the zona pellucidainduced acrosome reaction (16). Indeed, in another collaborative study, it was found that the immunoaffinity-purified FA-l mAb inhibits capacitation and acrosome reaction of human sperm in solution (in contrast to the present study indicating no effect of the FA-l polyclonal antibodies on acrosome reaction of sperm bound to zona pellucida) and completely blocks human sperm fusion with zona-free hamster oocytes (% penetration) (Kaplan P, Naz
A
PERCENT MOTILITY
100
o Control
80
• anti-FA-1 antibodies ~ anti-GA-1 antibodies Ii] anti-protamine antibodies
60 40 20 0
140
ohr B
5 hr
MOTILITY CHARACTERISTICS
120 100 80 60 40 20 0
VSl
vel
LINEARITY
AlH
MAD
Figure 1 Effects of antibodies to FA-I, GA-I, and protamine on percent motility (A) and motility characteristics (B) of human sperm cells. The swim-up sperm populations were incubated (37°C, 5% CO2:95% air mixture) with the antibody and the percent motility (A) was evaluated at 0 and 5 hours postincubation. The effects on motility characteristics (B) were studied 5 hours postincubation by CASA system. None of the antibody significantly affected the percent motility or the motility characteristics, namely the VSL, VCL, LIN, ALH, and MAD. The error bars represent SD of mean values.
Fertility and Sterility
RK, manuscript submitted, 1992). The effects ofthe antibodies were sperm -specific; the anti -FA-1 antibodies do not bind to the unfixed live or methanolfixed hamster and rabbit oocyte (2, 4), as well as human oocyte (Naz RK, unpublished data, 1992). Various sperm surface zona-binding proteins have been identified by Western blot procedure using either radiolabeled sperm/zona proteins or their bioeffective antibodies (5, 19). O'Rand et al. (20) have reported several proteins that are present on sperm of various mammalian species, including humans, and that bind to homologous zona pellucida. Additionally, a polypeptide of 53,000 d has been identified on the surface of boar sperm (21), and another antigen (PH -20) has been delineated on the guinea pig sperm (22); both of these antigens have been shown to be involved in sperm-zona binding. Several sperm proteins having enzymatic activity have been demonstrated to have zona-binding properties. The involvement of sperm enzymes such as galactosyltransferase (23) trypsin-like protease (24), sialytransferase (23), and fucosyltransferase (25) in zona binding has been reported in various mammalian species. Presently, it is not known whether or not FA-1 has any enzymatic (especially proteolytic) activity and what portion (carbohydrate or polypeptide) of the antigenic molecule is involved in binding to zona. However, preliminary data indicate that mAb to FA-1, which inhibits fertilization, seems to recognize the polypeptide rather than the carbohydrate moiety of the antigen. The antibodies to sperm surface GA-1 had high ELISA titers and showed specific binding on Western blot and 1FT but in contrast to FA-1 did not affect human sperm-human zona interaction. These results are in agreement with previous findings indicating that the tissue-specific but species-crossreactive GA-1 antibodies inhibit fertility by causing postfertilization preimplantation embryonic mortality (6-8). In another recent study, we found that purified GA-1 did not bind to ZP3 and did not neutralize its binding activity in the sperm-zona binding assay (18). These cumulative data indicate that GA-1 antibodies do not have any effect on fertilization in mammals, including humans. Antiprotamine antibodies did not bind to any sperm surface protein in 1FT and Western blot, and it is not surprising that these antibodies had no effect on sperm-oocyte interaction, acrosome reaction, and sperm motility. Protamine is a nuclear protein, and antibodies to it are not expected to bind to sperm surface proteins and thus affect fertilization. We have previously shown that active immunization Vol. 57, No.6, June 1992
with protamine does not affect fertilization of female rabbits in spite of formation of high titers of antibodies in serum and genital tract (11). In conclusion, our data demonstrate that the antibodies to sperm surface FA-1 can significantly reduce sperm binding to the human zona pellucida. The mechanism by which FA-1 antibodies inhibit sperm-zona binding is not entirely clear, but sperm motility is not affected and bound sperm are able to undergo the acrosome reaction. These data suggest that FA-1 will have applications in immunocontraception and in diagnosis and treatment of involuntary immunoinfertility in humans. Acknowledgment. We thank Ms. Daniela Pulitano for excellent typing assistance.
REFERENCES 1. Naz RK, Phillips TM, Rosenblum BB. Characterization of the fertilization antigen (FA-I) for the development of a contraceptive vaccine. Proc Natl Acad Sci USA 1986;83:57137. 2. Naz RK, Alexander NJ, Isahakia M, Hamilton MS. Monoclonal antibody against human germ cell glycoprotein that inhibits fertilization. Science 1984;225:342-4. 3. Naz RK. Involvement of the fertilization antigen (FA-I) in involuntary immunoinfertility in humans. J Clin Invest 1987;80:1375-83. 4. Naz RK, Bhargava KK. Antibodies to sperm surface fertilization antigen (FA-I): their specificities and site of interaction with sperm in male genital tract. Mol Reprod Dev 1990;26:175-83. 5. Naz RK. Sperm surface antigens involved in mammalian fertilization. Curr Opin ImmunoI1990;2:748-51. 6. Naz RK, Rosenblum BB, Menge AC. Characterization of a membrane antigen from rabbit testes and sperm isolated by using monoclonal antibodies and effect of its antiserum on fertility. Proc Natl Acad Sci USA 1984;8:857-61. 7. Naz RK, Saxe JK, Menge AC. Inhibition of fertility of rabbits by monoclonal antibodies against sperm. BioI Reprod 1983;28: 240-54. 8. Naz RK, Poffenberger RJ, Menge AC. Reduction of fertility in female rabbits and mice actively immunized with a germ cell antigen (GA-l) from the rabbit. J Reprod Immunol 1986;9:163-73. 9. Samuel T. Cross-reaction between protamine of different species: the role of arginine clusters. Immunol Commun 1980;9:283-8. 10. Naz RK, Deutsch J, Phillips TM, Menge AC, Fisch H. Sperm antibodies in vasectomized men and their effects on fertilization. BioI Reprod 1989;41:163-73. 11. Naz RK. Effects of sperm-reactive antibodies present in infertile sera on fertility of female rabbits. J Reprod Immunol 1990;18:161-77. 12. Friberg J. A simple and sensitive micromethod for demonstration of sperm-agglutinating activity in serum from infertile men and women. Acta Obstet Gynecol Scand 1974;74: 21-9. Naz et al.
Role of FA-l in human fertilization
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13. Isojima S, Li TS, Ashitaka Y. Immunological analysis of sperm -immobilizing factor found in sera of women and unexplained infertility. Am J Obstet Gynecol 1968;101:677-83. 14. Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW. Penetration of human spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertile donors and infertile patients. Fertil Steril 1980;33:534-42. ~ 15. Cross NL, Morales P, OverstreetJW, Hanson FW. Two simple methods for detecting acrosome-reacted human sperm. Gamete Res 1986;15:213-26. 16. Cross NL, Morales P, Overstreet JW, Hanson FW. Induction of acrosome reactions by the human zona pellucida. BioI Reprod 1988;38:235-44. 17. Boyers SP, Davis RO, Katz DF. Automated semen analysis. In: Barbieri RL, editor. Current problems in obstetrics and gynecology and fertility. New York: Year Book Medical Publishers, 1989:167-200. 18. Naz RK, Sacco AG, Yurewicz EC. Human spermatozoal FA1 binds with ZP3 of porcine zona pellucida. J Reprod Immunol 1991;20:43-58. 19. Saling PM, Lakoski KA. Mouse sperm antigens that participate in fertilization. II. Inhibition of sperm penetration
1310
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20.
21.
22.
23.
24.
25.
through the zona pellucida using monoclonal antibodies. BioI Reprod 1985;33:527-36. O'Rand MG, Matthews JE, Welch JE, Fisher SJ. Identification of zona binding-protein of rabbit, pig, human, mouse spermatozoa on nitrocellulose blots. J Exp Zool 1985;235: 423-8. Brown CR, Jones R. Binding of zona pellucida proteins to a boar sperm polypeptide of Mr 53,000 and identification of zona moieties involved. Development 1987;99:333-9. Primakoff PH, Hyatt H, Myles DG. A role of the migrating sperm surface antigen PH -20 in guinea pig sperm binding to the egg zona pellucida. J Cell BioI 1985;101:2239-44. Shur BD. Galactosyl transferase as a recognition molecule during fertilization and development. In: Schatten H, Schatten G, editors. Molecular biology of fertilization. New York: Academic Press, 1989:21-36. Saling PM. Involvement of trypsin-like activity in binding of mouse spermatozoa to zona pellucida. Proc Nat! Acad Sci USA 1985;78:6231-5. Ram PA, Cardullo RA, Millette CF. Expression and topographical localization of cell surface fucosyltransferase activity during epididymal sperm maturation in the mouse. Gamete Res 1989;22:321-32.
Fertility and Sterility
Vol. 57, No.6, June 1992
Copyright © 1992 The American Fertility Society
Printed on acid-free paper in U.S.A
Effects of antibodies to sperm surface fertilization antigen-l on human sperm-zona pellucida interaction*
Rajesh K. Naz, Ph.D.H Charlene Brazil, B.S.§ James W. Overstreet, M.D., Ph.D.§ Albert Einstein College of Medicine, Bronx, New York, and University of California, Davis, California
Objective: To investigate the effects of antibodies to well-defined sperm surface antigens (the fertilization antigen [FA-I] and germ-cell antigen [GA-I]) and nuclear antigen (protamine) on human sperm-zona interaction. Design: Number of total and acrosome-reacted human sperm bound to the human zona pellucida and the sperm movement characteristics assessed by computer-aided sperm analysis were evaluated after incubation of sperm with the antibodies. Setting: Academic research environment approved by the Institute Review Board. Patients: Human oocytes were obtained from ovaries removed at surgery. Semen from fertile donors was used in all assays. Interventions: Human oocytes were stored in salt solution at -80°C until used. Spermatozoa were treated with the antibodies to various sperm antigens. Main Outcome Measures: Total and acrosome-reacted sperm bound to zona pellucida and sperm movement characteristics were evaluated after 3 to 5 hours of incubation of the antibodies with human sperm. Results: Anti-FA-I antibodies significantly reduced human sperm fusion with zona-free hamster oocytes and sperm binding to the human zona pellucida but did not affect binding of acrosomereacted sperm and sperm movement characteristics. Anti-GA-I and antiprotamine antibodies did not affect sperm-oocyte interaction, acrosomal reaction, or sperm motility. Conclusions: Antibodies to FA-I but not to GA-I and protamine inhibit human sperm-zona Fertil SterilI992;57:1304-10 interaction. Key Words: Sperm antigens, antisperm antibodies, fertilization, sperm-zona interaction, human oocytes
We have isolated and characterized a sperm surface glycoprotein, the fertilization antigen (FA-1) from human and murine male germ cell plasma Received July 18, 1991; revised and accepted February 7, 1992.
* Supported by grant HD 24425 from the National Institutes of Health, Bethesda, Maryland (to R.K.N.). t Department of Obstetrics and Gynecology, Albert Einstein College of Medicine. § Department of Obstetrics and Gynecology, University of California. :j: Reprint requests: Rajesh K. Naz, Ph.D., Reproductive Immunology and Molecular Biology, Ullmann Research Building, Room 123, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. 1304
N az et at.
Role of FA -1 in human fertilization
membranes (1, 2). The antibodies (monoclonal and polyclonal) to FA-1 are germ-cell specific but crossreact among various mammalian species and are involved in involuntary immunoinfertility in humans (3, 4). Anti-FA-1 antibodies inhibit binding and penetration of sperm to zona-intact (but not zonafree) oocytes in mice and also block human sperm fusion with zona-free hamster oocytes (2, 5). In the experiments described in this communication, we evaluated the effects of anti-FA-1 antibodies on human sperm -zona pellucida interaction. For comparison, we also evaluated antibodies to two other sperm antigens that are not involved with sperm binding to the zona pellucida. The second Fertility and Sterility
antibody we examined was to the germ-cell antigen (GA-I), which we have also isolated from male germcell membranes (6). Antibodies to GA-I do not inhibit fertilization but cause postfertilization reduction of fertility in rabbits and mice by inhibiting early embryonic development (7, 8). Anti-GA-I antibodies bind to human sperm but do not inhibit their fusion with zona-free hamster oocytes. The third antibody that we studied was to the germ-cell specific intrinsic antigen, protamine. Protamine is a nuclear protein, and antibodies against this antigen cross-react with sperm from various species including salmon, rabbit, and human (9). Although antibodies to protamine have been found in the sera of infertile men (10, 11), this internal sperm antigen is not relevant to fertilization. The present study was conducted to investigate whether the antibodies to these three antigens (FA1, GA-I, and protamine) have any affect: (1) on the binding of human sperm to the human zona pellucida; (2) on human sperm capacitation and the acrosome reaction as induced by zona pellucida binding; and (3) on the percentage of motile sperm and sperm movement characteristics as assessed by computer-aided sperm analysis (CASA).
Protamine
Salmon protamine (salmine, free base, grade VI, Sigma Chemical Co., St. Louis, MO) was used for immunization. Protamines are small «10,000 d), strongly basic proteins (pH ~ 12) present exclusively in the nuclei of sperm. Salmon protamine immunologically cross-reacts with human protamine (9). The commercial protamine was tested for its homogeneity by size exclusion high-performance liquid chromatography before use (11). Antibodies
Antisera were raised against the three antigens (FA-I, GA-I, and protamine) and against lithium diiodosalicylate-solubilized human sperm preparation by immunizing sexually mature virgin female rabbits of New Zealand white strain (n = 3, for each group) as described elsewhere (1,3,8, 10, 11). The antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA) (1, 2, 10), Western blot procedure involving lithium diiodosalicylate-solubilized human sperm preparation (10), indirect immunofluorescence technique (1FT), using methanolfixed human sperm (11) and swollen-sperm head (9, 11), sperm agglutination technique (12), sperm immobilization technique (SIT) (13), and the sperm penetration assay (SPA) as described below.
MATERIALS AND METHODS
Sperm Penetration Assay Antigens
Fertilization Antigen
Fertilization antigen was purified from lithium diiodosalicylate-solubilized human testes by immunoaffinity chromatography involving monoclonal antibody (mAb) that inhibits fertilization (1, 2). Each batch of FA-I was tested for its homogeneity. Only those batches that showed specific bands of 23,000 d and/or 47,000 to 50,000 d in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) were used in the present study. Fertilization antigen showed its dimeric form in SDS-PAGE. Germ-Cell Antigen
Germ-cell antigen was purified from lithium diiodosalicylate-solubilized murine testes by immunoaffinity chromatography involving mAb that causes postfertilization inhibition of fertility (6, 7). Only those batches that showed a specific band in 63,000 d region in nonreduced SDS-PAGE were used. Vol. 57, No.6, June 1992
The effects of sera on fertilizing capacity of human sperm was assessed by the zona-free hamster egg human SPA as described elsewhere (10). Briefly, sperm were capacitated in TEST-yolk buffer for 42 to 48 hours at 4°C, washed three times, and resuspended in Biggers, Whitten, and Wittingham (BWW) medium containing 1% bovine serum albumin (BSA). The sperm suspension was adjusted to 5 X 106 sperm/mL, a 50-~L drop containing 7 ~L of the test antibody solution was mixed with 10 to 20 zona-free hamster eggs obtained from superovulated golden hamsters and incubated together for 2 to 3 hours at 37°C under oil. After incubation, the ova were washed, stained with acridine orange (0.2%), and penetration was determined by the presence of swollen sperm head in the cytoplasm of the ovum. The assay was repeated two to three times using three fertile donors, and each serum was tested with 20 to 50 oocytes. Preparation of Human Sperm and Oocytes for Sperm-Zona Pellucida Interaction
Spermatozoa from fertile donors were prepared by the swim-up technique in modified BWW meN az et al.
Role of FA -1 in human fertilization
1305
dium as described elsewhere (14), and after washing two times were resuspended in BWW containing human serum albumin (HSA) (35 mg/mL) at a concentration of 2 X 106 sperm/mL. The sperm suspension was divided into four aliquots of 380 ML, and each aliquot was incubated with 20 ML of the antibody (5% final concentration of anti-FA-1, antiGA-1, or anti-protamine) or with 20 ML of 3.5% HSA in BWW as a control. Sperm were incubated (37°C, in 5% CO 2 :95% air mixture) with the antibodies for 3 hours, and a 150 ML-drop of antibody-sperm suspension was transferred to a petri dish and covered with mineral oil for coincubation with oocytes. Human oocytes were obtained as previously described (14) from ovaries removed at surgery and were stored at -80°C until used. Such oocytes are not viable, but human sperm can bind to and penetrate the zona pellucida. The human oocytes were thawed, washed five times in BWW and suspended in 3.5% HSA-BWW. The oocytes were then added to 150 ML-sperm drops (4 to 5 oocytes per treatment) and coincubated for 25 to 30 minutes. The oocytes were washed three times in 3.5% HSA-BWW and passed repeatedly through a pipette to dislodge the more loosely adherent sperm. The oocytes were then fixed in cold 100% ethanol in small well dishes. The experiment was repeated three times on different days using sperm from different donors. Assessment of Sperm-Zona Binding and Acrosome Reaction
Fluorescence microscopy was used to count the number of sperm bound to the zona pellucida and to assess the acrosomal status of bound sperm. All sperm on the zonae were labeled using the stain Hoechst 33258 (H-258; Sigma Chemical Co., St. Louis, MO) that bound to the deoxyribonucleic acid of the ethanol-permeablized sperm. Sperm acrosomes were evaluated by 1FT after treatment with a polyclonal antisperm antiserum (15, 16). After removal from ethanol, the oocytes were transferred to a droplet of rabbit antisperm antiserum (15) for 60 minutes at room temperature, rinsed three times in Dulbecco's phosphate-buffered saline (DPBS; GIBCO, Grand Island, NY) containing 10 mg/mL BSA, incubated 30 minutes in 1:100 dilution of fluorescein isothiocyanate (FITC)-conjugated goat antirabbit IgG (Sigma Chemical Co.) and rinsed in DPBS. The oocytes were then transferred to H258 (0.5 mg/mL in DPBS) for 5 minutes and, after rinsing, they were mounted on slides in the glycerolmounting medium. The glycerol-mounting medium (pH 8.5 to 9.0) had 90% glycerol in DPBS containing 1306
N az et al.
Role of FA -1 in human fertilization
sodium azide (1 mg/mL) and 1,4-diazabicyclo (2,2,2) octane (100 mg/mL) (Sigma Chemical Co.) to reduce the photobleaching of the fluorescent probe during observation (15). Fluorescein isothiocyanate fluorescence was observed using an FITC excitation filter and a DM500 + 0515 dichroic mirror, with a 0530 barrier filter. A UG 1 excitation filter was used to visualize H-258. The number of bound sperm was first counted using the H258 label, and then the acrosomal status of the bound sperm was evaluated. Because of the difficulty in visualizing overlapping sperm, some bound sperm were not analyzed for their acrosomal status. Sperm with heavily labeled anterior heads were considered acrosome intact, and those with faintly labeled heads and/or an equatorial band were classified as acrosome reacted (see Fig. 1 of Cross et al. [16]). Sperm Motility Analysis
Sperm motility was evaluated in all sperm antibody suspensions and in controls immediately after preparation (0 hour) and after 5 hours of incubation at 37°C in 5% CO 2:95% air. The sperm that were assessed for motility were taken from the same suspensions that were used for coincubation with 00cytes. At each time point, an 8-ML aliquot of sperm suspension was observed at 37°C using a 20 Mmdeep microcell slide. The percentage motility was determined at each time by visually scoring 100 sperm as motile or immotile using a mechanical counter. At the 5 hours time point, >50 microscopic fields with one to five sperm/field were video recorded with a Cohn 48102 CCR video camera (Cohn Inc., San Diego, CA) and a Panasonic AG6300 VHS recorder (Panasonic, Secaucus, NJ) using an Olympus BH-2S microscope (Olympus, Tokyo, Japan) with lOX negative phase-contrast optics. Video tapes were analyzed by CellTrak System (Motion Analysis, Santa Rosa, CA). The parameter settings used for CASA analysis are given in Table 1. The following parameters of sperm motion were measured: straight line velocity (VSL), curvilinear velocity (VCL), linearity (LIN), amplitude of lateral head displacement (ALH), and mean angular deviation (MAD). The definitions of these parameters have been discussed previously (17). Statistical Analysis
Significance of differences between antibodytreated and controls was based on unpaired Student's t-test. Fertility and Sterility
Table 1 Parameter Settings Used for CASA Analysis of Sperm Motility Using Cell Trak Parameter
Setting
Frame rate (frames/s) Duration of data capture (frames) Minimum path length (frames) Minimum motile speed (!Lm/s) Maximum burst speed (!Lm/s) Centroid X search neighborhood (pixels) Centroid Y search neighborhood (pixels) Centroid cell size minimum Centroid cell size maximum Path maximum interpolation (frames) Path prediction (%) No. of edges
60 60 60 10 500 2 2
5 35 2
o 4
RESULTS
Anti-FA-1 antibodies having an ELISA titer of >1:4096 against FA-1 recognized a single band of 51,000 ± 3,000 d on Western blot of lithium diiodosalicylate-solubilized human sperm preparation (Table 2). Anti-FA-1 antibodies did not show any sperm agglutinating and immobilizing activity but reacted with postacrosome, midpiece, and tail regions of majority (60% to 80%) of methanol-fixed human sperm, and the remaining (20% to 40%) demonstrated binding with acrosomal, midpiece, and tail regions in 1FT. Anti-FA-1 antibodies inhibited
Table 2
human sperm penetration of zona-free hamster eggs (Table 2). Anti-GA-1 antibodies having an ELISA titer of >1:4096 against GA-1 recognized two bands in 63,000 ± 4,000 d region on Western blot of lithium diiodosalicylate-solubilized human sperm (Table 2). Anti-GA-1 antibodies did not have any sperm agglutinating and immobilizing activity but reacted with the acrosomal regions of methanol-fixed human sperm in 1FT. Anti-GA-1 antibodies did not inhibit human sperm penetration of zona -free hamster eggs (Table 2). Antiprotamine antibodies having ELISA titer of > 1:8192 against protamine did not react with any specific band corresponding to protamine on lithium diiodosalicylate-solubilized human sperm penetration (Table 2). Antiprotamine antibodies did not have sperm agglutinating or immobilizing activity and did not react with any region of methanol-fixed human sperm in 1FT. However, these antibodies reacted strongly with swollen human sperm heads in 1FT. These antibodies also did not inhibit human sperm penetration of zona-free hamster eggs (Table 2). When anti-FA-1, anti-GA-1, and antiprotamine antibodies were investigated for their effects on human sperm-human zona interaction, only the antiFA-1 antibodies significantly (P < 0.04) reduced the
Analysis of Antisperm Antibodies by Various Techniques SPA
Antibodies
ELISA titer
1. Anti-FA-1
>1:4096
2. Anti-GA-1
>1:4096
3. Anti-protamine
>1:8192
4. Negative control (HSA) 5. Positive control (antilithium diiodosalicylate sperm antibodies)1f
0 >1:8192
Western blot procedure
SATt
SIT:j:
Ova penetration
60% to 80% with post0 0 3.60 ± 2.20§ acrosome, midpiece, and tail regions; 20% to 40% with acrosome, midpiece, and tail regions 2 bands in 63,000 90% with acrosomal 0 0 13.80 ± 4.10 ± 4,000 d regions Bound only to swollen None 0 0 18.20 ± 4.90 sperm headll Negative None 0 0 19.30 ± 5.00 8 bands between Acrosome, midpiece, >1:2048 >1:128 2.38 ± 1.48§ 14,000 to and tail regions 92,000 d 1 band of 51,000 ± 3,000 d
• Indirect immunofluorescence technique was performed on methanol-fixed sperm from three different fertile donors. t Expressed as dilution titer in sperm agglutination technique (SAT). :j: Expressed as dilution titer in sperm immobilization technique (SIT). Vol. 57, No.6, June 1992
1FT·
Sperm penetration per egg
68§
100 100 100 47§
§ Values are means ± so. P < 0.01 to 0.001 versus negative control; others were insignificant versus negative control. II Antiprotamine antibodies did not bind to methanol-fixed sperm but bound to swollen sperm head in 1FT. 1f Positive control serum was raised against lithium diiodosalicylate-solubilized human sperm preparation.
Naz et aI. Role of FA-l in human fertilization
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Table 3 Effects of Antibodies on Human Sperm-Human Zona Interaction
Antibodies
Total sperm bound/egg
Acrosome-reacted sperm on zona surface %
1. 2. 3. 4.
Anti-FA-l Anti-GA-l Anti-protamine Control (HSA)
42.00 63.83 122.55 119.75
± ± ± ±
58.84* 79.51 95.62 92.45
26.0 21.3 16.3 20.5
± ± ± ±
7.0 11.7 10.8 5.0
* Values are means ± SD. P < 0.04 versus control; others were insignificant (P > 0.05) versus control.
number of sperm bound per oocyte (Table 3). The other antibodies did not significantly decrease the number of sperm bound to zona pellucida. None of the three antibodies demonstrated any significant effect on the percentages of acrosome-reacted human sperm bound to the zona pellucida (Table 3). These three antibodies were also studied for their effects on percent sperm motility and sperm movement characteristics. None of the antibodies showed any significant effect on sperm motility either initially or after 5 hours of incubation in vitro (Fig. 1). DISCUSSION
The FA-1 was immunogenic in rabbits and formed high titer antibodies that were monospecific, recognizing only FA-Ion the Western blot. These antibodies did not agglutinate or immobilize the sperm cells. These results are in agreement with the previous findings demonstrating the strong immunogenicity of the antigenic molecule (4, 5). Fertilization antigen is a sperm surface glycoprotein, and antibodies to it inhibit human sperm fusion with zona-free hamster oocytes and reduce binding and penetration of zona pellucida by murine sperm (1-3). In the murine in vitro fertilization assay, FA-l antibodies reduced sperm penetration of zona-encased but not zona-free oocytes. It was further found that the human spermatozoal FA -1 binds to purified zona pellucida antigen ZP3 and neutralizes its sperm ligand activity in sperm-zona binding assay in porcine system (18). These results suggested that FA-l may have zona ligand/receptor activity. The present findings in humans support the previous data. Fertilization antigen antibodies inhibited spermzona interaction in humans without affecting the motility characteristics of human sperm. These antibodies did not apparently affect the acrosome reaction of human sperm in the present study. However, they did reduce human sperm fusion with zona1308
Naz et al. Role of FA-l in human fertilization
free hamster oocytes. These findings are consistent with the effect of the antibody on the spontaneous acrosome reaction rather than the zona pellucidainduced acrosome reaction (16). Indeed, in another collaborative study, it was found that the immunoaffinity-purified FA-l mAb inhibits capacitation and acrosome reaction of human sperm in solution (in contrast to the present study indicating no effect of the FA-l polyclonal antibodies on acrosome reaction of sperm bound to zona pellucida) and completely blocks human sperm fusion with zona-free hamster oocytes (% penetration) (Kaplan P, Naz
A
PERCENT MOTILITY
100
o Control
80
• anti-FA-1 antibodies ~ anti-GA-1 antibodies Ii] anti-protamine antibodies
60 40 20 0
140
ohr B
5 hr
MOTILITY CHARACTERISTICS
120 100 80 60 40 20 0
VSl
vel
LINEARITY
AlH
MAD
Figure 1 Effects of antibodies to FA-I, GA-I, and protamine on percent motility (A) and motility characteristics (B) of human sperm cells. The swim-up sperm populations were incubated (37°C, 5% CO2:95% air mixture) with the antibody and the percent motility (A) was evaluated at 0 and 5 hours postincubation. The effects on motility characteristics (B) were studied 5 hours postincubation by CASA system. None of the antibody significantly affected the percent motility or the motility characteristics, namely the VSL, VCL, LIN, ALH, and MAD. The error bars represent SD of mean values.
Fertility and Sterility
RK, manuscript submitted, 1992). The effects ofthe antibodies were sperm -specific; the anti -FA-1 antibodies do not bind to the unfixed live or methanolfixed hamster and rabbit oocyte (2, 4), as well as human oocyte (Naz RK, unpublished data, 1992). Various sperm surface zona-binding proteins have been identified by Western blot procedure using either radiolabeled sperm/zona proteins or their bioeffective antibodies (5, 19). O'Rand et al. (20) have reported several proteins that are present on sperm of various mammalian species, including humans, and that bind to homologous zona pellucida. Additionally, a polypeptide of 53,000 d has been identified on the surface of boar sperm (21), and another antigen (PH -20) has been delineated on the guinea pig sperm (22); both of these antigens have been shown to be involved in sperm-zona binding. Several sperm proteins having enzymatic activity have been demonstrated to have zona-binding properties. The involvement of sperm enzymes such as galactosyltransferase (23) trypsin-like protease (24), sialytransferase (23), and fucosyltransferase (25) in zona binding has been reported in various mammalian species. Presently, it is not known whether or not FA-1 has any enzymatic (especially proteolytic) activity and what portion (carbohydrate or polypeptide) of the antigenic molecule is involved in binding to zona. However, preliminary data indicate that mAb to FA-1, which inhibits fertilization, seems to recognize the polypeptide rather than the carbohydrate moiety of the antigen. The antibodies to sperm surface GA-1 had high ELISA titers and showed specific binding on Western blot and 1FT but in contrast to FA-1 did not affect human sperm-human zona interaction. These results are in agreement with previous findings indicating that the tissue-specific but species-crossreactive GA-1 antibodies inhibit fertility by causing postfertilization preimplantation embryonic mortality (6-8). In another recent study, we found that purified GA-1 did not bind to ZP3 and did not neutralize its binding activity in the sperm-zona binding assay (18). These cumulative data indicate that GA-1 antibodies do not have any effect on fertilization in mammals, including humans. Antiprotamine antibodies did not bind to any sperm surface protein in 1FT and Western blot, and it is not surprising that these antibodies had no effect on sperm-oocyte interaction, acrosome reaction, and sperm motility. Protamine is a nuclear protein, and antibodies to it are not expected to bind to sperm surface proteins and thus affect fertilization. We have previously shown that active immunization Vol. 57, No.6, June 1992
with protamine does not affect fertilization of female rabbits in spite of formation of high titers of antibodies in serum and genital tract (11). In conclusion, our data demonstrate that the antibodies to sperm surface FA-1 can significantly reduce sperm binding to the human zona pellucida. The mechanism by which FA-1 antibodies inhibit sperm-zona binding is not entirely clear, but sperm motility is not affected and bound sperm are able to undergo the acrosome reaction. These data suggest that FA-1 will have applications in immunocontraception and in diagnosis and treatment of involuntary immunoinfertility in humans. Acknowledgment. We thank Ms. Daniela Pulitano for excellent typing assistance.
REFERENCES 1. Naz RK, Phillips TM, Rosenblum BB. Characterization of the fertilization antigen (FA-I) for the development of a contraceptive vaccine. Proc Natl Acad Sci USA 1986;83:57137. 2. Naz RK, Alexander NJ, Isahakia M, Hamilton MS. Monoclonal antibody against human germ cell glycoprotein that inhibits fertilization. Science 1984;225:342-4. 3. Naz RK. Involvement of the fertilization antigen (FA-I) in involuntary immunoinfertility in humans. J Clin Invest 1987;80:1375-83. 4. Naz RK, Bhargava KK. Antibodies to sperm surface fertilization antigen (FA-I): their specificities and site of interaction with sperm in male genital tract. Mol Reprod Dev 1990;26:175-83. 5. Naz RK. Sperm surface antigens involved in mammalian fertilization. Curr Opin ImmunoI1990;2:748-51. 6. Naz RK, Rosenblum BB, Menge AC. Characterization of a membrane antigen from rabbit testes and sperm isolated by using monoclonal antibodies and effect of its antiserum on fertility. Proc Natl Acad Sci USA 1984;8:857-61. 7. Naz RK, Saxe JK, Menge AC. Inhibition of fertility of rabbits by monoclonal antibodies against sperm. BioI Reprod 1983;28: 240-54. 8. Naz RK, Poffenberger RJ, Menge AC. Reduction of fertility in female rabbits and mice actively immunized with a germ cell antigen (GA-l) from the rabbit. J Reprod Immunol 1986;9:163-73. 9. Samuel T. Cross-reaction between protamine of different species: the role of arginine clusters. Immunol Commun 1980;9:283-8. 10. Naz RK, Deutsch J, Phillips TM, Menge AC, Fisch H. Sperm antibodies in vasectomized men and their effects on fertilization. BioI Reprod 1989;41:163-73. 11. Naz RK. Effects of sperm-reactive antibodies present in infertile sera on fertility of female rabbits. J Reprod Immunol 1990;18:161-77. 12. Friberg J. A simple and sensitive micromethod for demonstration of sperm-agglutinating activity in serum from infertile men and women. Acta Obstet Gynecol Scand 1974;74: 21-9. Naz et al.
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13. Isojima S, Li TS, Ashitaka Y. Immunological analysis of sperm -immobilizing factor found in sera of women and unexplained infertility. Am J Obstet Gynecol 1968;101:677-83. 14. Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hanson FW. Penetration of human spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertile donors and infertile patients. Fertil Steril 1980;33:534-42. ~ 15. Cross NL, Morales P, OverstreetJW, Hanson FW. Two simple methods for detecting acrosome-reacted human sperm. Gamete Res 1986;15:213-26. 16. Cross NL, Morales P, Overstreet JW, Hanson FW. Induction of acrosome reactions by the human zona pellucida. BioI Reprod 1988;38:235-44. 17. Boyers SP, Davis RO, Katz DF. Automated semen analysis. In: Barbieri RL, editor. Current problems in obstetrics and gynecology and fertility. New York: Year Book Medical Publishers, 1989:167-200. 18. Naz RK, Sacco AG, Yurewicz EC. Human spermatozoal FA1 binds with ZP3 of porcine zona pellucida. J Reprod Immunol 1991;20:43-58. 19. Saling PM, Lakoski KA. Mouse sperm antigens that participate in fertilization. II. Inhibition of sperm penetration
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through the zona pellucida using monoclonal antibodies. BioI Reprod 1985;33:527-36. O'Rand MG, Matthews JE, Welch JE, Fisher SJ. Identification of zona binding-protein of rabbit, pig, human, mouse spermatozoa on nitrocellulose blots. J Exp Zool 1985;235: 423-8. Brown CR, Jones R. Binding of zona pellucida proteins to a boar sperm polypeptide of Mr 53,000 and identification of zona moieties involved. Development 1987;99:333-9. Primakoff PH, Hyatt H, Myles DG. A role of the migrating sperm surface antigen PH -20 in guinea pig sperm binding to the egg zona pellucida. J Cell BioI 1985;101:2239-44. Shur BD. Galactosyl transferase as a recognition molecule during fertilization and development. In: Schatten H, Schatten G, editors. Molecular biology of fertilization. New York: Academic Press, 1989:21-36. Saling PM. Involvement of trypsin-like activity in binding of mouse spermatozoa to zona pellucida. Proc Nat! Acad Sci USA 1985;78:6231-5. Ram PA, Cardullo RA, Millette CF. Expression and topographical localization of cell surface fucosyltransferase activity during epididymal sperm maturation in the mouse. Gamete Res 1989;22:321-32.
Fertility and Sterility
