differences in immunogenicity indicating polymorphism of sperm antigens from mice of different...

Journal of Reproductive Immunology, 11 (1987) 167-180 167 Elsevier Scientific Publishers Ireland Ltd.

JRI 00480

Differences in immunogenicity indicating polymorphism of sperm

antigens from mice of different inbred strains

Chong Xu and Deborah J. Anderson Fearing Research Laboratory, Deparanem of Obstetrics, Gynecology and Reproductive Biology,

Harvard Medical School, Boston, MA 02115 (U.S.A.)

(Accepted for publication 29 April 1987)

Summary

Polymorphism of mouse sperm was investigated by analysis of immune sera generated in BALB/c female mice against sperm from 6 inbred strains. The immune sera were analyzed by immunofluorescence and Western blot techniques against sperm antigens from the 6 immunizing strains. Immunofluorescence revealed no differences in reactivity patterns or titers. However, several different reaction patterns were detected by Western blot technique which indicated that both the sperm extracts and t h e antisperm immune sera contained different components. Syngeneic (anti-BALB/c sperm) antisera showed far fewer reactive antibody species than allogeneic immune sera. The anti-BALB/c sera recognized an antigen of 23 kDa in sperm extracts from DBAJ2J and C57BL/6 mice, and failed to react with an antigen of the same molecular weight when applied to sperm from A/J and 129/J mice, indicating antigenic differences between sperm from these inbred strains. Anti-C57BL/6 sera contained a unique antibody which reacted with an antigen of 80 kDa in all of the 6 sperm extracts, whereas other antisera did not detect this antigen. These findings indicate antigenic and immunogenic polymorphism in sperm from different inbred strains of mice.

Key words: sperm; immunity; polymorphism; mouse.

Correspondence to: Dr. Deborah Anderson, Fearing Research Laboratory, 250 Longwood Ave., Rm 204, Boston, MA 02115.

0165-0378/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

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Introduction

A number of polymorphic genes, genes with allelic variation, have been described. Their products can have functional roles, and polymorphic variants of such gene products have been associated with impaired cellular functions such as immune responses and embryonic development (Bennett, 1975; Benacerraf, 1981; Longo and Paul, 1982). There is limited evidence for polymorphism in membrane structures of mammalian sperm. The t-complex loci on chromosome 17 of the mouse encode polymorphic gene products, some of which affect spermatogenesis and sperm function (Nadijeka and Hillman, 1980; Silver, 1985). Studies on sperm immunogenicity in inbred strains of mice have also indicated differences in sperm antigen immunogenicity. Snell (1944) reported that sperm from the C57 mouse strain induced antibodies in BALB/c mice that agglutinated C57 sperm following absorption with BALB/c sperm. In a recent study (Madrigal et al., 1986), sperm from 9 inbred strains of mice were injected into syngeneic female recipients, and dramatic differences in antibody responses were demonstrated by Western blot technique. This study, however, did not separate potential differences in sperm antigen immunogenicity in different inbred strains from possible immunogenetic differences in response between the strains. Another study (Tarter and Alexander, 1984) indicated that allogeneic mouse sperm is more immunogenic than syngeneic sperm, which provides further evidence for genetic differences in sperm immunogenicity.

In recent studies using monoclonal antibodies, histocompatibility antigens, a major class of polymorphic antigens, were not detected in significant quantities on the surface of human sperm (Brodsky et al., 1979; Anderson et al., 1982) or mouse sperm (Anderson, unpublished). Several earlier studies using HLA-A,B,C tissue-typing allosera indicated haploid expression of polymorphic antigens on human sperm with reactivity patterns resembling HLA antigen expression (Fellous and Dausset, 1970; Halim et al., 1974). However, in a later study (Anderson et al., 1982) it was found that platelet absorption of sperm-reactive tissue typing antisera to remove anti-HLA Class I-antibodies did not affect reactivity of the sera with sperm. Furthermore, the antisperm reactivity of tissue typing allosera did not exactly follow expected Class I or Class II MHC reactivity patterns, suggesting the presence of non-HLA-A,B,C polymorphic antigens on sperm. More recently, it has been reported that isoimmunity in women is significantly associated with sperm autoimmunity in their partners (Mathur et al., 1985) and that antisperm antibodies in sera from infertile women often react better with husband's sperm than with un- related donor sperm (Mathur et al., 1983; Witkin et al., 1986). These data

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indicate that sperm from some men may be more immunogenic and/or react better with antisperrn antibodies, providing evidence for polymorphism of human sperm antigens.

Polymorphic sperm antigens could affect fertility either by having functional roles in fertilization events that are affected by polymorphic variation, or by differences in immunogenicity which could promote immune responses which are negatively associated with fertility. The present study was performed to further study differences in immunogenicity and identify polymorphic antigens of mouse sperm. Ab- sorption, immunofluorescence and Western blot techniques were used to analyze antisperm antibody profiles in sera of age-matched virgin female BALB/c mice which were immunized with sperm from males of 6 different inbred strains.

Materials and Methods

Mice Male retired breeders of 6 inbred strains of mice (BALB/c, DBA/2J,

C57BL/6, C57L/J, A/J and 129/J) and 8-week-old virgin female BALB/c mice were obtained from the Jackson Laboratory, Bar Harbor, ME. Mice were housed in the Dana-Farber Cancer Institute animal facility, which was free of Sendal virus, mouse hepatitis virus and all other detectable mouse pathogens during the course of these experiments.

Immunization and bleeding Epididymal sperm were obtained from the inbred strains of mice listed

above by mincing the cauda epididymides in RPMI medium and passing the resultant cell suspension through a 20-gauge nylon mesh screen. The caput and corpus epididymis was not used because of a high number of contaminating somatic cells in minced preparations. Six groups of BALB/c mice were immunized subcutaneously with 2 x 107 washed epididymal sperm emulsified in Freund's complete adjuvant (CFA) from each inbred strain, respectively. Subsequently, three immunizations at 21- day intervals were performed with 2 x 107 sperm in incomplete adjuvant. Seven days after the final injection, mice were bled by retro-orbital puncture with a Pasteur pipette and bloods from each immunization group were pooled. Six BALB/c antisera, directed against sperm from different inbred strains of mice, were obtained by centrif-ugation after clotting. Control serum was obtained from age-matched CFA-immunized female BALB/c mice. The antisperm sera were termed anti-BALB/c, anti- DBA/2J, anti-C57BL/6, anti-C57L/J, anti-A/J, and anti-129/J sera, named for the strain that provided the immunizing sperm.

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Absorption Two hundred forty microliters of each antiserum were first absorbed

with 2.5 × 107 BALB/c sperm (freshly isolated from cauda epididymis) by incubation at 4°C for 2 h. Sperm were pelleted, and sera were treated with 20tt l of NP-40 BALB/c sperm extract (equivalent of 2× 107 sperm/antiserum). Antisera were spun at 10,000 × g for 10 min to remove immune complexes and the supernatant ("BALB/c-sperm-absorbed antisera") was aliquoted and stored at -70°C until use.

Immunofluorescence test Antisera were serially diluted (x4) into phosphate buffered saline/l%

bovine serum albumin (PBS/BSA) and applied to methanol-fixed sperm on teflon-coated slides as described previously (Madrigal et al., 1986). Antisera were tested against BALB/c sperm and sperm from all 5 of the allogeneic sperm donors used for immunization. Following the first incubation with primary antisera, slides were washed in PBS/BSA, and appropriately diluted secondary antibody (rabbit anti-mouse IgG, Fab' fragment, heavy and light chain specific, fluorescein isothiocyanate (FITC) conjugated (Cappel)) was applied for 30 min. Slides were washed, coverslips were mounted with glycerol-based mounting medium containing phenylene diamine, and fluorescence was assessed by reading slides on a Zeiss epifluorescence microscope. Recorded titer was the reciprocal of the last dilution that appeared as positive (+ green fluorescence) in the test.

Western blot SDS-polyacrylamide slab gel electrophoresis (PAGE) was carried out

as described previously (Madrigal et al., 1986). Washed sperm from 6 inbred strains of mice were extracted with lysis buffer containing 0.5% NP-40 (Sigma) for 1 h at 4°C. NP-40 extracts of thymic lymphocytes from each inbred strain were used as antigen controls. The extracts were denatured by treatment with sample buffer containing 5% 2-mercap- toethanol (2-ME) for 5 min at 100°C before loading onto 12.5% polyacrylamide gels.

Electrophoresis was performed at 20 V for 4h. The protein molecular weight standards used were: lysozyme, fl-lactoglobulin, 2-chymotryp- sinogen, ovalbumin, bovine serum albumin, phosphorylase B and myosin (H-chain) (Bethesda Research Labs). A narrow strip of the resultant electrophoretogram including protein molecular weight standards were stained with Coomassie's blue and the rest of the gel was applied to nitrocellulose (45/zm, Millipore Corp, Bedford, MA) for Western blot studies as described previously (Madrigal et al., 1986).

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The non-specific binding sites were blocked with 3% BSA (Sigma) in 10mMTBS (10mM Tris-HCl, 0.15 M NaCI, pH 7.4) for 3 h at 37°C. After washing three times with I0 mM TBS, the nitroeeilulo~ sheets were cut into 2-mm wide strips and each sperm type was ~ u b a t o d with 6 antisera and control sera (diluted 1:20 in TBS/BSA) overnight at 4"C on a rocker. The strips were thoroughly washed and incubateA with peroxi- dase-conjugated goat anti-mouse IgG (Cappel, heavy and light chain specific) for 2 h at room temperature on a rocker. The immune reactions were visualized with 4-chloro-naphthol (Sigma) and stopped by washing with water.

Results

Coomassie's blue staining of NP-40 sperm extracts from the 6 inbred mice following PAGE revealed identical protein patterns, with at least 47 distinct bands (Fig. 1). In immunofluorescence tests, all antisera had similar titers (2560 + one titer place) and reactivity patterns (whole sperm) regardless of whether tested against BALB/c sperm (syngeneic to immunized host) or against sperm syngeneic to the sperm donor.

Two approaches were used to analyze Western blot data. The first approach was to group the Western blot strips according to antiserum. This permitted direct comparison of reactivities of each antiserum against different sperm extracts, which was the clearest way to visualize differences in antigen profile between extracts (Fig. 2). The second approach was to group the Western blot strips according to sperm extract. This permitted direct assessment of differences in antibody profiles between antisera (Fig. 3).

In preliminary experiments it was determined that an antiserum dilution of 1:20 was required to visualize antigen bands clearly with all pooled antisera in Western blot technique. Control (adjuvant only) sera showed very weak reactivity with a few antigen bands at this dilution (Fig. 2, panel VII). A few antigen bands were also detected in thymocyte blots at this antiserum concentration, but they were visualized with both control and antisperm sera, and did not correspond in molecular weight to the sperm antigens detected by the antisera (data not shown). Whereas titer differences between antisera had not been detected on whole sperm by immunofluorescence assay, in Western blots, antisera from BALB/c female mice immunized with syngeneic sperm (anti-BALB/c sera) had much weaker activity against all sperm extracts than antisera from BALB/c mice immunized with allogeneic sperm (Fig. 2). Strongest reactivity was observed with anti-129/J sera (Fig. 2, panel VI). Anti-BALB/c sera also reacted with the least number of sperm antigen bands (2

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1 2 5 4 5 6

Fig. 1. PAGE of sperm extracts showing identical protein patterns by Commassie's blue stain. (1) BALB/c; (2) DBA/2J; (3) C57BL/6; (4) C57L/J; (5) A/J; (6) 129/£

prominent bands at 21 kDa and 45 kDa, and 7 weak bands) and anti- 129/J sera reacted with the most (24 antigen bands). The other antisera reacted with intermediate numbers of sperm antigens. Anti-C57BL/6 sera reacted with 80kDa and 27 kDa antigen bands found in all sperm extracts, whereas none of the other antisera contained significant levels of antibodies to these antigens. Anti-C57L/J sera was notably deficient in antibodies recognizing several antigens in the 50-90 kDa region and the 23--45 kDa regions, whereas other allogeneic antisera were reactive in this region. As already mentioned, anti-BALB/c sera contained few antibodies that reacted against any of the sperm extracts in the Western blot assay.

Overall comparison of Western blot profiles in Fig. 3 reveals some antigenic differences between sperm extracts from different strains. In contrast to the anti-BALB/c sera which was hyporeactive, the BALB/c

2 0 0

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m

m

26

18

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t 2 5 4 5 6

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1254.56 125456 |23456 125456 125456 125456

Fig. 2. Reaction patterns of antisera against different sperm extracts-revealing sperm antigen differences. (I) Anti-BALB/c serum; (II) anti-DBAJ2J serum; (m) anti-C57BL/6 serum; (IV) anti-C57L/J serum; (V) anti-AJJ serum; (VI) anti-129/6 serum; (VII) control (adjuvant alone) serum. (1) BALB/c sperm; (2) DBA/2J sperm; (3) C57BL/6 sperm; (4) C57L/J sperm; (5) A/J sperm; (6) 129/J sperm.

sperm extract contained more immunoreactive antigen bands than extracts from the other strains. Two sperm antigen bands (45 kDa and 2 fkDa) were considerably stronger in the BALB/c sperm extract. DBA/2J and 129/J sperm extracts appeared to be missing several immunoreactive bands (e.g. bands in the 19-21 kDa region for DBA/2J and some bands in the 50-65 kDa region for 129/J).

We followed two approaches to identify potential polymorphic sperm antigens using the allogeneic and syngeneic antisera reagents in Western blots. We first looked for antigen bands that were present when allogeneic antisera (e.g. anti-A/J sera) were applied to sperm extracts originating from the allogeneic immunizing strain (e.g. A/J) but not when allogeneic antisera (e.g. anti-A/J sera) were applied to syngeneic (i.e. BALB/c) sperm extracts (compare lanes 5 and 1 in Fig. 2, panel V).

Systematic comparison of reactivity of each aUogeneic antiserum with its

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200

97

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43

26

18

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I 2 3 4 5 6 7 1234 567 I 2 5 4 5 6 7 1234567 1234567 1254567

I II TIT I~ ~ 3ZI

Fig. 3. Reaction patterns of different antisperm antisera applied to sperm extracts from 6 different inbred strains of mice revealing differences in antibody content of the antisera. (I) BALB/c sperm; (II) DBA/2J sperm; (III) C57BL/6 sperm; (IV) C57L/J sperm; (V) A/J sperm; (VI) 129/J sperm. (1) Anti-BALB/c serum; (2) anti-DBA/2J serum; (3) anti-C57BL/6 serum; (4) anti-C57L/J serum; (5) anti-A/J serum; (6) anti-129/J serum; (7) control serum.

own versus BALB/c sperm extract revealed no distinct antigen bands in the allogeneic combination that were not detected when the allogeneic antiserum was applied to syngeneic BALB/c sperm (compare in Fig. 2: panel II, lane 2 vs. 1; panel III, lane 3 vs. 1; panel IV, lane 4 vs. 1; panel V, lane 5 vs. 1; panel VI, lane 6 vs. 1). In every case, in fact, more antigen bands were detected when allogeneic antisera were applied to the BALB/c sperm than to the specific allogeneic spen-n extract.

The other approach used to identify polymorphic sperm antigens was to compare reactivity of aUogeneic antisera to that of syngeneic (BALB/c) antisera when both were applied to the allogeneic sperm extract of the strain used to generate the alloimmune antiserum (compare in Fig. 3: panel II, lanes 2 and 1; panel III, lanes 3 and 1; panel IV, lanes 4 and 1, panel V, lanes 5 and 1; panel VI, lanes 6 and 1). In every case, more

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antigen bands were detected by alloimmune sera than by isoimmune (BALB/c) sera. As mentioned above, anti-BALB/c serum detected very few sperm antigens in any of the sperm extracts (most clearly seen in Fig. 2, panel I). However, the additional bands detected by allosera were present not only on allogeneic sperm of the immunizing strain, but also on BALB/c sperm (as noted above).

Although reactivity patterns clearly indicative of classic antigen polymorphism were not observed by applying the analysis techniques des- cfibed above, interesting differences in reactivity profiles were observed in this study. Some of the more striking examples are presented here, and their possible significance discussed below.

As clearly shown in Fig. 2, panel I, a prominent 23 kDa antigen band was detected when anti-BALB/c serum was reacted against extracts of DBA/2J and C57BL/6 sperm; a weak band at this molecular weight was detected by applying anti-BALB/c sera to extracts of BALB/c sperm and C57L/J sperm, and no band was detected at this molecular weight when anti-BALB/c serum was tested against extracts of A/J and 129/J sperm. However, by studying reactivity of other antisera to this antigen in Fig. 3, which displays blot data according to sperm extract groups for direct comparison of antiserum reactivity, it is clear that a prominent 23 kDa antigen is present in all sperm extracts. This prominent 23 kDa antigen is detected in BALBIc, C57BL/6 and C57L/J sperm extracts (panels I, III, IV) by all antisera, in A/J and 129/J sperm extracts (panels V and VI) by all antisera except anti-BALB/c serum, and in the DBA/2J sperm extract (panel II) intensely by anti-BALB/c and anti-DBA/2J sera, and only weakly by the other sera.

129/J antiserum contained high titers of antibodies that were reactive with an antigen family in the 50-55 kDa region. This antigen cluster appeared to be present in all sperm extracts (lane 6, Fig. 2), with slight variation in the positions of some of the antigen bands in different extracts. This antigen family was interesting because not all antisera contained antibodies directed against it, although the antigen cluster appears to have been present on all immunizing sperm. Anti-C57L/J and anti-BALB/c sera did not contain detectable levels of antibody to this antigen.

The absorption procedure used in combination with the Western blot technique was not helpful. Absorption removed most but not all reactive antibodies. All antigen bands visualized with BALB/c-sperm-absorbed antisera were weak and f,7~y. Prominent bands which remained were detected in all sperm extracts including BALB/c extracts; these, therefore, probably represented high titer antibodies that were not adequately removed by absorption with BALB/c sperm. Minor antibody populations,

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which may have possibly detected polymophic determinants in the sperm extracts, were undetectable following absorption, possibly due to decreased resolution following this procedure.

Discuss ion

Sperm express a number of differentiation antigens that can elicit antisperm immune responses in autologous, syngeneic and allogeneic hosts. Humoral immune responses to sperm vary in magnitude and antibody profiles; immunogenetic and environmental factors have been implicated to explain these qualitative and quantitative differences. To date, few studies have addressed the possibility that sperm immunogenicity may also be affected by sperm antigen polymorphism. A classical approach used to study other polymorphic antigen systems such as the HLA system in man, has been to absorb immune sera containing antibodies directed against polymorphic determinants with cells which express different variants of the polymorphic antigen. Antibodies remain- ing after absorption react with unique polymorphic determinants not expressed by the absorbing cells, and can be used as reagents to further study the expression patterns and other characteristics of the polymorphic antigenic determinant. In this study we applied this approach for the first time in combination with Western blot analysis to identify potential polymorphic antigenic determinants on mouse sperm. Western blots were used as a detection system because of the heterogeneity of sperm antigens, and the possibility that more than one polymorphic determinant exists on these cells. Furthermore, identification of polymorphic sperm antigens by Western blot technique would facilitate production of monoclonal antibodies to these antigens because specific molecular weight molecules could be used as immunogens. Monoclonal antibodies to polymorphic sperm antigens would be valuable reagents for further study of the structure and function of such antigenic determinants.

Failure in the combination of absorption and Western blot analysis used in this study to identify polymorphic antigens on mouse sperm, may have been attributable to technical difficulties encountered with this approach. Absorption lowered the titer of all allogeneic antisperm sera and seemed to affect the resolution of antigen bands in the Western blot assay. How- ever, analysis of patterns of unabsorbed sera tested against extracts of sperm from different inbred strains of mice indicated that both the sperm extracts and the antisperm immune sera contain different components detectable by Western blot analysis. One very apparent difference was the limited antibody profile in sera of BALB/c mice which had been ,ira-

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munized with syngeneic sperm. The titer of pooled anti-BALB/c sperm antisera was comparable by immunofluorescence assay to those of antisera raised against allogeneic sperm, but only three prominent antigen bands were visualized by Western blot analysis with the syngeneic antisera. Many more reactive antigen bands were observed with the other antisera.

Immunogenetic differences in response of immunized hosts were con- trolled in this study by using age-matched mice from one inbred strain. We chose the BALB/c strain because they are high responders to sperm antigens (Bigazzi, 1978), and because this was the strain used by Snell (1944) in his early studies of sperm antigen polymorphism. Virgin females were used to rule out effects of prior exposure to antigen, and strict attention was paid to the emulsification procedure to ensure that antigen batches were comparable. Somatic cell contamination was minimized by use of cauda epididymides only, and reactivity directed against somatic cell antigens such as H-2 antigens was monitored by performing Western blots against thymocyte extracts. The observation that BALB/c females produced more heterogeneous antibody profiles to allogeneic sperm than to syngeneic sperm, therefore, may indicate enhanced immunogenicity of antigenic determinants on allogeneic sperm. If this situation is similar to other situations of antigenic polymorphism, the mice are tolerant to many syngeneic sperm antigens; tolerance "is broken in allogeneic immunizations by either differences in the structure of the antigenic determinant itself, or differences in carrier determinants associated with the antigenic determinant eliciting the immune response.

Differences in antibody reactivity in the 5 different allosera, as discussed in the results section, were of two types: (1) allosera that reacted with an antigen band (identified by molecular weight) present in some but not all sperm extracts; and (2) allosera that contained antibodies that were not detectable in other sera. An example of case 1 was the reactivity of anti-BALB/c sera against a 23 kDa antigenic molecule, detectable by this reagent in some but not all sperm extracts. This reactivity pattern reflects either quantitative or qualitative differences in expression of the reactive antigenic determinant associated with a 23 kDa molecule, in different inbred strains of mice. The determinant recognized by BALB/c sera on the 23 kDa molecule was not detectable in all sperm extracts and therefore may be an example of a polymorphic determinant. Other alloimmune sera contained antibodies that reacted with a 23 kDa molecule present in all sperm extracts, and 23 kDa antigen-reactive antibodies were found in alloimmune sera of strains whose sperm extracts did not react with the BALB/c sperm immune sera. These data indicate that an immunogenic 23 kDa molecule was present in all sperm extracts. Antibodies in allosera apparently were directed against different antigenic determinant(s) on the

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23 kDa molecule or against other molecules comigrating at 23 kDa than were recognized by antibodies in syngeneic immune sera.

An example of the second case, where one immune sera contained antibodies that were not detectable in the other sera, was the anti- C57BL/6 allosera, which reacted with an antigen band found in all sperm extracts at 80 kDa. None of the other sera reacted with this antigen, although sperm extracts from the other strains which had been used to produce the other antisera contained the antigenic determinant recognized by the antibody. This finding that the 80 kDa antigen was only immunogenic in C57BL/6 mouse sperm, although the determinant was present in all mouse sperm extracts suggests polymorphism in the carrier (T cell dependent) portion of the antigenic molecule.

Western blot analysis is more powerful than tests such as immunofluorescence, sperm cytotoxicity or ELISA which have been previously used to monitor and characterize antisperm immune responses, because antisperm immune responses are highly heterogeneous, and separation of sperm antigens by molecular weight in part dissects out this system. However, there are shortcomings in this analysis system. Antigenic determinants are denatured by treatment with SDS and 2-ME, and only antibodies able to react with such denatured determinants are detected in the Western blot assay. Also, glycolipids and positively charged molecules are not clearly resolved by PAGE, and do not transfer efficiently to nitrocellulose paper; therefore these classes of antigens would not be identified in this study. Furthermore, as suggested by our data, reactivity detected by antibodies at certain .molecular weight locations in Western blots is often complex. Antibodies could be directed against any number of molecules comigrating in the same band, or various antigenic determinants associated with these molecules. Western blots of two-dimen- sional gels would provide better resolution of sperm antigens of similar molecular weight. However, despite the shortcomings, Western blot studies such as the one described in this report are useful in identifying immunogenic sperm molecules and in providing information on environmental and genetic factors affecting humoral immune responses directed against particular sperm antigens.

This study provides evidence for a new perspective on the immunogenicity of sperm antigens. The data indicate that there are inter- strain differences in sperm antigenicity, and further suggest that immunogenicity of sperm antigenic determinants may be affected by two patterns of polymorphism: (1) polymorphism in the carrier portion of the molecule (the region recognized by helper T cells); or (2) polymorphism in the antigenic determinant itself. Polymorphism of sperm antigenic structures could be genetic (allelic variation) or, as has been suggested by

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evidence in t-mutant strains (Shut, 1981), due to strain differences in post-transcriptionai modification (e.g. glycosylation) of sperm surface molecules.

In this study, only sperm from fertile mice were used. A similar approach could be applied to analyze differences in sperm antigen profiles between fertile and infertile animals or men, to possibly identify polymorphic sperm structures associated with fertilizing ability.

Acknowledgment

This research was supported by NIH grant CA 42738. Dr. Xu is the recipient of a training grant from the World Health Organization. The authors thank Ms. Martha Mann for her rigorous standards of animal care.

References

Anderson, D.J., Bach, D.L., Yunis, E.J. and Dewolf, W.C. (1982) Major histocompatibility antigens are not expressed on human epididymal sperm. J. Immunol. 129, 452--454.

Benacerraf, B. (1981) Role of MHC gene products in immune regulation. Science 212, 1229. Bennett, D. (1975) The T-locus of the mouse. Cell 6, 441--454. Bigazzi, P.E. (1978) Autoimmuue responses to spermatozoa in vasectomized rats and mice of

different inbred strains. In: Genetic Control of Aumimmune Disease (Rose N.R., Bigazzi P.E. and Warner, N.L., eds.), pp. 455-462. Elsevier/North-Holland, New York.

Brodsky, F.M., Parham, P., Barnstable, C.J., Crumpton, M.J. and Bodmer, W.F. (1979) Monoclonai antibodies for analysis of the HLA system. J. Immunol. Rev. 47, 3.

Fellous, M. and Dausset, J. (1970) Probable haploid expression of HLA on human sperm. Nature 225, 191.

Halim, A., Abbasi, K. and Festeustein, H. (1974) The expression of the HLA antigens on human spermatozoa. Tissue Antigens 4, 1.

Longo, D.L. and Paul, W.E. (1982) Immune response genes and Ia antigens. The relationship between them and their role in lymphocyte interactions. In: Histocompatibility Antigens: Struc- ture anct Function. Parhem P. and Strominger, J., eds.), p. 161. Chapman and Hall, London.

Madrigal, J.A., Yunis, E.J. and Anderson, D.J. (1986) Qualitative di~erences in sperm antibody responses in mice of different inbred strains and sexes. J. Reprod. lmmunol. 9, 175--186.

Mathur, S., Williamson, H.O. et al. (1985) Female isoimmunity to sperm is associated with sperm autoimmunity in their husbands. J. Clin. Immunoi. 5, 166-171.

Mathur, S., Wiiliamson, H.O., Geueo, PJ., Fndenherg, H.H. et al. (1983). Sperm immunity in infertile couples: antibody titers are higher against the husbands sperm than to sperm from controls. Am. J. Reprod. Immunol. 3, 18-22.

Nadijeka, M. and Hillman, N. (1980) Sterility in mutant (tt~/tLY) male mice. II. A morphological study of spermatozoa. J. EmbryoL Exp. Morphoi. 59, 39--67.

Shur, B.D. (1981) Galactosyltramferase activities on mouse sperm bearing multiple t-lethal and t-viabie hapiotypes of the T/t complex. Geuet. Res. 38, 225-236.

Silver, L.M. (1985) Mouse t hapiotypes. Am. Rev. Genet. 19, 179--208. Snell, G.D. (1944) Antigenic differences between the sperm of different inbred strains of mice.

Science 100, 272-273.

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Tarter, T.H. and Alexander, N.J. (1984) Genetic control of humoral immunity to sperm acrosomal and cell surface antigens. J. Reprod. Immunol. 6, 213-226.

Witkin, S.S., Berkeley, A., David, S.S., Goldstein, M. and Toth A. (1986) Antibodies to husband sperm but not to donor sperm in sera of infertile couples. (Abstract) Proceedings of Conjoint Annual Meeting, The American Fertility Society, The Canadian Fertility and Andrology Society, p. 35.

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