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Sperm and seminal plasma antigens relevant to contraceptive vaccine development
Sperm antigens and their potential for contraceptive vaccines*
Sperm and seminal plasma antigens relevant to contraceptive
vaccine development S. lsojima
Department
of Obstetrics
and Gynecology,
Current
Opinion
Hyogo Medical
in immunology
Introduction Sperm contain many antigenic molecules and epitopes, and animals probably generate many kinds of antibodies to them. The sperm antigens of mammals can be roughly divided into two groups: the sperm intrinsic membrane antigens (SM-Ags) and the sperm-coating antigens (SCAgs). The latter are present in the seminal plasma and adhere firmly to the sperm surface. Most SM-Ags are found in the cells of spermatozoa and spermatids in the testis, mainly in the acrosome or equatorial region; SCAgs, on the other hand, are usually excreted from the epithelial cells of the caput, corpus, cauda epidklymis and seminal vesicles, and become attached to sperm as they pass through the seminiferous tubes. In my opinion, antibodies to SC-Ags, which are composed of glycolipids or glycoproteins, probably cause sperm immobilization and agglutination whereas antibodies to SM-Ags, which are composed mainly of peptides, may be involved in the blocking of sperm binding or penetration of the zona pellucida. Here, I will discuss some recent advances in our understanding of these two types of sperm antigens and their potential for contraceptive vaccine development.
Sperm intrinsic membrane antigens Rabbit SM-Ags have been investigated by O’Rand and colleagues since 1976.They isolated a family of glycoproteins of molecular weights 13 RD [rabbit sperm antigen (RSA)-11, 11 kD (RSA-2) and 1OkD (RSA-3). Monoclonal antibodies (mAbs) were raised to epitopes on RSAl and these epitopes were found to be present on RSA-2 and
College,
Hyogo, Japan
1990, 2:752-756
RSA-3 as well. These mAbs inhibit in vitro fertilization, and it was therefore speculated that the RSA family has a central role in zona penetration (ORand et al, Biol Reprod 1984, 30:721-729). Human sperm extract binds mAbs to RSA-1, -2, and -3 with up to 75% of the maximum binding al&&y of rabbit sperm extract (O’Rand et aC, Biol Reprod 1984, 30:731-736). The antigens corresponding to these mAbs are concentrated in the middle region of the tail, with smaller amounts in the head. However, in rabbits, RSA are concentrated in the post-acrosomal equatorial region both before and after the acrosome reaction (Esaguy et al, Gamete Res 1988, 19:387-399). An examination of how RSA are distributed in somatic tissues in rabbits and humans [using a sensitive avidin-biotin complex (ABC) staining technique] is required to prove that RSA are sperm plasma membranespecilk antigens. Recently, O’Rand and Widgren [ll isolated members of the RSA family of molecular weights 14, 16, 17 and 18kD, and showed that some of them are resistant to boiling, reduction and sodium dodecyl sulphate (SDS) treatment. The 14 kD band was identified by western blotting with an anti-R&4 mAb, excised, and digested with Kallikrein. One particular peptide (PloC) of 10 amino acids was purifted by reverse-phase highperformance liquid chromatography (HPLC) and shown to react with anti-RSA serum. Antibodies to PlOG reacted specilically with low molecular weight RSA bands and with the sperm equatorial post-acrosomal border. O’Rand and Widgren [l] obtained the amino acid sequence of PlOG and showed that it blocks sperm binding to the zona pellucida. The question is now whether the PlOG peptide is actually present on human sperm. A human sperm acrosomal antigen has been isolated by Herr et al. [ 21. They had previously raised a mAb @IIS.
* This article and the preceding one offer two views on the use of sperm and related antigens in contraceptive vaccines.
Abbreviations ABC-avidinbiotin complex; anti-Id-anti-idiotypic polyclonal antibodies; ASP--acrosome-stabilizing factor; cDNA-complementary DNA; HPL-high-performance liquid chromatography; kb-kilobases; mAbs-monoclonal antibodies; mRNA-messenger RNA; PACIGpolyacrylamide gel electrophoresis; RSA-rabbit sperm antigen; SC-Ags-sperm-coating antigens; SDS-sodium dodecyl sulphate; SI-Abs-sperm-immobilizing antibodies; SM-Avperm intrinsic membrane antigens; TPM%trifluoromethasulphonic
75i
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acid.
Sperm
10) to a component of the human sperm membrane, and found that it reacts with the entire acrosomal region, and particularly with the inner aspect of the outer acrosomal membrane and the outer aspect of the inner acrosomal membrane. After an acrosome reaction caused by the calcium ionophore A23187, MHS-10 still stained the sperm head, particularly the inner acrosomal membrane and equatorial segment. The sperm membrane protein corresponding to MHS-10, SP-10, has now been purified and shown to be an antigenic peptide of 24-34kD, and to have a pI of 4.9 on two-dimensional polyacrylamide gel electrophoresis (PAGE). h&IS-IO was shown to Inhibit sperm binding and penetration in the zona-free hamster egg penetration assay. Using a radioactive probe corresponding to 628 nucleotides of the open reading frame of SP-10 on northern blots of poly(A) + RNA obtained from the testes of a baboon and two macaques, a messenger RNA &RNA) of 1.35 kb (the same size as the mRNA from human testes) was identilied [3]. Recently, three overlapping SP-10 complementary DNAs (cDNAS, SP-10-5, SP10-9, and SP-10-10) were isolated from a human testes cDNA expression library [4]. These cDNAs hybridized to a 1.35 kb mRNA present exclusively in the testes. Complete sequencing of these cDNAs produced a 1117nu cleotide sequence that contains a region coding for 265 amino acids of the SP-10 protein. A recombinant SP-10 fusion protein produced in an EscberiGbia coli expression vector was used to generate a polyclonal antiserum that stained the acrosomal cap in situ and reacted with a similar set of peptides on western blots as MHS-10. A search of three sequence databanks with the SP-10 cDNA sequence did not reveal any significant homology with other sequences, but this does not necessarily mean that SP-10 is sperm-specific. Therefore, ABC staining of human somatic tissues with MHS-10 is required to c0nIii-m that this is the case, although ABC staining might pick up some false positives. Herr’s group [4] also showed that the recombinant SP-10 fusion protein can act as an effective immunogen in humans or animals in viva to prevent fertilization. Prlmakoff et al. [5] have isolated a guinea pig sperm membrane protein called PH-20. It may have a role in spemgg adhesion and its surface expression may be regulated by the acrosome reaction. The PH-20 protein, purified by mob alEnity chromatography, retains the ability to bind three anti-PH.20 mAbs, and separates on SDS-PAGE into a major 64kD form and a minor 56kD form. An endoproteolytically cleaved form of the 64kD polypeptlde, composed of two disulphlde-linked fragments of 41-48kD and 27 kD, also shows up on SDSPAGE. Recently, Prlmakoff et al. [6] suggested that PH20 is spem-~specific, on the basis of absorption experiments with tissue extracts from guinea pigs. A mAb to PH-20 inhibited sperm binding and penetration of guinea pig oocytes in vitro, and immunization of both female and male guinea pigs with the purified PH-20 protein induced sterility in most animals. The mAb to PH-20 only reacted with guinea pig acrosome but polyclonal antibodies to the purihed protein also reacted with human sperm. Therefore, PH-20 may contaln an epitope whose
and seminal
plasma antigens
lsojima
antibodies cross-react with human sperm membrane, or there may be a small amount of contamination of the guinea pig sperm giving rise to cross-reaction. One possible pitfall in the development of PH-20 as a contraceptive vaccine is that antibodies to it may cross-react with human somatic tissues. Prlmakoff et al 161 claimed that PH-20 was sperm-specific because they used 100 times more guinea pig somatic tissue than sperm in absorption experiments with the anti-PH-20 rnAb; however, I think that absorption experiments may not be su&iently sensitive to determine sperm specificity and ABC staining of somatic tissues using anti-PH-20 mAbs is needed. A sperm-specific glycoprotein, FA-1, has been isolated from human and mouse male germ cell plasma membranes by Naz and Mehta [7]. They claim that it is a tissue-specific, species cross-reactive, antigen and that mAbs to it inhibit human sperm penetration of zonafree hamster ova and block mouse sperm penetration of mouse oocytes. They speculate that FA-1 is involved in unexplained infertility in humans. Naz and Mehta also suggest that FA-1 influences cell-mediated immunity because a lymphokine-like substance that immobilizes sperm and blocks embryonic development is released from FA-l-activated macrophages. However, their results are confusing and more precise deli&ion of FA-1 crossreactivity with human somatic tissues by sensitive ABC staining is therefore needed, as well as further analysis of its chemical characteristics.
Sperm-coating
antigens
Ever since the discovery by myself and colleagues (Isojima et aL, Am J Obstet Gynecoll968, lOk677-483) of sperm-immobilizing antibodies (SIAbs) in the sera of women with unexplained infertility, SI-Abs have been thought to be relevant to this phenomenon. Because most SI-Abs in the sera can be absorbed with azoospermic semen, it was thought that these antibodies might be generated in response to human seminal plasma antigens (Isojima et al, Am J Obstet Gynecd 1972, 112:19%207). Recently, azoospermic semen was found to lose its absorption capability for most of the SI-Abs in the sera of infertile women if the semmal plasma was treated with trifluoromethasulphonic acid (TFMS) [8]. It seems that most sterile women generate SI-Abs directed against the carbohydrate epitope of seminal plasma antigens. To identify these antigens, a human- mouse hybridoma, H63C4, was successfully established using cells from a sterile woman with a high titre of SIAbs (Isojlma et al, J Reprod ZmmunoZl987,1O:67-78). The hybrldoma secreted high titres of an antibody (I@) that immobilizes and agglutinates sperm. The antigenic epitope corresponding to mAb H63C4 was found to be localized on the surface of epithelial cells of the cauda epididymis and adhered firmly all over the motile sperm; it remained partly ln the sperm after capacitation and an ionophore-induced’ acrosome reaction. Tsuji et al. [9] showed that mAb H63C4 reacts with lactonorhexaosyl-ceramide (blood group i-Ag) regardless of whether sialic acid is present ln the
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terminal region; however, both sialyl i-Ag and sialyl I-Ag were found to be present on the surface of motile sperm. On the basis of these findings, molecules bearing i-Ag epitopes are thought to be excreted from the cauda epididymis and seminal vesicles and to adhere to the sperm surface after sialylation. In human seminal plasma, the molecules beating the epitopes recognized by mAb H63C4 were shown to have molecular weights of 15-25 kD by SDS-PAGE. If the carbohydrate chains of sialyl i-Ag can be synthesized and attached to an appropriate carrier, they will be good candidates for a contraceptive vaccine, particularly as there is a good example of a sterile woman with SI-Ab similar to mAb H6-3C4 who is very healthy. Kurpisz et al. [lo] raised 30 mouse mAbs to human sperm and found that they reacted with human lymphocytes, erythrocytes, bacteria and endotoxins; the spermassociated antigens dehned by these mAbs turned out to be glycoconjugates. There was some evidence that the predominant carriers of the carbohydrate antigens were glycolipids, with the terminal immunodominant monosaccharide being N-GlucNAC or N-GalcNAC. These results imply that the seminal plasma components that coat human sperm are predominantly carbohydrate but the terminal monosaccharide differs from that identified in my study [8]. An important question is whether these n-&s react with testicular germ cells because, according to our Iindings, the carbohydrate epitopes of sialylated i-Ag or sialylated I-Ag that coat the sperm surface are excreted from cauda epididymis or sernlnal vesicles and may not be sperm cell membrane components. Isahakia [ 111 has generated a mAb, called BSA-6, against an antigenic epitope secreted from baboon epididymis. The antigenic epitope corresponding to BSA-6 is excreted by the principal cells of the proximal corpus region, binds to the lateral acrosomal surfaces in the distal corpus region, and becomes unifomrly distributed over the acrosome in the cauda epididymis. The molecule contalnlng this epitope has a molecular weight of 82 kD and is specilic to spermatozoa and to the epithelial cells of the corpus epididymis. Isahakia did not investigate the chemical characteristics or biological role of the epitope and it is therefore not known whether this molecule could be a candidate for a contraceptive vaccine. Secreted glycollpids or glycoproteins from the male accessory organs such as the epidldymls or seminal vesicles may be important for spermatozoa. Therefore, an analysis of seminal plasma components may contribute to the development of a contraceptive vaccine. Iusem et al. [ 121 have identihed a purified 17 kD peptide as a major glycoprotein secreted from the rat epididymis. It has the biochemical characteristics of an N-glycosylated polypeptide and is actively biosynthesized and secreted by the rat epidldymis. Interestingly, testicular spermatozoa specifically bind this glycoproteln but epididymal spermatozoa do not. For more than a decade, it has been speculated that the interaction of secreted products with sperm causes the development of the zona pellucida recognition and fenillzlng ability of mature spermatozoa. If the 17 kD glycoprotein has an important role in sperm maturation, it would be worth using in a contraceptive vaccine.
Another seminal fluid component from rabbit epididymis is the acrosome-stabilizing factor (ASF) [13] which can reversibly decapacitate rabbit sperm in vivo and block the acrosome reaction in vitro. Epididymal maturation of sperm results in the progressive disappearance of-most of the surface testicular compounds, which are either renewed or masked by new permanent or transient low molecular weight polypeptides on the boar sperm surface membrane [ 141. Herr et al [ 151 have developed a method for purification of low molecular weight forms (912kD) of the seminal vesicle-specific antigen (SVSA) using CM cellulose chromatography followed by two cycles of mAb (MHS5) affinity chromatography. However, when the antibodies to this antigen coat the surface of sperm, they do not seem to have an effect on immobilization or agglutination.
Anti-idiotype
antibodies
In 1983, Wing and colleagues (r Exp Zool 1983, 227:481*) succeeded in generating a mAb (M4215) to mouse sperm that blocked fertilization in mice. They have since raised anti-idiotypic polyclonal antibodies (anti-Id) to M42-15 in rabbits (Carron et al, Biol Re prod l988,38:109~1103); these recognize determinants located close to or within the antigen-binding site of M42-15, and can inhibit M42-15 binding to the sperm antigen, Recently, Carron et al [I61 purl&d the anti-Id to M42-15 by al&&y chromatography. The purlhed antibody was shown to inhibit competitively M42-15 by a solid-phase radiolmrnune binding assay and also by speciIic immunoprecipitation of soluble M42-15. These results conlirmed that the anti-Id had antibodies directed to idiotopes within or adjacent to the antigen-binding site of the M42-15 mAb. When tested in in vitro fertilization assays, anti-Id, but not rabbit immunoglobulin, prevented M42-15-induced inhibition of fertilization. These reports have an important beating on the development of p immunocontraceptive vaccine. If the antigenic epitopes on the surface of sperm are composed of carbohydrate structures including glycoproteins, the conformation of carbohydrate chains and peptides could be important for antigenlcity, but analysis of the conformational status may be extremely di&ult. For this reason, anti-Ids that recognize dete rminants close to or within the antigen-binding site of antibodies directed against biologically active sperm antigens could function as antigens in a contraceptive vaccine.
Clinical
problems
of anti-sperm
antibodies
There seems to be some relationship between the presence of I~G antibodies directed against the sperm tail and a history of unexplained recurrent spontaneous abortion [ 171. Recently, my group discovered that the sialylated lactonorhexaosyl-ceramide residues (blood group i-Ag)
Sperm
and sialyl blood group I-Ag, which are secreted from the epididymis and coat the surface of sperm, are also present on the surface of human trophoblast. Therefore, antibodies against both sialyl neolactosamines in patients’ sera possess not only sperm immobilizing and agglutinating activities, but may also have some effect on trophoblast development. However, in our experiments the sIalyl i and I antigens coated the entire surface of the sperm and were not concentrated on the sperm tails, unlike the antibodies described by Witkin et al. [ 171.
Some progress has been made in the last year or so in characterizing aqtigenic epitopes that could have potential for contraceptive vaccine development. For both secreted SC-Ags and for those that are present in sperm membranes, the major problem at the moment is in demonstrating that they are spermspecific.
Annotated references and recommended reading Of interest Of outstanding interest
l
.a 1.
OXAND MG, WICCRENEE: Molecular biology of a sperm antigen: Identification of the sequence of an autoantigenic epitope. J R@rod Immunol 1989, suppl:6. One particular pepride of 10 amino acids, PlOG, putied from a 14 kD band by digestion with KaUkreii and HPLC, was shown to be immunoreactive. The antibodies to PlOG localized to the sperm equatorial postacmsomal border and sperm autoantibodies were shown to recognize PlOG. l
*
z. me
HERRJC, WRIGHTRM, FUCKINGER CJ: Biochemical, ultrastructural and genetic characterization of human acrosomal antigen SP-10. J Reprod Immunol 1989, suppL6. The human sperm peptide SP-10 which corresponds to a mAb fo human sperm (MHS-lo), is localized throughout the entire acrosomal region, particularly the acrosome membrane. Several cDNA clones encocing portions of SP-10 were isolated from a >ll human testis library and the mRNA for SP-10 was found to be 1.35 kb. The nucleotide se quence for 1.05 kb of SP-10 was obtained and SP-10 was shown to be present in monkeys and pigs. CJ: IdenHERRJC, WRIGHTRM, FOSTEREJJ, Karl T, FUCKINGER tllication of human acrosomal antigen SP-10 in primates and pigs. Biol Reprod 1990, 42:377-382. SP-10 was identified in sperm extracts from a baboon, two macaques and a pig using MHS-10. The monoclonai antibody did not react with acrosin and sperminogen in the pig; thus, SP-10 ls distinct from these components. Using a radioactive probe spanning 628 nucleotides of the open reading frame for SP-10 on northern blots of poly (A)+ RNA from baboon and macaque testes, a 1.35 mRNA was identified.
3. 0
4. 00
WRIGHTRM,JOHNE, KIMZ K, FLICKINGER CJ, HERRJC: Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10. Bid Reprod 1990, 42:693701 Three overlapping SP-10 cDNAs were isolated from a human testes cDNA expression library. These cDNAs hybridized to a 1.35 kb mRNA from human testes. The sequences did not show any signihcant homology to sequences in several databases. A recombinant SP-10 fusion protein was produced in an E. c&i expression vector and used to generate
plasma antigens
lsojima
a poIyclonaI antiserum. This antiserum showed similar reaction patterns to MHS-10. 5.
PRthf~~op~P, COWANA, HYA-ITH, TREDICK-KLINE J, A&LESDG:
Purlkation of the guinea pig sperm PH-20 antigen and detection of a site-specific endoproteolytic activity in sperm preparations that cleaves PH-20 into two disulEde-linked fragments. Biol Reprod 1988, 38~921-934. The PH.20 protein, purified by immunoaffinity chromatography, was separated into a major 64 kD and a minor 56 kD form by SDS-PAGE. The 64kD poIypeptide was cleaved info two disulphide-Iinked fragments of 414 kD and 27 kD by endoproteoIysis at a specilic site. This reaction may occur during the acrosome reaction and have biological significance. l
6.
Conclusion
and seminal
PRIMAKOFF P, UTHROP W, WOOLMANL, COWAN A, MYIES D:
Fully efkctive contraception in male and female guinea pigs immunized with the sperm protein PH-20. Nature 1988, 335:5435%. CompleteIy effective contraception was obtained by immunizing male and female guinea pigs with PH-20. Antisera from immunized females had high titres of antibodies that recognized PH.20 and blocked sperm binding to zona pellucida in vitro. l
.
NAZ RK, MEHTA K: Cell mediated immune responses to sperm antigens: effects on mouse sperm and embryos. Biol RqDrod 1989, 41:533542. A purified fertilization antigen (FA-1) activates presensitized lymphocytes to secrete soluble mediators that activate macrophages and inhibit sperm motility and embryonic development. 7. 0
8.
I~QJMA S: Chemical
structure of antigen epitope correspending to human sperm immobilizing monoclonal antibody H6-3C4 and the presence of sialyl neolactosandne structure as surface antigen of human sperm. J Reprod Immunol 1989, suppl:7. A mouse mAb, 2~6, directedto human seminal plasma, which strongIy immobilizes human sperm, also inhibits fertilization. This mAb recognizes a carbohydrate epitope closely related to that for human mAb H6-3C4. Clinica&, human seminal plasma loses the capability fo absorb the immobilizing activities of mAb H6-3C4 after TFMS treatment. l
S, ISOJMA TSUJIY, CIAUSENH, NUDEMANE, KALZUT, HAKOMORI S: Human sperm carbohydrate antigens defined by an antisperm human monoclonal antibody derived f?om an infertile woman bearing antispcrm antibodies in her serum. J E@ Med 1988, 168343356. Human monoclonal SI-Ab H6-3C4 defined a type 2 polylactosamine, irtern&y repetitive N-acetyflactosamine, i.e. sialyl-i, i, or fucosyl-i. Another mouse mAb, NUHZ, which can immobilize sperm, defined a binary a2-3 sialyl type 2 chain, i.e. sialyl-I. The SI-Abs in the sera of sterile women might be directed to sperm lactosaminogiycan or lactosaminolipid on the surface of sperm.
9. l
o
KURPISZM, Cwuc GF, MAHONYM, ANDERSONTL, ALEXANDER NJ: Mouse monoclonal antlbodies against human sperm: evidence for immunodo minant glycosylated antigenic sites. Clin .?kp Immunol 1989, 78:250-255. Studies of mouse mAbs raised against human sperm indicated that the sperm-associated antigens delined by these mAbs are gIycoconjuga@.. The predominant carriers of these carbohydrate antigens are glycolipids with N-GIucNAC or N-Gal&AC at the terminal. 10. 0
ISAHAKJA MA: Monoclonal antibody localization of sperk surface antigen secreted by the epididymis of the baboon. J Refn-od Fertal 1989, 86:51-58. A mAb, BSA6, was raised against an antigen secreted by the: epididymis of baboons. Its corresponding epitope is present on the acrosomaI surface of the sperm. The molecule bearing the epitope is secreted from the corpus epididymis and is an 82 kD molecule.
11. 0
IUSEMND, PJNEIROL, BIAQLIIER JA, BEKXOP~TOWE: Identification of a major secretory glycoprotein from rat epidldymis. Bid Reprod 1989, 40:307-316. The puri&d 17 kD secretory glycoprotein in the rat epididymis is phosphor@& and contains high mannose-type oligosaccharides. Specilic biding of the glycoprotein fo testicular sperm was demonstrated, but no binding to epididymal spermatozoa was detectable.
12. 0
755
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Reproduction 13. l
REYNOLDS AB, THOMAS‘IS, WIISON WI, OUPHANTG: Coflcennation of acrosome stabilizing factor (ASF) in rabbit epididymal fluid and species specificity of anti-ASF antibodies.
BiolReprod190!3, 40:675-680. Rabbit ASF in lumen fluids is concenttzted in the corpus, cauda epididymis and vas deferens. ASF has onIy been identifted in rabbits. 14. 0
DACHEUXJL, DACHEUXF, PAQLJIGNON M: Changes in sperm surface membrane and luminal protein fluid during epididymaI transit in the boar. BiolReprod1989, 40:63%51.
SequentiaIdramatic changes occur in the protein composition of sperm membrane and epididymaI fluids in the boar during epididymaI transit. The composition of the IuminaI proteins is akered throughout this process. HERRJC, CONKLIN DJ, MCGHEERS: Purilication of low molecl ular weight forms of seminal vesicle specific antigen by immunoatlinity chromatography on bound monoclonal antibody MHS-5. J ReprodImmurwll989,1699113. The low molecuIar weight forms of the seminai vesicle-specik antigen were puriIied by CM ceIIuIose chromatography followed by two cycles 15.
df immunoaftinity chromatography. The peptides were in the range of 1912kD. 16. **
CARRON CP, MA’rHIsA A, SAUNG PM: Anti-idiotype antibodies prevent antibody binding to mouse sperm and
antibody-mediated inhibition of fertilization. BiolReprod 1969, 40:153162. Anti-Id that recognize determmams located close to or within the antigen-binding site of an anti-sperm antibody were developed using an anti-sperm mAb, M42-15. The puriIied anti-Id competitIveIy inhibited the M42-15 antibody; thus, it could be used to neutrake the anti-fertility activity of M42-15. Wrna~ SS, C~AuDkntvA: Association between recurrent spontaneous abortions and circuIating IgG antibodks to sperm tails in women. / ReprodImmutwll989,15:151-158. There is a signikant association between the presence of IgG taildirected anti-sperm antibodies and a history of unexplained recurrent spontaneous abortion. 17. 0
Sperm and seminal plasma antigens relevant to contraceptive
vaccine development S. lsojima
Department
of Obstetrics
and Gynecology,
Current
Opinion
Hyogo Medical
in immunology
Introduction Sperm contain many antigenic molecules and epitopes, and animals probably generate many kinds of antibodies to them. The sperm antigens of mammals can be roughly divided into two groups: the sperm intrinsic membrane antigens (SM-Ags) and the sperm-coating antigens (SCAgs). The latter are present in the seminal plasma and adhere firmly to the sperm surface. Most SM-Ags are found in the cells of spermatozoa and spermatids in the testis, mainly in the acrosome or equatorial region; SCAgs, on the other hand, are usually excreted from the epithelial cells of the caput, corpus, cauda epidklymis and seminal vesicles, and become attached to sperm as they pass through the seminiferous tubes. In my opinion, antibodies to SC-Ags, which are composed of glycolipids or glycoproteins, probably cause sperm immobilization and agglutination whereas antibodies to SM-Ags, which are composed mainly of peptides, may be involved in the blocking of sperm binding or penetration of the zona pellucida. Here, I will discuss some recent advances in our understanding of these two types of sperm antigens and their potential for contraceptive vaccine development.
Sperm intrinsic membrane antigens Rabbit SM-Ags have been investigated by O’Rand and colleagues since 1976.They isolated a family of glycoproteins of molecular weights 13 RD [rabbit sperm antigen (RSA)-11, 11 kD (RSA-2) and 1OkD (RSA-3). Monoclonal antibodies (mAbs) were raised to epitopes on RSAl and these epitopes were found to be present on RSA-2 and
College,
Hyogo, Japan
1990, 2:752-756
RSA-3 as well. These mAbs inhibit in vitro fertilization, and it was therefore speculated that the RSA family has a central role in zona penetration (ORand et al, Biol Reprod 1984, 30:721-729). Human sperm extract binds mAbs to RSA-1, -2, and -3 with up to 75% of the maximum binding al&&y of rabbit sperm extract (O’Rand et aC, Biol Reprod 1984, 30:731-736). The antigens corresponding to these mAbs are concentrated in the middle region of the tail, with smaller amounts in the head. However, in rabbits, RSA are concentrated in the post-acrosomal equatorial region both before and after the acrosome reaction (Esaguy et al, Gamete Res 1988, 19:387-399). An examination of how RSA are distributed in somatic tissues in rabbits and humans [using a sensitive avidin-biotin complex (ABC) staining technique] is required to prove that RSA are sperm plasma membranespecilk antigens. Recently, O’Rand and Widgren [ll isolated members of the RSA family of molecular weights 14, 16, 17 and 18kD, and showed that some of them are resistant to boiling, reduction and sodium dodecyl sulphate (SDS) treatment. The 14 kD band was identified by western blotting with an anti-R&4 mAb, excised, and digested with Kallikrein. One particular peptide (PloC) of 10 amino acids was purifted by reverse-phase highperformance liquid chromatography (HPLC) and shown to react with anti-RSA serum. Antibodies to PlOG reacted specilically with low molecular weight RSA bands and with the sperm equatorial post-acrosomal border. O’Rand and Widgren [l] obtained the amino acid sequence of PlOG and showed that it blocks sperm binding to the zona pellucida. The question is now whether the PlOG peptide is actually present on human sperm. A human sperm acrosomal antigen has been isolated by Herr et al. [ 21. They had previously raised a mAb @IIS.
* This article and the preceding one offer two views on the use of sperm and related antigens in contraceptive vaccines.
Abbreviations ABC-avidinbiotin complex; anti-Id-anti-idiotypic polyclonal antibodies; ASP--acrosome-stabilizing factor; cDNA-complementary DNA; HPL-high-performance liquid chromatography; kb-kilobases; mAbs-monoclonal antibodies; mRNA-messenger RNA; PACIGpolyacrylamide gel electrophoresis; RSA-rabbit sperm antigen; SC-Ags-sperm-coating antigens; SDS-sodium dodecyl sulphate; SI-Abs-sperm-immobilizing antibodies; SM-Avperm intrinsic membrane antigens; TPM%trifluoromethasulphonic
75i
@ Current Biology Ltd ISSN 0952-7915
acid.
Sperm
10) to a component of the human sperm membrane, and found that it reacts with the entire acrosomal region, and particularly with the inner aspect of the outer acrosomal membrane and the outer aspect of the inner acrosomal membrane. After an acrosome reaction caused by the calcium ionophore A23187, MHS-10 still stained the sperm head, particularly the inner acrosomal membrane and equatorial segment. The sperm membrane protein corresponding to MHS-10, SP-10, has now been purified and shown to be an antigenic peptide of 24-34kD, and to have a pI of 4.9 on two-dimensional polyacrylamide gel electrophoresis (PAGE). h&IS-IO was shown to Inhibit sperm binding and penetration in the zona-free hamster egg penetration assay. Using a radioactive probe corresponding to 628 nucleotides of the open reading frame of SP-10 on northern blots of poly(A) + RNA obtained from the testes of a baboon and two macaques, a messenger RNA &RNA) of 1.35 kb (the same size as the mRNA from human testes) was identilied [3]. Recently, three overlapping SP-10 complementary DNAs (cDNAS, SP-10-5, SP10-9, and SP-10-10) were isolated from a human testes cDNA expression library [4]. These cDNAs hybridized to a 1.35 kb mRNA present exclusively in the testes. Complete sequencing of these cDNAs produced a 1117nu cleotide sequence that contains a region coding for 265 amino acids of the SP-10 protein. A recombinant SP-10 fusion protein produced in an EscberiGbia coli expression vector was used to generate a polyclonal antiserum that stained the acrosomal cap in situ and reacted with a similar set of peptides on western blots as MHS-10. A search of three sequence databanks with the SP-10 cDNA sequence did not reveal any significant homology with other sequences, but this does not necessarily mean that SP-10 is sperm-specific. Therefore, ABC staining of human somatic tissues with MHS-10 is required to c0nIii-m that this is the case, although ABC staining might pick up some false positives. Herr’s group [4] also showed that the recombinant SP-10 fusion protein can act as an effective immunogen in humans or animals in viva to prevent fertilization. Prlmakoff et al. [5] have isolated a guinea pig sperm membrane protein called PH-20. It may have a role in spemgg adhesion and its surface expression may be regulated by the acrosome reaction. The PH-20 protein, purified by mob alEnity chromatography, retains the ability to bind three anti-PH.20 mAbs, and separates on SDS-PAGE into a major 64kD form and a minor 56kD form. An endoproteolytically cleaved form of the 64kD polypeptlde, composed of two disulphlde-linked fragments of 41-48kD and 27 kD, also shows up on SDSPAGE. Recently, Prlmakoff et al. [6] suggested that PH20 is spem-~specific, on the basis of absorption experiments with tissue extracts from guinea pigs. A mAb to PH-20 inhibited sperm binding and penetration of guinea pig oocytes in vitro, and immunization of both female and male guinea pigs with the purified PH-20 protein induced sterility in most animals. The mAb to PH-20 only reacted with guinea pig acrosome but polyclonal antibodies to the purihed protein also reacted with human sperm. Therefore, PH-20 may contaln an epitope whose
and seminal
plasma antigens
lsojima
antibodies cross-react with human sperm membrane, or there may be a small amount of contamination of the guinea pig sperm giving rise to cross-reaction. One possible pitfall in the development of PH-20 as a contraceptive vaccine is that antibodies to it may cross-react with human somatic tissues. Prlmakoff et al 161 claimed that PH-20 was sperm-specific because they used 100 times more guinea pig somatic tissue than sperm in absorption experiments with the anti-PH-20 rnAb; however, I think that absorption experiments may not be su&iently sensitive to determine sperm specificity and ABC staining of somatic tissues using anti-PH-20 mAbs is needed. A sperm-specific glycoprotein, FA-1, has been isolated from human and mouse male germ cell plasma membranes by Naz and Mehta [7]. They claim that it is a tissue-specific, species cross-reactive, antigen and that mAbs to it inhibit human sperm penetration of zonafree hamster ova and block mouse sperm penetration of mouse oocytes. They speculate that FA-1 is involved in unexplained infertility in humans. Naz and Mehta also suggest that FA-1 influences cell-mediated immunity because a lymphokine-like substance that immobilizes sperm and blocks embryonic development is released from FA-l-activated macrophages. However, their results are confusing and more precise deli&ion of FA-1 crossreactivity with human somatic tissues by sensitive ABC staining is therefore needed, as well as further analysis of its chemical characteristics.
Sperm-coating
antigens
Ever since the discovery by myself and colleagues (Isojima et aL, Am J Obstet Gynecoll968, lOk677-483) of sperm-immobilizing antibodies (SIAbs) in the sera of women with unexplained infertility, SI-Abs have been thought to be relevant to this phenomenon. Because most SI-Abs in the sera can be absorbed with azoospermic semen, it was thought that these antibodies might be generated in response to human seminal plasma antigens (Isojima et al, Am J Obstet Gynecd 1972, 112:19%207). Recently, azoospermic semen was found to lose its absorption capability for most of the SI-Abs in the sera of infertile women if the semmal plasma was treated with trifluoromethasulphonic acid (TFMS) [8]. It seems that most sterile women generate SI-Abs directed against the carbohydrate epitope of seminal plasma antigens. To identify these antigens, a human- mouse hybridoma, H63C4, was successfully established using cells from a sterile woman with a high titre of SIAbs (Isojlma et al, J Reprod ZmmunoZl987,1O:67-78). The hybrldoma secreted high titres of an antibody (I@) that immobilizes and agglutinates sperm. The antigenic epitope corresponding to mAb H63C4 was found to be localized on the surface of epithelial cells of the cauda epididymis and adhered firmly all over the motile sperm; it remained partly ln the sperm after capacitation and an ionophore-induced’ acrosome reaction. Tsuji et al. [9] showed that mAb H63C4 reacts with lactonorhexaosyl-ceramide (blood group i-Ag) regardless of whether sialic acid is present ln the
753
754
Reproduction
terminal region; however, both sialyl i-Ag and sialyl I-Ag were found to be present on the surface of motile sperm. On the basis of these findings, molecules bearing i-Ag epitopes are thought to be excreted from the cauda epididymis and seminal vesicles and to adhere to the sperm surface after sialylation. In human seminal plasma, the molecules beating the epitopes recognized by mAb H63C4 were shown to have molecular weights of 15-25 kD by SDS-PAGE. If the carbohydrate chains of sialyl i-Ag can be synthesized and attached to an appropriate carrier, they will be good candidates for a contraceptive vaccine, particularly as there is a good example of a sterile woman with SI-Ab similar to mAb H6-3C4 who is very healthy. Kurpisz et al. [lo] raised 30 mouse mAbs to human sperm and found that they reacted with human lymphocytes, erythrocytes, bacteria and endotoxins; the spermassociated antigens dehned by these mAbs turned out to be glycoconjugates. There was some evidence that the predominant carriers of the carbohydrate antigens were glycolipids, with the terminal immunodominant monosaccharide being N-GlucNAC or N-GalcNAC. These results imply that the seminal plasma components that coat human sperm are predominantly carbohydrate but the terminal monosaccharide differs from that identified in my study [8]. An important question is whether these n-&s react with testicular germ cells because, according to our Iindings, the carbohydrate epitopes of sialylated i-Ag or sialylated I-Ag that coat the sperm surface are excreted from cauda epididymis or sernlnal vesicles and may not be sperm cell membrane components. Isahakia [ 111 has generated a mAb, called BSA-6, against an antigenic epitope secreted from baboon epididymis. The antigenic epitope corresponding to BSA-6 is excreted by the principal cells of the proximal corpus region, binds to the lateral acrosomal surfaces in the distal corpus region, and becomes unifomrly distributed over the acrosome in the cauda epididymis. The molecule contalnlng this epitope has a molecular weight of 82 kD and is specilic to spermatozoa and to the epithelial cells of the corpus epididymis. Isahakia did not investigate the chemical characteristics or biological role of the epitope and it is therefore not known whether this molecule could be a candidate for a contraceptive vaccine. Secreted glycollpids or glycoproteins from the male accessory organs such as the epidldymls or seminal vesicles may be important for spermatozoa. Therefore, an analysis of seminal plasma components may contribute to the development of a contraceptive vaccine. Iusem et al. [ 121 have identihed a purified 17 kD peptide as a major glycoprotein secreted from the rat epididymis. It has the biochemical characteristics of an N-glycosylated polypeptide and is actively biosynthesized and secreted by the rat epidldymis. Interestingly, testicular spermatozoa specifically bind this glycoproteln but epididymal spermatozoa do not. For more than a decade, it has been speculated that the interaction of secreted products with sperm causes the development of the zona pellucida recognition and fenillzlng ability of mature spermatozoa. If the 17 kD glycoprotein has an important role in sperm maturation, it would be worth using in a contraceptive vaccine.
Another seminal fluid component from rabbit epididymis is the acrosome-stabilizing factor (ASF) [13] which can reversibly decapacitate rabbit sperm in vivo and block the acrosome reaction in vitro. Epididymal maturation of sperm results in the progressive disappearance of-most of the surface testicular compounds, which are either renewed or masked by new permanent or transient low molecular weight polypeptides on the boar sperm surface membrane [ 141. Herr et al [ 151 have developed a method for purification of low molecular weight forms (912kD) of the seminal vesicle-specific antigen (SVSA) using CM cellulose chromatography followed by two cycles of mAb (MHS5) affinity chromatography. However, when the antibodies to this antigen coat the surface of sperm, they do not seem to have an effect on immobilization or agglutination.
Anti-idiotype
antibodies
In 1983, Wing and colleagues (r Exp Zool 1983, 227:481*) succeeded in generating a mAb (M4215) to mouse sperm that blocked fertilization in mice. They have since raised anti-idiotypic polyclonal antibodies (anti-Id) to M42-15 in rabbits (Carron et al, Biol Re prod l988,38:109~1103); these recognize determinants located close to or within the antigen-binding site of M42-15, and can inhibit M42-15 binding to the sperm antigen, Recently, Carron et al [I61 purl&d the anti-Id to M42-15 by al&&y chromatography. The purlhed antibody was shown to inhibit competitively M42-15 by a solid-phase radiolmrnune binding assay and also by speciIic immunoprecipitation of soluble M42-15. These results conlirmed that the anti-Id had antibodies directed to idiotopes within or adjacent to the antigen-binding site of the M42-15 mAb. When tested in in vitro fertilization assays, anti-Id, but not rabbit immunoglobulin, prevented M42-15-induced inhibition of fertilization. These reports have an important beating on the development of p immunocontraceptive vaccine. If the antigenic epitopes on the surface of sperm are composed of carbohydrate structures including glycoproteins, the conformation of carbohydrate chains and peptides could be important for antigenlcity, but analysis of the conformational status may be extremely di&ult. For this reason, anti-Ids that recognize dete rminants close to or within the antigen-binding site of antibodies directed against biologically active sperm antigens could function as antigens in a contraceptive vaccine.
Clinical
problems
of anti-sperm
antibodies
There seems to be some relationship between the presence of I~G antibodies directed against the sperm tail and a history of unexplained recurrent spontaneous abortion [ 171. Recently, my group discovered that the sialylated lactonorhexaosyl-ceramide residues (blood group i-Ag)
Sperm
and sialyl blood group I-Ag, which are secreted from the epididymis and coat the surface of sperm, are also present on the surface of human trophoblast. Therefore, antibodies against both sialyl neolactosamines in patients’ sera possess not only sperm immobilizing and agglutinating activities, but may also have some effect on trophoblast development. However, in our experiments the sIalyl i and I antigens coated the entire surface of the sperm and were not concentrated on the sperm tails, unlike the antibodies described by Witkin et al. [ 171.
Some progress has been made in the last year or so in characterizing aqtigenic epitopes that could have potential for contraceptive vaccine development. For both secreted SC-Ags and for those that are present in sperm membranes, the major problem at the moment is in demonstrating that they are spermspecific.
Annotated references and recommended reading Of interest Of outstanding interest
l
.a 1.
OXAND MG, WICCRENEE: Molecular biology of a sperm antigen: Identification of the sequence of an autoantigenic epitope. J R@rod Immunol 1989, suppl:6. One particular pepride of 10 amino acids, PlOG, putied from a 14 kD band by digestion with KaUkreii and HPLC, was shown to be immunoreactive. The antibodies to PlOG localized to the sperm equatorial postacmsomal border and sperm autoantibodies were shown to recognize PlOG. l
*
z. me
HERRJC, WRIGHTRM, FUCKINGER CJ: Biochemical, ultrastructural and genetic characterization of human acrosomal antigen SP-10. J Reprod Immunol 1989, suppL6. The human sperm peptide SP-10 which corresponds to a mAb fo human sperm (MHS-lo), is localized throughout the entire acrosomal region, particularly the acrosome membrane. Several cDNA clones encocing portions of SP-10 were isolated from a >ll human testis library and the mRNA for SP-10 was found to be 1.35 kb. The nucleotide se quence for 1.05 kb of SP-10 was obtained and SP-10 was shown to be present in monkeys and pigs. CJ: IdenHERRJC, WRIGHTRM, FOSTEREJJ, Karl T, FUCKINGER tllication of human acrosomal antigen SP-10 in primates and pigs. Biol Reprod 1990, 42:377-382. SP-10 was identified in sperm extracts from a baboon, two macaques and a pig using MHS-10. The monoclonai antibody did not react with acrosin and sperminogen in the pig; thus, SP-10 ls distinct from these components. Using a radioactive probe spanning 628 nucleotides of the open reading frame for SP-10 on northern blots of poly (A)+ RNA from baboon and macaque testes, a 1.35 mRNA was identified.
3. 0
4. 00
WRIGHTRM,JOHNE, KIMZ K, FLICKINGER CJ, HERRJC: Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10. Bid Reprod 1990, 42:693701 Three overlapping SP-10 cDNAs were isolated from a human testes cDNA expression library. These cDNAs hybridized to a 1.35 kb mRNA from human testes. The sequences did not show any signihcant homology to sequences in several databases. A recombinant SP-10 fusion protein was produced in an E. c&i expression vector and used to generate
plasma antigens
lsojima
a poIyclonaI antiserum. This antiserum showed similar reaction patterns to MHS-10. 5.
PRthf~~op~P, COWANA, HYA-ITH, TREDICK-KLINE J, A&LESDG:
Purlkation of the guinea pig sperm PH-20 antigen and detection of a site-specific endoproteolytic activity in sperm preparations that cleaves PH-20 into two disulEde-linked fragments. Biol Reprod 1988, 38~921-934. The PH.20 protein, purified by immunoaffinity chromatography, was separated into a major 64 kD and a minor 56 kD form by SDS-PAGE. The 64kD poIypeptide was cleaved info two disulphide-Iinked fragments of 414 kD and 27 kD by endoproteoIysis at a specilic site. This reaction may occur during the acrosome reaction and have biological significance. l
6.
Conclusion
and seminal
PRIMAKOFF P, UTHROP W, WOOLMANL, COWAN A, MYIES D:
Fully efkctive contraception in male and female guinea pigs immunized with the sperm protein PH-20. Nature 1988, 335:5435%. CompleteIy effective contraception was obtained by immunizing male and female guinea pigs with PH-20. Antisera from immunized females had high titres of antibodies that recognized PH.20 and blocked sperm binding to zona pellucida in vitro. l
.
NAZ RK, MEHTA K: Cell mediated immune responses to sperm antigens: effects on mouse sperm and embryos. Biol RqDrod 1989, 41:533542. A purified fertilization antigen (FA-1) activates presensitized lymphocytes to secrete soluble mediators that activate macrophages and inhibit sperm motility and embryonic development. 7. 0
8.
I~QJMA S: Chemical
structure of antigen epitope correspending to human sperm immobilizing monoclonal antibody H6-3C4 and the presence of sialyl neolactosandne structure as surface antigen of human sperm. J Reprod Immunol 1989, suppl:7. A mouse mAb, 2~6, directedto human seminal plasma, which strongIy immobilizes human sperm, also inhibits fertilization. This mAb recognizes a carbohydrate epitope closely related to that for human mAb H6-3C4. Clinica&, human seminal plasma loses the capability fo absorb the immobilizing activities of mAb H6-3C4 after TFMS treatment. l
S, ISOJMA TSUJIY, CIAUSENH, NUDEMANE, KALZUT, HAKOMORI S: Human sperm carbohydrate antigens defined by an antisperm human monoclonal antibody derived f?om an infertile woman bearing antispcrm antibodies in her serum. J E@ Med 1988, 168343356. Human monoclonal SI-Ab H6-3C4 defined a type 2 polylactosamine, irtern&y repetitive N-acetyflactosamine, i.e. sialyl-i, i, or fucosyl-i. Another mouse mAb, NUHZ, which can immobilize sperm, defined a binary a2-3 sialyl type 2 chain, i.e. sialyl-I. The SI-Abs in the sera of sterile women might be directed to sperm lactosaminogiycan or lactosaminolipid on the surface of sperm.
9. l
o
KURPISZM, Cwuc GF, MAHONYM, ANDERSONTL, ALEXANDER NJ: Mouse monoclonal antlbodies against human sperm: evidence for immunodo minant glycosylated antigenic sites. Clin .?kp Immunol 1989, 78:250-255. Studies of mouse mAbs raised against human sperm indicated that the sperm-associated antigens delined by these mAbs are gIycoconjuga@.. The predominant carriers of these carbohydrate antigens are glycolipids with N-GIucNAC or N-Gal&AC at the terminal. 10. 0
ISAHAKJA MA: Monoclonal antibody localization of sperk surface antigen secreted by the epididymis of the baboon. J Refn-od Fertal 1989, 86:51-58. A mAb, BSA6, was raised against an antigen secreted by the: epididymis of baboons. Its corresponding epitope is present on the acrosomaI surface of the sperm. The molecule bearing the epitope is secreted from the corpus epididymis and is an 82 kD molecule.
11. 0
IUSEMND, PJNEIROL, BIAQLIIER JA, BEKXOP~TOWE: Identification of a major secretory glycoprotein from rat epidldymis. Bid Reprod 1989, 40:307-316. The puri&d 17 kD secretory glycoprotein in the rat epididymis is phosphor@& and contains high mannose-type oligosaccharides. Specilic biding of the glycoprotein fo testicular sperm was demonstrated, but no binding to epididymal spermatozoa was detectable.
12. 0
755
756
Reproduction 13. l
REYNOLDS AB, THOMAS‘IS, WIISON WI, OUPHANTG: Coflcennation of acrosome stabilizing factor (ASF) in rabbit epididymal fluid and species specificity of anti-ASF antibodies.
BiolReprod190!3, 40:675-680. Rabbit ASF in lumen fluids is concenttzted in the corpus, cauda epididymis and vas deferens. ASF has onIy been identifted in rabbits. 14. 0
DACHEUXJL, DACHEUXF, PAQLJIGNON M: Changes in sperm surface membrane and luminal protein fluid during epididymaI transit in the boar. BiolReprod1989, 40:63%51.
SequentiaIdramatic changes occur in the protein composition of sperm membrane and epididymaI fluids in the boar during epididymaI transit. The composition of the IuminaI proteins is akered throughout this process. HERRJC, CONKLIN DJ, MCGHEERS: Purilication of low molecl ular weight forms of seminal vesicle specific antigen by immunoatlinity chromatography on bound monoclonal antibody MHS-5. J ReprodImmurwll989,1699113. The low molecuIar weight forms of the seminai vesicle-specik antigen were puriIied by CM ceIIuIose chromatography followed by two cycles 15.
df immunoaftinity chromatography. The peptides were in the range of 1912kD. 16. **
CARRON CP, MA’rHIsA A, SAUNG PM: Anti-idiotype antibodies prevent antibody binding to mouse sperm and
antibody-mediated inhibition of fertilization. BiolReprod 1969, 40:153162. Anti-Id that recognize determmams located close to or within the antigen-binding site of an anti-sperm antibody were developed using an anti-sperm mAb, M42-15. The puriIied anti-Id competitIveIy inhibited the M42-15 antibody; thus, it could be used to neutrake the anti-fertility activity of M42-15. Wrna~ SS, C~AuDkntvA: Association between recurrent spontaneous abortions and circuIating IgG antibodks to sperm tails in women. / ReprodImmutwll989,15:151-158. There is a signikant association between the presence of IgG taildirected anti-sperm antibodies and a history of unexplained recurrent spontaneous abortion. 17. 0