Eppin: a molecular strategy for male contraception.

New male contraceptives, both hormonal and non-hormonal, have many obstacles to overcome before they reach the market as a product. For hormonal contraceptives the long-term efficacy of oligospermia in a large population of unselected men remains to be determined. For nonhormonal contraception target selection remains a primary goal. Immunocontraception, which showed great promise for many years, has recently lost its appeal. Nevertheless, immunocontraception can be utilised as a strategy, particularly in primates, to discern the function of target molecules in the male. As an example, we discuss Eppin, an epididymal protease inhibitor that coats the surface of human spermatozoa. Because Eppin is predicted to be a serine protease inhibitor with chymotrypsin-like specificity and binds semenogelin, the natural substrate of PSA (prostate specific antigen, a serine protease), we investigated whether Eppin would modulate PSA activity and the hydrolysis of semenogelin. Additionally, because antibodies to Eppin provide contraception in male monkeys, we investigated whether antibodies to Eppin would inhibit the PSA hydrolysis of semenogelin. Eppin is a specific inhibitor of PSA activity that requires leucine 87, Eppin's P1 reactive site. Although Eppin modulates the hydrolysis of semenogelin by PSA, antibodies to Eppin do not inhibit PSA activity.

Eppin: an epididymal protease inhibitor and a target for male contraception.

Eppin (epididymal protease inhibitor) is one of several serine protease (or serine protease-like) inhibitors that are encoded by genes on human chromosome 20 and on mouse chromosome 2. Here we review our current knowledge of human and mouse Eppin genes and the Eppin protein in the context of protease inhibitors. Antibodies to Eppin in immunized male monkeys provide an effective and reversible contraceptive and these antibodies may be effective by interfering with Eppin's interaction with semenogelin during ejaculation. We review Eppin-semenogelin interaction and present a working model in the context of the hydrolysis of semenogelin by prostate specific antigen.

Eppin: an effective target for male contraception.

Eppin (epididymal protease inhibitor) is a member of the whey acidic protein (WAP)-type four-disulfide core (WFDC) gene family. This study provides updated information on Eppin and the Eppin-like genes within the Eppin cluster on human chromosome 20. A virtual structural model of the Eppin protein demonstrates that the C-terminal half of Eppin is structurally homologous to the Kunitz-type trypsin inhibitor. The Eppin N-terminal may have structural similarities to defensin-type molecules, rather than to that of the WAP consensus sequence. Human spermatozoa have a receptor for Eppin. When recombinant semenogelin (Sg) is digested with PSA many low molecular weight fragments are produced. However, when Eppin is bound to Sg, digestion by PSA is modulated. Addition of antibodies to the C-terminal of Eppin resulted in blocking PSA activity modulation. We can hypothesize from our analysis of anti-Eppin epitopes on Eppin that when anti-Eppin antibodies are bound to Eppin on the sperm surface they block the binding site for semenogelin.

Association of eppin with semenogelin on human spermatozoa.

Eppin (SPINLW1; GeneID, 57119) is a single-copy gene encoding a cysteine-rich protein found only in the testis and epididymis, which contains both Kunitz-type and WAP-type four disulfide core protease inhibitor consensus sequences. This study demonstrates that, in seminal plasma and on human spermatozoa following ejaculation, Eppin is bound to semenogelin I (Sg). Six different experimental approaches: 1) immunoprecipitation from spermatozoa and seminal plasma with anti-Eppin, 2) colocalization in semen and spermatozoa, 3) incubation of recombinant Eppin (rEppin) and rSg and immunoprecipitation with either anti-Eppin or anti-Sg, 4) far-Western blotting of Eppin and Sg, 5) Saturation binding of 125I-Sg to Eppin, which is competed by unlabeled Sg, and 6) direct binding of 125I-Sg to Eppin on a blot, all demonstrate that Eppin and Sg bind to each other. To study the specificity of binding, recombinant fragments of Eppin and Sg were made and demonstrate that the Eppin(75-133) C-terminal fragment binds the Sg(164-283) fragment containing the only cysteine in human Sg I (Cys-239). Reduction and carboxymethylation of Cys239 blocks binding of 125I-rEppin, indicating that a disulfide bond may be necessary for Eppin binding. The physiological significance of the Eppin-semenogelin complex bound on the surface of ejaculate spermatozoa lies in its ability to provide antimicrobial activity for spermatozoa, which has been reported for both Eppin and semenogelin-derived peptides, and in its ability to provide for the survival and preparation of spermatozoa for fertility in the female reproductive tract.

Reversible immunocontraception in male monkeys immunized with eppin.

Various forms of birth control have been developed for women; however, there are currently few options for men. The development of male contraceptives that are effective, safe, and reversible is desired for family planning throughout the world. We now report contraception of male nonhuman primates (Macaca radiata) immunized with Eppin, a testis/epididymis-specific protein. Seven out of nine males (78%) developed high titers to Eppin, and all of these high-titer monkeys were infertile. Five out of seven (71%) high-anti-Eppin titer males recovered fertility when immunization was stopped. This study demonstrates that effective and reversible male immunocontraception is an attainable goal. This method of immunocontraception may be extended to humans.

Characterization of Sp17: a ubiquitous three domain protein that binds heparin.

Sp17 is a protein that was originally thought to be expressed exclusively in the testis and whose primary function was binding to the extracellular matrix of the oocyte. Several recent reports have implicated Sp17 as having a role in cell-cell adhesion and/or cell migration in transformed, lymphocytic and haematopoietic cells, possibly through its interaction with extracellular heparan sulphate. In the present study, we report that Sp17's central domain (amino acids 61-117), spanning exon 3, is critical for heparin binding. Sp17 has two additional functional domains, an N-terminal domain similar to the dimer-interaction site in the cAMP-dependent protein kinase IIalpha regulatory subunit and a C-terminal calmodulin-binding domain. The mouse gene for Sp17 is 6.5 kb and contains four exons. Although Sp17 expression is highest in the testis, it is present in all of the mouse somatic tissues examined and is highly conserved throughout all mammalian species. Sp17's central domain, which is necessary for heparin binding, exhibits the greatest sequence divergence of all three domains. The Sp17 gene is induced in metastatic cells and during mucosal immune responses, and the protein appears to play an important role in cell migration and/or adhesion in somatic cells, as well as in male germ cells.

Analysis of the autoimmune epitopes on human testicular NASP using recombinant and synthetic peptides.

The human nuclear autoantigenic sperm protein, NASP, is a testicular histone-binding protein of 787 amino acids to which most vasectomized men develop autoantibodies. In this study to define the boundaries of antigenic regions and epitope recognition pattern, recombinant deletion mutants spanning the entire protein coding sequence and a human NASP cDNA sublibrary were screened with vasectomy patients' sera. Employing panel sera from 21 vasectomy patients with anti-sperm antibodies, a heterogeneous pattern of autoantibody binding to the recombinant polypeptides was detected in ELISA and immunoblotting. The majority of sera (20/21) had antibodies to one or more of the NASP fusion proteins. Antigenic sites preferentially recognized by the individual patients' sera were located within aa 32-352 and aa 572-787. Using a patient's serum selected for its reactivity to the whole recombinant protein in Western blots, cDNA clones positive for the C-terminal domain of the molecule were identified. The number and location of linear epitopes in this region were determined by synthetic peptide mapping and inhibition studies. The epitope-containing segment was delimited to the sequence aa 619-692 and analysis of a series of 74 concurrent overlapping 9mer synthetic peptides encompassing this region revealed four linear epitopes: amino acid residues IREKIEDAK (aa 648-656), KESQRSGNV (aa 656-664), AELALKATL (aa 665-673) and GFTPGGGGS (aa 680-688). All individual patients' sera reacted with epitopes within the sequence IREellipsis.GGS (aa 648-688). The strongest reactivity was displayed by peptides corresponding to the sequence AELALKATL (aa 665-673). Thus, multiple continuous autoimmune epitopes in NASP involving sequences in the conserved C-terminal domain as well as in the less conserved testis-specific N-terminal region comprising the histone-binding sites, as predicted for an antigen-driven immune response, may be a target of autoantibodies in vasectomized men and may provide a relevant laboratory variable to describe more accurately the spectrum of autoantibody specificities associated with the clinical manifestation of vasectomy.

Characterization of the histone H1-binding protein, NASP, as a cell cycle-regulated somatic protein.

Nuclear autoantigenic sperm protein (NASP), initially described as a highly autoimmunogenic testis and sperm-specific protein, is a histone-binding protein that is a homologue of the N1/N2 gene expressed in oocytes of Xenopus laevis. Here, we report a somatic form of NASP (sNASP) present in all mitotic cells examined, including mouse embryonic cells and several mouse and human tissue culture cell lines. Affinity chromatography and histone isolation demonstrate that NASP from myeloma cells is complexed only with H1, linker histones. Somatic NASP is a shorter version of testicular NASP (tNASP) with two deletions in the coding region arising from alternative splicing and differs from tNASP in its 5' untranslated regions. We examined the relationship between NASP mRNA expression and the cell cycle and report that in cultures of synchronized mouse 3T3 cells and HeLa cells sNASP mRNA levels increase during S-phase and decline in G(2), concomitant with histone mRNA levels. NASP protein levels remain stable in these cells but become undetectable in confluent cultures of nondividing CV-1 cells and in nonmitotic cells in various body tissues. Expression of sNASP mRNA is regulated during the cell cycle and, consistent with a role as a histone transport protein, NASP mRNA expression parallels histone mRNA expression.

A chimeric sperm peptide induces antibodies and strain-specific reversible infertility in mice.

The development of a contraceptive vaccine based on a gamete-specific antigen requires knowledge of the ability of the antigen to elicit an immune response that inhibits fertilization. A well-defined immune response, as elicited by a synthetic peptide comprising a dominant B-cell epitope coupled to a common promiscuous T-cell epitope, might be preferable. In this study, the immunodominant B-cell epitope of sperm antigen Sp17 has been identified and synthesized as a chimeric peptide with the promiscuous T-cell epitope bovine RNase[94-104] at the N terminal. Immunization of female BALB/c mice with this peptide induced a dose-dependent reduction in fertility. Although antibodies to recombinant and native Sp17 were elicited in these mice, there was no strict correlation between the level of these antibodies and the reduction in fertility. Moreover, the induction of infertility was strain-specific since no effect on fertility could be induced in B6AF1 mice. To understand the mechanism behind this apparent strain-specific infertility induction, a more extended study on both the humoral and the cellular immune response to the chimeric peptide was performed. The antigen-specific T-cell response and the levels of antigen-specific cytokines are the major factors that affect fertility outcome.

Sequence and characterization of the sperm protein Sp17 from the baboon.

In this study, cDNAs encoding the sperm protein Sp17 from the baboon (Papio papio) have been cloned and sequenced. Three clones, differing in the lengths of their 3' untranslated regions, were identified, which were encoded by mRNA transcripts of 0.8-1.35 kb. The open reading frame encodes 163 amino acids with a predicted molecular mass of 18.8 kDa. The baboon Sp17 protein sequence is 97% identical to human Sp17 but differs significantly by the addition of 12 amino acids at the C-terminal, providing an additional potential protein kinase C phosphorylation site. Northern blot analysis demonstrated that the baboon Sp17 mRNA was specific to the baboon testes and was not detected in the ovary, placenta, or any of the other somatic tissues tested. Western blot analysis using anti-Sp17 antibodies demonstrated that the native baboon sperm Sp17 protein consists of a doublet with an apparent M(r) of 26.5 and 27.2 kDa. Immunocytochemical staining of baboon testis with anti-Sp17 antibodies demonstrated Sp17 in spermatocytes, spermatids, and spermatozoa within the seminiferous epithelium. No specific staining was observed on spermatogonia, Sertoli cells, Leydig cells, or other somatic cell types.

Cloning and sequencing of cDNAs encoding the human sperm protein, Sp17.

In the present study we have cloned and sequenced two testis-specific cDNAs (1.3 kb and 1.6 kb) encoding a human sperm protein, designated HSp17. Each cDNA gave rise to identical protein sequences and differed only in the 5' untranslated region. The predicted amino-acid sequence revealed a protein of 17.5 kDa which exhibited a high degree of homology with both rabbit and mouse Sp17. Analysis of native and recombinant Sp17 by SDS-PAGE has shown the apparent molecular weight of the protein to be 24.5 kDa.

Sequence and localization of the mouse sperm autoantigenic protein, Sp17.

The present study characterizes the sperm protein Sp17 in the mouse. Sp17 is a mammalian testis- and sperm-specific protein that has been isolated, sequenced, and characterized from rabbit testis and spermatozoa. In this study, a rabbit Sp17 cDNA probe representing the entire protein coding region was used to screen a mouse testis cDNA library to obtain the mouse Sp17 sequence. The mouse mRNa for Sp17 encodes a 149-amino acid protein with a predicted molecular weight of 17,296. The mouse Sp17 (MSp17) cDNA sequence is 82% identical to the rabbit Sp17 cDNA sequence while the MSp17 protein sequence is 74% identical to the rabbit protein sequence. The presence of native Sp17 in mouse spermatozoa and testis was demonstrated by Western blot analysis, immunoprecipitation, and immunolocalization. After SDS-PAGE, the native Sp17 has an apparent molecular mass of 24 kDa. The sequence of the native Sp17 was confirmed by Western blots of mouse testis and spermatozoa probed with two anti-peptide antibodies--one, anti-G22C, made against amino acids 61-82 in the rabbit sequence (61-83 in the mouse), and a second, anti-K18C, made against amino acids 120-136 in the C-terminal region in the human sequence (118-134 in the mouse sequence). In the absence of proteolytic inhibitors, part of the C-terminal of native MSp17 is cleaved, giving rise to an 18-kDa band. Sp17 is present in spermatocytes and spermatids in the testis. In spermatozoa, Sp17 is not available to bind antibody on the surface of live, acrosome-intact spermatozoa, but it is present on the equatorial surface of live, acrosome-reacted spermatozoa. In fixed spermatozoa, staining is observed along the length of the principal piece, weakly along the midpiece, and over the acrosomal region of the head. When the acrosome reaction begins, acrosomal staining is seen throughout the equatorial region of the acrosome. Using mimotope analysis, this study has also demonstrated that native Sp17 is a sperm autoantigen and that recombinant mouse Sp17 is immunogenic in males with a highly restricted linear epitope.

Identification of sperm antigen targets for immunocontraception: B-cell epitope analysis of Sp17.

The selection of immunological targets present on gametes is an important first step in the successful production of animal or human vaccines for immunocontraception. One strategy with regard to sperm antigens is to select antigens with physiological roles in gamete interaction, obtain the mRNA sequence for such an antigen, and then determine which region or domain of the molecule is available to the immune system when spermatozoa are presented to the female reproductive system. To illustrate this strategy, we have used the rabbit sperm antigen Sp17 (RSA-3), which has been cloned and sequenced. The peptide pin-block method of Chiron Mimotopes, Australia, has been used to analyse the 146 amino acids of Sp17 with various homologous and heterologous antisera. This study demonstrates that such an analysis can lead to the identification of a B-cell epitope with strong immunocontraceptive potential.

Inhibition of fertility in female mice by immunization with a B-cell epitope, the synthetic sperm peptide, P10G.

Human patient sera containing antisperm antibodies (from vasectomized men and infertile women) immunologically react with the synthetic peptide P10G (PGGGTLPPSG), and affinity purified antibodies to P10G (anti-P10G) react with human spermatozoa (O'Rand et al., 1990). In this study P10G was used to elicit antibodies and the effect of the antibodies on fertility in female mice determined. The P10G sequence is derived from the 14-kDa rabbit sperm autoantigen, RSA (O'Rand and Widgren, 1990). The results of this study demonstrate that female mice can become infertile when immunized with the synthetic peptide P10G conjugated to the carrier protein keyhole limpet hemocyanin (KLH). However, the results also show that it is important to distinguish those mice with high serum antibody levels from those with lower levels. Infertility was clearly apparent in the high titer subgroup with an 80% decrease in pregnancy rate during the last of three matings and a 71% decrease in litter size over all three matings when compared to the low titer subgroup or the control groups. Significantly, mice immunized with P10G without carrier protein show no detectable antigen specific proliferation of lymph node cells in response to 100 microM of the peptide. P10G is not a T-cell epitope, but rather a B-cell epitope and it does not elicit an autoimmune response in the female mouse. This demonstration in mice is an important first step in the development of a safe human immunocontraceptive vaccine.

Sequence and localization of human NASP: conservation of a Xenopus histone-binding protein.

In this study the sequence and localization of human testicular NASP (nuclear autoantigenic sperm protein) are reported. NASP cDNA contains 2561 nt encoding a protein of 787 amino acids. The open reading frame contains 2446 nt followed by an ochre stop codon (TAA) and 104 nucleotides of untranslated sequence containing a poly(A) addition signal 10 bases upstream of the poly(A) tail. Northern blot analysis of human testis poly(A) mRNA indicates a message of approximately 3.2 kb. Multiple sequence alignment (MSA) analysis of the encoded human NASP amino acid sequence with the sequence for the Xenopus histone-binding protein N1/N2 and the rabbit NASP amino acid sequence demonstrates that the human sequence and the Xenopus sequence have extensive amino acid homology upstream of the rabbit initiation codon. Significantly, there is an 85% identity between the human and the rabbit NASP sequences when the alignment starts at the N-terminal of the rabbit sequence and at amino acid 101 of the human sequence. The nuclear translocation signal found in N1/N2 and rabbit NASP is completely conserved in human NASP. The first histone-binding domain of Xenopus is 70% identical and 90% similar to the human NASP domain. The second histone-binding domain of Xenopus is 48% identical and 71% similar to the human NASP domain. MSA analysis of the three sequences generated an unrooted ancestral tree with two branches, indicating that fewer amino acid changes have occurred between the Xenopus and the human sequences than between the Xenopus and the rabbit sequences. In the human testis, NASP is localized predominantly in primary spermatocytes and round spermatids. Spermatogonia, Sertoli cells, Leydig cells, peritubular cells, and other somatic cells do not stain. Human spermatozoa contain NASP in the acrosomal region. Following the acrosome reaction, some NASP remains in the equatorial and postacrosomal regions. We propose that mammalian testes and sperm contain a histone-binding protein which may play a role in regulating the early events of spermatogenesis.

A mammalian sperm lectin related to rat hepatocyte lectin-2/3. Purification from rabbit testis and identification as a zona binding protein.

In rat liver the asialoglycoprotein receptor is composed of three polypeptides, RHL-1, RHL-2 and RHL-3. In rat testis and spermatozoa a galactosyl receptor (RTG-r) which is immunologically related to RHL-2/3 has been described. We now report that in addition to its presence in the rat, an antigenic species of 54 kDa related to RHL-2/3 is present on rabbit, human, pig and mouse spermatozoa. Purified rabbit testis galactosyl receptor (RbTG-r) consists of two major proteins of 54 and 49 kDa, while purified rabbit liver galactose lectin consists of two major proteins of 43 and 40 kDa. In an ELISA the purified rabbit testis galactosyl receptor was shown to bind biotinylated heat solubilized rabbit zonae, while the purified liver galactose lectin did not. We conclude that one of the mammalian sperm's zona binding proteins is a galactose lectin of 54 kDa related to rat liver RHL-2/3.

Characterization of the rabbit sperm membrane autoantigen, RSA, as a lectin-like zona binding protein.

Adhesion between spermatozoa and the egg's extracellular coat, the zona pellucida, involves the sperm's zona binding proteins (ZBP) and their interaction with the carbohydrate residues of the zona. To investigate this interaction in more detail, a purified nonenzymatic ZBP, the rabbit sperm membrane autoantigen, RSA, was used. RSA-zona binding was demonstrated on nitrocellulose blots and by using the Denny-Jaffe crosslinking reagent which identified an 87,000 molecular weight zona component as the ligand for RSA. The RSA-zona binding was of high affinity with a dissociation constant of 5.6 X 10(-13) M. Furthermore, the binding of capacitated spermatozoa to intact zona was inhibited in the presence of RSA. Characterization of the RSA-zona interaction with a variety of simple and complex carbohydrates indicated that the sulfated, complex carbohydrates fucoidin, dextran sulfate, chondroitin sulfate B, and heparin strongly inhibited RSA-zona binding while chondroitin sulfates A and C, cholesterol-3-sulfate, and monosaccharides such as galactose inhibited RSA-zona binding only weakly. It is concluded that RSA functions as a sperm lectin-like molecule to bind the spermatozoon to the zona pellucida.

Characterization and derivation of the gene coding for mitochondrial carbamyl phosphate synthetase I of rat.

The nucleotide sequence of rat carbamyl phosphate synthetase I mRNA has been determined from the complementary DNA. The mRNA comprises minimally 5,645 nucleotides and codes for a polypeptide of 164,564 Da corresponding to the precursor form of the rat liver enzyme. The primary sequence of mature rat carbamyl phosphate synthetase I indicates that the precursor is cleaved at one of two leucines at residues 38 or 39. The derived amino acid sequence of carbamyl phosphate synthetase I is homologous to the sequences of carbamyl phosphate synthetase of Escherichia coli and yeast. The sequence homology extends along the entire length of the rat polypeptide and encompasses the entire sequences of both the small and large subunits of the E. coli and yeast enzymes. The protein sequence data provide strong evidence that the carbamyl phosphate synthetase I gene of rat, the carAB gene of E. coli, and the CPA1 and CPA2 genes of yeast were derived from common ancestral genes. Part of the rat carbamyl phosphate synthetase I gene has been characterized with two nonoverlapping phage clones spanning 28.7 kilobases of rat chromosomal DNA. This region contains 13 exons ranging in size from 68 to 195 base pairs and encodes the 453 carboxyl-terminal amino acids of the rat protein. Southern hybridization analysis of rat genomic DNA indicates the carbamyl phosphate synthetase I gene to be present in single copy.

Yeast carbamyl phosphate synthetase. Structure of the yeast gene and homology to Escherichia coli carbamyl phosphate synthetase.

A cloned fragment of yeast chromosomal DNA carrying the gene CPA2 coding for the large subunit of arginine-specific carbamyl phosphate synthetase has been sequenced. The cloned DNA has a 3,354-nucleotide long continuous reading frame coding for a polypeptide of 1,117 amino acids. The calculated molecular weight of the encoded polypeptide is 123,787, in good agreement with the reported molecular weight of the yeast carbamyl phosphate synthetase large subunit. The amino acid sequence of yeast carbamyl phosphate synthetase is homologous to the recently determined sequence of Escherichia coli carbamyl phosphate synthetase (Nyunoya, H., and Lusty, C. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4629-4633) over almost the entire length of the protein. Like the E. coli large subunit, the yeast enzyme exhibits an extensive internal homology between its NH2- and carboxyl-terminal halves. The internal homology in both the yeast and E. coli proteins indicates that the gene coding for the large subunit of carbamyl phosphate synthetase was derived from a tandem duplication which occurred prior to the divergence of eukaryotes and prokaryotes.