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.

Zonadhesin: characterization, localization, and zona pellucida binding.

Zonadhesin is a multiple-domain transmembrane protein that is believed to function as a sperm-zona pellucida binding protein. In this study we sequenced zonadhesin from rabbit testis and analyzed its processing, expression, localization, and zona pellucida binding. We show that the precursor protein occurs exclusively in the testis and that proteolytic processing results in the formation of three fragments: p43 (D1 domain), p97 (D2-D4 domains), and p58 (D4 domain-C-terminal). In mature spermatozoa the p43 and p97 fragments exist as disulfide-bonded dimers. During spermatogenesis, synthesis of zonadhesin mRNA chiefly occurs in primary spermatocytes, whereas the protein is abundant in both Sertoli cells and spermatids. In spermatozoa the protein is localized exclusively to the anterior acrosome but is not available for binding antibody on live spermatozoa. Once the acrosome reaction is induced, zonadhesin is lost from the spermatozoon, but remains with the acrosomal shroud. We show that recombinant D4 domain can bind zona pellucida, and we propose that zonadhesin functions after the acrosome reaction has been initiated to bind the acrosomal shroud to the zona pellucida.

Comparison of mouse and human NASP genes and expression in human transformed and tumor cell lines.

We previously cloned and sequenced cDNAs encoding mouse NASP (mNASP), a cell cycle regulated histone H1 binding protein. Here we report the genomic sequence and organization for mNASP along with its 5' regulatory region and compare these with human NASP (hNASP). The mNASP gene contains 16 exons interrupted by 15 introns. The sequence encoding testis mNASP uses all 16 exons while the somatic form uses 13 exons by differential splicing. All the exons conform to the AG/GT splicing rule. Putative TATA box-containing transcription initiation sites are present for somatic NASP in human and mouse and for testis hNASP. Comparison of the promoter regions of mNASP and hNASP approximately 1 kb upstream of the transcription start sites for the two splice variants revealed a number of possible transcription factor binding sites relevant to specific patterns of NASP tissue expression. The presence of single bands on Southern blots of mouse genomic DNA suggests that mNASP is a single copy gene although pseudogenes exist in both the mouse and human genomes. Chromosome fluorescence by in situ hybridization revealed that mNASP is present on chromosome 4, in an area that corresponds to band 4D1, a region syntenic to the locus of hNASP on chromosome 1. Additionally, we report that human somatic and testis NASP mRNAs are expressed at varying levels in all the transformed cell lines and human tumors tested, further supporting NASP's role in the cell cycle of dividing cells.

Primate epididymis-specific proteins: characterization of ESC42, a novel protein containing a trefoil-like motif in monkey and human.

Epididymal secreted proteins promote sperm maturation and fertilizing capacity by interacting with sperm during passage through the epididymis. Here we investigate the molecular basis of sperm maturation by isolating cDNA clones for novel epididymis-specific expressed sequences. Thirty-six novel cDNAs were isolated and sequenced from a subtracted Macaca mulatta epididymis library. The clones encode proteins with a range of motifs characteristic of protein-modifying enzymes, protease inhibitors, hydrophobic ligand-binding and transport proteins, extracellular matrix-interacting proteins, and transcription regulatory factors. The full length coding sequences were obtained for 11 clones representing a range of abundance levels. Expression of each is regionally localized and androgen regulated. The most abundant, ESC42, contains a cysteine-rich region similar to the signature binding domain of the trefoil family of motogenic wound repair proteins. The monkey and human proteins are nearly 90% identical. Immunohistochemical staining revealed that the protein is most abundant in the epithelium of the caput and is also present in the lumen and bound to sperm. The ESC42 gene, located on chromosome 20q11, contains two exons encoding two nearly identical predicted signal peptides and a third exon encoding the rest of the protein.

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.

HE2beta and HE2gamma, new members of an epididymis-specific family of androgen-regulated proteins in the human.

HE2 is an epididymis-specific sperm-binding secretory protein. We isolated a family of HE2-related complementary DNAs from a human caput/corpus library. The transcripts code for identical 71-amino acid N-termini and different C-termini, and 5'- and 3'-untranslated regions. Compared with the original HE2, HE2beta and HE2gamma proteins have a 25-amino acid deletion near the C-terminus, and HE2gamma isoforms have a second deletion. These frame-shifting deletions result in C-termini differing in length, amino acid sequence, including number of cysteines, and isoelectric point. Identical sequences and deletion start and stop points indicate the HE2 isoforms are derived from alternative splicing of 8 or more exons of a single gene. Northern hybridization revealed that the 0.9-kb messenger RNA (mRNA) is most abundant in human caput; there is much less of it (20%) in corpus and little (<5%) in cauda. In castrated Macaca mulatta, HE2 mRNA decreased to 10% of sham-operated levels. Testosterone replacement maintained HE2 mRNA 3- to 5-fold higher than castrate levels, indicating its androgen dependence. Immunohistochemical staining revealed that the beta1 form is highly expressed in principal cells of the initial segment and caput. It is secreted into the lumen and binds to the sperm surface in the postacrosomal and neck regions. The beta2 form is expressed in principal cells primarily in efferent ducts.

Cloning and characterization of an androgen-dependent acidic epididymal glycoprotein/CRISP1-like protein from the monkey.

A cDNA encoding an acidic epididymal glycoprotein (AEG)-like, CRISP1 (cysteine-rich secretory protein) protein from the monkey (Macaca mullata) epididymis has been cloned and sequenced. The monkey AEG (mAEG) has an open reading frame that encodes a protein containing 249 amino acids with a deduced molecular mass of 28 kDa. The mAEG protein sequence is 85% identical to human and 44% identical to mouse CRISP1, including all 16 conserved cysteine residues. mAEG also shows a significant amino acid homology with other CRISP proteins, rat AEG/DE, human TPX1/CRISP2, and guinea pig acrosomal autoantigen 1 (AA1). In addition, mAEG shows somewhat less homology to a toxin from the Mexican beaded lizard and to a human glioma pathogenesis-related protein. Northern blot analysis shows that the mRNA for mAEG is expressed in all the regions of the epididymis except the caput and was not detected in the testis, prostate, seminal vesicle, and brain. In castrated animals, mAEG gene expression in the epididymis is significantly diminished; however, testosterone enanthate replacement restored the normal level of expression, demonstrating that expression of mAEG is androgen dependent. Western blot analysis of monkey epididymal regions using mouse antirecombinant human AEG identified a 28-kDa protein only in the caudal region. Immunohistochemical analysis identified mAEG only in the principal cells of the cauda epididymal epithelium. Immunofluorescence analysis identified mAEG on the principal piece of the sperm tail and as small patches over the middle piece and head regions. The results described in the present study suggest that mAEG (CRISP1) is secreted in the monkey epididymis, regulated by androgens and present on epididymal spermatozoa.

Processing of the sperm protein Sp17 during the acrosome reaction and characterization as a calmodulin binding protein.

In this study we have demonstrated that the native rabbit sperm protein, Sp17, is a 22- to 24-kDa triplet of proteins in washed ejaculated rabbit spermatozoa and is unaffected by capacitation. However, during the acrosome reaction, Sp17 is processed from a 22- to 24-kDa triplet of proteins to a triplet of proteins at 17-19 kDa by the removal of amino acids from the C-terminal. Recombinant rabbit Sp17 (rRSp17) can also be proteolytically processed by acrosome-reacted spermatozoa in a similar manner. Protease inhibitors prevent the proteolytic processing of Sp17. Both forms of native Sp17 remain associated with acrosome-reacted spermatozoa and are solubilized by ionic detergents. Previously, sequence analysis of Sp17 revealed that Sp17 amino acids 108-137 were 52% identical to the calmodulin binding domain of neuromodulin and contained an IQ motif found in other calmodulin binding proteins. In this study, a truncated recombinant Sp17, rRSp17CB, which lacks amino acids 118-146, including the potential calmodulin binding site, was made. Recombinant rabbit Sp17, but not rRSp17CB, binds to calmodulin in the presence of Ca2+ or EDTA, under reduced or nonreduced conditions in biotinylated-calmodulin overlay assays. In DSS crosslinker experiments, calmodulin bound to rRSp17 in a 1:1 ratio but not to rRSp17CB. Additionally, biotinylated rRSp17 interacts with native sperm calmodulin. We propose that the processing of native Sp17, by removing a C-terminal fragment during the acrosome reaction, might be a mechanism to regulate the calmodulin binding activity of Sp17 and provide calmodulin at specific sites after the acrosome reaction.

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.

Immune response to immunization with sperm antigens in the macaque oviduct.

The oviduct of most mammalian species is the site where spermatozoa first encounter the oocyte and the process of fertilization is initiated. It should therefore be considered an important focus of study for the development of an effective gamete immunocontraceptive with the goal of immunologically interfering with spermatozoon function. The study reported here used the cynomolgus macaque as a nonhuman primate model in which to analyze the serum and oviductal fluid immune response to immunization with the human sperm protein Sp17 and synthetic peptides derived from Sp17. Human and macaque Sp17 were shown to share a very high degree of identity (96.7%), confirming this species as a suitable model in which to study the antibody response and recognition of spermatozoa. The oviductal fluid antibody titer varied with respect to serum titer both over time for individual monkeys and between different monkeys. In all cases, however, the antibody response was restricted solely to the immunoglobulin G class. Finally, both serum and oviductal fluid antibodies were directed against identical Sp17 B-cell epitopes and both recognized native macaque Sp17 in spermatozoa.

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.

Autoimmunogenicity of the human sperm protein Sp17 in vasectomized men and identification of linear B cell epitopes.

OBJECTIVE: To assess whether human sera positive for antisperm antibodies have detectable levels of Sp17 autoantibodies and to determine the linear B cell epitopes to which these are raised for both native and recombinant Sp17. DESIGN: Enzyme-linked immunoaborbent assays were performed against recombinant HSp17 on 15 serum samples from prevasovasostomy and postvasovasostomy patients. Positive sera then were used in mimotope analyses to determine HSp17 immunodominant linear B cell epitopes. These were compared with the linear B cell epitopes of recombinant HSp17. SETTING: University research laboratory. PATIENT(S): Fifteen vasectomized or vasovasostomized men. MAIN OUTCOME MEASURE(S): Serum antibody reactivity to human Sp17. RESULT(S): Sera from vasectomized and vasovasostomized men exhibit Sp17 antibodies raised predominantly to two immunodominant linear B cell epitopes (amino acids 4 to 19 and amino acids 118 to 127), which differed from those of recombinant HSp17 (amino acids 52 to 79 and amino acids 124 to 136). CONCLUSION(S): The results show that Sp17 is an antigen to which vasectomized men raise autoantibodies. Two linear B cell epitopes predominate in native Sp17 and these differ from (but overlap with) those of the bacterially expressed recombinant protein.

Designing an effective immunocontraceptive.

The immunological inhibition of fertilization is the goal of gamete immunocontraception. To achieve this goal a gamete-specific antigen target must be defined and the presentation of the immunogen to the immune system must be clearly understood in order to elicit a defined immune response which will target the native gamete molecule. Almost 20 years ago C.B. Metz suggested six studies which would answer the questions necessary for the development of a successful immunocontraceptive, and although much work has gone into answering each of these questions, none has been completed. Hyaluronidase is an example of a well studied sperm antigen whose native, membrane bound form (PH-20) is a successful immunocontraceptive in female guinea pigs. However, it remains to be demonstrated that a successful native antigen can be a successful synthetic or recombinant gamete immunocontraceptive (GAMICON). The problem of converting a successful native contraceptive antigen into an effective synthetic or recombinant GAMICON is at the heart of the problem of GAMICON design. If the epitopes of native and synthetic immunogens are not the same, then can a conversion ever be made? One approach to understanding how to make this conversion is to use defined, synthetic B- T-cell epitopes as specific B-cell epitopes on native antigens affect fertility.

Site-directed mutagenesis of rabbit proacrosin. Identification of residues involved in zona pellucida binding.

The mammalian acrosomal sperm protease proacrosin plays a role in fertilization by proteolysis of the oocyte's outer investments. In addition to its serine protease activity, acrosin from several species is known to have binding activity for the zona pellucida, and this action may serve to anchor sperm during zona penetration. In this study, proacrosin was purified from acid extracts of rabbit sperm and shown to bind to homologous zona pellucida using an in vitro assay. Measurement of this binding activity indicated a high affinity saturable interaction with a KD = 1.4 x 10(-8) M. Using cDNAs obtained from previously cloned and sequenced rabbit proacrosin and a splice variant that encodes a shorter form of acrosin (Richardson, R. T., and O'Rand, M. G. (1994) Biochim. Biophys. Acta 1219, 215-218), constructs of various sizes were produced using polymerase chain reaction and expressed as recombinant proteins. In the same in vitro zona binding assay, a construct representing residues 1-279 of rabbit proacrosin was found to bind to zona with a high affinity similar to that of native proacrosin, KD = 2.1 x 10(-8) M. By making smaller recombinant fragments and assaying them for zona binding activity, the location of the binding site was mapped to residues 47-94. Protein modeling of rabbit proacrosin using chymotrypsinogen A as a three-dimensional model indicated that an exposed loop Asp35 to His40 in chymotrypsinogen A is extended with an additional five amino acid residues in rabbit proacrosin from Ile43 to His53 containing arginine residues Arg47, Arg50 and Arg51. Site-directed mutagenesis of arginine residues Arg50 and Arg51 to alanine produced a recombinant without significant zona binding activity. These results are consistent with the hypothesis that rabbit proacrosin contains a specific zona pellucida binding site and that the loop containing arginine residues 50 and 51 is critical for zona binding activity.

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.