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.

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. (C) 1997 Wiley-Liss, Inc.

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.

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.

In one of our previous studies, the deduced amino acid sequence of the human nuclear autoantigenic sperm protein (hNASP) revealed two conserved histone-binding domains when compared to the Xenopus N1/N2 protein sequence. These histone-binding domains of Xenopus N1/N2 are known to be functional; however, their function in hNASP is unknown. In this study we have determined the number, location, and activity of the histone-binding domains on the primary sequence of hNASP. Purified recombinant polypeptides expressing the full-length hNASP and various deletion constructs covering the entire length of the hNASP sequence were tested by Western blotting and in ELISA for binding to biotin-labeled histones. A positive reaction was detected for the full-length recombinant protein and for the polypeptides spanning the N-terminal region (amino acids [aa] 32-192), and two additional regions: aa 193-352 and aa 353-572. The lack of binding to the expressed C-terminal (aa 573-787), which also contains polyacidic amino acids, suggests that the binding of hNASP to the somatic core histones is a sequence-specific as well as an electrostatic interaction. The removal of flanking sequences from the binding domains did not abrogate their ability to bind histones. We conclude that there are at least three functional histone-binding domains in hNASP, two of them encompassing the predicted histone binding sites homologous to the N1/N2 protein, and a third novel domain. Therefore, hNASP may be defined as a nuclear histone-binding protein found in human testis.

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.

This study extends our analysis of rabbit recombinant Sp17 (rSp17) by examining whether rSp17 synthesized in transfected COS cells will show a particular localization within the cell and whether the COS cell will bind with zona pellucida. We show, using the crosslinking, reagent DSS that rSp17 can bind to rabbit zona glycoprotein R45 or R55.

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.

The interaction of the mammalian spermatozoon with the oocyte's extracellular matrix or zona pellucida is a critical first step leading to successful fertilization. In this cell-extracellular matrix interaction it is the carbohydrate of the zona pellucida which serves as the sperm receptor and the surface of the spermatozoon which provides the lectin-like adhesion molecules. To better understand sperm-zona pellucida binding we have analyzed one specific zona binding protein (ZBP). This study has determined the mRNA sequence encoding a mammalian testis and sperm specific protein of 16,891 Da, which we have designated Sp17. Analysis of Sp17 revealed that the MRNA is present in rabbit, mouse, and human testes but not in any somatic tissue tested. In the rabbit, Sp17 is the 17-kDa member of the rabbit sperm autoantigen family of sperm specific autoantigens and is encoded by two mRNAs of 0.9 and 1.1 kb. Each mRNA has a unique 5′ untranslated region but both have identical coding regions. The deduced amino acid sequence of the Sp17 ZBP showed several interesting features, including a similarity to the N-terminal of human testis cAMP-dependent protein kinase. Localization of Sp17 on live spermatozoa using antibodies to recombinant Sp17 or to the Sp17 peptide, G22C, revealed that the peptide backbone of Sp17 is inaccessible until the acrosome reaction begins. However, an paraformaldehyde fixed, acrosome intact spermatozoa, the peptide backbone is accessible to the antibodies which localize Sp17 to the apical surface. In the rabbit as well us other similar species in which the corona radiata (granulosa) cells adhere tightly to the zona pellucida and synthesize zona glycoproteins, the fertilizing spermatozoon my have already begun the acrosome reaction within the cumulus oophorus. Thus, the rabbit sperm surface would be modified to expose the Sp17 polypeptide during the final phase of cumulus passage and consequently Sp17 would be available for initial zona binding. The present study has also demonstrated that recombinant Sp17 can bind zona pellucida, dextran, and dextran sulfate.

A 1414 bp cDNA for rabbit preproacrosin (RPA) and a related short preproacrosin (shRPA) cDNA of 951 bp were cloned and sequenced. RPA's 431 amino acid open reading frame encodes a 46,422 kDa protein. shRPA is identical to RPA except that it lacks an internal stretch of 468 bp, such that the encoded protein has a deduced molecular mass of 29,965 kDa. Antiserum against a synthetic peptide representing the light chain of rabbit proacrosin was used on Western blots of rabbit testis and sperm. Under reducing conditions, it revealed two major groups of bands at 50-57 and 29-32 kDa. Several lines of evidence suggest that shRPA is a splice variant of proacrosin and that it encodes a 30-33 kDa protein similar to sperminogen (Siegel, M. et al. (1987) Biol. Reprod. 36, 1063-1068), but apparently lacking proteinase activity.

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 μM 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.

Examination of the role of carbohydrates in specific recognition between spermatozoa and zona pellucida has focussed on understanding the interaction of sperm hydrolases or lectin-like molecules with zona pellucida ligands. To elucidate the role of specific spermatozoan hydrolases in gamete interaction, rabbit testis beta-galactosidase and arylsulfatase A were purified, characterized, and localized in spermatozoa. beta-Galactosidase and arylsulfatase A co-purified after affinity, size, or reverse-phase chromatography. N-Terminal amino acid analysis and enzymatic characterization suggested that neither enzyme is a testis-specific isozyme. Size chromatography indicated that both enzymes aggregated into macromolecular complexes at pH 4.0, while both dissociated at pH 8.0. beta-Galactosidase and arylsulfatase A co-localized on the sperm surface and in the acrosome and postacrosomal regions of spermatozoa. Throughout the zona-induced acrosome reaction, both enzymes remained associated with the detached acrosomal cap and postacrosomal region of acrosome-reacted spermatozoa. Because the acrosome is an acidic subcellular compartment, internal beta-galactosidase and arylsulfatase A are probably aggregated in acrosome-intact spermatozoa and dissociate as they are exposed to pH increases during the acrosome reaction.

To better understand the loss of the acrosomal cap on the surface of the zona pellucida and the function of the equatorial-postacrosomal region after the acrosome reaction, we have constructed an in vitro system using heat-solubilized zonae pellucidae dried onto a coverslip and incubated with capacitated spermatozoa. This system allows good optical resolution of spermatozoonzona interaction. Induction of the acrosome reaction by zonae on coverslips (30%) is comparable to the induction of the reaction reported previously for rabbit spermatozoa using solubilized zonae in solution. Antiserum to rabbit proacrosin, antiserum to a porcine 49-kDa proacrosin fragment, and antiserum to a porcine 14-kDa C-terminal acrosin fragment were utilized to monitor the acrosome reaction. Rabbit proacrosin/acrosin is not present on the surface of live, acrosome-intact, swimming spermatozoa. After contact with zona, the acrosome reaction begins and proacrosin/acrosin becomes available to bind antibody, first as a crescent in the apical region and then more posteriorly until the entire anterior acrosome is labeled. Proacrosin/acrosin remains on the equatorial and postacrosomal regions of acrosome-reacted spermatozoa and also remains associated with the acrosomal cap even after the spermatozoon is no longer associated with it. Further studies using zona-coated coverslips should lead to a more detailed understanding of the mechanism of zona penetration.

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.