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PubMed1994

Inhibition of in vivo fertilization by active immunization of male hamsters against a 26-kDa sperm glycoprotein.

We have identified a 26-kDa (P26h) epididymal hamster sperm glycoprotein with a species-specific affinity for zona pellucida glycoprotein. Two immunological procedures have been used to document the biological function of this sperm component; active immunization of males against P26h and inhibition of sperm-zona pellucida binding in vitro by anti-P26h antibodies. The immunized male hamsters produced circulating antibodies specific to P26h. Indirect immunofluorescence studies showed that these antibodies bind to the surface of the sperm covering the acrosome. These males were mated with superovulated females, and although spermatozoa were recovered from the genital tract, none of the 194 oocytes recovered were fertilized. In contrast, control males immunized with hamster albumin fertilized 97.4% of the oocytes. Unlike control spermatozoa, those recovered from the cauda epididymidis of males immunized with P26h were characterized by the presence of antibodies at the surface of the acrosome. To establish whether the inhibition of in vivo fertilization by active immunization was occurring at the level of sperm-zona pellucida interaction, a polyclonal antiserum against P26h was raised, and the IgG fraction was added to an in vitro sperm-zona pellucida assay. Compared to the preimmune serum, the IgG inhibited the binding of spermatozoa in a dose-dependent manner. The Fab fragments generated from these IgGs were almost as efficient in inhibiting the binding. These results are discussed with regard to a possible function of P26h in hamster gamete interaction.

Acrosome↗
PubMed2004

Studies with Synthetic Peptides of 80 kDa Human Sperm Antigen (80 kDa HSA).

PROBLEM: The 80 kDa human sperm antigen (HSA) is a sperm-specific and conserved antigen, capable of inducing immunological infertility. Partial N-terminal amino acid sequences of 80 kDa HSA (Peptide NT) and its peptides obtained by digestion with endoproteinase Lys-C (peptides 1-4) and endoproteinase Glu-C (peptides 5-6) did not show any sequence homology with reported known proteins deposited in the Gen-Bank. These sequenced peptides were synthesized and conjugated to key hole limpet haemocyanin (KLH) and evaluated for its antifertility effects. The present communication describes the characterization of these peptides and their antibodies. METHOD OF STUDY: Peptides NT, 1, 2, 3 and 4 were synthesized and conjugated to KLH. Antibodies to KLH conjugated peptides were raised in rabbits by active immunization and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) using sperm extract coated wells. The binding specificity of the synthetic peptides or purified 80 kDa HSA to their antibodies was assessed in the presence of various doses of respective synthetic peptides or 80 kDa HSA. The binding specificity was further confirmed by Western blot analysis. Antipeptide antibodies were also checked for sperm agglutinating activity, in-vitro. RESULTS: Active immunization of rabbits elicited significant antibody titers against the synthetic peptides, except for peptide 3. Antipeptide antibodies specifically recognized the native protein in an ELISA and induced in-vitro agglutination of human, rat and monkey sperm. In addition, Western blot analysis showed that these antipeptide antibodies specifically bind to the 80 kDa HSA band of the sperm extract. CONCLUSION: Synthetic peptides of 80 kDa HSA are immunogenic and antibodies raised against these peptides recognize the native protein detected by ELISA, Western blot analysis. In addition, they possess sperm agglutinating activity. These findings suggest that they are promising candidates in the development of immunocontraceptives.

Agglutination Tests↗
PubMed1997

Antiserum to the egg coats of the fat-tailed dunnart (Marsupialia, Dasyuridae) cross-reacts with egg coats of other marsupial and eutherian species.

We are examining the extracellular coats of the brush-tailed possum as a possible target for an immunocontraceptive vaccine for biocontrol of this pest species in New Zealand. In this study we have compared the composition of the extracellular coats of the fat-tailed and stripe-faced dunnarts, brush-tailed possum, domestic rabbit, and laboratory mouse using histochemistry, immunocytochemistry, and immunofluorescence. The histochemistry of the luminal epithelium of the oviduct and mucoid coats of the marsupials and rabbit indicated that they contain acidic glycoproteins. Immunofluorescence showed that polyclonal antiserum raised against the extracellular coats of the oocyte and early embryo of the fat-tailed dunnart, cross-reacted with the extracellular coats of the oocytes of all five species. These results suggest that there are common epitopes on the extracellular coats of oocytes and early embryos of distinctly related therian species. Further work to characterise these proteins is required to determine whether there is close homology between the oviductal glycoproteins of these species.

Animals↗
PubMed1997

Expression of bonnet monkey (Macaca radiata) zona pellucida-3 (ZP3) in a prokaryotic system and its immunogenicity.

An internal fragment (978 bp) corresponding to the bonnet monkey (Macaca radiata) ZP3, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by PCR from a full-length cDNA clone. The amplified Bam HI and SacI restricted fragment was cloned in frame downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector. Recombinant ZP3 (r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strains SG13009[pREP4] and BL-21(DE3). Immunoblot with a murine monoclonal antibody, MA-451 (raised against porcine ZP3 beta-a homologue of bonnet ZP3, and cross-reactive with bonnet zona pellucida) revealed a predominant band of 50 kDa besides degraded fragments. Optimum expression of r-ZP3 was observed at 0.5 mM IPTG. Antisera generated in monkeys against synthetic peptides from the N-(23-45 aa residues) and C-(300-322 and 324-347 aa residues) termini of the deduced bonnet monkey precursor ZP3 sequence reacted with the r-ZP3 protein in ELISA. The r-ZP3 expressed in SG13009[pREP4] was purified on Ni-NTA resin under denaturing conditions and conjugated with diphtheria toxoid (DT). Immunization of a female rabbit and six female bonnet monkeys with the r-ZP3-DT conjugate generated antibodies reactive with r-ZP3 in ELISA. Rabbit r-ZP3 antiserum reacted with porcine ZP3 beta and bonnet r-ZP3 but failed to react with porcine ZP3 alpha in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The availability of r-ZP3 will further help in evaluating its efficacy for fertility regulation and understanding the autoimmune oophoritis associated with ZP3 immunization in nonhuman primates.

Amino Acid Sequence↗
PubMed1997

Binding of rat and ovine epididymis-specific prealbumins (PES) to rat spermatozoa without effect of heterologous immunization on rat fertility.

The epididymis, under control of testosterone, secretes proteins which bind to the membrane of the spermatozoa during their passage through the lumen. One such class is termed PES (prealbumin epididymal specific). Injection of heterologous oPES (ovine PES) into male rats caused antibody production but failed to induce sterility, unlike results previously obtained when rat PES was injected into male rats. This suggests that only very restricted species-specific epitopes of PES might be useful for causing immunocontraception. Despite this, the sperm binding properties of PES purified from the rat (rat PES) and from the ram (oPES) were shown to be similar. When either rat PES or oPES, conjugated with a fluorescent probe (dimethylamino-fluorescein), was incubated with washed rat spermatozoa originating from the caput, corpus or cauda epididymis, results of flow cytometric analysis showed: (1) the number of spermatozoa bound to isologous or heterologous fluorescent PES, and (2) the binding-affinity of spermatozoa for PES was greater for sperm collected from more distal sites in the epididymis.

Animals↗
PubMed1997

Molecular cloning and sequencing of cDNA encoding for a novel testis-specific antigen.

cDNA encoding for a sperm antigen, designated NZ-1, was cloned and sequenced from murine testis cDNA-lambda gt11 expression library using antibodies to human sperm surface antigens belonging to 14-18 kD molecular region. These sperm antigens are involved in zona pellucida binding and have tyrosine phyosphorylation activity. Computer generated translation analysis of 1395-bp cDNA yielded an open reading frame (ORF) of 152 aa with first ATG, Met start codon at nt 32 and the stop codon TGA at nt 487. The translated protein has a calculated molecular weight of 17.9 kD and a potential tyrosine phosphorylation site at aa 46-54, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced aa sequence indicated it to be a membrane-anchored peptide with a hydrophobic NH2-terminus that is characteristic of a signal peptide. Extensive computer search in the GenBank, NBRF, and Swiss sequence banks, indicating it to be a novel protein. Northern blot analysis indicated testis-specific expression of NZ-1 antigen. The NZ-1 cDNA was subcloned into pGEX-1 lambda T vector and expressed in glutathione-S-transferase gene fusion system to obtain the recombinant protein. The recombinant protein specifically reacted with the original antibodies raised against the native 14-18 kD sperm proteins. These findings suggest that the sperm-specific recombinant NZ-1 may find applications in the development of a contraceptive vaccine, and in studying the normal and abnormal sperm function and the signal transduction mechanism.

Amino Acid Sequence↗
PubMed1999

Expression of recombinant mouse sperm protein sp56 and assessment of its potential for use as an antigen in an immunocontraceptive vaccine.

Recombinant mouse sp56 protein was produced for testing as an antigen in an immunocontraceptive vaccine. The coding sequence for the mature sp56 protein was cloned into the bacterial expression system pFLAG using a PCR-based method on mouse testis cDNA. Polyclonal antisera were raised in mice against affinity purified recombinant sp56 fusion protein (sp56FLAG) or an artificial sp56 peptide fused to a carrier protein (KLH) and shown to cross-react to a protein band of 75 kD in detergent extracts of mouse sperm by Western immunoblot analysis under reducing conditions. The antisera to sp56FLAG also immunolocalized over the entire acrosome of mouse sperm. Female BALB/c mice were immunized intraperitoneally with sp56FLAG in a fertility trial with 20 microg sp56FLAG in Freund's Complete Adjuvant and boosted three to five times with 20 microg sp56FLAG in Freund's Incomplete Adjuvant. Litter sizes of sp56FLAG-treated mice were significantly smaller than control-treated animals after five boosts.

Amino Acid Sequence↗
PubMed1999

Mammalian sperm-egg recognition: does fertilin beta have a major role to play?

The advent of simple in vitro fertilisation techniques has provided the reproductive biologist with an invaluable system for assaying sperm fertilising ability. In particular, they provide a useful way of identifying and characterising gamete-specific proteins that play a role in sperm-egg interactions, and in recent years, a growing number of sperm surface proteins have been identified that appear to be involved in these processes. Fertilin beta was one of the first sperm membrane proteins to be implicated in egg interactions and it has been proposed that this is mediated by means of binding of its disintegrin-like domain to cognate integrin receptors on the egg plasma membrane. A recent paper in Science by Cho and colleagues [Cho et al. 1998. Fertilisation defects in sperm from mice lacking fertilin beta. Science 281: 1857-1859 (Ref. 1)] provides preliminary data on a fertilin beta knockout mouse. Whilst fertilin beta null males had greatly reduced fertility, somewhat surprisingly, this could be largely attributed to causes other than impaired binding to the egg plasma membrane.

ADAM Proteins↗
PubMed2001

Differential extraction and enrichment of human sperm surface proteins in a proteome: identification of immunocontraceptive candidates.

The objective of this study was to discover previously unknown human sperm surface proteins that may be candidate contraceptive vaccinogens. To this end, methods of concentrating human sperm proteins for microsequencing by mass spectrometry were used, which increased the likelihood of identifying surface proteins. Vectorial labeling, differential extraction and two-dimensional (2-D) gel electrophoresis were employed to identify and isolate proteins accessible at the cell surface. Percoll harvested or swim-up sperm were either solubilized directly or solubilized after surface labeling with sulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin). Comparisons were made of proteins extracted with four lysis buffers: (i) Celis buffer containing 9.8 M urea and 2% Igepal CA-630; (ii) 1% Triton X (TX)-100; (iii) 1.7% TX-114 followed by phase partitioning; or (iv) 1 M NaCl. Blots of proteins separated by high-resolution 2-D electrophoresis were probed with avidin and antibodies to known proteins specific for three domains: the sperm surface (SAGA-1), the acrosome (SP-10), and the cytoskeleton (alpha-tubulin). Celis buffer (45 min) extracted proteins from all three major compartments. However, a 20-s extraction in Celis buffer enriched for several proteins and enabled the identification of several novel peptides by mass spectrometry. Mild extraction with TX-100 or 1 M NaCl solubilized mainly membrane and acrosomal proteins, but not cytoskeletal proteins. Comparison of biotinylated proteins extracted by each method showed that the major vectorially labeled proteins solubilized by Celis buffer were also solubilized by TX-100, TX-114, and 1 M NaCl. Extraction with TX-114 followed by phase-partitioning significantly enriched hydrophobic surface proteins and aided resolution and isolation. Eight protein spots microsequenced following all these extraction methods proved to be novel sperm molecules.

Acrosome↗
PubMed2001

Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein.

A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-(lambda)gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122-124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S-transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the approximately 17 kDa recombinant TSA-1, and a approximately 24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility.

Acrosome Reaction↗
PubMed1990

Molecular cloning of an acrosomal sperm antigen gene and the production of its recombinant protein for immunocontraceptive vaccine.

A monoclonal antibody, HS-63, which reacts specifically with a highly conserved sperm acrosome antigen, was shown to inhibit in vitro fertilization of mouse and human. The corresponding sperm antigen designated as MSA-63 was purified to homogeneity from mouse testes and used as an immunogen to generate polyclonal antisera in rabbits. The cDNA fragments of MSA-63 gene were cloned from mouse testis cDNA library by an immunoscreening method using polyclonal antisera specific for MSA-63. Using the established cDNA clone as a probe, the gene encoding for MSA-63 protein was found to be conserved among different mammalian species. Only one specific mRNA 1.5 kb in size was identified from the adult mouse testis among different mouse tissues. The recombinant fusion protein containing MSA-63 protein fragment was produced in Escherichia coli and used to immunize female mice. Similar to the original HS-63 monoclonal antibody, the antisera thus produced reacted only with the sperm acrosome and revealed significant inhibition to the in vitro fertilization of mouse oocytes. The results of this preliminary study suggest that it is feasible to mass produce sperm-specific antigens or their antigenic fragments by recombinant DNA technology for the development of sperm antigen-based immunocontraceptive vaccines.

Acrosome↗
PubMed1990

Antibodies to sperm surface fertilization antigen (FA-1): their specificities and site of interaction with sperm in male genital tract.

The fertilization antigen (FA-1) isolated from murine testes demonstrated its dimeric form of 49,000 +/- 2,000 molecular weight (M.W.) or a monomer of 23,000 M.W. on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The FA-1 was immunogenic in all three female rabbits tested and raised a high-titer antisera [enzyme-linked immunosorbent assay (ELISA) titers; 1:1,024 to 1:4,096]. The rabbit anti-FA-1 antisera predominantly recognized the dimeric form of 49,000 +/- 2,000 M.W. on the Western blot of lithium diiodosalicylate (LIS)-solubilized murine testes. None of the antisera reacted with any somatic tissue, indicating germ-cell specificity of FA-1. To determine the cellular localization of the immunoreactive FA-1, a novel ultrasensitive immunogold-silver staining (IGSS) procedure was developed. The anti-FA-1-IgG showed intense staining in the luminal region of the seminiferous tubules containing spermatids and spermatozoa. No reaction was observed in the peripheral area of the tubules containing Sertoli cells, spermatogonia, leptotene, and zygotene spermatocytes. The biodistribution studies of 125I-labeled anti-FA-1 IgG in mice revealed that the antibodies do not bind to somatic tissues such as blood cell, liver, heart, kidney, muscle, and gastrointestinal tissue and do not transudate into testes and seminal vesicle. However, the antibodies preferentially transudate into epididymis (especially corpus or cauda regions) and vas deferens to bind to sperm cells. In conclusion, our data indicate that FA-1 can induce an immune response that is germ cell-specific, directed against later stages of spermatogenesis. The antibodies to FA-1 interact with sperm after penetration through epididymis (especially corpus and cauda regions) and vas deferens rather than through testes and seminal vesicles.

Animals↗
PubMed1993

Evidence of new antigens in the mouse cumulus oophorus during preovulatory cumulus expansion.

The effects of an antiserum (anti-COC) against ovulated mouse cumulus-oocyte complexes (COC) on in vitro fertilization of mouse oocytes were studied. Preincubation of ovulated COC with various concentrations of anti-COC led to dose-dependent impairment of fertilization rates as well as to a decrease in the number of spermatozoa attached to the zona pellucida. Anti-COC was used to probe Western blots of cumulus proteins. These cumuli were obtained from 2 experimental groups of mice corresponding to 2 different maturational stages (preovulatory immature COC or preovulatory mature COC). Two antigens (70 and 80 kDa) present in cumulus intercellular matrix from mature COC were only found as traces in matrix from immature COC. In addition, the protein pattern of the cumulus intercellular matrix was different from that of cumulus cells, whatever the COC maturational stage. These results indicate the appearance of new proteins in the cumulus oophorus during preovulatory expansion and are consistent with the contraceptive action of anti-COC.

Animals↗
PubMed1993

Cloning and sequencing of baboon and cynomolgus monkey intra-acrosomal protein SP-10: homology with human SP-10 and a mouse sperm antigen (MSA-63).

In this study, cDNAs encoding the intraacrosomal protein SP-10 were cloned and sequenced from baboon (Papio papio) and macaque (Macaca fascicularis) testis libraries and the sequence compared to that of human SP-10. Two alternatively spliced SP-10 cDNAs were obtained from both baboon and macaque testis libraries. The two cDNAs in each species contained open reading frames encoding proteins of exactly 285 and 251 amino acids. A 98% homology between baboon and macaque SP-10 was found at the protein and DNA levels. An 85% and 89% homology between baboon and macaque SP-10 and human SP-10 was present at the protein and DNA level, respectively. A mouse intraacrosomal protein, MSA-63, considered to be an SP-10 homologue, exhibited an overall 53% homology to nonhuman primate SP-10 and a 60% homology to human SP-10 at the protein level. Polymerase chain reaction analysis of testis mRNA confirmed the existence of two alternatively spliced SP-10 mRNAs in both nonhuman primates. Primer extension analysis indicated a common major transcriptional start site in baboon, macaque, and human SP-10 67 nucleotides 5' to the ATG codon. The amino acid sequence data for nonhuman primate SP-10s suggest that antibodies generated by vaccinating baboons and macaques with human SP-10 will likely recognize nonhuman primate SP-10, supporting the testing of an SP-10 contraceptive vaccine based on human SP-10 in these nonhuman primate models.

Acrosome↗
PubMed2005

Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding.

Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction. Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries. In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT). Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E. coli in an ELISA. Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP. A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA). The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides. Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide. These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines.

Amino Acid Sequence↗
PubMed1986

Effects of anti-zona pellucida monoclonal antibodies on fertilization and early development.

The murine zona pellucida surrounds the growing oocyte, ovulated egg and dividing embryo. It is comprised of three sulfated glycoproteins designated ZP-1, ZP-2, and ZP-3 which have molecular weights of 185,000, 140,000 and 83,000 daltons respectively. We have isolated a series of cell lines that produce monoclonal antibodies to ZP-2 and to ZP-3. These immunogical probes indicate that the extracellular matrix proteins of the zona pellucida are found uniquely in the ovary where they surround maturing oocytes. We have demonstrated that passive immunization with antibodies specific either for ZP-2 or ZP-3 inhibit in vivo and in vitro fertilization. This effect is observed with ng/ml quantities of antibody. It appears that the antibodies do not preclude sperm binding but rather prevent sperm penetration of the zona by steric hinderance. Although long-term, the contraceptive effect is fully reversible and this reversibility is associated with loss of antibody from the zona pellucida surrounding intra-ovarian oocytes. Antibodies to ZP-2 or ZP-3 had no other adverse effect on in vivo or in vitro preimplantation development.

Animals↗