Proteomic identification of galectin-3 binding ligands and characterization of galectin-3 proteolytic cleavage in human prostasomes.

Galectin-3 is a multifunctional carbohydrate-binding protein that was previously characterized as a proteolytic substrate for prostate-specific antigen (PSA) and was shown to be associated with prostasomes in human semen. Prostasomes are exosome-like vesicles that are secreted by the prostatic epithelium and have multiple proposed functions in normal reproduction and prostate cancer. In the current study, galectin-3 binding ligands in human prostasomes were identified and characterized with the goal to investigate galectin-3 function in prostasomes. Galectin-3 binding proteins were isolated by affinity column chromatography. Candidate ligands identified by MS/MS were PSA, prostatic acid phosphatase (PAP), zinc alpha-2-glycoprotein (ZAG), dipeptidyl peptidase-4 (CD26), aminopeptidase N (CD13), neprilysin, clusterin, antibacterial protein (FALL-39) and alpha-1-acid glycoprotein (ORM1). Biochemical methods were used to characterize the ability of galectin-3 to bind to selected ligands, and galectin-3 cleavage assays were utilized to investigate the protease(s) in prostasomes that cleaves galectin-3. CD26, CD13, PSA, PAP and ZAG immunoreactivity were detected in extracts of purified prostasomes. One-dimensional electroblot analysis of prostasomes demonstrated that CD26, PAP and CD13 immunoreactivity co-migrated with galectin-3-reactive protein bands. PSA and ZAG were found to be associated with the surface of prostasomes. Both intact and cleaved galectin-3 were detected in prostate and prostasome extracts. Cleavage and inhibition assays indicated that PSA in prostasomes proteolytically cleaves galectin-3. The identification of these glycoproteins as galectin-3 ligands lays the groundwork for future studies of galectin-3 and prostasome function in reproduction and prostate cancer.

Hominoid-specific SPANXA/D genes demonstrate differential expression in individuals and protein localization to a distinct nuclear envelope domain during spermatid morphogenesis.

Human sperm protein associated with the nucleus on the X chromosome consists of a five-member gene family (SPANXA1, SPANXA2, SPANXB, SPANXC and SPANXD) clustered at Xq27.1. Evolved from an ancestral SPANX-N gene family (at Xq27 and Xp11) present in all primates as well as in rats and mice, the SPANXA/D family is present only in humans, bonobos, chimpanzees and gorillas. Among hominoid-specific genes, the SPANXA/D gene family is considered to be undergoing rapid positive selection in its coding region. In this study, RT-PCR of human testis mRNA from individuals showed that, although all SPANXA/D genes are expressed in humans, differences are evident. In particular, SPANXC is expressed only in a subset of men. The SPANXa/d protein localized to the nuclear envelope of round, condensing and elongating spermatids, specifically to regions that do not underlie the developing acrosome. During spermiogenesis, the SPANXa/d-positive domain migrated into the base of the head as the redundant nuclear envelope that protrudes into the residual cytoplasm. Post-testicular modification of the SPANXa/d proteins was noted, as were PEST (proline, glutamic acid, serine, and threonine rich regions) domains. It is concluded that the duplication of the SPANX-N gene family that occurred 6-11 MYA resulted in a new gene family, SPANXA/D, that plays a role during spermiogenesis. The SPANXa/d gene products are among the few examples of X-linked nuclear proteins expressed following meiosis. Their localization to non-acrosomal domains of the nuclear envelope adjacent to regions of euchromatin and their redistribution to the redundant nuclear envelope during spermiogenesis provide a biomarker for the redundant nuclear envelope of spermatids and spermatozoa.

Glycoconjugates in sperm function and gamete interactions: how much sugar does it take to sweet-talk the egg?

Glycoconjugates in the mammalian reproductive tract are critical components of the molecular mechanisms that control sperm maturation, sperm transport and gamete interactions. In the oviduct of many species, sperm transport and maturation are regulated by protein-carbohydrate interactions that form a sperm reservoir. Subsequently, gamete interactions are mediated by the binding of lectin-like sperm proteins with carbohydrate moieties on the zona pellucida. The sperm glycocalyx is extensively modified during sperm transport and maturation. Multiple functions have been proposed for this dense carbohydrate layer overlying the sperm plasmalemma, and sperm-surface carbohydrates have been implicated in immune-mediated human infertility. The structure and function of glycoconjugates in the oviductal sperm reservoir, the zona pellucida, and on the sperm surface are reviewed.

A male genital tract-specific carbohydrate epitope on human CD52: implications for immunocontraception.

The identification of unique sperm surface epitopes that are not expressed or exposed in the female reproductive tract is a key element in the development of antibody-based contraceptives. Western blotting and immunohistochemistry were performed to define the tissue distribution of the S19 epitope, which has been proposed as a target for immunocontraception. S19 is an IgG1 murine monoclonal antibody (mAb) directed to an N-linked carbohydrate epitope on a 15-25 kDa glycoprotein, sperm agglutination antigen-1 (SAGA-1), containing a peptide core identical to that of the lymphocytic surface protein CD52. In this study, the S19 epitope was shown to be absent from human lymphocytes, demonstrating a distinction between this epitope and the CAMPATH epitope that is recognized by an antibody against the terminal tripeptide and GPI-anchor of CD52. Further tissue specificity analysis identified the S19 epitope in the epithelium of the human epididymis and vas deferens, as well as on both epididymal and ejaculated spermatozoa. In contrast, the S19 epitope was absent in the five human female reproductive tract and 18 other somatic tissues tested. These results support the use of the S19 epitope as a contraceptive immunogen and the suitability of the S19 mAb as an intravaginal contraceptive. To test the agglutinating activity of the S19 mAb in a formulation designed for vaginal use, S19 mAb were bound to the surface of Novasomes, a multilamellar liposome delivery vehicle. S19-Novasome formulations agglutinated human spermatozoa and were as effective as unbound S19 mAb, demonstrating the feasibility of spermistatic contraceptives targeted to the male reproductive tract specific carbohydrate epitope.

Novel signaling pathways involved in sperm acquisition of fertilizing capacity.

Capacitation is a complex series of molecular events that occurs in sperm after epididymal maturation and confers on sperm the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of oviductal fluid. In most cases, capacitation media contain energy substrates, such as pyruvate, lactate and glucose, a cholesterol acceptor (usually serum albumin), NaHCO(3), Ca(2+), low K(+), and physiological Na(+) concentrations. The mechanism of action by which these compounds promote capacitation is poorly understood at the molecular level; however, some molecular events significant to the initiation of capacitation have been identified. For example, capacitation correlates with cholesterol efflux from the sperm plasma membrane, increased membrane fluidity, modulations in intracellular ion concentrations, hyperpolarization of the sperm plasma membrane and increased protein tyrosine phosphorylation. These molecular events are required for the subsequent induction of hyperactivation and the acrosome reaction. This review discusses the recent progress that has been made in elucidating mechanisms which regulate sperm capacitation.

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.

RASA, a recombinant single-chain variable fragment (scFv) antibody directed against the human sperm surface: implications for novel contraceptives.

BACKGROUND: A recombinant single-chain variable fragment (scFv) antibody was engineered to a tissue-specific carbohydrate epitope located on human sperm agglutination antigen-1 (SAGA-1), a sperm glycoform of CD52. METHODS AND RESULTS: cDNAs encoding the variable regions of the S19 [IgG(1)kappa] monoclonal antibody (mAb) were identified, linked, and cloned into the pCANTAB 5E vector. The recombinant anti-sperm antibody (RASA) was expressed in E. coli HB2151 cells as a 29 kDa monomer and, remarkably, also formed multimers of approximately 60 and 90 kDa. RASA reacted with the endogenous SAGA-1 antigen by Western blot analysis, labelled the entire human sperm surface by indirect immunofluorescence, and aggregated human spermatozoa in a tangled (head-to-head, head-to-tail, tail-to-tail) pattern of agglutination, as was also observed with the native S19 mAb. CONCLUSIONS: These results demonstrate that active recombinant antibodies can be produced to a tissue-specific carbohydrate epitope on the human sperm surface, thereby opening opportunities for novel contraceptive agents.

Differential nuclear localization of the cancer/testis-associated protein, SPAN-X/CTp11, in transfected cells and in 50% of human spermatozoa.

Cancer-testis antigens (CTAs) represent potential targets for cancer immunotherapy because these proteins are widely distributed in tumors but not in normal tissues, except testes. In this paper, we identify homology of the CTA CTp11 with SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome). On two-dimensional Western blots of human sperm extracts, SPAN-X antibodies recognized 19 spots ranging from 20 to 23 kDa with isoelectric points from 5.0 to 5.5. Differential extraction of spermatozoa demonstrated that the SPAN-X protein is highly insoluble. Only 50% of ejaculated spermatozoa exhibited SPAN-X immunofluorescent staining. Dual localization of the sex chromosomes and the SPAN-X protein demonstrated that an equal number of X- and Y-bearing spermatozoa exhibited SPAN-X staining. In transfected mammalian CV1 cells, the SPAN-Xa and SPAN-Xb proteins were localized to the nucleus and cytoplasm, respectively, by indirect immunofluorescence. On immunoblots of CV1 cells, the SPAN-Xa protein migrated at 15-20 kDa, whereas the SPAN-Xb protein migrated at a higher molecular weight of 21-22 kDa. The SPAN-X protein was ultrastructurally associated with nuclear vacuoles and the redundant nuclear envelope. SPAN-X is the first protein specifically localized to these poorly characterized structures of the mammalian sperm nucleus and provides a unique biochemical marker for investigation of their function in spermatozoa as well as the role of SPAN-X/CTp11 in human tumors.

Spermatid-specific expression of the novel X-linked gene product SPAN-X localized to the nucleus of human spermatozoa.

Formation of mature spermatozoa involves a series of dramatic molecular and morphological changes in the male germ cell lineage. These changes result from the temporally regulated transcription and translation of several testis-specific gene products. Here, we describe a novel, testis-specific protein designated SPAN-X for sperm protein associated with the nucleus on the X chromosome. SPAN-X sequences showed no significant similarity with known cDNA or peptide sequences. The SPAN-X peptide sequences contained three overlapping consensus nuclear localization signals, a high percentage (33%-37%) of charged amino acid residues, and a relatively acidic isoelectric point (pI; 4.88-6.05). Northern analysis of mRNA from multiple human tissues identified a SPAN-X transcript exclusively in the testis. In situ hybridization of human testes sections showed SPAN-X mRNA expression in haploid, round, and elongating spermatids. The SPANX gene was mapped to chromosome Xq27. 1 by fluorescence in situ hybridization and by Southern blot analysis of human/mouse somatic cell hybrids. On Western blots of human sperm proteins, antirecombinant SPAN-X antibodies reacted with broad bands migrating between 15-20 kDa. Immunofluorescent labeling of human spermatozoa demonstrated SPAN-X localization to nuclear craters and cytoplasmic droplets. Expression of SPAN-X, an X-linked gene product, exclusively in haploid spermatids leads to interesting questions regarding the transcription of sex-linked genes during spermiogenesis.

Anti-sperm antibodies from infertile patients and their cognate sperm antigens: a review. Identity between SAGA-1, the H6-3C4 antigen, and CD52.

PROBLEM: The correlation of anti-sperm antibodies (ASA) with some instances of unexplained infertility implicates a role for these antibodies in blocking fertilization. Improved diagnosis and treatment of immunologic infertility, as well as a more complete understanding of the mechanism behind this phenomenon, are dependent on the identification and characterization of relevant sperm antigens. METHOD OF STUDY: In this article, we review literature on methods employed to identify sperm antigens using anti-sperm polyclonal and monoclonal antibodies from infertile patients and vasectomized men. Particular focus is given to approaches using human and mouse monoclonal antibodies to define the SAGA-1 human sperm antigen. RESULTS: ASA present in sera and genital tract secretions from infertile patients and vasectomized men have been employed in a variety of methods to identify sperm antigens. In an alternate approach, a monoclonal antibody (mAb), H6-3C4, was immortalized from the lymphocytes of an infertile woman who exhibited sperm-immobilizing titers. Subsequently, the sperm-agglutinating, murine S19 mAb was shown to react with the H6-3C4 cognate antigen. The H6-3C4 S19 cognate antigen, designated Sperm Agglutination Antigen-1 (SAGA-1), was characterized as a polymorphic, highly acidic, GPI-anchored glycoprotein on the surface of human spermatozoa. Purification with the S19 mAb followed by microsequencing demonstrated that the SAGA-1 core peptide is identical to CD52, a glycoprotein on the surface of human lymphocytes. Immunoblot analysis demonstrated that these two glycoproteins differed in carbohydrate composition. Thus, sperm SAGA-1 and lymphocyte CD52 represent glycoforms, glycoproteins with the same core peptide but with different carbohydrate structures. CONCLUSIONS: Autoimmunity to the SAGA-1 and/or CD52 glycoforms may lead to infertility. Structural and immunologic differences between these glycoproteins may be important factors in the etiology of immunologic infertility and other autoimmune disorders.

Evidence for a unique N-linked glycan associated with human infertility on sperm CD52: a candidate contraceptive vaccinogen.

A major objective in developing a sperm antigen-based contraceptive vaccine for humans is the discovery of sperm surface immunogens that are functionally relevant and sperm specific. The latter criterion is deemed essential to avoid the possibility of inducing autoimmune disease upon vaccination. This review presents evidence that a unique carbohydrate epitope is synthesized in the human epididymis, is attached to the core peptide of CD52, a lymphocyte differentiation marker, and is subsequently inserted into the sperm membrane via a glycosylphosphatidylinositol anchor. This unique CD52 glycoform is localized to the entire sperm surface, functions as a potent target for agglutinating and cytotoxic antibodies, and is one of the few well-defined sperm surface glycoproteins indicated in human antibody-mediated infertility.

N-linked glycan of a sperm CD52 glycoform associated with human infertility.

In a benchmark study, Isojima and colleagues established H6-3C4, the first successful heterohybridoma immortalized from the peripheral blood lymphocytes of an infertile woman who exhibited high sperm-immobilizing antibody titers. The present report demonstrates the identity between the glycoprotein antigens recognized by the human H6-3C4 monoclonal antibody (mAb) and the murine S19 mAb, generated in our laboratory to sperm agglutination antigen-1 (SAGA-1). Both mAb's recognize N-linked carbohydrate epitopes on the 15-25 kDa, polymorphic SAGA-1 glycoprotein that is localized to all domains of the human sperm surface. Treatment with phosphatidylinositol-specific phospholipase C demonstrated that SAGA-1 is anchored in the sperm plasmalemma via a GPI-lipid linkage. Immunoaffinity purification and microsequencing indicated that the core peptide of the SAGA-1 glycoprotein is identical to the sequence of CD52, a GPI-anchored lymphocyte differentiation marker implicated in signal transduction. Comparison of anti-SAGA-1 and anti-CD52 immunoreactivities revealed that the sperm form of CD52 exhibits N-linked glycan epitopes, including the epitope recognized by the infertility-associated H6-3C4 mAb, which are not detected on lymphocyte CD52. Thus, the two populations of the CD52 glycoprotein on lymphocytes and spermatozoa represent glycoforms, glycoprotein isoforms with the same core amino acid sequence but different carbohydrate structures. Furthermore, mAb's to the unique carbohydrate epitopes on sperm CD52 have multiple inhibitory effects on sperm function, including a cytotoxic effect on spermatozoa in the presence of complement. These results are the first to implicate unique carbohydrate moieties of a sperm CD52 glycoform as target epitopes in the anti-sperm immune response of an infertile woman. Furthermore, localization of CD52 on all domains of the sperm surface coupled with the multiple sperm-inhibitory effects of antibodies to its unique carbohydrate moieties suggest opportunities for immunocontraceptive development.

Expression of the human antigen SPAG2 in the testis and localization to the outer dense fibers in spermatozoa.

ABSTRACT Antisperm antibodies (ASAs) have been implicated in some instances of infertility. To characterize sperm antigens relevant to immunologic and immunocontraceptive development, SPAG2 (sperm-associated antigen 2) was identified by screening a human testis cDNA library with human sera positive for ASAs. Subsequently, two isoforms, SPAG2-1 and SPAG2-2, were identified in testis and placenta libraries, respectively. In the current study, Southern analysis of human genomic DNA with a probe common to the two SPAG2 isoforms indicated a single SPAG2 gene; therefore, alternative splicing is a likely mechanism for production of variant mRNAs. In situ hybridization of human testis sections demonstrated the expression of SPAG2 in primary spermatocytes, with decreased or arrested expression in postmeiotic cells. Immunofluorescence of Triton X-100-extracted spermatozoa with an anti-SPAG2 peptide antiserum indicate that SPAG2 is an intracellular component of the sperm flagellum. Electron microscopy refined this localization to the outer dense fibers (ODFs), structural filaments associated with the mammalian sperm axoneme. The ODFs have been reported to be composed of keratin-like intermediate filament proteins. However, SPAG2 does not exhibit the molecular characteristics of such proteins, nor does SPAG2 demonstrate sequence homology with previously characterized ODF proteins. Therefore, SPAG2 represents a novel protein of human sperm ODFs. Characterization of SPAG2 will further our understanding of ODF function in normal sperm motility and of flagellar abnormalities that lead to male infertility.

Biochemical characterization of sperm agglutination antigen-1, a human sperm surface antigen implicated in gamete interactions.

The anti-sperm monoclonal antibody (mAb) S19 was previously demonstrated to agglutinate human spermatozoa, inhibit sperm penetration of cervical mucus, and inhibit sperm-zona pellucida binding. These results implicated the cognate S19 antigen, designated sperm agglutination antigen-1 (SAGA-1), in gamete interactions and identified SAGA-1 as an attractive candidate for immunocontraceptive development. In the present study, evaluation of sperm agglutination with video microscopy showed that the S19 mAb rapidly and completely agglutinated human spermatozoa in a "tangled" pattern of agglutination. One- and two-dimensional immunoblot analyses identified SAGA-1 as a highly acidic, polymorphic sperm protein with an apparent molecular mass of 15-25 kDa and an isoelectric point of 2.5-3.0. Periodate treatment abolished this immunoreactivity, demonstrating that the S19 mAb reacted with a carbohydrate epitope and indicating that SAGA-1 is a glycoprotein. Absence of S19 immunoreactivity in postvasectomy seminal fluid implicated the testis, epididymis, and/or proximal vas deferens in the expression of SAGA-1. In solubility and phase partitioning assays, SAGA-1 was extracted from spermatozoa in Triton X-114 and exhibited the hydrophobic characteristics of integral and glycosylphosphatidyl inositol-anchored membrane proteins. These results identify SAGA-1 as a hydrophobic, highly acidic sperm glycoprotein that is localized on the entire sperm surface and has potential significance as a target for antibodies that inhibit sperm function and gamete interactions.

Sperm antigens and their use in the development of an immunocontraceptive.

PROBLEM: Overpopulation is a global problem of significant magnitude, with grave implications for the future. World population is predicted to reach 10 billion people by the year 2050, an increase of more than 75% over the current population. Development of new contraceptives is necessary, since current forms of birth control are unavailable to many individuals due to sociological, financial, or educational limitations. METHODS: In an effort to identify new contraceptive alternatives, sperm-specific antigens are under the investigation as the basis for immunological regulation of fertility through contraceptive immunization. Anti-sperm antibodies (ASA) inhibit sperm function in vitro, induce infertility in experimental models, and have been implicated in some cases of clinical infertility. Furthermore, the development of ASA in vasectomized men does not lead to physiological complications despite the persistence of these antibodies for years; thus, ASA induced by immunization of men and women may be similarly harmless. Immunization has several conceptual advantages as a method of fertility regulation. Employment of non-pharmacologically active agents, convenience of administration, low cost, and the potential for relatively long-lasting yet reversible effects. RESULTS: Various approaches have been taken to identify candidate sperm antigens for immunocontraceptive development. Studies that utilized monoclonal antibodies and polyclonal antisera generated against sperm preparations have identified such promising candidates as PH-20, fertilin, SP-17, and SP-10. In animal model, 100% contraceptive effects were induced following PH-20 immunization and the effects were reversible. More recently, ASA from the sera of infertile patients were employed to identify several candidates as expressed by testis cDNA libraries. Perhaps the most extensively characterized and effective immunogen candidate identified thus far is lactate dehydrogenase-C4 (LDH-C4). Active immunization with LDH-C4 suppressed fertility in a variety of mammalian species, including primates, and the reversibility of these effects was demonstrated. CONCLUSION: The successful results obtained thus far support the feasibility of an effective immunocontraceptive and indicate the importance for continued investigation of additional sperm antigens as contraceptive immunogen candidates.

Characterization of a human antigen with sera from infertile patients.

We report the cDNA cloning and subsequent characterization of a novel antigen implicated in antibody-mediated human infertility. This antigen, designated AgX (unknown antigen), was identified originally by screening a human testis lambda gt11 cDNA expression library with infertile patients' sera known to contain anti-sperm antibodies. AgX cDNAs isolated from testis and placenta cDNA libraries (AgX-1 and AgX-2, respectively) differed by a 48-bp deletion in the open-reading frame (ORF). The AgX-1 and AgX-2 ORFs encoded putative peptide chains of 505 and 521 amino acids (approximately 55.5 and approximately 57.3 kDa), respectively. The AgX amino acid sequences contained consensus motifs indicative of NTP binding. However, computer homology searches did not identify any significant similarity with known sequences. Quantitative analysis using the reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that the AgX-1 mRNA was fiftyfold more abundant than AgX-2 in the testis, while AgX-2 was more abundant than AgX-1 in somatic tissues. An anti-AgX peptide antiserum identified two AgX isoforms on Western blots of human tissue extracts. An abundant 56-kDa isoform was detected only in testis and sperm. These data suggest that the 56- and 58-kDa isoforms are AgX-1 and AgX-2, respectively. AgX was localized by immunofluorescence to the principal piece of the sperm tail. Therefore, antibodies against an AgX isoform may reduce fertility by affecting sperm function.

Aminotransferase and the production of alanine during hyperosmotic stress in Paramecium calkinsi.

When Paramecium calkinsi encounter hyperosmotic stress, intracellular free alanine increases. In vivo assays indicate that the reaction catalyzed by alanine aminotransferase contributes to the build up of alanine in response to hyperosmotic shock. 14C-pyruvate is converted to 14C-alanine in cells grown axenically at 200 mosm. When shifted to 600 mosm, the rate of conversion of pyruvate to alanine increases, and conversion at either 200 or 600 mosm is blocked by 1 mM aminooxyacetic acid (AOA), an inhibitor of aminotransferase. Intracellular free alanine increase is partially inhibited by AOA, and AOA prevents cells living in fresh water from acclimating to higher salinities, an indication that the increase in intracellular alanine is physiologically significant.