Alan B. Diekman

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 (α‐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.

SLLP1, A Unique, Intra-acrosomal, Non-bacteriolytic, c Lysozyme-Like Protein of Human Spermatozoa

Abstract We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by β-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P ≤ 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, ∼1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.

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

Abstract. 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.

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.

Spermatid-Specific Expression of the Novel X-Linked Gene Product SPAN-X Localized to the Nucleus of Human Spermatozoa

Abstract 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.

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 mAbs 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, mAbs 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.—Diekman, A. B., Norton, E. J., Klotz, K. L., Westbrook, V. A., Shibahara, H., Naaby‐Hansen, S., Flickinger, C. J., Herr, J. C. N‐linked glycan of a sperm CD52 glycoform associated with human infertility. FASEB J. 13, 1303–1313 (1999)

Genomic organization, incidence, and localization of the SPAN-x family of cancer-testis antigens in melanoma tumors and cell lines.

Purpose: Members of the SPAN-X (sperm protein associated with the nucleus mapped to the X chromosome) family of cancer-testis antigens are promising targets for tumor immunotherapy because they are normally expressed exclusively during spermiogenesis on the adluminal side of the blood-testis barrier, an immune privileged compartment. Experimental Design and Results: This study analyzed the human SPANX genomic organization, as well as SPAN-X mRNA and protein expression in somatic and cancer cells. The SPANX family consists of five genes, one of which is duplicated, all located in a gene cluster at Xq27.1. From the centromere, the arrangement of the five SPANX genes mapped on one contiguous sequence is SPANXB, -C, -A1, -A2, and -D. Reverse transcription-PCR analyses demonstrated expression of SPAN-X mRNA in melanoma and ovarian cell lines, and virtual Northern analysis established SPANX gene expression in numerous cancer cell lines. Immunoblot analysis using polyclonal antisera raised against recombinant SPAN-X confirmed the translation of SPAN-X proteins in melanoma and ovarian tumor cell lines. The immunoreactive proteins migrated between Mr 15,000 and Mr 20,000 similar to those observed in spermatozoa. Immunoperoxidase labeling of melanoma cells and tissue sections demonstrated SPAN-X protein localization in the nucleus, cytoplasm, or both. Ultrastructurally, in melanoma cells with nuclear SPAN-X, the protein was associated with the nuclear envelope, a localization similar to that observed in human spermatids and spermatozoa. Significantly, the incidence of SPAN-X-positive immunostaining was greatest in the more aggressive skin tumors, particularly in distant, nonlymphatic metastatic melanomas. Conclusions: The data herein suggest that the SPAN-X protein may be a useful target in cancer immunotherapy.

Differential Nuclear Localization of the Cancer/Testis-Associated Protein, SPAN-X/CTp11, in Transfected Cells and in 50% of Human Spermatozoa

Abstract 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.

Identification of calcium-binding proteins associated with the human sperm plasma membrane

BackgroundThe precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.MethodsSurface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface.ResultsNine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm.ConclusionThe presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.