Gary F. Clark

Gender-specific Glycosylation of Human Glycodelin Affects Its Contraceptive Activity

We have recently demonstrated that a human amniotic fluid-derived glycoprotein, glycodelin-A (GdA; previously known as PP14 or PAEP), potently inhibits gamete binding in an established sperm-egg binding system and expresses immunosuppressive activities directed against a variety of different immune cell types. GdA has high mannose-, hybrid-, and complex-type biantennary oligosaccharides including structures with fucosylated or sialylated N,N′-diacetyllactosediamine (GalNAcβ1-4GlcNAc) sequences, which are rare in other human glycoproteins. We now report the characterization of glycodelin-S (GdS). This is a human seminal plasma glycoprotein that is immunologically indistinguishable from GdA, but unlike the latter, does not inhibit human sperm-zona pellucida binding under hemizona assay conditions. Analysis of the N-glycans of GdS by mass spectrometry revealed that all glycoforms of GdS are different from those of GdA. GdS glycans are unusually fucose-rich, and the major complex-type structures are biantennary glycans with Lewisx (Galβ1-4(Fucα1-3)GlcNAc) and Lewisy (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) antennae. It is probable that these highly fucosylated epitopes contribute to the immunosuppressive activity of human seminal plasma and to the low immunogenicity of sperm. This study provides the first evidence for gender-specific glycosylation that may serve to regulate key processes involved in human reproduction.

Nature of the inhibitory effect of complex saccharide moieties on the tight binding of human spermatozoa to the human zona pellucida

Fucoidin and heparin sulfate inhibit binding of human sperm to the human zona pellucida under hemizona assay (HZA) conditions. Here we used the HZA to further assess tight sperm binding with/without preincubation of the sperm with other sulfated and nonsulfated glycoconjugates and charged polymers. Fucoidin significantly inhibited binding compared with controls (greater than 75% inhibition), even if sperm were washed after preincubation with the saccharide. Dextran sulfate also produced significant inhibition, although to a lesser extent (54% inhibition). Chondroitin sulfates A and B, heparin, and dextran did not affect binding. Sodium sulfate and polyglutamic acid did not affect HZA results; polyphosphates produced only moderate inhibition. The potent inhibitory effect of the sulfated carbohydrates fucoidin and dextran is probably competitive (receptor-ligand type) in nature. However, the lack of significant effects of simple charged molecules (nonspecific effects) suggests that the degree of sulfation (charge) may not be crucial to its inhibitory action.

Fucoidin inhibits the zona pellucida-induced acrosome reaction in human spermatozoa

We recently reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits tight binding of human sperm to the human zona pellucida in vitro and that several oligosaccharides obtained after acid hydrolysis possess sperm-zona pellucida binding inhibitory activity equal to the original fucoidin. This inhibition may be specific to sperm-zona interactions or may be the consequence of the interruption of capacitation, a series of biochemical and physiological events leading to final sperm maturation, that must occur for successful fertilization. Completion of capacitation is most often determined by assessing two end-points of the process: acquisition of hyperactivated motility and ability to complete the acrosome reaction. Here, we examined the effects of fucoidin on these two end-points of capacitation in vitro. Fucoidin did not affect the proportion of sperm with hyperstimulated motility. Neither did fucoidin cause an increase in sperm that had spontaneously acrosome-reacted at 4.5 hours compared to controls as evaluated by indirect immunofluorescence using the acrosomal marker, monoclonal antibody, T-6. Comparable percentages of sperm had completed the acrosome reaction when exogenously stimulated by calcium ionophore A23187 with and without the addition of fucoidin. However, in the presence of fucoidin, stimulation of the acrosome reaction by acid solubilized human zonae pellucidae was significantly inhibited. These data indicate that fucoidin does not impede the normal progression of capacitation. These results provide strong evidence to support the hypothesis is that the inhibitory effect of fucoidin is at the level of the sperm membrane since inhibition can be bypassed by increasing intracellular calcium directly with a calcium ionophore.

Glycodelins as regulators of early events of reproduction

Markku Seppä lä*, Hannu Koistinen*, Riitta Koistinen*, Anne Dell†, Howard R. Morris†, Sergio Oehninger‡ and Gary F. Clark‡ *Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, FIN-00290 Helsinki, Finland; †Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK; ‡Departments of Obstetrics and Gynecology, and Biochemistry, Eastern Virginia Medical School, Norfolk, Va, USA

Involvement of selectin-like carbohydrate binding specificity in human gamete interaction

Summary The recognition of carbohydrate epitopes by complimentary protein receptors has been shown to be a critical factor in gamete interaction in many different animal species. In this study it was hypothesized that, in the human, gamete binding requires an interaction between selectin ligands on the zona pellucida and putative egg binding proteins on the sperm surface. The hemizona assay (a unique internally controlled bioassay that evaluates tight binding of sperm to the zona) and advanced methods of carbohydrate analysis were used to test this hypothesis. From these tests it was shown that oligosaccharide recognition is also required for initial human gamete binding. This study suggests the existence of distinct egg binding proteins on human sperm that can bind to selectin ligands. Additionally, the results suggest a possible convergence in the types of carbohydrate sequences recognized during initial human gamete binding and immune/inflammatory cell interactions. Glycoconjugates that manifest selectin‐ligand activity and that express specific carbohydrate epitopes have potent contraceptive and immunosuppressive effects. Such specific oligosaccharide sequences may provide an appropriate recognition signal for embryo development and protection.

The Species Recognition System: A New Corollary for the Human Fetoembryonic Defense System Hypothesis

We have previously suggested that the human fetus is protected during human development by a system of both soluble and cell surface associated glycoconjugates that utilize their carbohydrate sequences as functional groups to enable them to evoke tolerance. The proposed model has been referred to as the human fetoembryonic defense system hypothesis (hu-FEDS). In this paradigm, it has previously been proposed that similar oligosaccharides are used to mediate crucial recognition events required during both human sperm-egg binding and immune-inflammatory cell interactions. This vertical integration suggested to us that the sperm-egg binding itself is related to universal recognition events that occur between immune and inflammatory cells, except that in this case recognition of ‘species’ rather than recognition of ‘self’ is being manifested. In this paper, we have designated this component of hu-FEDS as the species recognition system (SRS). We propose that the SRS is an integral component of the hu-FEDS used to enable sperm-egg recognition and protection of the gametes from potential immune responses. Recent structural data indicates that the glycan sequences implicated in mediating murine gamete recognition are also expressed on CD45 in activated murine T lymphocytes and cytotoxic T lymphocytes. This overlap supports our contention that there is an overlap between the immune and gamete recognition systems. Therefore the hu-FEDS paradigm may be a subset of a larger model that also applies to other placental mammals. We therefore propose that the hu-FEDS model for protection should in the future be referred to as the eutherian fetoembryonic defense system hypothesis (eu-FEDS) to account for this extension. The possibility exists that the SRS component of eu-FEDS could predate eutherians and extend to all sexually reproducing organisms. Future investigation of the interactions between the immune and gamete recognition system will be required to determine the degree of overlap.

Fucoidin binding activity and its localization on human spermatozoa

We previously reported that fucoidin (a polymer of predominantly sulfated L-fucose) significantly inhibits: (1) tight binding of human sperm to human zona pellucida in vitro and (2) stimulation of the acrosome reaction by acid solubilized human zona pellucida. Here, we determined fucoidin binding activity on human spermatozoa and its localization on both live and permeabilized human sperm populations. A typical binding curve was demonstrated with biotinylated fucoidin. In competitive inhibition assays with unlabelled fucoidin or human sperm membrane extracts, IC50s were 4.0 micrograms/ml and 31.4 micrograms/ml, respectively. Fucoidin binding was localized over the acrosomal region of methanol-fixed human sperm and this pattern of binding significantly decreased from 92 +/- 3% to 74 +/- 6% with calcium ionophore A23187 treatment (p < 0.01). Binding of fucoidin-coated beads to live (non-permeabilized) human sperm was less than 1%. Addition of the detergent, Triton-X, to permeabilize sperm membranes resulted in a significant increase in binding (p = 0.001). These results provide evidence for the presence of a fucoidin binding compound in human spermatozoa that is localized to the membranes of the acrosomal region and can be extracted by a mild detergent extraction. Absence of binding by fucoidin to intact but not permeabilized spermatozoa suggests that the heteropolysaccharide binds to a receptor within the acrosomal matrix. However, further investigation is warranted to determine whether a fucoidin binding site is present both at the sperms surface for the initial contact with the zona pellucida, and also for secondary binding after exposure of the acrosomal membranes.

The Eutherian Fetoembryonic Defense System Hypothesis: An Update

All sexually reproducing organisms produce gametes that must be protected from immune challenge. Recent data indicates that the majority of the carbohydrate sequences that coat the murine zona pellucida are also upregulated on activated lymphocytes, and some participate in gamete binding. This overlap indicates that there may be a “species recognition system” (SRS) that is employed to identify both immune cells and gametes in the context of “species” rather than “self”. In eutherians, histoincompatible progeny must also be protected from the maternal immune response. The composite data indicates that several glycoconjugates produced in the pregnant can modify immune responses in vitro. We have previously referred to these collective factors as the “eutherian fetoembryonic defense system” or eu-FEDS. Based on the available results, it is very likely that these glycoconjugates are utilizing specific carbohydrate sequences as functional groups to mediate these activities. Many different persistent pathogens and tumor cells also either mimic or acquire these carbohydrate functional groups, indicating that they may be able to evoke similar effects. We outline new data that clearly implicate glycobiological subterfuge in the pathological effects associated with infection with lentiviruses like HIV-1 and simian immunodeficiency virus (SIV). In addition, recent data showing that the uterine mucin CA125 and the major surface glycoprotein of HIV-1 (gpl20) express the same N-glycans suggests a linkage to the pregnant uterus. Complete integration of this protective effect associated with eu-FEDS could also explain why the natural hosts of SIV (sooty mangabees and African green monkeys) are able to completely accommodate this virus without pathological effects. In summary, the requirements for accommodation of the germ cells and different stages of developing progeny could provide a very substantial “Achilles heel” for exploitation by pathogens and tumorcells. These more recent finding should provide a great impetus to further investigate the eu-FEDS hypothesis and its strong linkages to pathogenesis.