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Successful pregnancy depends largely on adequate placentation and maternal tolerance of the fetus. Glycodelin-A is a glycoprotein abundant in the decidua during early pregnancy. It plays an important role in placental development and fetomaternal defense. Glycodelin-A interacts by its unique carbohydrate side chains with the cell surface of various cell types in the human fetomaternal interface, particularly the trophoblasts and the immune cells, and modulates their functions and differentiation to permit successful pregnancy. Abnormal levels of glycodelin-A in the endometrium, uterine flushings, and/or maternal serum correlate with unexplained infertility, early pregnancy loss, and recurrent miscarriage. This review integrates recent studies on the role of glycodelin-A in placental development and fetomaternal tolerance in early pregnancy., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To study the effect of human chorionic gonadotropin (hCG) on olfactomedin-1 (Olfm1) expression and spheroid attachment in human fallopian tube epithelial cells in vitro., Design: Experimental study., Setting: Reproductive biology laboratory., Patient(s): Healthy nonpregnant women., Intervention(s): No patient interventions., Main Outcome Measure(s): Luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and Olfm1 expression in fallopian tube epithelium cell line (OE-E6/E7 cells). OE-E6/E7 cells treated with hCG, U0126 extracellular signal-regulated kinase (ERK) inhibitor, or XAV939 Wnt/β-catenin inhibitor were analyzed by Western blotting, real-time polymerase chain reaction, and in vitro spheroid attachment assay., Result(s): Human chorionic gonadotropin increased spheroid attachment on OE-E6/E7 cells through down-regulation of Olfm1 and activation of Wnt and mitogen-activated protein kinase (MAPK) signaling pathways. U0126 down-regulated both MAPK and Wnt/β-catenin signaling pathways and up-regulated Olfm1 expression. XAV939 down-regulated only the Wnt/β-catenin signaling pathway but up-regulated Olfm1 expression., Conclusion(s): Human chorionic gonadotropin activated both ERK and Wnt/β-catenin signaling pathways and enhanced spheroid attachment on fallopian tube epithelial cells through down-regulation of Olfm1 expression., (Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Macrophages represent the second major type of decidual leukocytes at the fetomaternal interface. Changes in macrophage number and activity are associated with fetal loss and pregnancy complications. Glycodelin-A (GdA) is an abundant glycoprotein in the first-trimester decidua. It is involved in fetomaternal defense and early placental development through its regulatory activities in various immune cells. The N-glycosylation of GdA mediates the binding and therefore the activities of the molecule. In this study, we studied the biological activities of GdA in the functions of human monocytes/macrophages. GdA was purified from amniotic fluid by affinity chromatography. GdA treatment did not affect the viability, cell death, or phagocytic activity of the monocytes/macrophages. GdA, but not recombinant glycodelin without glycosylation, induced IL-6 production as demonstrated by cytokine array, intracellular staining, and ELISA. GdA also induced phosphorylation of ERK in monocytes/macrophages. The involvement of ERKs in IL-6 induction was confirmed using pharmacological inhibitors. Co-immunoprecipitation showed that L-selectin on the monocytes/macrophages was the binding protein of GdA. Treatment with anti-L-selectin antibody reduced GdA binding and GdA-induced IL-6 production. GdA-treated macrophages suppressed IFN-γ expression by co-cultured T-helper cells in an IL-6-dependent manner. These results show that GdA interacts with L-selectin to induce IL-6 production in monocytes/macrophages by activating the ERK signaling pathway. In turn, the increased IL-6 production suppresses IFN-γ expression in T-helper cells, which may play an important role in inducing a Th-2-polarized cytokine environment that flavors the immunotolerance of the fetoplacental unit.

During placentation, the cytotrophoblast differentiates into the villous cytotrophoblast and the extravillous cytotrophoblast. The latter invades the decidualized endometrium. Glycodelin-A (GdA) is abundantly synthesized by the decidua but not the trophoblast. Previous data indicate that GdA suppresses the invasion of trophoblast cell lines by down-regulating proteinase expression and activities. This study addresses the signaling pathway involved in the above phenomenon. GdA was found to suppress phosphorylation of ERKs and expression of their downstream effector c-Jun, a component of the transcription factor activator protein-1 (AP-1). The involvement of ERKs and c-Jun in suppressing trophoblast invasion and biosynthesis of proteinases was confirmed by using siRNA knockdown and pharmacological inhibitors. Desialylation reduced binding affinity of GdA toward and invasion suppressive activities on the trophoblast. Co-immunoprecipitation showed that Siglec-6 on the trophoblast was the binding protein of GdA. The binding of GdA to Siglec-6 was sialic acid-dependent. Treatment with anti-Siglec-6 antibody abolished the invasion suppressive activities of GdA. These results show that GdA interacts with Siglec-6 to suppress trophoblast invasiveness by down-regulating the ERK/c-Jun signaling pathway.

Glycodelin-A (GdA) is a glycoprotein secreted from the endometrial glands and decidual glandular epithelium. Given its abundance and ubiquitous distribution in the first trimester uterus, GdA may be involved in early placental development via its modulatory effect on immune and trophoblast cells. GdA inhibits activation and proliferation, and induces apoptosis of T cells. By selectively inducing Th1 cell death, GdA may shift the Th1/Th2 ratio at the feto-maternal interface. This is also achieved indirectly through enhanced expression of Fas in the Th1 cells, thus making them vulnerable to cell death through Fas ligand expressed on trophoblast, endometrial, and activated T helper cells. GdA also promotes secretion of the Th2 cytokines IL-6 and IL-13 from NK cells, and induces immunological tolerance of dendritic cells and apoptosis of monocytes. Specific glycosylation is a prerequisite for the biological activities of GdA. Reduction in α2-6 sialylation of GdA, as in gestational diabetes, is associated with impairment of its T cell apoptosis-inducing activities. This review integrates recent studies on GdA and its role as a paracrine regulator in early pregnancy., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: The maternal-fetal interface has a unique immunological response towards the implanting placenta. It is generally accepted that a T-helper type-2 (Th-2) cytokine prevailing environment is important in pregnancy. The proportion of Th-2 cells in the peripheral blood and decidua is significantly higher in pregnant women in the first trimester than in non-pregnant women. Glycodelin-A (GdA) is a major endocrine-regulated decidual glycoprotein thought to be related to fetomaternal defence. Yet the relationship between its immunoregulatory activities and the shift towards Th-2 cytokine profile during pregnancy is unclear., Methods: GdA was immunoaffinity purified from human amniotic fluid. T-helper, T-helper type-1 (Th-1) and Th-2 cells were isolated from the peripheral blood. The viability of these cells was studied by XTT assay. Immunophenotyping of CD4/CD294, cell death and GdA-binding were determined by flow cytometry. The mRNA expression, surface expression and secretion of Fas/Fas ligand (FasL) were determined by quantitative polymerase chain reaction, flow cytometry and ELISA, respectively. The activities of caspase-3, -8 and -9 were measured. The phosphorylation of extracellular signal-regulated kinases (ERK), p38 and, c-Jun N-terminal kinase was determined by western blotting., Results: Although GdA bound to both Th-1 and Th-2 cells, it had differential actions on the two cell-types. GdA induced cell death of the Th-1 cells but not the Th-2 cells. The cell death was mediated through activation of caspase -3, -8 and -9 activities. GdA up-regulated the expression of Fas and inhibited ERK activation in the Th-1 cells, which might enhance the vulnerability of the cells to cell death caused by a trophoblast-derived FasL., Conclusions: The data suggest that GdA could be an endometrial factor that contributes to the Th-2/Th-1 shift during pregnancy.

Objective: Gestational diabetes mellitus (GDM) is a common metabolic disorder of pregnancy. Patients with GDM are at risk for high fetal mortality and gestational complications associated with reduced immune tolerance and abnormal carbohydrate metabolism. Glycodelin-A (GdA) is an abundant decidual glycoprotein with glycosylation-dependent immunomodulatory activities. We hypothesized that aberrant carbohydrate metabolism in GDM was associated with changes in glycosylation of GdA, leading to defective immunomodulatory activities., Research Design and Methods: GdA in the amniotic fluid from women with normal (NGdA) and GDM (DGdA) pregnancies was purified by affinity chromatography. Structural analysis of protein glycosylation was preformed by lectin-binding assay and mass spectrometry. Cytotoxicity, cell death, cytokine secretion, and GdA binding of the GdA-treated lymphocytes and natural killer (NK) cells were determined. The sialidase activity in the placental tissue from normal and GDM patients was measured., Results: GDM affected the glycosylation but not the protein core of GdA. Specifically, DGdA had a lower abundance of α2-6-sialylated and high-mannose glycans and a higher abundance of glycans with Sda (NeuAcα2-3[GalNAcβ1-4]Gal) epitopes compared with NGdA. DGdA had reduced immuosuppressive activities in terms of cytotoxicity on lymphocytes, inhibitory activities on interleukin (IL)-2 secretion by lymphocytes, stimulatory activities on IL-6 secretion by NK cells, and binding to these cells. Desialylation abolished the immunomodulation and binding of NGdA. Placental sialidase activity was increased in GDM patients, which may account for the reduced sialic acid content of DGdA., Conclusions: Taken together, this study provides the first direct evidence for altered enzymatic glycosylation and impaired bioactivity of GdA in GDM patients.

Background: Glycodelin-A interacts with spermatozoa before fertilization, but its role in modulating sperm functions is not known. Zona pellucida-induced acrosome reaction is crucial to fertilization and its dysfunction is a cause of male infertility. We hypothesized that glycodelin-A, a glycoprotein found in the female reproductive tract, potentiates human spermatozoa for zona pellucida-induced acrosome reaction., Methods: Glycodelin isoforms were immunoaffinity purified. The sperm intracellular cAMP concentration, protein kinase-A (PKA) and extracellular signal-regulated kinase (ERK) activities, and intracellular calcium were measured by ELISA, kinase activity assay kits and Fluo-4AM technique, respectively. The phosphorylation of inositol 1,4,5-trisphosphate type-1 receptor (IP3R1) mediated by ERK was determined by western blotting. Zona pellucida-induced acrosome reaction was detected by Pisum sativum staining., Results: Pretreatment of spermatozoa with glycodelin-A significantly up-regulated adenylyl cyclase/PKA activity and down-regulated the activity of ERK and its phosphorylation of IP3R1, thereby enhancing zona pellucida-induced calcium influx and zona pellucida-induced acrosome reaction. Glycodelin-F or deglycosylated glycodelin-A did not have these actions. Treatment of spermatozoa with a protein kinase inhibitor abolished the priming activity of glycodelin-A, whilst ERK pathway inhibitors mimic the stimulatory effect of glycodelin-A on zona pellucida-induced acrosome reaction., Conclusions: Glycodelin-A in the female reproductive tract sensitizes spermatozoa for zona pellucida-induced acrosome reaction in a glycosylation-specific manner through activation of the adenylyl cyclase/PKA pathway, suppression of extracellular signal-regulated kinase activation and up-regulation of zona pellucida-induced calcium influx. The action of glycodelin-A may be important in vivo to ensure full responsiveness of human spermatozoa to the zona pellucida.

We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC--with only a single sample injection event--as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.

Glycodelin-A increased the secretion of interleukin-6, interleukin-13, and granulocyte-macrophage colony-stimulating factor from natural killer cells in the peripheral blood but does not affect their viability, cell death, and cytotoxicity. These data suggest that glycodelin-A contributes to the cytokine shift in early pregnancy., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Glycodelin is a glycoprotein with 4 known glycoforms, namely glycodelin-S, glycodelin-A, glycodelin-F and glycodelin-C. The glycoforms are present in the female reproductive tract which the spermatozoa must pass through before fertilizing the oocyte. Thus the spermatozoa interact with each of these glycoforms in succession. The glycoforms have different effects on sperm function. Glycodelin-S in the seminal plasma suppresses albumin-induced cholesterol efflux from the spermatozoa and thereby regulates the initiation of capacitation. Glycodelin-A in the oviductal fluid suppresses extracellular signal-regulated kinase making the spermatozoa more sensitive to zona pellucida-induced acrosome reaction. Follicular fluid glycodelin-F suppresses progesterone-induced acrosome reaction and prevents premature acrosome reaction. Glycodelin-A and glycodelin-F also inhibit spermatozoa-zona pellucida binding by interacting with sperm surface fucosyltransferase-5, which also binds to zona pellucida glycoproteins. The physiological implication of this phenomenon remains to be determined. The inhibitory activities of glycodelin-A and glycodelin-F on spermatozoa-zona pellucida binding is removed by glycodelin-C in the cumulus matrix. Glycodelin-C not only displaces sperm-bound glycodelin-A and glycodelin-F, it also enhances spermatozoa-zona pellucida binding. These different biological activities of the glycodelin isoforms are determined by glycosylation of the proteins. Deglycosylation abolishes the binding and therefore the action of the glycodelins on spermatozoa. Knowledge of the mechanism of actions of glycodelins may enable development of novel strategies for fertility regulation.

Background: Glycodelin-C is a glycodelin isoform isolated from the cumulus matrix. It stimulates spermatozoa-zona pellucida binding. Here, we report the isolation and characterization of a novel glycodelin interacting protein (GIP) from human cumulus matrix., Methods: GIP was purified by liquid chromatograph and identified by mass spectrometry. The interaction of GIP with glycodelin, matrix molecule and spermatozoa were investigated., Results: Mass spectrometry analysis suggested that GIP contained the N-terminal region of alpha2-macroglobulin, confirmed by western blot with anti-alpha2-macroglobulin antibody. GIP bound to native but not deglycosylated glycodelin-C in native gel electrophoresis, suggesting that the binding was glycosylation-dependent. GIP did not bind to capacitated and uncapacitated human spermatozoa. The cumulus cells could convert exogenous labeled alpha2-macroglobulin into GIP in vitro. GIP interacted with hyaluronic acid, a major component of the cumulus matrix. Glycodelin-C bound to hyaluronic acid-coated agarose beads in the presence of GIP. Human spermatozoa acquired the hyaluronic acid-GIP-bound glycodelin-C during incubation in vitro., Conclusion: The hyaluronic acid-GIP complex formed in the cumulus matrix retains and concentrates glycodelin-C in the cumulus matrix for displacing sperm-bound glycodelin-A and -F and stimulating the zona binding activity of the spermatozoa traversing through the cumulus mass.

Background: Trophoblast invasion is crucial to placentation. The relationship between decidual glycodelin-A and trophoblast invasion is not known., Methods: Invasiveness of First trimester extravillous cytotrophoblast-1 (TEV-1) cell line, TEV-1, cells was determined by trans-well invasion assay. The gene expression, protein secretion and activities of the matrix metalloproteinase (MMP)-2 and -9, urokinase plasminogen activator (uPA), tissue inhibitor of metalloproteinase (TIMP)-1 and -2 and plasminogen activator inhibitor (PAI-1) of glycodelin-A-treated cells were measured by quantitative PCR, ELISA and gel zymography, respectively., Results: Glycodelin-A bound to TEV-1 cells. At a concentration of 1 microg/ml, glycodelin-A, but not other glycodelin isoforms, suppressed the invasion of TEV-1 cells. The effect was glycosylation-dependent and was associated with reduction (P < 0.05) of MMP2, MMP9 and uPA activities in the conditioned medium from the treated culture. Glycodelin-A treatment suppressed the amount of MMP2 protein in the conditioned medium (P < 0.05) and MMP2 mRNA in the cells (P < 0.05), but did not affect that of MMP9. Glycodelin-A also significantly reduced the expression, secretion and activity of uPA (P < 0.05). The treatment did not affect the expression of TIMP-1, TIMP-2 or PAI-1, cell proliferation or survival of the cells., Conclusions: Glycodelin-A inhibits the invasion of extravillous cytotrophoblasts mainly by suppressing the activity of MMP2 and MMP9 in a glycosylation-dependent fashion.

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galbeta1-4GlcNAc (lacNAc) and/or GalNAcbeta1-4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcalpha2-3(GalNAcbeta1-4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.

Spermatozoa have to swim through the oviduct and the cumulus oophorus before fertilization in vivo. In the oviduct, spermatozoa are exposed to glycodelin-A and -F that inhibit spermatozoa-zona pellucida binding. In this study, we determined whether these glycodelins would inhibit fertilization. The data showed that the spermatozoa without previous exposure to glycodelin-A and -F acquired glycodelin immunoreactivity during their passage through the cumulus oophorus. On the other hand, when glycodelin-A or -F-pretreated spermatozoa were exposed to the cumulus oophorus, the zona pellucida binding inhibitory activity of glycodelin-A and -F was not only removed, but the spermatozoa acquired enhanced zona pellucida binding ability. These actions of the cumulus oophorus were due to the presence of a cumulus isoform of glycodelin, designated as glycodelin-C. The cumulus cells could convert exogenous glycodelin-A and -F to glycodelin-C, which was then released into the surrounding medium. The protein core of glycodelin-C was identical to that in other glycodelin isoforms, as demonstrated by mass spectrum, peptide mapping, and affinity to anti-glycodelin antibody recognizing the protein core of glycodelin. In addition to having a smaller size and a higher isoelectric point, glycodelin-C also had lectin binding properties different from other isoforms. Glycodelin-C stimulated spermatozoazona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound glycodelin-A and -F. In conclusion, the cumulus cells transform glycodelin-A and -F to glycodelin-C, which in turn removes the spermatozoazona binding inhibitory glycodelin isoforms and enhances the zona binding capacity of spermatozoa passing through the cumulus oophorus.

Glycodelin is a glycoprotein with three isoforms, namely glycodelin-S, glycodelin-A and glycodelin-F. They have similar protein core but different glycan side chains. Spermatozoa are exposed to these isoforms during their passage to the fertilization site. They first encounter glycodelin-S in the seminal plasma. Data suggest that glycodelin-S suppresses albumin-induced cholesterol loss and maintains the spermatozoa in an uncapacitated state before they enter into the cervical canal where glycodelin-S is removed. This allows albumin in the uterine cavity to initiate capacitation. In the fallopian tube, the spermatozoa are exposed to glycodelin-A and -F produced by the fallopian tube. Glycodelin-A is an endogenous glycoprotein that inhibits the binding of spermatozoa to the zona pellucida. Glycodelin-A may protect the spermatozoa from maternal immune attack by its immunosuppressive activity. Glycodelin-F is the main glycodelin isoform in the follicular fluid. Similar to glycodelin-A, it inhibits spermatozoa-zona pellucida binding. The biological significance of the anti-fertilization activity of glycodelin-A and -F remains to be established. Glycodelin-F also suppresses progesterone-induced acrosome reaction. This is important to prevent premature acrosome reaction when the spermatozoa are swimming through the cumulus mass towards the oocyte and become exposed to progesterone produced by the cumulus cells. In summary, different isoforms of glycodelin act in succession to modulate different aspects of sperm function, and thereby, contribute to the success of fertilization.

Fertilization depends on successful binding of the spermatozoa to the zona pellucida of the oocyte. Glycodelin-A inhibits spermatozoa-zona pellucida binding. Previous data showed that glycodelin-A receptor(s) and zona pellucida protein receptor(s) on human spermatozoa are closely related. Using a chemical cross-linking approach, the glycodelin-A-sperm receptor complex was isolated. The receptor was identified to be fucosyltransferase-5 (FUT5) by mass spectrometry and confirmed with the use of anti-FUT5 antibodies. Sperm FUT5 was an externally oriented integral membrane protein in the acrosomal region of human spermatozoa. Biologically active FUT5 was purified from spermatozoa. Co-immunoprecipitation confirmed the interaction between glycodelin-A and sperm FUT5. Solubilized zona pellucida reduced the binding of glycodelin-A to sperm FUT5. An anti-FUT5 antibody and FUT5 acceptor blocked the binding of glycodelin-A to spermatozoa and the zona binding inhibitory activity of glycodelin-A. Sperm FUT5 bound strongly to intact and solubilized human zona pellucida. The equilibrium dissociation constant of sperm FUT5 binding to solubilized zona pellucida was 42.82 pmol/ml. These observations suggest that human sperm FUT5 is a receptor of glycodelin-A and zona pellucida proteins, and that glycodelin-A inhibits spermatozoa-zona binding by blocking the binding of sperm FUT5 to the zona pellucida.

Glycodelin is a glycoprotein with three well-defined isoforms. They are named as glycodelin-S, glycodelin-A and glycodelin-F. The three isoforms have similar protein core but different carbohydrate moieties. Glycodelin-S is abundant in the human seminal plasma. It suppresses sperm capacitation and in doing so, it maintains the spermatozoa in an uncapacitated state before they enter into the uterine cavity. Glycodelin-A is abundant in the amniotic fluid. It is also secreted from endometrial glands into uterine fluid and is produced by the fallopian tube. Glycodelin-A is the first endogenous glycoprotein that was found to inhibit the binding of spermatozoa to the zona pellucida. The immunosuppressive properties of glycodelin-A suggest that the molecule may protect the spermatozoa from immune attack in the maternal reproductive tract. Glycodelin-F was first found in the follicular fluid, hence its name. It also inhibits spermatozoa-zona pellucida binding. In addition, glycodelin-F suppresses progesterone-induced acrosome reaction, and may serve to prevent premature acrosome reaction. Preliminary findings suggest possible presence of yet another glycodelin isoform in the extracellular matrix of cumulus oophorus. Unlike glycodelin-A and -F, it stimulates spermatozoa-zona pellucida binding. In summary, different isoforms of glycodelin have different biological roles on sperm function, and they act in succession to contribute to the success of fertilization.

Tight control of sperm capacitation is important for successful fertilization. Glycodelin-S is one of the most abundant glycoproteins in the human seminal plasma. However, its function is unclear. We investigated the role of glycodelin-S on capacitation of human spermatozoa. Binding kinetics experiments demonstrated the presence of two saturable and reversible binding sites of glycodelin-S on human spermatozoa. Differently glycosylated other isoforms of glycodelin, glycodelin-A and -F, did not compete with glycodelin-S for these binding sites, suggesting that the glycodelin-S binding sites are different from those of the other isoforms. Indirect immunofluorescent staining revealed specific binding of glycodelin-S around the sperm head. This immunoreactivity was greatly reduced in spermatozoa that had migrated through the cervical mucus surrogates. Glycodelin-S at physiological concentrations significantly reduced the bovine serum albumin and cyclodextrin-induced cholesterol efflux and down-regulated the adenylyl cyclase/protein kinase A/tyrosine kinase signaling pathway, resulting in suppression of capacitation. Deglycosylation abolished glycodelin-S binding and the effect of glycodelin-S on bovine serum albumin-induced capacitation. This indicates that the carbohydrate moiety of glycodelin-S is critical for the function of the molecule. It is concluded that glycodelin-S in seminal plasma maintains the uncapacitated state of human spermatozoa.

Glycodelin is a glycoprotein produced in many glands, particularly those of reproductive tissues. It appears as different glycoforms in amniotic fluid (glycodelin-A) and seminal plasma (glycodelin-S), but only glycodelin-A inhibits gamete adhesion. In the present study, glycodelin from secretory-phase endometrium, first-trimester pregnancy decidua, and midtrimester amniotic fluid was studied with respect to physicochemical properties, including glycosylation patterns and inhibitory activity of sperm-egg binding. Purified glycodelins from all these sources were similar in isoelectric focusing and in lectin immunoassays using lectins from Wisteria floribunda and Sambucus nigra. Likewise, the glycodelins inhibited sperm-egg binding in a dose-dependent manner, as measured by hemizona-binding assay. However, subtle quantitative physicochemical and biological differences were found between glycodelins from different sources as well as within the same tissue/fluid between different individuals. Differences were most pronounced between endometrial glycodelins from nonpregnancy and first-trimester pregnancy. The glycan structures studied by fast-atom bombardment mass spectrometry of individual amniotic fluid glycodelin-A samples also showed interindividual quantitative differences. In conclusion, glycodelins from different female reproductive tract tissues and amniotic fluid share substantial similarity, allowing all of them to be called glycodelin-A. However, these glycodelins exhibit quantitative physicochemical and functional differences between different sources and individuals.

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.

The article discusses research that shows the binding of human sperm to eggs is attributed to the abundance of a sequence of sugar molecules called sialyl-Lewisx (SLeX) on the ends of oligosaccharides of zona pellucida (ZP) glycoproteins. According to the author, the ZP is a thick extracellular coat surrounding the eggs of all mammals that sperm must bind to and penetrate to fertilize the egg. He notes that the binding of sperm to an egg's outer coat occurs in a species-restricted manner and discusses both the involvement of SLeX sequences in sperm binding and whether sperm binds to all human ZP glycoproteins containing SLeX or is restricted to just a few of them.

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunulogical activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetraantennary complex-type glycans carrying Galβl-4GlcNAc (lacNAc) and/or GalNAcβ1-4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcα2-3(GalNAcβ1-4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitupe, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC. [ABSTRACT FROM AUTHOR]