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Relaxin stimulates glycodelin mRNA and protein concentrations in human endometrial glandular epithelial cells.

Human endometrium is the major organ that produces glycodelin A (GdA). The production of endometrial GdA causes a fluctuation of the peripheral glycodelin concentrations in women during the menstrual cycle and pregnancy. It has recently been reported that the rise of plasma concentrations of glycodelin is correlated with relaxin during the late luteal phase and early pregnancy. In addition, administration of relaxin increases glycodelin plasma concentrations, suggesting that relaxin induces GdA production in endometrium. To investigate whether relaxin regulates the GdA synthesis, human endometrial glandular epithelial cells were isolated and cultured with or without relaxin for up to 4 days. Western blot showed that GdA synthesized and secreted from epithelial glands had a major molecular weight of 28 kDa, i.e. the same as the GdA isolated from amniotic fluid. Cells incubated with relaxin consistently increased in GdA production rate (2-6-fold). The GdA mRNA concentrations increased 2-11-fold in cells incubated with relaxin for 2-4 days, as determined by solution hybridization/ribonuclease protection assay. The increase of the mRNA concentration indicates that relaxin activates GdA transcription.

Adult↗

Glycodelins: biological activity in reproduction and differentiation.

Studies published in recent years have elucidated the role of glycodelins in early human reproduction and the biological actions of glycodelins relative to their structure. New information has also emerged on the regulation of glycodelin synthesis and on the effects of glycodelin transfection on cellular growth and the expression of other epithelial markers.

Cell Differentiation↗

Glycodelin and amniotic fluid transferrin as inhibitors of E-selectin-mediated cell adhesion.

Human amniotic fluid contains a variety of glycoproteins. Several of these substances have been shown to exert immunomodulatory effects. Glycodelin, previously known as placental protein 14, is one of these glycoproteins. It has a unique carbohydrate configuration, consistent with fucosylated LacdiNAc structures that are very unusual for mammals. Oligosaccharides with fucosylated LacdiNAc antennae have previously been shown to block selectin-mediated cell adhesion. Another glycoprotein, human transferrin, is also present in amniotic fluid in relatively high concentrations. This transferrin shows a different glycosylation compared with serum transferrin. Amniotic fluid transferrin carries sialylated Lewis X antigens. Glycodelin and transferrin were isolated from amniotic fluid and for comparison from serum of pregnant women by chromatographic methods. The purified proteins were used as ligands to block E-selectin-mediated HepG2 cell adhesion. Two types of binding assays with distinct receptor accommodations (immobilised E-selectin and activated HUVECs) were used to quantify inhibition efficiencies of the different proteins. We found that glycodelin is a strong inhibitor with a 10(3)-fold potency compared to the monovalent tetrasaccharide sialyl Lewis X whereas the potency of transferrin is rather low.

Adult↗

Rapid purification and properties of human glycodelin (endometrial alpha2-globulin).

The method presented can easily produce milligram amounts of glycodelin from pregnancy endometrium, with a 19% yield. It involves anion-exchange chromatography, gel permeation and chromatofocusing; it results in one stainable band at Mr 28,000 after sodium dodecyl sulphate-polyacrylamide electrophoresis, as well as after immunoblot analysis, performed using an affinity-purified IgG fraction from an antiserum against glycodelin. In spite of this, the corresponding gel isoelectric focusing pattern gives four stainable bands with pI values between 4.55 and 5.2. Western immunoblot analysis of tissue extracts indicates the presence of glycodelin epitopes associated with materials heavier than the native protein. Circular dichroism spectra of the highly purified protein in water solutions indicate a large amount of beta-sheet conformation, whereas those obtained with different proportions of 2-propanol in water, show an increased proportion of alpha-helix conformation.

Anions↗

Oxidants and antioxidants affect the expression of glycodelin.

Glycodelin is a glycoprotein that has immunosuppressive activity. We have shown that K562 cells, hematopoitic progenitor cells, are capable of synthesizing glycodelin peptide (Gp) and, perhaps, contribute to Gp in tissues. In addition, several reproductive and nonreproductive tissues themselves are capable of synthesis of glycodelin. In this study, we report that lipid peroxides induce the synthesis of Gp. Antioxidants vitamin E and pyrrolidine dithiocarbamate (PDTC) and antioxidizing enzymes catalase and superoxide dismutase (SOD) effectively blocked phorbol myristate acetate- (PMA-) and lyso phosphatidic acid- (LPA-) induced synthesis of Gp. Dioctanoin (a mimic of diacylglycerol) activated Gp synthesis, and an inhibitor of protein kinase C (PKC) downregulated the response. Based on these observations, we postulate that oxidants by way of PKC might potentiate the angiogenic process.

Antioxidants↗

Meet the multifunctional and sexy glycoforms of glycodelin.

Glycodelin, a human-secreted glycoprotein that appears in a small number of glycoforms, exhibits diverse biological activities, such as in contraception and immunosuppression. Moreover, different tissue-specific glycoforms appear to mediate diverse functions. Quite unusually, the glycodelin N-linked glycans differ between the male and female glycoforms. The fact that these glycans are fundamental for exerting the physiological activities of the different glycoforms, makes them an interesting target for glycobiology research. This review will focus on the involvement of the glycans in glycodelin activity and compare between the several glycoforms.

Animals↗

Glycodelin A and differentiation of first trimester trophoblast cells in vitro.

AIM: The glycoprotein, glycodelin A (GdA) is a main product of the maternal decidua in the first trimester of pregnancy and is secreted into the amniotic fluid. The purpose of this study was to investigate the effect of GdA on secretion and surface markers of isolated first trimester trophoblasts in vitro. METHODS: Cytotrophoblasts were prepared from human first trimester placentae and incubated with varying concentrations of GdA or transfected separately with the expression plasmid of GdA. Supernatants were assayed for human chorionic gonadotropin (hCG) protein concentrations. Expression of human placental lactogen (hPL), mucin 1 (MUC1) and the Thomsen-Friedenreich (TF) epitope was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. RESULTS: Glycodelin A induced a reduced expression of hPL compared with unstimulated controls. Expression of MUC1 was not affected by GdA. Freshly isolated trophoblast cells showed no TF expression but became positive for this antigen after 96 h of cultivation. GdA-stimulated trophoblast cells inhibited TF expression after 96 h of cultivation. GdA plasmids induced a significantly higher hCG production in transfected cells than in cells transfected with the empty plasmid. CONCLUSIONS: The results obtained in this study suggest that GdA is involved in the differentiation of trophoblast cells. The treatment of GdA plasmid transfected trophoblast cells stimulated hCG production in isolated trophoblast cells and inhibited hPL and TF expression, suggesting a functional link between hCG and GdA.

Adult↗

Expression of cox-2, tie-2 and glycodelin by megakaryocytes in patients with chronic myeloid leukaemia and polycythaemia vera.

When evaluating bone marrow sections for markers of neo-angiogenesis, we found that megakaryocytes stained markedly positive for cyclooxygenase-2 (Cox-2), Tie-2 and glycodelin. This apparently novel finding was further evaluated for disease-specific variations. Bone marrow sections from two patient groups, known to be characterized by clonal megakaryocytopoiesis, viz. chronic myeloid leukaemia and polycythaemia vera, stained, however, similarly to healthy marrows for these markers. The biochemical background and clinical significance of Cox-2, Tie-2 and glycodelin remains to be elucidated.

Biomarkers↗

[Stimulation of HCG, estrogen and progesterone production in isolated trophoblast cells by glycodelin A or its N-glycans].

UNLABELLED: The immunosuppressive protein glycodelin A (GdA) is secreted by decidual tissue in high rates in the first trimester of pregnancy. GdA forms about 10 % of the total protein amount released by the decidua in the first trimester of pregnancy and has a unique glycosylation including fucosylated LacdiNAc structures. The purpose of this study was to investigate the effect of glycodelin A and its N-glycans on hCG, estrogen and progesterone release by cytotrophoblasts in vitro. Trophoblast cells were isolated from human term placenta by fragmentation of villous tissue. Trophoblast cells fuse in vitro to syncytiotrophoblast cells. Trophoblast cells were incubated with GdA and various glycans of GdA. The production of hCG and progesterone was measured after 24 and 48 hours. The production of hCG, estrogen and progesterone was increased in GdA and glycan-treated cell cultures as compared to untreated trophoblast cell cultures. CONCLUSION: HCG, estrogen and progesterone are markers for the differentiation process of cytotrophoblast cells. The results suggest that GdA with its distinct glycosylation modulates the differentiation of trophoblasts.

Cell Differentiation↗

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 (GalNAcbeta1-4GlcNAc) 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 (Galbeta1-4(Fucalpha1-3)GlcNAc) and Lewisy (Fucalpha1-2Galbeta1-4(Fucalpha1-3)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.

Contraceptive Agents↗

Analysis of the role of oligosaccharides in the apoptotic activity of glycodelin A.

Glycodelin A, also known as placental protein-14, is a multifunctional glycosylated protein secreted by the uterine endometrium during the early phases of pregnancy. It is a known suppressor of T cell proliferation, inducer of T cell apoptosis, and inhibitor of sperm zona binding. Unlike in contraceptive activity, where the glycans on the molecule have been shown to play a crucial role, mutagenesis of the asparagines at sites of N-linked glycosylation (Asn(28) and Asn(63)) to glutamine shows that the apoptogenic activity of glycodelin A is executed by the protein backbone. Glycosylation at Asn(28) appears to play a role in the extracellular secretion of the molecule, as mutation of Asn(28) resulted in a significant decrease in the amount of secreted protein, and loss of both glycosylation sites reduced the secretion drastically. Our results also suggest that the loss of glycosylation does not affect the dimerization status of the molecule.

Adult↗

Oligosaccharides modulate the apoptotic activity of glycodelin.

GlycodelinA (GdA), a multifunctional glycoprotein secreted at high concentrations by the uterine endometrium during the early phases of pregnancy, carries glycan chains on asparagines at positions N28 and N63. GdA purified from amniotic fluid is known to be a suppressor of T-cell proliferation, an inducer of T-cell apoptosis, and an inhibitor of sperm-zona binding in contrast to its glycoform, glycodelinS (GdS), which is secreted by the seminal vesicles into the seminal plasma. The oligosaccharide chains of GdA terminate in sialic acid residues, whereas those of GdS are not sialylated but are heavily fucosylated. Our previous work has shown that the apoptogenic activity of GdA resides in the protein backbone, and we have also demonstrated the importance of sialylation for the manifestation of GdA-induced apoptosis. Recombinant glycodelin (Gd) expressed in the Sf21 insect cell line yielded an apoptotically active Gd; however, the same gene expressed in the insect cell line Tni produced apoptotically inactive Gd, as observed with the gene expressed in the Chinese hamster ovary (CHO) cell line and earlier in Pichia pastoris. Glycan analysis of the Tni and Sf21 cell line-expressed Gd proteins reveals differences in their glycan structures, which modulate the manifestation of apoptogenic activity of Gd. Through apoptotic assays carried out with the wild-type (WT) and glycosylation mutants of Gd expressed in Sf21 and Tni cells before and after mannosidase digestion, we conclude that the accessibility to the apoptogenic region of Gd is influenced by the size of the glycans.

Animals↗

Glycodelins: role in regulation of reproduction, potential for contraceptive development and diagnosis of male infertility.

Glycodelins are glycoproteins synthesized in various glands, with sequence homology to beta-lactoglobulins, and named according to their unique oligosaccharide structures. We purified, cloned and sequenced endometrium- and seminal plasma-derived glycodelins (GdA and GdS respectively) and found that they are involved in various types of cell-cell communications. These include interactions between the spermatozoon and the egg, and between immune cells and their targets. Endometrial GdA inhibits sperm-egg binding, whereas the differently glycosylated GdS in seminal plasma does not. These observation are of interest for reproductive physiology, detection of causes of infertility, and they also may have potential for contraceptive development.

Biomarkers↗

Immunohistochemical analysis of steroid receptors and glycodelin A (PP14) in isolated glandular epithelial cells of normal human endometrium.

Highly purified fractions of isolated endometrial cells can be useful for investigating endometrial function. After a first collagenase digestion, normal human endometrial stromal and epithelial cells were separated by filtration. Glands were purified further by two collagenase digestion steps, filtration, differential sedimentations, and Ficoll gradient centrifugation. Epithelial cells were polyhedral and grew as islands in a whorl-like wavy pattern around glandular fragments. High cell culture purity was confirmed with the positive immunohistochemical reaction against cytokeratin 7,8,18,19. Isolated human glands had a similar distribution pattern of estrogen receptor (ER) and progesterone receptor (PR) as observed in vivo, suggesting that glands have a functional hormone receptor system at the time of plating. Using a specific monoclonal antibody against glycodelin A (GdA), a characteristic cyclical expression was demonstrated during the menstrual cycle. The GdA reaction was weak in the proliferative phase, increasing significantly till the late secretory phase, suggesting a similar GdA concentration in vitro as observed in vivo glands. In conclusion, this method could be a model for studying endometrial glandular cells from different menstrual phases, endometrial cell interactions, implantation mechanisms, GdA regulation mechanisms, and pharmacological or other influences on ER and PR alteration.

Adult↗

Enzyme-assisted synthesis of Asn-linked diantennary oligosaccharides occurring on glycodelin A.

The preparation of a series of sialylated and fucosylated N,N'-diacetyllactosediamine-type diantennary glycopeptides is reported. By sequential enzymatic action of jack bean beta-galactosidase, snail beta 4-N-acetyl-galactosaminyltransferase, bovine colostrum alpha 6-sialyltransferase and human milk alpha 3-fucosyltransferase, a diantennary glycopeptide obtained from asialo fibrinogen was converted at a 5-mumol scale to a series of structures occurring on the glycoprotein glycodelin A, which potentially inhibit human sperm-egg binding.

Animals↗

Endometrial glycodelin-A expression in the luteal phase of stimulated ovarian cycles.

OBJECTIVE: To determine if controlled ovarian hyperstimulation (COH) affects the endometrial expression of glycodelin-A (GdA). DESIGN: Prospective, controlled study. SETTING: Tertiary infertility clinic. PATIENT(S): Fifteen oocyte donors undergoing COH cycles and 19 natural-cycle control patients. INTERVENTION(S): COH, endometrial biopsies. MAIN OUTCOME MEASURE(S): Immunohistochemical scoring of endometrial GdA expression, morphologic endometrial dating, and serum E2, LH, and P4 concentrations. RESULT(S): GdA was detected in all subjects throughout the implantation window period. Immunolocalization was demonstrated in the endometrial glands and not in the stroma or on the surface. A significantly increased proportion of GdA-staining endometrial cells were noted in COH cycle patients as compared with natural-cycling control patients throughout the window of embryo implantation. Both cycle types demonstrated increasing GdA expression throughout the late luteal phase. A significant positive correlation was noted between GdA expression and serum E2 levels (r = 0.5, P<.001) in natural cycles and advanced histology in COH cycles (r = 0.63, P=.01). Neither LH nor P4 were correlated with endometrial GdA expression. CONCLUSION(S): COH cycles have a significantly increased endometrial GdA expression throughout the implantation phase of the luteal cycle when compared with normal menstrual cycles. The increased expression may affect implantation during COH cycles.

Adult↗

Effect of pH on the structure and aggregation of human glycodelin A. A comparison with beta-lactoglobulin A.

The effect of pH on the structure of glycodelin A (GdA) and of beta-lactoglobulin A (beta-LgA) has been investigated by means of circular dichroism, steady state fluorescence, synchrotron radiation small angle X-ray scattering (SR-SAXS) and gel permeation chromatography. The comparison between GdA and beta-LgA shows that, at pH 7.0, both proteins are dimers with an extended content of beta-sheet conformation, but pH 2.0 and 9.0 yield a different secondary, tertiary and quaternary structural organisation. Whilst beta-LgA is a monomer, that conserves beta-sheet conformation at pH 2.0 and 9.0, GdA has a stable dimeric structure at alkaline pH, but at pH 2.0 increases its alpha-helix content and it aggregates soon. SR beam has been used to perform SAXS comparative measurements of the two proteins. SR-SAXS data provide the radius of gyration and the radii of the cross-section and of the thickness. GdA aggregation at acid pH has been characterised by calculating the distance distribution function (P(r)). Isoelectric focusing and chromatofocusing data show a different charge distribution on the surfaces of the two proteins, supporting the hypothesis that the presence of oligosaccharides deeply influences the conformational state and the aggregation process of GdA at different pH values. In particular, the presence of sialic acid residues, within the oligosaccharide moiety of the GdA, might be responsible for the differences observed between the two proteins.

Animals↗