Molecular Human Reproduction, Vol. 8, No. 12, 1079-1086,
December 2002
© 2002 European Society of Human Reproduction and Embryology
Regulation of ovarian function |
High xylosyltransferase activities in human follicular fluid and cultured granulosa–lutein cells
1 Institut für Laboratoriums- und Transfusionsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstraße 11, 32545 Bad Oeynhausen and 2 Frauenklinik, Bielefelder Institut für Fortpflanzungsmedizin, Städtische Kliniken Bielefeld-Rosenhöhe, Bielefeld, Germany
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Follicular fluid proteoglycans play an important role in human oocyte maturation, including the development of a fluid-filled compartment and maintenance of the hypocoagulative state of the follicular fluid. Human xylosyltransferase (EC 2.4.2.26, XT) is the key enzyme in the biosynthesis of glycosaminoglycan chains in proteoglycans and is secreted into body fluids together with large proteoglycans. We investigated the XT activities in human follicular fluid and granulosa–lutein cells from women undergoing IVF procedures. The mean XT activity was determined as 17.7 mU/l, which is 20-fold higher than in serum and the highest XT activity ever found in body fluids. Cultured human granulosa–lutein cells secreted large amounts of XT (14.52 µU/106 cells), indicating that these cells are the main source of this enzyme in human follicular fluid. The XT from human follicular fluid was found to be associated with large chondroitin sulphate-containing proteoglycans. Furthermore, heparin was shown to bind strongly to the follicular fluid XT and to inhibit its enzyme activity. These findings indicate that XT may play a role in maintaining the haemostatic potential of the follicular fluid.
chondroitin sulphate/follicular fluid/heparan sulphate/proteoglycan/xylosyltransferase
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Development of human ovarian follicles and oocytes is a complex process involving tissue remodelling, cell replication and differentiation, and fluid accumulation. The follicular fluid plays an important biological role in folliculogenesis, oocyte maturation and ovulation, although the molecular composition of the human follicular fluid is not yet known in detail. This bodily fluid is mainly composed of plasma exudates and secreted products of the follicular cells, especially the granulosa cells. During follicular development, the fluid-filled compartment in the extracellular matrix surrounding the granulosa cells is created, without increasing the intrafollicular pressure, in the presence of large molecules capable of retaining water such as hyaluronate and proteoglycans (Yoshimura and Wallach, 1987
; Roughley and Lee, 1994).
Proteoglycans are polyanionic molecules widely expressed in animal cells and in virtually every tissue. These abundant molecules are present in the extracellular matrix and on the cell surface and serve a wide range of functions. They are increasingly implicated as important regulators in many biological processes, such as extracellular matrix deposition, cell membrane signal transfer, morphogenesis, cell migration, normal and tumour cell growth and viral infection (Ruoslahti, 1989; Herold et al., 1994; Silbert and Sugumaran, 1995). The large chondroitin sulphate proteoglycans in the extracellular matrix have a highly negative charge due to a large number of lateral polyanionic glycosaminoglycan chains. These unique structural properties result in a high water binding and tissue hydration capacity of these matrix molecules. Proteoglycans mediate diverse cellular processes through interaction with a variety of protein ligands. In most of these bindings, electrostatic interactions with the glycosaminoglycan chains attached to the core protein are involved (Kjellen and Lindahl, 1991). Thus, the biological activity of proteoglycans is intimately related to glycosaminoglycan biosynthesis.
The presence of proteoglycans in follicular fluid has been known for a long time, but recently studies have been trying to shed light on the molecular composition of the proteoglycans from human follicular fluid (Eriksen et al., 1999). The follicular fluid proteoglycans have been proposed to maintain (i) the fluid viscosity in the extended follicles during follicular maturation (Yanagishita and Hascall, 1979) and (ii) the hypocoagulable state of the pre-ovulatory ovarian follicle (Shimada et al., 2001). Furthermore, the variation observed in the concentration and structure of the proteoglycans at different stages of the follicular maturation has led to the hypothesis that the follicular fluid proteoglycans play an important role in folliculogenesis and fertilization (Bellin et al., 1986; Eriksen et al., 1997).
Proteoglycans are also functionally involved in other processes during mammalian conception such as the preservation of sperm motility and velocity and induction of the acrosome reaction as well as stimulation of capacitation (Yee and Cummings, 1988; Eriksen et al., 1994; Hamamah et al., 1996). Chondroitin sulphate-containing proteoglycans and hyaluronate isolated from human follicular fluid have been shown to stimulate sperm motility (Hamamah et al., 1996). Furthermore, pretreatment of sperm with glycosaminoglycans during IVF procedures results in a significant increase in oocyte cleavage and ongoing pregnancy rates (Hamamah et al., 1996).
Proteoglycans consist of a core protein which is post-translationally modified by the addition of lateral glycosaminoglycan chains. The glycosaminoglycans chondroitin sulphate, dermatan sulphate and heparan sulphate are bound to the proteoglycan core protein by a xylose–galactose–galactose binding region. Xylosyltransferase (UDP-D-xylose:proteoglycan core protein ß-D-xylosyltransferase, EC 2.4.2.26, XT) is the initiating and apparently rate-limiting enzyme in the biosynthesis of the glycosaminoglycan linkage region. It catalyses the transfer of D-xylose from UDP-D-xylose to specific serine residues of the core protein and is a regulatory factor in chondroitin sulphate biosynthesis (Rodén, 1980). We have demonstrated previously that XT is secreted into the extracellular space together with chondroitin sulphate proteoglycans (Kähnert et al., 1991; Götting et al., 1999), and that the XT activity in bodily fluids is an indicator for the actual proteoglycan biosynthesis rate. Furthermore, serum XT activity has been shown to be a biochemical marker for the determination of fibrotic activity in systemic sclerosis (Götting et al., 1999, 2000a).
In this study, we investigated the XT activities in human follicular fluid, the secretion of this enzyme by human granulosa–lutein cells, and some biochemical properties of XT in this body fluid. This is the first report on XT in mammalian follicular fluid.
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Materials and methods |
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Collection of follicular fluid samples
Human follicular fluid (n = 108) was collected from 53 women undergoing IVF and embryo transfer. Multiple follicular development was induced by a daily i.m. injection of 1–2 ampoules of hMG (Menogon; Ferring, Kiel, Germany) from day 3 of the menstrual cycle. One ampoule is the dosage equivalent to 75 IU of FSH and 75 IU of LH. Follicular development was monitored by sonography until a pre-ovulatory estradiol serum level of 200–300 ng/l was reached. Ovulation was then induced by administration of 5000 IU hCG, (Pregnesin 2500 IU; Serono, Unterschleißheim, Germany). 36 h later, aspiration of the follicular fluid was performed by an ultrasonically guided vaginal puncture. A sample of 10–15 ml of follicular fluid, including the oocytes and the granulosa cells, was obtained per puncture of each follicle. Oocytes were removed for the IVF procedures, and follicular fluid was centrifuged at 1000 g for 10 min. The supernatant was then stored at –70°C. All samples were collected with informed consent.
Collection of serum samples
Venous blood samples were collected in serum monovettes (Sarstedt, Nümbrecht, Germany). After clotting and centrifugation at 4000 g for 15 min, the serum was stored at –70°C until assayed. Samples were collected from the same 53 women undergoing IVF and embryo transfer as described above.
Isolation of human granulosa–lutein cells
Human granulosa–lutein cells were obtained from the follicular fluid samples after transvaginal puncture of superovulated follicles. Follicular fluid was centrifuged at 1000 g for 10 min, and the supernatant was removed. The cells were resuspended in McCoy’s 5A medium (Sigma, Deisenhofen, Gemany) and washed twice. The cell clusters were then dispersed by repeated pipetting with a sterile Pasteur pipette and resuspended in McCoy’s 5A medium in a total volume of 5 ml. For separation of granulosa–lutein cells from erythrocytes, 5 ml of Lymphoflot solution (Biotest, Dreieich, Germany) were added into a centrifugation vial and overlaid with the cell suspension. After centrifugation at 800 g for 20 min the red blood cells were pelleted, and the granulosa–lutein cells were layered over the Lymphoflot solution. The granulosa–lutein cells were aspirated from the interface with a Pasteur pipette, resuspended in medium and washed twice. Viability, as determined by trypan blue (Sigma) exclusion, was always >70%.
Cell culture
1x106 viable human granulosa–lutein cells were seeded in a 60 mm diameter cell culture dish containing 5 ml of McCoy’s 5A medium supplemented with 2 mmol/l L-glutamine, 1% of antibiotic/antimycotic solution (containing 10 000 IU/ml penicillin G, 10 mg/ml streptomycin sulphate, 25 µg/ml amphotericin B, (Sigma), 10% heat-inactivated fetal calf serum (FCS; Sigma) and 10-6 mol/l androstenedione (Sigma). After 24 h incubation at 37°C in a humidified atmosphere (95% air, 5% CO2), the cells were washed twice with Dulbecco’s phosphate-buffered saline (PBS) supplemented with 1% human serum albumin (HSA; Centeon Pharma, Marburg, Germany). To achieve serum-free conditions, FCS in the culture medium was then replaced by 5% HSA. Glucose and lactate concentrations of the cell culture medium were monitored using ACA (Dade Diagnostica, München, Germany) and Super G analyzers (RLT, Möhnesee, Germany) respectively. After incubation for 4 days, the spent medium was harvested and stored at –20°C after centrifugation at 1000 g for 10 min. For each condition, three or four replicates per culture were used. At the end of the experimental period, viability of the cells was determined by trypan blue exclusion. The viability of the cells was always >70%.
Human JAR placenta choriocarcinoma cells (ATCC HB-144; ATCC, Rockville, MD, USA), human HeLa cervix carcinoma cells (DSM ACC 57; DSMZ, Braunschweig, Germany), human EFO-21 ovary cystic adenocarcinoma cells (DSM ACC 235, DSMZ) and human EFO-27 ovary adenocarcinoma cells (DSM ACC 191, DSMZ) were cultivated using standard techniques (Butler and Dawson, 1992). The cell culture medium used was Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma) with 10% heat-inactivated FCS for all cell lines with the following exceptions: RPMI 1640 supplemented with 20% FCS, 1 mmol/l sodium pyruvate and 1xminimum essential medium non-essential amino acids solution [containing 0.89 g/l L-alanine, 1.5 g/l L-asparagine, 1.33 g/l L-aspartic acid, 1.47 L-glutamic acid, 0.75 g/l glycine, 1.15 g/l L-proline, 1.05 g/l L-serine, (Sigma)] was used for cultivation of EFO-21 and EFO-27. In addition, all media used were supplemented with antibiotic/antimycotic solution and 2 mmol/l L-glutamine. Cells were grown in media containing FCS in 60 mm diameter cell culture dishes until confluence was reached. The cell culture medium in each dish was then changed to the corresponding medium without supplementation with FCS. After incubation for 4 days, the spent medium was harvested and stored at –20°C after centrifugation at 1000 g for 10 min. The number of viable cells per dish was determined after detachment with trypsin/EDTA solution and trypan blue exclusion. Cell viability was always >70%.
Synthesis of recombinant bikunin and [Val36,Val38]
1,[Gly92,Ile94]2-bikunin
Recombinant bikunin and [Val36,Val38]1,[Gly92,Ile94]2bikunin were expressed in E.coli strain BL21(DE3) as described previously (Brinkmann et al., 1997). The purified proteins were then used as acceptors in the XT activity assay.
XT activity assay
The method for determination of XT activity is based on the incorporation of [14C]D-xylose with recombinant [Val36,Val38]1,[Gly92,Ile94]2bikunin as acceptor. The reaction mixture for the assay, in a total volume of 100 µl, was: 50 µl of XT solution, 25 mmol/l 4-morpholine ethanesulphonic acid (pH 6.5), 25 mmol/l KCl, 5 mmol/l KF, 5 mmol/l MgCl2, 5 mmol/l MnCl2, 1.0 µmol/l UDP-[14C]D-xylose (Du Pont, Homburg, Germany) and 1.5 µmol/l recombinant [Val36,Val38]1,[Gly92,Ile94]2bikunin (Weilke et al., 1997). After incubation for 1 h at 37°C, the reaction mixtures were placed on nitrocellulose discs. After drying, the discs were washed for 10 min with 10% trichloroacetic acid and three times with 5% trichloroacetic acid solution. Incorporated radioactivity was quantified after the addition of 5 ml of scintillation mixture (Beckman Coulter, Fullerton, CA, USA) using an LS500TD liquid scintillation counter (Beckman Coulter). The enzyme activity was expressed in units (1 U = 1 µmol of incorporated xylose*min-1). The linear range of the XT activity assay was determined as 0.02–3.5 mU/l (Weilke et al., 1997). To measure within the linear range, samples were diluted with PBS supplemented with 1% HSA. The assay linearity and the dilution procedure were validated using an enriched human XT solution which was prepared as described previously (Götting et al., 1998).
For the investigation of the influence of the glycosaminoglycans on the XT activity, commercial glycosaminoglycan preparations were used: chondroitin 6-sulphate from shark cartilage (Sigma), chondroitin 4-sulphate from bovine trachea (Sigma), heparan sulphate from porcine intestinal mucosa (Sigma), heparin from porcine intestinal mucosa (Sigma) and pharmaceutical heparin from porcine intestinal mucosa (Ratiopharm, Ulm, Germany). The protamine chloride preparation was isolated from salmon (Sigma). In order to investigate the influence of cationic ions, the samples were diluted in PBS supplemented with 1% HSA and with high manganese and magnesium concentrations. The final reaction mixtures contained 2-fold (10 mmol/l MgCl2 and 10 mmol/l MnCl2) or 3-fold (15 mmol/l MgCl2 and 15 mmol/l MnCl2) higher concentrations of manganese and magnesium in comparison with the standard reaction mixture. The influence of glycosaminoglycans on the XT activity was then assayed as described above.
Determination of total protein concentration in cell culture medium and follicular fluid
The total protein concentration was determined using the Bicinchoninic Acid Protein Assay Kit (Sigma). Free amino acids in the samples were removed prior to protein determination by ultrafiltration with Microcon 3000 tubes (Millipore, Eschborn, Germany) according to the manufacturer’s instructions.
Preparation of cell lysates
Cells were mechanically lysed in order to quantify intracellular XT activity. Cells were detached using trypsin/EDTA solution, centrifuged at 1000 g for 10 min and then gently washed twice with PBS supplemented with 1% HSA. The number of viable cells was determined using trypan blue exclusion, the typical viability at this stage was >70%. Glass beads (425–600 µm; Sigma) were added, and the samples were vigorously shaken on a mixer for 2 min followed by 10 min incubation on ice. This procedure was repeated twice and the degree of lysis was optically controlled. Samples were then centrifuged at 1000 g for 10 min, and the supernatant stored at –20°C.
Gel filtration chromatography
Samples were applied at 1 ml/min to a TSK G3000 SW column (30 cmx7.5 mm, 10 µm particle size TosoHaas, Montgomeryville, PA, USA) which had previously been equilibrated with buffer 1 (25 mmol/l sodium acetate, 150 mmol/l NaCl, pH 6.0). Proteins were eluted with the same buffer using the high performance liquid chromatography work station Biocad Sprint (Perseptive Biosystems, Framingham, MA, USA). 500 µl fractions were collected and tested for XT activity and glycosaminoglycans. Column calibration was performed using thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and ribonuclease A (13.7 kDa).
Ion exchange chromatography
Fractions obtained during size exclusion chromatography were applied to ion exchange chromatography after desalting by diafiltration with YM1 cellulose membranes (Millipore, Eschborn, Germany) and ultrafiltration cells. After concentration of the desalted protein solution using analogous techniques, the samples were subjected to ion exchange chromatography. The specimens were applied to the POROS HQ20 column (4.6x50 mm; Perseptive Biosystems) previously equilibrated with buffer 2 (25 mmol/l Tris–HCl, pH 9.0) at a low flow rate of 10 ml/min. The column was washed with 50 ml of buffer 2 and the adsorbed protein was eluted by a linear gradient using the same buffer containing 0.0–0.3 mol/l NaCl (50 ml), followed by another linear gradient using buffer 2 containing 0.3–1.0 mol/l NaCl (30 ml) and a step using buffer 2 with 2.6 mol/l NaCl (30 ml). Fractions were collected and assayed for XT activity and proteoglycans.
Heparin affinity chromatography
Follicular fluid samples were passed through a 0.2 µm filter and then applied to a POROS HE20 column (4.6x50 mm) equilibrated with buffer 3 (25 mmol/l sodium acetate, pH 6.0), at a flow rate of 10 ml/min. After washing the column with 5 ml of buffer 3, the XT activity was eluted with the same buffer containing NaCl. The NaCl concentration was increased by a linear gradient of 0.0–1.5 mol/l (25 ml) and a cleaning step with 1.5 mol/l NaCl (5 ml). Fractions of 1.5 ml each were collected, and the XT activity was measured.
Degradative methods
Proteoglycan-containing samples were digested with protease-free chondroitinase ABC (Seikagaku Kogyo, Tokyo, Japan) at 37°C for 8 h according to the manufacturer’s recommendations. The samples were then subjected to immunochemical dot blot analysis.
Immunochemical analysis
For the immunochemical analysis of the fractions obtained during size exclusion chromatography and ion exchange chromatography, the proteins were desalted and concentrated by ultrafiltration with Microcon 3000 tubes (Millipore) according to the manufacturer’s instructions. The samples were then spotted onto polyvinylidene difluoride membranes, and non-specific antibody-binding sites were blocked with 2% bovine serum albumin in 0.1 Tris–HCl, pH 7.5, for 1 h at 25°C. The membranes were then incubated for 1 h with the primary antibody in 50 mmol/l phosphate, 150 mmol/l NaCl, 0.05% Tween 20, pH 7.4, at a 1:500 dilution. For the detection of heparan sulphate-containing proteoglycans, a mouse anti-heparan sulphate monoclonal antibody (clone 7E12; Roche, Mannheim, Germany) was used as primary antibody. Chondroitin sulphate proteoglycans were detected after chondroitinase ABC digestion using mouse monoclonal antibodies raised against chondroitin 6-sulphate (Chemicon, Temecula, CA, USA) and chondroitin 4-sulphate (Chemicon). Versican and versican-like proteins were identified using the mouse monoclonal anti-human large proteoglycan antibody (clone 2-B-1; Seikagaku, Tokyo, Japan). After intensive washing steps, the bound antibody was detected using a horse-radish peroxidase-conjugated goat anti-mouse immunoglobulin at a 1:1000 dilution. The blot was developed using 4-chloro-1-naphthol (Sigma).
Statistical analysis
Statistical analysis was performed using t-test and Kolmogoroff–Smirnoff test. P 
0.05 was considered significant.
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Determination of XT activity in human follicular fluid
We determined XT activities in human follicular fluid samples (n = 108) obtained from 53 patients undergoing IVF procedures. The mean (SD) XT activity and 90% range were 17.7 (7.49) and 7.46–29.4 mU/l respectively. The total protein concentration in the follicular fluid samples was determined as 70.7 g/l (SD 7.2). In serum specimens obtained from all patients investigated, the XT activities were all within the normal range (mean value 0.93 mU/l, SD 0.21) as determined for blood donors (Weilke et al., 1997
; Götting et al., 1999).
Secretion of xylosyltransferase by cultivated human granulosa cells and other cell lines derived from the female reproductive tract
Granulosa–lutein cells isolated from follicular fluid samples were cultivated in 60 mm dishes, and the secretion of XT was determined after 4 days. XT activities were also analysed in other human cell lines derived from tissues from the female reproductive tract (Table I). The highest XT activity was observed in the culture medium conditioned by human granulosa–lutein cells (mean XT activity 14.52 µU/106 cells). Use of culture medium supplemented with androstenedione did not have any significant influence on the XT production of all cell lines investigated (data not shown). In all experiments, >90% of the total XT activity was found secreted in the culture supernatant and only 10% was located in the cell lysates.
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Partial purification of XT and proteoglycans from human follicular fluid
Follicular fluid was separated by size exclusion chromatography under non-reducing and non-denaturating conditions using a TSK G3000 SW column. Two XT activity maximums were detected at 600 and 120 kDa (Figure 1
). The glycosaminoglycan composition of each fraction was qualitatively determined after chondroitinase ABC treatment using antibodies directed against chondroitin 6-sulphate, chondroitin 4-sulphate and heparan sulphate. Heparan sulphate-containing proteoglycans were detected in fractions at medium molecular weight, whereas a strong signal with chondroitin sulphate antibodies was observed in fractions containing the large proteoglycans. In order to identify the main follicular fluid proteoglycan, which is associated with the XT, the fractions containing the large proteoglycans were separated by ion exchange chromatography on a POROS HQ20 column (Figure 2). After binding to the resin, the chondroitin sulphate-containing proteoglycans were eluted under conditions containing 0.5–0.7 mol/l NaCl. The condroitin sulphate proteoglycans were identified using immuno dot blot analysis after chondroitinase ABC treatment of the samples. All fractions with chondroitin sulphate proteoglycans also stained positive in an immunoblot using anti-versican antibodies.
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Effect of glycosaminoglycans and protamine chloride on the XT present in human follicular fluid
In order to investigate a potential inhibitory effect of the end products on the enzymatic activity of the XT present in follicular fluid, the glycosaminoglycans chondroitin 6-sulphate, chondroitin 4-sulphate, heparan sulphate and heparin were added to follicular fluid samples, and the XT activity was determined (Figure 3
). The effector concentrations used covered a broad range from 0.1 to 1000 µg/ml. The addition of chondroitin 4-sulphate at various concentrations had no effect on the XT activity, whereas the addition of chondroitin 6-sulphate resulted in a 36% inhibition of the enzyme activity at high concentrations (100 and 1000 µg/ml). However, this observed reduction was not statistically significant. Large amounts of heparan sulphate in the follicular fluid samples also revealed up to 70% inhibition of XT activity. The strongest inhibitory effect of glycosaminoglycans on the XT activity in follicular fluid was observed when coagulatory active heparin was applied. The addition of heparin at a concentration of 1 IU/ml (specific activity: 154 IU/mg; Sigma) resulted in >70% reduced enzyme activity, whereas heparin concentrations of 
10 IU/ml led to a complete loss of enzyme activity (Figure 3). Using a different heparin preparation (Ratiopharm), which has a higher specific activity and is used for therapeutic application in humans, similar results were obtained (data not shown).
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The addition of large negatively charged molecules at high concentrations may result in a potential complex formation with the cations leading to a lower concentration of free manganese and magnesium in the samples. In order to exclude an artificial inhibition of XT activity due to a lack of cationic ions, 2- and 3-fold higher concentrations of manganese and magnesium were added to the follicular fluid specimens and the XT activity was again determined. In no case was a significant restoration of XT activity observed (data not shown).
The addition of protamine chloride (10, 100 or 1000 µg/ml) resulted in a >2-fold increase (P < 0.05) in the XT activity in follicular fluid (Figure 4). Similar results were obtained using partially purified XT secreted from cultured JAR choriocarcinoma cells (unpublished data).
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Binding of XT from human follicular fluid to immobilized heparin
In order to investigate whether the XT binds to immobilized heparin, we subjected follicular fluid specimens to heparin affinity chromatography (Figure 5
). A total of 400 µl of a pooled follicular fluid sample was applied to a POROS HE20 column. More than 75% of the total protein but <2% of the XT activity passed through the column. The XT activity bound to the heparin matrix emerged at 
0.65 mol/l NaCl, whereas most of the other proteins bound to the matrix were eluted at lower salt concentrations (0.4–0.55 mol/l NaCl).
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The ovarian follicle is one of the most rapidly remodelled human organs undergoing a continuous series of events involving cell replication and differentiation, functional changes, apoptosis and fluid accumulation. The creation of a fluid-filled compartment during follicular development is mainly achieved by the enormous water-binding capacity of hyaluronate and proteoglycan molecules in the follicular fluid. XT is the initial enzyme involved in the biosynthesis of the polyanionic glycosaminoglycan chains in proteoglycans, and the activity of this enzyme is therefore closely related to the biological function of proteoglycans in the human follicular fluid. In contrast to almost all other glycosyltransferases, XT is secreted into the extracellular space (Götting et al., 1998
). More than 90% of the total XT activity of human cultured cells has been found to accumulate in the culture supernatant, whereas <10% is bound to intracellular membranes (Götting et al., 2000b). However, the addition of the glycosaminoglycan chains to the core protein in proteoglycans occurs in vivo in intracellular compartments such as the endoplasmatic reticulum and the Golgi apparatus (Hoffmann et al., 1984; Kimura et al., 1984; Vertel et al., 1993). In previous studies, we have shown that the XT is secreted into the extracellular matrix together with chondroitin sulphate proteoglycans (Kähnert et al., 1991; Götting et al., 1999) and, that the XT activity is a marker of the actual rate of proteoglycan biosynthesis (Götting et al., 1999).
In follicular fluid samples of women undergoing IVF procedures, the mean XT activity was determined as 17.7 mU/l (Table I
). This is the highest XT activity ever found in body fluids, and it is 19-fold higher than the XT activity in blood (Weilke et al., 1997; Götting et al., 1999) and 6-fold higher than the XT activity in seminal plasma (Götting et al., 2002). However, it has to be taken into consideration that the observed elevated XT activity might also be a result of the ovarian stimulation during the IVF procedures. We have shown in previous studies that XT is secreted into the extracellular matrix together with large proteoglycans and that an elevated proteoglycan biosynthesis during fibrotic and sclerotic processes results in an increased XT activity in blood (Götting et al., 1999). The XT activity in the blood of all patients investigated was within the normal range, and no differences between the blood XT activities in the IVF patients and normally cycling female blood donors were observed. Under the presumption that an elevation of XT activity in the follicular fluid due to the ovarian stimulation would also result in an increased XT activity in blood, our findings are an indicator that the observed high XT activities are not a consequence of the hormone treatment. However, in order to prove that the measured XT activities in the follicular fluid are not a side-effect of the ovarian stimulation, follicular fluid samples from normal antral follicles will have to be investigated in the future to determine the XT activity during normal folliculogenesis.
In order to localize the cells responsible for the high XT activities in follicular fluid, we isolated human granulosa–lutein cells and monitored the XT activity in the cell culture supernatant. The amount of XT secreted by granulosa cells was 5-fold higher than that of JAR choriocarcinoma cells which have been the cell line with the highest XT production investigated so far. This clearly indicates that granulosa–lutein cells are the main source for the high XT activities observed in human follicular fluid.
Separation of follicular fluid samples by size exclusion chromatography under non-reducing and non-denaturing conditions revealed two XT activity maximums at 600 and 120 kDa. The major activity peak at 120 kDa corresponds to the molecular mass of the XT which has been determined for the XT purified from JAR cell culture supernatant (Kuhn et al., 2001). In previous studies, we have shown that the XT is secreted into the extracellular space with large chondroitin sulphate proteoglycans. In order to determine the type of proteoglycan associated with the XT in human follicular fluid, the fractions containing the large proteoglycans were subjected to ion exchange chromatography, and the glycosaminoglycan chains were identified by immunoblot staining. Our results show that follicular XT is associated with large chondroitin sulphate proteoglycans. These large proteoglycans react strongly with antibodies raised against human versican, indicating that one of these proteoglycans might be versican or a versican-like chondroitin sulphate proteoglycan. These results are in accordance with recent results from another group, who identified and characterized a versican-like proteoglycan with chondroitin sulphate moieties in human follicular fluid (Eriksen et al., 1999). The XT binds to the glycosaminoglycan chains rather than to the core protein as it can be released from the proteoglycan by chondroitinase ABC digestion. Therefore, other large follicular fluid proteoglycans such as perlecan may also be associated with the XT, and future experiments will have to clarify which of the proteoglycans in human follicular fluid is the main binding partner.
XT was previously shown to be a regulatory factor of the chondroitin sulphate biosynthesis (Rodén, 1980). In order to investigate the end product inhibition of the XT in human follicular fluid, we applied the glycosaminoglycans chondroitin sulphate, heparan sulphate and heparin to follicular fluid samples and measured the XT activity. The addition of chondroitin 4-sulphate did not have any effect on the XT activity, whereas a slight inhibition of the enzyme activity was observed after the addition of high doses of chondroitin 6-sulphate. Reduced XT activities were also observed after the addition of heparan sulphate at high concentrations. Furthermore, an artificial inhibition of XT activity due to a lack of manganese and magnesium ions in the samples after addition of the polyanionic effector molecules could be excluded, as no restoration of XT activity was observed after the supplementation of additional cations.
The addition of coagulatory active heparin resulted in a complete inhibition of XT activity even at lower concentrations. Furthermore, the follicular fluid XT was eluted in the heparin affinity chromatography at a high salt concentration, indicating a very strong interaction of heparin with XT. The biological role of this strong influence of heparin on the follicular fluid XT is not yet known and will have to be clarified in future experiments. Recent studies have been focusing on the presence of haemostatic proteins in follicular fluid (Gentry et al., 2000) and the role of sulphated proteoglycans in maintaining the ovarian hypocoagulative state (Shimada et al., 2001). Tissue damage occurs during the mammalian ovulatory process, leading to an increased level of tissue factor in follicular fluid and a subsequent activation of the extrinsic coagulation pathway. However, follicular fluid must block this formation of fibrin and preserve the fluidity until the release of the oocyte. A combination of haemostatic proteins such as antithrombin, tissue factor pathway inhibitor (TFPI) and protein C and sulphated proteoglycans has been proposed to maintain the hypocoagulative state of the follicular fluid (Shimada et al., 2001). Furthermore, binding of sulphated proteoglycans isolated from follicular fluid to antithrombin has been shown to cause hypocoagulability (Stangroom and Weevers, 1962; Andrade-Gordon et al., 1992; Hosseini et al., 1996). Although little is known about the functional interaction of follicular fluid components during the coagulation pathway, a stringent end product inhibition of XT activity by coagulatory active heparin may play a role in the maintenance of the haemostatic potential of the follicular fluid.
Protamine chloride has been shown to serve as an activator of enzymatic activity in glycosyltransferases and sulfotransferases (Habuchi and Miyata, 1980; Habuchi et al., 1993, 1995). We have shown in previous studies that these arginine-rich proteins interact with XT secreted by JAR choriocarcinoma cells and that protamine chloride affinity chromatography could be employed for the purification of this enzyme (Kuhn et al., 2001). Therefore, we investigated the effect of protamine chloride on the XT activity in human follicular fluid. The addition of protamine chloride resulted in a dose-dependant increase in XT activity. Similar results were obtained when using the XT purified from JAR cell culture supernatant. This indicates that the xylosyltransferases from both sources show a similar response to stimulatory and inhibitory agents, although the molecular mechanisms for these properties are not understood.
Large chondroitin sulphate proteoglycans isolated from human follicular fluid have been shown to serve as enhancers of sperm motility and fertilization potential and to positively effect the outcome of in-vivo and in-vitro fertilization procedures. However, the functional role of proteoglycans in the seminal plasma and the follicular fluid is still under investigation and is not yet understood in detail. As we have demonstrated previously, XT is secreted into the extracellular space together with chondroitin sulphate proteoglycans. Thus, XT activity reflects the actual rate of proteoglycan biosynthesis. The recent isolation of the human XT (Kuhn et al., 2001) and the cloning of the corresponding genes (Götting et al., 2000b) are prerequisites for achieving detailed information on the function of XT in human follicular fluid.
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We would like to thank Anja Reuße-Kaup and Alexandra Adam for excellent technical assistance and Grainne Delany for linguistic advice. The work was supported by the Deutsche Forschungsgemeinschaft, grant BR1226/5-1.
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3 To whom correspondence should be addressed. E-mail: cgoetting{at}hdz-nrw.de
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Andrade-Gordon, H., Wang, S.Y. and Strickland, S. (1992) Heparin-like activity in porcine follicular fluid and rat granulosa cells. Thromb. Res., 66, 475–487.[Web of Science][Medline]
Bellin, M.E., Ax, R.L., Laufer, N., Tarlatzis, B.C., DeCherney, A.H., Feldberg, D. and Haseltine, F.P. (1986) Glycosaminoglycans in follicular fluid from women undergoing in vitro fertilization and their relationship to cumulus expansion, fertilization, and development. Fertil. Steril., 45, 244–248.[Web of Science][Medline]
Brinkmann, T., Weilke, C. and Kleesiek, K. (1997) Recognition of acceptor proteins by UDP-D-xylose: proteoglycan core protein ß-D-xylosyltransferase. J. Biol. Chem., 272, 11171–11175.
Butler, M. and Dawson, M. (1992) Cell Culture. Labfax, Bios Scientific Publishers, Oxford.
Eriksen, G.V., Malmström, A., Uldbjerg, N. and Huszar, G. (1994) A follicular fluid chondroitin sulfate proteoglycan improves the retention of motility and velocity of human spermatozoa. Fertil. Steril., 62, 618–623.[Web of Science][Medline]
Eriksen, G.V., Malmström, A. and Uldbjerg, N. (1997) Human follicular fluid proteoglycans in relation to in vitro fertilization. Fertil. Steril., 68, 791–798.[Web of Science][Medline]
Eriksen, G.V., Carlstedt, I., Mörglin, M., Uldbjerg, N. and Malmström, A. (1999) Isolation and characterization of proteoglycans from human follicular fluid. Biochem. J., 340, 613–620.
Gentry, P.A., Plante, L., Schroeder, M.O.B., LaMarre, J., Young, J.E. and Dodds, W.G. (2000) Human ovarian follicular fluid has functional systems for the generation and modulation of thrombin. Fertil. Steril., 73, 848–854.[Web of Science][Medline]
Götting, C., Kuhn, J., Brinkmann, T. and Kleesiek, K. (1998) Xylosylation of alternatively spliced isoforms of Alzheimer APP by xylosyltransferase. J. Prot. Chem., 17, 295–302.[Web of Science][Medline]
Götting, C., Sollberg, S., Kuhn, J., Weilke, C., Huerkamp, C., Brinkmann, T., Krieg, T. and Kleesiek, K. (1999) Serum xylosyltransferase: a new biochemical marker of the sclerotic process in systemic sclerosis. J. Invest. Dermatol., 112, 919–924.[Web of Science][Medline]
Götting, C., Kuhn, J., Sollberg, S. Huerkamp, C., Brinkmann, T., Krieg, T. and Kleesiek, K. (2000a) Elevated serum xylosyltransferase activity correlates with a high level of hyaluronate in patients with systemic sclerosis. Acta Derm. Venereol., 80, 60–61.[Web of Science][Medline]
Götting, C., Kuhn, J., Zahn, R., Brinkmann, T. and Kleesiek, K. (2000b) Molecular cloning and expression of human UDP-D-xylose:proteoglycan core protein ß-D-xylosyltransferase and its first isoform XT-II. J. Mol. Biol., 304, 517–528.[Web of Science][Medline]
Götting, C., Kuhn, J., Brinkmann, T. and Kleesiek, K. (2002) Xylosyltransferase activity in seminal plasma of infertile men. Clin. Chim. Acta, 317, 199–202.[Web of Science][Medline]
Habuchi, O. and Miyata, K. (1980) Stimulation of glycosaminoglycan sulfotransferase from chick embryo cartilage by basic proteins and polyamines. Biochem. Biophys. Acta, 616, 208–217.[Medline]
Habuchi, O., Matsui, Y., Kotoya, Y., Aoyama, Y., Yasuda, Y. and Noda, M. (1993) Purification of chondroitin 6-sulfotransferase secreted from cultured chick embryo chondrocytes. J. Biol. Chem., 268, 21968–21974.
Habuchi, H., Habuchi, O. and Kimata, K. (1995) Purification and characterization of heparan sulfate 6-sulfotransferase from the culture medium of Chinese hamster ovary cells. J. Biol. Chem., 270, 4172–4179.
Hamamah, S., Wittemer, C., Bethélemy, C., Richet, C., Zerimech, F., Royere, D. and Degand, P. (1996) Identification of hyaluronic acid and chondroitin sulfates in human follicular fluid and their effects on human sperm motility and the outcome of in vitro fertilization. Reprod. Nutr. Dev., 36, 43–52.[Web of Science][Medline]
Herold, B.C., Visalli, R.J., Susmarski, N., Brandt, C.R. and Spear, P.G. (1994) Glycoprotein C-independent binding of herpes simplex virus to cells requires cell surface heparan sulphate and glycoprotein B. J. Gen. Virol., 75, 1211–1222.
Hoffmann, H.P., Schwartz, N.B., Rodén, L. and Prockop, D.J. (1984) Location of xylosyltransferase in the cisternae of the rough endoplasmic reticulum of embryonic cartilage cells. Connect. Tissue Res., 12, 151–163.[Web of Science][Medline]
Hosseini, G., Liu, J. and de Agostini, A.I. (1996) Characterization and hormonal modulation of anticoagulant heparan sulfate proteoglycans synthesized by rat ovarian granulosa cells. J. Biol. Chem., 271, 22090–22099.
Kähnert, H., Paddenberg, R. and Kleesiek, K. (1991) Simultaneous secretion of xylosyltransferase and chondroitin sulphate proteoglycan in chondrocyte culture. Eur. J. Clin. Chem. Clin. Biochem., 29, 624–625.
Kimura, J.H., Lohmander, L.S. and Hascall, V.C. (1984) Studies on the biosynthesis of cartilage proteoglycan in a model system of cultured chondrocytes from the Swarm rat chondrosarcoma. J. Cell. Biochem., 26, 261–278.[Web of Science][Medline]
Kjellen, L. and Lindahl, U. (1991) Proteoglycans: structures and interactions. Annu. Rev. Biochem., 60, 443–475.[Web of Science][Medline]
Kuhn, J., Götting, C., Schnölzer, M., Kempf, T., Brinkmann, T. and Kleesiek, K. (2001) First isolation of human UDP-D-xylose:proteoglycan core protein ß-D-xylosyltransferase secreted from cultured JAR choriocarcinoma cells. J. Biol. Chem., 276, 4940–4947.
Rodén, L. (1980) Structure and metabolism of connective tissue proteoglycans. In Lennarz, W.J. (ed.), The Biochemistry of Glycoproteins and Proteoglycans. Plenum Press, New York, 269pp.
Roughley, P.J. and Lee, E.R. (1994) Cartilage proteoglycans: structure and potential functions. Microsc. Res. Tech., 28, 385–397.[Web of Science][Medline]
Ruoslahti, E. (1989) Proteoglycans in cell regulation. J. Biol. Chem., 264, 13369–13372.
Shimada, H., Kasakura, S., Shiotani, M., Nakamura, K., Ikeuchi, M., Hoshino, T., Komatsu, T., Ihara, Y., Sohma, M., Maeda, Y. et al. (2001) Hypocoagulable state of human preovulatory ovarian follicular fluid: role of sulfated proteoglycan and tissue factor pathway inhibitor in the fluid. Biol. Reprod., 64, 1739–1745.
Silbert, J.E. and Sugumaran, G. (1995) Intracellular membranes in the synthesis: transport, and metabolism of proteoglycans. Biochim. Biophys. Acta, 1241, 371–384.[Medline]
Stangroom, J.E. and Weevers, R.G. (1962) Anticoagulant activity of equine follicular fluid. J. Reprod. Fertil., 3, 269–282.[Medline]
Vertel, B.M., Walters, L.M., Flay, N., Kearns, A.E. and Schwartz, N.B. (1993) Xylosylation is an endoplasmic reticulum to Golgi event. J. Biol. Chem., 268, 11105–11112.
Weilke, C., Brinkmann, T. and Kleesiek K. (1997) Determination of xylosyltransferase activity in serum with recombinant human bikunin as acceptor. Clin. Chem., 43, 45–51.
Yanagishita, M. and Hascall, V.C. (1979) Biosynthesis of proteoglycans by rat granulosa cells cultured in vitro. J. Biol. Chem., 254, 12355–12364.
Yee, B. and Cummings, L.M. (1988) Modification of the sperm penetration assay using human follicular fluid to minimize false negative results. Fertil. Steril., 50, 123–128.[Web of Science][Medline]
Yoshimura, Y. and Wallach, E.E. (1987) Studies of the mechanism(s) of mammalian ovulation. Fertil. Steril., 47, 22–34.[Web of Science][Medline]
Submitted on June 2, 2002; accepted on September 30, 2002
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