2 University of Rostock, Institute of Medical Biochemistry and Molecularbiology, Schillingallee 70, 18057 Rostock, Germany; 3 Unit of Reproductive Biology, Research Institute of the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany; and 4 Unit of Molecular Biology, Research Institute of the Biology of Farm Animals, Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany
Received on February 19, 2004; revised on May 25, 2004; accepted on May 25, 2004
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Abstract |
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Key words: galectin-1 / granulosa cells / pig ovaries / progesterone synthesis
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Introduction |
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All galectins studied so far have the characteristics of cytoplasmic proteins, such as acetylated N-termini, the absence of a disulfide bond in spite of the presence of several cysteine residues, and a lack of glycosylation although a potential glycosylation site is available (Drickamer, 1993; Hirabayashi, 1993
). In general, galectins apparently are synthesized without any signal peptide sequence (Couraud et al., 1989
; Ohyama et al., 1986
) suggesting that they are designed as intracellular proteins. Although devoid of signal peptide, cytosolic gal-1 can be externalized from cells by a mechanism independent of the classical secretory pathway (Cooper and Barondes, 1990
; Lindstedt et al., 1993
) and is then bound by appropriate glycoepitopes on cell surface or extracellular matrix glycoproteins and glycolipids in an autocrine manner.
The binding of gal-1 to cell surface glycoconjugates suggests that the lectin is implicated in diverse cellular functions, such as cell adhesion to the endothelium (Allen et al., 1997; Barondes et al., 1994
), cell growth regulation (Adams et al., 1996
; Kopitz et al., 2001
; Wells and Mallucci, 1991
), immunosuppression (Offner et al., 1990
; Rabinovich et al., 1999
), and signaling events (Maeda et al., 2003
; Rabinovich et al., 2000
; Walzel et al., 2002
). Gal-1 is synthesized and quantitatively externalized by Chinese hamster ovary cells and associates with cell surface glycoconjugates where the carbohydrate-binding activity is stabilized (Cho and Cummings, 1995
). Gal-1 mRNA is abundantly expressed in mouse reproductive organs, such as uterus and ovary (Choe et al., 1997
). Uterine expression of gal-1 mRNA is differentially regulated by ovarian steroids progesterone and estrogen and correlated with the implantation process. The synthesis of gal-1 in the trophectoderm of expanded blastocyst immediately prior to implantation (Poirer et al., 1992
) suggests that it may be implicated in the interaction between the embryo and the extracellular matrix of uterine endometrium. Because gal-1 mRNA is abundantly expressed in mouse ovary (Choe et al., 1997
), it is entirely conceivable that the lectin can influence fertility by changing granulosa celloocyte interaction (Hirshfield, 1997
).
Progesterone is an important ovarian hormone to prepare the uterus for accepting the invading blastocyst/zygote for implantation and the subsequent maintenance of the pregnant stage (Hadley, 1995). Because granulosa cells secrete progesterone and estradiol (Campbell et al., 1996
; Gutierrez et al., 1997
), the study of granulosa cell function under defined conditions in vitro permits a detailed examination of the regulation of progesterone synthesis and its key enzymes by several trophic hormones and paracrine factors. Here we demonstrate the cytosolic and membrane-associated localization of gal-1 in granulosa cells from pig ovaries and provide evidence for the involvement of the lectin in regulation of progesterone synthesis of cultured ovarian granulosa cells. The gal-1-mediated decrease of follicle-stimulating hormone (FSH)-stimulated progesterone synthesis of granulosa cells and the absence of inhibitory effects on forskolin, dibutyryl cAMP (db-cAMP), and pregnenolone-enhanced progesterone production suggest that the lectin modulates the hormone receptor interaction.
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Results |
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To study whether the lectin interferes with FSH-enhanced progesterone production, granulosa cells were stimulated in serum-free medium with 10 ng/ml FSH in the presence of gal-1 at 3.3 µg/ml, 9.9 µg/ml, and 19.8 µg/ml. As demonstrated in Figure 6, gal-1 decreased the FSH-enhanced cellular progesterone production in a concentration-dependent manner. Gal-1 at 9.9 µg/ml and 19.8 µg/ml significantly reduced the cellular progesterone production to 39% and 28% relative to control cultures (c) stimulated with 10 ng/ml FSH (100%). In the presence of 30 mM lactose as a disaccharidic competitor, the FSH-stimulated progesterone production was restored and approximately in the range of control cultures.
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Discussion |
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LacNAc is the basic ligand recognized by conserved CRDs, however, gal-1 binds with increased avidity to multiple LacNAc sequences presented on branched N-linked or to poly-LacNAc chains on N- and/or O-linked glycans. The increased binding avidity for cellular glycoconjugates may be the reason that lactose nonsignificantly reduced the stimulating effects of gal-1 on cell proliferation. Gal-1 significantly reduced FSH-enhanced cellular progesterone synthesis in a concentration-dependent manner. Lactose as a disaccharidic competitor decreased this galectin-mediated inhibitory effect, and FSH-stimulated progesterone production was restored. Therefore we conclude that by recognition of low-affinity binding sites, gal-1 interferes the FSHreceptor interaction, leading to reduced FSH-enhanced cellular progesterone production. This assumption is supported by the data demonstrating no inhibitory effects of gal-1 when cellular progesterone production was enhanced by nonreceptor-mediated mechanisms.
Stimulation of cellular progesterone production with the cell-permeable derivative db-cAMP and forskolin believed to activate adenylate cyclase via its interaction with the catalytic subunit (Seamon and Daly, 1981) was not reduced by gal-1. On the other hand, binding of gal-1 to the glycosylated unique FSH ß-chain, which confers biological specificity to thyrotropin, lutropin, and FSH could hinder receptor recognition. As demonstrated in Figure 3, gal-1 failed to recognize FSH on blots. Furthermore, granulosa cells respond with increased progesterone production when exogenous pregnenolone was added as a substrate, and gal-1 did not reduce the stimulatory effect. From the absence of effects on basal, db-cAMP, and forskolin-enhanced progesterone synthesis we conclude that the lectin did not reduce the uptake of cholesterol into the mitochondria, where progesterone synthesis takes place. It is also conceivable that gal-1 inducing down-regulation of FSH receptors by endocytosis is a possible mechanism of action at the cell surface. Gal-3 mediates endocytosis of ß-1 integrins, advanced glycation end products, and acetylated low-density lipoproteins in Chinese hamster ovary cells. As an organizer of lipid rafts, a number of signaling molecules are sequestered in these raft domains (Ochieng et al., 2004
).
Although nonsignificantly inceased, there is evidence for gal-1 as an autocrine modulator because lactose in the absence of added gal-1 increased FSH-enhanced progesterone synthesis (Figure 6) and 3ß-HSD gene transcription (Figure 8). The gal-1-mediated inhibition of FSH-induced P450SCC and 3ß-HSD gene transcription identified the endogenous ß-galactoside-binding protein as a negative regulator of steroid synthesis in ovaries. It has been demonstrated that cAMP stimulates P450SCC mRNA accumulation in porcine granulosa cells (Picton et al., 1999; Urban et al., 1991
). In human (Inoue et al., 1988
) and bovine P450SCC genes (Ahlgren et al., 1990
) upstream regulatory elements have been identified to be responsible for cAMP-dependent transcription. The FSH receptor signaling pathway is coupled via heterotrimeric G-proteins to adenylate cyclase that increases intracellular cAMP levels (Conti, 2002
; Richards, 1994
) leading to enhanced P450SCC gene transcription and progesterone synthesis in granulosa cells (Brentano et al., 1992
). The data presented in Figure 8 that 3ß-HSD transcripts exceed those of P450SCC and granulosa cells did respond to pregnenolone as an exogenous substrate with increased progesterone synthesis are consistent suggesting that conversion of cholesterol to pregnenolone catalyzed by P450SCC is considered to be the rate-limiting step for progesterone synthesis.
The data presented here contribute to define a role for gal-1 as a modulator of FSH-enhanced progesterone production in porcine granulosa cells.
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Materials and methods |
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Purification of gal-1 from bovine placenta
Gal-1 was prepared from bovine placenta by lactose extraction with ethylenediaminetetraacetic acid (EDTA)-Me phosphate buffered saline (PBS) (20 mM sodium phosphate, pH 7.2, 150 mM NaCl, 4 mM 2-mercaptoethanol, 2 mM EDTA) and purified by sequential affinity chromatography on asialofetuin Sepharose 4B (Hirabayashi and Kasai, 1984) followed by lactosyl agarose. The protein was purified to homogeneity by anion exchange chromatography on a Resource Q column (Walzel et al., 2000
).
Generation and purification of gal-1 pAbs
Antibodies were produced in rabbits immunized with human placental gal-1. From serum the IgG fraction was separated by affinity chromatography on protein Aagarose. For separation of gal-1 specific antibodies, the IgG fraction was passed through gal-1 agarose prepared by coupling the lectin to NHS-activated Sepharose 4 Fast Flow according to the manufacturer's instructions. The immunoaffinity-purified pAb cross-reacts with gal-1 from pig and bovine as detected by antibody binding to the proteins on blots as well as after spotting the lectins to nitrocellulose membranes (data not shown).
Cell preparation and culture
Tissue collection and isolation of pig granulosa cells were carried out as described previously (Tiemann et al., 1996). The ovaries derived from pigs of a commercial slaughterhouse were collected in PBS (pH 7.4). Then the cells were aspirated from nonatretic preovulatory follicles (>3 mm in diameter) by means of a syringe and were flushed with PBS (pH 7.4). To disperse cell clusters, cell aggregates were resuspended several times through a pasteur pipette. The cells were collected by centrifugation at 200 x g for 5 min and resuspended in TCM 199 supplemented with 10% fetal calf serum (FCS) and 1% ABAM. Trypan blue exclusion revealed that cell viability was about 70%.
The cells were plated in 24-well plastic plates. After 24 h, the culture medium was changed, and the cells were cultured in complete medium at 37°C in humidified 95% air5% CO2 for a further 24 h (5070% confluency). Then the media were replaced with 1 ml/well serum-free medium supplemented with 1% ABAM and 1% ITS. The monolayers were incubated with either 10 ng/ml FSH in the absence or presence of 3.3 µg/ml, 9.9 µg/ml, or 19.8 µg/ml gal-1 ± 30 mM lactose, or with 10 µM forskolin, 2.5 µM pregnenolone, or 1 mM db-cAMP ± gal-1 as indicated in the figure legends. Control cultures were incubated in medium alone. After 48 h, the cells were centrifuged at 800 x g for 10 min, media were removed and stored at 80°C for progesterone determination. The cell number from each well was counted as will be described. For estimation of P450SCC and 3ß-HSD mRNA, media were removed for measuring progesterone accumulation and cell monolayers were stored at 80°C until use.
Cell viability test and counting
For estimation of cell viability, cells were plated in 24-well plates for up to 96 h. At the end of incubation, the cells were washed with Hank's balanced salt solution (without Ca2+ and Mg2+) twice, and all remaining cells attached to the plate were regarded as viable cells, when they did not take up Trypan blue. Cells were detached with 500 µl trypsinEDTA solution (0.020.05%) and the cell number was determined by a cell counter (Coulter-Multisizer, Krefeld, Germany). For counting, 100 µl cells were suspended in 9.9 ml 0.9% NaCl solution, and each sample was repeated twice.
Immunofluorescence
Indirect immunofluorescent staining for vimentin and cytokeratin was performed to discriminate between fibroblastic-like or epithelia-derived cell types as previously described (Tiemann et al., 2001). Briefly, granulosa cells were grown on chamber slides (Nunc, Naperville, IL) in complete TCM 199 until reaching confluence. Cells were washed with Tris-buffered saline (TBS) (20 mM Tris, pH 7.5, 0.5 M NaCl), fixed with acetone at 20°C, rinsed with TBS, and incubated with 4% (v/v) FCS in TBS for 30 min to block nonspecific binding sites. Then the cells were incubated with cytokeratin mAb or vimentin mAb (each at 10 µg/ml TBS containing 0.5% w/v bovine serum albumin [BSA]) for 60 min at 37°C (cytokeratin mAb) or at room temperature. After washing with TBS, cells were incubated with anti-mouse IgG F(ab')2FITC at 12 µg/ml in TBS containing 0.5% (w/v) BSA, and washed extensively with TBS. Immunofluorescence of granulosa cells was recorded with a Zeiss epifluorescence microscope.
For the detection and localization of gal-1, granulosa cells were grown on 18-mm glass coverslips coated with human fibronectin (25 µg/ml TBS) overnight at room temperature. Cells were allowed to spread on coverslips for 2 days. Then the cells were treated as described and incubated with a gal-1 pAb at 1 µg/ml TBS containing 0.25% BSA and 0.005% Tween 20 for 60 min at room temperature. The cells were rinsed with TBS/Tween 20 (0.5% BSA, 0.05% Tween 20) and incubated with anti-rabbit IgG-F(ab')2FITC (1:100 dilution in TBS/Tween 20) for 60 min at 37°C. After washing with TBS, coverslips were incubated with the DNA-binding dye propidium iodide at 1 µM for 20 min at room temperature. Then the cells were washed with TBS/Tween 20, coverslips were mounted in Mowiol, and the cells were analyzed for fluorescence using a Zeiss confocal laser scanning microscope. Incubations in the absence of gal-1 pAb with IgG-(Fab')2FITC (1:100 dilution) were used as controls.
Detection of gal-1 in granulosa cell lysates by immunoblotting
Granulosa cells cultured for 48 h in TCM 199 supplemented with 10% FCS and 1% ABAM (6 x 106 cells) were lysed in 300 µl Laemmli sample buffer (Laemmli, 1970) by treatment for 5 min at 100°C. Cell lysates (10 µl) were separated by SDSPAGE (12%) and electrophoretically transferred to Hybond ECL membranes. After blocking the blots with 4% BSA in TBS/Tween (20 mM TrisHCl, pH 7.2, 1 M NaCl, 0.05% Tween 20) for 2 h, the blots were probed with 1 µg/ml anti-gal-1 antibody or 1 µg/ml rabbit IgG (control) in TBS/Tween containing 1% BSA for 16 h at 6°C. Then the blots were washed and incubated with goat anti-rabbit IgG-HRP conjugate (1:1000 dilution in TBS/Tween with 1% BSA) for 2 h. Staining was performed with a mixture of 10 mg 3-amino-9-ethylcarbazole in 1 ml acetone, 25 ml 50 mM acetate buffer, pH 5.0, and 15 µl H2O2 (30%).
Radioimmunoassay for progesterone
Progesterone concentrations were measured in culture medium by specific radioimmunoassay as previously described (Tiemann et al., 1996). Intra- and interassay precision were 4.8% and 13.2% for progesterone. The number of cells was counted in each well. Steroidogenesis is expressed as ng hormone/1 x 105 cells.
Analysis of granulosa cell P450SCC expression by immunoblotting
Granulosa cells were cultured with 10 ng/ml FSH in the absence and presence of 9.9 µg/ml and 19.8 µg/ml galectin-1 with or without 30 mM lactose for 48 h. For analysis of P450SCC expression the cells were detached with 500 µl trypsin-EDTA solution, washed with ice-cold PBS, and lysed at 1 x 106 cells/100 µl Laemmli sample buffer (Laemmli, 1970) by treatment for 2 min at 100°C. After separation the lysates on 12.5% SDSPAGE slab gels, the proteins were transferred to polyvinylidene difluoride membranes. The membranes were saturated with 5% fat-free dry milk in TBS/Tween overnight at 4°C and, after washing with TBS/Tween, probed with a P450SCC pAb (1:1000 dilution in TBS/Tween with 5% BSA) for 3 h at room temperature. Then the blots were washed three times with TBS/Tween and incubated with a goat anti-rabbit secondary antibody conjugated to HRP (1:2000 dilution in TBS/Tween). The washed blots were treated with ECL reagents according to the manufacturer's instructions, and the bands were visualized luminographically on X-ray films (Kodak).
Quantification of mRNAs for P450SCC and 3ß-HSD by real-time PCR
Total RNA was prepared with the RNeasy mini kit. Briefly, cells stored frozen at 80°C were lysed and collected in 300 µl of a guanidine isothiocyanate containing buffer (lysis buffer) and subjected to homogenization by using QIAshredder Homogenizers (Qiagen). Subsequently an equal volume of 70% ethanol was added to the lysates, and the RNA was extracted from the samples by adsorption to silica-gel spin columns. After three washing steps and elution in 30 µl deionized RNase-free water (washing buffers and water provided by the kit) RNA was quantified in a GeneQuant II instrument (Pharmacia). Quality of RNA was monitored from randomly selected samples by denaturing agarose (1%) gel electrophoresis.
Primers for reverse transcription (RT) and PCR were derived from different exons to avoid amplification of residual genomic DNA. All primers were designed according to published sequences (P450SCC: EMBL/GenBank accession number, X13768, RT primer, 5'-CCAGGTCTTGGTCCTGAACAGAC-3', PCR primers, 5'-TTTACAGGGAGAAGCTCGGCAAC-3' and 5'-TTACCTCCGTGTTCAGGACCAAC-3'; 3ß-HSD: EMBL/GenBank accession number AF232699, RT primer, 5'-CTATGCTGCTGGTGTGGATGAAG-3', PCR primers, 5'-AGGGTTTCTGGGTCAGAGGATC-3' and 5'-CGTTGACCACGTCGATGATAGAG-3'). For cDNA synthesis, 0.15 µg total RNA were reversely transcribed in a 25 µl reaction volume using M-MLV reverse transcriptase, RNase H Minus, Point Mutant (Promega). Specifically primed reverse transcriptions were performed by simultaneously adding primers that bind to P450SCC and 3ß-HSD to avoid variations caused by different RT reactions.
The freshly synthesized cDNA samples were cleaned with the High Pure PCR Product Purification Kit and eluted in 50 µl elution buffer. The identity of products generated with different primer pairs had been controlled once by sequencing. For real-time PCR, 0.3 and 0.6 µl of purified cDNA samples were amplified with the LightCycler-FastStart DNA Master SYBR Green I Kit in 10 µl total reaction volume. Amplification and quantification of generated products were performed in a LightCycler instrument (Roche) under the following cycling conditions: preincubation at 95°C for 10 min, followed by 45 cycles denaturation at 95°C for 15 s, annealing at 60°C for 10 s, extension at 72°C for 10 s, and single-point fluorescence acquisition at 83°C for 6 s to avoid quantification of primer artifacts.
The melting peaks of all samples were routinely determined by melting curve analysis to ascertain that only the expected products had been generated. The melting peak of P450SCC-derived RT-PCR products was at 86.0°C and had a size of 251 bp and that of 3ß-HSD-derived products was at 87.7°C and had a size of 236 bp. Sizes of PCR products from randomly selected samples were monitored by agarose gel electrophoresis analysis (3% agarose, ethidium bromide stained).
To generate external standards for each gene, RT-PCR products of both genes were cloned into GEM-T plasmid vector (Promega). Routinely dilutions of standards covering five orders of magnitude (5 x 1016 to 5 x 1012 µg DNA/µl) were coamplified during each run. Fluorescence signals, which were recorded online during amplification, were subsequently analyzed using the Second Derivative Maximum method of the LightCycler Data Analysis software. Copy numbers were calculated relative to the amount of initially transcribed RNA. To normalize for variations between individual LightCycler runs, one or two arbitrarily selected samples were coamplified during all runs for each gene.
Statistical analysis
Data are expressed as the means ± SEM of triplicate measurements from separate granulosa cell preparations. A one-way analysis of variance with Student-Newman-Keuls test was used to determine significant differences. A p-value of less than 0.05 was considered significant.
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Footnotes |
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Abbreviations |
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References |
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