1 Reproductive Biology Unit, Dept of ObsGyn & Human Reproduction, CHU Tours, Faculté de Médecine, 37 044 Tours cedex, 2 INRA Xenobiotics, Toulouse and 3 INRAURA CNRS1291, PRMD, Nouzilly, France
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Abstract |
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Key words: aromatase/follicular fluid/gonadotrophins/granulosa cells/human
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Introduction |
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In addition to its effect on steroidogenesis, follicular fluid also has the ability to alter cell proliferation. On one hand, human follicular fluid has been shown to be mitogenic for endothelial cells (Bryant et al., 1988). On the other hand, ovine and bovine follicular fluid appear to contain factors which inhibit proliferation of fibroblasts (Carson et al., 1988
) or bovine granulosa cells (Hynes et al., 1996
). At present, however, the net effect of human follicular fluid on human granulosa cell proliferation is unknown.
The aims of this work were to validate a homospecific test system to analyse the effects of human follicular fluid on human granulosa cells. Hence the following steps were attempted: (i) optimization of an aromatase assay using the conversion of 3H-testosterone to 3H2O by human granulosa cells in vitro, (ii) description of the kinetics (Km, Vmax) of aromatase using this in-vitro model and (iii) use of this test to check whether human gonadotrophins and human follicular fluid modulate aromatase activity or cell proliferation.
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Materials and methods |
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Measurement of aromatase activity
Aromatase activity was assessed by measuring the conversion of [1ß, 2ß] 3H-testosterone to 3H2O as previously reported (Gore Langton and Dorrington, 1981).
After a 48 h culture period with the test substances (in the absence of the labelled substrate), cells were cultured for 12 h with 0.5 µmol of 1ß 2ß 3H-testosterone (Dupont de Nemours, Les Ulis, France). At the end of culture, medium was removed and stored at 20°C. Cell numbers in each well were measured (see below). To estimate aromatase activity, 100 µl of culture medium were loaded on C18-Sep Pak cartridges (Waters, Milford, MA, USA) and sequentially eluted with 3 ml of H2O and 3 ml of methanol to separate 3H2O from [1ß, 2ß] 3H-testosterone. Radioactivity in each fraction was counted in a scintillation counter (Packard Research, Rungis, France). Blanks were included in all cultures and radioactivity in blanks was deduced from the 3H2O counts. Aromatase activity was estimated as dpm 3H2O/(dpm 3H2O + [1ß, 2ß] 3H-testosterone) per 5x104 cells at the end of culture.
In order to determine cell numbers at the end of culture, cells were fixed by addition of 100 µl of 10% glutaraldehyde diluted in the culture medium and numbers then assessed according to Kueng et al. (1989). Briefly, following a 20 min fixation, the plates were washed with deionized water, dried and stained by addition of 100 µl of a 0.1% solution of crystal violet dissolved in 200 mmol/l 2[N-Morpholino]ethanesulphonic acid (Mes; Sigma-Aldrich Company), pH 6.0. The plates were again stirred for 20 min at room temperature, excess dye was removed by extensive washing with deionized water and plates were again dried. After crystal violet solubilization with 100 µl of 10% acetic acid in water, optical density (OD) was measured with a plate reader (Titertek Multiscan MKII®; Labsystems, Les Ulis, France) at a wavelength of 600 nm.
Chromatographic analysis of the samples
Radioactive steroids contained in the incubation medium were purified and concentrated on 1 g C18-Supelclean® LC-18 SPE cartridges (Supelco, St Germain-en-Laye, France) in order to discard 3H2O and specifically to elute retained steroids with methanol acidified with 1% acetic acid.
The purified radioactive steroids were analysed by high performance liquid chromatography (HPLC) on a Philips 4100 apparatus equipped with a rheodyne Model 7125 injector and connected to a Philips 4021 diode array detector (both from Philips, Eindhoven, Belgium). Testosterone metabolites were separated by HPLC on a reverse-phase ODS 2 column (180x4.6 mm, 3 µm) (Interchim, Montlucion, France) by means of a methodology derived from that of Wood et al. (Wood et al., 1983). Radioactive samples were collected on a Gilson-202® fraction collector (Gilson, Villiers de Bel, France) and were measured on a Packard 2200 Spectrophotometer® (Packard Research). Identification of the different steroidal products resulting from the metabolization (hydroxylation, aromatization or 17-oxidoreduction) was achieved by comparison with authentic standards.
In contrast, however, to what had been done previously with sheep follicular walls (Driancourt et al., 1996), attempts to purify sufficient amounts of hydroxylated and/or aromatized metabolites were unsuccessful.
Validation of the aromatase assay
Several specific points were tested to set up adequate culture conditions and validate the test: (i) whether 3H2O accumulation was linear at specific times following addition of [1ß, 2ß] 3H testosterone in the medium was assessed by obtaining samples at 312 h, following a 24 h plating period; (ii) whether aromatase activity was steady during the duration of culture was assessed by measurement of aromatase activity ([1ß, 2ß] 3H-testosterone added during 12 h), after 072 h following a 24 h plating period; (iii) to choose optimum cell numbers per well and optimum [1ß, 2ß] 3H-testosterone concentrations per well, the effects of cell numbers (6500100 000 cells per well) on aromatase activity were also checked, as were the effects of altering the amounts of substrate provided (109 mol/l to 106 mol/l). For this purpose, the duration of culture was 48 h followed by a 12 h incubation with 3H-testosterone.
Kinetic characteristics
Following a 24 h plating period, aromatase kinetics were measured by adding a range of [1ß, 2ß] 3H-testosterone concentrations (106 mol/l, 5x107 mol/l, 107 mol/l, 108 mol/l, or 109 mol/l) to wells containing 50 000 cells and obtaining samples of culture medium at 3 h intervals for 12 h. The amount of 3H2O and cell numbers were determined at each stage. Km and Vmax were calculated as described by Michaelis (1922). This experiment was replicated twice (pool 1 and pool 2).
Modulation of aromatase by gonadotrophins and follicular fluids
Study of the effects of gonadotrophins on aromatase activity
Granulosa cells were obtained from four women, pooled and seeded in triplicate at 50 000 cells per well. After a 24 h plating period, medium was aspirated and replaced with new medium containing 0, 12.5, 25, 50 ng/ml of human FSH (Metrodine HP®; Serono, France) or similar amounts of human LH (provided by Dr Y.Combarnous) or a combination of both hormones. After 48 h of treatment, 3H-testosterone was added for 12 h. Aromatase activity for each treatment was then determined and expressed according to the cell number found per well at the end of culture. This experiment was replicated twice.
Characterization of follicular fluids
Human follicular fluid was collected by laparoscopy, around the time a dominant follicle may have been present (according to the time lag from the previous menses), from the largest follicle in 10 regularly menstruating women who had tubal or unexplained infertility problems. Oestradiol and 4 androstenedione concentrations were measured in these follicular fluids by radioimmunoassays (125I-Estradiol Coatria®; 3H
4 androstenedione radioimmunoassay kit®; Biomerieux SA, Marcilly l'Etoile, France, Table I
). At the same time, blood samples were collected. Oestradiol, progesterone, and LH concentrations were measured in these samples by radioimmunoassays (125I Estradiol Coatria®; 125I Progesterone Coatria®; 125I human LH Coatria®; Biomerieux SA, Table I
). The stage of the cycle when the follicular fluids were obtained was established from 17ß-oestradiol, progesterone and LH concentrations in blood samples and from 17ß-oestradiol,
4 androstenedione concentrations in follicular fluids. Only dominant follicular fluid was used for the cultures. This was identified as follows: serum oestradiol concentrations were in excess of 5001000 pmol/l, those of progesterone were lower than 0.5 nmol/l, and those of LH lower than 180% of basal concentration, while the ratio between
4 androstenedione and oestradiol concentrations in follicular fluid was <1 (Gougeon, 1996
). Aliquots of 200 µl of follicular fluid of the largest follicle were prepared for each sample and stored at 20°C, after blood cells had been removed by centrifugation (400 g, 20 min).
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Specificity of follicular fluid effects
To ascertain whether follicular fluid effects detected in the previous studies were specific of aromatase, follicular fluid effects on two other steroidogenic enzymes were analysed. All reagents used were obtained from BioMerieux SA. Granulosa cells cultured for 48 h in the 10% serum or 10% dominant follicular fluid were immediately frozen in lysis medium (10 mmol/l KCl/10 mmol/l Tris/0.5 mmol/l EDTA), to which protease inhibitors had been added (1 µmol/l PMSF (phenyl-methyl-sulphonyl-fluoride), 100 µmol/l TPCK (N tosyl-L-phenylalanine-chloromethyl-ketone) and 10 µmol/l TLCK (N-P-tosyl-L-lysine-chloromethyl-ketone). Equal amounts of granulosa cell proteins (assessed according to Bradford, 1976
) were used for Western blot analysis. The samples were heated for 1 min with electrophoresis sample buffer [1.25 mol/l Tris containing 15% (v/v) glycerol, 10% (w/v) sodium dodecyl sulphate (SDS) pH6.8] at 90°C and subjected to electrophoresis using 15x13 cm polyacrylamide SDS gels (4% stacking gels and 10% separating gels). After electrophoresis, the proteins were transferred to a nitrocellulose membrane (Biorad, Ivry sur Seine, France) in a 1.2% (w/v) glycine/0.25% (w/v) Tris/20% (v/v) methanol/H2O buffer by incubation overnight at 4°C. After completion of transfer, the quality of each blot was checked by staining the nitrocellulose with 0.2% (w/v) Ponceau S stain (Sigma, France).
The membrane was then washed with Tris buffered saline (TBS: 10 mmol/l Tris containing 0.1% Tween 20; pH 7.4). The blot was then immersed in blocking buffer (1 mol/l Tris with 5% milk powder/0.2% Nonidet P-40; pH7.4) for 1 h. The membrane was then incubated with primary antibodies in blocking buffer for 1.5 h at 37°C and subsequently washed with TBS. The primary antibodies were polyclonal rabbit sera anti-P450scc (a gift from Dr B.Hales, Chicago University, USA) and anti-3 ßHSD (donated by Professor I.J.Mason, University of Edinburgh, UK) at a 1:1000 dilution. This was followed by incubation with a 1:1000 dilution of peroxidase-labelled goat anti-rabbit antibody (Pasteur, Sanofi, France). Bands corresponding to the steroidogenic enzymes were visualized using chemiluminescence (Amersham, Courtaboeuf, France) and were quantified by densitometric analysis (Kepler Software, Large Scale Biology, Rockville, TN, USA) as previously described (Driancourt et al., 1998).
Analysis of data
The data are expressed as mean ± SEM. Regression analysis was used to validate the aromatase test (effects of duration of culture, of 3H-testosterone concentrations, of cell number). One-way analysis of variance (ANOVA) and two-way ANOVA were used to compare effects of follicular fluids and of gonadotrophins on aromatase activity and on cell proliferation. Quantitative differences in the darkness of the bands found on the Western blots were identified by t-test.
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Results |
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The changes in aromatase activity according to the number of cells seeded showed a linear increase in aromatase activity (expressed per well), when cell numbers increased from 6500 to 100 000 cells (n = 21, r2 = 0.81, P < 0.01) (Figure 3). Hence, to allow space for the cells to proliferate during culture, 50 000 cells were seeded per well in all further experiments.
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Discussion |
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This conclusion is based on a bioassay which was developed and validated in the present study. Human follicular fluids and serum as well as granulosa cells from IVF patients were used to ensure the validation of this homospecific test. These cells, despite their earlier exposure to high concentrations of exogenous gonadotrophins, retain an active aromatase for a few days at least (Erickson et al., 1989; Wood et al., 1994
; Földesi et al., 1998
). Our bioassay used aromatase activity as an end point since aromatase is a key enzyme for granulosa cells which increases with follicle size and vanishes when follicles enter atresia (Driancourt et al., 1991
). Aromatase was measured using the tritiated water release assay (Gore Langton et al., 1981) and validated for other species (cattle, sheep) (Thatcher et al., 1991
; Driancourt et al., 1996
respectively). One of the pretrials of this study established that, in humans as well, 3H2O formation was indeed associated with formation of 17ß-Estradiol. All parameters of the culture system were adjusted following several pretrials to ensure optimal culture conditions. The conditions used were 50 000 cells plated for 24 h, an incubation of 48 h with test substances, and a concentration of 5x107 mol/l of testosterone. Culture duration was never longer than 84 h (24 h plating + 48 h of action of the test substances before addition of 3H-testosterone for 12 h) because aromatase tended to decrease for longer cultures. While this is in agreement with the data reported (Wood et al., 1994
), it contrasts with the data reported by Bernhisel et al. and Földesi et al. who demonstrated maintenance of aromatase for longer culture periods (Bernhisel et al., 1987
; Földesi et al., 1998
). However, serum-free conditions were used for plating in this study, while fetal bovine serum was used at the corresponding time in the above studies. Evidence that the granulosa cells remain functional in the conditions used in this study was provided (i) by the increase in cell number with time, (ii) by an aromatase activity well over background activity and (iii) by the sustained ability of gonadotrophins to stimulate aromatase activity. The magnitude of the increases in aromatase activity observed in the presence of LH and FSH were lower than the values reported using cells from unstimulated follicles (Mason et al., 1993
). Granulosa cells from IVF patients which have been exposed to large gonadotrophin concentrations in vivo may be partly refractory to additional gonadotrophins in vitro and appear to need a longer duration of culture (7 days) to recover a full ability to respond to FSH by an increased aromatase activity (Földesi et al., 1998
).
The approach selected in the present study used follicular fluid, a complex protein and steroid source, to check its effect on aromatase or cell proliferation. Numerous compounds present in follicular fluid (insulin-like growth factor I, activin, epidermal growth factor, interleukin-6 and leukaemia inhibitory factor) (Gougeon, 1996) have the ability positively or negatively to alter aromatase or cell proliferation. However, because compounds with positive and negative effects are mixed in follicular fluid, such studies do not provide clues to the net biological effect of follicular fluid on the cells of the follicle which contain it. In the present study, the dominant follicular fluid used was carefully selected to ensure it was aspirated while the follicle was in its dominance phase. Hence, the effect of follicular fluid from large (dominant) follicles was tested on granulosa cells from large (stimulated) follicles. This design was used because the question addressed dealt with the intrafollicular control of the maturation of the dominant follicle. In addition, earlier studies in swine (Ledwitz Rigby et al., 1985
; Ledwitz Rigby, 1987
) have underlined the complexity of interactions existing when follicular fluid obtained in a specific size group is added to granulosa cells originating from another size group.
The net effect of addition of dominant follicular fluid to human granulosa lutein cells was an inhibition of aromatase activity. Three lines of evidence support this claim: (i) aromatase in the presence of 20% dominant follicular fluid was significantly reduced compared to that observed in the presence of lower concentrations; (ii) aromatase in the presence of 20% dominant follicular fluid was three-fold lower than in the presence of 20% serum; (iii) contrasting with the effects observed with dominant follicular fluid, no change was observed in aromatase activity using non-dominant follicular fluid (Guet et al., 1997). This finding is in agreement with the earlier reports using heterospecific tests (di Zerega et al., 1982
, 1983a
,di Zerega et al., b
). The magnitude of the inhibition observed in the present study is close to what was reported previously (di Zerega et al., 1982
, 1983a
). A noticeable difference between the two approaches is that, in this study, basal conditions were used while in most studies of di Zerega's group, HMG or FSH stimulated conditions were used (di Zerega et al., 1982
, 1983b
). However, in one of their earlier studies, di Zerega et al. (1983b) demonstrated that the inhibition of aromatase induced by follicular fluid could also be observed when basal conditions were used.
Specificity of the effect on aromatase has been demonstrated in the present study by showing that other steroidogenic enzymes (namely P450scc and 3ß-HSD) were not affected by culture in the presence of follicular fluid compared to serum. In addition, dominant follicular fluids from sheep have no effect on progesterone production by luteal cells (M.A.Driancourt, unpublished data). Hence the effect of dominant follicular fluids on aromatase is likely to be specific.
As a decrease in aromatase activity at the higher concentration of follicular fluid used was observed, cell counts at the end of the culture were assessed to check whether this could be related to a toxic effect. Surprisingly, at all concentrations tested an increase in cell numbers both in the presence of follicular fluid and serum was observed. Such a finding strongly argues against a toxic effect.
The finding of a stimulatory action of dominant follicular fluid on granulosa cell proliferation is novel. Small amounts (2.5 and 5%) of dominant follicular fluid were very potent (six- to eight-fold increase in cell numbers) in stimulating cell proliferation and about two times more potent than similar concentrations of serum. Interestingly, concentrations in excess of 5% were not more efficient than 2.5 and 5%. However human follicular fluid is known to have the ability to stimulate proliferation of other cells (endothelial ones) (Bryant et al., 1988).
Because the estimates of aromatase were expressed per 50 000 cells, it may be argued that part of the changes in the index of aromatase activity may be due to increased cell proliferation. This is unlikely to be the case since (i) cell proliferation was stimulated by all concentrations of dominant follicular fluid tested while aromatase activity was only reduced at 20% dominant follicular fluid, (ii) at 20% serum or dominant follicular fluid, proliferation was similar while aromatase activity was reduced by dominant follicular fluid and (iii) non dominant follicular fluid has no ability to decrease aromatase activity (Guet et al., 1997) when added at a concentration of 20%. Whether the positive effect on cell proliferation is produced by the same compound or compounds as those negatively affecting aromatase activity is still unknown. Such a possibility might be unlikely since proliferation and differentiation are generally assumed to be regulated by distinct mechanisms (Monniaux et al., 1994
). In regard to the results of this study, endothelin-1 might be a suitable candidate for sustaining both inhibitory activity on aromatase and stimulating activity on proliferation of human luteinized granulosa cells (Kamada et al., 1995
). However, such a hypothesis looks unlikely since preliminary steps of characterization found that both activities were present after dialysis with a cut off of 5 kDa, which allowed low molecular weight compounds like endothelin to be excluded. Further purification of these compounds which might be important regulators of dominant follicle function will be the next step to understand their precise physiological role.
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Acknowledgments |
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Notes |
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References |
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Submitted on September 21, 1998; accepted on January 29, 1999.