Molecular Human Reproduction, Vol. 5, No. 2, 96-103,
February 1999
© 1999 European Society of Human Reproduction and Embryology
The mechanism of action of epidermal growth factor and transforming growth factor 
on aromatase activity in granulosa cells from polycystic ovaries*
1 Centre for Early Human Development, Institute of Reproduction and Development, 2 Centre for Inflammatory Diseases, Monash University, and 3 Monash IVF, Clayton, Victoria 3168, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We investigated aromatization and the mechanism of action of epidermal growth factor (EGF) and transforming growth factor
(TGF) on oestradiol biosynthesis in freshly prepared granulosa cells from polycystic ovaries. Freshly prepared granulosa cells from polycystic ovaries incubated for only 3 h under basal conditions secreted significantly (P < 0.001) greater amounts of oestradiol-17ß than that of granulosa cells from normal ovaries. 8-Bromo-cyclic adenosine monophosphate (8-Br-cAMP), but not follicle stimulating hormone (FSH) or luteinizing hormone (LH), further enhanced this activity. Both EGF and TGF inhibited gonadotrophin- or 8-Br-cAMP-stimulated, but not basal, oestradiol production. LH receptor (LHR) binding, estimated by immunolabelling the bound LH, was significantly (P < 0.001) reduced in granulosa cells from polycystic ovaries when compared with cells from normal ovaries. EGF or TGF significantly reduced the binding in cultured cells from all patient groups (P < 0.05). More interestingly, a further increase of the inhibitory effect was seen in granulosa cells from polycystic ovaries (P < 0.001). In conclusion, granulosa cells from polycystic ovaries contain high levels of basal aromatase activity in vitro, which is probably inherited from the in-vivo condition. EGF and TGF suppress oestradiol synthesis at a step beyond the production of cAMP and also LHR binding with more effect in granulosa cells from polycystic ovaries. aromatase/gonadotrophins/growth factors/PCOS
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Polycystic ovarian syndrome (PCOS), a common cause of anovulatory infertility, is a heterogenous and complex disease with unknown aetiology. This syndrome is characterized by a sharp reduction in ovarian cyclic oestradiol production (Erickson et al., 1992
; Mason et al., 1994). However, ovaries of patients with PCOS frequently respond to exogenous gonadotrophin administration by the excessive production of oestrogen and an increased risk of hyperstimulation syndrome (Armar et al., 1990). Moreover, dispersed granulosa cells from untreated polycystic ovaries (PCO) produce significantly more oestradiol in cultures than granulosa cells from normal ovaries (Haney et al., 1986; Almahbobi et al., 1996). These data suggest that aromatase is functional and highly active in PCO, but only after gonadotrophin stimulation and/or long-term granulosa cell culture in vitro. These apparent inconsistencies in aromatase activity in PCOS, in vivo and in vitro, raise questions regarding the control of the expression and activity of aromatase.
In previous reports, it has been suggested that PCOS is likely to be a result of an exaggerated action of intraovarian regulator(s) exerting dual effects, both stimulatory and inhibitory (Fauser 1994; Almahbobi and Trounson, 1996). Increasing evidence suggests that epidermal growth factor (EGF) and transforming growth factor (TGF) are putative candidates for such a role, enhancing early follicular growth (Bendell and Dorrington, 1990) but inhibiting aromatization (Franks et al., 1988; Almahbobi et al., 1995). We have recently demonstrated that granulosa cells from both anovulatory and ovulatory patients with PCO express significantly higher levels of EGF receptors than granulosa cells from size-matched follicles of normal ovaries, as further evidence for the possible role of EGF/TGF in the maintenance of PCOS (Almahbobi et al., 1998). However, the mechanism of action of EGF/TGF on ovarian oestradiol biosynthesis in PCOS has not been defined. It is well known that the expression and activity of the aromatase enzyme are mainly gonadotrophin-dependent. Therefore, it is of interest to determine whether the inhibitory effect of EGF/TGF is specifically related to interference in the action of gonadotrophins and their receptors, or directly related to enzyme expression and function. In a previous report, we have shown that granulosa cells from PCO contain significantly higher levels of follicle stimulating hormone (FSH) receptors than those from size-matched follicles of normal ovaries (Almahbobi et al., 1996). The aim of the present study was to investigate the mechanism which may mediate the action of EGF/TGF in the regulation of ovarian aromatization and luteinizing hormone (LH) receptor (LHR) formation leading to the maintenance of PCOS.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Patients and sample collection
Follicular aspirates were collected from patients with normal ovaries and PCO, involved in different treatments related to their fertility. The diagnosis of PCO in both patient groups (ovulatory and anovulatory) was based on ultrasonic evaluation of ovaries, together with clinical and endocrine manifestations of the syndrome (Trounson et al., 1981
). In PCOS, the ovaries are enlarged with more than 10 follicles of 
5 mm diameter in each. Other abnormalities of PCOS were anovulation, elevated androstenedione concentrations in serum (>6.5 nmol/l), LH:FSH ratios greater than 2 and reduced serum oestradiol concentrations (<0.2 nmol/l). These characteristics were variable in ovulatory patients with PCO and were frequently within the normal range for fertile women (Almahbobi et al., 1996).
Follicular aspirates were collected from anovulatory patients with PCOS just before laparoscopic electrocautery as part of treatment to induce ovulation for the purpose of conception in vivo. Samples were also collected from volunteers who were ovulatory with normal ovaries or PCO undergoing laparoscopic diagnosis for the investigation of their infertility. None of these patients received fertility drugs before follicular aspiration. Information on follicular number and size was recorded at the time of ultrasound examination 2 days before the sample collection. In anovulatory patients, follicular aspirates were collected when convenient and unrelated to any previous menses (Barnes et al., 1995; Szell et al., 1996). In the ovulatory groups of patients with normal ovaries or PCO, follicular aspiration was carried out on days 10–12 or 9–12 of the menstrual cycle respectively. The follicles were at immature stage with a size range of 2–9 mm (anovulatory patients with PCOS) and 2–10 mm (ovulatory patients with normal or PCO). However, not all follicles were necessarily aspirated.
In another group of patients, follicular aspirates were collected from mature pre-ovulatory follicles (Trounson et al., 1981) of patients undergoing ovulation induction with full stimulation with exogenous gonadotrophins for the recovery of mature oocytes for in-vitro fertilization (Calderon and Healy 1993). This group of patients is referred to as superovulated patients, regardless of their original aetiology. The treatments of these patients constituted pituitary down-regulation with gonadotrophin-releasing hormone (GnRH) agonist (Lucrin, 0.5–1 mg s.c.; Abbott Pharmaceuticals, Kurnell, Australia) and human menopausal gonadotrophin (HMG, Metrodin HP; Serono, French Forests, Australia) administration resulting in the growth and development of a cohort of ovulatory follicles. When at least three follicles of ~17 mm diameter were detected by vaginal guided ultrasound (Acuson, Melbourne, Australia) and peripheral plasma oestradiol concentrations were 3 nmol/l, 5000 IU of human chorionic gonadotrophin (HCG, Profasi; Serono) was given as a single i.m. injection is administered to complete follicular and oocyte maturation. Follicular aspiration (oocyte retrieval) was timed for 36 h after HCG administration. All other details of this treatment protocol were as previously described (Calderon and Healy, 1993). The oocytes were collected with sterile Pasteur pipettes, and the remainder of the follicular fluid was poured into a 50 ml bottle for subsequent isolation of granulosa cells. In all cases, aspirates from several small follicles of each patient were pooled together, the distribution of collected samples from different patient groups, numbers and days of collection are shown in Table I. These studies were approved by the Monash Private Hospital Research and Ethics Committee.
|
Granulosa cell preparation and culture
All chemicals and reagents were purchased from Sigma Chemical Co (St Louis, MO, USA), unless otherwise specified. Granulosa cells were prepared from follicular aspirates as previously described (Almahbobi et al., 1996
). In brief, follicular aspirates were centrifuged at 750 g for 5 min, the supernatant removed, the cell pellets pooled and then resuspended in 5 ml of Tissue Culture Medium 199 (TCM 199) supplemented with 25 mM HEPES, 50 mg/l penicillin, 50 mg/l streptomycin, 1.2 mg sodium bicarbonate and 0.1% bovine serum albumin (BSA). The granulosa cell suspension was dispersed by repeated pipetting for 30 s using a glass Pasteur pipette instead of an enzymatic method. This method allows only granulosa cells, but not (if any) thecal or stroma cells, to be dispersed and also reduces the damage to receptors on the cell membrane. The cells were then purified by centrifugation onto a 100% Ficoll gradient (Histopaque 1077) and then washed and resuspended in medium. An aliquot of dispersed cells was used to assess cell number and viability by Trypan Blue exclusion.
Granulosa cells were plated in TCM 199 and maintained at 37°C in a humidified atmosphere of 5% CO2 in air for different periods appropriate to the purpose of each experiment as indicated. The medium was carefully removed by aspiration and fresh pre-warmed medium was added. For the study of aromatase activity in culture, cells were plated at a concentration of 1–2x104 viable cells per 250 µl in 48-multiwell culture dishes (Falcon, Becton Dickinson, USA). Prior to testing for aromatase activity, the cells were pre-incubated for 0 (freshly prepared), 24 (D1), 48 (D2) or 120 h (D5). The test period was for 3 h in the presence or absence of 10–7 M testosterone as substrate, with or without 20 ng/ml FSH, 20 ng/ml LH, 2 mM 8-Br-cAMP and 10 ng/ml EGF or 10 ng/ml TGF. In some experiments, incubation time of freshly prepared cells was extended to 24 h in order to detect oestradiol production of granulosa cells from small follicles of normal ovaries. Purified testosterone, human pituitary FSH and LH, 8-Br-cAMP, EGF and TGF were diluted with sterile TCM 199 and added to appropriate wells in 10 µl volumes. The final volume of each well was 250 µl. Each test substance was repeated in five wells. At the end of the culture, the medium was collected for the measurements of oestradiol and the cells were lysed with 50 µl 0.1 M sodium hydroxide and harvested for assay of protein concentration.
In other experiments designed for the quantification of LHR using flow cytometry, granulosa cells were plated into 6-multiwell culture dishes (Falcon, Becton Dickinson, USA) at a concentration of ~4–5x105 cells/ml/well with media containing 10% fetal calf serum (FCS) and maintained for up to 5 days in the presence of testosterone (10–7 M), with or without EGF (10 ng/ml) or TGF (10 ng/ml). The wells were carefully washed with pre-warmed media every 48h. At the end of the culture periods, the spent media were removed and the cells were washed with medium containing no FCS. Trypsin versene (1 ml) was added to each well and the plate was gently shaken to allow the cells to detach. The detached granulosa cells were collected into tubes containing medium with FCS, washed and transferred to Eppendorf tubes in 100 µl media for LHR immunolabelling.
Oestradiol-17ß and protein assays
The measurements of oestradiol in cell-free culture spent media were based on a previously described radioimmunoassay (Carson et al., 1986) using 10 000 c.p.m. per tube 2,4,6,7-3H-oestradiol (Amersham International, Buckinghamshire, UK) and charcoal separation. Unlabelled oestradiol [1,3,5(10 estratriene-3, 17ß diol)] was used in serial dilution range of 800 pg/100 µl to 3.13 pg/100 µl. Diluted spent media (200 µl) was incubated with a rabbit anti-oestradiol antibody (Outch oestradiol Y17) at a dilution of 1:15 000 overnight at 4°C. After charcoal separation, the samples were counted with 2 ml scintillation liquid on a ß-counter (Packard Instrument Co, Meriden, USA). The sensitivity of the assay was 31.3 pg/ml. Proteins were determined using the Bio-Rad DC colorimetric protein assay with a BSA standard according to the instructions of the supplier (Bio-Rad Laboratories Ltd, Mercules, USA).
Immunofluorescence labelling and microscopy
The immunolocalization and quantification of LHR was carried out basically by a binding method with indirect detection through the immunolabelling of the receptor-bound ligands. Granulosa cells that were freshly prepared or following 5 days of culture were resuspended in pre-cooled phosphate-buffered saline (PBS) containing 5% FCS and left on ice for 30 min to allow the temperature of the cells to decrease. The cells were then incubated with an excess amount of human pituitary LH (200 ng/ml) for 45 min at 6°C with shaking every 10 min (Haigler et al., 1978). The procedure allowed the ligand to bind to the receptor but inhibited internalization of the receptor–ligand complex. This was followed by extensive washes to remove the unbound LH and the cells were processed, in suspension, for indirect immunofluorescence labelling at 4°C (Almahbobi and Hall 1993; Almahbobi et al., 1998). Non-specific binding of secondary antibody was reduced by incubation with 10% sheep serum (Oncogene Research Products, Cambridge, USA) diluted in PBS/FCS for 30 min. Monoclonal mouse anti-LH antibody was allowed to incubate for 30 min at a concentration of 10 µg/ml in PBS/FCS. Controls for non-specific binding were determined using mouse negative control immunoglobulin (Ig)G2a antibody (Dako, Glostrup, Denmark) instead of the specific antibody. After incubation with the control or specific antibody, granulosa cells within the Eppendorf tubes were thoroughly washed with PBS/FCS. This was followed by incubation with 5 µg/ml sheep anti-mouse [F(ab)2 fragments] antibody conjugated to fluorescein isothiocynate (FITC) (Silenus Laboratories, Melbourne, Australia) for 30 min. After extensive washes in PBS/FCS, the cells were fixed in 1% paraformaldehyde in PBS for 10 min for immediate analysis by flow cytometry, or mounted onto slides for microscopic examination. In some experiments, cells that had been cultured on coverslips (HD Scientific, NSW, Australia) over the 5 day period were labelled in the same manner for microscopy without trypsinization. Once the cells were fixed, a drop of Vectashield mounting media (Vector Laboratories, Inc, Burlingame, CA, USA) was placed on slides, on which the coverslips were placed upside down. Examinations were performed with a fluorescence microscope equipped for illumination using a mercury light source.
Flow cytometry
Labelled granulosa cells in 300 µl suspension were filtered through 75 µm mesh and analysed by flow cytometry basically as previously described (Almahbobi et al., 1996Almahbobi et al., 1998). Cell suspensions were run on a MO-FLO flow cytometer (Cytomation Inc, Fort Collins, CO, USA). An argon ion laser (Coherent, Palo Alto, CA, USA) operating at 488 nm was used to illuminate the cells. Forward angle light scatter, right angle light scatter and FITC fluorescence (525 ± 10 nm band pass filter) signals were collected for all samples. These were all processed through a 3-decade logarithmic amplifier prior to digital conversion. Fluorescence histograms of at least 5000 counts were generated from a gate set on the granulosa cells in the forward angle versus 90° light scatter histogram. Identification of granulosa cells among other contaminating cells was by their characteristic large size, high granularity, detectable autofluorescence (Almahbobi et al., 1996) together with the negative labelling with CD45 (specific surface marker of leukocytes), positive labelling with anti-EGF receptor antibody (Almahbobi et al., 1998) and LH antibody labelling (the present study). The mean fluorescence signals collected from a gated cell population are expressed in numerical values. The values for positive labelling were determined by subtraction of background values obtained in control samples incubated with the control antibody from the values obtained when the corresponding samples incubated with the specific anti-LH antibody.
Statistical analysis
The data from steroid production experiments were calculated using Microsoft Excel for Windows, version 5.0 for calculation of mean ± SD from five replicate wells of each treatment within patient groups. Comparisons between different groups of patients was undertaken using Student's paired t-test. Results of the LHR study, expressed as the mean ± SD using Microsoft Excel Version 6.0, were analysed by the Wilcoxen Signed Rank test for a non-parametric paired data analysis. SPSS (Software Packages for the Statistical Sciences) for Windows, version 6.0 (SPSS Inc, 1993) was used for both studies. The statistical significance of individual experiments is shown in the figure legends.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Aromatase activity in PCO
Aromatase activity in granulosa cells as expressed by oestradiol production was negligible without the addition of androgen substrates in all cultures at any given time. The addition of 10–7 M testosterone to freshly prepared granulosa cells and cells after 2 days in culture produced around 40- and 20-fold increase (P < 0.001) in oestradiol production respectively when compared with those without testosterone (data not shown). These results confirm that human granulosa cells do not synthesize androgens and that these granulosa cell cultures were free of contaminating stroma and thecal cells. It should be noted that there was no significant difference in basal and stimulated oestradiol production between granulosa cells from ovulatory and anovulatory patients with PCO (Figure 1A
) and therefore data were combined for subsequent analyses. Time-dependent changes in basal aromatase activity showed a sharp decline after 1 day of culture reaching about 15-fold less oestradiol production compared with fresh cells. Oestradiol production was maintained at this level during subsequent days (Figures 1B). Interestingly, the basal oestradiol production of freshly prepared and cultured granulosa cells from patients with PCO was comparable with that of granulosa cells from fully matured follicles of superovulated patients (Figure 1B). In addition, this ability of granulosa cells from patients with PCO to produce oestradiol was significantly (P < 0.001) greater than that of granulosa cells obtained from normal ovaries, incubated under both basal and stimulated conditions (Figure 1C).
|
Aromatase activity in response to gonadotrophins in culture
To determine whether granulosa cells from patients with PCO are responsive to gonadotrophins, freshly prepared granulosa cells were incubated with FSH or LH for 3 h in the presence of testosterone as a substrate. In these short-term incubations, the addition of gonadotrophins failed to stimulate oestradiol production by the granulosa cells from patients with PCO and superovulated patients (Figure 2
). In contrast, when granulosa cells were cultured for at least 2 days, the addition of FSH or LH to the cultures for 3 h induced a significant (P < 0.001) increase in oestradiol production. The stimulatory effect of gonadotrophins on aromatase activity was enhanced after 5 days in culture (Figure 2). Once again, the gonadotrophin-stimulated oestradiol production of granulosa cells from patients with PCO was identical to those of granulosa cells from superovulated patients.
|
Effects of EGF/TGF
on aromatase activityIn freshly prepared granulosa cells from patients with PCO and superovulated patients, there was no inhibitory effect of EGF or TGF
on basal aromatase activity (Figure 3A). Since there was no stimulation in the presence of gonadotrophins, the basal oestradiol production was unaffected by EGF/TGF. Because there was no difference between the effect of EGF and TGF on aromatase activity, the data were combined for subsequent analyses. In granulosa cells cultured for at least 2 days, EGF/TGF significantly (P < 0.001) inhibited gonadotrophin-stimulated oestradiol production but not basal aromatase activity and this was more pronounced in cultures after 5 days before adding EGF/TGF for the 3 h test (Figure 3B).
|
Mechanism of action of EGF/TGF
on aromatase activityMultiple lesions
In order to determine the mechanism which may regulate the effect of EGF/TGF
on gonadotrophin-induced aromatase activity, freshly prepared and cultured granulosa cells were incubated with or without EGF/TGF in the presence or absence of exogenous 8-Br-cAMP. In contrast to the lack of aromatase stimulation by gonadotrophins in freshly prepared cells (Figure 3A
), there was a significant (P < 0.001) increase in oestradiol production in response to 8-Br-cAMP of the cells obtained from the patient groups, with polycystic ovaries and who were superovulated (Figure 4). After 2 days in culture, the increased oestradiol production with 8-Br-cAMP was more pronounced than that with FSH or LH shown in Figure 2. Most interestingly, the increase in oestradiol production in response to 8-Br-cAMP was also significantly (P < 0.001) inhibited by EGF/TGF when added to either fresh or cultured cells (Figure 4).
|
EGF/TGF
inhibits LHR binding in vitroThe quantification of LHR in granulosa cells of different groups of patients was carried out using flow cytometric analysis. The identification and gating of the granulosa cell fraction within the suspended cell preparations were based on a previously described procedure (Almahbobi et al., 1996
Almahbobi et al., 1998). Since granulosa cell preparations were pre-cultured for several days before flow cytometry, most of the contaminating blood cells were removed, due to several washes, from these preparations as revealed by the forward light scatter analysis (Figure 5A). The labelling of LHR within the gated population of granulosa cells from both PCO and normal ovaries showed a heterogenous pattern of labelling. Figures 5B and 5C show representative histograms of fluorescence signals collected from granulosa cells of PCO. The histograms show the relative intensity of labelling and the number of labelled cells within the gated population of granulosa cells shown in Figure 5A. The levels of displacement of granulosa cell population indicate the intensity of the fluorescence signals collected per individual cell. Figure 5B shows non-specific binding using control antibody. When the specific antibody was used the whole cell population, which appeared on the left-hand side of the vertical cut-off line, was partially displaced to the right side on the fluorescence scale (Figure 5C). A fraction of the positively labelled cells showed strong labelling intensity with a maximum shift to the right hand side of the cut-off line (Figure 5C).
|
The mean ± SD of LHR-positive labelling in granulosa cells cultured for 5 days with or without EGF/TGF
is shown in Figure 6. Less consistant and significant changes in LHR were observed in 1 and 2 day cultures (data not shown). The positive LHR labelling was obtained by subtracting the control values from the values of corresponding samples incubated with the specific antibody. Interestingly, the LHR binding in granulosa cells from patients with PCO was significantly (P < 0.001) less than that seen in granulosa cells of size-matched follicles from normal ovaries or pre-ovulatory follicles of superovulated patients. In addition, both EGF and TGF significantly inhibited the binding of LHR in granulosa cells from all patient groups (superovulated and normal patients, P < 0.05; patients with polycystic ovaries, P < 0.001) (Figure 6). More interestingly, granulosa cells from PCO were more sensitive to the inhibitory effect of EGF and TGF on LHR (50%) compared with granulosa cells of matched-size follicles from normal ovaries (16%) and to a lesser extent to granulosa cells from superovulated patients (40%). Examination of the freshly prepared (Figure 6 inset) and cultured granulosa cells under the fluorescence microscope revealed that LHR-positive staining was confined to the cell membrane. This was supported by the staining of cell clumps characteristic of undispersed granulosa cells (data not shown). The staining intensity of individual cells within the granulosa cell population and amongst individual patients within a specific patient group was varied. All control samples revealed negative staining.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
PCOS is a heterogeneous disease with many contradictory features particularly the inhibition of ovarian cyclic oestrogen production. Although an intrinsic defect in aromatase expression or activity is unlikely to be the case in PCOS, the clear ambiguity in the function of this enzyme, in different in-vivo and in-vitro conditions, makes it difficult to determine the normality of aromatization in PCOS. It has been reported that within the range of PCO follicles (<10 mm), aromatase enzyme is not normally expressed (Erickson et al., 1979
; Jakimiuk et al., 1998) and that the in-vitro production of oestradiol by PCO granulosa cells is due to cell differentiation in long-term (48 h) cultures (Erickson et al., 1979). However, the aromatase enzyme was immunolocalized in human granulosa cells within the same range of follicle size (Sasano et al., 1989) and also those of <1 mm diameter (Inkster et al., 1991). It is also possible that differentiation of granulosa cells, or even development of new growing follicles may occur in vivo after the administration of gonadotrophins, explaining the response of PCO granulosa cells (Almahbobi and Trounson 1996). Nevertheless, differentiation of granulosa cells should not lead to excessive oestradiol production by PCO granulosa cells when compared with granulosa cells from normal ovaries as previously reported (Haney et al., 1986; Almahbobi et al., 1996; Pierro et al., 1997; for more references see Almahbobi and Trounson 1996). In order to clarify this confusion, we have investigated aromatase activity in freshly prepared cells incubated for short period (3 h) under basal conditions (no added serum or gonadotrophins). It was interesting to note that granulosa cells from immature follicles (2–10 mm) of PCO contain high aromatase activity, comparable with that seen in fully mature pre-ovulatory follicles of superovulated patients and as much as 13 times more activity than those of normal ovary. A recent report showing that PCO granulosa luteal cells, after treatment with gonadotrophins for the induction of superovulation, also had higher levels of aromatase activity in vitro when compared with non-PCO cells (Pierro et al., 1997).
Since the concentrations of bioactive FSH are normal in PCO (Erickson et al., 1992) and PCO granulosa cells contain significantly higher concentrations of FSH receptors than those of normal ovaries (Almahbobi et al., 1996), it is possible that granulosa cells of PCO express high values of aromatase enzyme. However, in-vivo aromatization is potentially inhibited, possibly by the intraovarian growth factors EGF/TGF.
In vitro, the inhibitory effect of EGF/TGF was removed. The values of oestradiol production were high and comparable with those of pre-ovulatory follicles from superovulated patients. Moreover, both types of cells did not respond to gonadotrophins during the first 24 h in culture. This may indicate that granulosa cells from PCO follicles may be functioning at near maximal aromatization. With subsequent time in culture, the cells lost the influence of the inherited in-vivo effects, aromatization sharply dropped to normal values and the cells recovered their normal responsiveness to gonadotrophins. In contrast to EGF, it has been shown that insulin-like growth factor I enhances the FSH-induced expression of aromatase in cultured human granulosa cells from superovulated patients (Steinkampf et al., 1988). Nevertheless, PCOS is a heterogenous syndrome whereby multiple factors may act separately or synergistically.
Three important points emerge from this study regarding the regulation of aromatase activity in human granulosa cells. First, in freshly prepared cells, only 8-Br-cAMP but not FSH or LH could stimulate aromatase activity. This indicates that the failure of gonadotrophins to induce aromatase stimulation in fresh cells is due to a lesion at a step preceeding cAMP production. Secondly, regardless of the culture period or the cell types, EGF/TGF suppressed only the stimulated aromatase activity but not the basal levels of aromatization (Steinkampf et al., 1988). This may indicate that the high levels of oestradiol production in both types of cells were in fact basal activity of highly expressed enzyme. Thirdly, the fact that EGF/TGF inhibited aromatase stimulation in the presence of an excess amount (or at least stimulatory amount) of 8-Br-cAMP indicates that the site of the inhibitory action of EGF/TGF is at a post-cAMP production step. In support of this, it has been shown that in cultured rat granulosa cells, EGF not only reduces the FSH-stimulated adenylate cyclase activity but also increased catabolism of cAMP (Knecht and Catt 1983), therefore exerting multiple sites of action. We suggest that while the levels of basal aromatization appears totally related to the amount of the expressed enzyme and are not affected by the intraovarian inhibitors such as EGF/TGF, the hormonally stimulated aromatization requires cAMP and is regulated by EGF/TGF.
The presence of LHR in human granulosa cells of follicles <10 mm is not surprising particularly during day 9–12 of the menstrual cycle (McNatty et al., 1992). LHR analysis showed that not all granulosa cells were positively labelled and the labelling intensity was variable. The intensely labelled cells were probably derived from the larger follicles within individual pooled samples. The inhibition of LHR binding, detected by immunolabelling, in human granulosa cells by EGF/TGF confirms previous studies using cultured rat granulosa cells (Mondshein and Schomberg, 1981; Knecht and Catt, 1983) and highlights the possible role of these growth factors in the maintenance of PCOS. This view was supported by the low levels of LHR labelling in PCO granulosa cells comparing to both stimulated and unstimulated cells from normal ovaries. This was confirmed by our previous study showing that the capacity of PCO granulosa cells to produce progesterone in culture was significantly less than those from normal ovaries (Almahbobi et al., 1996). Our data show that there is no significant difference between LHR detected in granulosa cells from small follicles of normal ovaries and those from mature follicles of superovulated patients. This is probably due to the rapid decline of LHR mRNA of pre-ovulatory follicles that normally occurs after the LH surge in vivo (Segaloff et al., 1990) in superovulated patients. An interesting and novel observation of the present study was that PCO granulosa cells were more sensitive to the inhibitory effects of EGF/TGF on LHR. It appears that the high levels of EGF receptors in granulosa cells of PCO (Almahbobi et al., 1998) may explain the hyper-sensitivity of these cells to the action of EGF/TGF. Because a similar hypersensitivity has not been found in terms of the inhibition of oestradiol production (Mason et al., 1990; the present results), it appears that the amplification of EGF/TGF action occurs in a selective manner.
The above results, together with others previously reported, suggest that in PCOS, FSH concentrations and their related ovarian functions are normal, including the selection of pre-ovulatory follicles (Erickson, 1996). However, the late follicular maturation of a selected follicle to achieve dominance is blocked. The latter process requires large amounts of oestrogen for multiple purposes such as follicular growth and the formation of LHR in granulosa cells (Richards et al., 1976; Segaloff et al., 1990). In fact, the blockade of follicular growth in PCO occurs around the time when granulosa cells acquire LHR and hence become responsive to this hormone (McNatty et al., 1992). LH is a principle regulator of aromatization (Hsueh et al., 1984) in the late follicular stage and its main role is to enhance late maturation of the selected follicle to ensure dominance and ovulation (Richards, 1994). In conclusion, most of the PCOS characteristics indicate hyperactivity of ovarian function (Almahbobi and Trounson, 1996) but the inhibition of cyclic ovarian oestradiol production and LHR formation by EGF/TGF is a mechanism likely to be operative in PCOS, leading to the blockade of follicular dominance and anovulation.
|
Acknowledgments |
|---|
The authors are grateful to the staff of Monash IVF Clinics and Carl Wood and Associates for their excellent collaboration in providing us with human follicular aspirates and the information regarding the patients pathology. The authors also like to thank the Department of Biochemistry at Monash Medical Centre for their generous gift of anti-oestradiol antibody. This work has been supported by grants from Monash IVF to GA and from NH&MRC No. 95/0790 to AOT and GA.
|
Notes |
|---|
*This paper was presented at the 13th Annual Meeting of the European Society of Human Reproduction and Embryology (ESHRE), Edinburgh, UK, 1997.
4 To whom correspondence should be addressed at: Institute of Reproduction and Development, Block B, Level 5, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria, 3168, Australia 
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Almahbobi, G., Anderiesz, C., Hutchinson, P. et al. (1996) Functional integrity of granulosa cells from polycystic ovaries. Clin. Endocrinol., 44, 571–580.[Medline]
Almahbobi, G. and Hall, P.F. (1993) Immunofluorescent method modified to display two antigens in one light filter. Histochem J., 25, 14–18.[Web of Science][Medline]
Almahbobi, G., Misajon, A., Hutchinson, P. et al. (1999) Hyper-expression of epidermal growth factor receptors in granulosa cells from women with polycystic ovarian syndrome. Fertil. Steril., 70, in press.
Almahbobi, G., Nagodavithane, A. and Trounson, A.O. (1995) Effects of epidermal growth factor, transforming growth factor and androstenedione on follicular growth and aromatizaton in culture. Mol. Hum. Reprod., 1, see Hum. Reprod., 10, 2767–2772.
Almahbobi, G. and Tounson, A.O. (1996) The role of intraovarian regulators in the aetiology of the polycystic ovarian syndrome. Reprod. Med. Rev., 5, 151–168.
Armar, N.A., McGarrigle, H.H.G., Honour, J. et al. (1990) Laparoscopic ovarian diathermy in the management of anovulatory infertility in women with polycystic ovaries: endocrinole changes and clinical outcome. Fertil. Steril., 53, 45–49.[Web of Science][Medline]
Barnes, F.I., Crombie, A., Gardner, D. et al. (1995) Blastocyst development and birth after in vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum. Reprod., 10, 3243–3247.
Bendell, J.J. and Dorrington, J.H. (1990) Epidermal growth factor influences growth and differentiation of rat granulosa cells. Endocrinology, 127, 533–540.
Calderon, I. and Healy, D. (1993) Endocrinology of IVF. In Trounson, A.O. and Gardner, D.G. (eds), (1993) Handbook of In Vitro Fertilisation. CRC Press Inc, Boca Raton, Florida, USA, pp. 1–16.
Carson, R.S., Salamonsen, L.A. and Findlay, J.K. (1986) Permeability of rat ovarian follicles to LH during development and luteinization. J. Reprod. Fert., 76, 663–676.
Erickson, G.F. (1996) The ovarian connection. In Adashi, E.Y., Rock, J.A. and Rosenwaks, Z. (eds), Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven Publishers, Philadelphia, USA, pp. 1141–1160.
Erickson, G.F., Hsueh, A.J.W., Quigley, M.E. et al. (1979) Functional studies of aromatase activity in human granulosa cells from normal and polycystic ovaries. J. Clin. Endocrinol. Metab., 49, 514–519.
Erickson, G.F., Magoffin, D.A., Garzo, V.G. et al. (1992) Granulosa cells of polycystic ovaries: are they normal or abnormal? Hum. Reprod., 7, 293–399.
Fauser, B.C.J.M. (1994) Observation in favour of normal early follicle development and disturbed dominant follicle selection in polycystic ovarian syndrome. Gynecol. Endocrinol., 8, 75–82.[Web of Science][Medline]
Franks, S., Mason, H.D., Polson, D.W. et al. (1988) Mechanism and management of ovulatory failure in women with polycystic ovary syndrome. Hum. Reprod., 3, 531–534.
Haigler, H., Ash, J.F., Singer, S.J. et al. (1978) Visualization by fluorescence of the binding and internalization of epidermal growth factor in human carcinoma cells A-431. Proc. Natl. Acad. Sci. USA, 75, 3317–3321.
Haney, A.F., Maxson, W.S. and Schomberg, D.W. (1986) Compartmental ovarian steroidogenesis in polycystic ovary syndrome. Obstet. Gynecol., 68, 638–644.[Web of Science][Medline]
Hsueh, A.J.W., Adashi, E.Y., Jones, P.B.C. et al. (1984) Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocrine Rev., 5, 76–126.
Inkster, S.E. and Brodie, A.M.H. (1991) Expression of aromatase cytochrome P-450 in premenopausal and postmenoposal human ovaries: an immunocytochemical study. J. Clin. Endocrinol. Metab., 73, 717–726.
Jakimiuk, A.J., Weitsman, S.R., Brzechffa, P.R. et al. (1998) Aromatase mRNA expression in individual follicles from polycystic ovaries. Mol. Hum. Reprod., 4, 1–8.
Knecht, M. and Catt, K.J. (1983) Modulation of cAMP-mediated differentiation in ovarian granulosa cells by epidermal growth factor and platelet-derived growth factor. J. Biol. Chem., 258, 2789–2794.
Mason, H.D., Margara, R., Winston, R.M.L. et al. (1990) Inhibition of oestradiol production by epidermal growth factor in human granulosa cells of normal and polycystic ovaries. Clin. Endocrinol., 33, 511–517.[Medline]
Mason, H.D., Willis, D.S., Beard, R.D. et al. (1994) Estradiol production by granulosa cells of normal and polycystic ovaries: relationship to menstrual cycle history and concentrations of gonadotropins and sex steroids in follicular fluid. J. Clin. Endocrinol. Metab., 79, 1355–1360.[Abstract]
McNatty, K.P., Smith, P., Hudson, N.L. et al. (1992) Follicular development and steroidogenesis. In Local Regulation of Ovarian Function. The Parthenon Publishing Group, London, UK, pp. 21–38.
Mondshein, J.S. and Schomberg, D.W. (1981) Growth factors modulate gonadotrophin receptor induction in granulosa cell cultures. Science, 211, 1179–1180.
Pierro, E., Andreani, C.L., Lazzarin, N. et al. (1997) Further evidence of increased aromatase activity in granulosa luteal cells from polycystic ovary. Hum. Reprod., 12, 1890–1896.
Richards, J.S. (1994) Hormonal Control of gene expression in the ovary. Endocrine Rev., 15, 725–751.
Richards, J.S., Ireland, J.J., Rao, M.C. et al. (1976) Ovarian follicular development in the rat: hormone receptor regulation by estradiol, follicle-stimulating hormone and luteinizing hormone. Endocrinology, 99, 1562.
Sasano, H., Okamoto, M., Mason, J.I. et al. (1989) Immunolocalization of aromatase, 17-hydroxylase and side-chain-cleavage cytochromes P-450 in the human ovary. J. Reprod. Fertil., 85, 163–169.
Segaloff, D.L., Wang, H. and Richards, J.S. (1990) Hormonal regulation of luteinizing hormone/chorionic gonadotropin receptor mRNA in rat ovarian cells during follicular development and luteinization. Mol. Endocrinol., 4, 1856–1865.
Steinkampf, M.P., Mendelson, C.R. and Simpson, E.R. (1988) Effects of epidermal growth factor and insulin-like growth factor I on the levels of mRNA encoding aromatase cytochrome P-450 of human ovarian granulosa cells. Mol. Cell. Endocrinol., 59, 93–99.[Web of Science][Medline]
Szell, A.Z., Kausche, A., May, K. et al. (1996) In vitro oocyte cultures: clinical aspects. In Filicori, M. and Flamigi, C. (eds), The Ovary, Regulation, Dysfunction and Treatment. Elsevier Science, Amsterdam, The Netherlands.
Trounson, A.O., Leeton, J.F., Wood, E.C. et al. (1981) Pregnancies in humans by fertilisation in vitro and embryo transfer in the controlled ovulatory cycle. Science, 12, 681–682.
Submitted on June 8, 1998; accepted on October 29, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  C. Glister, D. S. Tannetta, N. P. Groome, and P. G. Knight Interactions Between Follicle-Stimulating Hormone and Growth Factors in Modulating Secretion of Steroids and Inhibin-Related Peptides by Nonluteinized Bovine Granulosa Cells Biol Reprod, October 1, 2001; 65(4): 1020 - 1028. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||