Molecular Human Reproduction, Vol. 7, No. 4, 325-331,
April 2001
© 2001 European Society of Human Reproduction and Embryology
The balance between MMP-9 and MMP-2 and their tissue inhibitor (TIMP)-1 in luteinized granulosa cells: comparison between women with PCOS and normal ovulatory women
1 Laboratory for Research in Reproductive Sciences, Department of Obstetrics and Gynecology, Ha'Emek Medical Center, Afula and 2 Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel
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Polycystic ovarian syndrome (PCOS) involves follicular atresia, formation of multiple ovarian cysts and is frequently associated with a higher abortion rate. Follicular development, ovulation, formation of corpus luteum and its regression involve extensive tissue remodelling. Mammalian ovaries express a number of matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP). We assessed the differences in production of MMP-2, MMP-9 and TIMP-1 by cultured luteinized granulosa cells from women with PCOS and normal ovulatory women after ovarian stimulation for IVF treatment. In follicular fluid from women with PCOS, levels of MMP-9 and MMP-2 were higher than the normal group, as was the basal production of these proteins by cultured cells. Basal production of TIMP-1 by cultured cells was not different between PCOS and normal groups. A time-dependent increase in the production of MMP-9 was observed in cells from both normal and PCOS women, although the increase was more pronounced in the latter. Thus the MMP-TIMP balance is shifted toward greater MMP activity in luteinized granulosa cells from women with PCOS.
luteinized granulosa cells/MMP/polycystic ovarian syndrome/TIMP
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The polycystic ovarian syndrome (PCOS) is often associated with anovulatory infertility and frequent abortions. The main features of this disease state are anovulation, hyperandrogenism, disturbed hypothalamic function in the form of reversed LH:FSH ratio and decreased insulin sensitivity, and the primary underlying cause is still enigmatic (Burghen et al., 1980
; Balen et al., 1995; Ben-Shlomo et al., 1998). At the physiological level, there appears to be a deranged function of ovarian 17
-hydroxylase. Several studies have shown that granulosa cells obtained from PCOS patients differ functionally from normal cells as reflected by a lower insulin-like growth factor (IGF)-I and IGF-II production (Erickson et al., 1992; Barreca et al., 1996), and a higher rate of apoptosis (Anderson and Lee, 1997). The number of suggestions as to the exact mechanism, leading to the final clinical picture, seems to equal the number of defects described.
A fundamental aspect of follicular development, ovulation and subsequent formation and regression of the corpus luteum is dynamic and extensive tissue remodelling (Tsafriri, 1995; Liu et al., 1999). As hinted by the landmark of PCOS, the formation of multiple mid-size ovarian cysts and by the frequently insufficient luteal function, the mechanism(s) involved in tissue remodelling during the life cycle of an ovarian follicle may be affected in this pathology.
The role of extracellular matrix metalloproteinases (MMP) in ovarian tissue remodelling during the life span of the follicle has been documented in numerous studies. In these, the mammalian ovary has been shown to express MMP-1, MMP-2, MMP-3, MMP-9 and possibly more MMP (Cossins et al., 1996; Hulboy et al., 1997), as well as the naturally occurring tissue inhibitors of MMP, TIMP-1 and TIMP-2. Cultured luteinized granulosa cells from human ovaries have been shown to express MMP-9 and MMP-2 and TIMP-1 (Aston et al., 1996; Stamouli et al., 1996; Goldman et al., 1997). Human chorionic gonadotrophin (HCG) was shown to suppress the overall activity of granulosa cell-derived MMP by both suppression of their production by the cultured cells and enhancement of TIMP production (Stamouli et al., 1996). In addition to, and as a result of, hormonal regulation, a hierarchy has been suggested in the function of MMP, such as the case of membrane type MMP-1 (MT1-MMP) serving as the activator of MMP-2 (Liu et al., 1998; Hirsch et al., 1999). Furthermore, there are reasons to believe that the MMP-TIMP system, as a proteolytic cascade, may interact with other intraovarian regulatory systems, such as that of IGF-I and its respective binding proteins (Resnick et al., 1998).
In view of all the above, we set out to explore possible differences between women with PCOS and normal ovulatory women in the granulosa cell production and secretion of MMP and TIMP.
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Materials and methods |
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Patients and induction of ovulation
The present study involved 24 normal ovulatory women (controls; age 25-36 years; treated with assisted reproduction for male factor infertility) and 24 women affected with PCOS (study; age 26-36 years) diagnosed by endocrine profile (hyperandrogenism and LH:FSH ratio >2) and ultrasonographic criteria. Ultrasonic evidence was considered to be the presence of large ovaries with thickened cortex and multiple subcapsular follicles of 4-8 mm diameter before stimulation. These ultrasound criteria have been widely used in European studies to define polycystic ovarian morphology (Kyei-Mensah et al., 1996). Patients' details are shown in Table I
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Both types of patients underwent induction of ovulation for IVF/intracytoplasmic sperm injection-embryo transfer, as described previously. The local ethics committee formally approved the use of granulosa cells. Each participating patient consented to this as well. The protocols used for ovarian stimulation were as previously described (Shalev et al., 1995
; Ben-Shlomo et al., 1997): `long' or `short' protocols of gonadotrophin-releasing hormone (GnRH) agonist before administration of gonadotrophins. In each group of women, PCOS and normal controls, there were 12 women receiving long protocols and 12 women receiving short protocols. For the long protocol, DTRP-6-GnRH (Decapeptyl; Ferring, Malmö, Sweden) 3.75 mg controlled release was injected i.m. during the mid-luteal phase and ovarian down-regulation was verified 2 weeks later, before human menopausal gonadotrophin (HMG) 150–225 IU/day (Humegon; Organon, Os, The Netherlands) was started. For the short protocol, s.c. DTRP-6-GnRH 0.1 mg/day was started on the day of menstruation and HMG 150–225 IU/day was added on the third day. HCG (Pregnyl; Organon, Os, The Netherlands) 5000–10 000 IU i.m. was administered when at least three follicles reached a mean diameter of 18 mm. Oocyte retrieval was performed transvaginally under ultrasonographic guidance (5.0 MHz probe, SSD-1400; Aloka, Tokyo, Japan) 32–38 h later.
After oocyte removal, follicular fluid samples were centrifuged (800 g for 10 min) and supernatants were stored at –20°C until assays were performed. Only blood-free, yellow-coloured, undiluted follicular fluid samples were obtained from each patient. The protein content of each sample was determined by the Bio-Rad Laboratories, Inc. (Washington, DC, USA). Protein assay kit with bovine serum albumin (BSA) as the standard.
Cell culture
Human luteinized granulosa cells were harvested only from large follicles (>16 mm diameter). Cell preparation was as commonly used and was identical to that described in detail in our previous study (Goldman et al., 1997). Cells were cultured in triplicates at a density of 300 000 cells/well in 0.5 ml human tubal fluid medium (HTF; Irvine Scientific, Santa Ana, CA, USA) replete with 10% artificial serum substitute (SSR; Irvine Scientific).
For the time-course study, cells were cultured and at the termination of incubation (24–96 h) the medium was collected for substrate gel electrophoresis (zymography) and Western blot analyses.
Unless stated otherwise, all materials were purchased from Sigma Chemical Co. (St Louis, MO, USA).
Protein assay
The total protein content of luteinized granulosa cells was determined using protein assay kit with BSA as the standard (Bio-Rad). Samples of 5-20 µl were used in the assay.
Substrate gel electrophoresis (zymography)
In order to detect proteolytic activity within the follicular fluid and conditioned media (CM), substrate gel electrophoresis (zymography) on gels containing gelatin as the substrate were used (Salamonsen et al., 1993). CM (30 µl) diluted to contain 50 µg protein, a mass marker (10 µl), or standard commercial gelatinases A and B (7 µl; Oncogene Science, Cambridge, MA, USA) were diluted with 10 µl 4xsample buffer [5% sodium dodecyl sulphate (SDS), 20% glycerol in 0.4 mol/l Tris, PH 6.8 containing 0.02% Bromophenol Blue without 2-mercaptoethanol]. Samples were electrophoresed through a 10% polyacrylamide gel containing 0.5% gelatin (50 µg/ml). After electrophoresis, gelatin gels were washed twice in 2.5% Triton X-100 for 15 min. Gelatin gels were incubated for 24 h at 37°C in 0.2 mol/l NaCl, 5 mmol/l CaCl2, 0.02% Brij 35 and 50 mmol/l Tris, pH 7.5. The buffer was decanted and the gel stained with Coomassie Blue G in 30% methanol and 10% acetic acid for 10 min at room temperature on a rotary shaker. The stain was washed out with water until clear bands were seen. Finally, the gel was incubated for 30 min in 45% methanol, 5% glycerol prior to drying overnight between stretched sheets of cellophane. Areas where proteolytic activity degraded the gelatin are seen as absence of staining (`punch-out' bands). These were quantified using the BioImmaging gel documentation system (Dinco & Renium, Jerusalem, Israel) endowed with TINA software (Raytest, Staubenhardt, Germany). In addition, a validation/inhibition study with 10 mmol/l EDTA was performed (data not shown). This procedure chelates the Zn2+ at the active site of MMP and inhibits their activity (Rao et al., 1995).
Western blot analysis
To further verify the identity of MMP, we used Western blot analyses. CM (30 µl) diluted to contain 50 µg protein or a mass marker (10 µl) were diluted with 4xsample buffer (5% SDS, 20% glycerol in 0.4 mol/l Tris, pH 6.8 containing 0.02% Bromophenol Blue without 2-mercaptoethanol) and subjected to 10% polyacrylamide gel electrophoresis. After electrophoresis, the proteins (50 µg/lane) were blotted from the SDS–polyacrylamide gel electrophoresis (PAGE) gel onto 0.45 µm nitrocellulose membranes (Scheicher & Schuel, Dassel, Germany). Non-specific binding sites were blocked by incubating the nitrocellulose membranes overnight with 20% non-fat milk and Tris-buffered saline, containing 0.01% Tween-20. The membranes were then washed twice with Tris-buffered saline, containing 0.5% Tween-20, and incubated for 1 h with: mouse anti-human TIMP-1, mouse anti-human MMP-2 and rabbit anti-human MMP-9 monoclonal antibodies (Oncogene Science, Cambridge, MA, USA) in 10% non-fat milk and Tris-buffered saline, containing 0.01% Tween-20. The membranes were subsequently washed with Tris-buffered saline, containing 0.5% Tween-20 and incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit secondary antibody or horseradish peroxidase-conjugated anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) in 10% non-fat milk and Tris-buffered saline, containing 0.01% Tween-20, then detected by enhanced chemiluminescence (Amersham International) and quantified by densitometry as above.
Statistical methods
Statistical analysis of the data was performed using Student's t-test when two treatments were compared and analysis of variance when more than two treatments were evaluated (e.g. dose-dependent responses). P < 0.05 was considered significant.
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Cultured granulosa cells from women with PCOS appeared significantly different than cells from normal women
Figure 1
shows a representative photomicrograph of typical cells in monolayer culture from (A) a normal control and (B) a PCOS patient. The cells from normal ovulatory control women formed a fibroblast-like monolayer, whereas the cells from PCOS women assumed a more globular structure, lacking pseudopodia.
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Follicular fluid from women with PCOS contains high levels of MMP-9 and MMP-2, as compared to fluid from normal ovulatory women
Clear follicular fluids were aspirated from the collected follicles of the first eight women with PCOS and the first eight normal ovulatory women. Fluids were assayed as described in Materials and methods. Figure 2
displays a representative zymography for MMP-9 and MMP-2 (92 and 72 kDa respectively). Follicular fluids from women with PCOS showed 1.7-fold and 1.6-fold increased activities of MMP-9 and MMP-2 respectively, as compared to normal controls (1137 ± 213 and 773 ± 39 versus 641 ± 57 and 423 ± 63 arbitrary units respectively, P < 0.05).
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Cultured granulosa cells from women with PCOS secrete higher amounts of MMP-9, compared to cells from normal ovulatory controls
Granulosa cells from PCOS patients (n = 16) and normal controls (n = 16) were harvested and cultured as described in Materials and methods. After 48 h of incubation, 30 µl of conditioned media diluted to contain 50 µg protein was used for Western blot analysis and zymography. As shown in Figure 3B and C
, media conditioned by cells from women with PCOS contained 2.9-fold higher levels of immunoreactive MMP-9, (3948 ± 1469 versus 1353 ± 1438, P < 0.05), compared to media conditioned by cells from normal controls. On zymography (Figure 3A), gelatinolytic activity of a 92 kDa protein, corresponding to MMP-9 was clearly seen. The difference between PCOS and normal subjects was even more pronounced, 5.5-fold higher (3191 ± 2475 versus 571 ± 274, P < 0.05) by this method (Figure 3A and C).
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Cultured granulosa cells from women with PCOS secrete higher amounts of MMP-2, compared to cells from normal ovulatory controls
Granulosa cells from PCOS (n = 16) and normal controls (n = 16) were harvested and cultured as described in Materials and methods. After 48 h of incubation, 30 µl of conditioned media diluted to contain 50 µg protein was used for Western blot analysis and zymography. As revealed in Figure 4B
, media conditioned by cells from women with PCOS contained 3.26-fold higher levels of immunoreactive MMP-2 (319 ± 58 versus 1040 ± 113, P < 0.05), compared to media conditioned by cells from normal controls. On zymography (Figure 4A), this difference was 6.1-fold (1596 ± 527 versus 260 ± 67, P < 0.05).
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Cultured granulosa cells from women with PCOS and normal ovulatory controls secrete similar amounts of TIMP-1
As described earlier, 50 µg protein in 30 µl aliquots of media, conditioned by granulosa cells from PCOS (n = 16) and normal controls (n = 16), were processed for Western blot analysis. As shown in Figure 5
, there was no difference in the production of TIMP-1 between the two types of granulosa cells.
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The secretion of MMP-9 by cultured granulosa cells from women with PCOS increases during 96 h of incubation much more than in cells from normal ovulatory controls
Granulosa cells obtained from four normal and four PCOS patients were incubated for up to 96 h as described earlier. During these 96 h of incubation, the levels of MMP-9 increased slightly in the media conditioned by normal granulosa cells, and increased significantly in the media conditioned by PCOS granulosa cells (Figure 6A and B
). The levels of MMP-2 were initially higher in media conditioned by PCOS granulosa cells compared to controls, and increased only slightly and not significantly more than in the controls (Figure 6A).
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The secretion of TIMP-1 by cultured granulosa cells from women with PCOS and normal ovulatory controls increases during 48 h of incubation
Granulosa cells from four normal and four PCOS patients were incubated for up to 96 h as described earlier. TIMP-1 was detected by Western blot analysis as described above. During 96 h of incubation, the levels of TIMP-1 in the conditioned media increased with both normal and PCOS cells up to 48 h. Thereafter, its levels decreased in both normal and PCOS samples (Figure 7
).
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Discussion |
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The physiology of ovarian cells and its reflections in women with PCOS have been studied extensively. The most prominent direction of this investigative effort has been towards hormonal analyses (Willis and Franks, 1995
; Anderson and Lee, 1996). In this regard, it has been suggested that the failure of granulosa cells to proliferate and to produce adequate amounts of oestradiol results from androgen excess along with reduced levels of FSH and its cognate receptor (Balen et al., 1995). Nevertheless, a satisfactory explanation for the persistence of many mid-size follicles has not yet been suggested. Such an explanation may arise from putative differences in ovarian tissue remodelling between women with PCOS and normal ovulatory women.
The matrix degrading MMP and their physiological inhibitors, TIMP, are thought to play an important role in tissue remodelling under various physiological and pathological conditions (Birkedal-Hansen et al., 1993; Powell and Matrisian, 1996). The development of ovarian follicles and the breakdown of the follicular wall to release the mature oocyte at the time of ovulation as well as the formation of the corpus luteum from luteinizing follicular cells, all involve extensive angiogenesis and tissue remodelling (Tsafriri, 1995). If pregnancy does not occur, the corpus luteum will undergo regression which again involves extensive tissue degradation. Consistent with this, studies in rat, mouse and humans have shown that gelatinolytic (MMP-9 and MMP-2) and collagenolytic (MMP-1 and MMP-3) activities are present in the ovary (Tadakuma et al., 1993; Hulboy et al., 1997; Bagavandoss, 1998).
Three MMP have been detected in human luteinized granulosa cells. These are MMP-1, MMP-2 and MMP-9 (Aston et al., 1996; Stamouli et al., 1996; Zhao and Luck, 1996; Nothnick et al., 1997). Although several intraovarian regulators, such as IL-1, have been found to influence the level of expression of these respective MMP, it is not fully understood what may be the pathophysiological significance of their altered expression (Nothnick et al., 1997).
In the present study we have shown for the first time that MMP-9, MMP-2 and TIMP-1 are expressed differently in PCOS compared to normal human ovaries. The model accessible to us was that of luteinized granulosa cells, which are in some respect terminally differentiated. Nevertheless, in the absence of developmentally earlier forms of human granulosa cells, these cells are accepted to at least partially reflect preceding ovarian events. It is also important to note that both groups of women had undergone ovarian stimulation before removal of the follicles. While this would have affected the endocrinological status of the ovary, the differences observed between the PCOS and normal groups suggest that they could be due to the underlying pathological condition.
We found that cultured luteinized granulosa cells obtained from PCOS patients secrete higher levels of MMP-9 and MMP-2 compared to granulosa cells from normal ovulatory patients. The same trend was observed in the follicular fluid, indicating that the in-vitro finding is consistent with the in-vivo situation. In contrast to MMP-2 and MMP-9 activity, the secreted basal level of TIMP-1 was similar in both types of granulosa cells. Together these results indicate a higher net gelatinolytic activity within the luteinizing granulosa cells of patients with PCOS. One could therefore suggest that a high gelatinolytic activity could contribute to a rapid regression of the corpus luteum and consequently lead to insufficient luteal function, resulting in a higher than normal abortion rate, as has been well documented in PCOS patients (Tulppala et al., 1993). Stamouli et al. (1996) have shown that HCG decreases MMP-9 expression and increases TIMP expression and have suggested that the rescue of the corpus luteum in early pregnancy involves the maintenance of cellular function through the stabilizing of the ECM by increasing TIMP level. Moreover, it has recently been shown that in porcine luteinized granulosa cells, MMP are expressed and released in high amounts and that this is essential for the structural regression of the corpus luteum (Pitzel et al., 2000). While there are several types of TIMP, it is TIMP-1 that has been most consistently demonstrated in the ovary. However, it is possible that one of the other TIMP, for which we did not probe, could play a modulatory role in the collagenolytic balance in the ovary and corpus luteum.
It is well known that together with anovulation and increased androgen production, patients with PCOS portray a typical morphology of multiple mid-size ovarian cysts. These are believed to be follicles that have undergone early antral stage atresia but for some unknown reason did not shrink and disappear (Cano et al., 1997). It is conceivable that to a certain extent tissue remodelling in women with PCOS is different from normal, since, in normal ovulatory women, non-dominant follicles undergo atresia only at the pre-ovulatory stage. It has been shown that in sheep, diversion of normal follicles to atresia by hypophysectomy is followed by a significant increase of intrafollicular levels of MMP-2 and MMP-9 and the disappearance of connexin-43 (Huet et al., 1997). It is therefore reasonable to speculate that MMP-9 and MMP-2 may be associated with inappropriate atresia in PCOS.
It has been postulated that granulosa cells from women with PCOS are abnormal in many respects (Palumbo et al., 1993; Anderson and Lee, 1997). Our findings add another facet to these. Whether the difference in granulosa MMP/TIMP balance is secondary to the other abnormal phenomena, or primary to them, remains to be defined.
Our in-vitro time-course observations on the levels of MMP-9 and MMP-2, as well as of TIMP-1, indicate that after plating for culture, normal luteinized granulosa cells secrete MMP-9 and TIMP-1 for up to 48 h, while PCOS granulosa cells continue to secrete them even after 96 h. This may indicate a failure to shut down MMP production by these cells, in turn disclosing a possibly different set of regulatory events.
In conclusion, we found a remarkable difference in the production and secretion of MMP-9 and MMP-2 and TIMP-1 by luteinized granulosa cells from patients with PCOS when compared to those from normal ovulatory controls.
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3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Ha'Emek Medical Centre, Afula 18101, Israel. E-mail: shalev_e{at}clalit.org.il
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Submitted on September 12, 2000; accepted on January 26, 2001.
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