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Regular Article |
a Department of Obstetrics and Gynecology, Sapporo Medical University School of Medicine, Sapporo 060-0061, Japan
ABSTRACT
Polycystic ovary syndrome (PCOS) is characterized by cystogenesis; however, the cause of this cystogenesis is unknown. At ovulation, preovulatory collagenolytic activities in the ovarian follicles increase and various proteinases are needed to degrade the tissues surrounding the follicles. To clarify the roles of enzymes in collagen degradation of the follicular wall of polycystic ovary (PCO) in relation to the cystogenesis, we examined expression of lysyl oxidase (LOX), which initiates cross-link formation of the collagen and elastin in the extracellular matrix, and expression of matrix metalloproteinases (MMPs) in ovaries of model rats with PCO induced by dehydroepiandrosterone (DHEA) compared with MMP expression in control rats. DHEA treatment increased LOX mRNA expression to more than three times the control value (P < 0.01). MMP-2 mRNA expression in control rats was threefold greater than that in the DHEA-induced group (P < 0.05). Expression of both latent and active forms of MMP-2 in controls was more than twice that in the DHEA-induced group (P < 0.05) as shown by Western blotting, and expression of the active form of MMP-2 was also twice as high in the controls as in the DHEA-treated group (P < 0.05) as shown by zymography. Our results suggest that depression of MMP-2 activity and increased LOX expression may be one of the causes of the cystogenesis of PCO.
follicle, follicular development
INTRODUCTION
Polycystic ovary syndrome (PCOS) is a common disorder characterized by oligomenorrhea, bilateral polycystic ovaries with a thickened, fibrotic tunica albuginea and a subcortical band consisting of many cystic follicles in various stages of growth and atresia [1]. Hyperinsulinemia affects the development of cystic ovaries in the rat by causing an increase in the size of the cystic follicles and in the size of the ovary [2]. Since the first description in 1935 of Stein-Leventhal syndrome in women, a large number of clinical studies and experiments have been done to elucidate the pathophysiology of this disorder. Although it has been difficult to evaluate the etiology and development of PCOS in patients, the similarities in key steps of mammalian reproduction make animal models at[fp[gctractive for studying the pathogenesis of this syndrome. Polycystic ovary (PCO) can be induced in prepubertal rats by daily injection of dehydroepiandrosterone (DHEA) [3]. The polycystic ovaries of the DHEA-treated animals are steroidogenically more active than those of controls, raising the possibility that the DHEA acts directly on the ovary in addition to an action on the pituitary-hypothalamus axis [4]. In an effort to produce a model to study PCOs, DHEA was injected into immature rats, which resulted in ovarian cystic and hormonal changes [5]. This model exhibits many of the salient features of human PCOS [4, 6]. Under these conditions, prepubertal female rats develop cysts and become anovulatory and acyclic. Follicles after DHEA treatment underwent atresia or exhibited various stages of cystogenesis. Complete atresia or cystogenesis may complete the pathological transformation into a follicular cyst [7]. The cause of the lack of ovulation in PCOS remains unclear. Although the disruption of consistently appropriate hypothalamic-pituitary-ovarian interactions may explain the general concept of the lack of ovulation in PCOS, little attention has been given to the effect of such a state on ovarian follicular enzymes that are known to be involved in the process of ovulation. Collagen fibers in the ovarian follicles change during ovulation because of the preovulatory increase of collagenolytic activities. The collagenous components in the ovaries are interstitial collagen types I and III and type IV collagen in the basement membrane layer [8]. During follicular growth, this layer of connective tissue becomes thinner, but it remains intact until ovulation [9]. Recent studies on follicle rupture have focused on two proteolytic enzyme systems, the plasminogen-activating system and collagenase. Various proteinases are needed to degrade the connective tissue components surrounding the follicle. Matrix metalloproteinases (MMPs) are a group of matrix-degrading enzymes that degrade all the components of the extracellular matrix. The gelatinases (MMP-2 and MMP-9) degrade basement membrane collagens, gelatins, and elastin [10]. Lysyl oxidase (LOX) initiates cross-link formation of the collagen and elastin in the extracellular matrix [11]. The presence of 0.1 Schiff-base cross-links per collagen molecule results in twofold to threefold resistence to human synovial collagenase when compared with non-cross-linked controls or samples [12]. In the ovulatory process, collagen synthetic activity regulated by prolyl hydroxylase and LOX activities in the ovarian follicles of rabbits is activated after follicle rupture, resulting in reconstruction of collagen fiber [13]. The purpose of this study was to examine whether cystogenesis in PCOs might be involved in the process by producing components of the extracellular matrix. We examined the activity of MMPs, which might induce the rupture of collagen of follicle walls, and the expression of LOX, which might inhibit the rupture of collagen of follicles.
MATERIALS AND METHODS
Hormones and Drugs
DHEA was obtained from Wakou Hormone Manufactory Co. (Tokyo, Japan). Sesame oil was obtained from Teikoku Hormone Manufactory Co. (Tokyo, Japan).
Animal Treatments and Tissue Collection
Immature (21 day old) Sprague-Dawley female rats were purchased from Hokudo Co. (Sapporo, Japan) in the morning. To induce the hyperandrogenic PCO condition, 22-day-old rats were injected s.c. each day with DHEA (6 mg/100 g body weight/0.2 ml sesame oil) in the evening for 7 or 15 days. Control animals were injected with 0.2 ml of sesame oil in the evening for an equivalent length of time. These injections were done by gently holding the tail of the rat and catching the rat in the left hand. There was no sign of inflammation at the injection sites throughout the experiment. Rats were usually killed by decapitation in the evening of Day 8 or Day 16. The ovaries were dissected, trimmed, weighed, flash-frozen on dry ice, and stored at -80°C. Some ovaries were stained with hematoxylin and eosin. Trunk blood was collected for measurement of plasma hormone levels, and the plasma and red cells were separated by centrifugation. The plasma was stored at -20°C for subsequent RIA analysis. Animals used in this study were maintained in accordance with the guidelines of the Animal Resources Center of the Sapporo Medical University School of Medicine.
FSH, LH, Estradiol, Progesterone, DHEA-Sulfate, Androstendione, and Testosterone
Serum FSH, LH, estradiol, progesterone, DHEA-sulfate (DHEA-S), androstendione, and testosterone concentrations in control rats injected with sesame oil for 15 days and treated with DHEA for 15 days were determined by RIA. The five animals of each group were killed by decapitation in the evening of Day 16 after the start of injection, and the trunk blood was collected for measurement of plasma hormone levels. Plasma and red cells were separated by centrifugation and assayed using an RIA kit (DPC Co., Los Angeles, CA).
Gelatin Zymography and Western Blot Analysis
Ovaries were homogenized (100 mg wet weight/ml) in PBS (10 mM sodium phosphate and 150 mM sodium chloride, pH 7.8) containing 0.2% Triton X-100 with a Teflon glass tissue grinder on ice (15 strokes). Homogenates were centrifuged (12 000 x g at 4°C for 20 min), and the supernatant fractions were collected for protein assay and gelatin zymography as previously described [14]. Ovarian extracts (40 µg of protein) were subjected to electrophoresis in 10% polyacrylamide gels containing 1 mg/ml gelatin. Samples were diluted in nonreducing sample buffer (final concentration 1% SDS and 5% glycerol) and electrophoresed for 1–2 h at 10 mA. The gels were washed for 2 h in 2.5% Triton X-100 to remove SDS, rinsed three times with distilled water, and then incubated for 24 h in 50 mM Tris HCl at pH 8.0 with 5 mM CaCl2. The bands were revealed with Coomassie blue stain. Ovarian extract (20 µg of protein) was separated by 12.5% SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes using an electroblotting apparatus. Nonspecific binding sites were blocked by immersing the membrane overnight on an orbital shaker at room temperature in PBS containing 0.05% Tween 20 (TPBS) and 5% skim milk and then washing five times for 5 min each in TPBS. Rabbit serum anti-rat MMP-2 was obtained from Torrey Pines Biolabs (San Diego, CA). The membrane was incubated with the primary antibody (1:1000 diluted with PBS-BSA) for 90 min at room temperature in a humidfied chamber and washed five times for 5 min each in TPBS. The membrane was then incubated with horseradish peroxidase conjugate at 1:5000 dilution for 50 min at room temperature in a humidified chamber and washed five times for 5 min each in TPBS. The membrane was then incubated with Amersham enhanced chemiluminescence reagent (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK) for 1 min and exposed to x-ray film for 15 sec in a dark room. The densities of bands were measured with NIH Image (version 1.61; NIH, Bethesda, MD).
Northern Blot Hybridization
Total RNA (10 µg) of ovarian tissues was extracted by the guanidine-phenol method using Ultraspec RNA (Biotex Laboratories, Houston, TX). RNA was electrophoresed on 1% agarose-formaldehyde gel (100 V for 2 h), transferred onto nylon membranes (Nytran-Plus; Schleicher & Schuell, Keene, NH) in 20x standard saline citrate (SSC; 3 M sodium chloride, 0.3 M trisodium citrate) overnight, and then fixed with an ultraviolet light linker. Filters were prehybridized for 4 h and then hybridized overnight at 42°C with a radiolabeled cDNA probe. Human cloned MMP-2 cDNA probes [15, 16] and LOX cDNA probes [17] were radiolabeled with [32P]dCTP (Amersham) using Prime-It II random primer labeling kits (Stratagene, La Jolla, CA). Human MMP-2 and LOX sequences show >85% homology with rat MMP-2 and LOX sequences, respectively [18, 19]. Filters were washed in 2x SSC containing 0.1% SDS at 65°C for 15 min. Filters were exposed to x-ray film (Fuji RX; Fuji, Tokyo, Japan) at -80°C for 7 days. For quantitative analysis, the radioactivity was measured with a specific mRNA band analyzer (Fujix, Tokyo, Japan). Radioactivity was adjusted to that of L38 (ribosomal protein) RNA.
Statistics
Treatment effects were evaluated by one-way analysis of variance, followed by Scheffé's F-test post hoc analysis and the unpaired Student's t-test. A difference of P < 0.05 was considered significant.
RESULTS
Steroid Hormones
As shown in Table 1, the serum progesterone levels were higher in controls injected with vehicle for 15 days than in rats treated with DHEA for 15 days (P < 0.05).
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The serum DHEA-S, testosterone, androstendione, and estradiol levels were higher in DHEA-treated rats than in controls (P < 0.05) at 15 days. There was no significant difference in the serum FSH and LH levels between these two groups.
Northern Blot Hybridization of LOX and MMP-2
Expression of LOX mRNA (5.0 kilobases [kb]) was 50.5% greater in ovaries of animals treated with DHEA for 15 days than in ovaries of animals treated with DHEA for 7 days (n = 6 animals; P < 0.05). DHEA treatment for 15 days stimulated LOX mRNA expression to more than threefold the value of controls at 7 days and 15 days (n = 6 animals; P < 0.01). DHEA treatment for 7 days stimulated LOX mRNA expression to more than twofold the value of controls at 7 days (n = 6 animals; P < 0.05; Fig. 1). We also analyzed MMP-2 mRNA (3.2 kb) expression from ovaries of the DHEA-treated group and the untreated controls. The MMP-2 mRNA level of the rats treated with DHEA for 7 days was 93.5% greater than that of the group treated with DHEA for 15 days (n = 6 animals; P < 0.05). The MMP-2 mRNA level in control ovarian tissues for 7 days or 15 days was four times that of the group treated with DHEA for 15 days (n = 6 animals; P < 0.05; Fig. 2).
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Western Blot Analysis of MMP-2
Western blot analysis also revealed that these two bands corresponded to the active form (67 kDa) and latent form (72 kDa) of MMP-2 (Fig. 3). The expression of the active form of MMP-2 was 66.7% greater in ovaries of animals treated with DHEA for 7 days than in ovaries of animals treated with DHEA for 15 days (n = 4 animals; P < 0.05). The expression of the active form of MMP-2 in control ovarian tissues for 7 days and 15 days was almost twofold that of the group treated with DHEA for 15 days (n = 4 animals; P < 0.05). The expression of the active form of MMP-2 in control ovarian tissues for 7 days was 35.8% greater than that in ovaries of animals treated with DHEA for 7 days (n = 4 animals; P < 0.05). The expression of the latent form of MMP-2 in ovaries of animals treated with DHEA for 7 days was 95.4% greater than that in ovaries of animals treated with DHEA for 15 days (n = 4 animals; P < 0.05). The expression of the latent form of MMP-2 in control ovarian tissues for 7 days and 15 days was almost threefold that of the group treated with DHEA for 15 days (n = 4 animals; P < 0.01). The expression of the latent form of MMP-2 in control ovarian tissues at 7 days was 71.4% greater than that in ovaries of animals treated with DHEA for 7 days (n = 4 animals; P < 0.05).
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Gelatin Zymography
Gelatinase activity in extracts of ovarian tissues was examined by gelatin zymography (Fig. 4). The major gelatinase activity was present in bands of around 68 kDa, with less activity at around 62 kDa on the zymograms. Although not all MMPs can be detected by zymography, we suspected that the major MMP in ovarian tissues was MMP-2, present predominantly as the latent (68 kDa) form, with minor levels of activated MMP-2 (62 kDa). Gelatinase activity (62 kDa) in ovaries of animals treated with DHEA for just 7 days was 47.3% greater than that in ovaries of animals treated with DHEA for 15 days (n = 6 animals; P < 0.05). Gelatinase activities in control ovaries treated for 7 days and 15 days were 101.0% and 74.6% greater than gelatinase activities for ovarian tissue treated with DHEA for 7 days and 15 days, respectively (n = 6 animals; P < 0.01).
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DISCUSSION
The main criteria for determining the validity of an animal PCO model should include the maximum anatomic and physiologic similarities between that particular animal model and those of human PCO [20]. There are similarities in the alterations found in the patients with PCO and those described in rats injected with DHEA. The ovaries from DHEA-treated rats exhibit the formation of cysts from antral follicles [21]. The morphology of PCOs induced in treated ovaries by DHEA injection and in control ovaries by sesame oil injection in the present study was similar to that seen in other PCO model rats.
DHEA treatment increased LOX mRNA expression to more than threefold the control value (P < 0.01). MMP-2 mRNA expression in control rats was fourfold that of the DHEA-induced group (P < 0.05). Expression of both latent and active forms of MMP-2 was more than twice as high in controls as in the DHEA-treated group (P < 0.05), as shown by Western blotting. The active form of MMP-2 was more than twice as high in the controls as in the DHEA-treated group (P < 0.05), as indicated by zymography.
These results suggest that depression of MMP-2 activity and increased LOX expression may be one of the causes of the cystogenesis of PCOS.
The concentrations of serum DHEA-S, testosterone, and androstendione were higher in DHEA-treated rats than in control rats (P < 0.05). It appears that elevated testosterone and androstendione are important characteristics of ovarian cyst development [21]. Elevated levels of circulating androgens such as androstendione, testosterone, and DHEA are frequently associated with the presence of ovarian cysts and ovarian failure [22]. Testosterone treatment [23], old age [24], and exposure to constant light [25] are all examples of treatments that induce ovarian cysts in rats and are associated with elevated androgens. Elevated serum levels of androgens and estrogens were reported to increase ovarian steroidgenesis in DHEA-treated rats. Because DHEA is a weaker androgen than androstenedione, DHEA may be metabolized to aromatizable androgens and estrogen in some other tissues. Furthermore, the results showing high androgen and estradiol levels in the animals with cysts support the hypothesis that elevated levels of testosterone, androstendione, and estrogens may be important in the development and maintenance of PCOs in this animal model [5]. Animals treated with DHEA showed no changes in serum FSH and LH concentrations as compared with controls. Gonadotropins may play a secondary role in cyst formation in DHEA-treated rats.
Collagen fibers in ovarian follicles undergo drastic changes during ovulation because of the preovulatory increase of collagenolytic activities. The extracellular matrix surrounding ovarian follicles consists of the basement membrane under the germinal epithelium, the tunica albuginea, the theca externa, the theca interna, and the basement membrane adjacent to the granulosa cells. The collagenous components in the above structures are interstitial collagen types I and III, and type IV collagen in the basement membrane layers [8]. During follicular growth, this layer of connective tissue becomes thinner, but it remains intact until ovulation [9]. Various proteinases have been reported to be involved in the destruction of the follicle apex [26]. Collagenase activity increases toward the time of ovulation [27, 28]. Various proteinases are needed to specifically degrade the connective tissue components, types I, III, and IV collagen, surrounding the follicle. The significance of proteolytic enzymes during follicular maturation has been widely studied in animal models and in humans.
Cystogenesis and collagen have been studied extensively, but cystogenesis concerned with collagen of the follicles in PCOs has not. To clarify the regulatory roles of proteinases and enzymes in collagen modification of follicular walls of PCO rats in relation to cystogenesis, we measured expression of MMPs and LOX in ovaries of control rats injected with sesame oil and in rats with PCOs induced by DHEA. The experimental groups consisted of two control groups given sesame oil for 7 and 15 days and two PCO model groups given DHEA for 7 and 15 days.
MMPs are matrix-degrading enzymes that can degrade all matrix components. The 72-kDa gelatinase (MMP-2) and 92-kDa gelatinase (MMP-9) degrade denatured collagen (gelatin) and type IV collagen. MMPs have been identified in the ovaries of humans, rats, and mice [29–31] and play important roles in the extracellular matrix, including during ovulation [32, 33]. Zymographic analysis of this specific collagenolytic activity established the involvement of intersitial collagenase (MMP-1) and collagenase type IV (MMP-2) [14].
In this study, expression of MMP-2 mRNA and protein and gelatinase activity in the ovaries injected with sesame oil for 15 days were more than twofold higher in the model rats in which PCO was induced by DHEA for 15 days. These results suggested that decreases in the amount of MMP-2 expression in the ovaries of the PCO model rats might be correlated with DHEA treatment. Gelatinase activity of MMP-2 was higher in the PCOs induced with DHEA for 7 days and was more than twofold that in PCOs induced with DHEA for 15 days. This finding suggests that the activity of MMP-2 in the PCOs induced by DHEA might decrease as the period of DHEA treatment progresses and that the follicle walls of PCOs might be prevented from specifically degrading the connective tissue components surrounding the follicle because of the decrease in the activity of MMP-2 caused by DHEA treatment. Gelatinase activity of MMP-2 in the ovaries of rats injected with sesame oil for 7 days was equivalent to that in ovaries of rats injected with sesame oil for 15 days. Although it was possible that ovaries of rats injected with sesame oil for 15 days might partially include corpora lutea, the lack of significant differences in MMP-2 activity between rats injected with sesame oil for 15 days and and those injected for 7 days (whose ovaries included no corpora lutea) indicated that any difference in MMP-2 activity in the PCOs induced by DHEA was not correlated with the age of these rats but rather with the period of DHEA treatment.
LOX oxidizes peptidyl lysine and hydroxylysine residues to peptidyl aldehyde residues within collagen and elastin, thus initiating formation of the covalent cross-linkages that insolublilize these extracellular proteins. The known biochemical function of LOX is to catalyze the oxidative deamination of certain lysine and hydroxylysine (collagen only) residues in tropoelastin and tropocollagen before the formation of cross-links. The presence of 0.1 Schiff-base cross-links/collagen molecule results in a twofold to threefold resistance to human synovial collagenase when compared with non-cross-linked controls or samples [12]. In the ovulatory process, collagen synthetic activity is regulated by prolyl hydroxylase and lysyl oxidase activities in the ovarian follicles of rabbits [13].
Expression of LOX mRNA was increased in the ovaries of PCO model rats to more than threefold the control levels. These data suggest that the increase of expression of LOX in the ovaries of PCO model rats may form the cross-linkages within collagen and elastin that insolublilize the extracellular matrix in the follicle walls of PCOs and strengthen the defense of the collagen of these follicle walls during the process of cystogenesis.
DHEA treatment caused the formation of cysts from antral follicles in the ovaries of immature rats. DHEA treatment depressed MMP-2 activity, emphasizing collagen degradation as an important event in the process of ovulation and increased LOX expression, which activates the formation of cross-linkages that insolubilize these extracellular proteins. These changes in proteinases and enzymes may be one of the causes of cystogenesis in the ovaries of PCOS patients.
ACKNOWLEDGMENTS
The authors thank the staffs of Dr. Toshihiro Mitaka, Dr. Motoiki Koizumi, and Dr. Tsuyoshi Saito (Sapporo Medical University) for helpful discussion and suggestions.
FOOTNOTES
First decision: 24 April 2000.
1 This study was supported by Grant-in-Aid for Scientific Research-11671637 of the Japanese Ministry of Education, Science and Culture to T.E. 
2 Correspondence: T. Endo, Department of Obstetrics and Gynecology, Sapporo Medical University School of Medicine, S 1 W 16 Chuou-ku, Sapporo 060-0061, Japan. FAX: 011 614 0860; endot{at}sapmed.ac.jp
Accepted: August 15, 2000.
Received: March 15, 2000.
REFERENCES
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