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Endocrinology, doi:10.1210/en.2007-0168
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Endocrinology Vol. 148, No. 8 3781-3791
Copyright © 2007 by The Endocrine Society

A New Rat Model Exhibiting Both Ovarian and Metabolic Characteristics of Polycystic Ovary Syndrome

Louise Mannerås, Stefan Cajander, Agneta Holmäng, Zamira Seleskovic, Theodore Lystig, Malin Lönn and Elisabet Stener-Victorin

Institute of Neuroscience and Physiology, Department of Physiology (L.M., A.H., E.S.-V.), and Institute of Medicine, Department of Molecular and Clinical Medicine (T.L., M.L.), Sahlgrenska Academy, Göteborg University, 405 30 Göteborg, Sweden; Department of Pathology and Cytology (S.C., Z.S.), Akademiska Sjukhuset, 751 85 Uppsala, Sweden; and Department of Pathology, University Clinic Center (Z.S.), Tuzla 75000, Bosnia and Hercegovina

Address all correspondence and requests for reprints to: Elisabet Stener-Victorin, Ph.D. and associate professor, Institute of Neuroscience and Physiology, Department of Physiology, Sahlgrenska Academy, Göteborg University, Box 434, SE-405 30 Göteborg, Sweden. E-mail: elisabet.stener-victorin{at}neuro.gu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder associated with ovulatory dysfunction, hyperandrogenism, abdominal obesity, and insulin resistance. However, its etiology is unclear, and its management is often unsatisfactory or requires a diversified approach. Here, we describe a new rat PCOS model, the first to exhibit both ovarian and metabolic characteristics of the syndrome. Female rats received the nonaromatizable androgen dihydrotestosterone (DHT) or the aromatase inhibitor letrozole by continuous administration, beginning before puberty, to activate androgen receptors. Adult DHT rats had irregular cycles, polycystic ovaries characterized by cysts formed from atretic follicles, and a diminished granulosa layer. They also displayed metabolic features, including increased body weight, increased body fat, and enlarged mesenteric adipocytes, as well as elevated leptin levels and insulin resistance. All letrozole rats were anovulatory and developed polycystic ovaries with structural changes strikingly similar to those in human PCOS. Our findings suggest that the formation of a "hyperplastic" theca interna reflects the inclusion of luteinized granulosa cells in the cyst wall rather than true hyperplasia. We conclude that the letrozole model is suitable for studies of the ovarian features of human PCOS, while the DHT model is suitable for studies of both ovarian and metabolic features of the syndrome.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN ANOVULATORY WOMEN with polycystic ovaries (PCOs), the prominent ovarian sign is follicular maturation arrest that results in an abnormal ovarian endocrine environment (1). The major marker of PCO morphology is hyperandrogenism (2, 3), and the theca cells are the major source of androgen excess (4). About 50% of women with polycystic ovary syndrome (PCOS) are obese, and abdominal obesity is common, suggesting that elevated androgen levels might increase the amount of adipose tissue, particularly in the abdominal region, in these women (5). Other metabolic disturbances such as dyslipidemia, insulin resistance, and type 2 diabetes, which increase the risk of cardiovascular disease, are also common (6). Therefore, tools to gain a deeper understanding of the pathogenesis of PCOS and to evaluate treatment alternatives are important.

The etiology of PCOS is unclear. One hypothesis is that PCOS is a genetically determined ovarian disorder in which excessive androgen production early in life may provide a hormonal insult that leads to PCOS in adulthood (7, 8, 9). After fetal exposure to high levels of androgens, adolescent rhesus monkeys and sheep show many features of PCOS. However, their use to study the etiology of PCOS is prohibitively expensive (10, 11, 12).

Adiposity may play a central role in generating and maintaining the syndrome. Weight reduction often improves menstrual regularity (13). A lipolytic defect in sc adipocytes may contribute to accumulation of fat and obesity (14). In addition, adipocytes of nonobese women with PCOS are 25% larger than in matched controls (14). Enlargement of abdominal sc adipocytes is associated with insulin resistance and is an independent risk factor for type 2 diabetes (15).

We and others have used a rat model in which PCOs are induced with estradiol valerate (16) to study the effects and mechanisms of electroacupuncture and exercise in the treatment of PCOS (17, 18, 19). Estradiol valerate results in acyclicity and ovarian morphology resembling PCO (16, 17, 18, 19, 20, 21), but without the typical metabolic disturbances of human PCOS (21).

In another promising rat model, PCOS is induced by daily prepubertal exposure to testosterone for 7–35 d (22). In addition to typical PCO morphology and a majority of apoptotic follicles, the rats had disturbed glucose and insulin levels, indicating that high levels of androgens can lead to insulin resistance in this model. Letrozole, a nonsteroidal aromatase inhibitor that blocks the conversion of testosterone to estradiol, also induces PCOs in 6-wk-old female rats (23). Endocrine disturbances similar to those in human PCOS were observed, but the metabolic characteristics of the syndrome were not investigated (24).

The heterogeneity of the syndrome is reflected in the many animal models of PCOS. However, few rat models have focused on the metabolic disturbances that are a major feature of human PCOS. Therefore, new rat models exhibiting not only the ovarian but also the metabolic characteristics of the syndrome would be valuable. Such models would, for example, enable further evaluation of new treatments for PCOS.

PCOS is associated with excessive androgen production during early puberty (7). In this study we sought to determine if continuous administration of the nonaromatizable androgen 5{alpha}-dihydrotestosterone (DHT), specific for the androgen receptor (AR), from before puberty to adult age, induces both PCOs and metabolic abnormalities similar to those in human PCOS. The effects of letrozole, administered in the same fashion, were evaluated to determine if it produces the metabolic effects of PCOS. Ovarian morphology, hormonal and metabolic status, body composition, and adipocyte size in abdominal fat depots were investigated in all rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Five Wistar dams, each with 10 female pups, were purchased from Charles River Laboratories, Inc. (Munster, Germany). Pups were raised with the lactating dam (not the biological mother of all the pups) until 21 d of age and then housed four to five per cage under controlled conditions (21–22 C, 55–65% humidity, 12-h light/12-h dark cycle). Rats were fed commercial chow (18.7% protein, 4.7% fat, 63% carbohydrates, vitamins, and minerals; B&K Universal, Sollentuna, Sweden) and tap water ad libitum. The study was approved by the Animal Ethics Committee (Göteborg University). Accepted standards of animal care were used.

Study procedure
At 21 d of age, rats were randomly divided into three experimental groups [control (n = 13), DHT (n = 12), and letrozole (n = 11)] and implanted sc with 90-d continuous-release pellets (Innovative Research of America, Sarasota, FL) containing 7.5 mg DHT (daily dose, 83 µg) or 36 mg of letrozole (daily dose, 400 µg) (Novartis Pharma AG, Basel, Switzerland). The dose of DHT was chosen to mimic the hyperandrogenic state in women with PCOS, whose plasma DHT levels are approximately 1.7-fold higher than those of healthy controls (24, 25). The dose of letrozole was chosen according to a previous study (23). Controls received identical pellets lacking the bioactive molecule. A microchip (American Veterinary Identification Devices; Avid Identification Systems, Inc., Norco, CA) with an identification number was inserted sc in the neck along with the pellets. Rats were weighed weekly from 21 d of age. The study was concluded after 11–13 wk of drug administration, when the rats were 14–16 wk of age.

Vaginal smears
The stage of cyclicity was determined by microscopic analysis of the predominant cell type in vaginal smears obtained daily from 11 wk of age to the end of the experiment (26).

Body composition
Body composition was analyzed by dual-emission x-ray absorptiometry (DEXA) at 12 wk of age (i.e. 9 wk after implantation of the pellet) with a whole-body DEXA instrument (QDR-1000/W; Hologic, Waltham, MA). Rats were anesthetized by inhalation of isoflurane (2% in 1:1 mixture of oxygen and air; Abbott Scandinavia AB, Solna, Sweden) before scanning. Body fat, lean body mass (LBM), and bone mineral content (BMC) were determined for each rat. Body fat distribution was assessed by magnetic resonance imaging (MRI) (27). Rats were anesthetized as described previously and placed in a 7-T MRI system (Bruker BioSpin, Ettlingen, Germany). The distribution of sc and intra-abdominal fat was determined by analyzing the seventh axial slice and a coronal slice from the most caudal part of the kidney.

Blood sampling for lipid profile, leptin, and sex steroids
At 12 wk of age (9 wk after pellet implantation), tail blood was obtained after an overnight fast to assess the lipid profile and leptin concentrations. At 13–14 wk of age, blood samples were obtained in the estrus phase for analyses of progesterone, 17ß-estradiol, and testosterone. Plasma samples were stored at –20 C.

Euglycemic-hyperinsulinemic clamp
At 14–16 wk of age (11–13 wk after pellet implantation), rats were subjected to a euglycemic-hyperinsulinemic clamp (28) during the estrus phase. Rats were anesthetized with thiobutabarbital sodium (130 mg/kg ip; Inactin; Sigma, St. Louis, MO). Body temperature was maintained at 37 C with a heating blanket. Catheters were inserted into the left carotid artery for blood sampling, and into the right jugular vein for glucose and insulin infusions, and a tracheotomy was performed.

A baseline blood glucose sample was drawn directly into microcuvettes (10 µl) and analyzed with a B-glucose analyzer (HemoCue AB; Dronfield, Derbyshire, UK). Insulin (100 U/ml; Actrapid; Novo Nordisk, Bagsvaerd, Denmark) together with 0.2 ml albumin and 10 ml saline were infused at 24, 16, and 12 mU/min·kg for min 1, 2, and 3, respectively, followed by 8 mU/min·kg for the rest of the clamp. To maintain plasma glucose at a euglycemic level (6.0 mM), 20% glucose in saline was administered. The glucose infusion rate (GIR) was guided by glucose concentration measurements every 5 min. At steady-state (after 50–70 min), the mean GIR was normalized to body weight, and blood samples were taken to determine plasma insulin concentrations. An insulin sensitivity index was calculated (mean GIR/plasma insulin levels at steady-state x 102).

The rats were decapitated, and the ovaries were excised, fixed in neutral buffered 4% formaldehyde for 24 h, placed in 70% ethanol, dehydrated, and embedded in paraffin. The hind limb muscles (extensor digitorum longus, tibialis anterior, and soleus) and the parametrial, retroperitoneal, inguinal, and mesenteric fat depots were dissected and weighed.

Ovarian morphology
The ovaries were longitudinally and serially sectioned at 4 µm; every 10th section (n = 6 per ovary) was mounted on a glass slide, stained with hematoxylin and eosin, and analyzed under a conventional birefringence microscope by two persons blinded to the origin of the sections. For measurements and photographs, the slides were scanned with ScanScope (Aperio Technologies, Vista, CA) and analyzed with ImageScope virtual microscopy software (Aperio Technologies). The area of the ovary was determined with a calibrated scale tool in the virtual microscope. The area of the largest follicle and the thickness of its follicular wall, i.e. the theca cell layer and granulosa cell layer, were measured, and follicles were counted by two persons to avoid duplicate counting. The ovarian follicles and corpora lutea (CL) at different stages of development and regression, and the theca and granulosa cell layer were analyzed in detail.

Computerized determination of adipocyte size
Parametrial and mesenteric adipose tissues were cut into small pieces and treated with collagenase (Type A; Roche, Mannheim, Germany) in minimum essential medium (1.5 mg/ml; Invitrogen, Carlsbad, CA) containing 5.5 mM glucose, 25 mM HEPES, 4% bovine albumin (Fraction V; Sigma), and 0.15 µM adenosine (pH 7.4), for 50 min at 37 C in a shaking water bath (29). After filtration through a 250-µm nylon mesh, adipocytes were washed three times and suspended in fresh medium. The mean cell size and the size distribution were determined by computerized image analysis (KS400 software; Carl Zeiss, Oberkochen, Germany) (30). In brief, the cell suspension was placed between a siliconized glass slide and a coverslip, and transferred to the microscope stage. Nine random visual fields were photographed with a CCD camera (Axiocam; Carl Zeiss). Relevant surface areas were measured automatically, and diameters of the corresponding circles calculated. Uniform microspheres (diameter, 98.00 µm; Bangs Laboratories, Fishers, IN) served as a reference.

Analytical methods
Plasma concentrations of progesterone, testosterone, and 17ß-estradiol were determined with commercial double-antibody RIA kits (progesterone RIA kit, DSL-3400; testosterone RIA kit, DSL-4100; 3rd Generation Estradiol RIA kit, DSL-39100; Diagnostic Systems Laboratories, Webster, TX). Plasma concentrations of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TGs) were determined enzymatically with a Konelab autoanalyzer 2.0 (Thermo Clinical Labsystems, Espoo, Finland). Plasma leptin concentration was similarly determined (rat leptin RIA kit, RL-83K; Lincon Research, St. Charles, MO). Human insulin, given during the clamp, was measured with an ELISA kit (10-1113-01; Mercodia, Uppsala, Sweden). All analyses are from the same rat and assay.

The intraassay and interassay coefficients of variation and sensitivity were: 7.5%, 8.1%, and 0.05 ng/ml (testosterone); 3.6%, 6.0%, and 0.6 pg/ml (estradiol); 5.1%, 2.5%, and 0.1 ng/ml (progesterone); 1.1%, 2.0%, and 0.1 mmol/liter (TC); 1.6%, 2.2%, and 0.04 mmol/liter (HDL); 1.0%, 2.5%, and 0.02 mmol/liter (TG); 3.3%, 4.8%, and 0.5 ng/ml (leptin); and 3.4%, 3.0%, and 1 mU/liter (human insulin).

Statistical analyses
Most statistical evaluations were performed with SPSS software (version 13.0; SPSS, Inc., Chicago, IL). Effects of DHT or letrozole on body weight were analyzed by repeated-measures ANOVA, and effects on body weight, tissue weight, ovarian morphology, insulin sensitivity, body composition, adipocyte size, lipid profile, and sex steroid and leptin concentrations by one-way ANOVA. Differences between groups and controls were tested with Dunnett’s post hoc test. Values are mean ± SEM. P < 0.05 was considered significant.

Regression analyses were performed using linear regression. Adipocyte size distribution curves were compared with the Kolmogorov-Smirnov two-sample test (KS) (31). An exact P value for the comparison of two groups (A and B) was calculated through permutations. For "n" subjects in group A and "m" subjects in group B, KS statistics were calculated for all possible ways of splitting n + m subjects into two groups of sizes "n" and "m." The observed KS statistic was then ranked against the KS statistics from all of the possible permutations. The permutation P value is the percentage of possible KS statistics that are at least as extreme as the KS statistic from the original data. For these comparisons, statistical calculations were performed with the R language (http://www.R-project.org).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Increased body weight in DHT and letrozole rats
After 2 wk, DHT and letrozole rats had gained more weight than the controls (ANOVA, P < 0.001) (Fig. 1Go). The gain was more pronounced in the letrozole group.


Figure 1
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  		FIG. 1. Growth curves of rats exposed to placebo (control), DHT, or letrozole from pellet implantation (21 d of age) to 84 d of age. Values are mean ± SEM. ***, P < 0.001 vs. controls (repeated-measures ANOVA).

 
Disrupted estrous cyclicity and PCO morphology in DHT and letrozole rats
All control rats had a normal estrous cycle of 4 d (Fig. 2DGo). Light microscopic analysis showed no structural abnormalities, follicles, and CL in different stages of development and regression, and no ovarian cysts (Fig. 2AGo). The theca and granulosa cell layer were normal.


Figure 2
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  		FIG. 2. A–C, Survey views showing control, DHT, and letrozole-exposed rat ovaries in the same magnification (magnification, x0.8; distance bars, 2.0 mm). A, Ovary from a normal cycling control rat, showing CL and follicles at different stages. B, Ovary from a DHT-exposed rat. Arrows refer to Fig. 3Go, A–C. C, Cystic ovary from a letrozole-exposed rat. The box refers to Fig. 3DGo. D, Estrous cycle patterns at 77–90 d of age (i.e. 56–69 d after pellet implantation) in four representative rats from each group. D, Diestrus; E, estrus; M, metestrus; P, proestrus.

 
The letrozole rats were completely acyclic, while the DHT rats had irregular cycles (Fig. 2DGo). Vaginal smears of both groups showed leukocytes, the dominant cell type of the diestrus phase, indicating "pseudo diestrus." Ovary weight and area were lower in DHT rats and higher in letrozole rats than in controls (Table 1Go). The area of the largest follicle was greater in letrozole rats than controls; a trend toward follicular enlargement was observed in DHT rats. A cystic follicle was considered to be a large fluid-filled cyst with an attenuated granulosa cell layer and thickened theca interna cell layer. The number of cystic follicles was increased, and the cystic wall was thickened, characterized by a thickened theca interna cell layer and a diminished granulosa cell layer, in DHT and letrozole rats compared with controls (Table 1Go and Fig. 3Go). Consistent with the perturbed cyclicity in both groups, the ovaries displayed atretic antral follicles, follicular cysts with a thickened theca interna cell layer, and macrophages in the cyst fluid. In addition, letrozole rats had no CL.


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  		TABLE 1. Effects of DHT and letrozole on ovarian weight, ovarian area, and follicle development

 

Figure 3
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  		FIG. 3. A–C, Ovary from the DHT-exposed rat shown in Fig. 2BGo. A, High-power view of the healthy tertiary follicle (arrow 3A in Fig. 2BGo). The theca externa (TE), theca interna (TI), and membrana granulosa (MG) layers appear normal. A mitotic granulosa cell (arrow) can be seen, but no apoptotic bodies. B, A follicle in the early process of atresia (arrow 3B in Fig. 2BGo) with apoptotic granulosa cells (arrow), most of which are in the inner parts of the membrana granulosa. C, A cystic follicle with advanced atresia (arrow 3C in Fig. 2BGo). Thin and elongated epithelioid cells (thick arrows) form the inner surface of the wall. The cyst fluid contains macrophages (thin arrows). D and E, Ovary from a letrozole-exposed rat (seen in Fig. 2CGo). D, Higher magnification view of the boxed area in Fig. 2CGo shows a cystic follicle with the oocyte in the plane of section. E, Boxed area in D at higher magnification. The cyst wall consists of a vascularized and luteinized granulosa layer. The cells have almost spherical nuclei (10 µm in diameter) with a distinct nucleolus (thin arrow). Cells facing the central fluid are thin, elongated, and epithelioid in appearance. Theca interna cells (thick arrow) are present in the outer region. F, Normal 5-d-old CL demonstrating the cellular similarities (same magnification; bar, 20 µm) with the luteinized cells in the letrozole rats.

 
The granulosa cell layers in DHT rats, if present, were usually thin; cells facing the cyst were flat and epithelioid, and the cyst fluid invariably contained macrophages (Fig. 3Go, B and C). Some follicles were undergoing atresia and had many apoptotic granulosa cells, mainly in the inner layers of the membrana granulosa (Fig. 3BGo). Occasionally, apparently healthy tertiary follicles were present.

As in human PCOs, the increased number of cysts in letrozole rats was typically orientated in the periphery of the ovary (Figs. 2CGo and 4AGo). In some cysts, the inner wall was completely vascularized, and consisted of a thick layer of large and uniform luteinized cells with rounded nuclei containing a distinct central or pericentral nucleolus; occasionally two nucleoli were seen (Fig. 4Go, D–F). These cells were smaller and had less cytoplasm (Fig. 3EGo) than luteinized cells in the CL of cycling rats but were otherwise similar (Fig. 3FGo). In addition, luteinized granulosa cells in the letrozole group had nuclei identical in size (10 µm) to those in CL from normally cycling rats. In other cysts, the inner wall was formed by a partly vascularized cell layer composed of granulosa cells and luteinized cells (Fig. 4FGo). Mitotic endothelial cells, a sign of vascularization, were occasionally observed. There were no mitotic theca interna cells in any healthy tertiary follicles, or cystic follicles in the DHT or letrozole group.


Figure 4
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  		FIG. 4. Cystic ovary from a letrozole-exposed rat. A, Survey view showing cysts. The larger boxed area is shown at higher magnification in B; the smaller boxed area is shown in D–F. B, Follicle cyst with the oocyte in the plane of section, shown at higher magnification in the upper right corner. C, Higher magnification view of the smaller boxed area at the left in B. The cyst wall has a thick, vascularized layer of luteinized cells and groups of granulosa cells (arrow). D, Cyst wall with partly remaining membrana granulosa on the left side. E, Detail from D illustrating the transition (longarrow) from the nonvascularized membrana granulosa to a partly luteinized and vascularized granulosa. Short arrows indicate the sharp demarcation between the granulosa layer and the theca interna. F, Detail from E showing the partly luteinized (short thick arrow) granulosa layer and focal groups of nonluteinized granulosa cells (long arrow).

 
Steroid concentrations in DHT and letrozole rats
The plasma testosterone concentrations were similar in DHT rats and controls but were increased in the letrozole group (Table 2Go). Progesterone was decreased in both experimental groups; 17ß-estradiol was unaltered.


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  		TABLE 2. Effects of DHT and letrozole on plasma concentrations of estradiol, progesterone, and testosterone

 
Increased adiposity and mesenteric adipocyte size in DHT rats
Body composition was analyzed by DEXA, dissection of individual fat depots and muscles, MRI, and adipocyte size determination.

DEXA.
At the time of DEXA measurements, both DHT and letrozole rats were heavier than the controls. Both groups had more body fat than controls, but body fat in relation to body weight was increased only in DHT rats (Table 3Go). LBM was increased in both DHT and letrozole rats; however, in relation to body weight, LBM was decreased in DHT rats. BMC was increased in both DHT and letrozole rats, but BMC in relation to body weight was lower in both groups than in controls (Table 3Go).


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  		TABLE 3. Effects of DHT and letrozole on body composition estimated by DEXA1

 
Adipose tissue depots.
DHT and letrozole increased the weights of individual inguinal, parametrial, and retroperitoneal adipose depots; the increase in the mesenteric depot was of borderline statistical significance (Table 4Go). In relation to body weight, the weights of the inguinal, parametrial, and retroperitoneal depots were increased in DHT rats.


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  		TABLE 4. Effects of DHT and letrozole on the weight of dissected individual fat depots and hind limb muscles

 
Muscles.
The weights of individual extensor digitorum longus, soleus, and tibialis anterior were increased after DHT and letrozole exposure, except for the extensor digitorum longus in DHT rats (Table 4Go). However, the ratio of tibialis anterior weight to body weight was decreased in both DHT and letrozole rats.

MRI.
Fat distribution was visualized by MRI (Fig. 5Go, A and B). Consistent with the impression from the MRI, the weight of both the sc (inguinal) (P < 0.001, ANOVA) and the intra-abdominal (parametrial, retroperitoneal, and mesenteric) (P < 0.05, ANOVA) adipose tissue in relation to body weight was increased in the DHT group; in the letrozole group, the relative weights of adipose tissues did not differ from control (Fig. 5CGo).


Figure 5
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  		FIG. 5. Effects of DHT and letrozole on adipose tissue distribution. A, MRI sections at the seventh axial slice from the most caudal part of the kidney. B, MRI sections at the eighth coronal slice. White lines on the coronal slices depict the position where the representative axial slice was selected. C, Weight of sc and intra-abdominal (parametrial + mesenteric + retroperitoneal) adipose tissue depots in relation to body weight was significantly higher in DHT rats but unchanged in letrozole rats. Values are mean ± SEM. *, P < 0.05; ***, P < 0.001 vs. controls (one-way ANOVA followed by Dunnett’s post hoc test).

 
Adipocyte size.
The mean adipocyte size and size distribution were determined in mesenteric and parametrial adipose tissue. In DHT rats, the size distribution curve of mesenteric adipocytes was shifted to the right (Fig. 6AGo), and the mean mesenteric adipocyte size was larger than in controls (83.8 ± 3.74 vs. 71.6 ± 1.65 µm; P < 0.05, ANOVA). In letrozole rats, the size distribution curve (Fig. 6BGo) and mean adipocyte size (68.1 ± 4.01 µm) in mesenteric fat were similar to those in controls. In the parametrial fat depot, mean adipocyte size (DHT, 91.3 ± 6.01 µm; letrozole, 95.2 ± 7.88 µm; and control, 93.6 ± 4.16 µm) and adipocyte size distribution (data not shown) were unaltered. In each cell population, 359-7628 cells (mean 2685 ± 268) were analyzed. The mean diameter of the reference microspheres was 97.89 ± 0.050 µm (range 97.55–98.01; n = 9).


Figure 6
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  		FIG. 6. Effects of DHT and letrozole on mesenteric adipocyte size distribution in DHT rats (A) and letrozole rats (B). In each group, adipocytes were pooled from six rats.

 
The weight of mesenteric fat depots correlated with the mean size of mesenteric adipocytes, both in pooled rats (P < 0.01; R = 0.65; n = 18) and individual groups (DHT: P < 0.01, R = 0.92, n = 6; letrozole: P < 0.05, R = 0.91, n = 6; and controls: P = 0.0507, R = 0.81, n = 6). No such correlations were observed in the parametrial depot.

Decreased insulin sensitivity in DHT rats
The mean GIR was lower in DHT rats than in controls (14.0 ± 1.5 vs. 18.3 ± 1.5 mg/kg·min; P < 0.01), determined by a euglycemic-hyperinsulinemic clamp. Furthermore, insulin sensitivity index (ratio of mean GIR to steady-state plasma insulin level) was lower in DHT rats (ANOVA, P < 0.001) than in controls, indicating insulin resistance (Fig. 7Go). The mean GIR (21.4 ± 1.7 mg/kg·min) and the insulin sensitivity index (Fig. 7Go) were similar in letrozole rats and controls. At steady-state, the plasma glucose level was approximately 6 mmol/liter, and the plasma insulin levels were 193 ± 9 mU/liter (control), 270 ± 15 mU/liter (DHT), and 228 ± 11 mU/liter (letrozole).


Figure 7
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  		FIG. 7. Effects of DHT and letrozole on insulin sensitivity index. Values are mean ± SEM. ***, P < 0.001 vs. controls (one-way ANOVA followed by Dunnett’s post hoc test).

 
Normal lipid metabolism but altered leptin concentrations in DHT rats
Plasma concentrations of TC, TG, free fatty acids, and HDL-C were similar in all groups (data not shown). Leptin concentrations were higher in DHT rats than controls (ANOVA, P < 0.001), and similar in letrozole rats and controls (control, 4.99 ± 0.60 ng/ml; DHT, 10.93 ± 0.75 ng/ml; and letrozole, 5.67 ± 0.89 ng/ml). When values from all rats were pooled, the plasma leptin levels correlated with the amount of body fat (g) estimated by DEXA (P < 0.0001; R = 0.85; n = 27). Corresponding relationships were seen in each group (DHT, P < 0.001, R = 0.92, n = 9; letrozole, P = 0.0576, R = 0.65, n = 9; and controls, P < 0.01, R = 0.88, n = 9).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that DHT-induced PCOS in rats recapitulates both the ovarian and metabolic features of human PCOS, including PCO morphology and irregular cycles, increased body fat, enlarged adipocytes, elevated leptin levels, and insulin resistance. Exposure to letrozole induced PCOs with striking morphological similarities to human PCOs, including a thickened theca cell layer, anovulation, and increased ovarian weight and size. Our findings also suggest that the formation of a "hyperplastic" theca interna reflects the inclusion of luteinized granulosa cells in the cyst wall rather than true hyperplasia. Letrozole rats had increased body weight without other major body composition changes in relation to weight. Table 5Go summarizes similarities and dissimilarities between DHT and letrozole exposed rats vs. controls and PCOS women vs. controls.


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  		TABLE 5. Summary of similarities and dissimilarities between DHT, letrozole, and estradiol valerate exposed rats vs. controls and PCOS women vs. healthy women

 
Ovarian cyclicity, ovary size, and cell layer distribution
One intraovarian disturbance thought to cause PCOS is deficiency in aromatase activation. Decreased aromatase activity could increase ovarian production of androgen and decrease estrogen production, leading to PCOs. In the letrozole group, ovarian size was increased, as in women with PCOS (32). All rats were acyclic, and their ovaries had multiple cysts, with a relative thin granulosa cell layer and a thickened theca cell layer, consistent with previous findings (23). The thickening might reflect increased ovarian androgen synthesis.

DHT rats had irregular cycles. Their ovaries had large, atretic antral follicles, follicular cysts with a thickened theca interna cell layer, a diminished granulosa cell compartment, and few fresh CL, as in PCOs. Androgens may facilitate early follicular differentiation (33). In subhuman primates, androgens promote the differentiation of primordial to primary follicles; oocyte-derived IGF-I has been implicated in this activation (34). The findings in our models are in line with these observations, indicating that androgens are the major cause of PCOs.

Ovarian morphology
In both experimental groups, ovarian cysts had a flattened epithelioid cell layer facing the antrum, similar to that in rat models of PCOs induced by dehydroepiandrosterone (35) or letrozole (36). This "epithelialization" of inner cell layers may reflect the transformation of outer granulosa cells during atresia (35, 36). Alternatively, dying apoptotic cells in atretic follicles may secrete soluble factors that recruit macrophages (37), which appear in the follicular fluid during atresia. The macrophages must cross and, therefore, disrupt the basement membrane separating the vascular membrana granulosa from the theca cell layer. Growth factors and cytokines from macrophages contribute to angiogenesis and proliferation of fibroblasts and smooth muscle cells (38), and may be involved in the vascularization of the atretic granulosa layer we observed. Therefore, the flat epithelial-like cells could be a new population of cells accompanying the endothelial cells at vascularization. We hypothesize that vascularization of the remaining healthy granulosa cells causes luteinization of those cells, as in the letrozole group; however, the molecular mechanism remains to be elucidated.

The cysts in letrozole-exposed rats resembled those in human PCO cysts described by Green and Goldzieher (39). The cyst wall in Fig. 3Go is nearly identical to the vascularized wall they saw in an atretic follicle and interpreted as a thickened theca interna. We believe the thickened cyst wall consists of theca interna cells and a vascularized layer of luteinized granulosa cells. An "intermediate" stage of this process is suggested by the presence of granulosa cells imbedded in the cyst wall (Fig. 4Go). Therefore, the thickened "hyperplastic" theca interna in the cyst wall described in a letrozole PCO rat model similar to ours (23) may reflect luteinized granulosa cells rather than true hyperplasia.

Sex steroid concentrations
The plasma sex steroid profile of letrozole-exposed rats is consistent with findings in other rat models of letrozole-induced PCOs (23, 36). Testosterone levels were markedly higher in the letrozole rats than in controls, presumably because letrozole blocks the conversion of androgen substrates to estrogen, reflecting endogenous androgen accumulation. In both letrozole and DHT rats, progesterone production was decreased, indicating anovulation, as in human PCOS (40) and other rat models of letrozole-induced PCOs (23, 36). Estrogen levels were similar in all rats. Lower estradiol levels were expected because letrozole blocks aromatization of testosterone to estradiol, but were not observed. Estradiol levels vary considerably during the estrous cycle. We obtained blood samples for sex steroid measurements in the estrus phase in controls and in experimental rats with cyclic changes. Because estradiol levels are lowest in the estrus phase, this might explain the lack of difference between controls and letrozole-exposed rats.

DHT and letrozole could induce a hyperandrogenic status. But DHT rats had low plasma testosterone concentrations. However, because DHT was administered continuously, the endogenous production of androgens or testosterone was probably reduced in DHT-exposed rats; in men, androgen injections reduce endogenous androgen production (41). Furthermore, the DHT dose was chosen to initiate an androgenic status high enough to evoke a biological response.

We speculate that the effects in DHT rats are mediated by direct AR activation in target tissues, whereas those in letrozole rats are mediated by the accumulation of endogenous testosterone, which also results in pronounced activation and/or up-regulation of the AR.

Growth and body composition
In human PCOS, adiposity correlates with symptom severity (42, 43). DHT significantly increased the body weight of the rats, reflecting increased body fat, as well as intra-abdominal and sc adipose tissue depots in relation to body weight. Consistent with these findings, specific activation of the AR by DHT increases fat mass in orchidectomized (orx) male mice (44). As expected, the amount of body fat correlated with plasma leptin levels in pooled and individual groups of rats.

Sex steroids appear to be important in regulating adiposity and fat distribution. Visceral fat tends to accumulate in hyperandrogenic women (45), and fat mass, especially abdominal, in adolescents with PCOS is reduced by insulin-sensitizing treatment with metformin and specific AR blockade (46). PCOS women have increased fat mass and central adiposity related to hyperinsulinemia and hyperandrogenemia from prepuberty to postmenarche (47). These findings are consistent with the effects of DHT exposure in our study.

The increased body weight in letrozole rats was not related to relative changes in body fat or LBM. Thus, letrozole may stimulate general growth without affecting relative body composition. In combination with an aromatase inhibitor, testosterone increases the retroperitoneal fat mass in orx male mice, while testosterone itself does not (44).

Adipocyte size
Adipose tissue mass increases as a result of increased adipocyte size or number. The risk of metabolic complications is elevated by the amount and location of adipose tissue and adipocyte size. Adipocyte enlargement is associated with insulin resistance and is an independent predictor of type 2 diabetes (15, 48). In nonobese women with PCOS, sc adipocytes are about 25% larger, possibly as a result of lipolytic catecholamine resistance, than in healthy controls matched for body mass index (BMI) and body fat (14).

To analyze intra-abdominal adipose tissue depots, we used computerized image analysis that allows detailed assessment of mean adipocyte diameter and adipocyte size distribution (30). In parametrial adipose tissue, neither DHT nor letrozole influenced the mean size or size distribution of adipocytes. Thus, the increases in this depot likely reflect an increased number of adipocytes. However, in mesenteric adipose tissue, DHT increased mean adipocyte size. The borderline significance of the increase in mesenteric fat was probably mainly due to increased adipocyte size; however, we cannot exclude an increased number of adipocytes. Letrozole also tended to increase the mesenteric depot but did not influence adipocyte size, indicating that it, too, may increase the number of adipocytes in mesenteric fat.

In sum, in our rat model of DHT-induced PCOS, mesenteric adipocytes were larger than in controls or in letrozole rats with a similar amount of mesenteric fat. This enlargement may be analogous to the enlarged sc fat cells in nonobese women with PCOS (14).

Insulin sensitivity, leptin concentration, and lipid profile
In women with PCOS, insulin sensitivity is decreased by 35–40%, independently of obesity, although obesity further exacerbates insulin resistance (49), and an association between androgen and insulin levels has been suggested (50). DHT induced insulin resistance and increased abdominal fat mass and adipocyte size, as in women with PCOS and hyperandrogenemia (14, 49); both are independently associated with insulin resistance and prediction of type 2 diabetes (5). In female rats (51), testosterone administration induces insulin resistance, attributed to effects on glucose transport (52, 53); another finding was reduced capillary density in the muscle, despite increased muscle weight (51). Because DHT rats became insulin resistant, with increased body and muscle weight, they also might have had reduced capillary density in muscle. In line with our findings in DHT-exposed rats, elevated androgen levels are partly responsible for insulin resistance in muscle in PCOS women (54).

In contrast, letrozole did not decrease insulin sensitivity despite increasing testosterone levels. DHT might have a stronger, more direct effect than letrozole on insulin-signaling pathways. A challenge for future studies will be to elucidate the mechanisms.

Do women with PCOS have increased leptin levels? In one study, they had higher leptin levels than controls after adjustment for BMI (55). However, subsequent studies showed that leptin levels were similar in PCOS women and controls matched for age and weight or for BMI (56, 57). In our study, plasma leptin levels were higher in DHT rats than in controls and correlated with the amount of body fat. Hyperleptinemia is thought to indicate leptin resistance, which may contribute to the pathogenesis of obesity (58) and is strongly connected to insulin resistance (59).

Women with PCOS exhibit an abnormal lipoprotein profile, characterized by dyslipidemia (60); however, the results are not consistent. In orx mice, AR activation by DHT alters the serum lipid profile and increases fat mass (44). In the present study, both DHT and letrozole exposure increased fat mass but did not affect the lipid profile.

Conclusions
We believe the DHT model is preferable for studies of both ovarian and metabolic features, whereas the letrozole model is suitable for studies of the ovarian features of PCOS. Although none of the models address the etiology of PCOS, both models may be useful for assessing new treatments.


   Acknowledgments
 
We thank Elias Eriksson for valuable scientific discussions; Birgitta Oden, Britt-Marie Larsson, and Emma Gustafsson for technical assistance; and Novartis Pharma AG for the letrozole substrate.


   Footnotes
 
This study was supported by grants from the Swedish Medical Research Council (Project No. 12206, 2005-72VP-15445-01A, 2005-72VX-15276-01A, 2003-14735, 2003-14736), Novo Nordisk Foundation, Wilhelm and Martina Lundgrens’s Science Fund, Hjalmar Svensson, Magnus Bergwall Foundation, Tore Nilson Foundation, Åke Wiberg Foundation, Swedish Diabetes Association Research Foundation, Adlerbert Research Foundation, and the Swedish federal government under the ALF agreement.

Disclosure Statement: The authors have nothing to disclose.

First Published Online May 10, 2007

Abbreviations: AR, Androgen receptor; BMC, bone mineral content; BMI, body mass index; CL, corpora lutea; DEXA, dual-emission x-ray absorptiometry; DHT, dihydrotestosterone; GIR, glucose infusion rate; HDL-C, high-density lipoprotein cholesterol; KS, Kolmogorov-Smirnov two-sample test; LBM, lean body mass; MRI, magnetic resonance imaging; orx, orchidectomized; PCO, polycystic ovary; PCOS, polycystic ovary syndrome; TC, total cholesterol; TG, triglyceride.

Received February 6, 2007.

Accepted for publication April 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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