Rodent Models of Polycystic Ovary Syndrome

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Rodent Models of Polycystic Ovary Syndrome: Phenotypic Presentation, Pathophysiology, and the Effects of Different Interventions Manuel Maliqueo, PhD1

Anna Benrick, PhD1

Elisabet Stener-Victorin, PhD1

1 Department of Physiology, Institute of Neuroscience and Physiology,

The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

Address for correspondence Elisabet Stener-Victorin, PhD, Department of Physiology, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, Box 434, SE-405 30 Gothenburg, Sweden (e-mail: [email protected]).

Semin Reprod Med 2014;32:183–193

Abstract

Keywords

► ► ► ►

PCOS rodent models treatment phenotype

This review focuses on rodent models exposed to sex steroids prepubertally and describes their phenotypes and pathophysiology with specific focus on the estradiol valerate-, dihydrotestosterone-, and letrozole-induced rat polycystic ovary syndrome (PCOS) models. Phenotypic presentations are compared among models as a function of the timing and dose of the exposure. Furthermore, the use of these models to study the possible effects and mechanisms of different treatment modalities relevant for women with PCOS will be discussed. Importantly, we do not claim to review all available rodent models of PCOS. Despite the variety of rodent PCOS models currently available, there is no “gold standard” that mimics the complete range of abnormalities observed in women with PCOS. In this regard, it is important to select the most suitable model for the pathophysiological experiment to be performed or the treatment strategy to be tested. Important variables to take into consideration are dose, route of administration, timing and duration of exposure, and the relevance of the abnormalities of the reproductive and metabolic axes in the rodent model to those observed in human PCOS.

Polycystic Ovary Syndrome and Its Etiology Polycystic ovary syndrome (PCOS) is the most common endocrine and metabolic disorder affecting women of reproductive age.1 Endocrine and reproductive symptoms of PCOS are hyperandrogenism (hirsutism, acne, and alopecia), irregular menstrual cycles, subfertility, and polycystic ovary morphology. Women with PCOS also tend to suffer from insulin resistance with compensatory hyperinsulinemia, type 2 diabetes, and cardiovascular disease,2,3 but these symptoms is not part of the PCOS diagnosis. Obesity worsens all reproductive and metabolic symptoms of PCOS4 and demonstrates the importance of prevention and treatment of obesity in women with PCOS. Women with PCOS also often suffer from symptoms of anxiety and depression that lead to reduced healthrelated quality of life.

Issue Theme Developmental Origins and Future Fate in PCOS: Providence or Peril?; Guest Editor, Kathleen M. Hoeger, MD, MPH

There are currently three sets of criteria used in the clinical diagnosis of PCOS: the National Institute of Health (NIH) criteria that require both hyperandrogenism and chronic anovulation; the Rotterdam criteria that require two of the three symptoms of hyperandrogenism, chronic anovulation, and polycystic ovaries; and the Androgen Excess and PCOS Society criteria that require hyperandrogenism plus ovarian dysfunction as indicated by oligo/amenorrhea and/or polycystic ovaries.5 Due to the broad range of diagnostic criteria, as well as ethnic differences, it is difficult to establish a definitive prevalence for PCOS but current estimates range from 6 to 15%.6 The etiology of PCOS remains uncertain, and based on heterogeneity in its presentation it is sometimes viewed as a combination of several disorders. The most common features are high levels of circulating androgens and estrogens7 as well

Copyright © 2014 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.

DOI http://dx.doi.org/ 10.1055/s-0034-1371090. ISSN 1526-8004.

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as insulin resistance.8 Symptoms of PCOS manifest around puberty, but the origin might be programmed during fetal development. Prenatal androgenization in monkeys, sheep, and rodents is an established hypothesis of PCOS etiology and has been shown to result in many common features of PCOS in the offspring.9 Prepubertal exposure to androgens in rodents is another hypothesis arising from the pubertal manifestation of PCOS symptoms.10–12 The hypothesis that androgen excess can have a developmental programming effect is supported by the recent findings of epigenetic regulation in the visceral fat of prenatally androgenized monkeys.13 PCOS is associated with neuroendocrine dysfunction, including increased luteinizing hormone (LH) secretion and pulsatility and relatively decreased level of follicle stimulating hormone (FSH). The increased LH secretion stimulates androgen production in the theca cells, and the lower level of FSH impairs follicle maturation and ovulation.14 The altered follicular development is characterized by increased recruitment of primordial follicles and excessive early follicular growth followed at later stages by arrested follicle growth resulting in polycystic ovary morphology and altered ovulation.15 The impaired folliculogenesis and steroidogenesis are also affected by intrinsic local intraovarian factors as well as extraovarian factors including androgens, insulin, and neuroendocrine alterations. High sympathetic nerve activity has also been proposed as a causative mechanism of PCOS, and high muscle sympathetic nerve activity is related to high levels of circulating testosterone.16 A growing body of evidence suggests that the sympathetic nervous system is also important in the regulation of ovarian physiology.17 Although, the relevance of rodent PCOS models to understanding ovarian dysfunction in PCOS is limited because mice

and rats are polyovulatory, they can still provide valuable knowledge because most of them develop metabolic disturbances similar to those seen in women with PCOS. Important questions that can be addressed with rodent models are related to the source of the androgen excess and the timing as to when its effect is exerted.18,19 This review takes into account the clinical presentation and pathophysiology of PCOS and will focus on rodent models exposed to sex steroids prepubertally and describe their phenotypes and pathophysiology with specific attention to the estradiol valerate (EV)-, dihydrotestosterone (DHT)-, and letrozole-induced rat PCOS models. Phenotypic presentations will be compared among models, and timing of exposure, doses, etc., will be highlighted. In addition, the use of these models to study the effect and mechanism of different treatment modalities relevant for women with PCOS will be discussed. It is important to note that this review does not cover all available rodent models of PCOS.

Phenotypic Presentation of Sex SteroidExposed Rat PCOS Models Accumulating evidence indicates that reproductive and metabolic dysfunction in adult life might result from programming of developing systems during fetal or prepubertal life.20 There is a consistent evidence that androgen excess leads to the development of a PCOS-like phenotype in the adult life.9 Thus, the most commonly used models are the steroidinduced models (►Fig. 1). However, there are also genetically modified rat and mouse models as well as models developed by manipulation of environmental factors that all reflect at least some aspects of the syndrome (►Fig. 1). The prenatal,

Figure 1 Different interventions in rats and mice lead to a polycystic ovary syndrome (PCOS) phenotype.

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Rodent Models of Polycystic Ovary Syndrome neonatal, and prepubertal steroid-induced models give a clear indication that PCOS development involves exposure to abnormal sex steroid levels early in life (►Fig. 2). The typical phenotypic presentations of common rodent models are presented below and in ►Table 1 and in ►Fig. 2. Time points and durations of exposure, doses, and substances are given.

Estradiol Valerate-Induced PCOS (Rats) Adult rats are given a single injection of 2 mg EV in 0.2 mL sesame oil.21 Rats with EV-induced PCOS develop a polycystic ovary morphology including irregular estrous cycles and an increased androgen response to human chorionic gonadotropin stimulation although with smaller ovaries.22–24 The go-

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nadotropin secretion drops immediately after exposure but recovers after about 30 days and does not differ from controls thereafter. EV-induced PCOS rats do not develop insulin resistance or obesity but have higher blood pressure than controls.25

Dehydroepiandrosterone-Induced PCOS (Rats and Mice) Rats or mice exposed to daily subcutaneous (SC) dehydroepiandrosterone (DHEA) injections (4.5 or 6mg/100 g body weight) starting prepubertally on day 22 or day 23 after birth and lasting for 5 to 7 weeks develop a polycystic ovary-like morphology with smaller ovaries, disrupted estrous cycles, altered steroidogenesis, and increased levels of circulating

Table 1 Rat models with PCOS-like phenotypes Human PCOS

Sex steroid exposure EV

DHEA

DHT

Letrozole 200 µg

Letrozole 400 µg

Cyclicity and ovarian morphology Irregular cycles

þ

þ

þ

þ

þ

þ

Ovary size/weight

"

↓

↓

↓

"

"

Follicular cysts

þ

þ

þ

þ

þ

þ

Follicular atresia

þ

þ

þ

þ

þ

þ

Theca cell layer

"

"/

↓

"

"

"

Granulosa cell layer

↓

↓

"

↓

↓

Testosterone

"

↓/

"

"

"

Hormones DHT

"

n.d.

"

"

n.d.

n.d.

Estradiol

"/

"/

"

↓

Progesterone

↓

"

"

↓

↓

↓

GnRH

"

n.d.

n.d.

n.d.

n.d.

n.d.

LH

"

↓

"

n.d.

"

"

FSH

"/

n.d.

↓

LH/FSH

"

n.d.

n.d.

n.d.

"

n.d.

Leptin

"

n.d.

"

"

"

þ

þ

þ

þ

Metabolic and cardiovascular function Insulin resistance Dyslipidemia

þ

(þ)

(þ)

Adiponectin

↓

n.d.

n.d.

↓

n.d.

n.d.

Leptin

"

n.d.

n.d.

"

(")

Body weight

"

↓/

"

"

"

Adipocyte size

"

n.d.

n.d.

"

"

Obesity

þ

(þ)

n.d.

þ

þ

Altered adipose tissue function

þ

n.d.

þ

þ

n.d.

n.d.

Blood pressure

"

"

n.d.

"

n.d.

n.d.

Endothelial dysfunction

þ

n.d.

n.d.

þ

n.d.

n.d.

Abbreviations: þ, presence; , no changes; ", increased; ↓, decreased; n.d., not determined; (þ), trend; DHEA, dehydroepiandrosterone; DHT, dihydrostestosteron; EV, estradiol valerate; PCOS, polycystic ovary syndrome. Seminars in Reproductive Medicine

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gonadotropin.26–29 Rodents exposed to DHEA do not develop obesity and the metabolic dysfunction is not completely phenotyped.

Dihydrotestosterone-Induced PCOS (Rats and Mice) Continuous exposure to the nonaromatizable androgen 5αDHT at a daily dose of 83 µg (rats) or 27.5 µg (mice) from prepuberty (day 21) to adult age induces a polycystic ovary morphology, including disrupted estrous cycles and metabolic aberrations such as obesity, enlarged adipocytes, insulin resistance, and dyslipidemia, but with smaller ovaries.11,12,30 These rodent models also develop cardiovascular dysfunction such as hypertension.31

Letrozole-Induced PCOS (Rats) Continuous exposure of the nonsteroidal aromatase inhibitor letrozole at a daily dose of 400 µg from prepuberty (day 21) to adult age induces typical polycystic ovary morphology with enlarged ovaries and disrupted estrous cycles but no metabolic aberrations.11 When rats were exposed to a lower dose of letrozole (200 µg/d), the reproductive phenotype remained regarding ovarian morphology, disrupted cycles, elevated testosterone levels, higher circulating LH concentrations, and lower FSH concentrations but they also developed metabolic dysfunction including increased fat mass and adipocyte size and decreased insulin sensitivity.10,11 Taken together, the latter phenotype of letrozole-induced PCOS with the lower dose is promising and this model best reflects both the reproductive and the metabolic disturbances seen in women with PCOS.10

Endocrine and Reproductive Dysfunction Ovarian Morphology The histological analysis of ovaries from women with PCOS is characterized by ovarian hypertrophy with thickening of the capsule, increased ovarian stroma, and a scarcity of corpora lutea (CL). Moreover, ovaries from these women present with multiple subcapsular follicle cysts characterized by a thin granulosa cell layer with minimal cell proliferation and with thickening and luteinization of the internal theca layer.32 PCOS ovaries contain two- to threefold more small and usually atretic antral follicles.33,34 The presence of ovarian cysts is a hallmark in PCOS animal models. However, differences in the histological features can be seen among the different rodent models of PCOS. The small ovaries of EV-induced PCOS display multiple cysts with large cavities, a thin granulosa layer, and a normal or thickened theca internal cell layer.21,35 Moreover, few secondary follicles with different degrees of atresia are observed.21 This model is useful for understanding the contribution of sympathetic nerve activity in the genesis of PCOS23 and confirms observations in ovaries from women with PCOS.36,37 An increase in nerve growth factor (NGF) and its low-affinity receptor (p75NTR) plays a central role in cyst formation in this model.24 In this regard, the transection of the superior ovarian nerve or the blocking of NGF action by an anti-NGF antibody reduces the number of cysts and increases Seminars in Reproductive Medicine

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the number of CL indicating that the anovulation and cyst formation is mediated by high sympathetic nerve activity.22,38 In rats, the ovarian weight is decreased by DHEA exposure.39 Rats or mice exposed to DHEA show an increase in the number of cysts and atretic follicles.29,40 However, in mice there are no more than two cysts in each ovary.41 In both rats and mice, cysts show a thin granulosa cell layer and a nonvascularized internal theca cell layer. Interestingly, the presence of leukocytes in the ovarian stroma or inside the cyst have been observed and this indicates a possible role of the immune system in the cystogenic process.28,41 In some rats, a few CL are present in contrast to their total absence in mice. The ovarian capsule, however, does not increase in thickness as is observed in human polycystic ovaries.29 The DHEAinduced rodent PCOS model demonstrates that ovarian cysts are formed after the apoptosis of oocytes and mural granulosa cells and the epithelization of basal granulosa cells.42 Ovaries from rats and mice exposed to DHT from puberty show multiple large follicles with cystic appearance, hyperplasia of the theca cell layer, and a thin granulosa cell layer. In DHT-exposed rats, the ovarian weight is reduced whereas in mice it tends to be increased. These differences are probably attributable to the different species and doses used in the studies.11,12 In mice, DHT exposure does not modify the number of growing follicles, but the majority of the follicles are atretic and mainly small while antral follicles are large.12 This feature does not completely resemble human PCOS in which an increase in small antral follicles is observed.43 Exposure to letrozole in adult rats leads to the formation of ovarian cysts lined with a thin layer of granulosa cells, hyperplasia of the internal theca layer, and thickening of the capsule.10,11,44 In this model, it has been suggested that the cyst formation is related to an increase in the expression of cytoskeleton proteins and cellular adhesion molecules, such as vimentin, cytokeratin, and cadherin, and a reduction in granulosa cell proliferation.45 However, these studies do not show an increase in ovarian weight. Interestingly, rats exposed to letrozole for 90 days starting at a prepubertal age show an increase in ovarian weight.10,11 Moreover, the presence of a vascularized wall in the cyst resembles the cysts seen in human PCOS.34 However, the presence of granulosa cells in the thickened “hyperplastic” internal theca in the cyst wall might be indicative of luteinized granulosa cells rather than true hyperplasia.11

Estrous Cyclicity Disruption of estrous cyclicity is another essential feature observed in animal models of PCOS. In rats and mice, ovulation occurs every 4 to 5 days, and the phases of ovulation are characterized by the proportion of cornified cells and leukocytes among the epithelial cells in a vaginal smear.46 After 20 days of exposure, EV-induced PCOS rats exhibit a persistent estrous stage characterized by the presence of cornified cells in the vaginal smear.21 However, no oocytes are found in the oviducts and no CL are found in the ovaries and this is an indication of anovulation. Rats and mice exposed to DHEA are acyclic after a few normal estrous cycles.29,40 In rats with CL, a constant diestrous is observed after one complete cycle and

Rodent Models of Polycystic Ovary Syndrome rats without CL are in constant estrus.29 The letrozole-induced PCOS models, independent of the dose, route of administration, or age when exposure starts, are completely acyclic with a predominance of leukocytes in vaginal smears. These animals are in a constant “pseudodiestrous” phase with few or no CL in the ovaries.10,11 On the other hand, the DHTinduced rat PCOS model presents with irregular cycles and a predominance of the “pseudodiestrous” phase with a few fresh CL in ovarian morphology while mice are completely acyclic and devoid of CL.11,12

Neuroendocrine The neuroendocrine phenotype of PCOS is less consistent among the different rodent models of PCOS. LH and FSH secretion are lower in the EV-induced rat PCOS model,21 and this is associated with an attenuated LH pulsatility and response to gonadotropin releasing hormone (GnRH).47,48 These abnormalities can be the result of an increased opioid tone at the hypothalamic level or reduced secretion of gonadotropins at the pituitary level.49,50 However, this phenotype is in contrast to that observed in women with PCOS who exhibit elevated LH levels, accelerated frequency of the LH pulse, and an exaggerated response to GnRH.51 DHEA exposure increases LH levels but does not modify FSH levels.29 On the other hand, DHT-induced PCOS in mice does not alter circulating LH levels.12 Unfortunately, there is no data about the circulating gonadotropin levels in rats exposed to DHT. However, an increased expression of androgen receptor and colocalization with neurons immunoreactive to GnRH have been observed in the DHT-induced rat PCOS model indicating a possible role for androgens in regulating the function of GnRH neurons.52 The letrozole-induced PCOS model with exposure starting at adult age increases LH and FSH secretion likely through the feedback exerted by low estrogen levels.44 However, the continuous administration of letrozole starting before puberty induces an increase in LH and a decrease FSH levels that resembles the changes observed in women with PCOS.10 It has been suggested that the hyperandrogenism induced by letrozole before puberty modifies the transition from androgenic to estrogenic control of gonadotropin secretion and favors LH secretion.53

Sex Steroids Hyperandrogenism is a central feature in women with PCOS. In line with this, the letrozole-induced rat PCOS model exhibits a dramatic endogenous increase in serum testosterone concentration.10,11,44 However, low estradiol levels are observed in this model because letrozole is an inhibitor of aromatase activity.10,44 Women with PCOS present elevated circulating estrogen levels that can be explained by the aromatization of elevated androgens levels.54 However, lower aromatase activity has been suggested in ovarian follicles and adipose tissue.55,56 The analysis of steroidogenic enzyme expression in ovarian tissue from letrozole-induced PCOS rats shows elevated messenger RNA (mRNA) expression of CYP17a1,10,57 and this is in agreement with what is observed in women with PCOS.58 However, continuous administration of 400 μg letrozole per day for 90 days starting in prepubertal

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rats decreases the mRNA expression of StAR, Cyp11a1, and Hsd3b157 and this is in contrast to the increased protein expression of these steroidogenic enzymes.59 The administration of 200 μg letrozole per day for 90 days does not modify the mRNA expression of Cyp11a1 and Hsd3b110 indicating a possible dose–response effect in the expression of steroidogenic enzymes in the letrozole model. In the DHT-induced rat and mouse PCOS models, the hyperandrogenism comes from the exogenous administration of androgen with a three- to sixfold increase in circulating DHT compared with controls.12,31 The endogenous steroids in this model show low progesterone and normal or low testosterone and estradiol levels in rats.11 In this model, an increase in ovarian mRNA expression of Cyp17a1 and a decrease in StAR, Cyp11a1, Cyp19a1, and Hsd17b mRNA has been observed.60,61 In the EV-induced PCOS model, one study reported no changes in circulating androgen levels (androstenedione, DHEA, or testosterone) in rats treated with 2.0 mg of EV,47 but another showed low testosterone levels in rats exposed to 4.0 mg of EV.25 Rats exposed to 2.0 mg of EV show high estradiol levels.62 On the other hand, rats exposed to 4.0 mg of EV have normal to high estradiol levels and elevated progesterone levels.25 The activity of 3β-HSD is reduced and P450c17α is undetectable in ovaries from rats exposed to EV.63 Testosterone and DHT are increased in mice and rats exposed to DHEA, and estrogen in mice and estradiol in rats are also elevated.29,39,40 Progesterone serum concentrations are increased in DHEA-induced PCOS model.64

Metabolic and Cardiovascular Dysfunction Adipose Tissue Distribution Continuous exposure to both DHT and letrozole results in a significant increase in body weight compared with control animals.10–12,31 In rats given 400 μg of letrozole, the change in body weight is observed without any other major alteration in body composition. However, the increase in body weight seen after 200 μg of letrozole and in DHT-exposed rats is accompanied by an increase in body fat. In DHT-induced PCOS rats, both intra-abdominal and SC adipose tissue depots are increased in relation to body weight while letrozole-induced rats only have a pronounced SC obesity.10,11,31 EV-induced PCOS rats gain significantly less weight than controls, but the difference in body weight is no longer significant after 7 weeks and onwards. However, the SC fat depot is significantly heavier in the EV-group as compared to the controls while the intra-abdominal fat depots are not changed.25 DHEA exposure in rats and mice does not affect body weight, and there are no reports on altered fat depot weight.26,65

Adipocyte Size and Adipose Tissue Function Along with the differences in adiposity, 200 μg of letrozole exposure leads to enlarged adipocytes in both the SC and mesenteric fat depots compared with controls while 400 μg of letrozole does not alter adipocyte size.10,11 There are no publications, however, investigating whether letrozole exposure affects adipose tissue function. Increased adipocyte size Seminars in Reproductive Medicine

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in the mesenteric11 and in the SC fat60 has been seen in DHTexposed rats and mice.12 Along with enlarged adipocytes, DHT-exposure also leads to altered adipose tissue function in the form of increased gene expression of leptin, interleukin-6, and markers of sympathetic activity such as Adrb3, Ngf, and Npy.66,67 Adipocyte size has not been determined in DHEAinduced rats, but resistin mRNA levels are higher in the adipose tissue—pointing toward an altered function—and resistin secreted by adipose tissue might even mediate insulin resistance in PCOS.39 It is not known whether EV-induced rats have altered adipocyte size, and there is no data suggesting that the adipose tissue function is disturbed in this model. Serum leptin levels are elevated in DHT-exposed rodents,11,12,31 and adiponectin levels are reduced in DHT mice.12 There is a tendency to elevated leptin levels in rats exposed to the lower but not the higher dose of letrozole.10,11

Lipid Profiles In contrast to women with PCOS, most PCOS rat models do not have an established dyslipidemia. Exposure to EV, DHEA, or 400 μg of letrozole does not change the lipid profile—including plasma concentrations of total cholesterol, triglycerides, free fatty acids, and high-density lipoprotein—but triglyceride levels tend to be higher in rats exposed to 200 μg of letrozole. However, DHT-exposed rats show no change in circulating lipids in one study11 and increased cholesterol, low-density lipoprotein, and triglyceride levels in other studies.30,31,60

Insulin Resistance In female rats, testosterone administration induces insulin resistance partly via effects on glucose transport.68 In line with these findings, both letrozole-induced (200 μg) and DHT-induced PCOS in rats and mice show insulin resistance.12,31 In contrast, 400 μg of letrozole does not induce insulin resistance11 possibly due to a more anabolic effect when the dose is increased. Both DHEA-exposed rats39 and mice are more insulin resistant,41 while EV-induced rats have normal insulin sensitivity.25

Cardiovascular Disturbances Limited data are available on whether postnatal treatment with androgens, estrogens, or letrozole induces the cardiovascular and endothelial disturbances that are associated with PCOS. No cardiovascular effects have been determined in publications regarding DHEA- and letrozole-induced PCOS models. However, in DHT-induced PCOS in rats and mice hyperandrogenemia specifically impairs vascular function, alters markers of vascular endothelial function, and decreases endothelium-dependent vasorelaxation together with an elevated blood pressure.31,69 In addition, EV-induced PCOS rats have significantly higher mean systolic blood pressure than controls.25

Treatment Metformin Insulin-sensitizing agents, such as metformin, are used in women with PCOS and often improve the symptoms of hyperandrogenemia. In line with this, metformin decreases Seminars in Reproductive Medicine

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serum insulin and homeostatic model assessment (HOMA)index to control levels without any effect on body weight or body mass index in a DHEA-induced PCOS mouse model.65 Moreover, metformin reduces serum testosterone concentrations without improvement of ovarian morphology.70 Interestingly, at the ovarian level metformin increases the concentration of glutathione, increases nitric oxide synthase activity, and stimulates prostaglandin E production in DHEAinduced PCOS rats.71 However, it is unknown if there is a link between the changes in oxidative stress markers and the improvement in testosterone production in these animals.

Exercise and Acupuncture Several experimental observations in steroid-induced rat models of PCOS suggest that both manual stimulation and low-frequency electrical stimulation of acupuncture needles, so-called electroacupuncture (EA), exert long-lasting beneficial effects on reproductive and metabolic functions. Lowfrequency EA in DHT- and EV-induced rat PCOS models restores ovarian function, induces estrous cyclicity, increases progesterone levels, and improves ovarian morphology with the presence of growing follicles and CL.67,72,73 Similar findings have been observed in DHT-exposed rats treated with manual stimulation acupuncture.72 Interestingly, the improvement in ovarian function by acupuncture has also been demonstrated in a DHEA-induced PCOS rat model.74 Acupuncture with electrical and manual stimulation appears to have effects through fundamentally different mechanisms. Low-frequency EA regulates opioid peptides or their receptors,67,72,75 and it is well known that opioids can regulate the secretion of GnRH and modulate the secretion of gonadotropins and LH in particular.76 Interestingly, it has been demonstrated that EA reduces GnRH expression.52 On the other hand, manual stimulation affects the hypothalamic expression of steroid and kisspectin receptors that also affect the secretion of GnRH and LH.72 Moreover, in EV-induced PCOS rat model low-frequency EA regulates the ovarian expression of α1a-, α1b-, α1d-, and β2-adrenergic receptors together with the reduction of NGF and its receptor, p75NTR, suggesting that the effectiveness of EA is mediated by regulation of sympathetic inputs.35,73,77 In the EV-induced PCOS model, exercise improves the ovarian morphology with the presence of healthy and atretic primary, secondary, and tertiary follicles and CL without oocytes indicating a restoration of estrous cyclicity and ovulation.78 This effect is probably also mediated by the reduction of sympathetic outflow in a similar manner as low-frequency EA because the ovarian protein expression of NGF, p75NTR, and α1a-adrenergic receptor was almost normalized after exercise.78 In contrast, in the DHT-induced PCOS model exercise partially restores estrous cyclicity and decreases the proportion of atretic follicles.60,67 However, the lack of CL suggests that the cycles are anovulatory.67 Moreover, exercise normalizes the mRNA expression of Cyp11a1 suggesting an improvement in steroidogenic activity in the DHT-induced rat PCOS model.60 Exercise and acupuncture both improve insulin sensitivity, which is a hallmark of PCOS that worsens both endocrine and

Rodent Models of Polycystic Ovary Syndrome metabolic features. Exercise increases insulin sensitivity and lean body mass and reduces body fat and adipocyte size in DHT-induced PCOS rats,60,66 but it does not improve lipid profile.60 The decreased adiposity might partly explain the beneficial effects of exercise, but the insulin sensitizing effects of acupuncture occur in the absence of any change in fat mass or adipocyte size. This suggests that insulin sensitivity is somewhat independent of adiposity.30,67,79 Low-frequency EA with repetitive muscle contractions activates physiological processes similar to those resulting from physical exercise. In DHT-induced PCOS rats, EA stimulates glucose transport via increased GLUT4 translocation and restores insulin sensitivity without affecting body weight or food intake.30,67,79 However, one study has shown that EA reduces food intake and body weight possibly by increasing leptin levels.80 EA also partially improves the lipid profile in DHT-induced PCOS rats.30 Acupuncture with manual stimulation leads to improved whole body glucose clearance but does not affect molecular signaling pathways to the same extent as EA.79

Vertical Sleeve Gastrectomy Human PCOS patients have shown decreased androgen levels after bariatric surgery. Bariatric surgery (vertical sleeve gastrectomy) in DHT-induced PCOS rats causes loss of body weight and body fat but does not improve glucose tolerance or estrous cyclicity.81 Therefore, the lack of improvement in glucose tolerance and ovarian function might point to a direct effect of androgens as a mechanism for the improvements seen in human PCOS patients after bariatric surgery. Furthermore, the loss in body weight without improvement in glucose tolerance suggests that insulin sensitivity might be independent of body weight in women with PCOS.

Vitamin D Therapy Vitamin D has beneficial effects on metabolic and cardiovascular dysfunction related to hyperandrogenic status and insulin resistance in PCOS partly due to its antioxidant capacity. In DHT-induced PCOS rats, oral glucose tolerance is improved after vitamin D3 treatment. Vitamin D3 supplementation also leads to increased relaxation ability in the aorta and acts on pathways involved in the impaired endothelial function seen in DHT-induced rats.82,83 However, the vascular insulin resistance of the aorta that is induced by DHT exposure is not affected by vitamin D3 and thus vitamin D3 alone cannot resolve the aortic endothelial dysfunction caused by the hyperandrogenic state.84 Vitamin D3 therapy probably does not restore ovarian morphology because no changes in follicular diameter in rats exposed to DHT were observed.84

Affective Symptoms—Behavior Women with PCOS are more prone to develop affective symptoms including anxiety and depression.85 Whether symptoms of anxiety and/or depression are associated with circulating sex steroids and/or hyperinsulinemia is not clear.86 Androgen therapy in women improves depressive

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states87 even though women with PCOS and androgen excess display symptoms of anxiety and depression.88,89 Interestingly, the DHT-induced rat PCOS model displays an anxiety-like behavior as demonstrated by a general decrease in locomotor activity and a decrease in the time spent in the open arms in the elevated plus maze.90 These findings highlight the role of central androgens in behavioral function. Androgens also increase leptin synthesis in adipocytes and this stimulates central leptin signaling and might regulate anxiety-related behaviors. Thus, an interaction between peripheral and central androgens and leptin signaling might affect the neural mechanism of behavior in female rats exposed to DHT.90 None of the other models have been evaluated in terms of behavior.

Methodological Consideration We have reviewed rodent models that have been widely used in studies investigating the etiology of PCOS and mimic many of the features observed in women with PCOS. When selecting a model to be used, it is important to consider how the model has been developed with regard to dose, time point of exposure, method of exposure, duration of exposure, and so on. ►Fig. 2 summarizes the time points, doses, durations, and phenotypes of different rodent PCOS models. Models induced by the continuous exposure to 7.5 mg of DHT or 18.0 mg of letrozole administrated SC for 90 days starting at prepubertal age exhibit both reproductive and metabolic alterations.10,11 In this regard, higher doses of letrozole (36.0 mg administrated SC for 90 days starting at prepubertal age) or the treatment of adult rats with doses from 0.1 to 1.0 mg/kg body weight administrated orally (per os [PO]) for 21 days exhibit the ovarian phenotype of PCOS but not the metabolic phenotype. Only one study has reported glucose intolerance in adult rats exposed to PO letrozole at 0.5 mg/kg body weight.91 Recently, it was reported that prenatal exposure to 5.0 mg (SC) of DHT during day 16 to 19 of gestation induces insulin resistance in the offspring when they reach pubertal age.92 In another study, using 3.0 mg (SC) of DHT administered at the same gestational age results in irregular cycles, polycystic ovary morphology, and elevated circulating LH and testosterone levels that are associated with high LH pulsatility.93 These observations suggest that prenatal androgenization with DHT could be a new PCOS animal model that presents with a combination of reproductive and metabolic disturbances. Intramuscular injections of 2.0 mg or 4.0 mg EV to young or adult rats result in a similar ovarian phenotype and similar levels of steroid and gonadotropin production.21,35,94 Interestingly, the exposure to a single dose of 10.0 mg/kg body weight of EV given once between postnatal day 1 to 14 induces irreversible changes in polycystic ovary morphology.95 However, EV exposure after 2 weeks of age induce reversible polycystic ovary morphology,95 which is a useful information if the aim is to study the long-term effects of the ovarian abnormalities observed in PCOS and the effects of different treatments. EV- and DHEA-induced PCOS models give relevant information about the genesis of ovarian abnormalities; however, they are not the most suitable for Seminars in Reproductive Medicine

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Figure 2 Steroid-induced PCOS intervention at different developmental stages and the phenotypic results in the adult rats. Anov, anovulation; CVD, cardiovascular disturbances; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; EV, estradiol valerate; FSH, follicle stimulating hormone; HA, hyperandrogenism; IR, insulin resistance; LH, luteinizing hormone; PCOS, polycystic ovary syndrome; T, testosterone; TP, testosterone propionate; ", increase; ↓, decrease.

studying the metabolic disturbances associated with PCOS.21,28,29 In summary, despite the variety of rodent PCOS models currently available there is no “gold standard” that presents the complete range of abnormalities observed in women with PCOS. In this regard, it is important to select the most adequate model to answer the pathophysiological question at hand or the treatment strategy to be tested. Variables such as, dose, route of administration, timing, and duration of exposure seem to be of great importance and these factors are highly relevant in the development of abnormalities of the reproductive and metabolic axis resembling those observed in human PCOS.

Acknowledgments Manuel Maliqueo, PhD and Anna Benrick, PhD have contributed equally to this work.

2 Diamanti-Kandarakis E, Dunaif A. Insulin resistance and the

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Funding This study was financed by grants from the Swedish Research Council (Project No. K2012–55X-15276–08–3), The Jane and Dan Olsson Foundations, The Novo Nordisk Foundation, The Hjalmar Svensson Foundation, The Adlerbert Research Foundation, Wilhelm and Martina Lundgrens’s Science Fund, and the Swedish federal government under the LUA/ALF agreement (ALFGBG-136481).

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