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Animal Models for the Study of Polycystic Ovarian SyndromeOliver Oakley, Po-Ching Lin, Phillip Bridges1, CheMyong Ko2
Reproductive Sciences Graduate Program, Department of Clinical Sciences, University of Kentucky, Lexington KY; Department of Animal and Food Sciences1, University of Kentucky, Lexington KY; Department of Comparative Biosciences2, College of Veterinary Medicine University of Illinois at Urbana-Champaign, IL, USA
Corresponding author: CheMyong KoDepartment of Comparative Biosciences, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802, USATel: 217-333-9362, Fax: 217-244-1652, E-mail: [email protected]
Endocrinol Metab 26(3):193-202, September 2011http://dx.doi.org/10.3803/EnM.2011.26.3.193 REVIEW ARTICLE
INTRODUCTION
Polycystic ovarian syndrome (PCOS) remains one of the leading
endocrine disorders encountered by women of reproductive age;
however, the etiology and pathogenesis of this syndrome remain
an area of continued debate and controversy.
PCOS is considered a syndrome due to heterogeneity in the fea-
tures of this complex disorder. The Rotterdam criteria were estab-
lished to confirm diagnosis of PCOS in women who have the pres-
ence of at least two of the following symptoms; hyperandrogenism,
polycystic ovaries and oligo- and/or anovulation [1]. Hyperandrogen-
ism, an increase in circulating androgen levels such as testosterone,
androstenedione, or dehydroepiandrosterone, may lead to a variety
of clinical features such as hirsutism, a masculine pattern of facial
and body hair growth, presents as an increase in terminal hairs on
the lip, chin, chest, back, abdomen, or thigh and acne, inflamed se-
baceous glands of the skin [2]. Hyperandrogenemia also causes
hair loss (alopecia) on the scalp as a result of sensitivity of the hair
follicles to androgen [3]. Polycystic ovaries, the presence of multiple
(> 10) cysts in an ovary [4], is caused by the arrest of follicle devel-
opment at an immature stage (Fig. 1). PCOS is named in reference
to this morphological change. As the development of these follicles
is arrested well before the point of dominant follicle selection, and
therefore positive estrogen feedback to the hypothalamus and pi-
tuitary axis is lacking, the LH surge is absent in PCOS patients [5-7].
Consequently, ovulation and menstrual cycles are interrupted (oli-
govulation and oligoamenorrhea, respectively).
1. Endocrine features
PCOS is widely recognized as an endocrine disorder with numer-
ous metabolic consequences. Unfortunately, the metabolic conse-
quences of PCOS are interwoven with the metabolic consequences
of obesity and causal relationships are therefore hard to define (i.e.
while PCOS is classified as an endocrine disorder, obesity will affect
the endocrine parameters of this disorder). Insulin resistance is one
of the metabolic diseases associated with PCOS, and is perhaps the
most well documented symptom [8]. The clinical manifestation of
insulin resistance at a patient level reflects the complexities described
above. Approximately half the women with PCOS are obese [9] and
therefore prone to the presentation of insulin resistance [8]. However,
insulin resistance is also observed in women with PCOS of a nor-
mal body weight [10], together indicating that the hyperinsulinemia
associated with this syndrome is not specific to the stressors asso-
ciated with obesity. The relationship between glucose and insulin is
also somewhat patient-specific and affected by age, race, and phys-
ical condition. Therefore, insulin resistance/glucose tolerance is a
metabolic disease associated with PCOS that can manifest itself in
more severe outcomes such as diabetes. Cardiovascular risk is in-
creased in patients with PCOS [11-14] and hypertension [15] is the
most easily recognized manifestation. This metabolic disorder is
also associated with obesity and again, insulin resistance is a known
cardiovascular risk factor [11]. Insulin affects myocardial and skele-
tal energy metabolism and physical activity improves the insulin
sensitivity of skeletal muscle [10,16-18]. Downstream effects of ex-
ercise-dependent changes in insulin include better blood flow, more
efficient glycogen synthase activity and glucose transportation [18].
Similar to the individuality of glucose/insulin tolerances, measures of
cardiovascular function such as maximal O2 consumption (VO2max)
are also subject to age, physical condition and other diseases with
VO2max positively correlated to both testosterone and insulin sensi-
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Copyright © 2011 Korean Endocrine Society
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tivity [19].
The relationship between steroids, lipids and glucose metabolism
is not easily defined. Interactions between these parameters are
known and aberrations of one will affect the other as a metabolic
consequence will modulate the endocrine environment. As such,
many of the symptoms and features of PCOS play on one another
to exacerbate the presentation of the others. The interconnection
of the symptoms of PCOS has thus made it a large challenge to pin-
point the causal factor(s) as well as those that are secondary to that
initial aberration.
2. Clinical treatments
PCOS is a very complex disorder with heterogeneity of clinical as
well as endocrine features. To treat symptoms caused by the hyper-
androgenemia of PCOS (hirsutism, acne, seborrhea and hair loss),
antiandrogens such as flutamide and spironolactone are routinely
administered [20-27]. Most of all, however, treatment of PCOS pa-
tients has been centered on inducing ovulation. Due to an inability
to naturally ovulate, PCOS patients receive either pharmacological
or surgical treatments to induce ovulation together with other as-
sisted reproductive technology (ART) procedures [28-34]. The most
commonly used procedures are to increase sensitivity to insulin, in-
duce the gonadotropin surge, or lower circulating testosterone lev-
els. For each of the symptoms, the following treatments are used.
1) Metformin
While the etiology is unclear, it is well established that PCOS and
obesity/diabetes are tightly associated. In support of this physiolog-
ical relationship, weight loss not only improves insulin sensitivity
but also often restores ovulatory cycles in some women with PCOS
[35-40]. This finding has led to the application of a pharmacologi-
cal approach for recovering ovulatory cycles in PCOS patients us-
ing metformin, an insulin-sensitizing drug [41-45]. Metformin low-
ers blood sugar levels by decreasing the amount of sugar produced
by the liver, increasing the amount of sugar absorbed by muscle
cells and decreasing the body’s resistance to insulin. Recent studies
show that metformin is safe and effective in lowering insulin and
improving fertility [46,47].
2) Clomiphene/gonadotropins
For a PCOS patient seeking to become pregnant, the first line of
therapy may be to treat her with clomiphene citrate (CC). Clomi-
phene citrate is a selective estrogen receptor modulator (SERM) that
acts as an estrogen receptor antagonist in the hypothalamus [48-51].
When administered, CC prevents the negative feedback effect of
estrogen, thus stimulating gonadotropin releasing hormone (GnRH)
from the hypothalamus, which in turn increases the secretion of
gonadotropins from the pituitary. Therefore, CC is widely used to
stimulate folliculogenesis and ovulation in PCOS patients. However,
in some women, CC is not successful at inducing ovulation. In these
cases, the direct injection gonadotropins can be used to stimulate
follicle development and ovulation [52-54].
3) Laparoscopic surgery
In women for whom CC or gonadotropin treatment is unsuccess-
ful, another commonly used approach is ovarian surgery. Although
there are several different techniques, they all involve acute ovar-
ian tissue damage. Various types of ovarian surgery have been em-
ployed (wedge resection, electrocautery, laser vaporization, multiple
ovarian biopsies and others [55-60]. All of these procedures result
in a positively altered endocrine profile after surgery. While the cur-
rent hypothesis is that a small amount of damage to the ovary works
to break the cycle of excessive androgen production and abnormal
negative feedback, the mechanism behind the reversal of endocri-
nological dysfunction in PCOS after ovarian surgery remains incom-
pletely understood.
3. Proposed pathogenesis
While the initiating factor of PCOS has yet to be determined, we
can evaluate the collective pool of known and suspected features
of PCOS to better clarify how each symptom relates and what may
Fig. 1. Ultrasound image of human polycystic ovary. Ovarian cysts are shown in the periphery of the ovary as dark circles.
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be driving the symptoms as a whole. In Figure 2, we summarize
the known extraovarian and intraovarian factors, and propose causal
relationships among them and their effects on inducing hyperandr-
ogenemia and polycystic ovary (arrows). In this particular ‘hypo-
thetical’ diagram, theca cell hyperplasia, an established cause of
PCOS, is proposed as a driving factor for the development of hyper-
androgenemia and polycystic ovaries. Of the extraovarian factors,
metabolic disorders (abnormal regulation of insulin and/or IGFs)
and obesity may stimulate thecal hyperplasia, noting that approxi-
mately half of PCOS patients are obese [9]. Hypergonadism caused
by dysregulation of LH secretion may also induce thecal hyperpla-
sia, as LH stimulates theca cell proliferation [61-63]. Concurrently,
intraovarian factors such as defects in steroidogenesis, regulation
of the cell cycle, or ovulatory defects may result in thecal hyperpla-
sia and hyperandrogenemia.
In regard to the mechanism of how hyperandrogenism may lead
to ovulatory defect and therefore formation of follicular cyst in the
ovary, it has been proposed that elevated testosterone disrupts the
regulation of GnRH secretion [64]. In support of this, either GnRH
agonists or LH treatment induces ovulation in PCOS patients. A few
animal studies have shown that androgen suppresses progesterone
receptor (PR) expression in the hypothalamus [65-67]. As progester-
one down-regulates GnRH secretion by activating PR activity in the
hypothalamus [68-70], androgen-mediated suppression of PR expres-
sion may ameliorate GnRH secretion, and therefore LH secretion
(Fig. 3). This altered release of gonadotropins will lead to defects in
follicle development, the development may arrest outright leading
to failure to ovulate (anovulation) or be reduced leading to irregu-
lar ovulation (oligo-ovulation). The arrested follicle may thus form
a cystic structure, leading to the polycystic ovarian phenotype and
loss of fertility in PCOS patients.
Insulin resistance and obesity themselves are also known to neg-
atively affect the secretion of pituitary hormones, and therefore fol-
licular development [71-73]. It is not known whether altered ste-
roidogenesis, pituitary hormone regulation or the defects in follicu-
lar development lead to an alteration of metabolism such as insulin
Problems in estrogen feedback/LH secretion
Defects in steroidogenic pathways
POLYCYSTIC OVARY
OVULATORY DEFECT
HYPERANDOGEMEMIA
Increase serum LH (Hypergonadism)
Disruption of ovarian steroid receptor action
Insulin, IGFs Defects in theca cell cycle regulation
Glucose resistance (Hyperinsulinemia)
Increase in theca cell population
Liver or pancreatic malfunction/Obesity
Defects in follicular rupture or interstitial hyperplasia
Increased proliferation & activity
theca cell
Extra-ovarian factors Intra-ovarian factors
Fig. 2. Intra- and extra-ovarian fac-tors that may elevate ovarian tes-tosterone synthesis.
Fig. 3. Proposed dysregulation of HPO axis by elevated testosterone (T) level. The numbers represent sequence of events. (1) Positive estrogen (E2) feedback to E2 receptor (ER) in the hypothalamus to GnRH secretion and LH surge and progesterone (P) production in the ovary. Upon E2 stimulation, ER induces P re-ceptor (PR) in the hypothalamus. (2) P induced suppression of GnRH secretion. (3) Hyperandrogenism-induced suppression of P receptor expression in the hy-pothalamus.
Hypothalamus
Pituitary
Ovary
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resistance and obesity.
4. Animal models for the study of PCOS
To identify the complex nature of this disease, suitable animal
models are needed. Researchers during the past three decades have
identified different animal models that mimic many of the features
of PCOS in women (these are summarized in Table 1). These mod-
els have afforded valuable information into complex nature of PCOS.
Currently, although a genetic component to PCOS has been identi-
fied, a specific “PCOS gene” has not, and therefore a specifically
targeted gene deletion for PCOS as an animal model is not avail-
able. Therefore, the majority of PCOS models that are available to
date rely upon external chemical treatments with steroids, steroid
precursors or steroid receptor antagonist to achieve the pathology.
Many of these models do not produce consistent results or cease to
produce PCOS like symptoms once the treatment stops. Recently,
Hill et al. [74] noted that their model for cardiovascular disease, the
hypothalamic pro-opiomelanocortin (POMC) specific leptin-insulin
double receptor knockout mouse exhibited some similar features to
PCOS. Our recently developed theca specific Esr1 specific knockout
mouse model reproduces the clinical pathologic features of PCOS
in 100% of animals.
1) Prenatal androgen
Prenatal androgen (PNA) treatment in sheep and monkeys results
in multiple metabolic and reproductive abnormalities. In monkeys,
daily subcutaneous injections of 15 mg of testosterone propionate for
40-80 days gestation are needed to induce the syndrome. In ewes,
an injection of 100 mg of testosterone propionate twice a week for
60 days between days 30 and 90 of the 147-day pregnancy result in
the ovarian abnormalities. In both models, the abnormalities mirror
the symptoms found in women with PCOS. These models produce
a long lasting effect in the female offspring mimicking many simi-
lar features of PCOS in humans. However, ewes and monkeys in-
cur a large financial commitment for a long gestational period. How-
ever, it is noted that although PA monkeys exhibit hyperandrogen-
emia, the increases are not as extreme as in PCOS women 0.3-0.4
ng/mL (~50-100% elevation above normal) [76,90]; PCOS women,
0.5-0.7 ng/mL (~70-200% elevation above normal) [91-94] and that
although anovulation observed in PNA monkeys, its prevalence is
also significantly less than that of PCOS women (PA monkeys: ~40%;
PCOS women ~90%) [90].
2) Pre-pubertal androgen
This model exploits the association of elevated androgen levels
during puberty and PCOS. Immature rats (approximately 21 days
old) are treated for 7-35 days with ~100 μg/day testosterone propi-
onate or dihydrotestosterone. Similar to the PNA animal models, pre-
pubertal androgen (PPA) animal models of PCOS utilize a unique
window where administration of exogenous androgens results in
permanent damage to the ovarian tissue and recapitulated the hall-
mark symptoms of PCOS in an animal model. PPA model shows
many similar features to PCOS in women with the exception of the
hallmark increase in basal LH levels [80,81]. This model is reliant
on artificial hyperandrogenemia and therefore does not help iden-
tify abnormalities upstream of hyperandrogenemia.
3) Letrozole
Letrozole is an oral non-steroidal aromatase inhibitor. Inhibition
of aromatase prevents the conversion of androgens to estrogens and
therefore this model has similar features to the prepubertal andro-
gen treatment [80,82]. Immature rats (approximately 21 days old) are
Table 1. PCOS Animal Models
Models Hyper-androgenemia
Oligo- ovulation
Polycystic ovaries
Insulin resistance* Increased LH References Rotterdam
criteria**
Chemically-induced models Androgen (Prenatal) Androgen (Prepubertal) Letrozole DHEA RU486 Estradiol
√
N/A√√√x
√√√√√√
√√√√√√
√√x√√
N/A
√
N/AN/A
√√
N/A
[75-79][80,81][80,82][83-86]
[87][81,88]
√√√√√√
Transgenic models Leptin/Insulin Receptor KO Theca specific Esr1 KO
√√
N/A
√
√√
√√
x√
[74][89]
√√
√, present or yes; x, not present; N/A, data not available; *, or showing glucose tolerant; **, whether to be classified as PCOS; PCOS, polycystic ovarian syndrome.
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treated for 7-35 days with 400 μg/day letrozole. Similarly to pre-pu-
bertal androgen, this model is also reliant on artificial hyperandro-
genemia and does not help identify abnormalities upstream of hy-
perandrogenemia.
4) Dehydroepiandrosterone (DHEA)
Immature rats or mice (approximately 21-22 days old) are treated
with daily s.c. DHEA injections (rats; 6 mg/100 g body weight, mice
6 mg/kg body weight) for 15-20 days. This dose of DHEA is suffi-
cient to induce a hyperandrogenized state similar to that in PCOS
women. This model is also reliant on artificial hyperandrogenemia
and does not help identify abnormalities upstream of hyperandro-
genemia.
5) RU486
Mature cycling female rats are treated daily with RU486 (20 mg/
kg body weight) for more than 8 days starting on the day of estrus.
These animals exhibit increased basal LH, polycystic ovaries, ovu-
lation blockade and metabolic defects [87]. However, this model is
reversible and symptoms decrease upon cessation of the antipro-
gestin treatment.
6) Estradiol
The immune system is now a well-recognized component of re-
productive biology. Immune cells are involved in all aspects of nor-
mal reproductive function including ovulation, corpus luteal forma-
tion, uterine receptiveness and maintenance of pregnancy. The re-
cently developed PCOS model developed by Chapman and collea-
gues [88] now provides evidence that aberrant immune function
may play a role in the pathogenesis of PCOS and that PCOS may
result from as autoimmune disease. This model exploits a 2-3 day
window of neonatal period (5-7 days of age) when estradiol admin-
istration (20 μg/day) disrupts thymic maturation. As a consequence
of this, increased vascular permeability in the thymus allows auto-
reactive T-cells to escape into the circulation. The ultimate effect of
these “escapees” is a damage to tissues throughout the body includ-
ing the ovaries. This model offers an abrupt change in the direction
of PCOS research. The major deficiencies in this model are a lack
of hyperandrogenemia and the fact that the loss of regulatory T-cell
function would not only impact the ovary, but multiple other tissues
resulting in pathologies not associated with PCOS. It will be inter-
esting to follow the progress of this novel concept of autoimmune
responses resulting in PCOS in women and if, PCOS women have
deficiencies in regulatory T-cells.
7) Hypothalamic pro-opiomelanocortin (POMC) neuron specific
leptin and insulin receptor KO
Hill and her colleagues were interested in insulin resistance and
the development of type II diabetes when they developed their
Pomc-Cre, Leprflox/flox IRflox/flox mice, effectively removing both leptin
and insulin receptors specifically from the POMC [74]. However,
together with the anticipated glucose intolerance and insulin defi-
ciency these mice suffer from hyperandrogenemia and polycystic
ovaries. These two pathological conditions secure its eligibility as a
model for the study of human PCOS.
5. A novel hyperandrogenic animal model for the study of PCOS
The unraveling of a biological pathway for PCOS and assigning
etiologies to this syndrome requires the development of a novel an-
imal model that consistently present PCOS phenotypes. We there-
fore launched a project to develop a mouse model of PCOS that is
genetically modified and thus stably displays PCOS phenotypes. In
doing so, we utilized the underlying concept of a vicious cycle [95-
97], where the abnormalities are reinforced. As a way to initiate this
cycle, we targeted the ovarian steroidogenic pathway to induce a
preference to androgen synthesis and therefore create hyperandro-
genemia.
The ovary is comprised of follicles at different stages of develop-
ment. During follicular growth, the oocyte becomes surrounded
by the granulosa cells, a basement membrane forms, and layers of
theca cells develop, surrounding the follicle. The two cell layers act
cooperatively in the production of androgens and estrogens. Theca
cells produce androgens that traverse the basement membrane to
the granulosa cells, where aromatase converts the androgens to es-
trogens. The estrogens in turn negatively feedback to the theca cells
causing a decrease of androgen production. This negative feedback
action of estrogen is mediated by estrogen receptor 1 (Esr1; ERα) in
the theca cells, the prime site of Esr1 expression in the ovary. Upon
activation by the binding of estrogen, Esr1 suppresses the expres-
sion of Cyp17, a critical enzyme that catalyzes a rate-limiting step in
androgen production (Fig. 4), resulting in the decrease of androgen
production. Therefore, deleting Esr1 gene would be a logical choice
for achieving sustained high levels of androgens because loss of Esr1
will exclude negative regulation of Cyp17 gene expression by estro-
gen. This will ultimately result in increased production of androgens.
The deletion of Esr1 gene has to be, however, limited to theca cells
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because Esr1 is expressed in so many different tissues/organs in-
cluding all of the tissues of the hypothalamic-pituitary-ovary-uterus
axis [98,99]. Otherwise, the Esr1 deletion in non-theca cells would
cause other defects, making it difficult to isolate the Esr1 deletion
effect in the theca cells.
This goal was achieved by selectively knocking out Esr1in the ste-
roidogenic theca cells while keeping the gene intact elsewhere. In
principle, this approach would result in the increased expression of
Cyp17 gene that encodes a late-limiting enzyme (17apha-hydroxy-
lase) in androgen synthesis pathway, because deletion of Esr1 will
relieve Cyp17 transcription from the well-established suppression
of this gene by Esr1. We achieved this aim by first generating a
mouse line that expresses Cre recombinase under the regulation of
the Cyp17 promoter (Cyp17iCre) [100] and then breeding the mice
successively with floxed Esr1 (Esr1flox) mice [89]. The offspring with
the genotype Esr1flox/flox Cyp17iCre, as was expected, expresses higher
level of Cyp17 mRNA expression in the ovary and maintains higher
serum testosterone levels (1.5-2 folds over wild type littermates) (ref).
The Esr1flox/flox Cyp17iCre mice display other phenotypic symptoms
of PCOS patients: (i) irregular estrous cycles, (ii) an age-dependent
decrease in ovulatory capacity and fertility defect and (iii) arrest of
follicular development at the early antral stage (Figs. 5, 6). This novel
animal model generated from the proof-of-principal experiment
should bring a new opportunity for the study of PCOS as well as
for the study of hyperandrogenemia itself.
Fig. 5. Hyperandrogenic mouse ovary. 3-month old-mouse ovary stained with Hematoxylin and Eosin. Multiple early antral stage follicles and a cyst (C) are shown in the periphery of the ovary. Insert, age-matching wild type mouse ovary.
Fig. 6. Human polycystic ovary. A ovarian section of 41-year-old PCOS patients. Multiple cysts are shown in the periphery of the ovary. Insert, age-matching non-PCOS ovary.
Fig. 4. Targeted modulation of ovarian steroido-genesis. A. CYP17 expression pattern in the 2- month old mouse ovary. CYP17-positve cells (the-ca cells; TC) are shown in brownish color. A follicle is made of oocyte (O), granulosa cells (GC) and the-ca cells (TC). B. ovarian steroidogenic pathway. BA
TC
OGC
C
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6. Future direction
In contrast to the seriousness of the PCOS in women’s health, the
pathogenesis of this disease is poorly understood. In recent years,
efforts by multiple laboratories have led to the development of novel
animal models for this complicated disorder. In the next few years,
use of these animal models and those to be newly developed will
undoubtedly accelerate our understanding on PCOS pathogenesis,
and possibly lead to the development of new therapeutic protocols
for treating and preventing PCOS in women.
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