Letrozole - acupuncture, E2 and propranolol 2015

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Circulating gonadotropins and ovarian adiponectin system are
modulated by acupuncture independently of sex steroid or
β-adrenergic action in a female hyperandrogenic rat model of
polycystic ovary syndrome
Manuel Maliqueo a,b, Anna Benrick c, Asif Alvi c, Julia Johansson c, Miao Sun c,d,
Fernand Labrie e, Claes Ohlsson f, Elisabet Stener-Victorin a,d,*
aDepartment of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
bLaboratorio de Endocrinología y Metabolismo, Departamento de Medicina Occidente, Facultad de Medicina, Universidad de Chile, Santiago, Chile
cInstitute of Neuroscience and Physiology, Department of Physiology, Sahlgrenska Academy, University of Gothenburg, Sweden
dDepartment of Obstetrics and Gynecology, First Affiliated Hospital, Heilongjiang University of Chinese Medicine, Harbin 150040, China
eLaval University Research Center in Molecular Endocrinology, Oncology and Human Genomics, CHUL Research Center, Quebec G1V 4G2, Canada
fDepartment of Internal Medicine, Institute of Medicine, Centre for Bone and Arthritis Research, The Sahlgrenska Academy, University of Gothenburg,
Gothenburg 40530, Sweden
ARTICLE INFO
Article history:
Received 15 February 2015
Received in revised form 21 April 2015
Accepted 27 April 2015
Available online
Keywords:
Polycystic ovary syndrome
Animal model
Acupuncture
Estradiol
Sympathetic nervous system
ABSTRACT
Acupuncture with combined manual and low-frequency electrical stimulation, or electroacupuncture (EA),
reduces endocrine and reproductive dysfunction in women with polycystic ovary syndrome (PCOS), likely
by modulating sympathetic nerve activity or sex steroid synthesis. To test this hypothesis, we induced
PCOS in rats by prepubertal implantation of continuous-release letrozole pellets (200 μg/day) or vehicle.
Six weeks later, rats were treated for 5–6 weeks with low-frequency EA 5 days/week, subcutaneous in-
jection of 17β-estradiol (2.0 μg) every fourth day, or a β-adrenergic blocker (propranolol hydrochloride,
0.1 mg/kg) 5 days/week. Letrozole controls were handled without needle insertion or injected with sesame
oil every fourth day. Estrous cyclicity, ovarian morphology, sex steroids, gonadotropins, insulin-like growth
factor I, bone mineral density, and gene and protein expression in ovarian tissue were measured. Low-
frequency EA induced estrous-cycle changes, decreased high levels of circulating luteinizing hormone
(LH) and the LH/follicle-stimulating hormone (FSH) ratio, decreased high ovarian gene expression of
adiponectin receptor 2, and increased expression of adiponectin receptor 2 protein and phosphoryla-
tion of ERK1/2. EA also increased cortical bone mineral density. Propranolol decreased ovarian expression
of Foxo3,Srd5a1, and Hif1a. Estradiol decreased circulating LH, induced estrous cycle changes, and de-
creased ovarian expression of Adipor1,Foxo3, and Pik3r1. Further, total bone mineral density was higher
in the letrozole–estradiol group. Thus, EA modulates the circulating gonadotropin levels independently
of sex steroids or β-adrenergic action and affects the expression of ovarian adiponectin system.
© 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Polycystic ovary syndrome (PCOS), one of the most common en-
docrine and metabolic disorder in women, involves a wide
spectrum of endocrine and metabolic abnormalities in which
hyperandrogenism appears to be of central importance (Azziz, 2003).
Neuroendocrine abnormalities are reflected by an imbalance in go-
nadotropin secretion characterized by elevated levels of luteinizing
hormone (LH) and low levels of follicle-stimulating hormone (FSH),
leading to altered ovarian steroidogenesis and folliculogenesis,
anovulation, and infertility (Blank et al., 2007). Women with PCOS
also have high sympathetic nerve activity associated with
hyperandrogenism (Sverrisdottir et al., 2008).
Models of PCOS in rodents have helped to elucidate the patho-
physiological mechanisms of PCOS in human (Maliqueo et al., 2014).
Recently, we assessed the phenotype of a rat model of PCOS induced
by continuous administration of letrozole (200 μg/day), an inhib-
itor of aromatase activity, for 90 days starting before puberty
(Maliqueo et al., 2013). These rats have hyperandrogenemia asso-
ciated with low estrogen levels, anovulation and polycystic ovarian
Abbreviations: BMD, bone mineral density; DHT, dihydrotestosterone; EA,
electroacupuncture; FSH, follicle-stimulating hormone; IGF, insulin-like growth factor;
LH, luteinizing hormone; PCOS, polycystic ovary syndrome; sc, subcutaneous.
* Corresponding author. Karolinska Institutet, Department of Physiology
and Pharmacology, Von Eulersväg 4a, SE-171 77 Stockholm, Sweden. Tel.:
+46(0)705643655; fax: +46-8-31 11 01.
E-mail address: elisabet.stener-victorin@ki.se (E. Stener-Victorin).
http://dx.doi.org/10.1016/j.mce.2015.04.026
0303-7207/© 2015 Elsevier Ireland Ltd. All rights reserved.
Molecular and Cellular Endocrinology ■■ (2015) ■■–■■
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morphology, abnormal gonadotropin secretion, and metabolic dis-
turbances (Maliqueo et al., 2013). However, low estrogen activity
also alters endocrine and reproductive function in ovariectomized
rats and in estrogen receptor α (ERα) and aromatase knockout mice
(D’Eon et al., 2005; Jones et al., 2000; Song et al., 2005).
In several experimental and clinical trials, acupuncture with
needle placement in abdominal and leg muscle with somatic in-
nervations that correspond to the ovaries and uterus has been
effective in managing the reproductive derangements in women with
PCOS (Jedel et al., 2011; Johansson et al., 2013; Stener-Victorin et al.,
2000, 2013). In a rat model of PCOS induced by dihydrotestosterone
(DHT), acupuncture with low-frequency electrical stimulation of the
needles, the so-called electroacupuncture (EA), restored estrous cy-
clicity (Feng et al., 2009). However, the molecular mechanism
through which the acupuncture could induce ovulation is unclear.
Steroidogenesis in ovarian tissue is regulated by pathways related
to insulin sensitivity, sympathetic nerve activity, and lipid metab-
olism (Kraemer et al., 1991; Lara et al., 1993; Wu et al., 2014), and
molecules related to steroid action and tissue remodeling are in-
volved in the abnormal folliculogenesis in PCOS (Goldman and Shalev,
2004; Sproul et al., 2010). Therefore, it is likely that the effects of
low-frequency EA on ovarian function are mediated by these path-
ways. Of interest, the adiponectin pathway, monocyte chemotactic
protein-1, and peroxisome proliferator-activated receptor gamma,
among other molecules, could be the link between the metabolic
alterations and reproductive derangements in PCOS (Comim et al.,
2013; Figueroa et al., 2012; Komar, 2005; Sproul et al., 2010).
In rats with letrozole-induced PCOS, EA decreases the expres-
sion of P450c17α protein and increases expression of P450
aromatase, thereby lowering androgen levels, restoring estrous cy-
clicity, and normalizing ovarian morphology (Sun et al., 2013). Low-
frequency EA reduces high muscle sympathetic nerve activity in
women with PCOS and ovarian nerve growth factor content in rats
with PCO induced by estradiol valerate (Stener-Victorin et al., 2000,
2009). Interestingly, in ovariectomized rats, low-frequency EA stimu-
lates aromatase activity in the hypothalamus and increases
circulating estradiol levels (Zhao et al., 2004, 2005). In view of these
findings, EA exerts beneficial effects by restoring the complex balance
among sex steroids and sympathetic nerve activity.
In this study, we tested the hypothesis that low-frequency EA
alters the estrous cycle, reduces high circulating levels of testos-
terone and gonadotropins, and normalizes altered ovarian expression
of genes and proteins related to endocrine and reproductive func-
tion in rats with letrozole-induced PCOS. The effects of EA were
compared with those of β-adrenergic blockade (to elucidate the in-
volvement of sympathetic nerve activity) and those of cyclic estrogen
treatment (to mimic the physiological changes in estrogen concen-
tration during the estrous cycle) to determine whether the changes
induced by these treatments are similar to those induced by low-
frequency EA. Endocrine function was assessed by measuring
circulating sex steroids and gonadotropins. Reproductive function
was assessed by evaluating estrous cyclicity and ovarian morphol-
ogy. We also analyzed gene and protein expression of molecular
pathways related to steroidogenesis, steroid action, ovarian
folliculogenesis, tissue remodeling, and sympathetic nerve activi-
ty in ovarian tissue. Finally, we evaluated the effect of EA on bone
mineral density (BMD), which is modulated by the action of sex
steroid and β-adrenergic receptors.
2. Materials and methods
2.1. Animals
Eight Wistar dams, each with 7–9 female pups, were pur-
chased from Charles River Laboratories (Munster, Germany). Pups
were raised with a lactating dam until 21 days of age and then
housed three to five per cage under controlled conditions (21–
22 °C, 55–65% humidity, 12-h light/12-h dark cycle). They were fed
standard rat chow (Harlan Teklad Global Diet, Harlan, Germany) ad
libitum and had free access to water. Rats were cared for accord-
ing to the principles of the “Guide to the Care and Use of
Experimental Animals” (www.sjv.se). The study was approved by
the Animal Ethics Committee at the University of Gothenburg (Dnr:
364–2011).
2.2. Study procedure (Supplementary Fig. S1)
At 21 d of age, 50 rats were lightly anesthetized with isoflurane
(2% in a 1:1 mixture of oxygen and air) and implanted with sub-
cutaneous (sc) 90-d continuous-release pellets (Innovative Research
of America, Sarasota, FL) containing 18.0 mg (daily dose, 200 μg) of
letrozole (Novartis Pharma, Basel, Switzerland) (Maliqueo et al.,
2013). Twelve control rats received an identical pellet lacking the
bioactive molecule. A microchip (AVID, Norco, CA) with an identi-
fication number was inserted in the neck along with the pellets. Four
rats had normal estrous cyclicity and no changes in the body weight
after 5 weeks of letrozole exposure. Because all rats exposed to
letrozole in our previous study had anovulation and increased body
weight at this period (Maliqueo et al., 2013), these four rats were
not included in subsequent experiments.
Letrozole-exposed rats were randomly divided into groups
and treated with low-frequency EA (letrozole–EA, n =10), cyclic
estradiol every fourth day (letrozole–estradiol, n =10), or a
β-adrenoreceptor blocker (letrozole–propranolol, n =10). To control
for treatment effects on letrozole-exposed rats, rats were either
handled without needle insertion to control for the EA treatment
(n =8) or injected with sesame oil every fourth day to control for
estradiol/propranolol injections (n =8). As there were no differ-
ences between these control groups, they were pooled (letrozole,
n=16).
Treatments started at 63 days of age, 6 weeks after pellet im-
plantation. The study was concluded after 11–12 weeks of letrozole
exposure, including 5–6 weeks of treatment. All rats were weighed
weekly from 21 days of age. Rats were anesthetized with
thiobutabarbital sodium (Inactin; Sigma, St. Louis, MO) and killed
by decapitation during the estrus phase in control rats and rats
exposed to letrozole with evidence of estrous cycle change. The
ovaries and uterus were dissected and weighed. One ovary was
excised, fixed in neutral buffered 4% formaldehyde for 24 h, placed
in 70% ethanol, dehydrated, and embedded in paraffin; the other
ovary was frozen in liquid nitrogen for analysis of mRNA and pro-
teins. One femur was placed in 70% ethanol for analysis of BMD.
2.3. Treatments
2.3.1. EA
Acupuncture was performed daily in conscious rats from Monday
to Friday for 5–6 weeks; this schedule improves neuroendocrine,
reproductive, and metabolic function in rats with DHT-induced PCO
(Feng et al., 2009, 2012; Johansson et al., 2010). A group of letrozole-
exposed and control rats underwent the same handling procedure
but without the needle insertion to control for environmental factors
(Feng et al., 2009; Johansson et al., 2010; Manneras et al., 2009).
Briefly, two acupuncture needles 0.20 mm in diameter and 15 mm
long (HEGU Svenska, Landsbro, Sweden) were placed bilaterally at
a depth of 5–8 mm in the abdominal muscles, and two needles were
placed at a depth of 5–10 mm in each soleus and gastrocnemius
hindlimb muscle, in somatic segments (Th10 to L2) correspond-
ing to ovarian innervations. The needles were stimulated at 2.0 Hz
with 0.1-s, 80-Hz burst pulses with an electric stimulator (CEFAR
ACU II; Cefar-Compex Scandinavia, Malmo, Sweden). The intensi-
ty was 0.6–1.4 mA and was increased if no muscle contractions were
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observed. The treatment duration was 15 min in week 1, 20 min in
weeks 2 and 3, and 25 min thereafter. Before handling or needle
insertion, all rats assigned to EA treatment and controls were lightly
anesthetized with isoflurane (2% in a 1:1 mixture of oxygen and air)
for 2–3 min. During EA treatment and handling of controls, rats were
conscious and placed in a fabric harness and suspended above the
desk.
2.3.2. Cyclic estradiol and β-adrenoreceptor blockade
Estradiol was administrated once every fourth day by sc injec-
tion of 2 μg of 17β-estradiol-3-benzoate (E8515; Sigma) dissolved
in 100 μl of sesame oil, as described (Asarian and Geary, 2002).
β-Adrenoreceptors were blocked daily from Monday to Friday (same
schedule as EA) by sc injection of propranolol hydrochloride (0.1 mg/
kg body weight) (P0884; Sigma) dissolved in saline (Bonnet et al.,
2008). A group of letrozole-exposed and control rats received sc in-
jections of 100 μl of sesame oil according to the regimen for cyclic
estradiol. In both cases, subcutaneous intrascapular injections were
given between 09.00 and 10.00 h (estradiol) or 10.00 and 11.00 h
(propranolol). The dose and timing of estradiol were chosen because
2 μg of estradiol given once every fourth day produces a near-
physiological cyclic pattern of plasma estradiol concentration and
normalizes food intake and body weight in ovariectomized rats
(Asarian and Geary, 2002). The propranolol dose, 0.1 mg/kg body
weight, was chosen because it prevents the osteoporosis induced
by ovariectomy without affecting cardiac hemodynamic functions
(Bonnet et al., 2008).
2.4. Vaginal smears
The estrous cycle stage was determined by vaginal smears
(Marcondes et al., 2002). Vaginal smears were performed daily at
4–5 weeks after pellet implantation to evaluate the effectiveness
of letrozole exposure and after the first week of treatments (at 10
weeks of age, 8 weeks after pellet implantation) until the end of
the study (Supplementary Fig. S1). Estrous cyclicity was evaluated
as the percentage of the time in each phase (diestrous, proestrus,
estrus, and metestrus) 1 week after the start of treatments.
2.5. Blood sampling
At 7 weeks and 13 weeks of age (after 4 weeks of treatments)
(Supplementary Fig. S1), tail blood samples were obtained after
an overnight fast to assess circulating gonadotropins, insulin-like
growth factor I (IGF-I) and sex steroids. In cycling rats, blood
samples were obtained in the estrus phase, determined by vaginal
smear. The samples were centrifuged, and the serum was stored
at −80 °C.
2.6. Ovarian morphology
For assessment of ovarian morphology, two 4-μm sections of each
ovary were taken 40 μm apart at the largest diameter, mounted on
a glass slide, stained with hematoxylin and eosin, and analyzed by
conventional light microscopy.
2.7. Bone mineral density
BMD was analyzed ex vivo by peripheral quantitative comput-
erized tomography with a Stratec pQCT XCT Research M (v5.4B;
Norland-Stratec, Fort Atkinson, WI) with a resolution of 70 μm. BMD
(total, trabecular, and cortical volumetric) was analyzed as de-
scribed (Benrick et al., 2013).
2.8. Analytical methods
Plasma concentrations of progesterone, testosterone, 17β-
estradiol, and DHT were measured with a validated gas
chromatography–mass spectrometry system at Endoceutics (Quebec
City, Canada) (Stener-Victorin et al., 2010). Serum LH and FSH were
assayed with a rat pituitary magnetic bead panel (Milliplex Panel;
Millipore, Billerica, MA). IGF-I was assayed with a specific mouse/
rat ELISA kit (Quantikine, MG100,R&DSystems, Abingdon, UK).
The limits of detection were 0.25 ng/ml for progesterone, 0.025 ng/
ml for testosterone, 10 pg/ml for DHT, 1.00 pg/ml for 17β-estradiol,
3.28 pg/ml for LH, 7.62 pg/ml for FSH, and 3.5 pg/ml for IGF-I. The
intra- and interassay coefficients of variation were, respectively,
3.3% and 12.8% for LH, 2.8% and 12.3% for FSH, and 4.1% and 4.3%
for IGF-I.
2.9. RNA isolation and quantitative real-time RT-PCR
Total RNA was isolated from ovarian tissue with commercial kits
(74704, Qiagen, Hilden, Germany). RNA quality was evaluated in
random samples with an Experion electrophoresis system (Bio-
Rad Laboratories, Hercules, CA). First-strand cDNA was prepared from
1 μg of total RNA with Superscript VILO (Life Technologies, Paisley,
UK) following the manufacturer’s protocol.
For real-time RT-PCR, 500 ng of cDNA was analyzed with an ABI
Prism 7900HT Sequence Detection System, ABI Prism 7900HT SDS
Software 2.4 (Applied Biosystems, Carlsbad, CA), and a custom
TaqMan low-density array (Applied Biosystems) for 48 genes
(Supplementary Table S1). Five putative reference genes were in-
cluded: 18S ,Actb,Gapdh,Ppia, and Hprt. Thermal cycling conditions
were 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of
15 s at 95 °C and 1 min at 60 °C. Variability, calculated with the
NormFinder algorithm (Andersen et al., 2004), was lowest with the
combination of Actb and Gapdh. Gene expression values were cal-
culated with the ΔΔCq method (i.e., RQ =2–ΔΔCq)(Livak and
Schmittgen, 2001).
2.10. Western blot analysis
For western blot analyses, we used antibodies against the
adiponectin receptor 2 (ADIPOR2) (sc-46755; Santa Cruz Biotech-
nology, Santa Cruz, CA), phosphorylated-44/42 MAPK (Thr202/
Tyr204) (ERK1/2) (#9101; Cell Signaling, Beverly, MA), and β-actin
(A1978, Merck, St. Louis, MO).
Frozen ovarian tissues were homogenized in ice-cold buffer
(10 mM Tris·HCl; 100 mM NaCl; EDTA and EGTA at l mM each; 1%
Triton X-100; 10% glycerol; 0.1% sodium dodecyl sulfate; 0.5% de-
oxycholate; 1 mM dithiothreitol; 1.0 mM phenylmethylsufonyl
fluoride; 10 mM N-ethylmaleimide; 1 mM iodoacetamide; and 1×
protease inhibitor cocktail containing 1×complete protease inhib-
itor cocktail and phosphatase inhibitor cocktail (Roche Diagnostics,
Basel, Switzerland)). Homogenate samples were rotated on ice for
45 min and centrifuged (16,300 g) for 40 min at 4 °C. Superna-
tants were collected, and protein concentration was determined with
a spectrometer (Direct Detect; Millipore, MA).
Total protein (~30 μg) was separated on precast 4–12% Bis–Tris
gels (Invitrogen) and transferred to PVDF membranes with the iBlot
dry blotting system (Invitrogen). Membranes were blocked for 1 h
at room temperature in 5% nonfat dry milk, or 5×animal-free blocker
(Vector Laboratories, Burlingame, CA) for ADIPOR2, and incubated
with primary antibody overnight at 4 °C and then with secondary
antibody for 1 h at room temperature. Protein bands were devel-
oped with SuperSignal West Dura Extended Duration Substrate
(Pierce Biotechnology) and photographed with an LAS-1000 camera
system; ADIPOR2 protein signals were quantified by densitom-
etry with MultiGauge software Ver. 3.0 (Fujifilm, Tokyo, Japan).
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Phosphorylated ERK1/2 was analyzed with the ChemiDoc XRS+
System and Image Lab Software. β-Actin was used as a loading
control and for normalization. For each protein target, all individ-
ual density values in the letrozole groups were expressed relative
to the mean of the control, except for ADIPOR2, which was ex-
pressed relative to the mean of the letrozole group.
2.11. Statistical analyses
Statistical analyses were performed with SPSS (version 21.0; SPSS,
Chicago, IL) and Prism GraphPad (version 5.03, GraphPad Soft-
ware, La Jolla, CA). Letrozole-exposed rats handled without needle
insertion or injected with sesame oil every fourth day were pooled,
as no differences between them were observed. The normal distri-
bution of the data was tested by Shapiro–Wilk test. Differences
between groups were analyzed by one-way ANOVA followed by
post hoc Dunnett test for normal distributed data or Kruskal–
Wallis test followed by the Mann–Whitney Utest for skewed
data. Data are expressed as mean ±SEM. P <0.05 was considered
significant.
3. Results
3.1. Estrous cyclicity and ovarian morphology
The control rats had a normal estrous cycle of 4–5 days. Rats
exposed to letrozole were acyclic, and leukocytes were predomi-
nant in their vaginal smears, indicating pseudo-diestrus. On the other
hand, letrozole–EA resulted in a higher prevalence of estrus and a
shorter diestrus phase than in the letrozole group (P<0.01)
(Fig. 1C–D). Letrozole–propranolol did not modify the patterns of
pseudo-diestrus, but cyclic estradiol reduced the time in diestrus
and increase the time in proestrus, estrus and metestrus phases
(P<0.001, letrozole vs. letrozole–estradiol) (Fig. 1C–D).
The ovaries were heavier in letrozole rats than in controls
(0.236 ±0.01 g vs. 0.176 ±0.03 g; P=0.003) (Fig. 1B). Control rats had
normal ovarian morphology, with follicles and corpora lutea at dif-
ferent stages of development and regression. Rats exposed to
letrozole had atretic and cystic follicles in the periphery of the ovary
and no corpora lutea (Fig. 1A). Treatment with EA, cyclic estradiol,
or propranolol did not affect ovarian weight or morphology in the
letrozole-treated rats (Fig. 1A). The uterus weighed less in letrozole-
exposed rats than in controls (0.398 ±0.04 g, vs. 0.549 ±0.02 g;
P=0.013). However, the treatments did not modify uterus weight
in letrozole-exposed rats.
3.2. Circulating sex steroids (mass spectrometry)
At5weeks(
Table 1) and 10 weeks (Fig. 2A and B) after pellet
implantation, the circulating progesterone level was lower and the
testosterone level was higher in all groups of letrozole-exposed rats
than in controls. The DHT level was significantly higher in the
letrozole group than in controls at 5 weeks (Table 1;P=0.008). Cir-
culating estradiol concentrations did not differ between untreated
letrozole-exposed rats and controls at 5 or 10 weeks (Table 1). In
the letrozole–EA group, the estradiol level was lower than in the
letrozole group (P =0.006) (Fig. 2C). Cyclic estradiol reduced circu-
lating testosterone to levels lower than those in the letrozole group
(P =0.025) (Fig. 2B). However, circulating progesterone and DHT levels
did not differ between groups (Fig. 2A and D). Treatment with pro-
pranolol did not affect circulating sex steroids but tended to increase
circulating progesterone to concentrations higher than those in the
letrozole group (P =0.058) (Fig. 2A).
3.3. Circulating levels of gonadotropins
Circulating LH levels were higher and FSH levels were lower, re-
sulting in a higher LH/FSH ratio, in rats exposed to letrozole than
in controls at 10 weeks after pellet implantation (P <0.001)
(Fig. 2E–G). EA decreased LH (P =0.003) and tended to increase FSH
in letrozole–EA rats (P =0.053 vs. letrozole rats) (Fig. 2E and F).
Further, the LH/FSH ratio was lower in letrozole-EA than in letrozole
rats (P =0.007) (Fig. 2G). Letrozole–estradiol rats had lower circu-
lating LH levels than letrozole rats (P <0.001), but circulating FSH
levels did not differ (Fig. 2E and F). The LH/FSH ratio was also lower
in letrozole–estradiol than letrozole rats (Fig. 2G). Circulating LH and
FSH levels were not affected in the letrozole–propranolol group
(Fig. 2E–G).
3.4. Circulating levels of IGF-I
Circulating IGF-I levels were higher in rats exposed to letrozole
than in controls (P =0.026) and were not affected by treatment with
EA, propranolol, or cyclic estradiol (Fig. 2H).
3.5. BMD (Table 2)
In letrozole rats, the tibia was longer and total, trabecular, and
cortical BMD values were lower than in controls. Low-frequency EA
increased the cortical BMD (P =0.017 vs. letrozole group). On the
other hand, total BMD was higher in the letrozole–estradiol group
than in the letrozole group. The BMD values did not differ between
the letrozole–propranolol group and the letrozole group.
3.6. Ovarian gene expression
Ovarian mRNA expression of Adipor1,Adipor2,Amhr2,Cyp17a1,
Cyp19a1,Esr1,Esr2,Foxo3,Ngf, and Pik3r1 was higher and expres-
sion of Adrb2,Ar,Hif1a,Mmp2,Srd5a1, and Vegfa was lower in
letrozole-treated rats than in controls (Fig. 3A). mRNA expression
of Adipoq was higher and mRNA expression of Adipor2 and Hif1a
was lower in the letrozole–EA than in the letrozole group (Fig. 3B,
D, F). Moreover, Adipoq tended to be lower in letrozole group com-
pared to controls (P =0.058) (Fig. 3B). Propranolol reduced the mRNA
expression of Foxo3,Hif1a,and Srd5a1 (Fig. 3E, F, H).On the other
hand, cyclic estradiol treatment decreased mRNA expression of
Adipor1,Foxo3, and Pik3r1 (Fig. 3C, E, G).
3.7. Protein expression
Because EA normalized the mRNA expression of Adipoq and
Adipor2, we measured the ovarian expression of the correspond-
ing proteins. Unfortunately, we were not able to determine the
protein expression of adiponectin. Phosphorylated-ERK1/2 was mea-
sured because adiponectin activates ERK1/2 signaling regulating of
steroid production in ovarian cells (Chabrolle et al., 2007; Ledoux
et al., 2006; Pierre et al., 2009). ADIPOR2 protein was not ex-
pressed in control rat ovaries but was present in all letrozole-
exposed rats (Fig. 4A). EA increased expression of ADIPOR2 protein
(P =0.039 vs. letrozole group) (Fig. 4A). Phosphorylated ERK1/2 was
increased by EA (P =0.039 vs. letrozole group) and decreased by es-
tradiol (P =0.027 vs. letrozole group) (Fig. 4B).
4. Discussion
This study shows that repeated low-frequency EA significantly
affects the pituitary–ovarian axis by normalizing LH secretion and
increasing ovarian expression of ADIPOR2 and phosphorylation of
ERK1/2 in a rat model of letrozole-induced PCOS. Because there are
no inert placebo controls for acupuncture (Lund and Lundeberg,
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4M. Maliqueo et al./Molecular and Cellular Endocrinology ■■ (2015) ■■–■■

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Days old Days old Days old
Control
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Control
Letrozole
Letrozole-EA
Letrozole-propranolo
Letrozole-estradiol
Ovarian weight (g)
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
0.1
0.2
0.3
0.4
**
A
B
C
D
Diestrous
Proestrous
Estrous
Metaestrous
0
5
10
20
40
60
80
100
% of days after one week of treatments
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-Estradiol
*** *** ***
***
*** *** *** ***
**
**
Fig. 1. (A) Ovarian morphology of control (placebo) rat showing corpora lutea (CL) and follicles at different stages, and of letrozole, letrozole-EA, letrozole-propranolol, and
letrozole-estradiol rats (magnification, ×1.0; distance bars, 4.0 mm). (B) Ovarian weight in control (placebo), letrozole, letrozole-EA, letrozole-propranolol and letrozole-
estradiol rats. (C) Frequency of estrous changes in control (placebo) (n =12), letrozole (n =16), letrozole-EA (n =10), letrozole-propranolol (n =10), and letrozole-estradiol
(n =10) rats. Values are the percentage of the time in each phase (diestrus, proestrus, estrus, and metestrus) 1 week after the start of treatments. (D) Estrous cycle patterns
at 70–91 days of age (i.e., 50–71 days after pellet implantation) in three representative rats from each group. D, diestrus; E, estrus; M, metestrus; P, proestrus. P values were
calculated by ANOVA followed by Dunnett’s test. ***P<0.001, **P <0.01 vs. letrozole group.
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5M. Maliqueo et al./Molecular and Cellular Endocrinology ■■ (2015) ■■–■■

Estradiol (pg/ml)
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
1.0
2.0
3.0
4.0
5.0
6.0
**
DHT (pg/ml)
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0
20
40
60
80
100
Progesterone (ng/ml)
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0
5
10
15
20
25
***
[*]
Testosterone (ng/ml)
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
0.5
1.0
1.5
2.0
2.5
***
**
FSH(ng/ml)
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
2.5
5.0
7.5
10.0
**
[*]
LH (ng/ml)
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
2.0
4.0
6.0
8.0
** **
***
AB
CD
EF
IGF-I (
μg/ml)
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
0.5
1.0
1.5
2.0
*
LH/ FSH ratio
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
0.5
1.0
1.5
2.0
***
**
***
GH
Fig. 2. (A–D) Serum progesterone, testosterone, estradiol, and DHT concentrations assayed by mass spectrometry. (E–G) Serum gonadotropin concentrations (LH and FSH)
and LH/FSH ratio. (H) Serum IGF-1 concentrations in control (placebo) (n =8–12), letrozole (n =15–16), letrozole–EA (n=8–9), letrozole–propranolol (n =8–10) and letrozole–
estradiol (n =8–10) rats. P values were calculated by ANOVA followed by Dunnett’s test (testosterone, DHT, and IGF-1) and Kruskal–Wallis test followed by Mann–Whitney
Utest (progesterone, estradiol, LH, and FSH). ***P <0.001, **P <0.01, *P <0.05, [*]P<0.06 vs. letrozole group. Values are mean ±SEM.
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6M. Maliqueo et al./Molecular and Cellular Endocrinology ■■ (2015) ■■–■■

2006; Wu et al., 2002), we controlled for environmental factors by
using the same handling procedure for all letrozole-treated rats, a
well-documented way to control for acupuncture effects in rats and
humans (Feng et al., 2009; Johansson et al., 2010, 2013; Manneras
et al., 2009). Interestingly, β-adrenergic receptor blockade did not
affect the gonadotropin levels and only reduced the ovarian mRNA
expression of Foxo3,Hif1a, and Srd5a1, indicating that the effects
of EA are not mediated by β-adrenergic receptors. Cyclic estradiol
significantly altered estrous cyclicity and decreased circulating LH
and testosterone levels but did not affect FSH secretion. Moreover,
cyclic estradiol decreased and normalized the ovarian expression
of Adipor1,Foxo3, and Pik3r1 mRNA.
4.1. Circulating sex steroids analyzed by mass spectrometry
Cyclic estradiol decreased circulating testosterone levels but did
not increase estradiol levels. The lack of changes in the expression
of genes encoding other steroidogenic enzymes in ovarian tissue
suggests that estradiol affects the kinetics of P450-17 alpha-
hydroxylase/C17,20-lyase (P450c17), since the fall in androgen levels
before ovulation is mediated by an estrogen-receptor-induced re-
duction in P450c17 (Banks et al., 1991). However, the reduced
estradiol levels in the EA group could be mediated by changes in
testosterone metabolism, as DHT levels tend to be higher in rats
treated with low-frequency EA. It is highly unlikely that this effect
was mediated by β-adrenergic receptors, since propranolol did not
change the sex steroid profile in letrozole-treated rats.
In the ovaries of letrozole-treated rats, Cyp17a1 and Cyp19a mRNA
was upregulated and Srd5a1 mRNA was downregulated, probably
in response to high circulating levels of androgens (Fitzpatrick and
Richards, 1991). Previously, we suggested that the increase in ovarian
Cyp17a1 expression is related to thecal hypertrophy mediated by
hyperandrogenism, elevated LH levels, hyperinsulinemia due to
insulin resistance, or low estrogen action (Maliqueo et al., 2013).
Since cyclic estradiol normalized LH levels, it is likely that, in con-
trast to observations in co-cultures of isolated theca-granulosa
(Ortega et al., 2013), ovarian expression of Cyp17a1 mRNA is
upregulated as a result of elevated androgen levels induced by block-
ade of P450 aromatase activity by letrozole.
In rats with letrozole-induced PCOS, DHT levels were elevated
after 5 weeks of letrozole exposure, indicating an early increase in
the activity of 5-alpha reductase in peripheral tissue. But in con-
trast to our previous report (Maliqueo et al., 2013), the level of
circulating estradiol was not lower, most likely because the estro-
gen levels varied considerably in control rats with normal estrous
cyclicity.
4.2. Bone mineral density
The presence of the four stages of the estrous cycle and recov-
ery of total BMD in rats treated with cyclic estradiol revealed that
the estrogen response in peripheral tissue was intact. Interest-
ingly, although estradiol levels were lower, cortical BMD increased
in the letrozole–EA group. This finding could indicate higher sen-
sitivity of bone to estrogen or an effect on other modulators of bone
remodeling, such as growth factors or androgens. EA increases BMD
by promoting the IGF-I system in ovariectomized rats (Feng et al.,
2008). However, letrozole-exposed rats already had elevated cir-
culating IGF-I levels, probably reflecting the reduced estrogen action
(Borski et al., 1996). On the other hand, low-frequency EA induced
a small but not significant reduction in the IGF-I level, which did
not differ from that in the control group. Therefore, it is likely that
the changes in DHT and IGF-I levels induced by low-frequency EA
help maintain cortical BMD, as in ovariectomized rats (Coxam et al.,
1996). In women with PCOS, BMD is controversial and likely de-
pendent on body composition (Kassanos et al., 2010; Katulski et al.,
2014). Interestingly, cortical BMD is higher in PCOS women than
controls, and the difference seems to reflect the hyperandrogenic
condition in women with PCOS (Kassanos et al., 2010).
Low doses of β-adrenergic blocker, similar to those in our study,
maintain bone architecture in ovariectomized rats (Bonnet et al.,
2006). Nevertheless, we found that propranolol did not modify any
bone parameter, probably because of the steroid disruption in
letrozole-exposed rats.
4.3. Gonadotropin secretion
The letrozole-induced PCOS model has the advantage of main-
taining constantly low levels of estrogen and elevated levels of
androgens. The cyclic estradiol regimen reduced circulating LH levels
without modifying FSH levels; thus, suppression of FSH secretion
in letrozole-rats is mediated to a greater extent by a hyperandrogenic
action than by a reduced estrogenic action at the hypothalamic and/
or pituitary level (Blank et al., 2007). In turn, the improvement in
LH and, to a certain extent, FSH by low-frequency EA supports the
notion that acupuncture modulates the sensitivity of hypotha-
lamic cells to androgen (Feng et al., 2009). This effect was
independent of circulating estrogen levels and led to reduced se-
cretion of gonadotropin-releasing hormone, as in rats with DHT-
induced PCOS (Feng et al., 2009). It is unlikely that these effects are
mediated by β-adrenergic signaling, since blockade of β-adrenergic
receptors did not affect circulating gonadotropin levels. However,
we cannot exclude that the effect of low-frequency EA is medi-
ated via sympathetic nervous system as we demonstrated that high
Table 1
Serum testosterone, estradiol, progesterone, and dihydrotestosterone (DHT) con-
centrations assayed by mass spectrometry in control and letrozole-exposed rats 5
weeks after pellet implantation.
Control (n =12) Letrozole (n =45)
Progesterone (ng/mL) 10.35 ±1.19 *** 3.41 ±0.52
Testosterone (ng/mL) 0.37 ±0.05*** 1.53 ±0.12
Estradiol (pg/mL) 1.25 ±0.23 1.73 ±0.14
DHT (pg/mL) 49.38 ±6.86** 72.93 ±4.45
Values are mean ±SEM. P values were calculated by Mann–Whitney Utest.
*** P <0.001, ** P <0.01 vs. letrozole group.
Table 2
Bone mineral density (BMD) measured by peripheral quantitative computerized tomography.
Control (n =9) Letrozole
(n =15)
Letrozole–EA
(n =8)
Letrozole–propranolol
(n =8)
Letrozole–estradiol
(n =9)
Tibia length (mm) 34.16 ±0.13*** 37.11 ±0.23 36.91 ±0.19 36.90 ±0.24 37.23 ±0.26
Total BMD (mg/cm3) 727.3 ±11.9*** 642.4 ±10.1 641.2 ±12.9 657.5 ±19.1 697.2 ±12.4**
Trabecular BMD (mg/cm3) 418.6 ±17.8*340.6 ±15.5 306.6 ±16.1 345.0 ±26.8 366.6 ±19.4
Cortical BMD (mg/cm3) 1422.2 ±2.9** 1401.1 ±3.4 1413.5 ±3.4*1400.8 ±4.7 1405.1 ±3.9
Values are mean ±SEM. P values were calculated by ANOVA followed by Dunnett’s test (tibia length and trabecular BMD) or Kruskal–Wallis test followed by Mann–
Whitney Utest (total and cortical BMD).
*** P <0.001, ** P <0.01, * P <0.05 vs. letrozole group.
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muscle sympathetic nerve activity in women with PCOS and ovarian
nerve growth factor content in rats with PCO induced by estradiol
valerate is decreased by treatment (Stener-Victorin et al., 2000, 2009).
The opioid system may be an alternative pathway through which
EA modulates reproductive function in women with PCOS and in
animal models of PCOS (Feng et al., 2012; Lanzone et al., 1995).
Of interest, in women with PCOS, acupuncture with manual and
low-frequency electrical stimulation increases the frequency of ovu-
lation and normalizes circulating serum concentrations of ovarian
and adrenal sex steroids. However, LH levels and LH pulsatility are
unaltered (Johansson et al., 2013). These differences could indi-
cate that in women with PCOS, in contrast to rodent models of PCOS,
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
0.5
1.0
1.5
2.0
2.5
Adipor1 (RQ)
**
*
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
0.5
1.0
1.5
2.0
Adipor2 (RQ)
*
*
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
0.5
1.0
1.5
2.0
Foxo3 (RQ)
*
**
**
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
0.4
0.8
1.2
Hif1a (RQ)
*
*
*
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
0.5
1.0
1.5
2.0
2.5
Pik3r1 (RQ)
**
**
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
0.5
1.0
1.5
2.0
Srd5a1 (RQ)
*
**
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
0.5
1.0
1.5
2.0
2.5
Adipoq (RQ)
*
[*]
Adipor1
Adipor2
Adrb2
Amhr2
Ar
Cyp17a1
Cyp19a1
Esr1
Esr2
Foxo3
Hif1a
Mmp2
Ngf
Pik3r1
Srd5a1
Vegfa
0.0
2.0
4.0
6.0
8.0
10.0
100.0
Relative gene expression (RQ)
Control
Letrozole
** * *** *
***
*** *** ** *** ** ** **
**
*
A
CD E
Ovarian mRNA expression
GHF
B
Fig. 3. (A) Gene expression in ovarian tissue in control (placebo) (n =9) and letrozole rats (n =15–16). (B–H) Effect of EA (n =8–9), propranolol (n =8–9), and cyclic estra-
diol (n =8–9) on expression of Adipoq,Adipor1,Adipor2,Foxo3,Hif1a,Pik3r1, and Srd5a1 in ovarian tissue. P values were calculated by ANOVA followed by Dunnett’s test
(Adipoq,Adipor1,Adipor2, and Srd5a1) or Kruskal–Wallis test followed by Mann–Whitney Utest (Foxo3,Hif1a, and Pik3r1). ***P <0.001, **P <0.01, *P <0.05, [*]P<0.06 vs.
letrozole group. Values are mean ±SEM.
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acupuncture is more effective in regulating ovarian function than
neuroendocrine function.
4.4. Ovarian function and folliculogenesis
The upregulation of both isoforms of the estrogen receptor, ERα (Er1)
and ERβ (Er2), in ovarian tissue may favor estrogen signaling in letrozole-
exposed rats. Reduced expression of Pik3r1 and Foxo3 induced by cyclic
estradiol may reflect an improvement in folliculogenesis, as an in-
crease in the activity of PIK3 accelerates early follicular recruitment and
Foxo3 arrests follicular development (Reddy et al., 2008; Yang et al.,
2010). Interestingly, these two features are prominent ovarian changes
in women with PCOS (Franks et al., 2008).
The tendency toward decreased ovarian expression of adiponectin
mRNA and increased expression of adiponectin receptor mRNA in
rats with letrozole-induced PCOS is of interest because these genes
regulate the survival and steroidogenic function of ovarian granu-
losa cells (Pierre et al., 2009). In control rats, ADIPOR2 protein was
not expressed despite expression of Adipor2 mRNA. A similar dis-
crepancy has been reported in preovulatory granulosa, theca cells,
and corporea lutea from porcine ovaries (Maleszka et al., 2014). In-
terestingly, letrozole-exposed rats treated with estradiol exhibited
a strong tendency toward reduced ADIPOR2 protein expression
without changes in Adipor2 mRNA expression (P =0.061). Thus, the
discrepancy between mRNA and protein expression of ADIPOR2 can
be attributed to an effect of estradiol priming on the translation or
degradation of ovarian protein, suggesting that the reduction in
ADIPOR2 protein expression is associated with modifications due
to the estrous cycle.
In granulosa cells, adiponectin activates the ERK1/2 pathway
through ADIPOR1 and ADIPOR2, promoting estrogen synthesis in
the presence of FSH (Chabrolle et al., 2007; Pierre et al., 2009). In-
terestingly, we found that cyclic estradiol reduces phosphorylation
of ERK1/2 and expression of Adipor1, also supporting a potential reg-
ulatory action of estrogen on the ovarian adiponectin system.
The EA-induced increase in the expression of Adipoq mRNA re-
flects an increase in the regulation of the adiponectin system in the
ovaries. Unfortunately, we could not determine the expression of
adiponectin in ovarian samples. Moreover, low-frequency EA de-
creased ovarian expression of Adipor2 mRNA and increased ovarian
expression of ADIPOR2 protein, indicating that acupuncture could
favor translation of the protein. Interestingly, phosphorylated ERK1/2
was also increased in the letrozole–EA rats. The low serum estra-
diol concentration in letrozole-exposed rats treated with low-
frequency EA could explain the changes in the expression of Adipor2
mRNA and ADIPOR2 protein and the phosphorylation of ERK1/2.
42 kDa
42 kDa
44 kDa
Phosphorylated ERK1/2 (Relative density)
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
1.0
2.0
3.0
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
pERK1/2
β-actin
*
*
ADIPOR2 (Relative density)
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
0.0
1.0
2.0
3.0
4.0
42 kDa
44 kDa
Control
Letrozole
Letrozole-EA
Letrozole-propranolol
Letrozole-estradiol
ADIPOR2
β-actin
*
Adiponectin receptor 2 (ADIPOR2) Phosphorylated ERK1/2 (pERK1/2)
AB
Fig. 4. Ovarian expression of ADIPOR2 (A) and phosphorylated ERK1/2 (B) in control (placebo) (n =9), letrozole (n =15), letrozole–EA (n =8), letrozole–propranolol (n =8),
and letrozole–estradiol (n =8) groups. Representative immunoblots of each protein are shown. For ADIPOR2, individual density values were expressed relative to the mean
of letrozole-exposed rats. For phosphorylated ERK1/2, all individual density values in letrozole-exposed, letrozole–EA, letrozole–propranolol, and letrozole–estradiol groups
were expressed relative to the mean of the control. P values were calculated by Kruskal–Wallis test followed by Mann–Whitney Utest. *P <0.05 vs. letrozole group. Values
are mean ±SEM.
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It has been reported that adiponectin reduces androstenedi-
one synthesis in human theca cells (Comim et al., 2013). Low-
frequency EA altered the adiponectin system without causing major
changes in steroidogenesis and ovarian morphology. This might be
because letrozole strongly inhibits aromatase activity, and the dose
and intensity of EA we used cannot fully restore normal ovarian func-
tion. In this regard, we used the same treatment setting that
ameliorated the reproductive and metabolic disturbances in a rat
model of DHT-induced PCOS (Feng et al., 2009, 2012; Johansson et al.,
2010). Moreover, in one study, EA restored ovarian function in rats
with letrozole-induced PCOS; however, EA was given after the last
day of letrozole administration (Sun et al., 2013).
The effect of low-frequency EA on the adiponectin system does
not seem to be mediated by modulation of sympathetic activity, since
blockade of the β-adrenergic receptor did not affect ovarian gene
or protein expression. Interestingly, both low-frequency EA and
β-adrenergic receptor blockade reduced expression of Hif1a. This
hypoxia-induced angiogenic factor is important in the luteiniza-
tion of granulosa cells and in luteogenesis (Tam et al., 2010).
However, in our letrozole-induced PCOS model, no corpora lutea
were observed in any of the treatment groups. Thus, the reduc-
tion in Hif1a mRNA expression by β-adrenergic receptor blockade
might reflect an effect on the increased luteinization of granulosa
cells after letrozole exposure and related to the tendency of
propranalol to increase circulating progesterone levels (Maliqueo
et al., 2013; Manneras et al., 2007).
In conclusion, low-frequency EA normalized circulating levels
of gonadotropins, induced estrous-cycle changes, and stimulated the
ovarian adiponectin system in rats with letrozole-induced PCOS. The
effect of acupuncture in this model is most likely independent of
estrogen or β-adrenergic action (Fig. 5). These findings also show
that androgens are important in the establishment of the repro-
ductive PCOS phenotype induced by letrozole.
Authors’ roles
M.M., A.B., and E.S.-V. designed the study. M.M., A.B., A.A., M.S.,
J.J., and E.S.-V. performed the experiments. M.M., A.B., F.L, C.O., and
E.S.-V. analyzed and interpret the data. M.M. and E.S.-V. wrote the
manuscript. All the authors critically revised and approved the
manuscript.
Acknowledgments
We thank the Center for Physiology and Imaging and the Ge-
nomics Core Facility at the Sahlgrenska Academy, University of
Gothenburg, for the use of technical equipment and support. The
work was supported by the Swedish Medical Research Council
(Project No. 2014-2775); Wilhelm and Martina Lundgrens’s Science
Fund; Hjalmar Svensson Foundation (E. S.-V. and M.M.); Adlerbert
Research Foundation (ALFFGBG-136481); Swedish federal govern-
ment under the LUA/ALF agreement ALFGBG-429501. M.M. thanks
the Becas Chile Programme (Chile) and University of Chile for fi-
nancial support through a postdoctoral fellowship.
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.mce.2015.04.026.
References
Andersen, C.L., Jensen, J.L., Orntoft, T.F., 2004. Normalization of real-time quantitative
reverse transcription-PCR data: a model-based variance estimation approach to
identify genes suited for normalization, applied to bladder and colon cancer data
sets. Cancer Res. 64, 5245–5250.
Asarian, L., Geary, N., 2002. Cyclic estradiol treatment normalizes body weight and
restores physiological patterns of spontaneous feeding and sexual receptivity
in ovariectomized rats. Horm. Behav. 42, 461–471.
Azziz, R., 2003. Androgen excess is the key element in polycystic ovary syndrome.
Fertil. Steril. 80, 252–254.
Banks, P.K., Meyer, K., Brodie, A.M., 1991. Regulation of ovarian steroid biosynthesis
by estrogen during proestrus in the rat. Endocrinology 129, 1295–1304.
Benrick, A., Maliqueo, M., Miao, S., Villanueva, J.A., Feng, Y., Ohlsson, C., et al., 2013.
Resveratrol is not as effective as physical exercise for improving reproductive
and metabolic functions in rats with dihydrotestosterone-induced polycystic ovary
syndrome. Evid. Based Complement. Alternat. Med. 2013, 964070.
Blank, S.K., McCartney, C.R., Helm, K.D., Marshall, J.C., 2007. Neuroendocrine effects
of androgens in adult polycystic ovary syndrome and female puberty. Semin.
Reprod. Med. 25, 352–359.
Bonnet, N., Laroche, N., Vico, L., Dolleans, E., Benhamou, C.L., Courteix, D., 2006. Dose
effects of propranolol on cancellous and cortical bone in ovariectomized adult
rats. J. Pharmacol. Exp. Ther. 318, 1118–1127.
Bonnet, N., Benhamou, C.L., Malaval, L., Goncalves, C., Vico, L., Eder, V., et al., 2008.
Low dose beta-blocker prevents ovariectomy-induced bone loss in rats without
affecting heart functions. J. Cell. Physiol. 217, 819–827.
Borski, R.J., Tsai, W., DeMott-Friberg, R., Barkan, A.L., 1996. Regulation of somatic
growth and the somatotropic axis by gonadal steroids: primary effect on
insulin-like growth factor I gene expression and secretion. Endocrinology 137,
3253–3259.
Chabrolle, C., Tosca, L., Dupont, J., 2007. Regulation of adiponectin and its receptors
in rat ovary by human chorionic gonadotrophin treatment and potential
involvement of adiponectin in granulosa cell steroidogenesis. Reproduction 133,
719–731.
Comim, F.V., Hardy, K., Franks, S., 2013. Adiponectin and its receptors in the ovary:
further evidence for a link between obesity and hyperandrogenism in polycystic
ovary syndrome. PLoS ONE 8, e80416.
Coxam, V., Bowman, B.M., Mecham, M., Roth, C.M., Miller, M.A., Miller, S.C., 1996.
Effects of dihydrotestosterone alone and combined with estrogen on bone mineral
density, bone growth, and formation rates in ovariectomized rats. Bone 19,
107–114.
D’Eon, T.M., Souza, S.C., Aronovitz, M., Obin, M.S., Fried, S.K., Greenberg, A.S., 2005.
Estrogen regulation of adiposity and fuel partitioning. Evidence of genomic and
non-genomic regulation of lipogenic and oxidative pathways. J. Biol. Chem. 280,
35983–35991.
Feng, Y., Lin, H., Zhang, Y., Li, L., Wu, X., Wang, T., et al., 2008. Electroacupuncture
promotes insulin-like growth factors system in ovariectomized osteoporosis rats.
Am. J. Chin. Med. 36, 889–897.
Feng, Y., Johansson, J., Shao, R., Manneras, L., Fernandez-Rodriguez, J., Billig, H., et al.,
2009. Hypothalamic neuroendocrine functions in rats with dihydrotestosterone-
induced polycystic ovary syndrome: effects of low-frequency electro-acupuncture.
PLoS ONE 4, e6638.
Feng, Y., Johansson, J., Shao, R., Manneras-Holm, L., Billig, H., Stener-Victorin, E., 2012.
Electrical and manual acupuncture stimulation affect oestrous cyclicity and
neuroendocrine function in an 5alpha-dihydrotestosterone-induced rat polycystic
ovary syndrome model. Exp. Physiol. 97, 651–662.
Low-frequency
Electroacupunture
Hypothalamus
Ovary
Pituitary
Opioid system?
Estrous cycle
changes
GnRH pulsality?
↓LH and ↑FSH
ADIPOR2 ↑ p-ERK ↑
Sympathetic activity?
Fig. 5. A hypothetical model of how low-frequency EA may affect reproductive
function.
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Please cite this article in press as: Manuel Maliqueo, et al., Circulating gonadotropins and ovarian adiponectin system are modulated by acupuncture independently of sex steroid or
β-adrenergic action in a female hyperandrogenic rat model of polycystic ovary syndrome, Molecular and Cellular Endocrinology (2015), doi: 10.1016/j.mce.2015.04.026
10 M. Maliqueo et al./Molecular and Cellular Endocrinology ■■ (2015) ■■–■■

Figueroa, F., Davicino, R., Micalizzi, B., Oliveros, L., Forneris, M., 2012. Macrophage
secretions modulate the steroidogenesis of polycystic ovary in rats: effect of
testosterone on macrophage pro-inflammatory cytokines. Life Sci. 90, 733–739.
Fitzpatrick, S.L., Richards, J.S., 1991. Regulation of cytochrome P450 aromatase
messenger ribonucleic acid and activity by steroids and gonadotropins in rat
granulosa cells. Endocrinology 129, 1452–1462.
Franks, S., Stark, J., Hardy, K., 2008. Follicle dynamics and anovulation in polycystic
ovary syndrome. Hum. Reprod. Update 14, 367–378.
Goldman, S., Shalev, E., 2004. MMPS and TIMPS in ovarian physiology and
pathophysiology. Front. Biosci. 9, 2474–2483.
Jedel, E., Labrie, F., Oden, A., Holm, G., Nilsson, L., Janson, P.O., et al., 2011. Impact
of electro-acupuncture and physical exercise on hyperandrogenism and oligo/
amenorrhea in women with polycystic ovary syndrome: a randomized controlled
trial. Am. J. Physiol. Endocrinol. Metab. 300, E37–E45.
Johansson, J., Feng, Y., Shao, R., Lonn, M., Billig, H., Stener-Victorin, E., 2010. Intense
electroacupuncture normalizes insulin sensitivity, increases muscle GLUT4
content, and improves lipid profile in a rat model of polycystic ovary syndrome.
Am. J. Physiol. Endocrinol. Metab. 299, E551–E559.
Johansson, J., Redman, L., Veldhuis, P.P., Sazonova, A., Labrie, F., Holm, G., et al., 2013.
Acupuncture for ovulation induction in polycystic ovary syndrome: a randomized
controlled trial. Am. J. Physiol. Endocrinol. Metab. 304, E934–E943.
Jones, M.E., Thorburn, A.W., Britt, K.L., Hewitt, K.N., Wreford, N.G., Proietto, J., et al.,
2000. Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity.
Proc. Natl. Acad. Sci. U.S.A. 97, 12735–12740.
Kassanos, D., Trakakis, E., Baltas, C.S., Papakonstantinou, O., Simeonidis, G., Salamalekis,
G., et al., 2010. Augmentation of cortical bone mineral density in women with
polycystic ovary syndrome: a peripheral quantitative computed tomography
(pQCT) study. Hum. Reprod. 25, 2107–2114.
Katulski, K., Slawek, S., Czyzyk, A., Podfigurna-Stopa, A., Paczkowska, K., Ignaszak,
N., et al., 2014. Bone mineral density in women with polycystic ovary syndrome.
J. Endocrinol. Invest.
Komar, C.M., 2005. Peroxisome proliferator-activated receptors (PPARs) and ovarian
function–implications for regulating steroidogenesis, differentiation, and tissue
remodeling. Reprod. Biol. Endocrinol. 3, 41.
Kraemer, F.B., Tavangar, K., Hoffman, A.R., 1991. Developmental regulation of
hormone-sensitive lipase mRNA in the rat: changes in steroidogenic tissues.
J. Lipid Res. 32, 1303–1310.
Lanzone, A., Fulghesu, A.M., Cucinelli, F., Ciampelli, M., Caruso, A., Mancuso, S., 1995.
Evidence of a distinct derangement of opioid tone in hyperinsulinemic patients
with polycystic ovarian syndrome: relationship with insulin and luteinizing
hormone secretion. J. Clin. Endocrinol. Metab. 80, 3501–3506.
Lara, H.E., Ferruz, J.L., Luza, S., Bustamante, D.A., Borges, Y., Ojeda, S.R., 1993. Activation
of ovarian sympathetic nerves in polycystic ovary syndrome. Endocrinology 133,
2690–2695.
Ledoux, S., Campos, D.B., Lopes, F.L., Dobias-Goff, M., Palin, M.F., Murphy, B.D., 2006.
Adiponectin induces periovulatory changes in ovarian follicular cells.
Endocrinology 147, 5178–5186.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using
real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408.
Lund, I., Lundeberg, T., 2006. Are minimal, superficial or sham acupuncture procedures
acceptable as inert placebo controls? Acupunct. Med. 24, 13–15.
Maleszka, A., Smolinska, N., Nitkiewicz, A., Kiezun, M., Dobrzyn, K., Czerwinska, J.,
et al., 2014. Expression of adiponectin receptors 1 and 2 in the ovary and
concentration of plasma adiponectin during the oestrous cycle of the pig. Acta
Vet. Hung. 62, 386–396.
Maliqueo, M., Sun, M., Johansson, J., Benrick, A., Labrie, F., Svensson, H., et al., 2013.
Continuous administration of a P450 aromatase inhibitor induces polycystic ovary
syndrome with a metabolic and endocrine phenotype in female rats at adult age.
Endocrinology 154, 434–445.
Maliqueo, M., Benrick, A., Stener-Victorin, E., 2014. Rodent models of polycystic ovary
syndrome: phenotypic presentation, pathophysiology, and the effects of different
interventions. Semin. Reprod. Med. 32, 183–193.
Manneras, L., Cajander, S., Holmang, A., Seleskovic, Z., Lystig, T., Lonn, M., et al., 2007.
A new rat model exhibiting both ovarian and metabolic characteristics of
polycystic ovary syndrome. Endocrinology 148, 3781–3791.
Manneras, L., Cajander, S., Lonn, M., Stener-Victorin, E., 2009. Acupuncture and exercise
restore adipose tissue expression of sympathetic markers and improve ovarian
morphology in rats with dihydrotestosterone-induced PCOS. Am. J. Physiol. Regul.
Integr. Comp. Physiol. 296, R1124–R1131.
Marcondes, F.K., Bianchi, F.J., Tanno, A.P., 2002. Determination of the estrous cycle
phases of rats: some helpful considerations. Braz. J. Biol. 62, 609–614.
Ortega, I., Sokalska, A., Villanueva, J.A., Cress, A.B., Wong, D.H., Stener-Victorin, E.,
et al., 2013. Letrozole increases ovarian growth and Cyp17a1 gene expression
in the rat ovary. Fertil. Steril. 99, 889–896.
Pierre, P., Froment, P., Negre, D., Rame, C., Barateau, V., Chabrolle, C., et al., 2009. Role
of adiponectin receptors, AdipoR1 and AdipoR2, in the steroidogenesis of the
human granulosa tumor cell line, KGN. Hum. Reprod. 24, 2890–2901.
Reddy, P., Liu, L., Adhikari, D., Jagarlamudi, K., Rajareddy, S., Shen, Y., et al., 2008.
Oocyte-specific deletion of Pten causes premature activation of the primordial
follicle pool. Science 319, 611–613.
Song, D., Arikawa, E., Galipeau, D.M., Yeh, J.N., Battell, M.L., Yuen, V.G., et al.,
2005. Chronic estrogen treatment modifies insulin-induced insulin resistance
and hypertension in ovariectomized rats. Am. J. Hypertens. 18, 1189–
1194.
Sproul, K., Jones, M.R., Mathur, R., Azziz, R., Goodarzi, M.O., 2010. Association study
of four key folliculogenesis genes in polycystic ovary syndrome. BJOG 117,
756–760.
Stener-Victorin, E., Waldenstrom, U., Tagnfors, U., Lundeberg, T., Lindstedt, G.,
Janson, P.O., 2000. Effects of electro-acupuncture on anovulation in women
with polycystic ovary syndrome. Acta Obstet. Gynecol. Scand. 79, 180–
188.
Stener-Victorin, E., Lundeberg, T., Waldenstrom, U., Manni, L., Aloe, L., Gunnarsson,
S., et al., 2000. Effects of electro-acupuncture on nerve growth factor and ovarian
morphology in rats with experimentally induced polycystic ovaries. Biol. Reprod.
63, 1497–1503.
Stener-Victorin, E., Jedel, E., Janson, P.O., Sverrisdottir, Y.B., 2009. Low-frequency
electroacupuncture and physical exercise decrease high muscle sympathetic nerve
activity in polycystic ovary syndrome. Am. J. Physiol. Regul. Integr. Comp. Physiol.
297, R387–R395.
Stener-Victorin, E., Holm, G., Labrie, F., Nilsson, L., Janson, P.O., Ohlsson, C., 2010. Are
there any sensitive and specific sex steroid markers for polycystic ovary
syndrome? J. Clin. Endocrinol. Metab. 95, 810–819.
Stener-Victorin, E., Holm, G., Janson, P.O., Gustafson, D., Waern, M., 2013. Acupuncture
and physical exercise for affective symptoms and health-related quality of life
in polycystic ovary syndrome: secondary analysis from a randomized controlled
trial. BMC Complement. Altern. Med. 13, 131.
Sun, J., Jin, C., Wu, H., Zhao, J., Cui, Y., Liu, H., et al., 2013. Effects of electro-acupuncture
on ovarian P450arom, P450c17alpha and mRNA expression induced by letrozole
in PCOS rats. PLoS ONE 8, e79382.
Sverrisdottir, Y.B., Mogren, T., Kataoka, J., Janson, P.O., Stener-Victorin, E., 2008. Is
polycystic ovary syndrome associated with high sympathetic nerve activity and
size at birth? Am. J. Physiol. Endocrinol. Metab. 294, E576–E581.
Tam, K.K., Russell, D.L., Peet, D.J., Bracken, C.P., Rodgers, R.J., Thompson, J.G., et al.,
2010. Hormonally regulated follicle differentiation and luteinization in the mouse
is associated with hypoxia inducible factor activity. Mol. Cell. Endocrinol. 327,
47–55.
Wu, M.T., Sheen, J.M., Chuang, K.H., Yang, P., Chin, S.L., Tsai, C.Y., et al., 2002. Neuronal
specificity of acupuncture response: a fMRI study with electroacupuncture.
Neuroimage 16, 1028–1037.
Wu, S., Divall, S., Nwaopara, A., Radovick, S., Wondisford, F., Ko, C., et al., 2014.
Obesity-induced infertility and hyperandrogenism are corrected by deletion of
the insulin receptor in the ovarian theca cell. Diabetes 63, 1270–1282.
Yang, J.L., Zhang, C.P., Li, L., Huang, L., Ji, S.Y., Lu, C.L., et al., 2010. Testosterone induces
redistribution of forkhead box-3a and down-regulation of growth and
differentiation factor 9 messenger ribonucleic acid expression at early stage of
mouse folliculogenesis. Endocrinology 151, 774–782.
Zhao, H., Tian, Z., Cheng, L., Chen, B., 2004. Electroacupuncture enhances extragonadal
aromatization in ovariectomized rats. Reprod. Biol. Endocrinol. 2, 18.
Zhao, H., Tian, Z.Z., Chen, B.Y., 2005. Electroacupuncture stimulates hypothalamic
aromatization. Brain Res. 1037, 164–170.
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Please cite this article in press as: Manuel Maliqueo, et al., Circulating gonadotropins and ovarian adiponectin system are modulated by acupuncture independently of sex steroid or
β-adrenergic action in a female hyperandrogenic rat model of polycystic ovary syndrome, Molecular and Cellular Endocrinology (2015), doi: 10.1016/j.mce.2015.04.026
11M. Maliqueo et al./Molecular and Cellular Endocrinology ■■ (2015) ■■–■■