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ORIGINAL RESEARCH
In human granulosa cells from small
antral follicles, androgen receptor
mRNA and androgen levels in
follicular fluid correlate with FSH
receptor mRNA
M.E. Nielsen1, I.A. Rasmussen1, S.G. Kristensen2, S.T. Christensen3,
K. Møllga
˚rd4, E. Wreford Andersen 5, A.G. Byskov2, and
C. Yding Andersen2,*
1
The Fertility Clinic, Odense University Hospital, Odense, Denmark
2
Laboratory of Reproductive Biology, Section 5712, The Juliane Marie
Centre for Women, Children and Reproduction, University Hospital of Copenhagen, University of Copenhagen, Blegdamsvej 9,
Rigshospitalet, Copenhagen DK-2100, Denmark
3
Department of Cell and Developmental Biology, August Krogh Institute, University of
Copenhagen, Copenhagen, Denmark
4
Department of Cellular and Molecular Medicine, The Panum Institute, University of Copenhagen,
Copenhagen, Denmark
5
Department of Biostatistics, University of Copenhagen, Copenhagen, Denmark
*Correspondence address: Tel: +45-35455822; Fax: +45-35455824; E-mail: yding@rh.dk
Submitted on April 17, 2010; resubmitted on August 24, 2010; accepted on August 26, 2010
abstract: Human small antral follicles (diameter 3– 9 mm) were obtained from ovaries surgically removed for fertility preservation.
From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were
available for mRNA analysis (24 women). Expressions of androgen receptor (AR) mRNA levels in granulosa cells, and of androstenedione
and testosterone in follicular fluid, were correlated to the expression of the FSH receptor (FSHR), LH receptor (LHR), CYP19 and anti-
Mu
¨llerian Hormone-receptor II (AMHRII) mRNA in the granulosa cells and to the follicular fluid concentrations of AMH, inhibin-B,
progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly
significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated significantly
with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate
association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and
between the follicular fluid levels of androgen and FSHR expression. This suggests that follicular sensitivity towards FSH stimulation may
be augmented by stimulation of androgens via the AR.
Key words: AR / FSHR / LHR mRNA expression / steroids / small human antral follicles
Introduction
During folliculogenesis androgens serve an important function as sub-
strate for granulosa cell estradiol synthesis, and perform, in addition, a
number of trophic functions during follicular growth, especially in
earlier developmental stages prior to follicular selection. Studies in
rhesus monkeys almost a decade ago showed that androgens, adminis-
tered in pellets that provide slow release for 3–10 days, stimulate
growth in follicles until the early antral stage (Vendola et al., 1998,
1999a,b). Proliferation of granulosa cells was significantly increased
and the number of growing follicles augmented after exposure to tes-
tosterone or dihydrotestosterone treatment (Vendola et al., 1998,
1999a,b). Furthermore, androgen receptor (AR) gene expression in
granulosa cells from these primate ovaries, evaluated by in situ hybri-
dazation, correlated positively with proliferation and negatively with
apoptosis (Weil et al., 1998).
Subsequent studies demonstrated interactions between FSH and
androgens in the follicular development and revealed that follicles
demonstrate a highly significant positive correlation between the
FSH receptor (FSHR) and AR mRNA levels when comparing the
mean expression from each group of follicles in each individual
monkey (Weil et al., 1999). Furthermore, testosterone administration
that resulted in supra-physiological levels augmented granulosa
cell FSHR expression and it was suggested that androgens promote
&The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Molecular Human Reproduction, Vol.17, No.1 pp. 63– 70, 2011
Advanced Access publication on September 14, 2010 doi:10.1093/molehr/gaq073
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follicular growth by sensitizing granulosa cells to FSH action (Weil
et al., 1999).
In the ovary, the AR seemed predominantly to be expressed by the
granulosa cells and in particular by the smaller follicles (rats: Harlow
et al., 1986;Tetsuka and Hillier, 1997; marmoset: Hillier et al.,
1997). However, in humans and sheep, the pattern of AR expression
in granulosa cells appears to be regulated differently from that in rat
and monkey, since granulosa cell AR immunostaining was reported
to be stronger in pre-ovulatory follicles than in smaller follicles
(human: Horie et al., 1992, sheep: Campo et al., 1985). A recent
study in mouse that used granulosa cells and oocyte-specific AR
knockout mice found that such mice had premature ovarian failure
and were subfertile, with longer oestrous cycles and fewer ovulated
oocytes (Sen and Hammes, 2010). It was concluded that the AR
appears to promote pre-antral follicle growth and prevent follicular
atresia and are essential for normal follicular development and fertility
(Sen and Hammes, 2010).
At the clinical level, the above observations may explain the
response women with hyper-androgenism often show when treated
with exogenous FSH administration for ovulation induction. These
women may react with an exaggerated follicular response, suggesting
that their granulosa cells are especially sensitive to FSH stimulation
(Farhi and Jacobs, 1997;Thessaloniki ESHRE/ASRM-Sponsored
PCOS Consensus Workshop Group, 2008). Indeed, a number of
studies have shown that granulosa cells from PCOS women during
culture are hyper-responsive to FSH and possess an augmented
capacity for synthesizing estrogens (Mason et al., 1994). In a group
of women who showed a moderate or poor response to ovarian
stimulation with exogenous gonadotrophins the use of locally pro-
duced androgens to augment sensitivity to FSH stimulation and to
increase the number of retrieved mature oocytes has recently been
attempted (Løssl et al., 2006). Prior to controlled ovarian stimulation
with exogenous gonadotrophins local ovarian androgen production
was enhanced by pre-treating women with a bolus of hCG, thereby
stimulating local androgen production and by co-administering an
aromatase inhibitor to prevent the conversion of androgens to estro-
gens (Løssl et al., 2006). Compared with a control group the androgen
pretreatment group developed significantly more embryos following
ovarian stimulation and IVF treatment, supporting the concept that
androgens may also enhance follicular responsiveness and FSHR
expression in humans. However, since the AR appears to be mainly
expressed in pre-ovulatory follicles in women (Horie et al., 1992), it
is unclear at what developmental stage FSH sensitivity is enhanced
and whether it is feasible to augment follicular responsiveness to
smaller follicles.
Human small antral follicles are scarce material and not many
studies have been performed on such individual small follicles. In the
present study, we took advantage of the presence of normal small
antral follicles in ovaries surgically removed for fertility preservation
by cryopreservation of the ovarian cortex. The antral follicles
located in the medually part cannot sustain freezing and are normally
discarded in this procedure. In order to characterize human antral fol-
licles and evaluate whether AR expression played similar roles in
humans as found in primates and other species, the aim of the
present study was to determine AR mRNA expression in granulosa
cells of normal small human antral follicles and to correlate that
with the mRNA expression of FSHR, LH receptor (LHR), AMH
receptor II (AMHRII) and aromatase (CYP19). In addition, the hormo-
nal characteristics of the corresponding follicular fluid were also
evaluated.
Materials and Methods
Patients and collection of small antral
follicular fluid
Granulosa cell and follicular fluid samples were obtained from individual
small antral follicles present in ovaries surgically removed for fertility pres-
ervation. Cryopreservation of the ovarian cortex was offered as means of
fertility preservation to patients in whom the appropriate treatment posed
a high risk of eliminating the ovarian follicle pool.
A total of 64 follicles were obtained from 24 women aged 15– 37 years
(median 29 years) at various times during their menstrual cycle. Diagnosis
for ovarian cryopreservation included mammary cancer (9), Hodgkin’s and
non-Hodgkin’s disease (5), Ewing and oestogenic sarcoma (3), lymphoma
(2), rectum cancer (1), cervical cancer (2), medulla blastoma (1) and der-
matomyositis (1). None of the women received GnRH agonists and two
women received oral contraception. All ovaries appeared normal by (i)
visual inspection in connection with the cryopreservation procedure and
(ii) by subsequent microscopic evaluation of histological sections from a
small piece of the ovarian cortex.
The follicles were collected immediately after recovery of the ovary
prior to, or during, isolation of the ovarian cortex. Each antral follicle
visible on the surface of the ovary or observed during preparation of
cortex was aspirated with a 1 ml syringe with a 26-gauge needle
(Becton Dickinson, Brøndby, Denmark). The granulosa cells of each
small follicle were isolated from the follicular fluid by centrifugation
(2000g, 2 min). After aspirating the follicular fluid from the granulosa
cells in the pellet, each ampoule of granulosa cells and follicular fluid
were snap-frozen in liquid nitrogen and stored at – 808C until RNA puri-
fication or until measurement of hormones.
One to eight antral follicles were collected per patient (one follicular
fluid, five patients; two follicular fluids, eight patients; three follicular fluids,
seven patients; four follicular fluids, two patients; six follicular fluids, one
patient; eight follicular fluids, one patient). The volume of each follicle
was estimated from the volume in the syringe and only those with a
volume from 30 to 350 ml corresponding to 3 –9 mM in diameter
were included in the study. The ethical committee of the municipalities
of Copenhagen and Frederiksberg approved the project. Results from
this material have been used in a previous publication (Nielsen et al.,
2010).
RNA purification
Total RNA was purified under RNase-free conditions at room tempera-
ture using Tri Reagent (Sigma-Aldrich, St Louis, MO, USA) in combination
with the RNAeasy Mini Kit (Qiagen, Hilden, Germany). Each cell sample
was lysed in 1 ml Tri Reagent according to the Tri Reagent protocol and
incubated at room temperature for 5 min. Following addition of 200 ml
1-bromo-3-chloropropane (Sigma-Aldrich), the cell sample solutions
were shaken vigorously for 15 s and incubated at room temperature for
5 min. Following centrifugation at 15 000gfor 15 min at 48C, the
mixture separated into three phases and the upper aqueous phase con-
taining the RNA was transferred to a clean, RNase-free Eppendorf tube.
The following steps of RNA purification was carried out according to
the RNeasy Mini Kit protocol. The final elution step was repeated and
the purified total RNA was subsequently stored at 2608C.
The samples were analysed for total RNA quality and level of degra-
dation using an Agilent 2100 Bioanalyzer and RNA 6000 Pico LabChip
64 Nielsen et al.
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according to the manufacturer’s instructions (RNA 6000 Pico assay kit,
Agilent Technologies, Waldbronn, Germany). RNA samples which did
not show two peaks representing 18 and 28 s rRNA were excluded.
Total RNA quantity was evaluated using the Nanodrop
w
ND1000 Spec-
trophotometer (NanoDrop Technologies, Wilmington, DE, USA).
DNA synthesis and RT–PCR analysis
First-strand cDNA was synthesized using the High-Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) according to the
manufacturer’s manual. A master mix containing 2.0 ml10×RT Buffer,
0.8 ml25×dNTP Mix (100 mM), 2.0 mlof10×RT random primers,
1.0 ml of MultiScribeTM Reverse Transcriptase (50 U/ml), 1.0 mlofRNase
Inhibitor and 3.2 ml of nuclease-free (Diethylpyrocarbonate, DEPC) water
was prepared and for each 20 ml of reaction 10 ml of master mixture was
added to an equal volume of total RNA (concentrations ranging from 5
to 10 ng/ml). All steps were performed on ice. Samples were centrifuged
briefly at 12 000gand then incubated at room temperature for 10 min, fol-
lowed by 378C for 2 h and finally, the reaction was stopped at 858Cfor5s.
First-strand cDNA was stored at 2208C until real-time PCR analysis.
Evaluation of gene expression levels was achieved by relative quantifi-
cation (RQ) real-time PCR analysis using TaqMan
w
technology (Applied
Biosystems). FSHR, LHR, CYP19a1 and AMH-r2 TaqMan
w
Gene Expres-
sion Assays (pre-designed) as well as the Endogenous Control Assays
for human b-actin and glyceraldehyde 3-phosphatdehydrogenase
(GAPDH) were purchased from Applied Biosystems (Assay id-no.:
b-actin:#4326315E; GAPDH:#4333764F; AR:#Hs00171172_m1 FSHR:#
Hs00174865_m1; LHR:#Hs00174885_m1; AMHRII:#Hs00179718_m1;
CYP19a1:#Hs00903413_m1). Sample triplicates were prepared according
to the manufacturer’s instructions. A total reaction volume of 25 mlwas
prepared on ice containing 12.5 ml TaqMan
w
Universal PCR Master Mix,
No AmpErase
w
UNG(2X), 1.25 ml20XTaqMan
w
Gene Expression
Assay Mix, 1.7 –1.85 ml undiluted cDNA and RNase-free water up to
25 ml. The samples were then centrifuged at 2800gat 48Cfor5min.
cDNA was amplified using the 7500 Real-time PCR system (Applied
Biosystems) under the following thermal cycling conditions: 958C for
10 min, 45 cycles of 958C for 15 s and 608C for 1 min. The data were sub-
sequently analysed using human b-actin and GAPDH as housekeeping
genes and the SDS Software for Relative Quantification (Applied Biosys-
tems). The calculation of the expression levels of each individual gene
was carried out according to the Comparative CTMethod for RQ as
described by the manufacturer (Relative Quantification of Gene
Expression: User Bulletin #2, Applied Biosystems). The expression levels
were related to the housekeeping genes human b-actin and GAPDH.
The expression level for each gene was calculated according to human
b-actin and GAPDH separately, and the DDC
T
values were compared. If
no deviation was observed, the housekeeping gene expression was con-
sidered to be constant. As another test for variation in housekeeping
gene expression, the ratios between the RQ values for the five target
genes calculated according to b-actin or GAPDH, were compared. On
the basis of these two tests, the housekeeping genes were considered
to remain sufficient constantly expressed. Often, expression levels are
expressed as 2−DDCTwhen compared with the expression of a calibrator
or another target gene. Normalized expression levels can also be
expressed as 2−DDCT, for example, when expression levels are not
related to a calibrator. The expression levels in this study are therefore
presented as 2−DCTand normalized to GAPDH (Applied Biosystems
Support; Schmittgen and Livak, 2008).
Hormone measurements
Estradiol and progesterone were measured using commercially available
RIA kits (DSL-43100 & DSL-3400; Diagnostic System Laboratories,
Webster, TX, USA). Samples for both assays were diluted 1:50 in steroid-
free serum just prior to measurement. Androstenedione was measured
using an RIA kit (DSL-3800) with samples being diluted 1:200 in steroid-
free serum and testosterone using an RIA kit (DSL-4000) diluted 1:100
in steroid-free serum.
The steroid-free serum was produced in-house by stripping a pool of
serum with repeated washings of charcoal particles. The charcoal was
removed through several filtering steps ending with a sterile filter to
ensure total removal of charcoal particles. Blank samples were tested
for the absence of steroids prior to the running hormone assays.
AMH was measured using a specific ELISA-kit according to the manufac-
turer’s instructions (DSL-10-14400; Diagnostic System Laboratories). Fol-
licular fluid samples from small antral follicles were diluted either 1:500 or
1:3000 in the zero standard provided by the manufacturer. Inter-assay
variation of a sample containing 7.6 ng AMH/ml was 4.4 % (n¼12) and
intra-assay variation was 3.3% (n¼5) of sample containing 0.45 ng/ml.
Dilution curves of follicular fluid samples proved parallel to the standard
curve.
Inhibin-B was measured using a specific ELISA-kit according the manu-
facturer’s instructions (The Oxford Bio-innovation kit; Biotech-IgG,
Copenhagen, Denmark). Prior to measurement, all follicular fluid
samples irrespective of whether they derived from small antral or pre-
ovulatory follicles were diluted 1:100 or 1:500 in serum obtained from a
pool of five post-menopausal women (who had no inhibin-B activity).
The follicular fluids were pre-treated with SDS, heated, and exposed to
hydrogen peroxide before they were applied to the wells of the plate
and incubated overnight at room temperature. Subsequently, the plates
were washed and incubated with detection antibody for 3 h at room
temperature. Substrate solution was applied and incubated for 1 h. The
amplifier solution was added, and the plates were read at 490 nm
with an ELISA reader with its reference at 620 nm (coefficient of
variation ,7%).
SDS–polyacrylamide gel electrophoresis and
western blot analysis
Cell lysates were prepared by using 0.5% sulphSDS lysis buffer. Lysates
were sonicated and centrifuged at 20 000gto precipitate debris. Protein
concentrations were compared with a BCA protein standard (Pierce Bio-
technology, Rockford, IL, USA) and measured with a ‘ Protein assay
(BioRAD Laboratories, CA, USA, DC based on Lowry’s method) ensuring
that equal amounts of protein could be loaded in each lane. Proteins were
analysed by SDS–polyacrylamide gel electrophoresis (PAGE) and western
blot analysis using the XCell and XCell II blot module systems (Novex) as
described (Christensen et al., 2001). Briefly, proteins were separated by
gel electrophoresis under denaturing and reducing conditions on 10%
NuPAGE Bis–Tris gels (NP0303BOX, Invitrogen, Taastrup, Denmark)
using NuPAGE MOPS SDS running buffer (NP0001, Invitrogen) and Fer-
mentas protein standards. Separated proteins were electrophoretically
transferred to nitrocellulose membranes (LC2000, Invitrogen), which
were stained in 1% Ponceau S red solution (P7170, Sigma-Aldrich, Copen-
hagen, Denmark), and incubated for 1 h at room temperature in blocking
buffer [5% non-fat dry milk in TBST (0.01 M Tris–HCl, 0.15 M NaCl, 0.1%
Tween 20, pH 7.4)] before incubation with primary antibodies in blocking
buffer overnight at 48C. The antibodies used were rabbit polyclonal
anti-AR (N-20) (1:200; Santa Cruz Biotechnology, CA, USA), rabbit poly-
clonal anti-AR (N-20) blocking peptide (40:1; Santa Cruz Biotechnology)
and mouse monoclonal anti-b-actin (1:5000; Sigma-Aldrich). The
membranes were washed several times in TBST, incubated for 1 h with
alkaline phosphatase-coupled secondary antibodies diluted 1:5000 in
blocking buffer, and washed in TBST prior to development with
S-bromo-D-chloro-3-indolyl phosphate/nitro blue tetrazolium solution
Androgen receptor and FSHR in human follicles 65
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(KLP, Gaithersburg, MD, USA). The developed blots were scanned and
processed for publication in Adobe Photoshop version 6.
Immunohistochemistry
For immunohistochemistry ovarian medulla tissue containing an antral fol-
licle was dissected free and fixed in Bouin’s fixative. The tissue was pre-
pared for histology. Five micrometre paraffin sections were dewaxed,
rehydrated and washed in TBS (0.05 M Tris, pH 7.6, 0.15 M NaCl) with
0.01% Nonidet P-40 (TBS/Nonidet). Sections were incubated in 0.45%
H
2
O
2
in TBS/Nonidet for 15 min to block endogenous peroxidase
activity, and then in 10% normal goat serum in TBS/Nonidet for 30 min
at room temperature to block non-specific binding. All sections were incu-
bated overnight at 48C with the primary antibody raised against the human
AR (#SC-816, Santa Cruz Biotechnology) diluted 1:50 in 10% goat serum.
The primary antibody was detected using DakoCytomation EnVision+
DualLink System, Peroxidase (DAB+) code K4065, following the manu-
facturer’s instructions. As a negative control, sections were incubated
with Rabbit IgG (X0903 DakoCytomation) instead of the primary anti-
body. As a positive control, sections from fetal testes tissues with
known occurrence of the antigen were used.
Statistics
For evaluation of a possible association between the levels of the
measured substances, normal random effects models were used with a
random effect of women to take into account that results from the
same women may be more similar than results from different women.
AR mRNA expression was grouped into four approximately equally
sized groups (,20, 20 –45, .45 – 130, .130) and similarly testosterone
concentration was grouped (,45, 45–210, .210 – 350, .350 nmol/l).
The outcomes were log-transformed to approximate normality leading
to geometric means being calculated in each group on the original scale.
Likelihood ratio tests were conducted for equality over groups as well
as trend tests. P-value of ,0.05 was accepted as statistical significance.
Results
The quality of the extracted total RNA allowed evaluation of the ana-
lysed genes in 55 of the 64 samples. In the remaining nine samples the
concentration of the extracted total RNA was too low or not of
appropriate quality for quantification of the gene expression levels.
Table Ishows the mRNA expression levels of AR divided into four
groups of approximately equal size in relation to mRNA expression of
FSHR, LHR, CYP19 (aromatase) and AMHRII expression. AR mRNA
expression was highly significantly associated with the FSHR
expression (Table II). A significant linear trend was shown between
the mRNA expression of AR and FSHR (P,0.0001; Fig. 1). In
addition, AMHRII mRNA expression was also significantly associated
with the AR mRNA expression (Fig. 1). In contrast, LHR and
CYP19 mRNA expression did not show any significant associations
with AR mRNA expression (Table I).
The correlation between mRNA levels of AR in granulosa cells and
the corresponding follicular fluid levels of hormones showed no signifi-
cant associations (Table II).
.......................................................................................................
.............................................................................................................................................................................................
Table II AR mRNA expression in granulosa cells of small antral follicles in relation to the hormone concentration of the
corresponding follicular fluid.
Androgen receptor mRNA expression [geometric mean (95% CI)] Overall test Test for trend
<20 20–45 >45–130 >130
No. of follicular fluid 11 16 14 14
AMH (ng/ml) 399 (226; 701) 394 (243; 639) 813 (508; 1300) 525 (312; 881) 0.0967 0.1903
Inhibin-B (ng/ml) 58 (34; 99) 59 (38; 94) 62 (40; 97) 52 (32; 86) 0.9346 0.7683
Estradiol (nmol/l) 26 (10; 69) 27 (12; 61) 15 (7; 34) 22 (9; 53) 0.7381 0.5734
Progesterone (nmol/l) 245 (157; 382) 281 (192; 411) 191 (132; 276) 309 (206; 464) 0.2378 0.7244
Androstenedione (nmol/l) 1860 (1336; 2589) 2785 (2091; 3710) 2618 (1984; 3456) 2593 (1894; 3551) 0.0977 0.1904
Testosterone (nmol/l) 139 (93; 207) 189 (133; 268) 178 (126; 252) 226 (155; 329) 0.1494 0.0587
......................................................................................................
.............................................................................................................................................................................................
Table I Estimated level of mRNA expression of FSHR, CYP19, LHR and AMHRII in relation to AR mRNA expression in
granulosa cells of small antral follicles as geometric mean and 95% confidence interval, with a random effect of woman.
Androgen receptor mRNA expression [geometric mean (95% CI)] Overall test Test for trend
<20 20–45 >45–130 >130
No. of follicular fluid 11 16 14 14
FSHR 8 (3; 19) 20 (9; 42) 76 (37; 157) 107 (48; 237) ,0.0001 ,0.0001
CYP19 9 (3; 24) 10 (4; 24) 10 (4; 23) 18 (7; 47) 0.6632 0.2999
LHR 0.33 (0.14; 0.78) 0.21 (0.10; 0.43) 0.53 (0.26; 1.07) 0.76 (0.34; 1.70) 0.1011 0.0485
AMHRII 3.1 (1.6; 5.7) 10.2 (6.1; 17.1) 10.7 (6.4; 17.9) 19.6 (11.3; 33.9) 0.0003 0.0001
Test for same level in all four groups and test for trend over groups.
66 Nielsen et al.
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The diameter of the majority of the follicles was present in a relative
narrow range of around 4–7 mm (estimated from the aspirated
volume) and the AR mRNA expression did not show any significant
correlation to the follicular volume (data not shown).
Table III shows the follicular fluid concentrations of testosterone
divided into four groups of approximately equal size in relation to
the mRNA expression of FSHR, LHR, CYP19 (aromatase) and
AMHRII expression. Intrafollicular concentrations of testosterone
were highly significantly associated with FSHR mRNA expression in
the corresponding granulosa cells, whereas LHR, CYP19 (aromatase)
and AMHRII expression showed no significant associations. As
expected, follicular fluid concentrations of testosterone showed a
highly significant association with androstenedione, but no significant
correlation to levels of inhibin-B.
Intrafollicular concentrations of androstenedione did not show
significant correlations to mRNA expression levels for any of the
evaluated substances except for the FSHR mRNA expression, which
proved to be significant (P,0.05; data not shown).
In patients in whom granulosa cells from more than one follicle were
included, the FSHR mRNA levels were mostly seen to vary as much as
between patients (Fig. 2). Within patient variation reached one order
of magnitude with the remaining variability being related to between
patient variation (Fig. 2). The other granulosa cell measured mRNAs
showed a range similar to that of FSHR.
Figure 3shows a representative result of western blots of granulosa
cells from small human antral follicles and demonstrated that the AR
protein itself was expressed. A similar result was obtained with mul-
tiple samples. Expression levels of AR were higher in granulosa cells
from antral follicles than in medulla tissue from human ovaries. As a
control an extract from undifferentiated human embryonic stem
cells showed no expression. The western blot result is supported by
immunohistochemical staining of histological sections of a human
antral follicle, which showed expression of the AR protein mainly on
the granulosa cells but also some staining on the theca cells.
Discussion
The present study comprises material from human antral follicles with
diameters from 3 to 9 mm obtained from normal ovaries. The data
showed that granulosa cell FSHR gene expression was significantly
associated with the expression of AR on the granulosa cells, and, in
addition, significantly associated with the intrafollicular concentrations
of androgens (i.e. androstenedione and testosterone). This suggests
that the collective effect of androgens acting through the AR
appears to promote gonadotrophins-responsiveness of developing
human follicles. Although the present results should be interpreted
with caution due to their correlative nature, they are in accordance
with a number of previous studies on primates and rodents.
Further, ovaries from rhesus monkeys showed that the AR gene
was most abundantly expressed on granulosa cells in the ovary and
augmented in healthy follicles. It was suggested that androgens stimu-
late early primate follicle development (Weil et al., 1998). Subsequent
Figure 1 Correlation between mRNA expression of the FSHR and
the AR and between FSHR and AMHRII in granulosa cells from
normal small human antral follicles.
.......................................................................................................
.............................................................................................................................................................................................
Table III Follicular fluid concentrations of testosterone in relation to mRNA expression of FSHR, LHR, CYP19 and
AMHRII genes in the corresponding granulosa cells and to other selected follicular fluid hormones.
Testosterone concentration (nmol/l) [geometric mean (95% CI)] Overall test Test for trend
<145 145–210 211 –350 >350
No. of follicular fluid 15 13 14 13
FSHR 24 (11; 49) 13 (6; 29) 98 (46; 211) 105 (44; 250) 0.0030 0.0052
LHR 0.5 (0.2; 1.1) 0.5 (0.2; 1.2) 0.4 (0.2; 0.8) 0.4 (0.2; 0.9) 0.8814 0.5484
CYP19 10 (4; 26) 14 (6; 37) 11 (4; 28) 11 (4; 32) 0.9465 0.9078
AMHRII 6 (3; 12) 16 (8; 31) 11 (5; 21) 9 (4; 20) 0.1393 0.4312
Androstenedione (nmol/l) 1541 (1206; 1970) 2262 (1762; 2904) 3418 (2664; 4385) 3509 (2643; 4660) ,0.0001 ,0.0001
Inhibin-B (ng/ml) 53 (33; 84) 48 (30; 78) 67 (41; 107) 84 (49; 143) 0.3081 0.1048
Androgen receptor and FSHR in human follicles 67
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studies found that exogenously administered androgens promote
FSHR expression in vivo explaining earlier studies on the androgen-
induced enhanced estrogen biosynthesis and partly explained the
amplifying effect that androgens exert on FSH action (Weil et al.,
1999). Finally, a recent study on conditional granulosa cells and
oocyte-specific AR knockout mice saw that increasing androgen and
AR gene expression by small follicles was associated with increased
FSHR expression, which adds to the perception that androgens via
their receptors may stimulate pre-antral follicle growth, prevent
atresia and are essential for normal follicular development (Sen and
Hammes, 2010).
Taken together, the present results confirm and extends previous
studies and suggest that androgens also in humans exert important
functions in small antral follicles and indicate that proper androgen
priming of such follicles enhance FSHR expression and make them
more sensitive to FSH stimulation and subsequent growth.
Previous studies have shown that androgens enhance granulosa cells
expression of FSHR in vitro resulting in an increased aromatase
Figure 2 Concentration of FSHR mRNA in individual granulosa cell samples from small antral follicles given for each individual patient.
Figure 3 AR expression in granulosa cells from small human antral follicles. (A) Western blot analysis of AR (N-20, 110 kDa), blocking peptide
control (BP, 40×of Ab concentration) and b-actin (43 kDa) reactivity on human ovarian medulla tissue, granulosa cells from small human antral
follicles and human embryonic stemcells (hESCs). (Band C) Histological sections of human ovarian tissue showing immunohistochemical staining of
granulosa cells in antral follicles. GC, granulosa cells; BM, basal membran; TC, theca cells.
68 Nielsen et al.
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expression and an augmented conversion of androgen to estrogen
(rat: Hillier and De Zwart, 1981,1982;Daniel and Armstrong,
1984; primate: Harlow et al., 1986). However, in rhesus monkeys
treated with large doses of exogenous androgens, testosterone was
without effect on granulosa cell expression of aromatase and circulat-
ing levels of estradiol became reduced (Weil et al., 1999). The present
study of naive follicles support the in vivo data, since we were also
unable to show an association between neither AR gene expression
and aromatase expression, nor between follicular fluid androgens
and aromatase expression. The fact that androgens and AR gene
expression appear to promote FSHR expression in human follicles
adds another regulatory element to the complex 2-cell two gonado-
trophins regulation of follicular estradiol synthesis, but does suggest
that proper FSH stimulation is a prerequisite for the androgen
action to be effective. It is unlikely that the AR directly regulates the
FSHR gene since no androgen response element on the FSHR gene
promoter has been identified. It may be that the AR or follicular
fluid androgens influence FSHR mRNA stability or may work indirectly,
perhaps by causing enhanced local production of IGF1 or inhibin-B
(Vendola et al., 1999a). The significant association between the
expression of AR and FSHR with AMHRII in the present study
showed that the AMH signalling system also might affect the inter-
action between AR and FSHR expression.
Studies in post-pubertal gilts treated with dihydrotestosterone
demonstrated a dose-dependant increase in ovulation rate (Ca
´rdenas
et al., 2002). The androgen treatment resulted in an increased amount
of FSHR mRNA in pre-ovulatory follicles and it was suggested that
the androgen effect might be caused by an up-regulation of FSHR
expression (Ca
´rdenas et al., 2002). Although an increased number
of pre-ovulatory follicles were not reported in primate studies
(Vendola et al., 1998), the results of the present study suggest an
increased FSH responsiveness of follicles that have a high expression
of AR and contain high concentrations of androgen. Collectively,
these results encourage pursuing clinical studies to evaluate whether
androgen priming of patients who show a poor or inadequate follicular
response following controlled ovarian stimulation can enhance follicu-
lar responsiveness and the number of recruitable follicles.
In fact several studies described administration of dehydroepian-
drosterone (DHEA) to patients in order to increase the systemic
levels of androgens and reported an enhanced harvest of mature
oocytes subsequent to ovarian stimulation (Barad and Gleicher,
2005,2006;So
¨nmezer et al., 2009). Whether androgens augmented
systematically by administration of DHEA or increased locally
by stimulating the ovaries own androgen production (i.e. LH or
hCG stimulation possible in combination with aromatase inhibitors
that block the conversion of androgen to estrogen), it appears to
make follicles more sensitive to FSH. To what extent an enhanced
FSH responsiveness of follicles augment the quality of the enclosed
oocyte and subsequently results in more fit embryos with a higher
implantation potential is not yet clear. Further studies are needed to
define the conditions by which androgen priming may improve clinical
results, but the present study does encourage this approach.
It is further of interest that mRNA expression of AMHRII also was
significantly associated with AR mRNA expression. In a previous study
we found that FSHR was significantly associated with AMHRII mRNA
expression (Nielsen et al., 2010). The present study is unable to
address whether AR and androgens affect AMHRII expression or
whether it occurs via the FSHR or a different signal transduction
pathway, but it does demonstrate that AMH, which occurs in concen-
trations of several hundreds ng/ml (Yding Andersen et al., 2008), and
its receptor AMHRII are likely to be involved in the complicated regu-
lation of follicular growth and development.
This study was unable to demonstrate significant correlations
between the follicular diameter and the AR mRNA expression. This
may relate to the fact that the diameters of a majority of the follicles
included had a relatively narrow range of 4 –7 mm. In addition, the
procedure by which the follicular fluid was aspirated may not have
yielded the total volume of each individual follicle fluid in all cases.
Further, the health status of the follicle may be expected to influence
the results. In order to compensate for these shortcomings future
studies should increase numbers in combination with assessment of
the follicular health status.
In conclusion, using fluid and granulosa cells from normal human fol-
licles with a diameter of 3–9 mm, the present study found a highly sig-
nificant association between the expression of AR on granulosa cells,
and the expression of FSHR, and between the follicular fluid content of
androgens and FSHR expression. These results encourage further
studies to improve results of already-employed strategies to
enhance the outcome of ovarian stimulation by performing androgen
priming prior to the administration of exogenous gonadotrophins.
Authors’ roles
M.E.N. performed all the mRNA purification and quantification and
participated in planning the study, evaluating the results and writing
the paper. I.A.R. assisted M.E.N. and participated in evaluating the
results. S.G.K. performed W.B. and participated in writing the
paper. S.C. performed W.B. and participated in writing the paper.
K.M. performed immunohistochemical staining and participated in
writing the paper. E.W.A. performed all statistical analyses and partici-
pated in writing the paper. A.G.B. participated in planning the study
and in writing the paper. C.Y.A. collected all the small antral follicles,
participated in planning the study, performed hormone analysis of the
follicular fluids, evaluated the results and wrote the paper.
Acknowledgements
The excellent technical assistance by Tiny Roed and Maria Westfall
is gratefully acknowledged. Cryopreservation of ovarian tissue is
performed in a much appreciated collaboration with the following,
Anders Nyboe Andersen, The Fertility Clinic, University Hospital of
Copenhagen, Denmark, Erik Ernst, Skejby University Hospital,
Aarhus, Denmark and Per Emil Rasmussen, Odense University
Hospital, Odense, Denmark.
Conflict of interest: none declared.
Funding
Cryopreservation of ovarian tissue would not have been possible
without support from the Danish Cancer Foundation Grant
(DP05112/R2-A41-09-S2) and the Novo Nordisk Foundation,
which is hereby acknowledged.
Androgen receptor and FSHR in human follicles 69
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