269
Gene expression differences induced by equimolar low doses of LH
or hCG in combination with FSH in cultured mouse antral follicles
Ingrid Segers, Tom Adriaenssens, Sandra Wathlet and Johan Smitz
Follicle Biology Laboratory, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Jette, Belgium
(Correspondence should be addressed to I Segers; Email: segersingrid@gmail.com)
Abstract
In a natural cycle, follicle growth is coordinated by FSH and
LH. Follicle growth stimulation in Assisted Reproductive
Technologies (ART) requires antral follicles to be exposed to
both FSH and LH bioactivity, especially after GNRH analog
pretreatment. The main aim was to detect possible
differences in gene expression in granulosa cells after
exposing the follicle during antral growth to LH or hCG,
as LH and hCG are different molecules acting on the same
receptor. Effects of five gonadotropin treatments were
investigated for 16 genes using a mouse follicle culture
model. Early (day 6) antral follicles were exposed to high
recombinant FSH combined or not with equimolar
concentrations of recombinant LH (rLH) or recombinant
hCG (rhCG) and to highly purified human menopausal
gonadotropin (HP-hMG) for 6 h, 12 h, or 3 days. Expression
differences were tested for genes involved in steroidogenesis:
Introduction
The final stages of follicle development leading to ovulation
are dependent on two pituitary glycoproteins: FSH and LH.
Granulosa cells of primary follicles start expressing FSH
receptors (Fshr) and the theca cells of secondary follicles start
expressing LH receptors (Lhcgr) (Erickson et al. 1985, Oktay
et al. 1997). Early antral follicle growth is only sustained under
rising FSH serum concentrations, which induce a rise in
estradiol (E2) and inhibin B (Inhb) (Groome et al. 1996),
followed by expression of Lhcgr on granulosa cells (Peng et al.
1991). Negative feedback mechanisms of E2 and INHB on
FSH concentrations select out a dominant follicle, which can
survive due to its LH responsiveness (McGee & Hsueh 2000).
Stimulation of ovarian follicle growth in human ART practice
involves the use of gonadotropin preparations containing
either FSH alone or combinations of FSH plus LH activity.
LH activity in highly purified human menopausal gonado-
tropin (HP-hMG) is mainly represented by hCG stimulation
(Wolfenson et al. 2005). More recently, recombinant LH
(rLH) became available to complement recombinant FSH
(rFSH) during stimulation (Mochtar et al. 2007). Differences
in gene expression in human follicle cells were shown in
Journal of Endocrinology (2012) 215, 269280
00220795/12/0215269 q 2012 Society for Endocrinology Printed in Great Britain
Mvk, Lss, Cyp11a1, Hsd3b1, Cyp19a1, Nr4a1, and Timp1;
final granulosa differentiation: Lhcgr, Oxtr, Pgr, Egfr, Hif1a,
and Vegfa; and cytokines: Cxcl12, Cxcr4, and Sdc4. Lhcgr was
present and upregulated by gonadotropins. Nr4a1, Cxcl12,
and Cxcr4 showed a different expression pattern if LH
bioactivity was added to high FSH in the first hours after
exposure. However, no signs of premature luteinization were
present even after a 3-day treatment as shown by Cyp19a1,
Oxtr, Pgr, and Egfr and by estrogen and progesterone
measurements. The downstream signaling by rhCG or rLH
through the LHCGR was not different for this gene
selection. Granulosa cells from follicles exposed to
HP-hMG showed an enhanced expression level for several
genes compared with recombinant gonadotropin exposure,
possibly pointing to enhanced cellular activity.
Journal of Endocrinology (2012) 215, 269280
relation to the type of gonadotropin preparation used
(Grondahl et al. 2009, Adriaenssens et al. 2010), and these
might influence clinical outcome (Afnan 2009, van Wely et al.
2011). Also the origin of the gonadotropin preparation,
urinary or recombinant, might, via isoform composition
differences, affect bioavailability and bioactivity in relation to
the species (de Leeuw et al. 1996).
Gonadotropin receptors typically activate adenylyl cyclase
through G-proteins and thereby induce cAMP production.
However, the cellular response upon FSH cannot entirely
substitute for LHCGR signaling during the final stages of
follicle growth and ovulation as shown in the Lhcgr knockout
mouse (Zhang et al. 2001) and in women with inactivating
mutations of Lhcgr (Huhtaniemi & Alevizaki 2006). Hence,
stimulation of FSHR or LHCGR during antral follicle
growth leads to a specific differential response.
The large N-terminal ectodomain of the LHCGR is
responsible for the high affinity and selective binding of its
two ligands LH and hCG (Caltabiano et al. 2008). The LH
and hCG b subunit share 80% sequence homology but greatly
differ in carbohydrate additions (for review Fares (2006)).
Where the conserved amino acid composition of LH and
hCG determines the ligandreceptor specificity, the divergent
DOI: 10.1530/JOE-12-0150
Online version via http://www.endocrinology-journals.org
270 I SEGERS and others . Gonadotropins influence gene expression
carbohydrate chains of LH and hCG interact differently with
the LHCGR changing their affinity to the receptor and affect
half-life and hence bioactivity in living organisms (Galet &
Ascoli 2005). Although in vivo bioactivity will largely
determine differences in the extent of LHCGR stimulation
upon LH or hCG stimulation, it remains to be determined
whether LH and hCG can elicit intrinsically different
responses at the level of the Lhcgr.
Our primary interest was to differentiate low-dose effects of
LH and hCG on in vitro cultured follicles exposed to a high-
FSH dose (25 mIU/ml) comparable with a superovulation
condition. The validated mouse model used is physiologically
relevant as it allows natural interactions between the three
cell types of the follicle (Cortvrindt & Smitz 2002). The
responsiveness and sensitivity for FSH and LH in this in vitro
follicle model has already been studied (Cortvrindt et al. 1998,
Adriaens et al. 2004). Different gonadotropin regimens were
shown to induce differences in mRNA expression in follicle
cells (Adriaenssens et al. 2009, Sanchez et al. 2011) and in
secretion of proteins (Foster et al. 2010). Therefore, in the
current study, early antral follicles were exposed to five
different gonadotropin regimens relevant to the ART clinic.
A low rFSH tonus (10 mIU/ml) was used during the
preantral follicle stage (from days 1 to 6), followed by a high
FSH supplementation (as in clinical ART, 25 mIU/ml) in
combination or not with low doses of LH or hCG or by
HP-hMG. The doses of rLH or recombinant hCG (rhCG)
included (5.35 pM, equivalent to 5 mIU/ml rhCG) were
equimolar and similar to the amount of LH/hCG present in
HP-hMG, when the dose used in medium is 25 mIU/ml.
These doses are 240 times less than the ovulatory trigger
(1.2 IU/ml hCG) and w100 times less than the minimal
effective dose (0.4 IU/ml rhCG, historical laboratory data) to
induce mucification and maturation in the applied follicle
culture system.
Downstream acute effects of gonadotropin exposure were
studied by quantitative gene expression (day 6 at 6 and 12 h)
and the chronic effects after 3 days of exposure. The 16 genes
chosen for analysis involve LH-induced processes,
e.g. steroidogenesis: Mvk, Lss, Cyp11a1, Hsd3b1, Cyp19a1,
Nr4a1, and Timp1; final differentiation of granulosa cells:
Lhcgr, Oxtr, Pgr, Egfr, Hif1a, and Vegfa; and cytokine
expression: Cxcl12, Cxcr4, and Sdc4 (Table 1).
Materials and Methods
Mice and follicle culture
F1 mice (C57BL/6J!CBA/Ca; Charles River, Brussel,
Belgium), housed and bred according to the national
standards for animal care and approved by the Ethical
Committee for animal experiments of the Free University
Brussels (Project 09-216-1), were used in this study. Preantral
follicles (110130 mm) were mechanically isolated from
ovaries from 13- to 14-day-old F1 mice in L15 Leibovitz
Journal of Endocrinology (2012) 215, 269280
glutamax-I medium supplemented with 10% heat-inactivated
fetal bovine serum (HIA FBS), 100 IU/ml penicillin, and
100 mg/ml streptomycin (all Gibco; Invitrogen) and placed as
single units in half area 96-well plates (Costar; Elscolab,
Kruibeke, Belgium). On the first day of culture, intact
oocytegranulosa cell connections and presence of theca cells
were ascertained. At days 6, preantral follicles that developed
into early antral follicles were considered for exposure to the
five treatments during their antral growth phase. At day 9,
these follicles had developed into late antral follicles that
produced mature oocytes and expanded cumulus cells
16 h after maturation induction. Culture medium was
refreshed every 3 days by sampling 30 ml from 75 ml culture
medium and adding 30 ml fresh medium. Preantral follicles
were cultured for 6 days in a basal culture medium
supplemented with 10 mIU/ml rFSH (Gonal F, Ares
Serono). Basal culture medium consists of a-minimal essential
medium with glutamax-I (Gibco; Invitrogen) supplemented
with 5% HIA FBS, 5 mg/ml insulin, 5 mg/ml transferrin, and
5 ng/ml selenium (Sigma). Gonadotropin products were
dissolved in basal medium. At day 9 of culture, follicles in
reference plates received 1.2 IU/ml rhCG (Ovidrel, Ares
Serono) and 4 ng/ml r-EGF (Roche) to induce meiotic
resumption. All manipulations were done on a heated stage
and cultured follicles were grown at 37 8C, 100% humidity,
and 5% CO2 in air.
Experimental design
After the initial growth period of 6 days from the preantral to
the early antral follicle stage under a gonadotropin concen-
tration of 10 mIU/ml rFSH, early antral follicles were
exposed to five different gonadotropin regimens:
10 mIU/ml rFSH, 25 mIU/ml rFSH, 25 mIU/ml rFSHC
5.35 pM rLH (Luveris, Ares Serono), 25 mIU/ml rFSHC
5.35 pM rhCG, or 25 mIU/ml HP-hMG (Menopur,
Ferring). The 10 mIU/ml rFSH condition is a continuation
of the minimal effective dose of FSH needed in culture
(Adriaens et al. 2004, Segers et al. 2012). The 25 mIU/ml
rFSH condition is considered slightly supraphysiological in
accordance with the supraphysiological FSH injections in
assisted reproductive technologies (ART) patients (Sanchez
et al. 2011). Supplementation of 25 mIU/ml rFSH with
equimolar concentrations of 5.35 pM of rLH (3.66 mIU/ml)
or rhCG (5 mIU/ml) was chosen to ensure identical ligand
availability (equal to the identical amount of molecules) for
the LHCGR in this in vitro system.
The effects of different gonadotropin regimens on gene
expression in the mural cell compartment were investigated.
Early response on increased doses of gonadotropins (acute
phase) was measured at 0, 6, and 12 h after the medium
refreshment on day 6. Chronic effects of gonadotropin
treatment during early antral to late antral follicle growth were
studied by exposure for 3 days up to day 9 of culture. Cells
were collected in four sets of experiments. Follicle culture
repeats consisted of one of these possibilities: acute phase of
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 Gonadotropins influence gene expression . I SEGERS and others 271

Table 1 Overview of the genes analyzed for gene expression in relation to different gonadotropin treatments during antral growth. Gene
name, symbol, and known function in the ovary are listed. The effect of high doses of LH or hCG, as in the ovulation trigger, on the gene
expression or protein level is given

Effect of high doses of LH/hCG

Gene name (gene symbol) Function in the ovary (ovulation trigger)

LH/choriogonadotropin Receptor for both LH and hCG, important during antral follicle Lhcgr mRNA is downregulated in rat
receptor (Lhcgr) growth, maturation, and ovulation (McGee & Hsueh 2000) ovaries (Hoffman et al. 1991)
Mevalonate kinase (Mvk) Catalyzes conversion of mevalonic acid to mevalonate-5- Mvk mRNA is transiently upregulated in
phosphate, a step in lanosterol and subsequent cholesterol rat, human, and mouse granulosa cells
biosynthesis (Wang & Menon 2005, Wang et al.
MVK binds Lhcgr mRNA and hereby negatively affect its stability 2007)
(Wang & Menon 2005, Wang et al. 2007)
Lanosterol synthase (Lss) Catalyzes conversion of 2,3-oxidosqualene to lanosterol, a step Lanosterol enhanced meiotic resumption
in cholesterol biosynthesis in porcine oocytes (Marco-Jimenez et al.
Induced by FSH and inhibited by forkhead box O1 (FOXO1; 2010)
Liu et al. 2009)
Cytochrome P450, family 11, Catalyzes conversion of cholesterol to pregnenolone, the first Cyp11a1 mRNA is upregulated through
subfamily a, polypeptide 1 and rate-limiting step in the synthesis of steroid hormones. EGFRERK1/2 signaling in mouse COC
(Cyp11a1) Increased by FSH and E2 and decreased by FOXO1 (Hernandez-Gonzalez et al. 2006)
(Liu et al. 2009)
Hydroxy-d-5-steroid Catalyzes conversion of pregnenolone to progesterone Hsd3b1 mRNA is upregulated in mouse
dehydrogenase, 3b- and Upregulated by FSH and insulin (McGee et al. 1995) COCs (Hernandez-Gonzalez et al.
steroid d-isomerase 1 2006)
(Hsd3b1)
Cytochrome P450, family 19, Catalyzes conversion of C19 androgens to aromatic Cyp19a1 mRNA is downregulated through
subfamily a, polypeptide 1 C18 estrogens EGFR signaling (Hernandez-Gonzalez
(Cyp19a1) Induced by FSH through the A-kinase pathway (Fitzpatrick & et al. 2006, Andric et al. 2010)
Richards 1991)
Nuclear receptor subfamily 4, Orphan receptor of the steroid hormone receptor superfamily Nr4a1 mRNA is rapidly and transiently
group A, member 1 (Nr4a1) that acts as a nuclear transcription factor implicated in expressed in rat granulosa cells (Park
immediate-early response (Stocco et al. 2000) et al. 2001), which inhibits Cyp19a1
expression in a human granulosa-like
tumor cell line (Wu et al. 2005)
Progesterone receptor (Pgr) Essential for follicle rupture during the ovulatory process but Pgr mRNA and protein is rapidly and
does not affect follicle growth, development, differentiation, transiently expressed in mouse mural
or luteinization (Robker et al. 2009) granulosa cells (Robker et al. 2000)
Chemokine (C-X-C motif) Chemokine that mediates chemotaxis and homing of primordial Cxcl12 mRNA and protein is present in
ligand 12 (Cxcl12) germ cells (Ara et al. 2003), granulosa cell survival in the human follicle fluid granulosa cells
pre- and periovulatory period (Kryczek et al. 2005), and isolated at oocyte pick up (Kryczek et al.
implantation (Tapia et al. 2008) 2005)
Chemokine (C-X-C motif) Receptor for CXCL12 Cxcr4 mRNA is transiently induced in
receptor 4 (Cxcr4) Human CXCR4 expression in cumulus cells is negatively mouse COCs (Hernandez-Gonzalez
correlated with embryo cleaving capacity (van Montfoort et al. 2006)
et al. 2008)
Induced by hypoxia and EGFR signaling (Phillips et al. 2005)
Syndecan 4 (Sdc4) Proteoglycan that directly binds CXCL12 and is involved in Sdc4 mRNA is progressively upregulated
Cxcr4-mediated cell invasion (Charnaux et al. 2005) in mouse cumulus cells (Adriaenssens
et al. 2010)
Vascular endothelial growth Affects follicle growth (Danforth et al. 2003) and survival (Irusta Vegfa mRNA shows sustained increase in
factor A (Vegfa) et al. 2010), ovulation, and corpus luteum formation (Fraser human luteinized granulosa cells
et al. 2005) (Koos 1995)
Tissue inhibitor of metallo- Secreted protein that inhibits matrix metalloproteinases (MMPs) Timp1 mRNA is rapidly and transiently
proteinase 1 (Timp1) at ovulation and at corpus luteum formation and regression induced in periovulatory rat granulosa
(Li & Curry 2009) cells (Li & Curry 2009)
Hypoxia inducible factor 1a Forms with HIF-1B subunit: HIF1 transcription factor, stabilized HIF1A is induced in human luteinized
subunit (Hif1a) by hypoxia, inducing, e.g. VEGF (Forsythe et al. 1996) and granulosa cells (van den Driesche et al.
Cxcr4 (Phillips et al. 2005). Under normal oxygen levels, 2008)
Hif1a can be induced by growth factors (Semenza 2003)
Epidermal growth factor Receptor for several EGF-like factors (e.g. EGF, EREG, and Egfr mRNA and protein are downregulated
receptor (Egfr) AREG), important during follicle growth and differentiation, in mouse cumulus (Romero et al. 2011)
steroidogenesis and maturation/ovulation, where it mediates
the LH stimulus (Conti et al. 2006)
Oxytocin receptor (Oxtr) G-protein-coupled receptor influencing steroidogenesis, Oxtr mRNA is induced in mouse mural
ovulation, luteinization, and luteal regression granulosa cells but not in cumulus cells
(Gimpl & Fahrenholz 2001) (Segers et al. 2012)

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272 I SEGERS and others . Gonadotropins influence gene expression

treatment rFSH10, rFSH25CLH, rFSH25ChCG, and
HP-hMG25 (A); acute phase of treatment rFSH10 and
rFSH25 (B); chronic phase of treatment rFSH10, rFSH25CLH,
rFSH25ChCG, and HP-hMG25 (C); or chronic phase of
treatment rFSH10 and rFSH25 (D). In each follicle culture
experiment, follicles were harvested from the ovaries of
several mice and randomly distributed to different culture
plates, resulting in on average ten follicles per culture plate.
The acute phase A was collected from in total seven follicle
culture repeats using 49 prepubertal mice and the acute phase B
from two follicle culture repeats using 16 mice. The chronic
phase C was collected from four culture repeats using 28 mice
and the chronic phase D from two culture repeats using
14 mice. Only follicles that already showed an antral cavity
were withheld for gonadotropin exposure (around 40% of the
initially cultured follicles). After gonadotropin exposure, cells
were immediately snap frozen in liquid nitrogen pooled per
culture plate. To obtain a pool of cells of on average eight
follicles (to use as one sample in further gene expression
analysis), the cells collected from two culture plates were
often combined within each treatment and time point.
One gonadotropin treatment consisted mostly of six
samples per time point for further use in gene expression
analysis, as detailed in Table 2. Based on the common control
condition rFSH10 and for the sake of clarity, acute (ACB)
and chronic (CCD) sets of experiments were merged for
data representation and analysis.
Collection of mural granulosa cells was performed after
cumulusoocyte complex (COC) removal by securely
pipeting with a pulled pasteur pipette. As in this culture
system, the theca cells are firmly attached onto the bottom of
the well as a monolayer with a multilayer-mounted mural
granulosa cell stack on top; the dense mass of mural cells
was collected with a Pasteur pipette. Although it cannot be
100% excluded that also a few theca cells might have been
aspirated, we estimate that the granulosa mass is nevertheless
very pure.
Gene expression analysis
Total RNA was extracted using RNeasy Micro kit (Qiagen)
on the QiaCube (Qiagen) following the manufacturers
Table 2 Gene expression results for the acute phase: early response in early antral follicles (day 6) to different gonadotropin regimes for 6 and
12 h. Values represent relative expression ratios of the specific gene and the endogenous control Rn18s (meanGS.E.M.)
instructions with addition of an on-column DNase I treatment
(27 U/reaction; Qiagen). Total RNA (14 ml) in water was
obtained. cDNA synthesis was performed on 10 ml total RNA
using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories)
using the blend of oligo(dT) and random hexamers. After 30 0
at 48 8C, the obtained 20 ml cDNA was diluted four times
with RNase-free water and stored at K80 8C until expression
analysis. Negative controls were generated in each run of
cDNA synthesis by omitting RT enzyme or RNA.
The PCR primer design was performed using Universal
Probe Library (UPL) Software (Roche Diagnostics, Roche
Applied Science). Gene expression was detected using SYBR
green or UPL fluorescent probes. Primer specificity was
ascertained by melting curve analysis if SYBR green
detection was performed and by sequencing analysis for
both SYBR green and probe detection. Primer and reference
sequences are detailed in Supplementary Table 1, see section
on supplementary data given at the end of this article.
Real-time PCR was performed on LightCycler 480 with
0.6 mM primers (Eurogentec, Liege, Belgium) in LC480
SYBR Master or in LC480 Probes Master (Roche
Diagnostics) with 0.01 mM probe (UPL, Roche Diagnostics)
and 2 ml cDNA into a total volume of 15 ml. PCR conditions
were 10 0 at 95 8C followed by 45 cycles of 10 00 at 95 8C and 30 00
at 60 8C. A log10 dilution series of a synthetic oligonucleotide
(Eurogentec) corresponding to the amplicon sequence was
simultaneously run to enable quantitative measurement of
expression. Samples were run in triplicate for each gene. As
endogenous control, Rn18s was detected with SYBR green
(Adriaenssens et al. 2009). Relative expression values were
calculated for all samples. All control samples appeared
negative for real-time PCR assessment of Rn18s and specific
genes. The different genes were quantified in the most
relevant culture setting: acute phase, chronic phase, or both,
based on the literature and in-house micro-array data.
Steroid measurements
Spent medium of follicle culture was pooled per plate at each
refreshment day or at the collection time point. Samples were
frozen at K20 8C. E2-17b production was measured with the
RIA E2RIA-CT (Biosource, Nivelles, Belgium) having a

0h 6h 12 h
rFSH10 rFSH10 rFSH25 rFSH25C rFSH25C HP-hMG25 rFSH10 rFSH25 rFSH25C rFSH25C HP-hMG25
(6) (6) (6) rLH (5) rhCG (6) (6) (6) (6) rLH (6) rhCG (6) (6)

Mvk 7.3G0.6A 12.3G1.0B 14.8G1.1 13.3G2.5 13.3G1.5 9.9G0.7 6.2G0.2A 6.9G0.3 7.7G1.0 7.7G0.7 7.9G1.1
Lss 1.4G0.2A 2.7G0.2B 3.0G0.2 3.1G0.5 2.9G0.2 2.4G0.2 1.4G0.1A 1.6G0.1 1.4G0.2 1.5G0.2 1.6G0.2
Timp1 2.2G0.2A 4.6G0.6B 5.6G0.7 5.5G0.9 4.3G0.4 3.5G0.2 1.6G0.2A 1.6G0.2 1.9G0.2 2.3G0.4 1.8G0.2
Vegfa 3.2G0.4A 4.7G0.6A,B 4.2G0.5 4.7G0.9 4.7G0.5 4.1G0.3 4.9G0.2B 6.3G0.3 4.3G0.5 4.7G0.6 5.3G0.8
Sdc4 9.2G1.2A,B 13.8G1.8A 12.4G1.6 12.4G1.6 11.1G0.8 9.0G0.4 8.0G0.1B 8.8G0.1 9.4G1.5 10.5G1.3 9.0G1.5

A,B
Different capital letters in a row define statistical differences between the time points 0, 6, and 12 h in rFSH10 treatment (P!0.05, ANOVACTukey). Between
brackets, the amount of samples analyzed for gene expression is given.

Journal of Endocrinology (2012) 215, 269280 www.endocrinology-journals.org

 Gonadotropins influence gene expression .
sensitivity of 20 pg/l and a total imprecision profile
!10% coefficient of variation (CV). Progesterone secretion
was determined with Prog-CTRIA (Cis bio International,
Gif-sur-Yvette Cedex, France) with a functional sensitivity of
0.5 mg/l and a total imprecision profile !10% CV. E2 was
measured on day 6 of culture before and after acute exposure
for 6 and 12 h to the different gonadotropin treatments. The
average E2 production per hour was calculated, taking into
account the residual fraction of E2 present in the well after
medium change, e.g. after 6 h: ((E2 on 6 h)K(E2 on
0 h!0.6))/6 h. E2 and progesterone were measured after
chronic exposure for 3 days from days 6 to 9 of culture.
Statistical analysis
Relative expression ratios were log2 transformed to ensure
Gaussian distribution. Per time point, different gonadotropin
conditions were subjected to one-way ANOVACTukey
posttest or unpaired test using GraphPad Prism 4.0 Software
(La Jolla, CA, USA). Differences with a P!0.05 are
considered statistically significant.
Results
Follicle culture
Early antral follicles on day 6 developed into late antral
follicles on day 9 as expected, with extensive granulosa cell
proliferation and enlargement of follicular structure. There
were no obvious morphological differences between the fully
grown follicles generated from any of the gonadotropin
regimens at day 9. Antral follicle survival up to day 9 was
equally successful in all conditions (O96%; PO0.05) and
meiotic maturation observed at 16 h after rhCG plus rEGF
was O80% in control plates grown for 9 days in all condtions
(PO0.05).
LH/choriogonadotropin receptor expression
All follicles included in our analysis had from day 6 onward a
clear delineated COC with mural granulosa and theca cells
attached to the plate. All samples expressed Lhcgr.
In the acute exposure experiment, there was no change
in Lhcgr expression within the first 6 h of exposure to the
different gonadotropin conditions (Fig. 1). After 12 h, Lhcgr
expression remained low for rFSH10, while high
FSH25 increased Lhcgr expression (rFSH10 vs rFSH25
and rFSH25CrhCG, P!0 . 05). In the large antral
follicles (rFSH10, day 9), Lhcgr was significanlty increased
by threefold compared with day 6 (rFSH10). On day 9,
chronic exposure to HP-hMG25 induced significantly more
Lhcgr mRNA compared with rFSH10 (P!0.05). rFSH25,
rFSH25CrLH, and rFSH25CrhCG showed intermediate
Lhcgr expression (Fig. 2).
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I SEGERS and others 273
Acute phase: early response in early antral follicles (day 6) to
different gonadotropin regimens for 6 and 12 h
After 6 h of exposure, expression of Cxcl12, Cxcr4, and Hif1a
were differentially regulated by the different gonadotropin
preparations (Fig. 1). Their expression was not influenced
by increasing the FSH dose (rFSH10 vs rFSH25). Cxcl12
expression was decreased if high FSH25 was combined with
LH bioactivity (rFSH10 vs rFSH25CrhCG and HP-hMG25,
P!0.05). Cxcr4 showed an inverse reaction and was
upregulated by high FSH25 combined with LH bioactivity
(rFSH10 vs rFSH25CLH, rFSH25ChCG, and HP-hMG25,
P!0.05). Hif1a expression was similar in all recombinant
gonadotropin treatments but reduced in HP-hMG25 (P!0.05).
After 12 h of exposure, Cxcr4, Cyp19a1, and Nr4a1 showed
gonadotropin-dependent expression (Fig. 1). Their expression
was significantly induced by increasing the FSH dose (rFSH10
vs rFSH25, P!0.05). If high FSH25 was combined with LH
bioactivity, expression of Cxcr4 (rFSH25 vs rFSH25ChCG,
P!0.05) and Nr4a1 (rFSH25 vs rFSH25ChCG and
HP-hMG25, P!0.05) was further increased.
Mvk, Lss, Timp1, Vegfa, and Sdc4 expression was not
influenced by gonadotropin treatment. The baseline of
rFSH10 was upregulated from 0 to 6 h and returned back
to initial values at 12 h for Mvk, Lss, Timp1, Hif1a, Cxcl12,
Cxcr4, and Nr4a1 (Table 2, ANOVACTukey, P!0.05).
Chronic phase: continuous exposure to different gonadotropin
regimens during antral growth from days 6 to 9
During antral follicle growth in rFSH10 from days 6 to 9,
Lhcgr was threefold upregulated and Egfr was 1.5-fold
downregulated (Fig. 2, t-test, P!0.05). After a 3-day
exposure of the growing follicles to different gonadotropin
treatments, Cxcr4, Cyp19a1, and Cyp11a1 expression was
gradually increasing from rFSH10 to rFSH25, to rFSH25C
rLH/hCG, and highest in HP-hMG25. Statistical significance
was obtained between rFSH10 and HP-hMG25 (P!0.05)
for Cxcr4, Cyp19a1, and Cyp11a1 (Fig. 2). A tendency for
increased expression in HP-hMG25 was present for Hsd3b1,
Cxcl12, Oxtr, Pgr, and Sdc4 (Table 3).
Steroid production
The immediate effects by different gonadotropin treatments
on E2 production on day 6 showed highest E2 production/
hour in HP-hMG25 both at 6 h (Table 4, P!0.01 vs
rFSH10) and 12 h (tendency). By day 9 of culture, the E2
concentration of rFSH10 was significantly lower compared
with the other four conditions (Table 5, P!0.001). The basal
progesterone production was elevated in rFSH25CLH
bioactivity compared with rFSH10 and rFSH25 (rFSH10 vs
rFSH25CrLH, P!0.05). Sixteen hours after the ovulatory
trigger, no differences in progesterone production were found
between the different gonadotropin preparations (PO0.05,
data not shown).
Journal of Endocrinology (2012) 215, 269280
274 I SEGERS and others . Gonadotropins influence gene expression

Lhcgr Cxcl12 Cxcr4

b B
120 35 a 5 c c
ab
ab
100 30
b 4
ab 25 c

Cxcl12/Rn18s

Cxcr4/Rn18s
ac
Lhcgr/Rn18s

80 bc
20 3
60 A c bc A
a 15 2 b
40 b b
10 B ab
A
20 1 a
05 A a
0 00 0
rFSH10 0 h
rFSH10 6 h
rFSH25 6 h
rFSH25+rLH 6 h
rFSH25+rhCG 6 h
HP-hMG25 6 h
rFSH10 12 h
rFSH25 12 h
rFSH25+rLH 12 h
rFSH25+rhCG 12 h
HP-hMG25 12 h

rFSH10 0 h
rFSH10 6 h
rFSH25 6 h
rFSH25+rLH 6 h
rFSH25+rhCG 6 h
HP-hMG25 6 h
rFSH10 12 h
rFSH25 12 h
rFSH25+rLH 12 h
rFSH25+rhCG 12 h
HP-hMG25 12 h

rFSH10 0 h
rFSH10 6 h
rFSH25 6 h
rFSH25+rLH 6 h
rFSH25+rhCG 6 h
HP-hMG25 6 h
rFSH10 12 h
rFSH25 12 h
rFSH25+rLH 12 h
rFSH25+rhCG 12 h
HP-hMG25 12 h
B Hif1a Cyp19a1 Nr4a1
a c
35 a 25 b 35 c
ab ab bc
30 b 30
20 b
Cyp19a1/Rn18s

25 b 25

Nr4a1/Rn18s
Hif1a/Rn18s

b B b
20 15 20
A A
15 a 15 A
10 A
a
10 10
5
5 5
0 0 0
rFSH10 0 h
rFSH10 6 h
rFSH25 6 h
rFSH25+rLH 6 h
rFSH25+rhCG 6 h
HP-hMG25 6 h
rFSH10 12 h
rFSH25 12 h
rFSH25+rLH 12 h
rFSH25+rhCG 12 h
HP-hMG25 12 h

rFSH10 0 h
rFSH10 6 h
rFSH25 6 h
rFSH25+rLH 6 h
rFSH25+rhCG 6 h
HP-hMG25 6 h
rFSH10 12 h
rFSH25 12 h
rFSH25+rLH 12 h
rFSH25+rhCG 12 h
HP-hMG25 12 h

rFSH10 0 h
rFSH10 6 h
rFSH25 6 h
rFSH25+rLH 6 h
rFSH25+rhCG 6 h
HP-hMG25 6 h
rFSH10 12 h
rFSH25 12 h
rFSH25+rLH 12 h
rFSH25+rhCG 12 h
HP-hMG25 12 h
Figure 1 Gene expression results during the acute exposure for 6 and 12 h to different gonadotropin regimens. Bars represent mean
expression ratios of the specific gene over the Rn18s endogeneous control with S.E.M. error bars. Statistical analysis was performed by
ANOVACTukey posttest in the rFSH10 condition over the three time points 0, 6, and 12 h, indicated by capital letters above columns.
ANOVACTukey posttest was also applied to the different gonadotropin preparations per time point (separated by full lines), indicated by
small letters above columns. Statistical differences have at least P!0.05.

Discussion et al. 2007). Lhcgr downregulation was not observed in the

current study using high FSH25 supplemented with
The effects of FSH, LH, and hCG during antral follicle 5.35 pM of rLH or rhCG during antral growth and no
growth were investigated on genes involved in Lhcgr signaling correlation with Mvk expression was found.
both immediately after exposure (acute phase) and 3 days after The growth of the follicle from the early (day 6) to the late
exposure (chronic phase). (day 9) antral stage coincided with more Lhcgr expression. The
highest level of Lhcgr mRNA was found after exposure to
HP-hMG25 for 3 days. Equal levels of E2 were found in all
Lhcgr expression in the acute and chronic phase
conditions containing high gonadotropin concentrations;
The increase in FSH at day 6 was followed 12 h later by an hence, E2 cannot be responsible for higher Lhcgr expression
induction of Lhcgr expression. This was expected as FSH and in HP-hMG25.
estrogen (already increased at 6 h in the spent medium)
induce Lhcgr during normal antral follicle growth (Richards
Acute phase (6 and 12 h): Expression levels of Hif1a, Nr4a1,
et al. 1976, Ikeda et al. 2008). Ovulatory doses of LH are
and Cyp19a1 are influenced by gonadotropins
known to downregulate the expression of Lhcgr (Hoffman
et al. 1991) already after 4 h (Wang & Menon 2005). This is The increase in Hif1a mRNA expression after 6 h seen in
directly caused by Lhcgr mRNA destabilization through early antral follicles in this study was independent of
MVK, an immediate effect that can be detected by gene increasing doses of recombinant gonadotropins but was
expression as soon as 6 h after the ovulatory stimulus (Wang absent in HP-hMG25. In all conditions, Hif1a returned to

Journal of Endocrinology (2012) 215, 269280 www.endocrinology-journals.org

 Gonadotropins influence gene expression . I SEGERS and others 275

Lhcgr Egfr Cxcr4

b A
600 175 25
b
500 150
20
125 ab
ab B ab

Cxcr4/Rn18s
Lhcgr/Rn18s

400

Egfr/Rn18s
ab 100 15
300 B ab
ab
a 75 10 a
200
50
100 A 05
25
0 00 00
rFSH10 Day 6

rFSH10 Day 9

rFSH25 Day 9

rFSH25+rLH Day 9

rFSH25+rhCG Day 9

HP-hMG25 Day 9

rFSH10 Day 6

rFSH10 Day 9

rFSH25 Day 9

rFSH25+rLH Day 9

rFSH25+rhCG Day 9

HP-hMG25 Day 9

rFSH10 Day 6

rFSH10 Day 9

rFSH25 Day 9

rFSH25+rLH Day 9

rFSH25+rhCG Day 9

HP-hMG25 Day 9
Cyp19a1 Cyp11a1
b
70 b 1400
60 1200
ab
ab
Cyp11a1/Rn18s
Cyp19a1/Rn18s

50 1000
ab ab
40 800
a
30 ab 600
a a
20 400
10 200
0 0
rFSH10 Day 6

rFSH10 Day 9

rFSH25 Day 9

rFSH25+rLH Day 9

rFSH25+rhCG Day 9

HP-hMG25 Day 9

rFSH10 Day 6

rFSH10 Day 9

rFSH25 Day 9

rFSH25+rLH Day 9

rFSH25+rhCG Day 9

HP-hMG25 Day 9

Figure 2 Gene expression results during the chronic exposure for 3 days (from days 6 to 9 of culture) to different gonadotropin regimens.
Bars represent mean expression ratios of the specific genes over the Rn18s endogeneous control with S.E.M. error bars. Statistical analysis
was performed by ANOVACTukey posttest on day 9 (separated by full lines from the day 6 result) with different small letters indicating
statistical differences with at least P!0.05. In capital letters, statistics (t-test, P!0.05) are shown for the regulation of the genes over the
two time points (days 69) in the rFSH10 condition.

baseline levels after 12 h. The transient nature of Hif1a as a An ovulatory dose of LH/hCG causes an immediate
rapid response mediator of FSH action (Alam et al. 2009) and response of Nr4a1 expression within 1 h in granulosa cells
the equivalent level of induction of its downstream target (Carletti & Christenson 2009) through cAMP-mediated
Vegfa by all gonadotropin treatments after 6 and 12 h could signaling and returns back to basal levels after 9 h (Wu et al.
indicate that the total level of induction of Hif1a had been 2005). This stimulates HSD3B2 (Havelock et al. 2005) and
similar in all conditions but differed in kinetics. represses Cyp19a1 (Wang & Menon 2005), shifting the steroid

Table 3 Gene expression results for the chronic phase: continuous exposure to different gonadotropin regimens during antral growth from
days 6 to 9. Values represent relative expression ratios of the specific gene and the endogenous control Rn18s (meanGS.E.M.)

Day 6 Day 9
FSH10 (6) rFSH10 (6) rFSH25 (6) rFSH25CrLH (6) rFSH25CrhCG (4) HP-hMG25 (6)

Hsd3b1 629G112 812G102 650G82 743G139 754G138 1118G196

Cxcl12 1.26G0.24 0.84G0.23 0.59G0.16 1.20G0.35 0.98G0.33 1.40G0.38
Oxtr 2.31G0.43 2.59G0.48 3.10G0.57 3.10G0.54 2.93G0.46 5.33G1.09
Pgr 0.42G0.10 0.36G0.07 0.32G0.07 0.37G0.05 0.41G0.09 0.60G0.13
Sdc4 15.6G5.0 25.5G3.9 23.0G3.5 27.4G4.9 26.9G6.1 42.3G8.3

Between brackets, the amount of samples analyzed for gene expression is given.

www.endocrinology-journals.org Journal of Endocrinology (2012) 215, 269280

276 I SEGERS and others . Gonadotropins influence gene expression
Table 4 Estradiol-17b production on day 6 at 6 and 12 h after
different gonadotropin exposures (meanGS.E.M.)
Mean increase/h (ng/l per h)
n 6h n 12 h
rFSH10 10 11G4A 6 12G5
rFSH25 4 30G7A,B 4 14G9
rFSH25CrLH 6 31G4A,B 6 8G3
rFSH25CrhCG 6 28G5A,B 6 11G2
HP-hMG25 6 49G13B 6 30G7
A,B
Different letters in a column define statistical differences between
treatments (P!0.05, ANOVACTukey).
secretion profile from estrogenic to progestagenic. In the
current experimental setup on day 6, Nr4a1 showed a late
induction at 12 h by FSH further elevated by LH activity,
which coincided with an induction of Cyp19a1. FSH is
believed to induce a slow but sustained cAMP signal during
growth while an ovulatory LH surge would elicit a rapid but
transient cAMP signal (Conti 2002). Our findings could
suggest that during antral growth, Nr4a1 expression is cAMP
mediated through Fshr and mild stimulation of Lhcgr.
Steroidogenesis: expression of key genes and steroid production
in the acute and chronic phase
Cyp19a1 mRNA induction by high FSH after 12 h and the
activity of the present CYP19A1 protein (E2 production,
Table 4) was kept intact after administration of a small dose of
LH or hCG. In rat, cultured granulosa cells exposed to FSH
also showed E2 production within a 6-h period (Daniel &
Armstrong 1984), suggesting that FSH in our culture model is
indeed both activating the existing CYP19A1 protein and
inducing its mRNA expression only by 12 h. The E2
production occurred mainly during the first 6 h and was
most evident for HP-hMG. Coincidently, an induction of
Mvk, Lss, and Timp1 expression was seen after 6 h of
exposure, independent of gonadotropins. The temporary
depletion of steroids after medium refreshment induced Mvk
and Lss expression (Wang et al. 2007, Ikeda et al. 2008),
providing the necessary precursors for E2 synthesis. Timp1
knockout mice showed increased E 2 and decreased
progesterone (Nothnick 2000) and TIMP1 stimulated
progesterone production in testis (Boujrad et al. 1995). This
could make Timp1 another target upregulated to bring the
follicular steroidogenic environment in balance after medium
refreshment.
After 3 days, exposure to different gonadotropin treatments
Cyp11a1 tended to be upregulated by low-dose LH activity.
The potential to convert pregnenolone into progesterone was
comparable as Hsd3b1 expression was not significantly altered.
Cyp19a1, converting androgens into estrogens, was again
highly expressed in HP-hMG25. Steroid measurements on
day 9 corroborated the gene expression patterns, where the E2
Journal of Endocrinology (2012) 215, 269280
and progesterone content for rFSH25Clow-dose LH
bioactivity was higher compared with rFSH25 alone. This
indicates that the presence of a low dose of LH or hCG
bioactivity during follicle growth induced a more active
steroidogenesis (Wolfenson et al. 2005), which could be a
result of a more active transcription of Cyp11a1 and Cyp19a1.
Secondly, the additional supply of precursors through
LH-stimulated theca cells, which express Cyp11a1 and
Hsd3b1 (Nimz et al. 2009), could also provide an elevated
steroid level. Basal progesterone levels in low-dose LH-sup-
plemented conditions remained at least 15 times lower than
the progesterone levels 16 h after the ovulatory hCG stimulus,
on average 104 mg/l.
Cytokines Cxcl12 and Cxcr4 in the acute and chronic phase
The targets from our selection most influenced by a change in
gonadotropin regimen were Cxcl12 and its receptor Cxcr4.
An alternative (co)-receptor for Cxcl12 and Sdc4 showed no
differences. Cytokines and other immune-related processes
have been described during ovulation (Richards et al. 2008).
Cumulus cells acquire some gene expression patterns specific
for immune-like cells, like Cdc34 antigen, myelin basic
protein (Mbp), and Cxcr4 (Hernandez-Gonzalez et al. 2006).
Here was shown that Cxcr4 and Cxcl12 are present and
regulated by gonadotropins in mural/theca cells already
during antral follicle growth. Cxcl12 was elevated in
rFSH10/25 at 6 h after exposure to different treatments,
while reduced in rFSH25CLH bioactivity. As suggested for
Hif1a, this could be a matter of kinetics, where additional LH
activity enhances the general activity of the cell and hereby
more rapidly recovers from medium change.
Cxcr4 was induced by HP-hMG25 but even more
by rFSH25CrLH/rhCG at 6 and 12 h. This pointed to a
different reaction through the Lhcgr if ligands are from
recombinant or urinary purified origin. High FSHCLH
bioactivity induced already at 6 h a massive induction of
Cxcr4; however, Cxcr4 levels only increased under the
influence of high rFSH25 alone after 12 h. This suggested a
synergy in LHCGR and FSHR signaling, both using cAMP
as second messenger in inducing Cxcr4 expression. No clear
Table 5 Estradiol-17b and progesterone content on day 9
(meanGS.E.M.)
Estradiol-17b (ng/l) Progesterone (mg/l)
n Day 9 n Day 9
rFSH10 13 3273G591 A
10 0.20G0.08A
rFSH25 2 7300G2100B 3 0.35G0.04A,B
rFSH25CrLH 9 12 540G1959B 9 6.79G2.16B
rFSH25CrhCG 8 12 390G1458B 8 5.37G2.34A,B
HP-hMG25 8 12 640G1197B 8 2.94G1.22A,B
A,B
Different letters in a column define statistical differences between
treatments (P!0.05, ANOVACTukey).
www.endocrinology-journals.org
 Gonadotropins influence gene expression .
interrelation between Cxcl12 and Cxcr4 expression was
found. Preliminary laboratory data suggested a cell-type-
and cell-stage-specific expression pattern for both Cxcl12 and
its receptors; hence, the presented data in mural cells could be
too fragmentarious to reveal complex interrelations.
In the current study, exposure to HP-hMG25 for 3 days
generated the highest level of Cxcr4 expression, where the
immediate response after 6 and 12 h on day 6 was higher if
exposed to high FSH and rLH bioactivity. This suggests that
slight differences in hormone composition like in the
recombinant or urinary purified hormones could affect
downstream signaling kinetics.
Chronic phase (days 69): no differences due to gonadotropin
treatment for Oxtr, Pgr, and Egfr
Three receptors implicated in ovulation and luteinization
were studied: Egfr, Oxtr, and Pgr. All three genes were not
affected by gonadotropins. Absence of significant Oxtr, Pgr,
and Egfr expression induction indicated that mural cells did
not express early signs of luteinization (Fujinaga et al. 1994,
Clemens et al. 1998, Segers et al. 2012), which was reinforced
by the endocrine profile on day 9.
Differences in expression induced by rLH, rhCG, or urinary
purified products
For all genes in this study, equimolar doses of rLH or rhCG
did not lead to differences in response in the early or late antral
granulosa cells (t-test, PO0.05). Hence, it could be
considered that in an in vitro culture system, rLH and rhCG
are activating Lhcgr signaling equally.
However, HP-hMG25 did elicit some different responses
compared to the recombinant products. Some tendencies
were noticed. Six hours after exposure of early antral follicles
to rFSH25 with or without LH activity, six of 11 genes (Mvk,
Lss, Cxcl12, Cxcr4, Hif1a, and Timp1) were upregulated by
the medium refreshment. The exposure to HP-hMG25
attenuated the initial response or accelerated the decrease to
basal levels as seen for all conditions at 12 h (Mvk, Lss, Cxcl12,
Hif1a, and Timp1). Secondly, after 3 days of growth in
HP-hMG25, nine of ten of the studied genes (all except
Cxcl12) showed a tendency for elevated expression in
HP-hMG25 compared with the rFSH25/rLH/rhCG treat-
ments. An overall higher activity status of granulosa cells
under HP-hMG treatment has also been described for protein
levels: higher levels of cytokines were found in mouse follicle
culture in HP-hMG if compared with rFSH treatment only
(Foster et al. 2010). The differences in gene expression
patterns seen in HP-hMG25 are (most likely) an effect of
both differences in the FSH and the LH/hCG component.
For FSH, it was already shown that recombinant vs
urinary purified FSH contains different subsets of isoforms
and that acidity of isoforms influenced capacity to induce
meiosis in COC, to stimulate proliferation in granulosa cells
www.endocrinology-journals.org
I SEGERS and others 277
(GC) (Barrios-De-Tomasi et al. 2002), and to influence
embryonic development when applied in follicle culture
(Vitt et al. 2001). Like FSH, both LH (Burgon et al. 1996) and
hCG (Lopata et al. 1997) consist out of different isoforms
in vivo with different pI and hence implications in bioactivity.
Conclusions
Lhcgr was present and regulated by gonadotropin type and
dose in early and late antral follicles. A low dose of LH or
hCG applied during early antral follicle growth did not
induce premature luteinization effects in both gene expression
patterns and steroid production. Gene expression responses
were identical after rLH or rhCG stimulation, suggesting an
equal in vitro biopotency. However, the urinary-derived
HP-hMG did systematically show the highest gene induction
after 3-day exposure. Nr4a1, Cxcl12, and Cxcr4 were most
influenced by introducing gonadotropin treatment in early
antral follicles. This study emphasizes the complexity of
LHCGR and FSHR signaling during folliculogenesis, where
in-depth ligandreceptor interaction studies are needed to
understand the origin of the differential expression patterns
induced by the different ligands.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/
JOE-12-0150.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived
as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Agentschap voor Innovatie door Wetenschap
en Technologie, Brussels, Belgium (grant no. IWT70719), Fonds
Wetenschappelijk Onderzoek, Vlaanderen, Brussels, Belgium (grant no.
KN1.5.040.09) and Ferring Pharmaceuticals, Saint-Prex, Switzerland.
Acknowledgements
The authors would like to thank Ms Katy Billooye for excellent technical
assistance in the follicle culture experiments and RIA, Mr Johan Schiettecatte
for technical assistance with hormone measurements, and Ms Sandra
De Schaepdryver for editorial support.
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Received in final form 13 August 2012
Accepted 17 August 2012
Made available online as an Accepted Preprint
20 August 2012
www.endocrinology-journals.org