Gene expression differences induced by equimolar low doses of LHn

nd

0 Je

com

hCG (rhCG) and to highly purified human menopausal genes compared with recombinant gonadotropin exposure,

involves the use of gonadotropin preparations containing

(rLH) became available to complement recombinant FSH

growth leads to a specific differential response.

differ in carbohydrate additions (for review Fares (2006)).

269Journal of Endocrinology (2012) 215, 269280(rFSH) during stimulation (Mochtar et al. 2007). Differences

in gene expression in human follicle cells were shown in

Where the conserved amino acid composition of LH and

hCG determines the ligandreceptor specificity, the divergent

DOI: 10.1530/JOE-12-0150either 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

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 greatlyFSH 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

mouse (Zhang et al. 2001) and in women with inactivating

mutations of Lhcgr (Huhtaniemi & Alevizaki 2006). Hence,

stimulation of FSHR or LHCGR during antral follicledifferences 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 on00220795/12/0215269 q 2012 Society for Endocrinology Printed in GreatJournal 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 knockoutgonadotropin (HP-hMG) for 6 h, 12 h, or 3 days. Expression possibly pointing to enhanced cellular activity.or hCG in combination with FSH i

Ingrid Segers, Tom Adriaenssens, Sandra Wathlet a

Follicle Biology Laboratory, Vrije Universiteit Brussel, Laarbeeklaan 101, 109

(Correspondence should be addressed to I Segers; Email: segersingrid@gmail.

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 recombinantcultured mouse antral follicles

Johan Smitz

tte, Belgium

)

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 severalBritain Online version via http://www.endocrinology-journals.org

condition. The validated mouse model used is physiologically

relevant as it allows natural interactions between the three

After the initial growth period of 6 days from the preantral to

I SEGERS and others . Gonadotropins influence gene expression270cell 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) wereequimolar 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 minimaleffective dose (0.4 IU/ml rhCG, historical laboratory data) toinduce 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 fromovaries from 13- to 14-day-old F1 mice in L15 Leibovitzcarbohydrate 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 superovulationJournal of Endocrinology (2012) 215, 269280the 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 rFSHC5.35 pM rLH (Luveris, Ares Serono), 25 mIU/ml rFSHC5.35 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 ofglutamax-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 assingle 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 culturemedium and adding 30 ml fresh medium. Preantral follicleswere 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 essentialmedium with glutamax-I (Gibco; Invitrogen) supplemented

with 5% HIA FBS, 5 mg/ml insulin, 5 mg/ml transferrin, and5 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, AresSerono) 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 designwww.endocrinology-journals.org

Table 1 Overview of the genes analyzed for gene expression in relation to different gonadotropin treatments during antral growth. Genename, 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 geneexpression or protein level is given

Gene name (gene symbol) Function in the ovaryEffect of high doses of LH/hCG(ovulation trigger)

LH/choriogonadotropinreceptor (Lhcgr)

Receptor for both LH and hCG, important during antral folliclegrowth, maturation, and ovulation (McGee & Hsueh 2000)

Lhcgr mRNA is downregulated in ratovaries (Hoffman et al. 1991)

Mevalonate kinase (Mvk) Catalyzes conversion of mevalonic acid to mevalonate-5-phosphate, a step in lanosterol and subsequent cholesterolbiosynthesis

Mvk mRNA is transiently upregulated inrat, human, and mouse granulosa cells(Wang & Menon 2005, Wang et al.2007)MVK binds LhcgrmRNA and hereby negatively affect its stability

(Wang & Menon 2005, Wang et al. 2007)Lanosterol synthase (Lss) Catalyzes conversion of 2,3-oxidosqualene to lanosterol, a step

in cholesterol biosynthesisLanosterol enhanced meiotic resumption

in porcine oocytes (Marco-Jimenez et al.2010)Induced by FSH and inhibited by forkhead box O1 (FOXO1;

Liu et al. 2009)Cytochrome P450, family 11,

subfamily a, polypeptide 1(Cyp11a1)

Catalyzes conversion of cholesterol to pregnenolone, the firstand rate-limiting step in the synthesis of steroid hormones.Increased by FSH and E2 and decreased by FOXO1(Liu et al. 2009)

Cyp11a1 mRNA is upregulated throughEGFRERK1/2 signaling in mouse COC(Hernandez-Gonzalez et al. 2006)

Hydroxy-d-5-steroiddehydrogenase, 3b- andsteroid d-isomerase 1(Hsd3b1)

Catalyzes conversion of pregnenolone to progesterone Hsd3b1 mRNA is upregulated in mouseCOCs (Hernandez-Gonzalez et al.2006)

Upregulated by FSH and insulin (McGee et al. 1995)

Cytochrome P450, family 19,subfamily a, polypeptide 1(Cyp19a1)

Catalyzes conversion of C19 androgens to aromaticC18 estrogens

Cyp19a1 mRNA is downregulated throughEGFR signaling (Hernandez-Gonzalezet al. 2006, Andric et al. 2010)Induced by FSH through the A-kinase pathway (Fitzpatrick &

Richards 1991)Nuclear receptor subfamily 4,

group A, member 1 (Nr4a1)Orphan receptor of the steroid hormone receptor superfamily

that acts as a nuclear transcription factor implicated inimmediate-early response (Stocco et al. 2000)

Nr4a1 mRNA is rapidly and transientlyexpressed in rat granulosa cells (Parket al. 2001), which inhibits Cyp19a1expression in a human granulosa-liketumor cell line (Wu et al. 2005)

Progesterone receptor (Pgr) Essential for follicle rupture during the ovulatory process butdoes not affect follicle growth, development, differentiation,or luteinization (Robker et al. 2009)

Pgr mRNA and protein is rapidly andtransiently expressed in mouse muralgranulosa cells (Robker et al. 2000)

Chemokine (C-X-C motif)ligand 12 (Cxcl12)

Chemokine that mediates chemotaxis and homing of primordialgerm cells (Ara et al. 2003), granulosa cell survival in thepre- and periovulatory period (Kryczek et al. 2005), andimplantation (Tapia et al. 2008)

Cxcl12 mRNA and protein is present inhuman follicle fluid granulosa cellsisolated at oocyte pick up (Kryczek et al.2005)

Chemokine (C-X-C motif)receptor 4 (Cxcr4)

Receptor for CXCL12 Cxcr4 mRNA is transiently induced inmouse COCs (Hernandez-Gonzalezet al. 2006)

Human CXCR4 expression in cumulus cells is negativelycorrelated with embryo cleaving capacity (van Montfoortet al. 2008)

Induced by hypoxia and EGFR signaling (Phillips et al. 2005)Syndecan 4 (Sdc4) Proteoglycan that directly binds CXCL12 and is involved in

Cxcr4-mediated cell invasion (Charnaux et al. 2005)Sdc4 mRNA is progressively upregulated

in mouse cumulus cells (Adriaenssenset al. 2010)

Vascular endothelial growthfactor A (Vegfa)

Affects follicle growth (Danforth et al. 2003) and survival (Irustaet al. 2010), ovulation, and corpus luteum formation (Fraseret al. 2005)

Vegfa mRNA shows sustained increase inhuman luteinized granulosa cells(Koos 1995)

Tissue inhibitor of metallo-proteinase 1 (Timp1)

Secreted protein that inhibits matrix metalloproteinases (MMPs)at ovulation and at corpus luteum formation and regression(Li & Curry 2009)

Timp1 mRNA is rapidly and transientlyinduced in periovulatory rat granulosacells (Li & Curry 2009)

Hypoxia inducible factor 1asubunit (Hif1a)

Forms with HIF-1B subunit: HIF1 transcription factor, stabilizedby hypoxia, inducing, e.g. VEGF (Forsythe et al. 1996) andCxcr4 (Phillips et al. 2005). Under normal oxygen levels,Hif1a can be induced by growth factors (Semenza 2003)

HIF1A is induced in human luteinizedgranulosa cells (van den Driesche et al.2008)

Epidermal growth factorreceptor (Egfr)

Receptor for several EGF-like factors (e.g. EGF, EREG, andAREG), important during follicle growth and differentiation,steroidogenesis and maturation/ovulation, where it mediatesthe LH stimulus (Conti et al. 2006)

Egfr mRNA and protein are downregulatedin mouse cumulus (Romero et al. 2011)

Oxytocin receptor (Oxtr) G-protein-coupled receptor influencing steroidogenesis,ovulation, luteinization, and luteal regression(Gimpl & Fahrenholz 2001)

Oxtr mRNA is induced in mouse muralgranulosa cells but not in cumulus cells(Segers et al. 2012)

Gonadotropins influence gene expression . I SEGERS and others 271

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

treatment rFSH10, rFSH25CLH, rFSH25ChCG, andHP-hMG25 (A); acute phase of treatment rFSH10 and

rFSH25 (B); chronic phase of treatment rFSH10, rFSH25CLH,rFSH25ChCG, and HP-hMG25 (C); or chronic phase oftreatment 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

follicles (to use as one sample in further gene expression

instructions with addition of an on-column DNase I treatment

(27 U/reaction; Qiagen). Total RNA (14 ml) in water wasobtained. cDNA synthesis was performed on 10 ml total RNAusing the iScript cDNA Synthesis Kit (Bio-Rad Laboratories)

using the blend of oligo(dT) and random hexamers. After 30 0at 48 8C, the obtained 20 ml cDNA was diluted four timeswith RNase-free water and stored atK80 8C until expressionanalysis. Negative controls were generated in each run of

cDNA synthesis by omitting RTenzyme 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

SYBR Master or in LC480 Probes Master (Roche

n eaan

HP-(6)

9.9G2.4G3.5G4.1G9.0G

e p

I SEGERS and others . Gonadotropins influence gene expression272analysis), 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 fordata 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 i12 h. Values represent relative expression ratios of the specific gene

0 h 6 h

rFSH10(6)

rFSH10(6)

rFSH25(6)

rFSH25CrLH (5)

rFSH25CrhCG (6)

Mvk 7.3G0.6A 12.3G1.0B 14.8G1.1 13.3G2.5 13.3G1.5Lss 1.4G0.2A 2.7G0.2B 3.0G0.2 3.1G0.5 2.9G0.2Timp1 2.2G0.2A 4.6G0.6B 5.6G0.7 5.5G0.9 4.3G0.4Vegfa 3.2G0.4A 4.7G0.6A,B 4.2G0.5 4.7G0.9 4.7G0.5Sdc4 9.2G1.2A,B 13.8G1.8A 12.4G1.6 12.4G1.6 11.1G0.8

A,BDifferent capital letters in a row define statistical differences between the timbrackets, the amount of samples analyzed for gene expression is given.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 eightJournal of Endocrinology (2012) 215, 269280Diagnostics) with 0.01 mM probe (UPL, Roche Diagnostics)and 2 ml cDNA into a total volume of 15 ml. PCR conditionswere 100 at 95 8C followed by 45 cycles of 1000 at 95 8C and 3000at 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 atK20 8C. E2-17b production was measured with theRIA E2RIA-CT (Biosource, Nivelles, Belgium) having a

rly antral follicles (day 6) to different gonadotropin regimes for 6 andd the endogenous control Rn18s (meanGS.E.M.)

12 h

hMG25 rFSH10(6)

rFSH25(6)

rFSH25CrLH (6)

rFSH25CrhCG (6)

HP-hMG25(6)

0.7 6.2G0.2A 6.9G0.3 7.7G1.0 7.7G0.7 7.9G1.10.2 1.4G0.1A 1.6G0.1 1.4G0.2 1.5G0.2 1.6G0.20.2 1.6G0.2A 1.6G0.2 1.9G0.2 2.3G0.4 1.8G0.20.3 4.9G0.2B 6.3G0.3 4.3G0.5 4.7G0.6 5.3G0.80.4 8.0G0.1B 8.8G0.1 9.4G1.5 10.5G1.3 9.0G1.5

oints 0, 6, and 12 h in rFSH10 treatment (P!0.05, ANOVACTukey). Betweendetection 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 LC480www.endocrinology-journals.org

conditions were subjected to one-way ANOVACTukey

regimens at day 9. Antral follicle survival up to day 9 was

equally successful in all conditions (O96%; PO0.05) and

attached to the plate. All samples expressed Lhcgr.

gonadotropin-dependent expression (Fig. 1). Their expression

During antral follicle growth in rFSH10 from days 6 to 9,

Gonadotropins influence gene expression . I SEGERS and others 273In 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 antralfollicles (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 intermediateLhcgr expression (Fig. 2).meiotic maturation observed at 16 h after rhCG plus rEGF

wasO80% 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 cellsposttest or unpaired test using GraphPad Prism 4.0 Software

(La Jolla, CA, USA). Differences with a P!0.05 areconsidered 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 gonadotropinsensitivity of 20 pg/l and a total imprecision profile

!10% coefficient of variation (CV). Progesterone secretionwas 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 wasmeasured 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 on0 h!0.6))/6 h. E2 and progesterone were measured afterchronic 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 gonadotropinwww.endocrinology-journals.orgLhcgr was threefold upregulated and Egfr was 1.5-folddownregulated (Fig. 2, t-test, P!0.05). After a 3-dayexposure of the growing follicles to different gonadotropin

treatments, Cxcr4, Cyp19a1, and Cyp11a1 expression was

gradually increasing from rFSH10 to rFSH25, to rFSH25CrLH/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 vsrFSH10) and 12 h (tendency). By day 9 of culture, the E2concentration of rFSH10 was significantly lower compared

with the other four conditions (Table 5, P!0.001). The basalprogesterone production was elevated in rFSH25CLHbioactivity compared with rFSH10 and rFSH25 (rFSH10 vs

rFSH25CrLH, P!0.05). Sixteen hours after the ovulatorytrigger, no differences in progesterone production were found

between the different gonadotropin preparations (PO0.05,data not shown).was significantly induced by increasing the FSH dose (rFSH10

vs rFSH25, P!0.05). If high FSH25 was combined with LHbioactivity, expression of Cxcr4 (rFSH25 vs rFSH25ChCG,P!0.05) and Nr4a1 (rFSH25 vs rFSH25ChCG andHP-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 gonadotropinregimens during antral growth from days 6 to 9Acute phase: early response in early antral follicles (day 6) todifferent 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 wasupregulated by high FSH25 combined with LH bioactivity

(rFSH10 vs rFSH25CLH, rFSH25ChCG, and HP-hMG25,P!0.05). Hif1a expression was similar in all recombinantgonadotropin treatments but reduced in HP-hMG25 (P!0.05).

After 12 h of exposure, Cxcr4, Cyp19a1, and Nr4a1 showedJournal of Endocrinology (2012) 215, 269280

rFS

SH2

HP

FSH

H25

HP-

rFSH

25+r

LH 6

h

rFSH

25+r

hCG

6 h

c

1 aaA

C

I SEGERS and others . Gonadotropins influence gene expression274rF r

rFS

Hif1a

25

30

35Ba

ab

a

ab

b18s

15

20

25

n18sLhcgr

rFSH

10 0

h

rFSH

10 6

h

rFSH

25 6

h

H25+

rLH

6 h

5+rh

CG 6

h

-hM

G25

6 h

rFSH

10 1

2 h

rFSH

25 1

2 h

25+r

LH 1

2 h

+rhC

G 1

2 h

hMG

25 1

2 h

rFSH

10 0

h

rFSH

10 6

h

rFSH

25 6

h

0

20

40

60

80

100

120

a

b

abb

ab

Lhcg

r/Rn1

8s

00

05

10

15

20

25

30

35Ba

ab

ac

A

Cxcl1

2/Rn

18sDiscussion

The effects of FSH, LH, and hCG during antral follicle

growth were investigated on genes involved in Lhcgr signaling

both immediately after exposure (acute phase) and 3 days after

exposure (chronic phase).

Lhcgr expression in the acute and chronic phase

The increase in FSH at day 6 was followed 12 h later by an

induction of Lhcgr expression. This was expected as FSH and

estrogen (already increased at 6 h in the spent medium)

induce Lhcgr during normal antral follicle growth (Richards

et al. 1976, Ikeda et al. 2008). Ovulatory doses of LH are

known to downregulate the expression of Lhcgr (Hoffman

et al. 1991) already after 4 h (Wang & Menon 2005). This is

directly caused by Lhcgr mRNA destabilization through

MVK, an immediate effect that can be detected by gene

expression as soon as 6 h after the ovulatory stimulus (Wang

rFSH

10 0

h

rFSH

10 6

h

rFSH

25 6

h

rFSH

25+r

LH 6

h

rFSH

25+r

hCG

6 h

HP-

hMG

25 6

h

rFSH

10 1

2 h

rFSH

25 1

2 h

rFSH

25+r

LH 1

2 h

rFSH

25+r

hCG

12

h

HP-

hMG

25 1

2 h

rFSH

10 0

h

rFSH

10 6

h

rFSH

25 6

h

rFSH

25+r

LH 6

h

rFSH

25+r

hCG

6 h

0

5

10

15

20 A A

Hif1

a/Rn

0

5

10

Cyp1

9a1/

R

Figure 1 Gene expression results during the acute exposure for 6 anexpression ratios of the specific gene over the Rn18s endogeneous conANOVACTukey posttest in the rFSH10 condition over the three time pANOVACTukey posttest was also applied to the different gonadotropinsmall letters above columns. Statistical differences have at least P!0.0

Journal of Endocrinology (2012) 215, 269280HP-

hMG

25 6

h

rFSH

10 1

2 h

rFSH

25 1

2 h

rFSH

25+r

LH 1

2 h

rFSH

25+r

hCG

12

h

HP-

hMG

25 1

2 h

rFSH

10 0

h

rFSH

10 6

h

rFSH

25 6

h

rFSH

25+r

LH 6

h

rFSH

25+r

hCG

6 h

HP-

hMG

25 6

h

rFSH

10 1

2 h

rFSH

25 1

2 h

rFSH

25+r

LH 1

2 h

rFSH

25+r

hCG

12

h

HP-

hMG

25 1

2 h

0

yp19a1

b

bb b

Nr4a1

25

30

35

b

bcc

c

B18sCxcl12

bc A

Cxcr4

2

3

4

5

B ab

c

c

b

Ab

bc

c

bCxcr

4/Rn

18set al. 2007). Lhcgr downregulation was not observed in the

current study using high FSH25 supplemented with

5.35 pM of rLH or rhCG during antral growth and nocorrelation with Mvk expression was found.

The growth of the follicle from the early (day 6) to the late

(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

conditions containing high gonadotropin concentrations;

hence, E2 cannot be responsible for higher Lhcgr expression

in HP-hMG25.

Acute phase (6 and 12 h): Expression levels of Hif1a, Nr4a1,and Cyp19a1 are influenced by gonadotropins

The increase in Hif1a mRNA expression after 6 h seen in

early antral follicles in this study was independent of

increasing doses of recombinant gonadotropins but was

absent in HP-hMG25. In all conditions, Hif1a returned to

HP-

hMG

25 6

h

rFSH

10 1

2 h

rFSH

25 1

2 h

rFSH

25+r

LH 1

2 h

rFSH

25+r

hCG

12

h

HP-

hMG

25 1

2 h

rFSH

10 0

h

rFSH

10 6

h

rFSH

25 6

h

rFSH

25+r

LH 6

h

rFSH

25+r

hCG

6 h

HP-

hMG

25 6

h

rFSH

10 1

2 h

rFSH

25 1

2 h

rFSH

25+r

LH 1

2 h

rFSH

25+r

hCG

12

h

HP-

hMG

25 1

2 h

a

0

5

10

15

20Aa

A

Nr4a

1/Rn

d 12 h to different gonadotropin regimens. Bars represent meantrol with S.E.M. error bars. Statistical analysis was performed byoints 0, 6, and 12 h, indicated by capital letters above columns.preparations per time point (separated by full lines), indicated by

5.

www.endocrinology-journals.org

Lhcgr

rFSH

10 D

ay 6

rFSH

10 D

ay 9

rFSH

25 D

ay 9

rFSH

25+r

LH D

ay 9

SH25

+rhC

G D

ay 9

HP-

hMG

25 D

ay 9

rFSH

10 D

ay 6

rFSH

10 D

ay 9

rFSH

25 D

ay 9

rFSH

25+r

LH D

ay 9

SH25

+rhC

G D

ay 9

HP-

hMG

25 D

ay 9

rFSH

10 D

ay 6

rFSH

10 D

ay 9

rFSH

25 D

ay 9

rFSH

25+r

LH D

ay 9

SH25

+rhC

G D

ay 9

HP-

hMG

25 D

ay 9

0

100

200

300

400

500

600

aab

abab

b

A

B

Lhcg

r/Rn1

8sEgfr

00

25

50

75

100

125

150

175A

B

Egfr/

Rn18

s

Cxcr4

00

05

10

15

20

25

a

ab

ab ab

b

Cxcr

4/Rn

18s

C

a

Gonadotropins influence gene expression . I SEGERS and others 275rF

Cyp19a1

30

40

50

60

70

a abab ab

b

9a1/

Rn18

s

600

800

1000

1200

1400

a1a1

/Rn1

8sbaseline levels after 12 h. The transient nature of Hif1a as a

rapid response mediator of FSH action (Alam et al. 2009) and

the equivalent level of induction of its downstream target

Vegfa by all gonadotropin treatments after 6 and 12 h could

indicate that the total level of induction of Hif1a had been

similar in all conditions but differed in kinetics.

rFSH

10 D

ay 6

rFSH

10 D

ay 9

rFSH

25 D

ay 9

rFSH

25+r

LH D

ay 9

rFSH

25+r

hCG

Day

9

HP-

hMG

25 D

ay 9

rFSH

10 D

ay 6

rFSH

10 D

ay 9

0

10

20Cyp1

0

200

400Cyp1

Figure 2 Gene expression results during the chronic exposure for 3 daBars represent mean expression ratios of the specific genes over the Rnwas performed by ANOVACTukey posttest on day 9 (separated by fulstatistical differences with at least P!0.05. In capital letters, statisticstwo time points (days 69) in the rFSH10 condition.

Table 3 Gene expression results for the chronic phase: continuous expodays 6 to 9. Values represent relative expression ratios of the specific ge

Day 6 Day 9

FSH10 (6) rFSH10 (6) rFSH25 (6)

Hsd3b1 629G112 812G102 650G82Cxcl12 1.26G0.24 0.84G0.23 0.59G0.16Oxtr 2.31G0.43 2.59G0.48 3.10G0.57Pgr 0.42G0.10 0.36G0.07 0.32G0.07Sdc4 15.6G5.0 25.5G3.9 23.0G3.5

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

www.endocrinology-journals.orgrF rF

yp11a1

ab ab

bAn ovulatory dose of LH/hCG causes an immediate

response of Nr4a1 expression within 1 h in granulosa cells

(Carletti & Christenson 2009) through cAMP-mediated

signaling and returns back to basal levels after 9 h (Wu et al.

2005). This stimulates HSD3B2 (Havelock et al. 2005) and

repressesCyp19a1 (Wang & Menon 2005), shifting the steroid

rFSH

25 D

ay 9

rFSH

25+r

LH D

ay 9

rFSH

25+r

hCG

Day

9

HP-

hMG

25 D

ay 9

ys (from days 6 to 9 of culture) to different gonadotropin regimens.18s endogeneous control with S.E.M. error bars. Statistical analysisl lines from the day 6 result) with different small letters indicating(t-test, P!0.05) are shown for the regulation of the genes over the

sure to different gonadotropin regimens during antral growth fromne and the endogenous control Rn18s (meanGS.E.M.)

rFSH25CrLH (6) rFSH25CrhCG (4) HP-hMG25 (6)

743G139 754G138 1118G1961.20G0.35 0.98G0.33 1.40G0.383.10G0.54 2.93G0.46 5.33G1.090.37G0.05 0.41G0.09 0.60G0.1327.4G4.9 26.9G6.1 42.3G8.3

Journal of Endocrinology (2012) 215, 269280

also showed E2 production within a 6-h period (Daniel &

and progesterone content for rFSH25Clow-dose LHbioactivity 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.

I SEGERS and others . Gonadotropins influence gene expression276Armstrong 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 E2production 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 E synthesis. Timp1secretion 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 productionin 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

Table 4 Estradiol-17b production on day 6 at 6 and 12 h afterdifferent gonadotropin exposures (meanGS.E.M.)

Mean increase/h (ng/l per h)

n 6 h n 12 h

rFSH10 10 11G4A 6 12G5rFSH25 4 30G7A,B 4 14G9rFSH25CrLH 6 31G4A,B 6 8G3rFSH25CrhCG 6 28G5A,B 6 11G2HP-hMG25 6 49G13B 6 30G7

A,BDifferent letters in a column define statistical differences betweentreatments (P!0.05, ANOVACTukey).2

knockout mice showed increased E2 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, 269280Table 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 3273G591A 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,BDifferent letters in a column define statistical differences betweentreatments (P!0.05, ANOVACTukey).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 forHif1a, 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 adifferent reaction through the Lhcgr if ligands are from

recombinant or urinary purified origin. High FSHCLHbioactivity 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 clearwww.endocrinology-journals.org

are activating Lhcgr signaling equally.

Cxcl12) showed a tendency for elevated expression in

Lhcgr was present and regulated by gonadotropin type and

dose in early and late antral follicles. A low dose of LH or

after 3-day exposure. Nr4a1, Cxcl12, and Cxcr4 were most

induced by the different ligands.

Gonadotropins influence gene expression . I SEGERS and others 277HP-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 cellsHowever, 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 exceptinterrelation 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 gonadotropintreatment 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 urinarypurified 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 beconsidered that in an in vitro culture system, rLH and rhCGwww.endocrinology-journals.orgAdriaens I, Cortvrindt R & Smitz J 2004 Differential FSH exposure in

preantral follicle culture has marked effects on folliculogenesis and

oocyte developmental competence. Human Reproduction 19 398408.

(doi:10.1093/humrep/deh074)

Adriaenssens T, Mazoyer C, Segers I, Wathlet S & Smitz J 2009 Differences

in collagen expression in cumulus cells after exposure to highly purifiedDeclaration 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 2012Accepted 17 August 2012Made available online as an Accepted Preprint20 August 2012

I SEGERS and others . Gonadotropins influence gene expression280Journal of Endocrinology (2012) 215, 269280 www.endocrinology-journals.org

Outline placeholderIntroductionMaterials and MethodsMice and follicle cultureExperimental designGene expression analysisSteroid measurementsStatistical analysis

ResultsFollicle cultureLH/choriogonadotropin receptor expressionAcute phase: early response in early antral follicles (day 6) to different gonadotropin regimens for 6 and 12hChronic phase: continuous exposure to different gonadotropin regimens during antral growth from days 6 to 9Steroid production

DiscussionLhcgr expression in the acute and chronic phaseAcute phase (6 and 12h): Expression levels of Hif1a, Nr4a1, and Cyp19a1 are influenced by gonadotropinsSteroidogenesis: expression of key genes and steroid production in the acute and chronic phaseCytokines Cxcl12 and Cxcr4 in the acute and chronic phaseChronic phase (days 6-9): no differences due to gonadotropin treatment for Oxtr, Pgr, and EgfrDifferences in expression induced by rLH, rhCG, or urinary purified productsConclusions

Declaration of interestFundingAcknowledgementsReferences