Published November 24, 2014
Effects of fibroblast growth factor 9 on steroidogenesis
and control of FGFR2IIIc mRNA in porcine granulosa cells1,2
J. R. Evans, N. B. Schreiber, J. A. Williams, and L. J. Spicer3
Department of Animal Science, Oklahoma State University, Stillwater 74078
ABSTRACT: The objectives of this study were to
investigate the effects of fibroblast growth factor 9
(FGF9) on hormone-stimulated porcine granulosa cell
proliferation and steroid production and to further elucidate the hormonal and developmental control of
FGFR2IIIc gene expression in granulosa cells. Porcine
ovaries were collected from a local slaughterhouse and
granulosa cells were collected from small to medium (1
to 5 mm) follicles for 5 in vitro studies that were conducted. Cells were cultured for 48 h in 5% fetal calf
serum plus 5% porcine serum and then treated with various combinations of FSH, IGF-I, FGF9, Sonic hedgehog (SHH), cortisol, PGE2, and/or wingless-type mouse
mammary tumor virus integration site family member
5A (WNT5A) in serum-free medium for an additional
24 or 48 h. Medium was collected for analysis of steroid concentration via RIA, or RNA was collected for
gene expression analysis of FGFR2IIIc via quantitative
reverse transcription PCR. Fibroblast growth factor 9
stimulated (P < 0.05) IGF-I-induced estradiol production in the presence of FSH and testosterone. However,
FGF9 had inconsistent effects on progesterone production, stimulating progesterone production in the pres-
ence of FSH and testosterone but inhibiting progesterone production in the presence of IGF-I, FSH, and
testosterone. Cell numbers were increased (P < 0.05) by
FGF9 in the presence of IGF-I and FSH but not in the
presence of FSH and absence of IGF-I. For FGFR2IIIc
mRNA studies, granulosa cells were treated with FSH,
IGF-I, FGF9, SHH, cortisol, PGE2, or WNT5A. Follicle-stimulating hormone alone had no effect (P > 0.10)
whereas IGF-I increased (P < 0.05) FGFR2IIIc mRNA
abundance. Cortisol, PGE2, SHH, and WNT5A had no
effect (P > 0.10) on FGFR2IIIc gene expression whereas
FGF9 in the presence of FSH and IGF-I inhibited (P <
0.05) FGFR2IIIc gene expression. In an in vivo study,
granulosa cells from large (7 to 14 mm) follicles had
greater (P < 0.05) abundance of FGFR2IIIc mRNA than
small (1 to 3 mm) or medium (4 to 6 mm) follicles. In
conclusion, IGF-I-induced FGFR2IIIc mRNA may be a
mechanism for increased responses to FGF9 in FSH plus
IGF-I-treated granulosa cells. Fibroblast growth factor 9
and IGF-I may work together as amplifiers of follicular
growth and granulosa cell differentiation by stimulating estradiol production and concomitantly stimulating
granulosa cell growth in pigs.
Key words: fibroblast growth factor 9,
fibroblast growth factor receptor 2IIIc, granulosa cell, insulin-like growth factor I, pigs
© 2014 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2014.92:511–519
doi:10.2527/jas2013-6989
INTRODUCTION
1This
work supported in part by: the Lew Wentz Foundation;
the NICHD, National Institutes of Health, through Agreement R15HD-066302, and the Oklahoma Agricultural Experiment Station
under project H-2510.
2The authors thank: the Oklahoma State University Wentz Project
scholarship program for financial support of J. R. Evans and J. A.
Williams, Ralph’s Packing Plant (Perkins, OK) for donation of porcine
ovaries, Dr. A. F. Parlow, National Hormone & Pituitary Program
(Torrance, CA) for purified FSH, and the Oklahoma State University
Recombinant DNA/Protein Core Facility for use of their equipment.
3Corresponding author: [email protected]
Received August 2, 2013.
Accepted November 24, 2013.
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Fibroblast growth factor 9 (FGF9) is part of the
fibroblast growth factor (FGF) family of hormones
consisting of 22 members (Moroni et al., 2002;
Chaves et al., 2012). A role for FGF9 in ovarian function was first reported by Drummond et al. (2007)
who showed that FGF9 mRNA and protein are localized in rat granulosa and theca cells, receptors exist
for FGF9 including fibroblast growth factor receptor
(FGFR) 2 and FGFR3, and FGF9 stimulates granulosa cell progesterone production. More recently, it
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Evans et al.
was discovered that FGF9 acts as a dedifferentiation
factor inhibiting IGF-I-induced granulosa and theca
cell steroidogenesis while stimulating cell proliferation in cattle (Schreiber and Spicer, 2012; Schreiber et
al., 2012). Using microarray technology, FGF9 mRNA
was found to be downregulated in cystic follicles as
compared to noncystic follicles of cattle (Grado-Ahuir
et al., 2011). In another study, gene expression of 1 of
the receptors for FGF9, FGFR2IIIc, was upregulated
by IGF-I in FSH-treated porcine granulosa cells (Grado-Ahuir et al., 2009). What other hormones regulate
FGFR2IIIc mRNA in granulosa cells is unknown. We
hypothesized that hormones in addition to IGF-I may
be regulating FGFR2IIIc mRNA in porcine granulosa
cells, and therefore we evaluated the effects of FSH,
PGE2, Sonic hedgehog (SHH), wingless-type mouse
mammary tumor virus integration site family member
5A (WNT5A), and cortisol on FGFR2IIIc mRNA expression in porcine granulosa cells. These hormones
have been shown to interact with the FGF system
(St-Jacques et al., 1999; Li et al., 2002; Salaria et al.,
2006; Finetti et al., 2009; Schreiber and Spicer, 2012).
We also hypothesized that IGF-I will enhance the response of granulosa cells to FGF9, a major ligand for
FGFR2IIIc (Santos-Ocampo et al., 1996; Kinkl et al.,
2003). Therefore, we evaluated the effects of FGF9 on
IGF-I-stimulated porcine granulosa cell proliferation
and steroidogenesis in addition to determining the hormonal and developmental control of FGFR2IIIc gene
expression in porcine granulosa cells.
MATERIALS AND METHODS
Reagents and Hormones
The reagents used in cell culture were Ham’s F-12,
Dulbecco modified Eagle medium (DMEM), gentamicin,
sodium bicarbonate, trypan blue, collagenase, deoxyribonuclease, and porcine serum (PS) from Sigma-Aldrich
Chemical Company (St. Louis, MO) and fetal calf serum
(FCS) from EquiTech-Bio, Inc. (Kerrville, TX).
The reagents used in RNA extraction were TRI
Reagent Solution and diethylpyrocarbonate (DEPC)treated water from Life Technologies, Inc. (Gaithersburg, MD), chloroform from Sigma-Aldrich Chemical Co., isopropyl alcohol, and ethanol from Pharmco
Products Inc. (Brookfield, CT).
The hormones used in cell culture were ovine FSH
(NIDDK-oFSH-20; activity: 175 X NIH-FSH-S1 units/
mg) from the National Hormone and Pituitary Program
(Torrance, CA), recombinant human IGF-I, recombinant
human FGF9, recombinant human SHH, and recombinant human WNT5A from R&D Systems (Minneapolis, MN), and cortisol and PGE2 from Sigma-Aldrich
Chemical Co. Amino acid sequence homology between
human and porcine IGF-I, FGF9, SHH, and WNT5A is
98, 99, 91, and 99%, respectively.
The reagents used for radioimmunoassays were
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[ I]iodo-progesterone (MP Biomedicals, Solon, OH),
anti-progesterone rabbit antiserum (X-16) provided by
Dr. P. Natashima Rao (Southwestern Foundation for Research Education, San Antonio, TX), and normal rabbit
serum (Invitrogen Corp., Carlsbad, CA), 125I-estradiol
(MP Biomedicals), anti-estradiol rabbit antibody (Lilly
Research Laboratories, Indianapolis, IN), and goat antirabbit antibody (Equitech-Bio, Inc., Kerrville, TX) as
previously described (Spicer and Enright, 1991).
Cell Culture
Ovaries from nonpregnant gilts were collected from
a local abattoir and transported to the lab in 0.9% saline
with 1% streptomycin/penicillin on ice. Follicular fluid
was aspirated from small to medium (1 to 5 mm) follicles and granulosa cells isolated as previously described
(Ranzenigo et al., 2008). The small to medium size category selected was based on previous studies (Garmey et
al., 2000; Ranzenigo et al., 2008). Granulosa cells were
washed twice in short-term medium (1:1 DMEM and
Ham’s F12, sodium bicarbonate, and gentamicin) and resuspended in serum-free medium containing collagenase
and deoxyribonuclease (Sigma-Aldrich Chemical Co.)
at 1.25 and 0.5 mg/mL, respectively, to prevent clumping. Viability of granulosa cells was determined by trypan
blue exclusion method on a 0.1 mm deep hemocytometer
(American Optical Corporation, Buffalo, NY). Viability
of the various pools of porcine granulosa cells used for
cell culture averaged 40.6 ± 3.5%.
On average, 3.0 × 105 viable cells per well were
plated on 24-well Falcon multiwell plates (number 3047;
Becton Dickinson, Lincoln Park, NJ) in 1 mL of medium
composed of a mixture of 1:1 DMEM and Ham’s F-12
containing 2.0 mM of glutamine, 0.12 mM of gentamicin, 38.5 mM sodium bicarbonate, 5% FCS, and 5% PS
(Ranzenigo et al., 2008). As previously described by
Ranzenigo et al. (2008), cells were cultured in an environment of 5% CO2 and 95% air at 38.5°C for the first
48 h with a medium change at 24 h. Cells were then
washed twice with 0.5 mL of serum-free medium followed by the addition of different hormonal treatments
applied in serum-free medium for either 24 or 48 h.
Ribonucleic Acid Extraction and Real-Time PCR
At the end of the treatment period, medium was
either aspirated or collected from each well depending
on the experiment and cells from 2 replicate wells were
lysed in 0.5 mL of TRI reagent solution (Life Technolo-
Fibroblast growth factor 9 effects in pigs
gies, Inc.) as previously described (Ranzenigo et al.,
2008). Briefly, 0.25 mL TRI reagent was added to all
wells and cells were lysed by repeated pipetting and then
combined with their respective replicates. Combined
wells were then transferred to 1.5 mL Eppendorf tubes.
Each treatment containing 4 wells generated 2 duplicate
samples of RNA. The RNA was suspended in 16.5 μL of
DEPC-treated water and stored at –80°C.
Ribonucleic acid was quantitated by spectrophotometry at 260 nm using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington,
DE). Aliquots of 1.5 μL of RNA were used to determine
the concentration in nanograms per microliter as well as
the purity given as a ratio of 260:280 nm where values
between 1.8 and 2.2 were acceptable. Ribonucleic acid
was then diluted to 10 ng/μL in DEPC-treated water.
Relative abundance of FGFR2IIIc mRNA was determined by fluorescent quantitative real-time PCR using
an ABI Prism 7500 sequence detection system (Applied
Biosystems, Foster City, CA). Primer and probe sequences used were previously described in Grado-Ahuir et al.
(2009). The dual labeled probes (FAM-TAMRA) were
obtained from Applied Biosystems as well as the 18S
primers and probe (VIC) for TaqMan polymerase as previously described (Ranzenigo et al., 2008; Grado-Ahuir et
al., 2009). For all RT-PCR runs, a no template control and
a no reverse transcriptase control were included to insure
the lack of contaminants in the master mix and the absence of any genomic DNA contamination, respectively.
Furthermore, the RT-PCR products were run on agarose
gels to verify the length and size of the expected target
genes, and the same RT-PCR cDNA samples were sequenced to verify the amplified product. Target gene expression was normalized to constitutively expressed 18S
rRNA and relative quantity of FGFR2IIIc mRNA was expressed as 2–ΔΔCt using the relative comparative threshold cycle (CT) method as described previously (Lagaly et
al., 2008; Grado-Ahuir et al., 2009, 2011).
Radioimmunoassay and Cell Counting
A double antibody progesterone RIA was conducted
as previously described (Baraño and Hammond, 1985;
Ranzenigo et al., 2008). Intra-assay CV averaged 5.9%.
A double antibody estradiol RIA was conducted as previously described (Spicer and Enright, 1991; Ranzenigo
et al., 2008). Intra-assay CV averaged 6.8%.
To determine cell numbers, culture medium was
aspirated and all wells were washed twice with 0.5 mL
saline (0.9%). Trypsin (0.25% solution; 0.5 mL) was
added and allowed to incubate for 20 min at room temperature. Wells were then scraped, aspirated, washed an
additional time, and diluted 1:10 in saline as previously
described (Ranzenigo et al., 2008). Cell number was de-
513
termined using a Z2 Coulter Particle Counter and Size
Analyzer (Beckman Coulter, Hialeah, FL).
Experimental Design
Experiment 1 was designed to evaluate the effect
of IGF-I on the dose response of FGF9 on steroidogenesis and cell numbers in FSH- and testosterone-treated
granulosa cells. Cells were cultured as described above
and treatments applied in triplicate culture wells for 48
h as follows (all treatments included 30 ng/mL of FSH
and 500 ng/mL of testosterone): 0, 3, 10, or 30 ng/mL of
FGF9 with or without 30 ng/mL of IGF-I. After 48 h of
treatment, medium was collected for estradiol and progesterone determinations and cells were counted. Medium was changed 24 h after initial treatment.
Experiment 2 was designed to evaluate the effect
of FSH, IGF-I, and PGE2 on abundance of FGFR2IIIc
mRNA in granulosa cells. Cells were cultured as described above in serum-containing medium for 48 h and
then washed and cultured for 24 h in serum-free medium
containing no additions (control), 30 ng/mL FSH, or 30
ng/mL IGF-I with or without PGE2 (300 ng/mL). Doses
of FSH, IGF-I, and PGE2 were selected based on previous
studies (Kage et al., 1999; Lagaly et al., 2008; Ranzenigo
et al., 2008; Grado-Ahuir et al., 2009). Prostaglandin E2
was evaluated because it has been shown to interact with
the FGF system (Kage et al., 1999; Finetti et al., 2009).
Experiment 3 was designed to evaluate the effect of
WNT5A and cortisol on FGFR2IIIc mRNA abundance
in FSH plus IGF-I-treated granulosa cells. Cells were
cultured as described in Exp. 2 and then cultured for an
additional 24 h in serum-free medium as follows: control
(30 ng/mL FSH plus 30 ng/mL IGF-I) with and without
WNT5A (300 ng/mL) or cortisol (300 ng/mL). Doses of
FSH, IGF-I, WNT5A, and cortisol were selected based
on previous studies showing that these doses significantly alter granulosa cell function (Spicer and Chamberlain, 1998; Spicer et al., 2002, 2009; Grado-Ahuir et
al., 2009) and that these hormones interact with the FGF
system (Li et al., 2002; Salaria et al., 2006; Pond et al.,
2010; Schreiber and Spicer, 2012).
Experiment 4 was designed to evaluate the effect
of SHH and FSH on abundance of FGFR2IIIc mRNA
in IGF-I-treated granulosa cells. Cells were cultured as
described in Exp. 2 and then cultured for an additional
48 h in serum-free medium as follows (all treatments
included 500 ng/mL testosterone): 30 ng/mL IGF-I and
30 ng/mL FSH plus IGF-I with or without SHH (500 ng/
mL). Medium was changed 24 h after initial treatment.
Doses of FSH, IGF-I, and SHH were selected based on
previous studies (Spicer et al., 2002, 2009; Grado-Ahuir
et al., 2009; Lagaly et al., 2008). Sonic hedgehog was
evaluated because previous studies indicated that hedge-
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Evans et al.
hog proteins interact with the FGF system (Belaoussoff
et al., 1998; St-Jacques et al., 1999).
Experiment 5 was designed to evaluate the effect
of FGF9 on abundance of FGFR2IIIc mRNA in granulosa cells. Cells were cultured as described for Exp. 2
and then cultured for an additional 48 h in serum-free
medium containing FSH (30 ng/mL) and IGF-I (30 ng/
mL) with or without FGF9 (30 ng/mL). Medium was
changed 24 h after initial treatment. Doses of FGF9,
FSH, and IGF-I were selected based on previous studies (Spicer et al., 2002; Grado-Ahuir et al., 2009; Schreiber and Spicer, 2012).
Experiment 6 was designed to determine if FGFR2IIIc gene expression changes during follicle development. Granulosa cells were collected from small (1–3
mm), medium (4–6 mm), and large (7–14 mm) porcine
follicles (n = 6 to 8 pools of cells per size group) as described earlier except that cells were not cultured and
immediately after isolation, cells were lysed with TRI
reagent and frozen for later extraction of RNA. The
use of 1 to 3, 4 to 6, and 7 to 14 mm follicles to define
small, medium, and large size categories, respectively,
was to match classifications of previous studies (Channing et al., 1976; Baraño and Hammond, 1985; Grasselli et al., 2003).
Statistical Analyses
For Exp. 1 to 5, 3 different pools of porcine granulosa
cells were used as experimental replicates. Each pool of
cells originated from a volume of 1 to 2 mL of follicular
fluid (from 2 to 4 gilts) per pool within each experimental replicate. At least 1 of the replicate experiments was
conducted using cells collected from a separate trip to the
slaughterhouse. Ovaries from a total of 21 gilts were used
for these experiments. Steroid production was expressed
as nanograms or picograms/105 cells per 24 h, and cell
numbers determined at the end of the experiment were
used for this calculation. For RNA experiments, medium was applied to 4 wells and duplicate samples for
each pool of cells were derived by combining RNA from
2 wells. The treatment effects of the dependent variables
(e.g., steroid production, FGFR2IIIc mRNA abundance)
were determined using ANOVA and the general linear
models (GLM) procedure of SAS for Windows (version
9.2; SAS Inst. Inc., Cary, NY). Data from Exp. 1 were
analyzed as a 2 × 4 factorial ANOVA whereas Exp. 2 data
were analyzed as a 2 × 3 factorial ANOVA and Exp. 4 data
were analyzed as a 2 × 2 factorial ANOVA. Data from
Exp. 3, 5, and 6 were analyzed as a 1-way ANOVA. Mean
differences were determined by Fisher’s protected least
significant differences test (Ott, 1977) if significant main
effects (i.e., P < 0.05) in the ANOVA were detected. Data
were presented as the least square means ± SEM.
RESULTS
Experiment 1: Effect of Fibroblast Growth
Factor 9 and IGF-I on Steroidogenesis
and Numbers of Granulosa Cells
Fibroblast growth factor 9 increased (P < 0.05) FSHinduced estradiol production in a dose-dependent manner
with or without IGF-I in porcine granulosa cells (Fig. 1A).
Maximal stimulation by 30 ng/mL of FGF9 increased estradiol production by 4.6-fold and 8.6-fold in the presence
and absence of IGF-I, respectively. The estimated effective
dose (ED50) of FGF9 stimulating 50% of the maximal aromatase response (calculated from stimulation curves that
were linearized using a semi-log plot) averaged 13 ng/mL
in IGF-I-treated cells. Fibroblast growth factor 9 at 3 and
30 ng/mL had no effect on progesterone production in the
presence of IGF-I plus FSH, but FGF9 at 30 ng/mL weakly
increased (P < 0.05) progesterone production (by 20%) in
the presence of FSH and testosterone (Fig. 1B). At 10 ng/
mL, FGF9 decreased (P < 0.05) progesterone production
by 20% in the presence of FSH, IGF-I, and testosterone
(Fig. 1B). Insulin-like growth factor-I inhibited (P < 0.05)
progesterone production by 18 and 33% in the presence
of 10 and 30 ng/mL of FGF9, respectively, but had no effect in the presence of 0 or 3 ng/mL of FGF9 (Fig. 1B). In
the presence of IGF-I and FSH, FGF9 at 10 and 30 ng mL
stimulated (P < 0.05) granulosa cell numbers by 85 and
76%, respectively, but had no significant effect at 3 ng/mL
(ED50 of 8 ng/mL in IGF-I-treated cells; Fig. 2). In the presence of FSH but absence of IGF-I, FGF9 had no significant
effect on granulosa cell numbers (Fig. 2).
Experiment 2: Effect of FSH, IGF-I, and PGE2 on
Abundance of FGFR2IIIc mRNA in Granulosa Cells
Treatment of granulosa cells with PGE2, FSH, or
both had no significant (P > 0.10) effect on FGFR2IIIc
mRNA abundance (Fig. 3A). However, treatment of
granulosa cells with IGF-I increased (P < 0.05) FGFR2IIIc mRNA abundance by 1.8-fold (Fig. 3A) in the
presence and absence of PGE2.
Experiment 3: Effect of Wingless-Type Mouse
Mammary Tumor Virus Integration Site Family
Member 5A and Cortisol on mRNA Abundance of
FGFR2IIIc in FSH plus IGF-I-Treated Granulosa Cells
Treatment of granulosa cells with either WNT5A or
cortisol had no effect (P > 0.10) on FGFR2IIIc mRNA
abundance in FSH plus IGF-I-treated granulosa cells.
Relative abundance of FGFR2IIIc mRNA in control,
WNT5A, and cortisol treated cultures averaged 2.03,
2.20, and 1.82 ± 0.26, respectively.
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Fibroblast growth factor 9 effects in pigs
Figure 2. Effect of fibroblast growth factor 9 (FGF9; 0, 3, 10, or 30 ng/
mL) on numbers of porcine granulosa cells treated with FSH and FSH plus
IGF-I (30 ng/mL) in vitro (Exp. 1). All cells were concomitantly treated with
30 ng/mL of FSH and 500 ng/mL of testosterone. a,b,c,dMeans (±SEM; n = 9)
without a common letter differ (P < 0.05).
Experiment 5: Effect of Fibroblast Growth Factor 9 on
Abundance of FGFR2IIIc mRNA in Granulosa Cells
Treatment of granulosa cells with FGF9 in the presence of FSH plus IGF-I decreased (P < 0.05) abundance
of FGFR2IIIc mRNA by 44% compared to treatment of
FSH plus IGF-I without FGF9 (Fig. 3B).
Experiment 6: Changes in FGFR2IIIc Gene Expression during Follicle Development
Figure 1. Effect of fibroblast growth factor 9 (FGF9) on steroidogenesis
in porcine granulosa cells (Exp. 1). Panel A: Effect of FGF9 (0, 3, 10, or 30
ng/mL) on estradiol production induced by FSH and FSH plus IGF-I (30 ng/
mL) in cultured granulosa cells. Panel B: Effect of FGF9 (0, 3, 10, or 30 ng/
mL) on progesterone production induced by FSH and FSH plus IGF-I (30 ng/
mL) in cultured granulosa cells. All cells were concomitantly treated with 30
ng/mL of FSH and 500 ng/mL of testosterone. a,b,c,d,e,fWithin a panel, means
(±SEM; n = 9) without a common letter differ (P < 0.05).
Experiment 4: Effect of Sonic Hedgehog
and FSH on Abundance of FGFR2IIIc mRNA
in IGF-I-Treated Granulosa Cells
Treatment of granulosa cells with 500 ng/mL of
SHH with IGF-I or IGF-I plus FSH did not affect (P >
0.10) FGFR2IIIc mRNA abundance. Furthermore,
FSH did not influence (P > 0.10) abundance of FGFR2IIIc mRNA in the presence of IGF-I or IGF-I plus
SHH. Relative abundance of FGFR2IIIc mRNA in
IGF-I and IGF-I plus FSH treated cultures averaged
1.47 and 1.50 ± 0.12, respectively, in the absence of
SHH and 1.55 and 1.85 ± 0.12, respectively, in the
presence of SHH.
Abundance of FGFR2IIIc mRNA did not differ (P >
0.10) between small- and medium-sized follicles but
was 100-fold greater (P < 0.05) in large follicles as compared to either small or medium follicles (Fig. 4).
DISCUSSION
The effects of FGF9 on ovarian cell steroidogenesis
in pigs have not been studied previously. We found a dramatic dose-dependent increase in estradiol production induced by FGF9 in the presence of IGF-I in granulosa cells
treated with FSH and testosterone. To a much lesser extent,
FGF9 at 30 ng/mL stimulated progesterone production in
the absence of IGF-I and is in agreement with Drummond
et al. (2007) who found that treatment of cultured rat granulosa cells with 1 to 50 ng/mL of FGF9 increases basal and
FSH-induced progesterone production. At 5 and 50 ng/mL,
FGF9 also stimulates basal but not hCG-induced testosterone production by mouse Leydig cells (Lin et al., 2010)
and a related FGF, FGF2 (at 0.64 to 10 ng/mL), stimulates
both basal and hCG-induced testosterone production in
cultured porcine Leydig cells treated with insulin (Sordoillet et al., 1992). In contrast, FGF9 (at 10 and 30 ng/mL) is
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Evans et al.
Figure 4. Effect size of follicle on abundance of granulosa cell FGFR2IIIc mRNA in porcine follicles (Exp. 6). Granulosa cells from small (1 to
3 mm), medium (4 to 6 mm), and large (7 to 11 mm) follicles were collected,
RNA isolated, and quantitative reverse transcription-PCR used to quantify
mRNA levels. Values are normalized to constitutively expressed 18S ribosomal
RNA. a,bWithin a panel, means (n = 6 to 8 pools per mean) without a common
superscript differ (P < 0.05).
Figure 3. Effect of FSH, IGF-I, and PGE2 (Exp. 2) and fibroblast growth
factor 9 (FGF9; Exp. 5) on abundance of FGFR2IIIc mRNA in porcine granulosa cells. Panel A: Granulosa cells were treated with control (no additions),
FSH (30 ng/mL), IGF-I (30 ng/mL), and/or PGE2 (300 ng/mL) in Exp. 2. Panel
B: Granulosa cells were treated with FSH (30 ng/mL) and IGF-I (30 ng/mL)
with and without FGF9 (30 ng/mL) in Exp. 5. Values are means of 3 replicate
experiments and normalized to constitutively expressed 18S ribosomal RNA.
a,bMeans (±SEM; n = 6) without a common superscript differ (P < 0.05).
a potent inhibitor of IGF-I-induced steroidogenesis in gonadotropin-treated bovine granulosa (Schreiber and Spicer,
2012) and theca (Schreiber et al., 2012) cells. Why FGF9
stimulates progesterone production in the absence of IGFI but inhibits it in the presence of IGF-I is unclear but may
be related to the increase in cell numbers induced by FSH
plus IGF-I. Previously, bovine granulosa cell proliferation
and progesterone production has been shown to require
de novo cholesterol synthesis (Spicer et al., 1996). Perhaps granulosa cells’ priority is to use cholesterol for cell
proliferation rather than progesterone production. Interestingly, FGF2 inhibits steroidogenesis in porcine (Biswas et
al., 1988) and bovine (Vernon and Spicer, 1994) granulosa
cells. Because multiple receptors for FGF exist, a specific
cell type and species response to a specific FGF is likely
determined by the cadre of FGFR that are present in that
cell type. For the 22 FGF ligands, there are a total of 7
FGF receptors (which includes splice variants of 3 of the
4 main FGF receptor subtypes) each of which bind some
but not all of the FGF ligands (Ornitz et al., 1996; Moroni
et al., 2002; Chaves et al., 2012). The preferred receptors
for FGF9 are FGFR2c, FGFR3c, FGFR3b, and FGFR4
(Santos-Ocampo et al., 1996; Kinkl et al., 2003).
In the present study, abundance of FGFR2IIIc mRNA
did not differ between small and medium-sized follicles
but large follicles had 100-fold greater abundance of FGFR2IIIc mRNA than either small or medium follicles.
Similarly, in cattle, granulosa cells from large follicles
have 3-fold greater FGFR2IIIb mRNA abundance than do
those from small or medium follicles (Parrott and Skinner,
1998). In contrast, FGFR2IIIc mRNA abundance in bovine theca and granulosa cells did not differ among follicles with various estradiol levels (Berisha et al., 2004)
or change between 0 and 60 h post-GnRH (Berisha et al.,
2006). However, FGFR3 mRNA increased with follicle
estradiol content in cattle (Buratini et al., 2005). Therefore, the specific FGFR that is present and physiologically
regulated within follicles may depend upon species and
cell type. In support of the idea that abundance of FGF
receptors changes during follicle growth in pigs, FGFR2IIIc and FGFR2IIIb mRNA abundance in theca cells
increased during follicle growth but neither changed in
porcine granulosa cells (Schams et al., 2009). Why FGFR2IIIc mRNA was greater in large vs. medium and small
follicles in the present study and not in a previous study
(Schams et al., 2009) is unclear but may be due to methodological differences. In vitro, FGF2 inhibits progesterone production in porcine granulosa cells from small fol-
Fibroblast growth factor 9 effects in pigs
licles but stimulates progesterone production in granulosa
cells from medium follicles (Biswas et al., 1988; Grasselli
et al., 2003) further suggesting that changes in FGF responses occur during follicular growth in pigs.
Similar to previous studies in nonovarian tissue (Cohen and Chandross, 2000), we found that FGF9 downregulated abundance of mRNA for 1 of its own receptors, FGFR2IIIc, suggesting that an autocrine FGF9-FGFR2IIIc signaling loop may exist in porcine granulosa
cells. This autocrine feedback inhibition would presumably operate to prevent overstimulation by FGF9. A similar effect has been reported during teratocarcinoma cell
differentiation, further suggesting that cell surface FGF
receptors are downregulated as an autocrine response to
FGF ligand binding (Moscatelli, 1994). Consistent with
the present study, IGF-I increased FGFR2IIIc mRNA
in FSH-treated porcine granulosa cells (Grado-Ahuir
et al., 2009), and based on results of the present study,
FSH does not appear to influence the stimulatory effect
of IGF-I on FGFR2IIIc gene expression. However, FSH
and IGF-I synergistically stimulate steroidogenesis and
mitogenesis in porcine granulosa cells (Baraño and Hammond, 1985; Ranzenigo et al., 2008). Therefore, as follicles grow, stimulation of FGFR2IIIc gene expression by
IGF-I would enhance FGF9 signaling, further promoting
the differentiation effects of FGF9 as well as stimulating angiogenesis (Gómez-Raposo et al., 2009; Sakurai
and Kudo, 2011), an important event as follicles grow
larger (Jiang et al., 2004; Martelli et al., 2009; Chaves
et al., 2012). In contrast, FGF9 has been proposed as a
dedifferentiation factor in bovine follicles as FGF9 is a
potent inhibitor of IGF-I-induced steroidogenesis of both
granulosa and theca cells in cattle (Schreiber and Spicer,
2012; Schreiber et al., 2012).
The present studies also indicate that WNT5A and
SHH have no regulatory effect on FGFR2IIIc gene expression in porcine granulosa cells although winglessrelated integration site proteins control cell differentiation and proliferation in mouse chondrocytes (Yang et
al., 2003) and regulate bovine granulosa cell steroidogenesis (Castañon et al., 2012) while hedgehog proteins (SHH or Indian hedgehog) control proliferation
of granulosa (Russell et al., 2007) and theca (Spicer et
al., 2009) cells. Both cortisol (Spicer and Chamberlain,
1998; Viveiros and Liptrap, 1999) and PGE2 (Tsang
and Carnegie, 1984; Moon et al., 1986) stimulate
granulosa cell steroidogenesis, but like WNT5A and
SHH, neither cortisol nor PGE2 affected FGFR2IIIc
gene expression. In contrast to the present study, dexamethasone significantly decreased FGFR2 and FGFR1
mRNA expression in rat gastric epithelial cells (Luo et
al., 2008), supporting the idea that species and/or celltype differences likely exist in terms of the hormonal
control of FGFR2IIIc gene expression.
517
Similar to what we have reported for bovine granulosa cells (Schreiber and Spicer, 2012), we found that
FGF9 stimulated cell numbers in a dose-dependent
manner in FSH- plus testosterone-treated granulosa
cells. In the presence of IGF-I and FSH, FGF9 had an
ED50 of 8 ng/mL, which is similar to its ED50 of 6 ng/
mL reported for bovine granulosa cells (Schreiber and
Spicer, 2012) and further suggests that the FGF9 effect
in bovine and porcine granulosa cells is being mediated by high affinity receptors because these values
approximate the dissociation constant (Kd) values obtained for FGF9 and FGF2 binding to their respective
receptors (Neufeld and Gospodarowicz, 1985; Hecht
et al., 1995). Unfortunately, concentrations of FGF9
in serum or follicular fluid have not been reported.
Therefore, FGF9 may dually support follicular development in pigs by stimulating granulosa cell proliferation and differentiation. Previous evidence indicated
that FGF9 acts as a potential embryonic growth factor
during placental attachment in pregnant sows (Østrup
et al., 2010), as an endometrial stromal growth factor in humans (Tsai et al., 2002), and as an embryonic
stem cell growth factor in mice (Peterslund and Serup,
2011). In the absence of IGF-I, FGF9 had no significant effect on granulosa cell numbers, suggesting that
IGF-I may increase the sensitivity of granulosa cells to
the mitogenic effects of FGF9. Indeed, the present experiments evaluating FGFR2IIIc mRNA indicated that
IGF-I may increase FGF9 sensitivity by increasing
numbers and/or sensitivity of 1 of the FGF9 receptors
in porcine granulosa cells. Studies using rat skeletal
muscle satellite cells indicate IGF-I can induce FGFR1
mRNA (Sheehan and Allen, 1999). Previously, FGF2
has been shown to be a potent stimulator of granulosa
cell proliferation in pigs (Gospodarowicz and Bialecki, 1979), rats (Anderson and Lee, 1993), and cattle
(Hoshi et al., 1995; Rodgers et al., 1996), suggesting
multiple FGF may stimulate proliferation of granulosa
cells in vertebrates.
Our results show that FGF9 upregulates estradiol
production and cell numbers induced by IGF-I in FSHplus testosterone-treated porcine granulosa cells. We
also show developmental and hormone (i.e., IGF-I and
FGF9) sensitive changes in FGFR2IIIc mRNA abundance in porcine granulosa cells that are consistent
with the notion that IGF-I may enhance FGF9-induced
granulosa cell differentiation via increased FGFR2IIIc
gene expression. More research is needed to elucidate
the role of the various types of FGF receptors and their
ligands during follicular growth in pigs. An understanding of FGF9’s mechanism of action and its receptor regulation may also lead to a better understanding
of the difference in follicular development between
monovular and polyovular species.
518
Evans et al.
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