Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells

Abstract

Context
Human granulosa cells (hGCs) produce and respond to insulin-like growth factor 2 (IGF2) but whether the oocyte participates in IGF2 regulation in humans is unknown.

Objective
To determine the role of oocyte-secreted factors (OSFs) such as growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in IGF2 production by hGCs.

Design
Primary human cumulus GCs in culture.

Setting
University infertility center.

Patients or Other Participants
GCs of women undergoing in vitro fertilization.

Intervention(s)
Cells treated with GDF9 and BMP15 in the presence of vehicle, FSH, dbcAMP, or SMAD inhibitors.

Main Outcome Measure(s)
Quantification of mRNA, protein, promoter activity, and DNA methylation.

Results
FSH-stimulation of IGF2 (protein and mRNA) was significantly potentiated by the GDF9 and BMP15 (G+B) combination (P<0.0001) in a concentration-dependent manner showing a maximal effect at five ng/ml each. However, GDF9 or BMP15 alone or in combination (G+B) have no effect on IGF2 in the absence of FSH. FSH stimulated IGF2 promoter 3 activity, but G+B had no effect on promoter activity. G+B potentiated IGF2 stimulation by cAMP. SMAD3 inhibitors inhibited G+B enhancement of IGF2 stimulation by FSH (P<0.05) but had no effect on FSH induction. Moreover, inhibition of insulin-like growth factor receptor partially blocked G+B potentiation of FSH actions (P<0.009).

Conclusions
For the first time, we show that the oocyte actively participates in the regulation of IGF2 expression in hGCs, an effect that is mediated by the specific combination of G+B via SMAD2/3, which in turn target mechanisms downstream of the FSH receptor.

Full text

doi:10.1210/clinem/dgz057 J Clin Endocrinol Metab, January 2020, 105(1):327–335 https://academic.oup.com/jcem 327
Abbreviations: AMH, anti-Müllerian hormone; ANOVA, analysis of variance; BMP15,
bone morphogenetic protein 15; ELISA, enzyme-linked immunosorbent assay; FSH,
follicle-stimulating hormone; GDF9, growth differentiation factor 9; hGC, Human
granulosa cell; IGF2, insulin-like growth factor 2; IVF, in vitro fertilization; LSD, least
significant difference; OSF, oocyte-secreted factor; qPCR, quantitative real-time poly-
merase chain reaction; SEM, standard error of the mean
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in USA
© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.
permissions@oup.com
Received 11 July 2019. Accepted 20 September 2019.
First Published Online 7 October 2019.
CLINICAL RESEARCH ARTICLE
Regulation of Insulin-Like Growth Factor 2 by Oocyte-
Secreted Factors in Primary Human GranulosaCells
Elie Hobeika,1 Marah Armouti,2 MicheleA. Fierro,1 Nichola Winston,1
Humberto Scoccia,1 AlberuniM. Zamah,3 and Carlos Stocco2
1Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology,
University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612; 2Department of Physiology
and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612; and 3Division of Reproductive
Endocrinology and Infertility, Department of Obstetrics and Gynecology, the University of Chicago
Medicine, Chicago, Illinois 60637
ORCiD number: 0000-0001-7961-6681 (Carlos Stocco).
Context: Human granulosa cells (hGCs) produce and respond to insulin-like growth factor 2
(IGF2) but whether the oocyte participates in IGF2 regulation in humans is unknown.
Objective: To determine the role of oocyte-secreted factors (OSFs) such as growth
differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in IGF2 production
by hGCs.
Design: Primary human cumulus GCs in culture.
Setting: University infertility center.
Patients or Other Participants: GCs of women undergoing in vitro fertilization.
Intervention(s): Cells treated with GDF9 and BMP15 in the presence of vehicle, follicle-
stimulating hormone (FSH), dibutyryl cyclic-AMP (dbcAMP), or mothers against decapentaplegic
homolog (SMAD) inhibitors.
Main Outcome Measure(s): Quantification of mRNA, protein, promoter activity, and DNA
methylation.
Results: FSH stimulation of IGF2 (protein and mRNA) was significantly potentiated by the GDF9
and BMP15 (G+B) combination (P<0.0001) in a concentration-dependent manner showing a
maximal effect at 5ng/mL each. However, GDF9 or BMP15 alone or in combination (G+B) have
no effect on IGF2 in the absence of FSH. FSH stimulated IGF2 promoter 3 activity, but G+B had
no effect on promoter activity. G+B potentiated IGF2 stimulation by cAMP. SMAD3 inhibitors
inhibited G+B enhancement of IGF2 stimulation by FSH (P<0.05) but had no effect on FSH
induction. Moreover, inhibition of insulin-like growth factor receptor partially blocked G+B
potentiation of FSH actions (P<0.009).
Conclusions: For the first time, we show that the oocyte actively participates in the regulation
of IGF2 expression in hGCs, an effect that is mediated by the specific combination of G+B via
SMAD2/3, which in turn target mechanisms downstream of the FSH receptor. (J Clin Endocrinol
Metab 105: 327–335, 2020)
Downloaded from https://academic.oup.com/jcem/article-abstract/105/1/327/5582040 by lib-electronic@uic.edu user on 05 June 2020

Folliculogenesis is a long process lasting several
months in humans that transforms a primordial fol-
licle into a dominant preovulatory follicle (1). The suc-
cess of folliculogenesis depends on a close interaction
between the two main components of the follicle, the
granulosa cells (GCs) and the oocyte. The active role of
the oocyte in folliculogenesis was described in the early
1970s, when it was observed that oocyte ablation leads
to impaired folliculogenesis and follicle luteinization
(2). Later, Nekola etal. (3) conrmed these ndings and
showed that GCs cultured near oocytes appeared to be
less luteinized than those cultured without oocytes. It
was not until two decades later that landmark studies
identied two oocyte-specic growth factors (OSFs),
growth differentiation factor 9 (GDF9) and bone mor-
phogenetic protein 15 (BMP15) (4, 5). The inuence of
these factors on the process of folliculogenesis is now
well accepted.
GDF9 and BMP15 have a high degree of homology
in their sequence and structure. They have similar ex-
pression patterns and functions in the ovary (6–8).
Moreover, recent evidence suggests that GDF9 and
BMP15 form heterodimers, which mediate some of
their actions (9, 10). However, the role of GDF9 and
BMP15 in the control of folliculogenesis has been elu-
cidated exclusively using animal models and cell lines.
These reports showed that GDF9 is a critical player in
the follicular development of mice and sheep, whereas
BMP15 is not essential for fertility in mice but critical
in sheep (4, 5, 8). However, GDF9 and BMP15 function
in the human ovary and in human primary ovarian cells
remains unexplored, mainly due to the lack of appro-
priate experimental approaches.
We previously validated the use of cumulus cells
obtained from patients undergoing in vitro fertiliza-
tion (IVF) as a proxy of undifferentiated GCs to study
follicle-stimulating hormone (FSH) actions in humans
(11, 12). Also, we examined the interaction between
FSH, GDF9, and BMP15 on GC function and showed
that, whereas FSH inhibits anti-Müllerian hormone
(AMH) production in primary human GCs, the com-
bination of GDF9 and BMP15 (GB) potentiates the pro-
duction of AMH (13). The interaction between FSH and
OSFs is not always antagonistic. For instance, our most
recent report shows that GDF9 and BMP15 potentiate
FSH stimulation of aromatase and estrogen production,
two hallmarks of GC differentiation (14).
Previous studies demonstrated an essential role of
the insulin-like growth factor (IGF) system on the in-
duction of aromatase and estradiol synthesis in human
GCs (11, 15, 16). Interactions between FSH and IGFs
have also been shown to upregulate the production of
estradiol and progesterone in several species such as ro-
dent (17), porcine (18), and bovine (19), beyond that
of either factor alone. However, the IGF system differs
signicantly between rodents and humans, making the
use of animal models unsuitable for studies aiming to
elucidate the regulation of the IGF system in humans.
For instance, while IGF1 is mostly expressed in mouse
GCs, IGF2 is the only IGF expressed in human GCs (11,
16, 20). We have also demonstrated that FSH inhibits
IGF1 expression in rodent GCs, whereas FSH stimu-
lates IGF2 expression in human GCs (11, 15, 16). These
ndings were conrmed in a recent report showing
that IGF2 expression in human GCs increases approxi-
mately 64-fold as follicles progress from the small an-
tral to the preovulatory stage, a process that is entirely
regulated by FSH (21). However, the mechanisms con-
trolling the expression of IGF2 in human GCs remains
unexplored. Here, we examined the interaction between
GDF9, BMP15, and FSH on the regulation of IGF2 in
human primary cumulus GCs. We tested the hypothesis
that GDF9 and BMP15 interact with FSH to stimulate
the expression of IGF2 and that this effect mediates, at
least in part, the enhancing effect of OSFs on aromatase
and estradiol production.
Materials and Methods
Human cumulus cell culture
Human cumulus cells were collected from patients under-
going IVF at the University of Illinois IVF Center under an
Institutional Review Board-exempt protocol. No patient
information was collected for reporting. After controlled
ovarian stimulation, mature follicles were aspirated from
women undergoing IVF. The cumulus–oocyte complexes were
then removed from the follicular aspirates, and the cumulus
cells were separated from the oocyte manually. The cumulus
cells from all follicles for a single patient were then pooled and
transported immediately to the laboratory where they were
dispersed by hyaluronidase digestion (80IU/μL) and then cen-
trifuged at 500× g for 2 minutes. Cells were incubated at room
temperature in red blood cell lysis buffer for 5 minutes to elim-
inate contaminating erythrocytes, centrifuged again at 500×
g for 2 minutes. Cells were suspended in 0.4mL of serum-free
and phenol red-free DMEM/F12-0.25% BSA (Sigma-Aldrich)
media supplemented with penicillin (50IU/mL), streptomycin
(50g/mL), and sodium bicarbonate (1.2g/L; Sigma-Aldrich).
Cells were then cultured on plates precoated with BD Matrigel
(BD Biosciences) at a density of 6× 104/mL. Cells were cultured
for at least 48 hours, followed by treatment with various com-
binations of hormones and signaling inhibitors. Treatments
included human recombinant FSH (Serono), GDF9 (R&B),
BMP 15 (R&B), inhibitors of SMAD2/3 (SB431542, Tocris),
SMAD 3 (SIS3; Cayman Chemical Company), or SMAD1/5/8
(LDN-193189, Selleck Chemicals), IGF1R inhibitor AEW
(NVP-AEW451) (Calbiochem), and dbcAMP (Sigma). Cells
of different individuals were not pooled together.
328 Hobeika et al. GDF9 and BMP15 Enhance FSH-Induced IGF2 Expression J Clin Endocrinol Metab, January 2020, 105(1):327–335
Downloaded from https://academic.oup.com/jcem/article-abstract/105/1/327/5582040 by lib-electronic@uic.edu user on 05 June 2020

Polymerase chain reaction
Total RNA was isolated using TRIzol Reagent (Invitrogen)
as recommended in the manufacturer’s protocol. Total RNA
(1 µg) was reverse-transcribed using anchored oligo-dT pri-
mers (Integrated DNA Technologies), and reverse transcriptase
from Moloney murine leukemia virus (Invitrogen) at 37°C for
2 hours. The resulting cDNA was diluted to a nal concentra-
tion of 10ng/µL. Quantitative real-time polymerase chain re-
action (qPCR) was performed using intron-spanning primers
specic for the detection of ribosomal protein L19 (RPL19)
and promoter 3-specic IGF2 transcripts. The number of
copies for each gene was calculated using a standard curve
made with a serial dilution of the respective cDNA (22). Once
the number of copies of the gene of interest was obtained,
the relative expression is calculated as the ratio between the
copy number of the gene of interest and the copy number of
the housekeeping gene Rpl19. Primer sequences are available
upon request.
Promoter activityassay
The IGF2p3–Luc reporter was cloned into a lentivirus
reporter vector using the method described previously (14).
Lentiviruses containing this construct were generated as de-
scribed (14). Empty plasmids were used as controls. Cells were
infected with lentivirus and after overnight incubation treated
with FSH, GDF9, BMP15, or their combination. At 48 hours,
luciferase activity was determined in 50μL of lysates as previ-
ously described (14).
Promoter 3 methylation
We examined the methylation status of the 37 CpG sites
found in the IGF2 promoter 3 using bisulte sequencing.
Cumulus cells were treated with vehicle or G+B in the pres-
ence or absence of FSH for 48 hours before genomic DNA iso-
lation. An aliquot of DNA was treated with sodium bisulte
and puried using Wizard DNA clean-up system (Promega).
To quantify CpG methylation, IGF2 promoter 3 was ampli-
ed using primers specic for bisulte-treated DNA. Then,
next-generation sequencing libraries were prepared from the
original PCR amplicon using primers that introduce Illumina
adaptors. Samples were then sequenced using Illumina
NextSeq. Athreshold of 98% conversion of no-CpG C to
uracil was set for the sample to be included in the analysis. The
sequences were aligned to the IGF2-P3 sequence and 5 mC
percentage calculated.
Enzyme-linked immunosorbentassay
Enzyme-linked immunosorbent assay (ELISA) for IGF2
protein secretion was performed on the supernatant of human
GCs treated with the different combinations of FSH, GDF9,
and BMP15 using the Quantikine® ELISA kit for Human
IGF2 (R&D Systems).
Statistical analysis
Each experiment was run at least in duplicate, and data for
continuous variables are presented as mean values±standard
error of the mean (SEM). Statistical comparisons of mean
values between groups were performed with paired t-tests,
and multiple comparisons were performed with one-way ana-
lysis of variance (ANOVA) with repeated measures followed
by Bonferroni adjustment, Fisher’s least signicant difference
(LSD) test or Tukey’s multiple comparisons test, where appro-
priate. Differences were considered to be statistically signi-
cant if P<0.05.
Results
The combination of GDF9 and BMP15 potentiates
FSH induction ofIGF2
We previously demonstrated that the combination of
GDF9 and BMP15 potentiates the stimulatory effect of
FSH on IGF2 expression (14). To further evaluate the
impact of GDF9 and BMP15 on the expression of IGF2
mRNA, primary hGCs were treated with increasing
concentrations of GDF9 and BMP15 in the presence
of 50 ng/mL of FSH. The concentration of FSH was
initially chosen based on that used in previous reports
(13, 14). As previously shown, FSH signicantly in-
creased IGF2 mRNA transcripts when compared with
nontreated cells using the t-test (P <0.05) (Fig. 1A). This
stimulatory effect of FSH on IGF2 was signicantly
augmented by the addition of the combination GDF9
and BMP15 (G+B) to the media (P<0.01). As shown in
Fig. 1A, IGF2 mRNA levels were signicantly increased
in human GCs treated with G+B at concentrations of
2.5 and 10 ng/mL in the presence of FSH compared
with other treatments and the untreated control cells.
Thus, treatment with FSH and either GDF9 or BMP15
at concentrations of 10ng/mL, or treatment with 10ng/
mL G+B in the absence of FSH did not stimulate IGF2
compared with FSH-only treated cells. To maintain con-
sistency with previous reports, a dose of 5ng/mL for
each GDF9 and BMP15 was used in the remainder of
the experiments (13, 14).
Next, we examined whether G+B was able to en-
hance the effect of increasing concentrations of FSH. As
shown in Fig. 1B, although G+B cotreatment enhanced
the effect of FSH, increasing concentrations of FSH did
not further increase IGF2 expression, suggesting that
the stimulation of IGF2 appears to be maximal in the
presence of FSH and G+B.
GDF9 and BMP15 potentiate FSH stimulation of
IGF2 proteinlevels
Next, we examined whether the effect of G+B on
IGF2 mRNA levels translates into an increase in IGF2
protein. IGF2 was measured in the supernatant of cul-
tures treated as indicated above using ELISA. In con-
cordance with ndings at the mRNA levels, cotreatment
of FSH with G+B potentiated the stimulatory effect of
FSH on IGF2 protein expression (Fig. 2). IGF2 concen-
tration in the supernatant of cells treated with FSH and
G+B cotreatment was increased 1.9-fold compared with
doi:10.1210/clinem/dgz057 https://academic.oup.com/jcem 329
Downloaded from https://academic.oup.com/jcem/article-abstract/105/1/327/5582040 by lib-electronic@uic.edu user on 05 June 2020

the control group (66.67±5.5 vs 33.99±0.6 pg/mL,
P<0.01) and 1.5-fold compared with the group treated
with FSH alone (66.67 ± 5.5 vs 43.15 ± 0.4 pg/mL,
P < 0.05). A paired t-test comparing the FSH-treated
group with the untreated control group showed a stat-
istically signicant increase in IGF2 protein production
(43.15±0.4 vs 33.99±0.6pg/mL, P<0.01). Taken to-
gether, these ndings indicate a robust positive effect of
the GDF9 and BMP15 combination on the stimulatory
effect of FSH on IGF2 protein production.
GDF9 and BMP15 potentiate cAMP stimulation
ofIGF2
Next, we determined whether G+B interacts with
cAMP, the primary second messenger of the FSH re-
ceptor, to stimulate IGF2. As shown in Fig. 3, treat-
ment with dbcAMP (a cAMP analog) alone stimulated
IGF2 mRNA levels compared with the control group
(P<0.05). However, IGF2 mRNA expression was sig-
nicantly higher in cells treated with GDF9, BMP15,
and dbcAMP than in cells treated with dbcAMP alone
Figure 1. The combination of FSH, GDF9, and BMP15 (GB)
potentiate FSH-induced IGF2 expression in a dose-dependent manner.
A. Primary human cumulus cells were treated for 48 hours with
vehicle (C), GDF9 (G: 10ng/mL), BMP15 (B: 10ng/mL), GDF9 and
BMP15 combined (GB: 0.6, 2.5, and 10ng/mL for each of G and
B), in the presence or absence of follicle-stimulating hormone (FSH)
(F: 50ng/mL). IGF2 mRNA levels were determined by quantitative
real-time polymerase chain reaction (qPCR) and expressed relative
to Rpl19. Columns represent the mean±SEM (standard error of the
mean). Columns with different letters differ significantly by one-way
analysis of variance (ANOVA) analysis with Fisher’s least significant
difference (LSD) test, a–b P<0.05, a–c P<0.01, b–c P<0.05,
n=9. Control and FSH groups were compared by t test; *P<0.05,
n=9. B. Primary human cumulus cells were treated for 48 hours
with vehicle (C), GDF9 (G: 5ng/mL), BMP15 (B: 5ng/mL), in the
presence or absence of FSH (F: 5, 25, 100ng/mL). IGF2 mRNA levels
were determined by qPCR and expressed relative to Rpl19. Columns
represent the mean±SEM. Columns with different letters differ
significantly by one-way ANOVA analysis with Fisher’s LSD test, a–b
P<0.05, a–c P<0.01, b–c P<0.05, n=5.
Figure 2. The combination of GDF9 and BMP15 potentiates follicle-
stimulating hormone (FSH)-induced IGF2 expression and protein
synthesis. IGF2 protein levels were quantified by enzyme-linked
immunosorbent assay. Columns represent the mean±standard error
of the mean, columns with different letters differ significantly by
one-way analysis of variance analysis with repeated measures and
Bonferroni correction a–b P<0.001. One-way paired t-test was used
to measure the difference between C and F, (**) for P<0.01, n=3.
Figure 3. The potentiation of FSH actions by GDF9 and BMP15 (GB)
occurs downstream of cAMP. Primary human cumulus cells were
treated for 48 hours with vehicle (C) or the combination of GDF9
and BMP15 (GB: 5ng/mL) in the presence or absence of dbcAMP
(cAMP: 2mM) and the absence of FSH.mRNA levels were determined
by quantitative real-time polymerase chain reaction and expressed
relative to Rpl19. One-way ANOVA analysis with repeated measures
and Bonferroni correction. Columns represent the mean±SEM,
columns with different letters differ significantly, a–b P<0.05, a–c
P<0.01, b–c P<0.05, n=5.
330 Hobeika et al. GDF9 and BMP15 Enhance FSH-Induced IGF2 Expression J Clin Endocrinol Metab, January 2020, 105(1):327–335
Downloaded from https://academic.oup.com/jcem/article-abstract/105/1/327/5582040 by lib-electronic@uic.edu user on 05 June 2020

(P<0.05) or the control (C) group (P<0.01). Thus, as
in the case of FSH, the stimulatory effect of dbcAMP
was potentiated by the presence of G+B in the media,
suggesting that the crosstalk between FSH and G+B oc-
curs downstream of the FSH receptor.
GDF9 and BMP15 increase IGF2 transcripts
derived from the promoter 3 but have no effect
on promoter activity
IGF2 gene transcription is driven by four different
promoters (23). The activity of each promoter gener-
ates IGF2 mRNA with promoter-specic untranslated
exons upstream of the protein-coding exons 7 to 9 that
are common to all transcripts. We have demonstrated
that IGF2 expression in human GCs is controlled by
promoters 3 and 4, of which only promoter 3 is regu-
lated by FSH (16). For this reason, the results listed
in Figs. 1 and 3 were performed using primers that
amplify transcripts derived from promoter 3. To fur-
ther examine the regulation of promoter 3 by G+B, we
cloned 511bp of the IGF2 promoter 3 into a lentiviral
reporter plasmid (IGF2p3–LUC). To determine whether
G+B regulates IGF2p3–LUC, cumulus cells were plated
in 48-well plates and immediately infected with the
lentivirus for 24 hours. At this time, cells were treated
with vehicle, FSH, G+B, or FSH plus G+B. Luciferase
activity was quantied 48 hours after the addition of
FSH. FSH treatment alone increased the activity of
the IGF2p3–LUC construct compared with the control
group (P<0.05) (Fig. 4A). In contrast to the ndings
observed on promoter 3 transcript levels, treatment
with FSH plus G+B did not increase the activity of
the IGF2p3–LUC construct above the levels observed
with FSH alone. This lack of agreement between the
observed regulation of promoter 3 transcripts and the
promoter 3 luciferase reporter by G+B suggests alterna-
tive mechanisms by which GDF9 and BMP15 regulate
IGF2 expression.
IGF2 mRNA upregulation in cancer cells correlates
with demethylation of CpG islands in the genomic re-
gion corresponding to promoter 3, which contains
37 CpG islands (24, 25). Therefore, we examined the
methylation status of all CpG sites in this genomic re-
gion using bisulte sequencing. Cumulus cells were
treated with vehicle or G+B in the presence or absence
of FSH for 48 hours before genomic DNA isolation
and bisulte sequencing. The results demonstrated that
treatment with G+B in the presence or absence of FSH
had no effect on the percentage of methylation of the
IGF2 promoter 3 (data not shown), suggesting that pro-
moter 3 methylation is unlikely to mediate the effects
of G+B.
To examine other possible mechanisms for the regula-
tion of IGF2 by G+B, we examined the expression of the
H19 gene. H19 and IGF2 form a reciprocally imprinted
cluster (IGF2/H19) located on human chromosome 11
(26). It is well known that IGF2 and H19 expression
is inversely regulated via a differentially methylated
region upstream of the H19 gene (27). Therefore, we
quantied the mRNA levels of H19 in cells treated with
vehicle or G+B in the presence or absence of FSH. The
results demonstrated that treatment with G+B and FSH
strongly stimulated the expression of H19 reaching
levels that were signicantly higher than those found in
cells treated with FSH or GB alone (Fig. 4B).
Figure 4. Effect of GDF9 and BMP15 (GB) on IGF2 promoter 3
activity. A. Cells were infected with lentivirus carrying the IGF2p3–
LUC reporter and after overnight incubation treated for 48 hours
with vehicle (C), follicle-stimulating hormone (FSH), FSH+GB, or
GB. Luciferase activity was quantified and expressed relative to the
control. Columns represent the mean±SEM, columns with different
letters differ significantly, n=4. B. H19 transcripts were measured in
cumulus cells treated with increasing doses of G+B and FSH (50ng/
mL) for 48 hours. Expression of H19 was determined by quantitative
real-time polymerase chain reaction and expressed relative to
Rpl19. Columns represent the mean±standard error of the mean.
Columns with different letters differ significantly by one-way analysis
of variance analysis followed by Tukey’s multiple comparisons test
(n=5).
doi:10.1210/clinem/dgz057 https://academic.oup.com/jcem 331
Downloaded from https://academic.oup.com/jcem/article-abstract/105/1/327/5582040 by lib-electronic@uic.edu user on 05 June 2020

GDF9 and BMP15 regulate IGF2 via
SMAD signaling
GDF9 and BMP15 activate SMAD2/3 and
SMAD1/5/8 signaling pathways, respectively (28–30).
To investigate the mechanism of G+B-induced IGF2 ex-
pression in the presence of FSH, we used inhibitors of
SMAD2/3 (SB431542), SMAD3 (SIS3), or SMAD1/5/8
(LDN-193189). The concentration of SMAD inhibi-
tors used was based on previous publications in mouse
and humans GCs (10, 30). The inhibitors were added
to the media 1 hour before the addition of FSH or FSH
plus GB. Forty-eight hours later, cells were harvested
for IGF2 mRNA determination. The results show that
inhibition of SMAD1/5/8 had no signicant effects on
the induction of IGF2 mRNA levels by FSH plus G+B
(Fig. 5). However, the potentiation of IGF2 expression
by G+B in the presence of FSH was inhibited by the add-
ition of SB431542 and SIS3 (P=0.018 and P=0.007,
respectively).
IGF1R activity is necessary for GDF9 and BMP15
potentiation of IGF2 expression
In human GCs, we previously reported that FSH
stimulation of IGF2 requires IGF1R activity (16). To
determine whether the stimulatory effect of G+B on
IGF2 also requires the IGF1R, human GCs were treated
with NVP-AEW451, a known inhibitor of IGF1R ac-
tivity, at a concentration of 1μM for one hour before
the addition of FSH, G+B, or both to the media. IGF2
mRNA levels were quantied 48 hours after the add-
ition of FSH. The potentiation of FSH plus G+B on
IGF2 mRNA was signicantly decreased by the pres-
ence of NVP-AEW451 in the media when compared
with cells treated with FSH plus G+B alone (P= 0.02)
(Fig. 6). However, G+B was still able to potentiate the
effect of FSH on IGF2 mRNA levels in the presence of
NVP-AEW451(P=0.008, ANOVA).
Discussion
We have shown for the rst time that GDF9 and
BMP15, acting in coordination with FSH, maximally
stimulate IGF2 in primary human GCs. The enhancing
effect of GDF9 and BMP15 occurs downstream of
the FSH receptor and is inhibited by SMAD2/3 and
SMAD3 pathway inhibitors. Only the combination of
GDF9 and BMP15 enhances IGF2 expression. These
ndings support the hypothesis that GDF9 and BMP15
form heterodimers (9, 10). However, GDF9:BMP15
heterodimers have not been detected in either animal
or human models. On the other hand, a recent report
suggested that monomers of BMP15 and GDF9 acti-
vate cell-surface receptors initiating a synergistic ef-
fect without the need for heterodimer formation (31).
Therefore, additional experiments are needed to deter-
mine the mechanism of the collaborative effect between
GDF9 and BMP15 on the regulation ofGCs.
Figure 5. GDF9 and BMP15 regulate IGF2 via SMAD signaling.
Primary human cumulus cells were treated for 48 hours with vehicle
(C) or the combination of GDF9 and BMP15 (GB: 5ng/mL) in the
presence or absence of FSH (50ng/mL), SMAD2/3 inhibitor SB431542
(SB: 1µM), SMAD3 inhibitor SIS3 (SIS: 5µM), or SMAD1/5/8 inhibitor
LDN-193189 (LDN: 100nM). IGF2 mRNA levels were determined by
qPCR and expressed relative to Rpl19. t-Test was used to evaluate
the difference between mRNA expression in the F+GB with and
without the different SMAD inhibitors. Columns represent the
mean±standard error of the mean, columns with different letters
differ significantly, a–b P<0.05, a–c P<0.01, b–c P<0.05, n=8.
Figure 6. The combination of GDF9 and BMP15 potentiates FSH
stimulation of IGF2 mRNA partially through IGF1R signaling. Primary
human cumulus cells were treated for 48 hours, as described in
Fig. 2A in the presence or absence of the IGF1R inhibitor NVP-
AEW451(1μM). mRNA levels were determined by qPCR and
expressed relative to Rpl19. One-way ANOVA analysis with repeated
measures and Bonferroni correction indicated a global P=0.008
for the group. Columns represent the mean±SEM, columns with
different letters differ significantly. At-test revealed that the addition
of NVP-AEW451inhibits GDF9 and BMP15 potentiation of IGF2
mRNA (**P=0.02), n=4.
332 Hobeika et al. GDF9 and BMP15 Enhance FSH-Induced IGF2 Expression J Clin Endocrinol Metab, January 2020, 105(1):327–335
Downloaded from https://academic.oup.com/jcem/article-abstract/105/1/327/5582040 by lib-electronic@uic.edu user on 05 June 2020

The use of human cumulus GCs collected from cu-
mulus–oocyte complexes isolated from women under-
going IVF has been adopted by other groups and us
to investigate human GC biology (11, 13–16, 32). We
demonstrated that cumulus cells have distinct properties
compared with mural cells such as lack of luteinization
and the ability to respond to FSH and IGFs (12). The use
of human cells is highly relevant because of the specic
characteristics of the IGF system in the human ovary
that are not conserved in other species (see earlier) (11,
16, 20).
IGF2 plays a central role in the differentiation of
human GCs. Our group was the rst to show that FSH
enhances IGF2 mRNA expression in human GCs (16)
and showed that IGF2 is necessary for FSH-induced
upregulation of steroidogenic enzymes such as Cyp19a1,
Cyp11a1, and Star (11). Together, these ndings and the
current report support the hypothesis that the IGF2
system is also involved in the selection of the dominant
follicle. Thus, considering that IGF2 is vital for GC
proliferation and that OSFs increase IGF2 expression,
it is reasonable to propose that a healthy oocyte will
secrete higher quantities of GDF9 and BMP15, hence
sensitizing GCs to FSH. An oocyte producing high levels
of GDF9 and BMP15 will enhance the survival of its
surrounding GCs in the presence of low levels of FSH,
leading to its selection from the cohort.
We proved previously and conrmed in this report
that FSH stimulates IGF2 expression by increasing pro-
moter 3-derived transcripts in a v-akt murine thymoma
viral oncogene homolog 1 (AKT) activation dependent
manner (16). However, the molecular mechanisms in-
volved in the transcriptional activation of IGF2 in hu-
mans are unknown. We show here for the rst time that
FSH increases IGF2 transcription using a reporter vector
in which luciferase activity is controlled by 511bp of
the genomic region of human P3 promoter. The mech-
anisms and factors participating in the stimulation of
promoter 3 activity by FSH remain to be determined. Of
note, in silico analysis of the promoter showed the pres-
ence of binding sites for specicity protein 1 (SP1), early
growth response protein 1 (ERG1), activation protein 2
(AP2), andCCAAT enhancer binding protein beta (C/
EBPb). However, this analysis revealed the absence of
binding sites for cAMP/CREB, which mediates FSH ac-
tions, suggesting an atypical regulation of IGF2 expres-
sion by FSH in humanGCs.
Inhibition of SMAD2/3 activation abolishes IGF2
stimulation by G+B, suggesting a major role of SMAD2/3
in IGF2 regulation. Upon phosphorylation by type 1 re-
ceptor kinase, Smads form heteromeric complexes with
co-Smads and translocate into the nucleus to activate
gene transcription. Additionally, a well-documented
mechanism of R-Smad regulation is through phosphor-
ylation of the linker region found between the MH1 and
MH2 domains (33), which profoundly affects SMAD
signaling. Among the kinases targeting this region is
AKT. We have previously demonstrated that AKT is es-
sential for the induction of IGF2 by FSH (16). Thus,
in human GCs, the activation of SMAD by G+B could
be further enhanced by FSH via activation of AKT.
We speculate that this mechanism could play a central
role in mediating the crosstalk between the signaling
activated by oocyte-secreted factors and FSH. Future
studies will be needed to test this hypothesis.
How GDF9 and BMP15 regulate the expression of
P3 transcripts is also unclear. Our ndings suggest that
combined these factors enhance promoter 3 IGF2 tran-
script expression in the presence of FSH. Strikingly, our
promoter studies showed that GDF9 and BMP15 do not
enhance the stimulation of the P3-Luc reporter by FSH.
Thus, GDF9 and BMP15 may regulate the steady-state
levels of IGF2 by affecting gene transcription indirectly,
or by regulating the stability of the IGF2 mRNA. For
the rst of these possibilities, we considered two pos-
sible mechanisms: (1) changes occur in the methyla-
tion status of promoter 3, or (2) methylation changes
occur upstream of the H19 gene (27), which is known
to upregulate IGF2 and reduce H19 expression. Our re-
sults suggest that the methylation of promoter 3 is not
affected by GDF9+BMP15 treatment. Moreover, IGF2
and H19 are regulated similarly by GDF9 and BMP15,
indicating that methylation on the imprinting-control
region (ICR) region upstream of the H19 gene is most
probably not affected by these factors. Therefore, the
evidence points to the idea that GDF9 and BMP15 are
not involved in the regulation of IGF2/H19 methylation.
However, further studies will be required to evaluate
this possibilityfully.
Changes in mRNA stability may also play a role in
the regulation of IGF2 mRNA by GDF9 and BMP15.
For instance, it will be of great interest to determine if
GDF9 and BMP15 regulate the expression of mRNA
binding proteins known as IGF2 mRNA-binding pro-
teins (IMPs). IMPs have been shown to participate in
the post-transcriptional regulation of IGF2 (34). Are-
cent report demonstrated that the expression of all
IMP isoforms is relatively constant during human fol-
licular development (21). This report also showed a
64-fold increase in IGF2 expression from small antral
to preovulatory follicle maturation (21). Moreover, we
observed previously that inhibition of IGF1 receptor ac-
tivity abolishes IGF2 induction by FSH (16). However,
the lack of IGF1R activity does not prevent the
doi:10.1210/clinem/dgz057 https://academic.oup.com/jcem 333
Downloaded from https://academic.oup.com/jcem/article-abstract/105/1/327/5582040 by lib-electronic@uic.edu user on 05 June 2020

enhancement of IGF2 expression by GDF9 and BMP15.
Taken together, our ndings and previous reports sug-
gest that IGF2 regulation in human GCs may involve
several interdependent mechanisms, which agrees with
our hypothesis that IGF2 plays a central role in follicle
maturation and selection in humans.
A limitation of our study includes the under-represen-
tation of women with diminished ovarian reserve or
low oocyte yield at the time of oocyte retrieval, which
would yield a suboptimal number of GCs. Thus, the
possibility of a variable response of GCs from patients
with different etiologies of infertility cannot be ruled
out. However, the strength of the study is that GCs from
different patients were not pooled together, suggesting
that the mechanisms activated by OSFs on the regula-
tion of human GCs are conserved despite human subject
variability.
In conclusion, GDF9 and BMP15 play a signicant
role in follicle development. In addition to their part-
nership with FSH in the regulation of aromatase, we
present evidence of their synergism in the stimulation of
IGF2. This in itself sensitizes the follicle to FSH-induced
aromatase expression, which is of utmost importance
in follicular growth in humans. This report adds to
our understanding of the unique role that OSFs play in
folliculogenesis. Furthermore, these ndings can be used
in efforts to develop new strategies to improve in vitro
maturation of follicles.
Acknowledgments
Financial Support: NIH grant R56HD086054 (C.S.).
Additional Information
Correspondence: Carlos Stocco, 835 S Wolcott Ave, Chicago,
Illinois, 60612. E-mail: costocco@uic.edu.
Disclosure Summary: The authors declare that there is no
conict of interest that could be perceived as prejudicing the
impartiality of the research reported.
References
1. EricksonGF, ShimasakiS. The physiology of folliculogenesis: the
role of novel growth factors. Fertil Steril. 2001;76(5):943–949.
2. el-Fouly MA, Cook B, Nekola M, Nalbandov AV. Role
of the ovum in follicular luteinization. Endocrinology.
1970;87(2):286–293.
3. NekolaMV, NalbandovAV. Morphological changes of rat fol-
licular cells as inuenced by oocytes. Biol Reprod. 1971;4(2):
154–160.
4. DongJ, AlbertiniDF, NishimoriK, KumarTR, LuN, MatzukMM.
Growth differentiation factor-9 is required during early ovarian
folliculogenesis. Nature. 1996;383(6600):531–535.
5. GallowaySM, McNattyKP, CambridgeLM, etal. Mutations in
an oocyte-derived growth factor gene (BMP15) cause increased
ovulation rate and infertility in a dosage-sensitive manner. Nat
Genet. 2000;25(3):279–283.
6. McIntosh CJ, Lun S, Lawrence S, Western AH, McNatty KP,
Juengel JL. The proregion of mouse BMP15 regulates the co-
operative interactions of BMP15 and GDF9. Biol Reprod.
2008;79(5):889–896.
7. PauliniF, MeloEO. The role of oocyte-secreted factors GDF9 and
BMP15 in follicular development and oogenesis. Reprod Domest
Anim. 2011;46(2):354–361.
8. Yan C, Wang P, DeMayoJ, etal. Synergistic roles of bone mor-
phogenetic protein 15 and growth differentiation factor 9 in
ovarian function. Mol Endocrinol. 2001;15(6):854–866.
9. MottersheadDG, SugimuraS, Al-MusawiSL, etal. Cumulin, an
oocyte-secreted heterodimer of the transforming growth factor-β
family, is a potent activator of granulosa cells and improves oo-
cyte quality. J Biol Chem. 2015;290(39):24007–24020.
10. PengJ, LiQ, WigglesworthK, etal. Reply to Mottershead etal.:
GDF9:BMP15 heterodimers are potent regulators of ovarian
functions. Proc Natl Acad Sci U S A. 2013;110(25):E2258.
11. BaumgartenSC, ConvissarSM, FierroMA, WinstonNJ, ScocciaB,
StoccoC. IGF1R signaling is necessary for FSH-induced activa-
tion of AKT and differentiation of human Cumulus granulosa
cells. J Clin Endocrinol Metab. 2014;99(8):2995–3004.
12. StoccoC, BaumgartenSC, ArmoutiM, etal. Genome-wide inter-
actions between FSH and insulin-like growth factors in the regu-
lation of human granulosa cell differentiation. Hum Reprod.
2017;32(4):905–914.
13. Convissar S, Armouti M, Fierro MA, Winston NJ, Scoccia H,
Zamah AM, Stocco C. Regulation of AMH by oocyte-specic
growth factors in human primary cumulus cells. Reproduction.
2017;154(6):745–753.
14. HobeikaE, ArmoutiM, KalaH, etal. Oocyte-secreted factors synergize
with FSH to promote aromatase expression in primary human cu-
mulus cells. J Clin Endocrinol Metab. 2019;104(5):1667–1676.
15. Zhou P, BaumgartenSC, WuY, etal. IGF-I signaling is essential
for FSH stimulation of AKT and steroidogenic genes in granulosa
cells. Mol Endocrinol. 2013;27(3):511–523.
16. Baumgarten SC, Convissar SM, Zamah AM, Fierro MA,
WinstonNJ, ScocciaB, StoccoC. FSH regulates IGF-2 expression
in human granulosa cells in an AKT-dependent manner. J Clin
Endocrinol Metab. 2015;100(8):E1046–E1055.
17. AdashiEY, Resnick CE, BrodieAM, SvobodaME, VanWykJJ.
Somatomedin-C-mediated potentiation of follicle-stimulating
hormone-induced aromatase activity of cultured rat granulosa
cells. Endocrinology. 1985;117(6):2313–2320.
18. Maruo T, Hayashi M, Matsuo H, Ueda Y, Morikawa H,
Mochizuki M. Comparison of the facilitative roles of insulin
and insulin-like growth factor Iin the functional differentiation
of granulosa cells: in vitro studies with the porcine model. Acta
Endocrinol (Copenh). 1988;117(2):230–240.
19. Spicer LJ, Chamberlain CS, Maciel SM. Inuence of
gonadotropins on insulin- and insulin-like growth factor-I (IGF-
I)-induced steroid production by bovine granulosa cells. Domest
Anim Endocrinol. 2002;22(4):237–254.
20. HernandezER, RobertsCT Jr, LeRoithD, AdashiEY. Rat ovarian
insulin-like growth factor I(IGF-I) gene expression is granulosa
cell-selective: 5’-untranslated mRNA variant representation and
hormonal regulation. Endocrinology. 1989;125(1):572–574.
21. Bøtkjær JA, Pors SE, Petersen TS, et al. Transcription prole
of the insulin-like growth factor signaling pathway during
human ovarian follicular development. J Assist Reprod Genet.
2019;36(5):889–903.
22. StoccoC. In vivo and in vitro inhibition of cyp19 gene expression
by prostaglandin F2alpha in murine luteal cells: implication of
GATA-4. Endocrinology. 2004;145(11):4957–4966.
23. Sussenbach JS, Steenbergh PH, Jansen E, et al. Structural and
regulatory aspects of the human genes encoding IGF-I and -II.
Adv Exp Med Biol. 1991;293:1–14.
334 Hobeika et al. GDF9 and BMP15 Enhance FSH-Induced IGF2 Expression J Clin Endocrinol Metab, January 2020, 105(1):327–335
Downloaded from https://academic.oup.com/jcem/article-abstract/105/1/327/5582040 by lib-electronic@uic.edu user on 05 June 2020

24. Raizis AM, Eccles MR, Reeve AE. Structural analysis of the
human insulin-like growth factor-II P3 promoter. Biochem J.
1993;289 (Pt 1):133–139.
25. Eriksson T, FriskT, GraySG, etal. Methylation changes in the
human IGF2 p3 promoter parallel IGF2 expression in the pri-
mary tumor, established cell line, and xenograft of a human
hepatoblastoma. Exp Cell Res. 2001;270(1):88–95.
26. ReikW, WalterJ. Genomic imprinting: parental inuence on the
genome. Nat Rev Genet. 2001;2(1):21–32.
27. Davis TL, Yang GJ, McCarrey JR, Bartolomei MS. The H19
methylation imprint is erased and re-established differentially on
the parental alleles during male germ cell development. Hum Mol
Genet. 2000;9(19):2885–2894.
28. MooreRK, OtsukaF, ShimasakiS. Molecular basis of bone mor-
phogenetic protein-15 signaling in granulosa cells. J Biol Chem.
2003;278(1):304–310.
29. Mazerbourg S, Klein C, Roh J, et al. Growth differentiation
factor-9 signaling is mediated by the type I receptor, activin
receptor-like kinase 5. Mol Endocrinol. 2004;18(3):653–665.
30. Liu C, Yuan B, Chen H, et al. Effects of MiR-375-BMPR2 as
a key factor downstream of BMP15/GDF9 on the Smad1/5/8
and Smad2/3 signaling pathways. Cell Physiol Biochem.
2018;46(1):213–225.
31. HeathDA, PitmanJL, McNattyKP. Molecular forms of ruminant
BMP15 and GDF9 and putative interactions with receptors.
Reproduction. 2017;154(4):521–534.
32. Pogrmic-MajkicK, SamardzijaD, Stojkov-MimicN, etal. Atrazine
suppresses FSH-induced steroidogenesis and LH-dependent
expression of ovulatory genes through PDE-cAMP signaling
pathway in human cumulus granulosa cells. Mol Cell Endocrinol.
2018;461:79–88.
33. Kamato D, Burch ML, Piva TJ, et al. Transforming growth
factor-β signalling: role and consequences of Smad linker
region phosphorylation. Cell Signal. 2013;25(10):2017–
2024.
34. HammerNA, HansenTv, ByskovAG, etal. Expression of IGF-II
mRNA-binding proteins (IMPs) in gonads and testicular cancer.
Reproduction. 2005;130(2):203–212.
doi:10.1210/clinem/dgz057 https://academic.oup.com/jcem 335
Downloaded from https://academic.oup.com/jcem/article-abstract/105/1/327/5582040 by lib-electronic@uic.edu user on 05 June 2020