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REPRODUCTION
RESEARCH
Expression and effect of resistin on bovine and rat granulosa cell
steroidogenesis and proliferation
Virginie Maillard, Pascal Froment, Christelle Rame
´, Svetlana Uzbekova, Se
´bastien Elis
and Joe
¨lle Dupont
Unite
´de Physiologie de la Reproduction et des Comportements, UMR85, Equipe Me
´tabolisme et Reproduction,
Institut National de la Recherche Agronomique, F-37 380 Nouzilly, France
Correspondence should be addressed to J Dupont; Email: jdupont@tours.inra.fr
Abstract
Resistin, initially identified in adipose tissue and macrophages, was implicated in insulin resistance. Recently, its mRNA was found in
hypothalamo–pituitary axis and rat testis, leading us to hypothesize that resistin may be expressed in ovary. In this study, we determined
in rats and cows 1) the characterization of resistin in ovary by RT-PCR, immunoblotting, and immunohistochemistry and 2) the effects of
recombinant resistin (10, 100, 333, and 667 ng/ml)GIGF1 (76 ng/ml) on steroidogenesis, proliferation, and signaling pathways of
granulosa cells (GC) measured by enzyme immunoassay, [
3
H]thymidine incorporation, and immunoblotting respectively. We observed
that resistin mRNA and protein were present in several bovine and rat ovarian cells. Nevertheless, only bovine GC abundantly expressed
resistin mRNA and protein. Resistin treatment decreased basal but not IGF1-induced progesterone (P!0.05; whatever the dose) and
estradiol (P!0.005; for 10 and 333 ng/ml) production by bovine GC. In rats, resistin (10 ng/ml) increased basal and IGF1-induced
progesterone secretion (P!0.0001), without effect on estradiol release. We found no effect of resistin on rat GC proliferation.
Conversely, in cows, resistin increased basal proliferation (P!0.0001; for 100–667 ng/ml) and decreased IGF1-induced proliferation of
GC (P!0.0001; for 10–333 ng/ml) associated with a decrease in cyclin D2 protein level (P!0.0001). Finally, resistin stimulated AKT and
p38-MAPK phosphorylation in both species, ERK1/2-MAPK phosphorylation in rats and had the opposite effect on the AMPK pathway
(P!0.05). In conclusion, our results show that resistin is expressed in rat and bovine ovaries. Furthermore, it can modulate GC functions
in basal state or in response to IGF1 in vitro.
Reproduction (2011) 141 467–479
Introduction
Resistin is an adipocyte-derived cytokine that plays an
important role in the development of insulin resistance
and obesity in rodents (Lazar 2007). This finding was
supported by in vitro experiments using cultured cells as
well as animal experiments (Steppan et al. 2001a,Moon
et al. 2003,Banerjee et al. 2004,Kim et al. 2004,
Rangwala et al. 2004,Satoh et al. 2004). Resistin is a
cysteine-rich protein of around 12 kDa that belongs to a
family of polypeptides named resistin-like molecules
(RELMs). These molecules contain three domains: an
N-terminal signal sequence, a variable middle portion,
and a highly constant C-terminal sequence (Holcomb
et al. 2000,Steppan et al. 2001b). Resistin is secreted in
circulation mainly as a homodimer (Banerjee & Lazar
2001,Rajala et al. 2002). Several types of cell can
express resistin. In mice, adipocytes may be the major
source of resistin (Steppan et al. 2001a,2001b), whereas
in humans, resistin mainly come from monocytes and
macrophages (Nagaev & Smith 2001,Savage et al. 2001,
Patel et al. 2003). Beside its effects on glucose
metabolism and insulin sensitivity, resistin regulates a
plethora of various functions through its action on
multiple cell targets in both rodents and humans. Thus,
resistin is able to exert proinflammatory processes in
adiposetissue(Nagaev et al.2006)andvascular
endothelium (Li et al. 2007), promote vascular smooth
muscle cell proliferation (Calabro et al. 2004), and
stimulate in vitro angiogenesis (Di Simone et al. 2006,
Mu et al. 2006,Robertson et al. 2009). Despite much
research on resistin’s action, the receptor(s) mediating its
biological effects has not yet been identified, and little is
known on the intracellular signaling pathways activated
by this protein.
Recently, some data have suggested that resistin could
affect male and female fertility. Indeed, expression of
resistin (mRNA and protein) has been reported in several
reproductive tissues including hypothalamus (Morash
et al. 2002,Tovar et al. 2005,Wilkinson et al. 2005),
pituitary (Morash et al. 2002,Brown et al. 2005), and
testis (Nogueiras et al. 2004). In hypothalamus, resistin
has been shown to inhibit feeding (To v a r et al. 2005,
Vazquez et al. 2008). In pituitary, resistin expression is
q2011 Society for Reproduction and Fertility DOI: 10.1530/REP-10-0419
ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org
regulated in a nutritional-, age-, and gender-specific
manner (Morash et al. 2004). Resistin mRNA expression
increases to a peak level in pituitary of pre-pubertal mice
(Morash et al. 2002,2004). Furthermore, at this stage,
pituitary mRNA resistin levels are strongly stimulated by
corticosteroids (Brown et al. 2005). Administration of
resistin to dispersed rat anterior pituitary cells increases
GH release (Rodriguez-Pacheco et al. 2009). In rat testis,
resistin protein is detectable throughout postnatal
development, and its mRNA is under the control of
several hormones and mediators such as gonadotropins,
leptin, and nutritional status (Nogueiras et al. 2004). In
this organ, resistin is more precisely present in interstitial
Leydig cells and, to a lesser extent, in Sertoli cells within
seminiferous tubules (Nogueiras et al. 2004). Further-
more, in rodent testis, resistin is able to induce in vitro
testosterone secretion in the basal state and in response
to hCG (Nogueiras et al. 2004). In cultured human theca
cells, 17a-hydroxylase activity is increased by resistin in
the presence of forskolin or forskolinGinsulin,
suggesting a role of resistin in stimulation of androgen
production by theca cells (Munir et al. 2005). Moreover,
some studies have shown elevated concentrations of
serum resistin in women with polycystic ovary syndrome
(Panidis et al. 2004,Carmina et al. 2005,Munir et al.
2005,Yilmaz et al. 2009), which is known to be
associated with hyperinsulinemia, hyperandrogenism,
and insulin resistance (Gambineri et al. 2002). In
mammals, insulin as well as insulin-like growth factor
1 (IGF1) is well known to play a key role on the
development of antral follicles, i.e. steroidogenesis and
granulosa cell (GC) proliferation (Silva et al. 2009). We
and others reported that adipokines, such as adiponectin
and leptin, influence GC function (Zachow & Magoffin
1997,Spicer et al. 2000,Chabrolle et al. 2007a,2007b,
2009,Maillard et al. 2010). Some studies showed that
resistin could interfere with some components of IGF1
receptor signaling, such as insulin receptor substrate 1
(IRS1; Barnes & Miner 2009). All these findings led us to
hypothesize that resistin could be expressed in the ovary
and could modulate IGF1 effects on ovarian cells.
The objectives of this study were to investigate in two
different species, bovine and rat, 1) the mRNA and protein
expression of resistin in ovary and 2) the effect of
recombinant resistin with or without IGF1 on prolifer-
ation, steroidogenesis, and different signaling pathways
(AKT, AMPK, and pERK1/2 and p38-MAPK) in GCs in vitro.
Results
Characterization of resistin in bovine and rat ovaries
In bovine ovary, RT-PCR analysis revealed the amplifi-
cation of one cDNA corresponding to a fragment of
resistin (300 bp) in whole ovary, corpus luteum, small and
large follicles, GCs, immature and in vitro matured
cumulus cells, and oocytes (Fig. 1A). More precisely, we
determined by real-time PCR the resistin mRNA levels in
fresh and cultured GCs from small follicles. As shown in
Fig. 1A, resistin mRNA expression was about fivefold
higher in fresh than in cultured GCs. In rat ovary, RT-PCR
analysis also resulted in the amplification of one cDNA,
which corresponds to a fragment of resistin (321 bp) in
whole ovary and corpus luteum, but not in GCs (Fig. 1C).
As shown in Fig. 1C, the expression of rat resistin mRNA is
very low compared with those of rat cyclophilin A in fresh
GCs. Furthermore, it is undetectable in cultured rat GCs.
As shown in Fig. 1B, resistin protein (as the dimer form,
about 23 kDa) was expressed in bovine large and small
follicles, whole ovary, corpus luteum, cumulus cells,
immature and in vitro matured oocytes, and fresh isolated
and cultured GCs. In rat species, immunoblotting of
protein extracts revealed the presence of resistin (as the
dimer form, about 23 kDa) in whole ovary, corpus luteum
and a weak expression in cultured GCs (Fig. 1D).
Immunohistochemistry analyses confirmed the
immunoblot results. Resistin was localized in bovine
primary and antral follicles, and equally expressed
in oocyte and theca, granulosa and cumulus cells
(Fig. 2). In rat ovary, resistin was present more
abundantly in oocyte, theca cells, and corpus luteum
than in GCs (Fig. 3).
Effect of recombinant resistin on basal and
IGF1-induced steroid production by primary GCs
We next investigated whether the supplementation of
various doses of recombinant human (rh) resistin or
recombinant rat (rr) resistinGexogenous rh IGF1
(76 ng/ml) affected the steroidogenesis of primary bovine
and rat GCs respectively. The progesterone and estradiol
secretions were measured by EIA protocols in the culture
medium after 48 h of treatment.
We found that rh resistin decreased the basal
progesterone and estradiol secretions by primary bovine
GCs whatever the dose used (10–667 ng/ml; Fig. 4A
and C). The decrease in progesterone secretion was
significant for the four doses of rh resistin (10, 100, 333,
and 667 ng/ml; about K22%, P!0.05; Fig. 4A), whereas
only the treatment with 10 and 333 ng/ml of resistin
significantly decreased the estradiol production (about
K30%, P!0.005; Fig. 4C). As expected, the pro-
gesterone and estradiol secretions by cultured bovine
GCs were significantly increased by IGF1 treatment
compared with the basal release, by 2.3- and 1.7-fold
respectively (P!0.0001 and P!0.05; Fig. 4B and D). In
the presence of IGF1, no significant effect of rh resistin
treatment was observed on bovine GC steroidogenesis
(Fig. 4B and D). In the rat, the resistin supplementation at
10 ng/ml significantly increased the progesterone
secretion in the presence or not of IGF1 (about 1.9-fold,
P!0.0001 for both; Fig. 5B and A). No significant effect
of rr resistin was found on both basal and IGF1-induced
estradiol secretion by primary rat GCs (Fig. 5B).
468 V Maillard and others
Reproduction (2011) 141 467–479 www.reproduction-online.org
Effect of recombinant resistin on bovine and rat
GC proliferation
We examined whether the treatment with various doses
of recombinant resistin affected the DNA synthesis of
primary GCs. [
3
H]thymidine incorporation by bovine or
rat GCs was measured after 24 h of culture in serum-free
medium in the presence or absence of rh (10, 100, 333,
and 667 ng/ml) or rr (10 and 100 ng/ml) resistin
respectively Grh IGF1 (76 ng/ml). Resistin did not
modify the basal DNA synthesis of rat GCs (data not
shown). Conversely, resistin (100, 333, and 667 ng/ml)
significantly increased the basal DNA synthesis of
bovine GCs by about 1.3-fold (P!0.0001; Fig. 6A).
We observed in the two species that IGF1 significantly
increased [
3
H]thymidine incorporation induced by
about 5.8- and 1.4-fold in bovine and rat GCs
respectively (P!0.0001; Fig. 6B and data not shown
for rat GCs). Whereas no effect of resistin was shown on
rat GC proliferation induced by IGF1 (data not shown),
resistin at 10, 100, and 333 ng/ml doses significantly
decreased [
3
H]thymidine incorporation induced by IGF1
in bovine GCs (Fig. 6B): this decrease in cell proliferation
was more marked for the dose of 10 ng/ml resistin
(K21.2%, P!0.0001). At this latter dose of resistin, we
observed that the protein level of cyclin D2 induced by
IGF1 (76 ng/ml, 3 h) was significantly reduced (K45%,
P!0.0001) compared with the stimulation with IGF1
alone on bovine GCs (Fig. 6C).
Various signaling pathways modulated by recombinant
resistin treatment in bovine and rat GCs
The pattern of AKT, ERK1/2, p38-MAPK, and AMPKa
phosphorylation from 1 to 120 min was analyzed in
primary bovine and rat GCs, which were overnight
serum starved and then supplemented with rh and rr
resistin respectively. In both species, a rapid and
transient increase in phosphorylated AKT (Figs 7Aand8A)
and phosphorylated p38-MAPK (Figs 7C and 8C) was
observed after 1 min of recombinant resistin treatment
mRNA (Bovine) mRNA (rat)
SF LF CL O
Cum
Im
Oo
Im
GC AT
Resistin (300 bp)
Resistin (300 bp)
Actin (188 bp)
Cum
IVM
Oo
IVM
OGCCL AT
Resistin (321 bp)
Actin (188 bp)
Protein (bovine)
γ-Tubulin γ-Tubulin
γ-Tubulin
LF SF hATOCLMGAT kDa
48
~ 23
Oo
Im
Oo
IVM
hAT hATCum fGC cGC
Resistin dimer
Protein (rat)
Resistin dimer
48
~ 23
UT O ATCLcGC
kDa
Resistin dimer
kDa
48
~ 23
0
0.1
0.2
0.3
fGC cGC
***
Ratio resistin
/cycloA
0
0.001
0.002
0.003
0.004
fGC cGC
Ratio resistin
/cycloA
Undetectable
A
BD
C
Figure 1 Expression of resistin in bovine and rat ovaries. (A and C) RT-PCR analysis of the mRNA for resistin in ovary. In the cow (A), the analysis was
conducted in small (SF) and large (LF) follicles, corpus luteum (CL), whole ovary (O), cumulus cells from immature (Cum Im) and 24 h IVM (Cum
IVM) cumulus–oocyte complexes (COC), immature (Oo Im) and 24 h IVM (Oo IVM) oocytes, fresh granulosa cells (GC), and adipose tissue (AT).
Real-time RT-PCR in fresh and cultured bovine granulosa cells from small follicles. Each reaction was run in duplicates, and threshold (C
t
) values for
resistin mRNA were subtracted from that of cyclophilin A and converted from log linear to linear term. The graph represents mean values from three
different pools of granulosa cells(one pool is representative of one bovine ovary containing aboutten small follicles). ***P!0.001. In the rat (C), the
analysis was performed in O, CL, GC, and AT. DNA fragments were electrophoresed in 1.5% agarose gel stained with ethidium bromide for the two
species. The level of rat resistin mRNAwas also determined by real-time quantitative PCR in fresh and cultured rat granulosa cells as described in the
Materials and Methods section. The graph represents mean values from two different pools of granulosa cells (one pool is representative of 20 rate
ovaries). In cultured rat granulosa cells, the mRNA resistin expression was undetectable. (B and D) Detection of resistin in ovary by immunoblotting.
In cow (B), resistin dimer (w23 kDa) was observed in LF, SF, O, CL, mammary gland (MG), AT, Oo Im (nZ50 per lane), Oo IVM (nZ50 per lane),
cumulus cells (Cum, from 60 24-h IVM COC per lane), and fresh (fGC) and 48 h cultured (cGC) granulosa cells from small follicles (!6 mm).
Human adipose tissue (hAT) was used as positive control for the presence of resistin, since the antibody cross-reacts with the human resistin. In rat
(D), resistin dimer (w23 kDa) was detected in 48 h cGC, uterusCoviduct (UT), O, CL, and AT. g-Tubulin protein was used as a loading control in the
two species (nZ3).
Role of resistin in granulosa cells 469
www.reproduction-online.org Reproduction (2011) 141 467–479
(P!0.05 and P!0.1 respectively for cows, and P!0.005
and P!0.05 respectively for rats). Recombinant resistin
significantly and transiently increased phosphorylation
of ERK1/2-MAPK in rat GCs after 1 min (P!0.005;
Fig. 8B), but not in bovine ones (Fig. 7B). Finally,
phosphorylated AMPKawas significantly increased in
cultured bovine GCs by resistin treatment from 1 to
120 min (P!0.05; Fig. 7D), whereas a significant and
transient decrease in AMPKaphosphorylation was found
in primary rat GCs after 1 min of supplementation
(P!0.05; Fig. 8D).
Discussion
In this study, we reported for the first time that resistin
mRNA and protein were present in various structures of
bovine and rat ovaries. Besides, we showed that a 48 h
treatment with rh resistin decreased progesterone and
estradiol secretions by primary bovine GCs. In the rat, rr
resistin induced the progesterone production by GCs,
without effect on estradiol release. Whereas resistin
treatment did not affect the rat GC proliferation, in the
cow it increased the basal proliferation and decreased
the IGF1-induced proliferation of GCs associated with a
decrease in cyclin D2 protein level. Finally, in both
species, recombinant resistin stimulated AKT and p38-
MAPK phosphorylation and had the opposite effect on
the AMPK pathway in GCs. ERK1/2-MAPK phosphoryl-
ation was only affected in rat GCs.
Protein and mRNA expression of resistin have been
described in several tissues of the reproductive axis
(Morash et al. 2002,Nogueiras et al. 2004,Brown et al.
2005,Tov ar et al. 2005,Wilkinson et al. 2005).
Furthermore, resistin mRNA is expressed in the bovine
mammary gland, and its expression in this tissue
decreases during lactation (Komatsu et al. 2003). Our
results of RT-PCR, immunoblotting, and immunohisto-
chemistry showed the presence of resistin in rat and
bovine whole ovary. In the cow, resistin was widely
expressed in small and large follicles, corpus luteum,
oocyte and cumulus, theca and GCs. In their communi-
cation, Jones et al. (2009) also revealed the mRNA
expression of resistin in rat whole ovary. We demon-
strated in addition that resistin mRNA was present in rat
corpus luteum but very weakly in fresh GCs (and
undetectable in cultured GCs), and that resistin protein
was localized in rat oocyte, theca cells, corpus luteum
and weakly present in GCs. Previously, we found a
similar distribution of adiponectin, another adipokine, in
rat (Chabrolle et al. 2007b), chicken (Chabrolle et al.
2007a), and human (Chabrolle et al. 2009) ovarian cells,
whereas it was equally expressed in all structures
(including fresh and cultured GCs) in bovine ovary
(Maillard et al. 2010). If we hypothesize that the
specificity of primary antibodies used in our study is
the same in each species, our results are in favor of a
specific expression of adipokines in the bovine ovary.
Thus, our present data underline an in situ production of
resistin in ovarian cells and a different expression of this
adipokine in rat and bovine GCs. This local production
of resistin in bovine cultured GCs and not in rat cultured
GCs could contribute to ‘species’ differences in
responses including steroidogenesis and proliferation.
Subsequently, we explored the effects of recombinant
resistin on steroidogenesis and proliferation of primary
bovine and rat GCs in the presence or absence of IGF1.
We tested a physiological dose of resistin (10 ng/ml),
referring to published resistin concentrations in human
(Munir et al. 2005,Asimakopoulos et al. 2009,Hansen
et al. 2010) and rodent (Chabrolle et al. 2008,Shankar
et al. 2010) plasma (between around 5 and 20 ng/ml)
and higher resistin supplementations (100, 333, or
667 ng/ml), since resistin concentration in bovine
plasma is still unknown. Moreover, we chose rr resistin
for tests on rat GCs and rh resistin for tests on bovine
cells, since the protein sequence of Bos taurus resistin is
AF AF
Control IgGResistin
50 µM
50 µM
GC
TC
50 µM
50 µM
50 µM
O
O
Cum
Cum
50 µM
GC
TC
PrF
FF
FF
Figure 2 Localization of resistin in bovine ovary by
immunohistochemistry. Resistin was detected in
primary (PrF) and antral (AF) follicles, granulosa
cells (GC), cumulus cells (Cum), theca cells (TC),
and oocyte (Oo). Follicular fluid (FF). Negative
controls included a section incubated with rabbit
IgG (nZ3).
470 V Maillard and others
Reproduction (2011) 141 467–479 www.reproduction-online.org
closer to the sequence of Homo sapiens resistin
(similarity: around 83%) than that of other species.
In this study, in the cow, the basal but not the IGF1-
induced progesterone and estradiol productions by
primary GCs from small follicles were significantly
decreased by resistin without any dose effect (at the
four doses and only at the two doses 10 and 333 ng/ml
respectively), while resistin at the physiological dose
enhanced progesterone but not estradiol release by
cultured rat GCs. As suggested previously, these different
responses observed between the bovine and rat cultured
GCs could be explained by species differences in resistin
protein expression. Leptin, another adipokine, has been
shown to exert an inhibitory effect on steroidogenesis in
rat (Zachow & Magoffin 1997) and bovine (Spicer et al.
2000) cultured GCs. In cultured rat GCs, leptin inhibited
the increase induced by IGF1 in FSH-stimulated
estradiol production. This inhibitory effect of leptin was
specific for estradiol production, since there was no
effect on basal, FSHK,orFSHCIGF1-dependent
progesterone levels (Zachow & Magoffin 1997). In
bovine cultured GCs, leptin had also weak inhibitory
effects on gonadotropin-and/or IGF1-induced steroido-
genesis (Spicer et al. 2000). In this study, resistin did not
significantly regulate the estradiol secretion of primary
rat GCs in the presence of IGF1, and the progesterone
release in this model was not influenced by IGF1
treatment. Further investigations are required to eluci-
date the role of resistin in the presence of IGF1 on GCs of
different species. Other ovarian cells have also been
described to response to a resistin stimulation. Munir
et al. (2005) had demonstrated in primary human
theca cells that recombinant resistin (notably 10 and
100 ng/ml) increased 17a-hydroxylase activity and
CYP17 (steroidogenic enzyme) mRNA expression
induced by forskolin alone or in combination with
insulin. All these data suggest that physiological levels of
resistin could affect ovarian functions.
Resistin has been reported to stimulate proliferation of
different cellular types (Calabro et al. 2004,Ort et al.
2005,Mu et al. 2006,Park et al. 2008). Conversely,
resistin-13 peptide, a fragment of human resistin
possessing some biological activity of the entire
hormone, inhibited the proliferation of the breast cancer
hCG
(resistin)
PMSG
(resistin)
Immature
(resistin) Control (IgG)
TC
GC
O
TC
GC
O
FF
GC
TC
GC CL
CL
CL
CL
GC
CL
CL
CL
CL
Cum
100 µM
100 µM
100 µM 50 µM
25 µM
250 µM
250 µM
250 µM
AB
CDE
HGF
Figure 3 Localization of resistin in rat ovary by immunohistochemistry. Resistin was detected in ovary from immature rat (A), in large follicles in ovary
from rat treated with PMSG for 48 h (C, D, and E) and in corpus luteum (CL) in ovary from rat treated withPMSG for 48 h and then with hCG for 24 h
(F, G, and H). Resistin was localized more precisely in theca cells (TC), CL and oocyte (O), and weakly in cumulus (Cum) and granulosa (GC) cells.
Follicular fluid (FF). Negative controls (B) included a section incubated with rabbit IgG (nZ3).
Role of resistin in granulosa cells 471
www.reproduction-online.org Reproduction (2011) 141 467–479
MDA-MB-231 cells (Pan et al. 2007). Moreover, other
adipokines, such as adiponectin and leptin, were able to
modulate basal or hormone-induced proliferation of GCs
from different species (Spicer et al. 2000,Sirotkin &
Grossmann 2007,Sirotkin et al. 2008,Maillard et al.
2010,Sirotkin & Meszarosova 2010). For these reasons,
we evaluated the potential effects of several doses of
recombinant resistinGIGF1 (76 ng/ml) on the prolifer-
ation of primary bovine and rat GCs for 24 h. As already
shown for adiponectin (Chabrolle et al. 2007b) and leptin
(Duggal et al. 2002) on rat GC proliferation, no effectof rr
resistin (10 and 100 ng/ml) was observed on basal and
IGF1-induced mitosis of rat GCs in our study, suggesting
that adipokines including resistin do not play a role in rat
GC growth. On the other hand, we found in cows that
resistin was able to affect GC proliferation: the basal cell
proliferation was stimulated with high levels of resistin
(100, 333, and 667 ng/ml) but not with the physiological
dose (10 ng/ml), whereas the IGF1-induced proliferation
was significantly decreased with 10, 100, and 333 ng/ml
resistin. This partial inhibitory effect of resistin was
greater for the physiological supplementation. To inves-
tigate the molecular mechanism involved in this
impaired mitotic response of IGF1-stimulated bovine
GCs to resistin, we evaluated the expression of the cyclin
D2 in GCs treated with resistin (10 ng/ml) for 24 h and
then with IGF1 (76 ng/ml) for 3 h. The cyclin D2 is a well-
established marker of mammalian cell proliferation
and more particularly one of the crucial factors of the
G1/S transition of the cell cycle (Sherr 1993,
Sicinski et al. 1996,Sherr & Roberts 1999). We observed
a significantly decrease in cyclin D2 protein expression
induced by IGF1, suggesting that reduction in IGF1-
induced proliferation of bovine GCs by resistin could be
due to in part an inhibition of cyclin D2.
Until now, few data were available on the intracellular
signaling pathways activated by resistin. This hormone
has been shown to modulate phosphorylation of MAPK
(ERK1/2, p38, and JNK; Di Simone et al. 2009,Chen
et al. 2010), AKT (Palanivel et al. 2006), and AMPK
(Satoh et al. 2004) in different cell types. These signaling
pathways have been described to play a role in
steroidogenesis and/or proliferation of GCs in response
to various hormones such as FSH, IGF1, and insulin, as
well as more recently to some adipokines including
leptin and adiponectin (Moore et al. 2001,Kayampilly &
Menon 2004,Tosca et al. 2005,Yu et al. 2005,Ryan
et al. 2008,Kayampilly & Menon 2009). Since the
majority of resistin effects on rat and bovine steroidogen-
esis and proliferation were observed from the physio-
logical level (10 ng/ml) in our study, we then examined
the effect of this dose on phosphorylation of AKT,
ERK1/2, p38-MAPK, and AMPKain bovine and rat
GCs for 1–120 min. We chose these times of stimulation
because some studies showed that other adipokines
including adiponectin or leptin activate rapidly these
signaling pathways in GCs (Chabrolle et al. 2007b,Lin
et al. 2009). Resistin supplementation led to different
and even opposite effects on ERK1/2-MAPK and AMPK
pathways. Indeed, phosphorylation of ERK1/2-MAPK
A
C
0
0.2
0.4
0.6
0.8
1.0
1.2 a
b
a,b
b
a,b b,c
b,c
c
a,b a,b
a
0
0.5
1
1.5
2
2.5
3
Resistin (ng/ml) 0 10 100 333 667 Resistin (ng/ml) 00 10 100 333 667
b
a
b
bb
0
0.2
0.4
0.6
0.8
1.0
1.2
IGF1–+++++
Resistin (ng/ml) 00 10 100 333 667Resistin (ng/ml) 100 100 333 667
IGF1–+++++
b,c
b,c b,c b
c
a
0
0.5
1
1.5
2
2.5
3
B
D
Bovine steroidogenesis
Ratio progesterone secretion/
basal amount
Ratio estradiol secretion/
basal amount
Ratio estradiol secretion/
basal amount
Ratio progesterone secretion/
basal amount
Figure 4 Effect of rh resistin on progesterone (A and B) and estradiol (C and D) secretions by bovine granulosa cells. Progesterone (A and B) and
estradiol (C and D) secretions were measured by EIA protocol in culture medium of granulosa cells after 48 h of culture in enriched McCoy’s 5A
medium (without FBS) with different concentration of rh resistin (10, 100, 333, and 667 ng/ml)GIGF1 (76 ng/ml; B and D). The data are expressed as
the amount of steroids (pg/ml) secreted per 48 h per basal amount. The concentration of progesterone in the culture medium at the basal state (no
resistin or IGF1 treatment) was 998.6G75.5 ng/ml, and the concentration of estradiol was 73.8G9.7 pg/ml. The results, expressed as meansGS.E.M.,
are representative of three to four independent cultures with each condition in quadruplet. Bars with different superscripts are significantly
different (P!0.05).
472 V Maillard and others
Reproduction (2011) 141 467–479 www.reproduction-online.org
was transiently induced in rat GCs after 1 min of resistin
treatment, whereas no effect was observed in bovine
GCs. The activation of ERK1/2-MAPK pathway may
explain in part the increased secretion of progesterone
measured in medium of rat GCs after 48 h of resistin
treatment, since the use of the specific U0126 inhibitor
of ERK1/2-MAPK has been described to significantly
decrease progesterone secretion (Tosca et al. 2005).
Finally, we found that resistin induced a prolonged
phosphorylation of AMPKafrom 1 to 120 min in bovine
GCs, while resistin transiently decreased the protein
quantity of phosphorylated AMPKain rat GCs after 1 min
of supplementation. In previous studies, we showed that
activation of AMPK with 5-aminoimidazole-4-carbox-
amide-1-d-ribonucleoside decreased progesterone
secretion in basal state in both rat and bovine species
(Tosca et al. 2005,2007). In this study, we observed an
inhibitory and a stimulatory effect of resistin treatment
on basal progesterone secretion by bovine and rat GCs
respectively. Thus, we can postulate that opposite
resistin effects on AMPK phosphorylation found in both
species could take part in this difference. Our data also
showed in both species a rapid and transient increase
in phosphorylated AKT and p38-MAPK after 1 min of
recombinant resistin treatment. Similar results have been
shown in rat GCs in response to adiponectin (Chabrolle
et al. 2007b). These two signaling pathways are involved
in the cell survival and apoptosis of GCs (Westfall et al.
2000,Peter & Dhanasekaran 2003). Thus, we can
speculate that resistin through activation of p38-MAPK
and AKT pathways treatment could regulate follicular
atresia. The specific inhibition of each one of these
intracellular signaling pathways could allow us to really
understand the role of resistin on rat and bovine GC
steroidogenesis and survival.
In conclusion, this study shows the presence of resistin
in bovine and rat ovarian cells. More precisely, resistin
expression is species-dependent in GCs. Furthermore, in
these cells, we observed that recombinant resistin can
modulate steroidogenesis and proliferation in basal state
or in response to IGF1 in vitro. Thus, resistin could be a
metabolic signal involved in the reproductive functions.
Further investigations in several species are required to
understand the different molecular mechanisms involved
in resistin effects and its possible interaction with
different hormones (such as FSH, insulin, IGF1, and
other adipokines) on steroidogenesis and proliferation of
cultured ovarian cells. Particularly, it seems important to
measure plasma resistin in different species in order to
compare the ovarian local and plasma concentration of
this adipokine. However, the role of resistin will not be
able to be fully understood until its receptor is identified.
Materials and Methods
Chemicals, hormones, and antibodies
Unless otherwise stated in the text, chemicals were obtained
from Sigma–Aldrich.
Rh and rr resistins produced in Escherichia coli were purchased
from Biovendor Research and Diagnostic Products (Heidelberg,
Germany). Rh IGF1 was obtained from Sigma–Aldrich.
Rabbit polyclonal antibodies to rat resistin and human
resistin were purchased from Chemicon International (Milli-
pore, Guyancourt, France) and Biovendor Research and
Diagnostic Products respectively. Rabbit polyclonal antibodies
to cyclin D2 (C-17), p38a(C-20), ERK2 (C-14), and phospho-
AKT1/2/3 (Ser473)-R were from Santa Cruz Biotechnology
(Euromedex, Souffelweyersheim, France). Rabbit polyclonal
antibodies to phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204),
phospho-p38 MAP kinase (Thr180/Tyr182), phospho-AMPKa
(Thr172), AMPKa, and AKT were obtained from Cell Signaling
Technology (Ozyme, Saint Quentin Yvelines, France). Mouse
MABs to vinculin (clone hVIN-1) and g-tubulin (clone GTU-88)
were purchased from Sigma–Aldrich. HRP-conjugated anti-
rabbit and anti-mouse IgG were purchased from Eurobio
(Les Ulis, France).
A
B
IGF1 (76 ng/ml)
Resistin (ng/ml)
Resistin (ng/ml)
0100 0 10 10010 667 667
–– +++–– +
IGF1 (76 n
g
/ml)
0100 0 10 10010 667 667
–– +++–– +
0
0.5
1
1.5
2
2.5
a,b
c
a,b a
b
d
a,d a,d
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
aaa,b a,b
b,c,d
a,c
d
d
2.0
Rat steroidogenesis
Ratio progesterone secretion/
basal amount
Ratio estradiol secretion/
basal amount
Figure 5 Effect of rr resistin on progesterone (A) and estradiol (B)
secretions by rat granulosa cells. Progesterone (A) and estradiol (B)
amounts were determined by EIA protocol in culture medium of
granulosa cells after 48 h of culture in enriched McCoy’s 5A medium
(without FBS) with different concentration of rh resistin (10, 100, and
667 ng/ml)GIGF1 (76 ng/ml). The data are expressed as the amount of
steroids (pg/ml) secreted per 48 h per basal amount. The concentration
of progesterone in the culture medium at the basal state (no resistin or
IGF1 treatment) was 20G6 ng/ml, and the concentration of estradiol
was 42.4G4.9 pg/ml. The results, expressed as meansGS.E.M., are
representative of three to four independent cultures with each
condition in quadruplet. Different letters indicate significant
difference (P!0.05).
Role of resistin in granulosa cells 473
www.reproduction-online.org Reproduction (2011) 141 467–479
Ethics, animals, and tissues samples
All procedures were approved by the Agricultural Agency and
the Scientific Research Agency, and conducted in accordance
with the guidelines for Care and Use of Agricultural Animals in
Agricultural Research and Teaching.
Mature (60-day old) and immature (21-day old) female rats
of the Wistar strain were purchased from Charles River
(L’Arbresle, France). Protein and mRNA characterization of resistin
were performed on ovaries, uterus (Coviduct), and adipose
tissue from mature female rats. For immunohistochemistry
analyses, ovaries were collected from immature (21-day old)
rats that were treated with 25 IU pregnant mares serum
gonadotropin (PMSG) alone for 48 h to induce follicular
growth (nZ6) or from immature rats that received a single
i.p. injection of 25 IU hCG after 48 h of PMSG treatment to
induce ovulation and luteinization (nZ6).
Bovine ovaries, mammary gland, and abdominal adipose
tissue were collected at a local slaughterhouse.
Tissue samples for mRNA and protein characterization were
frozen in liquid nitrogen and stored at K80 8C.
Isolation and culture of GCs and cumulus–oocyte
complexes
Bovine ovaries from a slaughterhouse were transferred to saline
solution until the dissection. Immature female Wistar rats were
injected subcutaneously with diethylstilboestrol (DES,
1 mg/day) for 3 days. On the third day of DES treatment, the
animals were killed, and the ovaries were removed aseptically.
GCs were isolated by puncturing follicles (follicles !6mm
for cow), allowing the expulsion of cells in modified McCoy’s
5A medium, supplemented with androstenedione (0.1 mmol/l).
The composition of the complete medium was previously
described (Maillard et al.2010). After several steps of
centrifugation/washes in fresh medium, recovered cells were
incubated in modified McCoy’s 5A medium with amphotericin
B (2.7 mmol/l; Eurobio) and 10% fetal bovine serum (FBS, PAA
Laboratories, Les Mureaux, France) for 24 or 48 h, followed by
an overnight serum starvation. Then, cells were cultured in the
presence or absence of test reagents for different time according
to the analyzed biological function. Cultures were performed in
water-saturated atmosphere containing 5% CO
2
in air at 37 8C.
Cumulus–oocyte complexes (COC) were aspirated from 3
to 6 mm antral follicles. COC with a compact and complete
cumulus were selected and washed three times in HEPES-
buffered tissue culture medium-199 (TCM-199) with gentamy-
cin (50 mg/l). Groups of 50 COC were matured in vitro in 500 ml
serum-free TCM-199 supplemented with ascorbic acid
(75 mg/ml), L-cysteine (90 mg/ml), epidermal growth factor
(EGF) (10 ng/ml), and fibroblast growth factor (FGF) from
bovine pituitary (mainly FGF2, 2.2 ng/ml), b-glycine
(720 mg/ml), glutamine (0.1 mg/ml), hCG (5 IU/ml), IGF1
(19 ng/ml), insulin (5 mg/ml), mercaptoethanol (0.1 mM),
PMSG (10 IU/ml), pyruvate (110 mg/ml), selenium (5 ng/ml),
and transferrin (5 mg/ml) (Donnay et al. 2004). In vitro
maturation (IVM) was performed for 24 h at 38.8 8Cin
humidified atmosphere consisting of 5% CO
2
and 95% air.
Oocytes and cumulus cells (Cum) were mechanically separated
Cell proliferation and cyclin D2
expression in bovine granulosa cells
IGF1 (76 ng/ml)
a
d
0
10
20
30
40
50
60
70
80
90
bb,c
cc
Resistin (ng/ml)
0
2
4
6
8
10
12
14
16
18
20
0
a
10
a
100
b
333
c
667
Resistin (ng/ml) 0 0 10 100 333 667
–+++++
–
–
+
–
–
+
+
+
b
A
Resistin (10 ng/ml, 24 h)
IGF1 (76 n
g
/ml, 3 h)
Cyclin D2
Vinculin
Ratio cyclin
D2/vinculin
0
1
2
3
4
5
6
a
a
b
a
kDa
116
34
C
[3H] thymidine incorporation
(CPM × 103)
B
[3H] thymidine incorporation
(CPM × 103)
Figure 6 Effect of rh resistin on cell proliferation (A and B) and cyclin D2
expression (C) in bovine granulosa cells. (A and B) [
3
H]thymidine
incorporation was determined in bovine granulosa cells cultured for
24 h in enriched McCoy’s 5A medium (without FBS) with various doses
of rh resistin (10, 100, 333, and 667 ng/ml)GIGF1 (76 ng/ml) as
described in the Materials and Methods section. The data are expressed
as meanGS.E.M., and the measurement unit of [
3
H]thymidine
incorporation is counts per min (c.p.m.). The results are representative
of three independent cultures with each condition in triplicate.
Bars with different superscripts are significantly different (P!0.05).
(C) Serum-starved granulosa cells were pre-incubated or not for 24 h
with rh resistin (10 ng/ml) and then stimulated or not with IGF1
(76 ng/ml) for 3 h. Protein extracts were separated by electrophoresis
on 12% (w:v) SDS-polyacrylamide gel. After transfer to nitrocellulose
membranes, the proteins were probed with anti-cyclin D2. The blots
were stripped and reprobed with antibodies against vinculin. Bands on
the blots were quantified, and the cyclin D2/vinculin ratio was
calculated. Values represent meansGS.E.M. relative to the basal state
from three independent experiments. Different letters indicate
significant difference (P!0.05).
474 V Maillard and others
Reproduction (2011) 141 467–479 www.reproduction-online.org
and collected after COC aspiration (immature stage, germinal
vesicle stage for oocyte) or after 24 h of IVM (mature stage).
They were then frozen in liquid nitrogen and stored at K80 8C
before RNA or protein analyses.
RNA isolation, RT-PCR, and real-time RT-PCR
As described previously (Maillard et al. 2010), total DNA-free
RNA was extracted from whole ovary, dissected healthy
follicles (small follicles !6mm%large follicles), corpus luteum,
oocytes, cumulus and GCs, and adipose tissue (as positive
control), and was stored at K80 8CuntilRT.
RTand PCR were carried out according to previous protocols
(Maillard et al. 2010). Briefly, total RNA (1 mg or from 50
oocytes) was reverse transcribed from oligo(dT) 15 Primer
(Promega) in a 20 ml final volume of reaction mixture for 1 h at
37 8C. PCR was performed with 2 mM of each specific primer
designed on bovine or rat resistin (Table 1) and purchased from
Invitrogen (Fischer Scientific, Strasbourg, France). The PCR
conditions were 94 8C for 6 min, 58 8C for 1 min, 72 8C
for 1 min, and 72 8C for 7 min for 35 cycles. After visualization
on 1.5% agarose gel stained with ethidium bromide, all
PCR products were sequenced by Genome Express
(Meylan, France).
For real-time RT-PCR, 1 mg total RNA of fresh or cultured GCs
was reverse transcribed in a final volume of 20 ml using RNase
H–MMLV reverse transcriptase (Superscript II, Invitrogen) and
oligo(dT) 15 primers (Promega). cDNA was then diluted to
1:20. A 15 ml master mix containing 10 ml iQ SYBR Green
supermix (Bio-Rad), 0.25 ml forward primer (10 mM), 0.25 ml
reverse primer (10 mM), and 4.5 ml water was then prepared to
perform real-time PCR. Specific sets of primer pairs used are
shown in Table 1. cDNA dilution (5 ml) was added to the PCR
master mix to a final volume of 20 ml. The following PCR
protocol was used on MyiQ Cycler system (Bio-Rad): initial
denaturation (5 min at 95 8C), followed by a three-step
0
0.5
1
1.5
2
2.5
3
Ratio pAKT/AKT
a
b
a,b a,b a,b
Phospho AKT
AKT
60
60
kDa
Ratio pp38/p38
0
0.4
0.8
1.2
1.6
2
a
b
P
<0.1
a,b
a,b a,b
38
38
kDa
Phospho p38
p38
0
0.5
1
1.5
2
Ratio pAMPK/AMPK
a
bbbb
Phospho AMPKα
AMPKα
62
62
kDa
Phospho ERK1/2
ERK2
44
42
42
kDa
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Ratio pERK1/2/ERK2
Time of stimulation (min)
resistin 10 ng/ml
0
a
1
a
5
a
30
a
120
Time of stimulation (min)
resistin 10 ng/ml
0 1 5 30 120
Time of stimulation (min)
resistin 10 n
g
/ml
0 1 5 30 120
Time of stimulation (min)
resistin 10 n
g
/ml
0 1 5 30 120
a
ACow B
CD
Figure 7 Effect of rh resistin on phosphorylation of AKT (A), ERK1/2 (B)
and p38 (C) MAPK, and AMPKa(D) in bovine granulosa cells. After
overnight serum starvation, granulosa cells from small follicles were
incubated in serum-free medium with rh resistin (10 ng/ml) from 1 to
120 min. Protein extracts were separated by electrophoresis on 12%
(w:v) SDS-polyacrylamide gel. After transfer to nitrocellulose mem-
branes, theproteins were probed with anti-phospho-AKT1/2/3 (A)or anti-
phospho-ERK1/2 (B) or anti-phospho-p38 (C) or anti-phospho-AMPKa
(D). The blots were stripped and reprobed with antibodies against AKT,
ERK2, p38, or AMPKarespectively. The immunoblots shown are
representative of three independentexperiments. Bands on theblots were
quantified, and the phosphorylated/total protein ratio was calculated.
The results are reported as meansGS.E.M. Different letters indicate
significant difference (P!0.05).
AB
CD
Phospho AKT
Rat
AKT
60
60
kDa Phospho ERK1/2
ERK2
44
42
42
kDa
38
38
kDa
Phospho p38
p38
Phospho AMPKα
AMPKα
62
62
kDa
Time of stimulation (min)
resistin 10 ng/ml
0
0.5
1
1.5
2
2.5
3
Ratio pAKT/AKT
0
a
1
b
5
a
120
a
0
0.5
1
1.5
2
2.5
3
Ratio pERK1/2/ERK2
Time of stimulation (min)
resistin 10 ng/ml
0
a
1
b
5
a
120
a
Time of stimulation (min)
resistin 10 n
g
/ml
0
0.5
1
1.5
2
2.5
0
a
1
b
5
a
120
a,b
Ratio pAMPK/AMPK
Time of stimulation (min)
resistin 10 n
g
/ml
Ratio pp38/p38
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0
a
1
b
5
a
120
a
Figure 8 Effect of rr resistin on phosphorylation of AKT (A), ERK1/2 (B)
and p38 (C) MAPK, and AMPKa(D) in rat granulosa cells. After overnight
serum starvation, granulosa cells were stimulated with rh resistin
(10 ng/ml) in serum-free medium from 1 to 120 min. Protein extracts
were separated by electrophoresis on 12% (w:v) SDS-polyacrylamide
gel. After transfer to nitrocellulose membranes, the proteins were probed
with anti-phospho-AKT1/2/3 (A) or anti-phospho-ERK1/2 (B) or
anti-phospho-p38 (C) or anti-phospho-AMPKa(D). The blots were
stripped and reprobed with antibodies against AKT, ERK2, p38, or
AMPKarespectively. Bands on the blots were quantified, and the
phosphorylated/total protein ratio was calculated. Values represent
meansGS.E.M. from at least three independent experiments. Bars with
different superscripts are significantly different (P!0.05).
Role of resistin in granulosa cells 475
www.reproduction-online.org Reproduction (2011) 141 467–479
amplification program (30 s at 95 8C, followed by 30 s at 60 8C
and 30 s at 72 8C) repeated 40 times. At the end of the PCR,
dissociation was performed by slowly heating the samples from
60 to 95 8C and continuous recording of the decrease in SYBR
Green fluorescence resulting from the dissociation of double-
stranded DNA. The threshold cycle (C
t
), defined as the cycle at
which an increase in fluorescence above a defined baseline
can be first detected, was determined for each sample. Bovine
and rat resistin mRNA levels were estimated on the basis of PCR
efficiency and C
t
deviation of an unknown sample versus a
control according to the equation proposed by Pfaffl (2001):
EDCTtarget ðcontrolKsampleÞ
target . Cyclophilin A was chosen as the
reference gene. The results were expressed as the bovine or
the rat resistin mRNA/cyclophilin A mRNA ratio. Each PCR run
included a no template control and replicates of control and
unknown samples. Runs were performed in duplicates.
Protein isolation and immunoblotting
Protein extraction and separation and immunoblotting were
performed as reported previously (Maillard et al.2010).
Analyses were conducted on total protein extracts from
whole ovary, dissected healthy follicles (small follicles
!6mm %large follicles), corpus luteum, fresh isolated and
cultured GCs, cumulus cells, oocytes and mammary gland,
uterus (Coviduct), and adipose tissue (as positive controls).
Protein lysates were subjected to electrophoresis on 12 or
15% (w:v) SDS-polyacrylamide gel and electrotransferred. The
membranes were then incubated overnight at 4 8C with
appropriate primary antibodies at a 1/1000 final dilution. The
blots were washed with Tris-buffered saline containing 0.1%
Tween 20 several times and were further incubated for 2 h at
room temperature with a HRP-conjugated anti-rabbit or anti-
mouse or anti-goat IgG (dilution 1/5000). The signal of specific
bands, detected by ECL (Western Lightning Plus-ECL, Perkin
Elmer, Life and Analytical Sciences, Courtaboeuf, France), was
quantified with the software Scion Image for Windows (Scion
Corporation, Frederick, MA, USA).
Immunohistochemistry
After fixation and dehydration, rat and cow ovaries were
embedded in paraffin and serially sectioned (7 mm thickness).
Immunohistochemistry was performed as described previously
(Maillard et al. 2010). Sections were incubated overnight at
48C with rabbit antibody raised against either human resistin
(dilution 1/200) for bovine ovaries or rat resistin (dilution
1/200) for rat ovaries or with rabbit IgG (dilution 1/200, as
negative control) in PBS with 5% lamb serum. After several
washes in PBS, sections were incubated for 30 min at room
temperature with a ready-to-use labeled polymer HRP anti-
rabbit antibody (Kit DakoCytomation EnVision Plus System-
HRP; Dako, Trappes, France). Immunoreactivity was revealed
by incubation in DAB at room temperature (Kit DakoCytoma-
tion EnVision Plus System-HRP). The slides were counter-
stained with hematoxylin and observed using an Axioplan
Zeiss transmission microscope.
Progesterone and estradiol measurements
The steroid concentration was determined in serum-free
medium from bovine and rat GCs after 48 h of culture in the
presence or absence of several doses of resistin (10, 100, 333,
or 667 ng/ml)GIGF1 (76 ng/ml). Initially, GCs were grown in
48-well dishes (1.25!10
5
viable cells/250 ml medium/well) in
modified McCoy’s 5A medium with androstenedione
(0.1 mmol/l), amphotericin B (2.7 mmol/l), and 10% FBS for
24 h. After an overnight serum starvation, GCs were incubated
with the different treatments for 48 h. The concentration of
progesterone in the culture medium from bovine and rat GCs
was measured by an EIA protocol as described previously
(Canepa et al. 2008). For a range of progesterone concen-
trations between 0.4 and 10 ng/ml, the intra-assay coefficients
of variation (CV) were in the majority inferior to 10%. The
concentration of estradiol was performed by using the Estradiol
EIA kit from Cayman Chemical (Interchim, Montluc¸on, France)
according to the manufacturer’s procedure. The intra-assay CV
ranged from 22% to about 12% for estradiol concentrations
between 16.4 and 256 pg/ml. The results are expressed as the
amount of steroids (pg/mL) secreted per 48 h per basal amount.
They are representative of three to four independent cultures
with each condition in quadruplicate.
Measurement of GC proliferation and cyclin D2
expression in GCs
Cell proliferation was assessed by the measurement of
[
3
H]thymidine incorporation after 24 h of culture. GCs (2!10
5
viable cells/400 ml medium/well) were cultured into 24-well
dishes in modified McCoy’s 5A medium supplemented with
amphotericin B (2.7 mmol/l) and 10% FBS for 48 h. Cells were
then serum starved overnight followed by the addition of
Table 1 Oligonucleotide primer sequences for RT-PCR amplification.
Primer name Primer sequence Accession number Product size (bp)
Bovine resistin
Sense 50-TGT GCC CCA TAG ATA AAG CC-30NM_183362 300
Antisense 50-CAG GCC TGC AGC AGT CTT AG-30
Rat resistin
Sense 50-CCT CCT TTT CCT TTT CTT CC-30NM_144741 321
Antisense 50-AAC CAA CCC GCA GGG TAC AG-30
Cyclophilin A
Sense 50-GCA TACA GGT CCT GGC ATC T-30NM_178320 217
Antisense 50-TGT CCA CAG TCA GCA ATG GT-30
476 V Maillard and others
Reproduction (2011) 141 467–479 www.reproduction-online.org
0.25 mCi [
3
H]thymidine (Perkin Elmer; Life and Analytical
Sciences) in the presence or absence of resistin (10, 100, 333,
or 667 ng/ml)GIGF1 (76 ng/ml). Cultures were maintained at
37 8C under 5% CO
2
in air. After 24 h of culture, excess of
thymidine was removed by washing twice with PBS, fixed
with cold 50% trichloroacetic acid for 15 min, and lysed by
0.5 mol/l NaOH. The radioactivity was determined in Ultima
Gold MV scintillation fluid (Perkin Elmer) by counting in a
b-photomultiplier. The values, expressed as counts per min
(c.p.m.), are representative of three independent cultures with
each condition in triplicate.
For the determination of the protein levels of cyclin D2,
bovine GCs were starved overnight. Cells were then pre-
incubated in new serum-free medium without or with rh
resistin (10 ng/ml) for 24 h, and then IGF1 (76 ng/ml) was
added or not for 3 h in the serum-free medium. Cells were then
lysed, and immunoblots were performed.
Statistical analysis
All experimental results are presented as meansGS.E.M.
Statistical analyses were carried out using an one-way (for
data on signaling pathways and cyclin D2 expression) or a two-
way (for results on steroidogenesis and cell proliferation)
factorial ANOVA test followed by Fisher’s PLSD test, when
the ANOVA revealed significant effects. In the various graphs,
bars with different superscripts were considered statistically
significant at P!0.05.
Declaration of interest
The authors declare that there is no conflict of interest that
could be perceived as prejudicing the impartiality of the
research reported.
Funding
This study was supported by the GIS-AGENAE Programme,
ANR, and APIS-GENE. V Maillard is a recipient of a grant
from INRA.
Acknowledgements
We thank Thierry Delpuech and Pascal Papillier for the
collection of bovine ovaries; Claude Cahier and his staff for
the rat care; and Gilles Gomot, Jean-Philippe Dubois, and
Albert Arnoud for the collection of bovine tissue samples.
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Received 7 October 2010
First decision 8 November 2010
Revised manuscript received 26 December 2010
Accepted 14 January 2011
Role of resistin in granulosa cells 479
www.reproduction-online.org Reproduction (2011) 141 467–479
