# Role of the Peroxisome Proliferator-Activated Receptors, Adenosine

Authors: Joëlle Dupont; Christine Chabrolle; Christelle Ramé; Lucie Tosca; Stéphanie Coyral-Castel
Journal: PPAR Research
 (2008)

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## Abstract

The mechanisms controlling the interaction between energy balance and reproduction are the subject of intensive investigations. The integrated control of these systems is probably a multifaceted phenomenon involving an array of signals governing energy homeostasis, metabolism, and fertility. Two fuel sensors, PPARs, a superfamily of nuclear receptors and the kinase AMPK, integrate energy control and lipid and glucose homeostasis. Adiponectin,
one of the adipocyte-derived factors mediate its actions through the AMPK or PPARs pathway. These three molecules are expressed in the ovary, raising questions about the biological actions of fuel sensors in fertility and the use of these molecules to treat fertility problems. This review will highlight the expression and putative role of PPARs, AMPK, and adiponectin in the ovary, particularly during folliculogenesis, steroidogenesis, and oocyte maturation.


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## Body

## 1. Introduction

The levels of various molecules, including metabolites (glucose, fatty acids, amino acids) and hormones (adiponectin, insulin, leptin, ghrelin, etc.),  are modulated by nutrition and energy supply. Most of these molecules are known to be directly involved, through a fuel sensor, in the regulation of fertility at each level of the hypothalamo-pituitary-gonad axis (for review see
[1, 2]). For example, mice
lacking insulin-signalling pathway components, such as insulin receptor
substrate 2 (IRS-2) or insulin receptor, display female and male infertility
[3, 4].In humans, a close link between energy status and reproductive function
has been found in some diseases. Polycystic ovary syndrome (PCOS), which is
frequently associated with insulin resistance, affects 5 to 10% of women of reproductive age [5]. Women with
PCOS present with ovulation problems, which may be associated with infertility. The
treatment of PCOS patients with insulin-sensitising agents of
various drug families, such as thiazolidinediones (TZDs) or metformin (a derivative of biguanide), restores the menstrual cycle [6] and increases ovulation (by improving follicular growth), fertilization,
and pregnancy rates [7]. TZDs bind to the nuclear peroxisome proliferator-activated
receptor gamma (PPARγ) and metformin activates the AMP-activated protein kinase (AMPK) pathway [8, 9]. In women with PCOS, plasma adiponectin is also
significantly decreased independently of obesity [10]. Adiponectin plasma
levels seem to be related to TZDs or Metformin treatment. Adiponectin is an
adipokine known to increase sensitivity to insulin and vasodilatation (for
review [11]). Adiponectin could also be involved in the regulations of some reproductive functions [12, 13]. In mammals, and particularly in cattle, dietary fats also influence reproductive function. For example, fatty acid supplementation in the diet
increases the total number of follicles and stimulates growth of the
preovulatory follicle [14]. In cows, the availability of fatty acid precursors
is coupled with an increase in sexual steroid levels and eicosanoid secretion, potentially
affecting ovarian and uterine function and embryo implantation [15]. These phenomena
may involve several hormones including insulin, IGFs, leptin, adiponectin, and
some factors such as PPARs and AMPK. Indeed, these molecules are known to play
a role in energy control and lipid metabolism. They may hypothetically play a role as fuel sensors in
reproductive compartments, providing the cells with information about energy
status. However, how metformin and TZDs influence ovarian function is
still under investigation. The functions of PPARs, AMPK, and adiponectin in the
ovary also remain unclear. In this review, we will describe the expression and potential
implications of these fuel sensors in the
ovary.


## 2. PPARs and AMPK Structures and Implications

The PPAR family (α, β/δ and γ) integrates energy control with
lipid and glucose metabolism and affects insulin sensitivity [16]. Like PPARs, AMPK plays a key role in regulating lipid and glucose metabolism in response to metabolic stress and energy demand [17]. AMPK acts at various steps and plays a
central role in controlling fatty acid, triglyceride, and cholesterol
synthesis, and the oxidation of fatty acids, through direct phosphorylation and
control over gene transcription [17].PPARs and AMPK have similar effects and close links have been found between these molecules. Indeed, it is generally assumed that TZDs
activate PPARγ and AMPK independently [18–20]. The inhibition of AMPK expression by siRNA abolishes the inhibitory effects of rosiglitazone and
15d-PGJ2 (two PPARγ ligands, see below) on iNOS expression and activity [21]. The mitochondria may house a pathway common to PPARγ and AMPK.
Indeed, both metformin and TZDs cause a rapid increase in cellular ADP:ATP
ratio, probably by inhibiting the respiratory chain, leading to the
phosphorylation and activation of AMPK [22]. PPARs and AMPK also participate in the molecular action of adiponectin, an adipocytokine involved in the insulin
sensitivity of tissues [7].


### 2.1. Structure and Mechanisms of Action of PPARs

The PPARs are transcription factors that share a common structure with steroid
hormone receptors: the N-terminal A/B domain responsible for
ligand-independent transactivation function, the C domain containing the
DNA-binding domain, the D domain involved in the receptor dimerization, and the
C-terminal E/F domain containing the ligand binding domain (for review [23]).
The members of the nuclear PPAR (α, β/δ, and γ) family bind to specific regions of
DNA in heterodimers with the retinoid X receptors (RXRs) [24]. These DNA
sequences are known as PPREs (peroxisome proliferator response elements). The
transcription is activated subsequent to heterodimerisation of PPAR and
retinoid receptors (RXR). Furthermore, PPARs are able to indirectly regulate
gene expression through transrepression mechanisms by linking some cofactors (reviewed
in [23]). In this review, we focus on the PPARα and PPARγ isoforms.The stimulation of PPARγ by TZDs
modifies the transcription and/or the activity of several key regulators of
energy homeostasis, including several glucose regulators (glucose transporters,
insulin receptor, IRS, etc.), which it stimulates (for review see [25, 26]). PPARs regulate the transcription of
a number of target genes involved in ovarian functions such as steroidogenesis,
ovulation, oocyte maturation, and maintenance of the corpus luteum (cyclooxygenase-2
(COX-2), nitric oxide synthase (NOS), several proteases, including matrix
metalloprotease-9, plasminogen activator, and vascular endothelial growth factor
(VEGF), reviewed in [23]). PPARγ
activity is governed by binding to small lipophilic ligands, such as
polyunsaturated fatty acids and eicosanoids derived from the diet or metabolic
pathways (e.g., the prostaglandin D2 metabolite 15-deoxy-12, 14-prostanglandin
J2 (PGJ2)) [27]. PPARγ is also activated by synthetic compounds
called thiazolidinediones (TZDs), a class of insulin-sensitising agents. PPARγ
may also be regulated by AMPK. Indeed, AMPK can phosphorylate PPARγ, repressing
both the ligand-dependent and ligand-independent transactivating functions of
this receptor [28].PPARα is another isoform of PPAR expressed in the ovary. It regulates genes responsible for
the uptake into cells and beta-oxidation of fatty acids [29]. Hypolipidaemic
fibrate drugs, phthalate esters (plasticisers, herbicides), and long-chain
polyunsaturated fatty acids and their lipooxygenase-derived metabolites (e.g.,
leukotriene) have been described as agonists of PPARα [30–32]. In vivo,
fibrates are currently administrated alone or in combination with statins to
patients with increased cardiovascular risk to impede the progression of
atherosclerotic lesions. Insulin increases the transcriptional activity of
PPARα by activating the MAPK pathway [33]. New therapeutics agents, such as
glitazar, may activate both PPARα and PPARγ [34].


### 2.2. Structure and Mechanisms of Action of AMPK

Unlike PPARs, AMPK is a kinase comprised of three subunits: a catalytic subunit alpha
and two regulatory subunits, beta and gamma [35]. The alpha subunit contains the catalytic core and binds, via its C-terminal tail, to the beta subunit,
which serves as a docking subunit for the alpha and gamma subunits. AMPK is
activated by a change in the AMP : ATP ratio within the cell and therefore acts
as an efficient sensor of cellular energy state. This change in AMP : ATP ratio
may result from exercise [36], hypoxia [37], hormones [38, 39], or the effects
of pharmacological drugs, such as 5-aminoimidazole-4-carboxamide-riboside-5-phosphate (AICAR) [40]. Binding to AMP activates AMPK
allosterically and induces phosphorylation of the threonine 172 residue of the α subunit by upstream kinases,
including the tumour suppressor LKB1 [41, 42].AMPK phosphorylates target proteins (including PPARγ) involved in a number of metabolic pathways, including lipid and cholesterol metabolism (adipocytes, liver, and muscle), glucose transport, glycogen, and protein metabolism (see review
[35, 41]).


### 2.3. Involvement of PPARs and AMPK in the Adiponectin Action

AMPK and PPARα are both activated by
adiponectin [11, 43] (Figure 1). Adiponectin (also known as apM1,
AdipoQ, Gbp28, and Acrp30) is an adipocyte-derived factor [44, 45]. It is present as a multimer at high concentrations in the circulation (5 to 25 μg/ml in human [46]). In obese and type 2 diabetic humans, plasma adiponectin is strongly reduced suggesting that circulating adiponectin may be related to
the development of insulin resistance [11]. Two adiponectin receptors (AdipoR1
and AdipoR2) have been identified in different tissues of various species. They
each contain seven transmembrane domains, but are structurally and functionally
different from G protein-coupled receptors. Adiponectin plays an important role
in insulin sensitisation in mammals (inhibition of gluconeogenesis and
stimulation of fatty acid oxidation) by activating AMPK [47] and PPARα proteins
in skeletal muscle, liver, and adipocytes [43]. Thus, both TZDs and adiponectin
have been shown to activate AMPK. Moreover, the promoter of the adiponectin
gene contains a PPRE [48] and TZDs increase the production and plasma
concentration of adiponectin [49]. TZDs have weaker antidiabetic effects in ob/ob mice lacking adiponectin gene
than in ob/ob mice with
adiponectin, and the activation of AMPK by TZDs is also attenuated in these
mice, suggesting that adiponectin is required for the activation of AMPK by
TZDs [50].Figure 1
Schema illustrating the putative functional interactions between PPARs, AMPK, and adiponectin. PPARγ
is activated by binding with PGJ2or TZDs and
PPARα with fibrates or WY 14 463. They control gene transcription, and, in particular, PPARγ ligands increase adiponectin expression [49]. Metformin and TZDs activate AMPK probably via the respiratory chain in mitochondria [22],
and AICAR stimulates AMPK. AMPK controls protein activity by phosphorylation
(e.g., inhibits PPARγ by
phosphorylation [35]). Adiponectin activates
AdipoR1 and AdipoR2 receptors which act on metabolism via AMPK (AdipoR1) or
PPARα (AdipoR2) [43].In porcine granulosa cells, adiponectin treatment induces the expression of genes
associated with periovulatory remodeling of the ovarian follicle
(cyclooxygenase-2, prostaglandin E synthase, and vascular endothelial growth
factor [51]). Some of these genes are also activated by PPARγ. Furthermore, adiponectin receptors, PPARs, and AMPK are expressed in reproductive tissues, including the ovary.


## 2.1. Structure and Mechanisms of Action of PPARs

The PPARs are transcription factors that share a common structure with steroid
hormone receptors: the N-terminal A/B domain responsible for
ligand-independent transactivation function, the C domain containing the
DNA-binding domain, the D domain involved in the receptor dimerization, and the
C-terminal E/F domain containing the ligand binding domain (for review [23]).
The members of the nuclear PPAR (α, β/δ, and γ) family bind to specific regions of
DNA in heterodimers with the retinoid X receptors (RXRs) [24]. These DNA
sequences are known as PPREs (peroxisome proliferator response elements). The
transcription is activated subsequent to heterodimerisation of PPAR and
retinoid receptors (RXR). Furthermore, PPARs are able to indirectly regulate
gene expression through transrepression mechanisms by linking some cofactors (reviewed
in [23]). In this review, we focus on the PPARα and PPARγ isoforms.The stimulation of PPARγ by TZDs
modifies the transcription and/or the activity of several key regulators of
energy homeostasis, including several glucose regulators (glucose transporters,
insulin receptor, IRS, etc.), which it stimulates (for review see [25, 26]). PPARs regulate the transcription of
a number of target genes involved in ovarian functions such as steroidogenesis,
ovulation, oocyte maturation, and maintenance of the corpus luteum (cyclooxygenase-2
(COX-2), nitric oxide synthase (NOS), several proteases, including matrix
metalloprotease-9, plasminogen activator, and vascular endothelial growth factor
(VEGF), reviewed in [23]). PPARγ
activity is governed by binding to small lipophilic ligands, such as
polyunsaturated fatty acids and eicosanoids derived from the diet or metabolic
pathways (e.g., the prostaglandin D2 metabolite 15-deoxy-12, 14-prostanglandin
J2 (PGJ2)) [27]. PPARγ is also activated by synthetic compounds
called thiazolidinediones (TZDs), a class of insulin-sensitising agents. PPARγ
may also be regulated by AMPK. Indeed, AMPK can phosphorylate PPARγ, repressing
both the ligand-dependent and ligand-independent transactivating functions of
this receptor [28].PPARα is another isoform of PPAR expressed in the ovary. It regulates genes responsible for
the uptake into cells and beta-oxidation of fatty acids [29]. Hypolipidaemic
fibrate drugs, phthalate esters (plasticisers, herbicides), and long-chain
polyunsaturated fatty acids and their lipooxygenase-derived metabolites (e.g.,
leukotriene) have been described as agonists of PPARα [30–32]. In vivo,
fibrates are currently administrated alone or in combination with statins to
patients with increased cardiovascular risk to impede the progression of
atherosclerotic lesions. Insulin increases the transcriptional activity of
PPARα by activating the MAPK pathway [33]. New therapeutics agents, such as
glitazar, may activate both PPARα and PPARγ [34].


## 2.2. Structure and Mechanisms of Action of AMPK

Unlike PPARs, AMPK is a kinase comprised of three subunits: a catalytic subunit alpha
and two regulatory subunits, beta and gamma [35]. The alpha subunit contains the catalytic core and binds, via its C-terminal tail, to the beta subunit,
which serves as a docking subunit for the alpha and gamma subunits. AMPK is
activated by a change in the AMP : ATP ratio within the cell and therefore acts
as an efficient sensor of cellular energy state. This change in AMP : ATP ratio
may result from exercise [36], hypoxia [37], hormones [38, 39], or the effects
of pharmacological drugs, such as 5-aminoimidazole-4-carboxamide-riboside-5-phosphate (AICAR) [40]. Binding to AMP activates AMPK
allosterically and induces phosphorylation of the threonine 172 residue of the α subunit by upstream kinases,
including the tumour suppressor LKB1 [41, 42].AMPK phosphorylates target proteins (including PPARγ) involved in a number of metabolic pathways, including lipid and cholesterol metabolism (adipocytes, liver, and muscle), glucose transport, glycogen, and protein metabolism (see review
[35, 41]).


## 2.3. Involvement of PPARs and AMPK in the Adiponectin Action

AMPK and PPARα are both activated by
adiponectin [11, 43] (Figure 1). Adiponectin (also known as apM1,
AdipoQ, Gbp28, and Acrp30) is an adipocyte-derived factor [44, 45]. It is present as a multimer at high concentrations in the circulation (5 to 25 μg/ml in human [46]). In obese and type 2 diabetic humans, plasma adiponectin is strongly reduced suggesting that circulating adiponectin may be related to
the development of insulin resistance [11]. Two adiponectin receptors (AdipoR1
and AdipoR2) have been identified in different tissues of various species. They
each contain seven transmembrane domains, but are structurally and functionally
different from G protein-coupled receptors. Adiponectin plays an important role
in insulin sensitisation in mammals (inhibition of gluconeogenesis and
stimulation of fatty acid oxidation) by activating AMPK [47] and PPARα proteins
in skeletal muscle, liver, and adipocytes [43]. Thus, both TZDs and adiponectin
have been shown to activate AMPK. Moreover, the promoter of the adiponectin
gene contains a PPRE [48] and TZDs increase the production and plasma
concentration of adiponectin [49]. TZDs have weaker antidiabetic effects in ob/ob mice lacking adiponectin gene
than in ob/ob mice with
adiponectin, and the activation of AMPK by TZDs is also attenuated in these
mice, suggesting that adiponectin is required for the activation of AMPK by
TZDs [50].Figure 1
Schema illustrating the putative functional interactions between PPARs, AMPK, and adiponectin. PPARγ
is activated by binding with PGJ2or TZDs and
PPARα with fibrates or WY 14 463. They control gene transcription, and, in particular, PPARγ ligands increase adiponectin expression [49]. Metformin and TZDs activate AMPK probably via the respiratory chain in mitochondria [22],
and AICAR stimulates AMPK. AMPK controls protein activity by phosphorylation
(e.g., inhibits PPARγ by
phosphorylation [35]). Adiponectin activates
AdipoR1 and AdipoR2 receptors which act on metabolism via AMPK (AdipoR1) or
PPARα (AdipoR2) [43].In porcine granulosa cells, adiponectin treatment induces the expression of genes
associated with periovulatory remodeling of the ovarian follicle
(cyclooxygenase-2, prostaglandin E synthase, and vascular endothelial growth
factor [51]). Some of these genes are also activated by PPARγ. Furthermore, adiponectin receptors, PPARs, and AMPK are expressed in reproductive tissues, including the ovary.


## 3. Expression of PPARs and AMPK in the Ovary

### 3.1.  Expression of PPARs in the Ovary

All the PPAR isoforms are expressed in the ovary. The PPARα and PPARβ/δ isoforms are expressed primarily in
the theca and stroma tissues [52], reviewed by [23], (see Table 1). The
deletion of PPARα has no apparent effect on the fertility of mice, whereas PPARβ/δ-null mice present placental malformations leading to embryo death during early pregnancy [53–55]. PPARγ
is expressed strongly in granulosa cells, and less strongly in the theca cells
and corpus luteum, in the ovaries of rodents and ruminants (see Table 1) [52, 56, 57]. PPARγ is detected early in
folliculogenesis (at the primary/secondary follicle stage) [58], and its expression
increases until the large follicle stage and then decreases after the LH surge [58].
In mice, the absence of PPARγ in the ovaries results in lower levels of
fertility [59]. No effect on folliculogenesis or ovulation rate has been
observed, but fewer embryos implant, probably due to lower levels of progesterone
production by the corpus luteum [59].Table 1
Location of PPARs, AMPK, and adiponectin in ovary.
SpeciesLocationmRNA or ProteinReferencesPPARαRatTheca and stroma[52]PPARβ/δRatThroughout the ovary[52]PPARγMouse, rat, pig, sheep, cow, and humanGranulosa, corpus, luteum, porcine theca and granulosa cells oocytesReviewed by [23]AMPKRat, cow, chicken, pig, mouseGranulosa cells, oocyte, corpus luteum (weaker in rat theca cells for AMPKα1)mRNA and protein[60–64]AdiponectinRat, chicken, pigTheca cells, oocyte, and corpus luteum, Follicular liquidmRNA (chicken) mRNA and protein (rat)[12, 13, 51]Adiponectin receptor IRat, chicken, pigGranulosa and theca cells, oocyte and corpus luteum (rat)mRNA (chicken) mRNA and protein (rat)[12, 13, 51]Adiponectin receptor IIRat, chicken, pigGranulosa cells, oocyte and corpus luteum (rat)mRNA (chicken) mRNA and protein (rat)[12, 13, 51]


### 3.2. Expression of AMPK and Adiponectin in the Ovary

AMPK expression has been studied in the ovaries of various species, including rat [60, 65], mouse [61], cow [62], pig [63], and chicken [64]. RT-PCR has shown the mRNAs of all the AMPK subunits to be present in granulosa cells, the corpus luteum, oocyte, and cumulus-oocyte-complexes in rodent and bovine ovaries (Table 1) [60, 62]. We have shown, by immunohistochemical analyses, that the AMPK α-subunit, like PPARγ, is more
strongly expressed in granulosa cells than in theca cells in rats and cows [60, 62]. In cows, levels of AMPKα- and β-subunits were similar in small and large follicles. In hens, the activation of AMPK by its phosphorylation on the Thr172
residue increased during follicle development [64]. In mice, the absence of the catalytic AMPK alpha 2 subunit does not affect female fertility [66]. Until
now, no data are available on the reproductive functions of the transgenic or
knockout mice for the other subunits of AMPK.In chicken ovary, adiponectin mRNA is more abundant in theca cells than in
granulosa cells (Table1) [13]. In porcine ovary, adiponectin is detected at
similar concentrations in the follicular fluid and serum [51]. Both receptors
are expressed in ovarian follicles. In chicken, the adiponectin type I receptor
(AdipoRI) is twice as abundant in granulosa cells as in theca cells, and the
type II receptor (AdipoR2) is expressed equally strongly in granulosa and
thecal cells (Table 1) [13]. Studies in mice have shown that AdipoR1 may be more tightly linked to AMPK pathway activation, whereas AdipoR2 seems to be associated with PPARα activation [43].
However, mice lacking adiponectin [67], AdipoR1, AdipoR2, or both receptors
[43] are fertile, which suggests that this signalling is not absolutely
essential for ovarian function. However, it may be required for ovulation in
other species or may simply be an additional component for fine-tuning ovarian
function.


## 3.1.  Expression of PPARs in the Ovary

All the PPAR isoforms are expressed in the ovary. The PPARα and PPARβ/δ isoforms are expressed primarily in
the theca and stroma tissues [52], reviewed by [23], (see Table 1). The
deletion of PPARα has no apparent effect on the fertility of mice, whereas PPARβ/δ-null mice present placental malformations leading to embryo death during early pregnancy [53–55]. PPARγ
is expressed strongly in granulosa cells, and less strongly in the theca cells
and corpus luteum, in the ovaries of rodents and ruminants (see Table 1) [52, 56, 57]. PPARγ is detected early in
folliculogenesis (at the primary/secondary follicle stage) [58], and its expression
increases until the large follicle stage and then decreases after the LH surge [58].
In mice, the absence of PPARγ in the ovaries results in lower levels of
fertility [59]. No effect on folliculogenesis or ovulation rate has been
observed, but fewer embryos implant, probably due to lower levels of progesterone
production by the corpus luteum [59].Table 1
Location of PPARs, AMPK, and adiponectin in ovary.
SpeciesLocationmRNA or ProteinReferencesPPARαRatTheca and stroma[52]PPARβ/δRatThroughout the ovary[52]PPARγMouse, rat, pig, sheep, cow, and humanGranulosa, corpus, luteum, porcine theca and granulosa cells oocytesReviewed by [23]AMPKRat, cow, chicken, pig, mouseGranulosa cells, oocyte, corpus luteum (weaker in rat theca cells for AMPKα1)mRNA and protein[60–64]AdiponectinRat, chicken, pigTheca cells, oocyte, and corpus luteum, Follicular liquidmRNA (chicken) mRNA and protein (rat)[12, 13, 51]Adiponectin receptor IRat, chicken, pigGranulosa and theca cells, oocyte and corpus luteum (rat)mRNA (chicken) mRNA and protein (rat)[12, 13, 51]Adiponectin receptor IIRat, chicken, pigGranulosa cells, oocyte and corpus luteum (rat)mRNA (chicken) mRNA and protein (rat)[12, 13, 51]


## 3.2. Expression of AMPK and Adiponectin in the Ovary

AMPK expression has been studied in the ovaries of various species, including rat [60, 65], mouse [61], cow [62], pig [63], and chicken [64]. RT-PCR has shown the mRNAs of all the AMPK subunits to be present in granulosa cells, the corpus luteum, oocyte, and cumulus-oocyte-complexes in rodent and bovine ovaries (Table 1) [60, 62]. We have shown, by immunohistochemical analyses, that the AMPK α-subunit, like PPARγ, is more
strongly expressed in granulosa cells than in theca cells in rats and cows [60, 62]. In cows, levels of AMPKα- and β-subunits were similar in small and large follicles. In hens, the activation of AMPK by its phosphorylation on the Thr172
residue increased during follicle development [64]. In mice, the absence of the catalytic AMPK alpha 2 subunit does not affect female fertility [66]. Until
now, no data are available on the reproductive functions of the transgenic or
knockout mice for the other subunits of AMPK.In chicken ovary, adiponectin mRNA is more abundant in theca cells than in
granulosa cells (Table1) [13]. In porcine ovary, adiponectin is detected at
similar concentrations in the follicular fluid and serum [51]. Both receptors
are expressed in ovarian follicles. In chicken, the adiponectin type I receptor
(AdipoRI) is twice as abundant in granulosa cells as in theca cells, and the
type II receptor (AdipoR2) is expressed equally strongly in granulosa and
thecal cells (Table 1) [13]. Studies in mice have shown that AdipoR1 may be more tightly linked to AMPK pathway activation, whereas AdipoR2 seems to be associated with PPARα activation [43].
However, mice lacking adiponectin [67], AdipoR1, AdipoR2, or both receptors
[43] are fertile, which suggests that this signalling is not absolutely
essential for ovarian function. However, it may be required for ovulation in
other species or may simply be an additional component for fine-tuning ovarian
function.


## 4. Function of PPARs, AMPK, and Adiponectin in the Ovary

### 4.1. Regulation of Steroidogenesis by PPARγ, PPARα, AMPK, and Adiponectin

TZDs modulate cell proliferation and steroidogenesis in granulosa cells in vitro (reviewed by [23]). Sex steroid secretion
(progesterone, oestradiol) may be inhibited by TZDs in sows and in women
undergoing in vitro fertilization
[56, 68] or stimulated (progesterone and oestradiol), as in rats and ewes [52, 57]). The effects of TZDs depend on the species and the status of granulosa
cell differentiation (follicular phase, before or after the gonadotropin surge
in human granulosa cells). TZDs could regulate their target genes at the
transcriptional level (reviewed by [23, 68]). However, several studies
have suggested that TZDs could also exert their effects by modifying the activity
of steroidogenic enzymes (3-beta-hydroxysteroid-dehydrogenase (3-βHSD) and
aromatase) [56, 69]. Indeed, the concentrations of Cyp11a1 and 3-βHSD mRNA in porcine
granulosa cells and the levels of the corresponding proteins in ovine granulosa
cells are not affected by TZD treatment [56, 57]. Moreover, TZDs increase the
release of pregnenolone, a substrate of 3β-HSD, from porcine granulosa cells
into the medium, whereas progesterone production decreases [56]. Ligands for
PPARα are also known to alter ovarian steroidogenesis. For example, in vivo. fenofibrate, through PPARα-dependent mechanism, inhibits aromatase cytochrome
P450 expression and activity in the ovary of mouse [70]. Another PPARα
synthetic ligand, Wy-14 463, suppresses also aromatase transcript levels
and oestradiol production in cultured rat granulosa cells [71].AMPK, like PPARγ and PPARα, may influence ovarian function by modifying the synthesis of
progesterone and oestradiol. Studies
based on AICAR and the adenovirus-mediated expression of dominant negative AMPK
have demonstrated that AMPK reduces progesterone production, but not oestradiol
production, in rat granulosa cells [60]. This decrease is associated with a
decrease in 3β-HSD
mRNA and protein levels and a decrease in MAPK ERK1/2 phosphorylation [60].
Furthermore, the activation of AMPK by metformin decreases basal and
FSH-induced progesterone secretion by decreasing the levels of proteins
involved in steroidogenesis: (3βHSD, CYP11a1, STAR)
[65]. In granulosa cells from humans and cows, metformin strongly decreases the
secretion of progesterone and oestradiol [62, 72]. In bovine granulosa cells,
this effect is mediated by AMPK activation, and leads to a decrease in MAPK
activation. In human
granulosa cells, metformin also decreases androgen synthesis, by directly
inhibiting Cyp17 activity [73].Thus, AMPK activation decreases steroidogenesis in the granulosa cells
of various species. The effects of AMPK on steroid secretion, like those of
PPARγ, depend on the species and the stimulator of AMPK (AICAR versus metformin). Several
results suggest that metformin-induced AMPK activation could act through
transcriptional mechanism. Further investigations are needed to determine the
molecular mechanism of metformin.Women treated for in vitro fertilization (IVF) present
an increase in serum adiponectin concentration after the administration of
human chorionic gonadotropin, this increase being correlated with progesterone
levels [74]. In cultured porcine granulosa cells, adiponectin modulates the
expression of genes encoding proteins involved in steroid production,
increasing the abundance of transcripts for the steroidogenic acute regulatory
protein, and decreasing the abundance of cytochrome P450 aromatase transcripts
[51]. The MAPK pathway, rather than protein kinase A or
AMPK, mediates the adiponectin signal in ovarian granulosa cells, by ERK1/2
phosphorylation [51]. Surprisingly, adiponectin alone does not affect steroid
production in rat granulosa cells [12]. However, it approximately doubled the
IGF-1-induced secretion of progesterone. These effects may be due to an
increase in IGF-1 receptor beta subunit tyrosine phosphorylation and ERK1/2
phosphorylation [12]. A schema illustrating the effects of PPARα and γ, AMPK and adiponectin activation on the steroidogenesis of rat granulosa
cells is shown in Figure 2.Figure 2
Schema illustrating the effects of (a) metformin- or AICAR-induced AMPK activation, (b) adiponectin, and (c) TZDs or PPAR alpha ligands on the rat granulosa cell steroidogenesis. (a) Metformin or AICAR treatment decreases MAPK ERK1/2 phosphorylation and progesterone secretion through AMPK activation [60, 65]. Metformin decreases also oestradiol secretion through an independent AMPK pathway [60]. (b) Adiponectin treatment increases IGF-1-induced IGF-1R β-subunit tyrosine phosphorylation and MAPK ERK1/2 phosphorylation and progesterone secretion [12]. (c) The PPARα ligand, Wy-14 463, inhibits oestradiol secretion whereas TZDs or PGJ2 increases progesterone secretion and inhibits estradiol secretion in eCG-primed immature rats or increases estradiol
secretion in gonadotropin-primed immature rat [23, 52]. 3βHSD: 3β-hydroxysteroiddehydrogenase, STAR: Steroidogenic acute regulatory protein, CYP11a1: P450 sidechain cleavage, Adipo R1/2: Adiponectin receptor type I and II, MAPK ERK1/2: Mitogen Activated protein kinase Extracellular Regulated kinase,1/2, PGJ2: prostaglandine J2.


### 4.2. Regulation of Granulosa Cell Proliferation

In addition to their effects on steroidogenesis, TZDs decrease the
proliferation of granulosa cells in sheep (PPARγ, [57]). These data are in good
agreement with those obtained in bovine lutein cells since an
aurintricarboxylic acid-mediated decrease of PPARγ is accompanied by a progression of the cell cycle [75]. In our
knowledge, there are no data on the effects of PPARα ligands on granulosa cell
proliferation. In contrast, AMPK and adiponectin are not essential for
granulosa cell proliferation in rat [12, 60].


### 4.3. Regulation of Oocyte Maturation

PPARγ, AMPK, and adiponectin are all expressed in mammalian oocytes [12, 23, 60, 76].
However, AMPK has been studied in more detail than PPARγ, PPARα, and adiponectin. PPARγ may regulate the expression of genes involved in the meiotic maturation
of oocytes (e.g., nitric oxide synthase (NOS)) [23]. Wood et al. recently identified putative binding sites for PPARγ/RXR in the
proximal promoters of several genes differentially expressed in oocytes from
women with PCOS and known to play a role in the meiotic cell cycle [77]. All
these results suggest that PPARγ/RXR may be active in the oocyte. The two
adiponectin receptors, AdipoR1 and AdipoR2, are also expressed in rat oocytes,
and AMPK activity has also been detected in oocytes of several species (see
above), suggesting that adiponectin may play a role through AMPK in determining
oocyte quality (cited by [78]). In addition, women with PCOS showing impairment
in the final maturation of oocytes and in ovulation, present lower circulating
concentrations of adiponectin [10, 79].In vivo, the oocyte remains at the immature stage or germinal vesicle stage (GV, i.e., prophase of meiosis I) until the preovulatory
LH surge [79]. However, if cumulus-oocyte complexes (COCs) are removed from the
follicles and cultured in vitro, oocytes may spontaneously resume meiosis [80, 81]. During nuclear maturation,
immature oocytes undergo germinal vesicle breakdown (GVBD) and proceed through metaphase
II of meiosis. The pharmacological activation of AMPK, by AICAR injection,
in mouse oocytes leads to the induction of oocyte maturation in arrested cumulus-enclosed
oocytes [82]. Metabolic
stresses (oxidative or osmotic) known to activate
AMPK accelerate meiosis in oocytes in which meiosis was previously
arrested by cAMP analogues [83]. However,
the data for mice conflict with those obtained with porcine and bovine oocytes
[84, 85]. Indeed, in these two latter species, AICAR
and metformin significantly increase phosphorylation/activation of AMPK and the percentage of COCs arrested at the GV stage. Thus, AMPK activation has opposite effects in
the control of oocyte maturation in cows, sows and mice. This could be
explained by the important differences that exist in the regulation of oocyte
meiotic resumption between rodent and nonrodent animals such as for example the
time taken for oocytes to undergo meiotic resumption (2 to 3 hours of in vitro
maturation in rodent, 20 hours in pig, and 22 hours in bovine species). Interestingly,
in women with PCOS treated with metformin, the number of mature oocytes
retrieved and oocytes fertilized has been shown to increase after gonadotropin
stimulation for IVF [86]. However, recent data indicate that clomiphene is
superior to metformin in achieving live birth in infertile women with PCOS
[87].


## 4.1. Regulation of Steroidogenesis by PPARγ, PPARα, AMPK, and Adiponectin

TZDs modulate cell proliferation and steroidogenesis in granulosa cells in vitro (reviewed by [23]). Sex steroid secretion
(progesterone, oestradiol) may be inhibited by TZDs in sows and in women
undergoing in vitro fertilization
[56, 68] or stimulated (progesterone and oestradiol), as in rats and ewes [52, 57]). The effects of TZDs depend on the species and the status of granulosa
cell differentiation (follicular phase, before or after the gonadotropin surge
in human granulosa cells). TZDs could regulate their target genes at the
transcriptional level (reviewed by [23, 68]). However, several studies
have suggested that TZDs could also exert their effects by modifying the activity
of steroidogenic enzymes (3-beta-hydroxysteroid-dehydrogenase (3-βHSD) and
aromatase) [56, 69]. Indeed, the concentrations of Cyp11a1 and 3-βHSD mRNA in porcine
granulosa cells and the levels of the corresponding proteins in ovine granulosa
cells are not affected by TZD treatment [56, 57]. Moreover, TZDs increase the
release of pregnenolone, a substrate of 3β-HSD, from porcine granulosa cells
into the medium, whereas progesterone production decreases [56]. Ligands for
PPARα are also known to alter ovarian steroidogenesis. For example, in vivo. fenofibrate, through PPARα-dependent mechanism, inhibits aromatase cytochrome
P450 expression and activity in the ovary of mouse [70]. Another PPARα
synthetic ligand, Wy-14 463, suppresses also aromatase transcript levels
and oestradiol production in cultured rat granulosa cells [71].AMPK, like PPARγ and PPARα, may influence ovarian function by modifying the synthesis of
progesterone and oestradiol. Studies
based on AICAR and the adenovirus-mediated expression of dominant negative AMPK
have demonstrated that AMPK reduces progesterone production, but not oestradiol
production, in rat granulosa cells [60]. This decrease is associated with a
decrease in 3β-HSD
mRNA and protein levels and a decrease in MAPK ERK1/2 phosphorylation [60].
Furthermore, the activation of AMPK by metformin decreases basal and
FSH-induced progesterone secretion by decreasing the levels of proteins
involved in steroidogenesis: (3βHSD, CYP11a1, STAR)
[65]. In granulosa cells from humans and cows, metformin strongly decreases the
secretion of progesterone and oestradiol [62, 72]. In bovine granulosa cells,
this effect is mediated by AMPK activation, and leads to a decrease in MAPK
activation. In human
granulosa cells, metformin also decreases androgen synthesis, by directly
inhibiting Cyp17 activity [73].Thus, AMPK activation decreases steroidogenesis in the granulosa cells
of various species. The effects of AMPK on steroid secretion, like those of
PPARγ, depend on the species and the stimulator of AMPK (AICAR versus metformin). Several
results suggest that metformin-induced AMPK activation could act through
transcriptional mechanism. Further investigations are needed to determine the
molecular mechanism of metformin.Women treated for in vitro fertilization (IVF) present
an increase in serum adiponectin concentration after the administration of
human chorionic gonadotropin, this increase being correlated with progesterone
levels [74]. In cultured porcine granulosa cells, adiponectin modulates the
expression of genes encoding proteins involved in steroid production,
increasing the abundance of transcripts for the steroidogenic acute regulatory
protein, and decreasing the abundance of cytochrome P450 aromatase transcripts
[51]. The MAPK pathway, rather than protein kinase A or
AMPK, mediates the adiponectin signal in ovarian granulosa cells, by ERK1/2
phosphorylation [51]. Surprisingly, adiponectin alone does not affect steroid
production in rat granulosa cells [12]. However, it approximately doubled the
IGF-1-induced secretion of progesterone. These effects may be due to an
increase in IGF-1 receptor beta subunit tyrosine phosphorylation and ERK1/2
phosphorylation [12]. A schema illustrating the effects of PPARα and γ, AMPK and adiponectin activation on the steroidogenesis of rat granulosa
cells is shown in Figure 2.Figure 2
Schema illustrating the effects of (a) metformin- or AICAR-induced AMPK activation, (b) adiponectin, and (c) TZDs or PPAR alpha ligands on the rat granulosa cell steroidogenesis. (a) Metformin or AICAR treatment decreases MAPK ERK1/2 phosphorylation and progesterone secretion through AMPK activation [60, 65]. Metformin decreases also oestradiol secretion through an independent AMPK pathway [60]. (b) Adiponectin treatment increases IGF-1-induced IGF-1R β-subunit tyrosine phosphorylation and MAPK ERK1/2 phosphorylation and progesterone secretion [12]. (c) The PPARα ligand, Wy-14 463, inhibits oestradiol secretion whereas TZDs or PGJ2 increases progesterone secretion and inhibits estradiol secretion in eCG-primed immature rats or increases estradiol
secretion in gonadotropin-primed immature rat [23, 52]. 3βHSD: 3β-hydroxysteroiddehydrogenase, STAR: Steroidogenic acute regulatory protein, CYP11a1: P450 sidechain cleavage, Adipo R1/2: Adiponectin receptor type I and II, MAPK ERK1/2: Mitogen Activated protein kinase Extracellular Regulated kinase,1/2, PGJ2: prostaglandine J2.


## 4.2. Regulation of Granulosa Cell Proliferation

In addition to their effects on steroidogenesis, TZDs decrease the
proliferation of granulosa cells in sheep (PPARγ, [57]). These data are in good
agreement with those obtained in bovine lutein cells since an
aurintricarboxylic acid-mediated decrease of PPARγ is accompanied by a progression of the cell cycle [75]. In our
knowledge, there are no data on the effects of PPARα ligands on granulosa cell
proliferation. In contrast, AMPK and adiponectin are not essential for
granulosa cell proliferation in rat [12, 60].


## 4.3. Regulation of Oocyte Maturation

PPARγ, AMPK, and adiponectin are all expressed in mammalian oocytes [12, 23, 60, 76].
However, AMPK has been studied in more detail than PPARγ, PPARα, and adiponectin. PPARγ may regulate the expression of genes involved in the meiotic maturation
of oocytes (e.g., nitric oxide synthase (NOS)) [23]. Wood et al. recently identified putative binding sites for PPARγ/RXR in the
proximal promoters of several genes differentially expressed in oocytes from
women with PCOS and known to play a role in the meiotic cell cycle [77]. All
these results suggest that PPARγ/RXR may be active in the oocyte. The two
adiponectin receptors, AdipoR1 and AdipoR2, are also expressed in rat oocytes,
and AMPK activity has also been detected in oocytes of several species (see
above), suggesting that adiponectin may play a role through AMPK in determining
oocyte quality (cited by [78]). In addition, women with PCOS showing impairment
in the final maturation of oocytes and in ovulation, present lower circulating
concentrations of adiponectin [10, 79].In vivo, the oocyte remains at the immature stage or germinal vesicle stage (GV, i.e., prophase of meiosis I) until the preovulatory
LH surge [79]. However, if cumulus-oocyte complexes (COCs) are removed from the
follicles and cultured in vitro, oocytes may spontaneously resume meiosis [80, 81]. During nuclear maturation,
immature oocytes undergo germinal vesicle breakdown (GVBD) and proceed through metaphase
II of meiosis. The pharmacological activation of AMPK, by AICAR injection,
in mouse oocytes leads to the induction of oocyte maturation in arrested cumulus-enclosed
oocytes [82]. Metabolic
stresses (oxidative or osmotic) known to activate
AMPK accelerate meiosis in oocytes in which meiosis was previously
arrested by cAMP analogues [83]. However,
the data for mice conflict with those obtained with porcine and bovine oocytes
[84, 85]. Indeed, in these two latter species, AICAR
and metformin significantly increase phosphorylation/activation of AMPK and the percentage of COCs arrested at the GV stage. Thus, AMPK activation has opposite effects in
the control of oocyte maturation in cows, sows and mice. This could be
explained by the important differences that exist in the regulation of oocyte
meiotic resumption between rodent and nonrodent animals such as for example the
time taken for oocytes to undergo meiotic resumption (2 to 3 hours of in vitro
maturation in rodent, 20 hours in pig, and 22 hours in bovine species). Interestingly,
in women with PCOS treated with metformin, the number of mature oocytes
retrieved and oocytes fertilized has been shown to increase after gonadotropin
stimulation for IVF [86]. However, recent data indicate that clomiphene is
superior to metformin in achieving live birth in infertile women with PCOS
[87].


## 5. Conclusion

The nuclear PPARs and the fuel sensor AMPK are expressed in the ovary of various species. Several
studies have shown that they modulate ovarian cell proliferation and
steroidogenesis and could be involved in oocyte maturation. Both PPARα and AMPK mediate the effects of hormones involved in lipid and glucose
metabolism, including adiponectin. Thus, PPARs, AMPK, and adiponectin may be
key signals regulating the amount of energy required for the growth of
follicles, oocytes, and embryos. Further investigations are necessary to assess
the exact importance and mechanisms of action of these molecules in some
ovarian dysfunctions including for example PCOS syndrome.



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*Source: 176275-2007-08-23.xml*