# PPAR Gamma: Coordinating Metabolic and Immune Contributions to Female Fertility

Authors: Cadence E. Minge; Rebecca L. Robker; Robert J. Norman
Journal: PPAR Research
 (2008)

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## Abstract

Peroxisome proliferator-activated receptor gamma (PPARG) regulates cellular functions such as adipogenesis and immune cell activation. However, new information has indicated additional roles of PPARG directing the cyclic changes that occur within ovarian tissue of female mammals, including those that facilitate the release of oocytes each estrous cycle. In addition to ovarian PPARG expression and function, many PPARG actions within adipocytes and macrophages have additional direct and indirect implications for ovarian function and female fertility. This encompasses the regulation of lipid uptake and transport, insulin sensitivity, glucose metabolism, and the regulation of inflammatory mediator synthesis and release. This review discusses the developing links between PPARG activity and female reproductive function, and highlights several mechanisms that may facilitate such a relationship.


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## Body

## 1. Introduction

Since its initial identification in the early 1990’s, peroxisome
proliferator-activated receptor gamma (PPARG) has been primarily recognised as
a regulator of cellular functions such as adipogenesis and immune cell
activation. However, some recent reviews have discussed additional roles of
PPARG directing the cyclic changes that occur within ovarian tissue of female
mammals, including those that facilitate the release of oocytes each estrous cycle [1–4]. In addition to ovarian PPARG expression and function, many PPARG
actions within adipocytes and macrophages have additional direct and indirect
implications for ovarian function and female fertility. For instance, PPARG,
through activation by thiazolidinediones (TZDs), is known to regulate the
metabolism of lipids, providing both self-regulatory PPARG transcriptional
mechanisms, and stimulating an increase in adipogenesis. Whilst the net volume
of adipose tissue carried within an individual can influence reproductive
potential, genes associated with lipid metabolism are also important for
ovarian cells directly. As a result, PPARG has the potential to influence the
cellular operations of follicles containing oocytes and, consequently, the
health of those oocytes released. Likewise, the PPARG regulation of insulin
sensitivity, downstream signalling pathways, and ultimately glucose uptake are
likely to be also  vitally important for normal ovarian function and overall female
fertility.Similarly, PPARG regulation of macrophage function has been
addressed in vitro and within the context of the adipose tissue for many years;
but appropriate activity of resident immune cells is also a prerequisite for
normal ovarian function, as they are required for tissue remodelling
facilitating ovulation, luteinization, and luteolysis [5]. Therefore, not only are adipose/circulating macrophage-sourced
inflammatory mediators sensed by ovarian cells, but these mediators, when
produced locally by the ovary, may  influence the ovarian
function in an autocrine fashion.This review aims to provide evidence for how PPARG-regulated pathways influence the
female’s ability to produce healthy, developmentally competent oocytes. This is
impacted by cellular function operating primarily at the local ovarian level,
either directly acting upon the oocyte itself, or influencing the supporting
ovarian cells that supply the oocyte with hormonal signals and nutrients. In
addition, signals from extraovarian tissues, in particular adipose tissue and
the circulating and/or resident immune cells, also exert powerful influences
over the normal function of the ovary.These concepts of overlapping influence on female fertility are particularly
important when we consider conditions of reduced and impaired fertility such as
polycystic ovary syndrome (PCOS), as well as reduction of reproductive function
associated with excessive bodyweight and insulin resistance. In these
situations, profound dysregulation of both metabolic and immune signalling
pathways exacerbate ovarian perturbations, which are often successfully treated
with administration of PPARG-activating pharmaceuticals.


## 2. PPARG Gene Expression

Successful
mammalian reproduction requires a female body adequately, but not excessively,
nourished, equipped to produce healthy eggs and to supply a growing fetus with sufficient energy. In this way, many tissues within
the female body are able to influence the level of fertility. The extent of
PPARG expression and its temporal regulation within these tissues can provide
an interesting insight into the role of PPARG in female fertility.


### 2.1. Ovarian PPARG

Within the ovary, processes that are modulated by the PPAR superfamily, particularly
PPARG, are among the most critical to normal ovarian function (Figure1).
Steroidogenesis, tissue remodelling, angiogenesis, lipid metabolism, immune
cell activation, and production of proinflammatory mediators are all, to some
extent, controlled by the presence and activity of the PPAR nuclear receptors.
All three PPAR isotypes have been identified in the ovary of many species
including the rat [2, 6], mouse [7], pig [8], sheep [9], cow [10, 11], and human [12, 13]. Localisation of
these nuclear receptors has been established by both in situ hybridisation and
immunohistochemistry [6]. Transcripts for
PPAR alpha (PPARA) have been identified in immune cells and cells in the theca
and stroma, whilst PPAR delta (PPARD) is found across all ovarian compartments [2]. Ovarian
expression of both PPARA and PPARD is relatively stable across the ovulatory
cycle, which suggests these isotypes are likely involved in regulating basal
ovarian functions. PPARG is expressed strongly in the granulosa cells
(primarily responsible for both estradiol
production and the regulation of follicular fluid content), and lessstrongly
in the thecal-region (site of androgen precursor production for granulosa estradiol synthesis) and luteal cells (postovulatory progesterone
production) in the ovaries of rodents and ruminants [1, 2, 9, 14]. PPARG is
detected earlyin folliculogenesis, and in contrast to PPARA and
PPARD isotypes, its expression is dynamic, increasing until the large folliclestage [9], followed by downregulation
in response to the LH surge [2].Figure 1
Overview of PPARG expression by specific ovarian cell types, as follicular development progresses
from early antral and preovulatory follicle to postovulatory corpus luteum.Within the oocyte itself, PPARG expression seems to be dependent upon species, as
moderate expression has been reported in ruminants [16], trace levels in
Xenopus oocytes [17], and undetectable
expression in rodents [1, 18]. It has not yet
been investigated within the human oocyte.


### 2.2. Extraovarian PPARG

The highest level of mammalian PPARG expression is found within adipose tissue [19, 20], and activation
of this adipose PPARG is sufficient [21] and essential to
induce adipogenesis [22, 23]. Adiposity is
also a key regulator of female fertility, affecting multiple aspects of the
reproductive axis in women [24, 25]. Many of the
adipocyte-sourced factors that are under PPARG control, such as the production
of non-esterified free fatty acids, have widespread effects including ovarian
targets [26–28]. In addition, any
activation of adipose PPARG that may influence the amount and activity of
adipocytes/adipokines can subsequently impact upon reproductive potential [29].Both the ovary and adipose tissue are comprised of a considerable proportion of
immune cells, in particular macrophages. Macrophages recruited into tissues are
an important source of many inflammatory mediators that have functions both
locally and systemically. Within the ovary, macrophage contribution to the pool
of functional PPARG has been assessed [1]. TZD treatment
has also been found to affect adipose-recruited macrophages, by increasing the
rate of apoptosis, providing a subsequent reduction in the number of proinflammatory
cytokine-producing cells [30]. Improvements to
the chronically inflamed profile of women with PCOS may well go some way in
explaining the beneficial systemic effects of PPARG activation in such patients
(see Sections 3.2 and 4.4).


### 2.3. Mutations in PPARG Negatively Influence Female Fertility

The PPARG gene contains 9 exons, and spans more than 100 kb [31] (Figure 2(a)).
There are at least 4 isoforms of PPARG, resulting from the use of different
initiator methionines [31–33], which are
believed to be involved in regulated gene expression in specific cells and
tissues. PPARG1, expressed utilizing the untranslated exons A1 and A2, is 477
amino acids long, and is expressed at low levels in many tissues [34]. PPARG2 contains
the translated exon B, and as a result is 28 amino acids longer than PPARG1 [35]. This isotype is
expressed selectively in white adipose tissue, colonic epithelium, and
macrophages [36]. PPARG3, which
contains only exon A2, is found only in the large intestine and macrophages [31]. PPARG4 is
limited to exon 1-6 common to all isotypes [33]. There have been
numerous studies into the effects of genetic variability of PPARG gene sequence
and expression, in both rodent models and human patients (Figure 2(b), Table 1).Table 1
Phenotypes and reproductive effects associated with PPARG mutations in mice
and humans. Abbreviations
used: ART: artificial reproductive technology; BAT: brown adipose tissue; BMI:
body mass index; HbA(1C): haemoglobin A1C; KO: knock-out; PCOS: polycystic
ovary syndrome; T2DM: Type 2 Diabetes Mellitus; TG: Triglycerides; WAT: white
adipose tissue.
SpeciesGenetic AbberationOutcomeEffect on female fertilityReferenceMouseGlobal PPARG-/-Neonatal death—[23]Global PPARG-/+Improved insulin sensitivityFertile[39]Mammary, epithelium, ovary, B- and
T-cell nullOvarian dysfunction and abrogated
mammary development30% of animals completely infertile,
remainder had delayed conception, reduced litter size[7]PPARGhyp/hyp: WAT BAT,
liver, and muscle null.Normal birthweight but subsequent
growth retardation, lipodystrophy, hyperlipideamia, and mild glucose
intoleranceHeterozygote matings produce normal
sized litters, but homozygote matings result in reduced litter size.[40]HumanPro12Ala (34C > G), PPARG2 only.Ala allele↓PPREs affinity, ↓PPARG transactivation. ↑Insulin sensitivity in some studies,
conflicting reports on association with BMI.Possible
relationship with PCOS. In wider, non-PCOS population Ala allele associated
with↓testosterone production[41–43]Pro115Gln
(344G > T), PPARG2 only.Constitutively
activated PPARG,↑adipocyte differentiation. Severe obesity, 3/4 subjects T2DM.Fertility not
assessed.[44]His447His
(1431C > T)T allele may
increase adipocyte differentiation. Presence of T allele associated with↑BMI, and insulin sensitivity.T allele more
common in PCOS compared to BMI-matched controls. T allele associated with↓testosterone.[43, 45]Pro467Leu
(1647C > T)Mutation in LBD,↓coactivator recruitment and downstream
transactivation. ↓Basal gene activity. Lipodystrophy but
normal BMI, severe insulin resistance and hypertension. One carrier (from 4)
responsive to rosiglitazone therapy.Oligomenorrhoea
and hirtsutism, required ART for 1st pregnancy, complicated by
pre-eclampsia and induced labour. 2nd pregnancy spontaneously
conceived, with pre-eclampia, preterm emergency caesarean, and neonatal
infant death.[46, 47]Val290Met
(1115G > A)Mutation
affects LBD, profound blockage of transcriptional activation. Similar
phenotype to P467L. Unresponive to rosiglitazone therapy.Primary
amenorrhoea, hirsutism, acanthosis nigricans, and hypertension.[46, 47]Phe388Leu
(1164T > A)↓PPARG-ligand binding, ↓basal transcriptional activity.
Lipodystrophic and hypertensive with ↑TG. Hyperinsulinemic, later T2DM.Irregular
menses, and bilateral polycystic ovaries treated with salpingo-oopherectomy.
Prior to this carried two pregnancies.[48]Arg397Cys
(1273C > T)Mutation in LBD,
unknown effect on PPARG function. Lipodystrophic,↑TG and T2DM.Hirsutism but no
other indications of hyperandrogenism. Delayed menarche, but regular menses.[49](a) The genomic structure of the5′ end of the human PPARG gene. Exons 1-6 are common. Exons A1
and A2 are untranslated, and exon B is translated, giving rise to two different
proteins corresponding to the G1 or G2 transcripts. (b) The domain structure of
PPARG1 and G2 isoforms with the positioning of mutations or polymorphisms
resulting in substituted amino acid residues, and altered protein functions.
DBD, DNA-binding domain; LBD, Ligand-binding domain. (Figure adapted from
Sundvold and Lien[33], Tsai and Maeda [37], and Stumvoll and Häring [38]).
(a)(b)Work initiated in rodent knockout models revealed
that total PPARG−/− mutants display two independent lethal
phases [23]. Firstly, PPARG
deficiency interferes with terminal differentiation of the trophoblast cells
and with placental vascularization, leading to myocardial thinning, and death
by embryonic day 10. When PPARG null embryos are provided with a wild-type
placenta, this cardiac defect was corrected permitting delivery, although
postnatal pathologies (including multiple haemorrhages and lipodystrophy)
resulted in lethality. To circumvent such restrictions, the Cre-loxP system can
be applied, where Cre recombinase was under the control of the whey acidic
protein (WAP) or mouse mammary tumour virus (MMTV) promoters. This causes PPARG
gene deletions specific to secretory and hematopoietic tissues (alveolar
epithelial cells of mammary tissue, salivary gland cells, oocytes, granulosa
cells, megakaryocytes, and B- and T-cells) [50]. The results of
this study revealed an important PPARG role in fertility: although the mutant
mice appeared to ovulate normally, they exhibited reduced progesterone
secretion as well as impaired implantation. Interestingly, fertility is
affected even when the lesion in PPARG expression is restricted to extraovarian
sites, as homozygote matings of PPARhyp/hyp mutants, lacking PPARG
expression in white and brown adipose tissue, liver, and muscle, had reduced
litter size [40].Examinations of naturally occurring human polymorphisms have focussed on susceptibility to
Type II diabetes, insulin sensitivity, and obesity, and to date at least seven
polymorphisms within the PPARG gene have been described.The Pro12Ala polymorphism is located in exon2, and is only translated within the
adipose tissue-, macrophage-, and colonic epithelium-specific PPARG2 isotype.
The Pro12 allele is carried by approximately 85% of certain regional
populations [51], and a single
nucleotide mutation (C→G) leads to the substitution of an Ala amino
acid [41]. PPARG protein
produced by the Ala12 allele shows reduced in vitro affinity for PPAR response elements
(PPREs) in target gene proximal promoters, and subsequently has reduced PPARG
transactivation [41]. This PPARG SNP
was extensively studied, following initial reports that it was strongly
associated with bodyweight and insulin sensitivity [41], and the effect of the Ala12 mutation on PCOS symptoms has been
closely studied, although some specific conclusions are difficult to reach.
There are conflicting reports regarding the effect of this allele on BMI:
either linked with increased BMI [45, 52–55],
lower BMI [41, 56–59], or not associated
at all [45, 60–64]. Current assumptions are that differential environmentalinteractions between populations can modify the function of this polymorphism.
However, the relationship between Pro12Ala and insulin sensitivity appears more
conclusive. Populations of women positive for Ala12 and PCOS have lower fasting
insulin, reduced measures of systemic insulin resistance, lower insulin
secretion, and lower hirsutism scores than women without the allele [54, 65, 66]. Consequently, the frequency of this allele is much lower in
groups categorised as PCOS [54, 65, 67]. It appears that the Pro12Ala polymorphism of the PPARG gene
may be a modifier of insulin resistance in women with PCOS, which can have a
profound influence on fertility (see Section 4.1).Another PPARG2-specific polymorphism is the rare Pro115Gln substitution in exon 3 that
results in permanent, ligand independent activation [44]. This induces
excessive adipocyte differentiation, and as a result the 4 individuals known to
carry this (nonfamilial) SNP suffer extreme obesity [44], although present
with only moderate metabolic complications including Type 2 Diabetes. The reproductive
implications of hyperactive PPARG2 have not been addressed in these subjects.All other reported polymorphisms are located in regions
common to both PPARG1 and PPARG2. The His447His polymorphisms resulting from a
C to T substitution at nucleotide 1431 in exon 6 is a silent polymorphism that
encodes histidine with either allele [55]. Also referred to as the C161T polymorphism, it is proposed that
this substitution may modulate expression of PPARG by
altering mRNA processing or translation, leading to increased adipocyte
differentiation. Subsequently, carriers of the T allele have elevated BMI. The
T allele is also more common in women with PCOS compared to non-PCOS
BMI-matched controls [45], and therefore
has suspected involvement in the high incidence of obesity in PCOS population.
However, both PCOS subjects and controls with T allele appear to be protected
from other complicating symptoms of obesity, having better insulin sensitivity
in addition to lower circulating testosterone.The remaining polymorphisms are all extremely rare and restricted to single
families.Reported by Barroso et al. [46] and Savage et al. [47], there is a
PPARG1 Pro467Leu substitution in the region required for ligand-dependent
transactivation (PPARG2 residue 495) which results in impaired coactivator
recruitment and downstream transactivation. This mutation also inhibits basal
gene activity and has been found within 4 members of a single family spanning 3
generations. Medical histories reveal that in addition to lipodystrophy and
hypertension (both frequently associated with PPARG mutations), the female
carrier also experienced oligomenorrhea and hirsutism, and required ART
intervention to conceive. This, and a subsequent spontaneously conceived
pregnancy were both complicated with severe pre-eclampsia. Treatment of the
male carrier with rosiglitazone (8 mg/day) was found to normalise chronic
hyperglycaemia after 6 months, suggesting that the mutant PPARG protein is
still able to be activated by exogenously sourced ligands, indicating the
phenotypic profile of these subjects results from abnormal basal and
endogenously activated PPARG activity.Also identified by this study was a similarly positioned PPARG1 Val290Met mutation
(PPARG2 residue 318) in a single female individual. This mutation results in a
profound loss of PPARG function evidenced by both in vitro reporter gene
activity, and in vivo response to rosiglitazone. Experiencing comparable
metabolic complications to subjects with the Pro467Leu substitution, this
individual also reported primary amenorrhoea, hirsutism, and acanthosis
nigricans. Implications of these gynaecological and endocrine aberrations
relating to conception and pregnancy have not been reported.Another loss-of-function mutation is the phenylalanine to leucine substitution at
position 388 (reported with respect to PPARG2, the substitution corresponds to
residue 360 in PPARG1) found in 4 individuals from 3 generation of a single
family [48]. Despite the
reduction in normal PPARG function, concurrent treatment of one individual with
both metformin and rosiglitazone (8 mg daily) provided effective glycemic
control. Two of the affected individuals were female (46-year-old mother and
her 22-year-old daughter), with the older individual experiencing a history of
irregular menses and polycystic ovarian disease, eventually treated with
bilateral salpingo-oophorectomy. At the time of study, the daughter did not
have any significant medical problems (other than diet-controlled
hyperinsulinemia and mild type IV hyperlipoproteinia), with regular menses and
no polycystic ovarian pathology observed.A heterozygous arginine to cysteine mutation at position 397 in PPARG1
(corresponding to residue 425 in PPARG2) was identified in a 64-year-old woman
in 2002 by Argarwal and Garg [49]. Although the
effect on PPARG functionality has not been explicitly described, but the
mutation lies in a region of the protein that forms a salt-bridge, and as a
result, the mutated form may lack proper protein configuration. The Arg397Cys
substitution was associated with lipodystrophy, elevated triglycerides, and
early-onset Type 2 Diabetes. In addition, although pregnancy was never sought,
moderate hirsutism as well as a history of delayed menarche (age 18) and
subsequently irregular menstrual cycles were reported.Overall, these studies demonstrate that PPARG precisely controls various aspect of
systemic metabolism in humans. As female fertility is also disrupted in a
significant number of these patients, it is likely that PPARG regulates female
reproduction either directly, by intrinsic actions within reproductive organs
such as the ovary, or indirectly via the myriad effects on metabolic tissues
such as adipose and liver. To better define links between the metabolic and
reproductive consequences observed in so many of these PPARG mutations, it
would be interesting to recapitulate, in a tissue-specific manner, some of
these PPARG genetic aberrations in mice.


## 2.1. Ovarian PPARG

Within the ovary, processes that are modulated by the PPAR superfamily, particularly
PPARG, are among the most critical to normal ovarian function (Figure1).
Steroidogenesis, tissue remodelling, angiogenesis, lipid metabolism, immune
cell activation, and production of proinflammatory mediators are all, to some
extent, controlled by the presence and activity of the PPAR nuclear receptors.
All three PPAR isotypes have been identified in the ovary of many species
including the rat [2, 6], mouse [7], pig [8], sheep [9], cow [10, 11], and human [12, 13]. Localisation of
these nuclear receptors has been established by both in situ hybridisation and
immunohistochemistry [6]. Transcripts for
PPAR alpha (PPARA) have been identified in immune cells and cells in the theca
and stroma, whilst PPAR delta (PPARD) is found across all ovarian compartments [2]. Ovarian
expression of both PPARA and PPARD is relatively stable across the ovulatory
cycle, which suggests these isotypes are likely involved in regulating basal
ovarian functions. PPARG is expressed strongly in the granulosa cells
(primarily responsible for both estradiol
production and the regulation of follicular fluid content), and lessstrongly
in the thecal-region (site of androgen precursor production for granulosa estradiol synthesis) and luteal cells (postovulatory progesterone
production) in the ovaries of rodents and ruminants [1, 2, 9, 14]. PPARG is
detected earlyin folliculogenesis, and in contrast to PPARA and
PPARD isotypes, its expression is dynamic, increasing until the large folliclestage [9], followed by downregulation
in response to the LH surge [2].Figure 1
Overview of PPARG expression by specific ovarian cell types, as follicular development progresses
from early antral and preovulatory follicle to postovulatory corpus luteum.Within the oocyte itself, PPARG expression seems to be dependent upon species, as
moderate expression has been reported in ruminants [16], trace levels in
Xenopus oocytes [17], and undetectable
expression in rodents [1, 18]. It has not yet
been investigated within the human oocyte.


## 2.2. Extraovarian PPARG

The highest level of mammalian PPARG expression is found within adipose tissue [19, 20], and activation
of this adipose PPARG is sufficient [21] and essential to
induce adipogenesis [22, 23]. Adiposity is
also a key regulator of female fertility, affecting multiple aspects of the
reproductive axis in women [24, 25]. Many of the
adipocyte-sourced factors that are under PPARG control, such as the production
of non-esterified free fatty acids, have widespread effects including ovarian
targets [26–28]. In addition, any
activation of adipose PPARG that may influence the amount and activity of
adipocytes/adipokines can subsequently impact upon reproductive potential [29].Both the ovary and adipose tissue are comprised of a considerable proportion of
immune cells, in particular macrophages. Macrophages recruited into tissues are
an important source of many inflammatory mediators that have functions both
locally and systemically. Within the ovary, macrophage contribution to the pool
of functional PPARG has been assessed [1]. TZD treatment
has also been found to affect adipose-recruited macrophages, by increasing the
rate of apoptosis, providing a subsequent reduction in the number of proinflammatory
cytokine-producing cells [30]. Improvements to
the chronically inflamed profile of women with PCOS may well go some way in
explaining the beneficial systemic effects of PPARG activation in such patients
(see Sections 3.2 and 4.4).


## 2.3. Mutations in PPARG Negatively Influence Female Fertility

The PPARG gene contains 9 exons, and spans more than 100 kb [31] (Figure 2(a)).
There are at least 4 isoforms of PPARG, resulting from the use of different
initiator methionines [31–33], which are
believed to be involved in regulated gene expression in specific cells and
tissues. PPARG1, expressed utilizing the untranslated exons A1 and A2, is 477
amino acids long, and is expressed at low levels in many tissues [34]. PPARG2 contains
the translated exon B, and as a result is 28 amino acids longer than PPARG1 [35]. This isotype is
expressed selectively in white adipose tissue, colonic epithelium, and
macrophages [36]. PPARG3, which
contains only exon A2, is found only in the large intestine and macrophages [31]. PPARG4 is
limited to exon 1-6 common to all isotypes [33]. There have been
numerous studies into the effects of genetic variability of PPARG gene sequence
and expression, in both rodent models and human patients (Figure 2(b), Table 1).Table 1
Phenotypes and reproductive effects associated with PPARG mutations in mice
and humans. Abbreviations
used: ART: artificial reproductive technology; BAT: brown adipose tissue; BMI:
body mass index; HbA(1C): haemoglobin A1C; KO: knock-out; PCOS: polycystic
ovary syndrome; T2DM: Type 2 Diabetes Mellitus; TG: Triglycerides; WAT: white
adipose tissue.
SpeciesGenetic AbberationOutcomeEffect on female fertilityReferenceMouseGlobal PPARG-/-Neonatal death—[23]Global PPARG-/+Improved insulin sensitivityFertile[39]Mammary, epithelium, ovary, B- and
T-cell nullOvarian dysfunction and abrogated
mammary development30% of animals completely infertile,
remainder had delayed conception, reduced litter size[7]PPARGhyp/hyp: WAT BAT,
liver, and muscle null.Normal birthweight but subsequent
growth retardation, lipodystrophy, hyperlipideamia, and mild glucose
intoleranceHeterozygote matings produce normal
sized litters, but homozygote matings result in reduced litter size.[40]HumanPro12Ala (34C > G), PPARG2 only.Ala allele↓PPREs affinity, ↓PPARG transactivation. ↑Insulin sensitivity in some studies,
conflicting reports on association with BMI.Possible
relationship with PCOS. In wider, non-PCOS population Ala allele associated
with↓testosterone production[41–43]Pro115Gln
(344G > T), PPARG2 only.Constitutively
activated PPARG,↑adipocyte differentiation. Severe obesity, 3/4 subjects T2DM.Fertility not
assessed.[44]His447His
(1431C > T)T allele may
increase adipocyte differentiation. Presence of T allele associated with↑BMI, and insulin sensitivity.T allele more
common in PCOS compared to BMI-matched controls. T allele associated with↓testosterone.[43, 45]Pro467Leu
(1647C > T)Mutation in LBD,↓coactivator recruitment and downstream
transactivation. ↓Basal gene activity. Lipodystrophy but
normal BMI, severe insulin resistance and hypertension. One carrier (from 4)
responsive to rosiglitazone therapy.Oligomenorrhoea
and hirtsutism, required ART for 1st pregnancy, complicated by
pre-eclampsia and induced labour. 2nd pregnancy spontaneously
conceived, with pre-eclampia, preterm emergency caesarean, and neonatal
infant death.[46, 47]Val290Met
(1115G > A)Mutation
affects LBD, profound blockage of transcriptional activation. Similar
phenotype to P467L. Unresponive to rosiglitazone therapy.Primary
amenorrhoea, hirsutism, acanthosis nigricans, and hypertension.[46, 47]Phe388Leu
(1164T > A)↓PPARG-ligand binding, ↓basal transcriptional activity.
Lipodystrophic and hypertensive with ↑TG. Hyperinsulinemic, later T2DM.Irregular
menses, and bilateral polycystic ovaries treated with salpingo-oopherectomy.
Prior to this carried two pregnancies.[48]Arg397Cys
(1273C > T)Mutation in LBD,
unknown effect on PPARG function. Lipodystrophic,↑TG and T2DM.Hirsutism but no
other indications of hyperandrogenism. Delayed menarche, but regular menses.[49](a) The genomic structure of the5′ end of the human PPARG gene. Exons 1-6 are common. Exons A1
and A2 are untranslated, and exon B is translated, giving rise to two different
proteins corresponding to the G1 or G2 transcripts. (b) The domain structure of
PPARG1 and G2 isoforms with the positioning of mutations or polymorphisms
resulting in substituted amino acid residues, and altered protein functions.
DBD, DNA-binding domain; LBD, Ligand-binding domain. (Figure adapted from
Sundvold and Lien[33], Tsai and Maeda [37], and Stumvoll and Häring [38]).
(a)(b)Work initiated in rodent knockout models revealed
that total PPARG−/− mutants display two independent lethal
phases [23]. Firstly, PPARG
deficiency interferes with terminal differentiation of the trophoblast cells
and with placental vascularization, leading to myocardial thinning, and death
by embryonic day 10. When PPARG null embryos are provided with a wild-type
placenta, this cardiac defect was corrected permitting delivery, although
postnatal pathologies (including multiple haemorrhages and lipodystrophy)
resulted in lethality. To circumvent such restrictions, the Cre-loxP system can
be applied, where Cre recombinase was under the control of the whey acidic
protein (WAP) or mouse mammary tumour virus (MMTV) promoters. This causes PPARG
gene deletions specific to secretory and hematopoietic tissues (alveolar
epithelial cells of mammary tissue, salivary gland cells, oocytes, granulosa
cells, megakaryocytes, and B- and T-cells) [50]. The results of
this study revealed an important PPARG role in fertility: although the mutant
mice appeared to ovulate normally, they exhibited reduced progesterone
secretion as well as impaired implantation. Interestingly, fertility is
affected even when the lesion in PPARG expression is restricted to extraovarian
sites, as homozygote matings of PPARhyp/hyp mutants, lacking PPARG
expression in white and brown adipose tissue, liver, and muscle, had reduced
litter size [40].Examinations of naturally occurring human polymorphisms have focussed on susceptibility to
Type II diabetes, insulin sensitivity, and obesity, and to date at least seven
polymorphisms within the PPARG gene have been described.The Pro12Ala polymorphism is located in exon2, and is only translated within the
adipose tissue-, macrophage-, and colonic epithelium-specific PPARG2 isotype.
The Pro12 allele is carried by approximately 85% of certain regional
populations [51], and a single
nucleotide mutation (C→G) leads to the substitution of an Ala amino
acid [41]. PPARG protein
produced by the Ala12 allele shows reduced in vitro affinity for PPAR response elements
(PPREs) in target gene proximal promoters, and subsequently has reduced PPARG
transactivation [41]. This PPARG SNP
was extensively studied, following initial reports that it was strongly
associated with bodyweight and insulin sensitivity [41], and the effect of the Ala12 mutation on PCOS symptoms has been
closely studied, although some specific conclusions are difficult to reach.
There are conflicting reports regarding the effect of this allele on BMI:
either linked with increased BMI [45, 52–55],
lower BMI [41, 56–59], or not associated
at all [45, 60–64]. Current assumptions are that differential environmentalinteractions between populations can modify the function of this polymorphism.
However, the relationship between Pro12Ala and insulin sensitivity appears more
conclusive. Populations of women positive for Ala12 and PCOS have lower fasting
insulin, reduced measures of systemic insulin resistance, lower insulin
secretion, and lower hirsutism scores than women without the allele [54, 65, 66]. Consequently, the frequency of this allele is much lower in
groups categorised as PCOS [54, 65, 67]. It appears that the Pro12Ala polymorphism of the PPARG gene
may be a modifier of insulin resistance in women with PCOS, which can have a
profound influence on fertility (see Section 4.1).Another PPARG2-specific polymorphism is the rare Pro115Gln substitution in exon 3 that
results in permanent, ligand independent activation [44]. This induces
excessive adipocyte differentiation, and as a result the 4 individuals known to
carry this (nonfamilial) SNP suffer extreme obesity [44], although present
with only moderate metabolic complications including Type 2 Diabetes. The reproductive
implications of hyperactive PPARG2 have not been addressed in these subjects.All other reported polymorphisms are located in regions
common to both PPARG1 and PPARG2. The His447His polymorphisms resulting from a
C to T substitution at nucleotide 1431 in exon 6 is a silent polymorphism that
encodes histidine with either allele [55]. Also referred to as the C161T polymorphism, it is proposed that
this substitution may modulate expression of PPARG by
altering mRNA processing or translation, leading to increased adipocyte
differentiation. Subsequently, carriers of the T allele have elevated BMI. The
T allele is also more common in women with PCOS compared to non-PCOS
BMI-matched controls [45], and therefore
has suspected involvement in the high incidence of obesity in PCOS population.
However, both PCOS subjects and controls with T allele appear to be protected
from other complicating symptoms of obesity, having better insulin sensitivity
in addition to lower circulating testosterone.The remaining polymorphisms are all extremely rare and restricted to single
families.Reported by Barroso et al. [46] and Savage et al. [47], there is a
PPARG1 Pro467Leu substitution in the region required for ligand-dependent
transactivation (PPARG2 residue 495) which results in impaired coactivator
recruitment and downstream transactivation. This mutation also inhibits basal
gene activity and has been found within 4 members of a single family spanning 3
generations. Medical histories reveal that in addition to lipodystrophy and
hypertension (both frequently associated with PPARG mutations), the female
carrier also experienced oligomenorrhea and hirsutism, and required ART
intervention to conceive. This, and a subsequent spontaneously conceived
pregnancy were both complicated with severe pre-eclampsia. Treatment of the
male carrier with rosiglitazone (8 mg/day) was found to normalise chronic
hyperglycaemia after 6 months, suggesting that the mutant PPARG protein is
still able to be activated by exogenously sourced ligands, indicating the
phenotypic profile of these subjects results from abnormal basal and
endogenously activated PPARG activity.Also identified by this study was a similarly positioned PPARG1 Val290Met mutation
(PPARG2 residue 318) in a single female individual. This mutation results in a
profound loss of PPARG function evidenced by both in vitro reporter gene
activity, and in vivo response to rosiglitazone. Experiencing comparable
metabolic complications to subjects with the Pro467Leu substitution, this
individual also reported primary amenorrhoea, hirsutism, and acanthosis
nigricans. Implications of these gynaecological and endocrine aberrations
relating to conception and pregnancy have not been reported.Another loss-of-function mutation is the phenylalanine to leucine substitution at
position 388 (reported with respect to PPARG2, the substitution corresponds to
residue 360 in PPARG1) found in 4 individuals from 3 generation of a single
family [48]. Despite the
reduction in normal PPARG function, concurrent treatment of one individual with
both metformin and rosiglitazone (8 mg daily) provided effective glycemic
control. Two of the affected individuals were female (46-year-old mother and
her 22-year-old daughter), with the older individual experiencing a history of
irregular menses and polycystic ovarian disease, eventually treated with
bilateral salpingo-oophorectomy. At the time of study, the daughter did not
have any significant medical problems (other than diet-controlled
hyperinsulinemia and mild type IV hyperlipoproteinia), with regular menses and
no polycystic ovarian pathology observed.A heterozygous arginine to cysteine mutation at position 397 in PPARG1
(corresponding to residue 425 in PPARG2) was identified in a 64-year-old woman
in 2002 by Argarwal and Garg [49]. Although the
effect on PPARG functionality has not been explicitly described, but the
mutation lies in a region of the protein that forms a salt-bridge, and as a
result, the mutated form may lack proper protein configuration. The Arg397Cys
substitution was associated with lipodystrophy, elevated triglycerides, and
early-onset Type 2 Diabetes. In addition, although pregnancy was never sought,
moderate hirsutism as well as a history of delayed menarche (age 18) and
subsequently irregular menstrual cycles were reported.Overall, these studies demonstrate that PPARG precisely controls various aspect of
systemic metabolism in humans. As female fertility is also disrupted in a
significant number of these patients, it is likely that PPARG regulates female
reproduction either directly, by intrinsic actions within reproductive organs
such as the ovary, or indirectly via the myriad effects on metabolic tissues
such as adipose and liver. To better define links between the metabolic and
reproductive consequences observed in so many of these PPARG mutations, it
would be interesting to recapitulate, in a tissue-specific manner, some of
these PPARG genetic aberrations in mice.


## 3. Ligands

Together with expression of PPARG
itself, availability of ligands is a primary regulating factor determining the
ability of PPARG to influence target gene expression. Ligands can be produced
endogenously, providing physiological significance, or sourced exogenously, as
therapeutic factors given to target specific metabolic and reproductive
symptoms.


### 3.1. Endogenous Ligands: Physiological Function  of PPARG

All PPARs bind and are activated by naturally occurring fatty
acids and their metabolites [68], thus acting as fatty acid-activated receptors that function as
key regulators of glucose and cholesterol metabolism. The precise nature of
endogenous PPARG ligand binding and activation remains poorly defined and more
research is needed in this area. However, the potential for important
physiological ovarian PPARG activation is considerable, as many natural ligands
have been shown to be present within the ovary, and produced locally by ovarian
cells. Included in this list are ω3- and ω6-polyunsaturated fatty acids (PUFAs) such as
the essential fatty acids linoleic acid, linolenic acid, arachidonic acid, and
eicosapentanoic acid ([69] and reviewed [34]). Additional PPARG agonists such as prostaglandin metabolites of
these substances and immunologically-derived eicosanoids are also produced
within the ovarian environment in a hormonally regulated manner, with elevated
production as ovulation progresses [70–73]. It is possible that PPARG may have a role in the feed-forward
production of eicosanoid ligands, based on identification of a PPRE in the prostaglandin-endoperoxide
synthase 2 (a.k.a. COX-2) promoter [74], which would facilitate amplified production of pro-ovulatory
prostaglandins.


### 3.2. Exogenous Ligands: Therapeutic Application  of PPARG Activation

As information emerges regarding the endogenous roles for naturally
activated PPARG within the ovarian follicular environment, other evidence of
PPARG involvement with ovarian function comes from reports utilising synthetic
PPARG ligands, specifically, administration of TZDs to women diagnosed with
PCOS (Table2).Table 2
Summary of reports published within the past 2 years on
the use of PPARG activating agents for reproductive symptoms. Abbreviations used: AUC,
area under the curve; BMI body mass index; CC clomiphene citrate; DHEA-S
dehydroepiandrosterone sulfate; E2, estradiol; FAI, free androgen index; FSH, follicle-stimulating
hormone; GnRH,
gonadotropin releasing hormone; HbA(1C), haemoglobin A1C; HDL-C, high density
lipoprotein-cholesterol; HOMA, homeostasis model of assessment for insulin
sensitivity; IGF1 insulin-like growth factor 1; IGFBP-1/3, insulin-like growth
factor binding protein 1 or 3; LDL-C, low density lipoprotein-cholesterol; LH,
luteinizing hormone; OGTT, oral glucose tolerance test; PCOS, polycystic ovary
syndrome; QUICKI,
quantitative insulin-sensitivity check index; SHBG, sex hormone binding globulin; T, testosterone; WHR, waist to
hip ratio.
Reference:Rautio et al. [75] and Rautio et al. [76]Patient
profile:Overweight
but not obese PCOS (n = 30)PPAR
agonist:Rosiglitazone
(4 mg once daily for 2 weeks then 4 mg twice daily for 16 weeks)Metabolic
outcomes:Serum
C-reactive protein levels, leukocyte count, and alanine aminotransferase
enzyme activity decreased, but lipid and blood pressure did not change. Glucose
tolerance and peripheral insulin response normalized in the rosiglitazone
group.Reproductive
outcomes:Rosiglitazone
improved menstrual cyclicity, SHBG levels; and decreased serum levels of
androstenedione, 17-hydroxyprogesterone (17-OHP), DHEA and DHEA-S.Reference:Rouzi and Ardawi [77]Patient
profile:Obese
PCOS (n = 12)PPAR
agonist:Rosiglitazone
(4 mg twice daily for 3 cycles, CC administered for 5 days starting 3 days
after rosiglitazone initiated)Metabolic
outcomes:No changes in fasting plasma glucose or HbA(1C) or
IGFBP-3 values. Fasting serum insulin, DHEA-S, androstenedione, and IGF-1
levels decreased significantly and IGFBP-1 exhibited significant increases.Reproductive
outcomes:Total-T, free-T, LH, and SHBG decreased. Follicular
development and ovulation rate increased, trend for increased pregnancy rate
in group receiving short-term administration of rosiglitazone compared to
matched control receiving metformin.Reference:Mitkov et al. [78]Patient
profile:Obese,
insulin resistant PCOS (n = 15)PPAR
agonist:Rosiglitazone
(4 mg/day for 12 weeks)Metabolic
outcomes:Hyperinsulinemia
and insulin resistance normalized.Reproductive
outcomes:Total-T
and FAI profile tended to normalise. Number of women with oligomenorrhea was
reduced by 67%Reference:Cataldo et al. [79]Patient
profile:Insulin
resistant PCOS (n = 11–16/group)PPAR
agonist:Rosiglitazone
(2, 4 or 8 mg/day for 12 weeks)Metabolic
outcomes:Steady
state plasma glucose declined and hyperinsulinemia fell in a dose-dependent
manner.Reproductive
outcomes:Serum
LH, total-T, and free-T were unchanged; SHBG increased. Ovulation occurred in
55%, without significant dose dependence. Before and during treatment,
ovulators on rosiglitazone had lower circulating insulin and free-T and
higher SHBG than nonovulators.Reference:Lemay et al. [80]Patient
profile:Overweight,
insulin resistant PCOS (n = 15)PPAR
agonist:Rosiglitazone
(4 mg/day for 6 months)Metabolic
outcomes:Plasma
insulin, insulin resistance indices and insulin AUC in response to OGTT all
decreased compared to controls receiving antiandrogenic estrogen-progestin.
Effect on lipids was limited.Reproductive
outcomes:No
significant effect on androgens or hirsutism.Reference:Garmes et al. [81]Patient
profile:Obese
insulin resistant PCOS (n = 15)PPAR
agonist:Pioglitazone
(30 mg/day for 8 weeks)Metabolic
outcomes:Insulin
response to OGTT significantly decreased.Reproductive
outcomes:Total-T
and free-T levels decreased, SHBG increased, and LH response to GnRH
stimulation decreased.Reference:Yilmaz et al. [82–84]Patient
profile:Obese
or lean PCOS
(n = 20 obese, n = 20 lean)PPAR
agonist:Rosiglitazone
(4 mg/day for 12 weeks)Metabolic
outcomes:Indices
of oxidative stress improved. HOMA, insulin AUC, fasting insulin and
C-peptide levels decreased significantly. Glucose/insulin ratio and BMI
increasedReproductive outcomes:Seurm
levels of free-T, androstenedione, and DHEA-S decreased significantly.
Menstrual disturbances improved in 61.5% of lean and 53.8% of obese
patients. In a second cohort of patients, menstrual cycles became regular in
87.8%.Reference:Tarkun et al. [85]Patient
profile:Young,
lean PCOS (n = 31)PPAR
agonist:Rosiglitazone
(4 mg/day for 12 months)Metabolic
outcomes:Fasting
insulin and insulin resistance indices significantly improved. No changes in
BMI, waist circumference, serum total cholesterol, or LDL-C. Serum C-reactive
protein levels decreased; and endothelium-dependent vascular responses
improved.Reproductive
outcomes:Significant
decreases in serum T, although no change in FSH and LH levels. Hirsutism
score decreased significantly after treatment. 77.4% of women reverted to
regular menstrual cycles. Levels of SHBG increased significantly after
treatment.Reference:Dereli et al. [86]Patient
profile:Nonobese
PCOS (n = 20/group)PPAR
agonist:Rosiglitazone
(2 mg/day or 4 mg/day for 8 months)Metabolic
outcomes:75%
of women in the 2 mg group and 95% in the 4 mg group achieved normal glucose
tolerance. Improved insulin resistance in a dose-related fashion, without  adverse events
or liver enzyme elevations.Reproductive
outcomes:Decreased
free-T levels were better in the 4 mg group than the 2 mg group, and 70% of
women in the 2 mg group and 85% of women in the 4 mg group achieved ovulatory
menses.Reference:Mehta et al. [87]Patient
profile:Obese
PCOS (n = 9)PPAR
agonist:Pioglitazone
(45 mg/day for 20 weeks)Metabolic
outcomes:Significant
improvement in insulin sensitivityReproductive
outcomes:LH
levels, LH pulse frequency and amplitude, as well as gonadotropin responses
to GnRH were not influenced.Reference:Ortega-González et al. [88]Patient
profile:Obese,
insulin resistant PCOS (n = 25)PPAR
agonist:Pioglitazone
(30 mg/day for 6 months)Metabolic
outcomes:Body
weight, BMI, and WHR increased significantly. Fasting insulin and insulin AUC
during a 2-h OGTT decreased. Insulin resistance decreased and insulin
sensitivity increased after treatment with either pioglitazone or metformin
received by control group.Reproductive
outcomes:Hirsutism,
free-T and androstenedione declined to a similar extent after treatment with
either drug. Treatment with both drugs was associated with the occurrence of
pregnancyReference:Sepilian and Nagamani [89]Patient
profile:Obese
insulin resistant PCOS (n = 12)PPAR
agonist:Rosiglitazone
(4 mg/day for 6 months)Metabolic
outcomes:Fasting
insulin, insulin AUC, fasting glucose, and glucose AUC significantly decreased.
No significant change in BMIReproductive
outcomes:Both
total-T, free-T and DHEA-S levels decreased significantly. No significant
change in LH levels. Levels of SHBG increased significantly after treatment,
91.7% of women reverted to regular ovulatory cycles during the treatment
periodPCOS is the leading cause of infertility and menstrual irregularities in women of
reproductive age and is characterised by chronic hyperandrogenic anovulation [90].
This is thought to be due, in general, to hypothalamic-pituitary axis
dysregulation causing elevated basal LH levels that overstimulate cells of the
theca interna [91].
Insulin resistance also appears to contribute to the syndrome in many instances
[92],
as the pituitary responds to elevated plasma levels of insulin to augment LH
release [91].The potential merits of applying TZDs to improve reproductive outcomes in infertile
PCOS women was first demonstrated by Azziz et al. [93]. Since then,
treatment of PCOS patients with the TZDs rosiglitazone or pioglitazone have
been shown to not only improve insulin action in peripheral tissues, attenuate
hyperinsulinemia, and lower circulating levels of lipids [92, 94], but also to
improve a range of reproductive outcomes particularly circulating sex hormone
levels, and ovulation rate [77, 88, 90, 95–97] (see recent
reports summarised in Table 2).The beneficial effects of TZDs on ovarian PCOS symptoms were first attributed to
improvements in defective insulin action and secretion [92].
However, actions upon various ovarian cells directly
illustrated both in vitro [1, 9, 14, 98, 99] and in vivo [100, 101] confirms a direct interaction between these compounds and ovarian
PPARG.Particular focus has been directed upon the effect of PPARG
activation on the synthesis of ovarian steroid hormones and the expression of
many rate-limiting steroidogenic enzymes has been investigated.(1)
Steroidogenic acute regulatory protein (StAR): facilitates that
rapid mobilization of cholesterol for initial catalysis to pregnenolone by the
P450-side chain cleavage enzyme located within the mitochondria [102]. It has been recently reported that both rosiglitazone and
pioglitazone significantly up regulate StAR protein synthesis by human
granulosa cells in vitro [103].(2)
3β-hydroxysteroid dehydrogenase (3β-HSD):
catalyses the conversion of pregnenolone to progesterone by luteal cells [104]. Work on porcine granulosa cells has found that troglitazone
competitively inhibits 3β-HSD enzyme
activity within these cells [99].(3)
Steroid 17-alpha-hydroxylase (P450c17): converts progesterone to
androgen within ovarian theca cells [105]. Conflicting reports have arisen regarding the effect of TZDs on
the expression and activity of this enzyme, many of which may be artefacts of
various culture conditions. P450c17 mRNA production has been found to increase
following porcine thecal cell exposure to TZDs [8], whilst other reports indicate suppression of enzymatic
expression and/or activity in primary porcine thecal cells or human cell lines [106–108].(4)
P450 aromatase (P450arom): aromatises androgen precursor to
estradiol, and is expressed by ovarian granulosa [109] and luteal [110] cells. Although there is no correlation between the expression of
the P450arom enzyme and PPARG itself during folliculogenesis, many reports have
described the down-regulation of P450arom following TZD exposure in human
ovarian cell cultures [13, 111–113].
Taken together, these findings provide strong evidence for the direct effect of TZD
administration and PPARG activation on ovarian hormonal synthesis and
secretion. Specifically the following.(1)
Androgen:
TZDs found to inhibit LH- and
insulin-stimulated androgen biosynthesis by purified porcine thecal [108], and mixed human ovarian [98] cells. They have also been found to reduce plasma
testosterone levels in women with PCOS [76, 77, 81–84, 86, 89, 91, 97, 114, 115].(2)
Estrogen: While it is accepted that TZDs indeed influence estrogen
secretion, estrogenic responses to TZDs appear to be dependent on confounding
factors such as species, age, and endocrine setting. For instance, TZDs have
been found to increase estradiol secretion [2], and decrease estradiol production [116]. PPARG activation by TZDs and phthalate toxins are believed to
mediate the antiestrogenic effects of these agents in cultured rat granulosa
cells [116], and TZDs have also been found to suppress stimulated estradiol
secretion in human granulosa cell cultures [98].(3)
Progesterone: As for estrogen, progesterone
responses to PPARG activation via natural or endogenous ligands are unclear,
and are probably regulated by species, and stage of folliculogenesis. Most
publications investigating a range of species, including primary bovine, ovine,
porcine, or rodent cell cultures, report increases in progesterone secretion
following administration of PPARG activators in vitro [2, 8–10],
whilst some others suggest inhibition of stimulated progesterone secretion by
porcine granulosa cells [14].
The net influence of TZD treatment on ovarian PPARG activation and subsequent
steroidogenesis in vivo remains poorly defined across all species investigated.
The most conclusive evidence for an advantageous outcome on hormonal
(specifically androgen) profile following treatment is observed in women with
PCOS, as overviewed in Table 2. As a result, increasing attention may be paid
towards the application of these drugs in such conditions of significant
hormonal perturbations.Rosiglitazone
and pioglitazone are currently listed as a Pregnancy Category C drug (i.e., not
tested for use during human pregnancy), and some side effects of TZD
administration, such as weight gain, fluid retention (reviewed in [117]), and possible
bone demineralisation [118], preclude their
widespread use during pregnancy. However, in vitro treatment of 2 cell mouse
embryos, or in vivo treatment of pregnant mice with rosiglitazone was not found
to impact upon normal blastocyst development, or litter rates and sizes [119]. In situations
where conception has occurred following TZD treatment for PCOS, no adverse
fetal outcomes have been observed [88, 96, 120]. Also in a recent
study, examining tissue obtained from women with scheduled pregnancy
terminations, it was found that placental transfer of maternally administered
rosiglitazone to fetal tissues is minimal in the first 10
weeks of pregnancy [121].


## 3.1. Endogenous Ligands: Physiological Function  of PPARG

All PPARs bind and are activated by naturally occurring fatty
acids and their metabolites [68], thus acting as fatty acid-activated receptors that function as
key regulators of glucose and cholesterol metabolism. The precise nature of
endogenous PPARG ligand binding and activation remains poorly defined and more
research is needed in this area. However, the potential for important
physiological ovarian PPARG activation is considerable, as many natural ligands
have been shown to be present within the ovary, and produced locally by ovarian
cells. Included in this list are ω3- and ω6-polyunsaturated fatty acids (PUFAs) such as
the essential fatty acids linoleic acid, linolenic acid, arachidonic acid, and
eicosapentanoic acid ([69] and reviewed [34]). Additional PPARG agonists such as prostaglandin metabolites of
these substances and immunologically-derived eicosanoids are also produced
within the ovarian environment in a hormonally regulated manner, with elevated
production as ovulation progresses [70–73]. It is possible that PPARG may have a role in the feed-forward
production of eicosanoid ligands, based on identification of a PPRE in the prostaglandin-endoperoxide
synthase 2 (a.k.a. COX-2) promoter [74], which would facilitate amplified production of pro-ovulatory
prostaglandins.


## 3.2. Exogenous Ligands: Therapeutic Application  of PPARG Activation

As information emerges regarding the endogenous roles for naturally
activated PPARG within the ovarian follicular environment, other evidence of
PPARG involvement with ovarian function comes from reports utilising synthetic
PPARG ligands, specifically, administration of TZDs to women diagnosed with
PCOS (Table2).Table 2
Summary of reports published within the past 2 years on
the use of PPARG activating agents for reproductive symptoms. Abbreviations used: AUC,
area under the curve; BMI body mass index; CC clomiphene citrate; DHEA-S
dehydroepiandrosterone sulfate; E2, estradiol; FAI, free androgen index; FSH, follicle-stimulating
hormone; GnRH,
gonadotropin releasing hormone; HbA(1C), haemoglobin A1C; HDL-C, high density
lipoprotein-cholesterol; HOMA, homeostasis model of assessment for insulin
sensitivity; IGF1 insulin-like growth factor 1; IGFBP-1/3, insulin-like growth
factor binding protein 1 or 3; LDL-C, low density lipoprotein-cholesterol; LH,
luteinizing hormone; OGTT, oral glucose tolerance test; PCOS, polycystic ovary
syndrome; QUICKI,
quantitative insulin-sensitivity check index; SHBG, sex hormone binding globulin; T, testosterone; WHR, waist to
hip ratio.
Reference:Rautio et al. [75] and Rautio et al. [76]Patient
profile:Overweight
but not obese PCOS (n = 30)PPAR
agonist:Rosiglitazone
(4 mg once daily for 2 weeks then 4 mg twice daily for 16 weeks)Metabolic
outcomes:Serum
C-reactive protein levels, leukocyte count, and alanine aminotransferase
enzyme activity decreased, but lipid and blood pressure did not change. Glucose
tolerance and peripheral insulin response normalized in the rosiglitazone
group.Reproductive
outcomes:Rosiglitazone
improved menstrual cyclicity, SHBG levels; and decreased serum levels of
androstenedione, 17-hydroxyprogesterone (17-OHP), DHEA and DHEA-S.Reference:Rouzi and Ardawi [77]Patient
profile:Obese
PCOS (n = 12)PPAR
agonist:Rosiglitazone
(4 mg twice daily for 3 cycles, CC administered for 5 days starting 3 days
after rosiglitazone initiated)Metabolic
outcomes:No changes in fasting plasma glucose or HbA(1C) or
IGFBP-3 values. Fasting serum insulin, DHEA-S, androstenedione, and IGF-1
levels decreased significantly and IGFBP-1 exhibited significant increases.Reproductive
outcomes:Total-T, free-T, LH, and SHBG decreased. Follicular
development and ovulation rate increased, trend for increased pregnancy rate
in group receiving short-term administration of rosiglitazone compared to
matched control receiving metformin.Reference:Mitkov et al. [78]Patient
profile:Obese,
insulin resistant PCOS (n = 15)PPAR
agonist:Rosiglitazone
(4 mg/day for 12 weeks)Metabolic
outcomes:Hyperinsulinemia
and insulin resistance normalized.Reproductive
outcomes:Total-T
and FAI profile tended to normalise. Number of women with oligomenorrhea was
reduced by 67%Reference:Cataldo et al. [79]Patient
profile:Insulin
resistant PCOS (n = 11–16/group)PPAR
agonist:Rosiglitazone
(2, 4 or 8 mg/day for 12 weeks)Metabolic
outcomes:Steady
state plasma glucose declined and hyperinsulinemia fell in a dose-dependent
manner.Reproductive
outcomes:Serum
LH, total-T, and free-T were unchanged; SHBG increased. Ovulation occurred in
55%, without significant dose dependence. Before and during treatment,
ovulators on rosiglitazone had lower circulating insulin and free-T and
higher SHBG than nonovulators.Reference:Lemay et al. [80]Patient
profile:Overweight,
insulin resistant PCOS (n = 15)PPAR
agonist:Rosiglitazone
(4 mg/day for 6 months)Metabolic
outcomes:Plasma
insulin, insulin resistance indices and insulin AUC in response to OGTT all
decreased compared to controls receiving antiandrogenic estrogen-progestin.
Effect on lipids was limited.Reproductive
outcomes:No
significant effect on androgens or hirsutism.Reference:Garmes et al. [81]Patient
profile:Obese
insulin resistant PCOS (n = 15)PPAR
agonist:Pioglitazone
(30 mg/day for 8 weeks)Metabolic
outcomes:Insulin
response to OGTT significantly decreased.Reproductive
outcomes:Total-T
and free-T levels decreased, SHBG increased, and LH response to GnRH
stimulation decreased.Reference:Yilmaz et al. [82–84]Patient
profile:Obese
or lean PCOS
(n = 20 obese, n = 20 lean)PPAR
agonist:Rosiglitazone
(4 mg/day for 12 weeks)Metabolic
outcomes:Indices
of oxidative stress improved. HOMA, insulin AUC, fasting insulin and
C-peptide levels decreased significantly. Glucose/insulin ratio and BMI
increasedReproductive outcomes:Seurm
levels of free-T, androstenedione, and DHEA-S decreased significantly.
Menstrual disturbances improved in 61.5% of lean and 53.8% of obese
patients. In a second cohort of patients, menstrual cycles became regular in
87.8%.Reference:Tarkun et al. [85]Patient
profile:Young,
lean PCOS (n = 31)PPAR
agonist:Rosiglitazone
(4 mg/day for 12 months)Metabolic
outcomes:Fasting
insulin and insulin resistance indices significantly improved. No changes in
BMI, waist circumference, serum total cholesterol, or LDL-C. Serum C-reactive
protein levels decreased; and endothelium-dependent vascular responses
improved.Reproductive
outcomes:Significant
decreases in serum T, although no change in FSH and LH levels. Hirsutism
score decreased significantly after treatment. 77.4% of women reverted to
regular menstrual cycles. Levels of SHBG increased significantly after
treatment.Reference:Dereli et al. [86]Patient
profile:Nonobese
PCOS (n = 20/group)PPAR
agonist:Rosiglitazone
(2 mg/day or 4 mg/day for 8 months)Metabolic
outcomes:75%
of women in the 2 mg group and 95% in the 4 mg group achieved normal glucose
tolerance. Improved insulin resistance in a dose-related fashion, without  adverse events
or liver enzyme elevations.Reproductive
outcomes:Decreased
free-T levels were better in the 4 mg group than the 2 mg group, and 70% of
women in the 2 mg group and 85% of women in the 4 mg group achieved ovulatory
menses.Reference:Mehta et al. [87]Patient
profile:Obese
PCOS (n = 9)PPAR
agonist:Pioglitazone
(45 mg/day for 20 weeks)Metabolic
outcomes:Significant
improvement in insulin sensitivityReproductive
outcomes:LH
levels, LH pulse frequency and amplitude, as well as gonadotropin responses
to GnRH were not influenced.Reference:Ortega-González et al. [88]Patient
profile:Obese,
insulin resistant PCOS (n = 25)PPAR
agonist:Pioglitazone
(30 mg/day for 6 months)Metabolic
outcomes:Body
weight, BMI, and WHR increased significantly. Fasting insulin and insulin AUC
during a 2-h OGTT decreased. Insulin resistance decreased and insulin
sensitivity increased after treatment with either pioglitazone or metformin
received by control group.Reproductive
outcomes:Hirsutism,
free-T and androstenedione declined to a similar extent after treatment with
either drug. Treatment with both drugs was associated with the occurrence of
pregnancyReference:Sepilian and Nagamani [89]Patient
profile:Obese
insulin resistant PCOS (n = 12)PPAR
agonist:Rosiglitazone
(4 mg/day for 6 months)Metabolic
outcomes:Fasting
insulin, insulin AUC, fasting glucose, and glucose AUC significantly decreased.
No significant change in BMIReproductive
outcomes:Both
total-T, free-T and DHEA-S levels decreased significantly. No significant
change in LH levels. Levels of SHBG increased significantly after treatment,
91.7% of women reverted to regular ovulatory cycles during the treatment
periodPCOS is the leading cause of infertility and menstrual irregularities in women of
reproductive age and is characterised by chronic hyperandrogenic anovulation [90].
This is thought to be due, in general, to hypothalamic-pituitary axis
dysregulation causing elevated basal LH levels that overstimulate cells of the
theca interna [91].
Insulin resistance also appears to contribute to the syndrome in many instances
[92],
as the pituitary responds to elevated plasma levels of insulin to augment LH
release [91].The potential merits of applying TZDs to improve reproductive outcomes in infertile
PCOS women was first demonstrated by Azziz et al. [93]. Since then,
treatment of PCOS patients with the TZDs rosiglitazone or pioglitazone have
been shown to not only improve insulin action in peripheral tissues, attenuate
hyperinsulinemia, and lower circulating levels of lipids [92, 94], but also to
improve a range of reproductive outcomes particularly circulating sex hormone
levels, and ovulation rate [77, 88, 90, 95–97] (see recent
reports summarised in Table 2).The beneficial effects of TZDs on ovarian PCOS symptoms were first attributed to
improvements in defective insulin action and secretion [92].
However, actions upon various ovarian cells directly
illustrated both in vitro [1, 9, 14, 98, 99] and in vivo [100, 101] confirms a direct interaction between these compounds and ovarian
PPARG.Particular focus has been directed upon the effect of PPARG
activation on the synthesis of ovarian steroid hormones and the expression of
many rate-limiting steroidogenic enzymes has been investigated.(1)
Steroidogenic acute regulatory protein (StAR): facilitates that
rapid mobilization of cholesterol for initial catalysis to pregnenolone by the
P450-side chain cleavage enzyme located within the mitochondria [102]. It has been recently reported that both rosiglitazone and
pioglitazone significantly up regulate StAR protein synthesis by human
granulosa cells in vitro [103].(2)
3β-hydroxysteroid dehydrogenase (3β-HSD):
catalyses the conversion of pregnenolone to progesterone by luteal cells [104]. Work on porcine granulosa cells has found that troglitazone
competitively inhibits 3β-HSD enzyme
activity within these cells [99].(3)
Steroid 17-alpha-hydroxylase (P450c17): converts progesterone to
androgen within ovarian theca cells [105]. Conflicting reports have arisen regarding the effect of TZDs on
the expression and activity of this enzyme, many of which may be artefacts of
various culture conditions. P450c17 mRNA production has been found to increase
following porcine thecal cell exposure to TZDs [8], whilst other reports indicate suppression of enzymatic
expression and/or activity in primary porcine thecal cells or human cell lines [106–108].(4)
P450 aromatase (P450arom): aromatises androgen precursor to
estradiol, and is expressed by ovarian granulosa [109] and luteal [110] cells. Although there is no correlation between the expression of
the P450arom enzyme and PPARG itself during folliculogenesis, many reports have
described the down-regulation of P450arom following TZD exposure in human
ovarian cell cultures [13, 111–113].
Taken together, these findings provide strong evidence for the direct effect of TZD
administration and PPARG activation on ovarian hormonal synthesis and
secretion. Specifically the following.(1)
Androgen:
TZDs found to inhibit LH- and
insulin-stimulated androgen biosynthesis by purified porcine thecal [108], and mixed human ovarian [98] cells. They have also been found to reduce plasma
testosterone levels in women with PCOS [76, 77, 81–84, 86, 89, 91, 97, 114, 115].(2)
Estrogen: While it is accepted that TZDs indeed influence estrogen
secretion, estrogenic responses to TZDs appear to be dependent on confounding
factors such as species, age, and endocrine setting. For instance, TZDs have
been found to increase estradiol secretion [2], and decrease estradiol production [116]. PPARG activation by TZDs and phthalate toxins are believed to
mediate the antiestrogenic effects of these agents in cultured rat granulosa
cells [116], and TZDs have also been found to suppress stimulated estradiol
secretion in human granulosa cell cultures [98].(3)
Progesterone: As for estrogen, progesterone
responses to PPARG activation via natural or endogenous ligands are unclear,
and are probably regulated by species, and stage of folliculogenesis. Most
publications investigating a range of species, including primary bovine, ovine,
porcine, or rodent cell cultures, report increases in progesterone secretion
following administration of PPARG activators in vitro [2, 8–10],
whilst some others suggest inhibition of stimulated progesterone secretion by
porcine granulosa cells [14].
The net influence of TZD treatment on ovarian PPARG activation and subsequent
steroidogenesis in vivo remains poorly defined across all species investigated.
The most conclusive evidence for an advantageous outcome on hormonal
(specifically androgen) profile following treatment is observed in women with
PCOS, as overviewed in Table 2. As a result, increasing attention may be paid
towards the application of these drugs in such conditions of significant
hormonal perturbations.Rosiglitazone
and pioglitazone are currently listed as a Pregnancy Category C drug (i.e., not
tested for use during human pregnancy), and some side effects of TZD
administration, such as weight gain, fluid retention (reviewed in [117]), and possible
bone demineralisation [118], preclude their
widespread use during pregnancy. However, in vitro treatment of 2 cell mouse
embryos, or in vivo treatment of pregnant mice with rosiglitazone was not found
to impact upon normal blastocyst development, or litter rates and sizes [119]. In situations
where conception has occurred following TZD treatment for PCOS, no adverse
fetal outcomes have been observed [88, 96, 120]. Also in a recent
study, examining tissue obtained from women with scheduled pregnancy
terminations, it was found that placental transfer of maternally administered
rosiglitazone to fetal tissues is minimal in the first 10
weeks of pregnancy [121].


## 4. Mechanisms: PPARG Regulation of  Metabolic and Immune Factors  Influencing Female Fertility

PPARG is known to regulate many
pathways involving insulin sensitivity, glucose metabolism, adipokine
signalling, lipid uptake and metabolism, and secretion of inflammatory
mediators. As a result, PPARG is being revealed as a key mediator of the
fundamental metabolic and immune contributions that are required for normal female fertility.


### 4.1. Insulin Sensitivity

Normal insulin sensitivity
and subsequently efficient metabolism of glucose are essential for healthy
reproduction in the female. Conditions of hyperinsulinemia can interfere with
normal ovarian cell function or be indirectly associated with other hormonal
conditions detrimental to optimal fertility [122–124]. Also,
exposure to high levels of glucose can have a deleterious effect on the oocyte [125, 126]. By normalising peripheral insulin signalling, PPARG
activation can circumvent many of these adverse effects of hyperinsulinemia, as
well as those detrimental outcome associated with persistently elevated blood
glucose levels.The genetic studies
detailed above, and the pharmacokinetics of TZD treatment improving insulin
sensitivity are both consistent with a direct role for PPARG in the regulation
of cellular insulin utilization. Despite this, it remains to be determined
exactly how TZD treatment and subsequent PPARG activation impacts gene
expression directly related to insulin signalling and glucose uptake (through
genes such as the insulin receptor (IR), IR-substrates, and glucose
transporters), as a range of conflicting results have emerged. Suggested
mechanisms include increases in glucosetransport protein 4 (GLUT4),
stimulation of phosphatidyl-3-kinase andmodified phosphorylation of
insulin receptor substrates [127–133]. In addition, it is well accepted that activation of PPARG does
improve not only basal hepatic glucose secretion, but also peripheral
insulin-stimulated glucose uptake, potentially indirectly via reduction of FFA,
TNFα, plasminogen activator inhibitor-1, and other
autocrine/endocrine signalling molecules which otherwise interfere with
efficient insulin signalling (reviewed in [134]). In this way, PPARG activation may improve female infertility
exacerbated by obesity and insulin resistance [25, 135–141].New reports are also
describing some of the first investigations into the ovarian-specific responses
to TZD that facilitate insulin sensitivity in this tissue. The work of
Seto-Young et al. [103] has shown that ovarian cells directly respond to TZDs to increase
transcription of insulin signalling components including IR alpha and beta
subunits and IRS-1, which would subsequently provide more efficient signal
transduction and cellular response to insulin.


### 4.2. Adipokines: Leptin and Adiponectin

Produced primarily by adipose tissue,
leptin and adiponectin are “adipokines” with contrasting actions on insulin
sensitivity. Whilst other adipokines such as visfatin and retinol-binding protein
4 (RBP-4) are also linked with
insulin sensitivity [142, 143] and the incidence of PCOS [144, 145], leptin and adiponectin are of
particular interest to those investigating female reproduction as it is known their presence can be detected by
ovarian cells which express leptin and adiponectin receptors. In addition,
although only the adiponectin promoter has been shown to contain a PPRE [146], transcriptional activity of both
leptin and adiponectin genes is
known to be decreased and increased, respectively, in the presence of
PPARG-activating ligands [147–151]. In this way, they can operate as
secondary messengers of signals initiated by PPARG activation.Leptin receptors are present in the granulosa
and thecal layers of the ovary [152, 153], and have been shown to be cyclically
regulated [154]. Leptin appears to influence ovarian
gonadotropin and steroid secretion [152, 153, 155], and affect oocyte quality and
developmental potential [156, 157].Adiponectin receptors AdipoR1 and
AdipoR2 are also both expressed by ovarian tissue [158] and adiponectin itself has been
identified within the follicular fluid of developed follicles in similar
concentrations to that observed in the serum [159]. Adiponectin appears to be involved in
many processes including those essential for ovulation, such as induction of COX-2 and prostaglandin E synthase expression in ovarian
granulosa cells [159].As the entire range of leptin and
adiponectin effects on ovarian cellular functions, including the outcomes of
PPARG activation (including enhancement of insulin sensitivity), are gradually
established, it is likely we will find that the improvements to reproductive
profiles and ovarian function of sub-fertile or infertile women treated with TZDs are mediated, at
least in part, through modulation of these two adipokines.


### 4.3. Lipid Uptake: CD36 and SCARB1

PPARG has a critical role in the regulation of adipocyte differentiation [94]. Among the best
characterised PPARG target genes are those involved in lipid metabolism,
including phosphoenolpyruvate carboxykinase [160], lipoprotein
lipase [161], fatty acid
binding protein [162, 163], and CD36 and
SCARB1 [164, 165]. CD36 and SCARB1,
class B scavenger receptors that mediate the endocytosis or selective
cholesterol uptake from oxLDL and HDL lipoproteins, are also both strongly
expressed by the ovary. The CD36 antigenis highly expressed by
granulosa cells of preantral and earlyantral follicles, with
moderatestaining also evident in the vascular thecal layers [166].In
this context, CD36 has been reported as a facilitator of thrombospondins-1 and
-2 activities [166, 167], influencing cell adhesion, wound healing, and angiogenesis [168, 169]; important components of the tissue and cellular changes that
occur during the ovarian cycle. CD36 is upregulated following
activation of PPARG in macrophages [164, 170], and a summary of
PPARG control of gene expression [171] suggested  this might act as a
positive feedback mechanism, such that more potential PPARG ligands can be
imported, enhancing expression of both PPARG and CD36.Ovarian SCARB1 expression appears to be strongly associated with HDL-cholesterol
ester requirement for production of androgen for aromatase-mediated conversion
to estradiol by the granulosa cells, and progesterone synthesis by luteal
cells. Thecal cells consistently express high levels of SCARB1 at all stages of
both healthy and atretic follicle development [172], and high
expression is also found within luteal structures [173].In these respects, PPARG activation may have profound influence on ovarian
function through the regulation of these genes or others regulating lipid
metabolism, by affecting availability of substrate for hormone synthesis, and
the remodelling of tissue structures required for oocyte release, luteinization,
and luteolysis.


### 4.4. Suppression of Chronic Inflammation

An important role for PPARG is the
suppression of immune cell synthesis and secretion of proinflammatory mediators
[174–182] (reviewed [183–185]). The role of the
immune system in female fertility is critical, both systemically, and locally
at the ovarian level.In addition, there are also interesting correlations between the development of adiposity, insulin
resistance and, chronic inflammation. Increased serum concentrations of TNF, NO,
and IL-6 are strongly associated with obesity [186, 187], and proinflammatory cytokines sourced from adipose tissue
including TNF, and IL-6 are among several important factors that participate in
the development of insulin resistance and type 2 diabetes mellitus [188–191]. Interestingly, together with central adiposity and insulin
resistance, we also find aspects of systemic inflammation independently
associated with impaired female fertility and PCOS [192, 193]. PPARG is implicated in improvements to the systemic inflammation
observed in obese and insulin resistant individuals treated with TZDs. These
studies describe reductions in serum C-reactive protein, IL-6, and soluble TNF
receptor 2 [194–198]. Other studies investigating the chronically inflamed profile
of PCOS patients support these findings, reporting that in addition to
restoring menstrual cyclicity and improving markers of hyperandrogensism, TZD
treatment is able to lower circulating C-reactive protein levels and the number
of circulating leukocytes [75, 85].


### 4.5. Ovarian Macrophages

Macrophages, dendritic cells, lymphocytes, and neutrophils have unique roles in the context
of ovarian physiology, and are essential for the normal regulation of ovulation
and control of the reproductive cycle [5, 199–201].
Macrophage distribution and numbers within the ovary varies across the cycle,
influenced by gonadotrophins and ovarian steroidogenic hormones. Resident
macrophages are present in the theca and stroma of the ovary during the late
stages of folliculogenesis [202].
Once the LH surge commences prior to ovulation, there is a massive recruitment
of new leukocytes from the circulation into the theca of the preovulatory
follicle [202, 203],
where they function to release proinflammatory cytokines and mediators assisting
the breakdown of the ovarian epithelium at ovulation. Their presence persists
until after ovulation, further increasing in number in the developing and
regressing corpus luteum [204].Ovarian macrophages maintain high levels of PPARG transcript expression until a
significant reduction in response to the proovulatory LH surge [1]. Immediately
following ovulation, expression is restored to high preovulatory levels [1]. In vitro
treatment of purified ovarian macrophages with the TZD troglitazone has been
shown to significantly alter proinflammatory gene expression [1]. Specifically,
these cells respond to TZD exposure by significantly suppressing mRNA
production of NOS2 (or inducible Nitric Oxide Synthase, iNOS), the enzyme that
catalyses the reaction producing the potent vasodilator, nitric oxide (NO). In
the human, NO seems to direct follicular selection and maturation [205], and application
of this NO property to IVF patients, deemed “poor responders”, has been found
to increase the number of oocytes retrieved [206]. This is an indication
that recruited and specialized ovarian macrophages can potentially respond
directly to TZDs administered systemically, and can regulate the availability
of ovulatory mediators. Such responses parallel the anti-inflammatory effects
of PPARG activation in nonovarian-activated macrophages [171], but was here
found to be specific to macrophages closely associated with the ovarian
environment (distinct to those located in the peritoneal cavity for instance).
This illustrates the unique influence of the ovarian milieu on normal PPARG
function and effects.


## 4.1. Insulin Sensitivity

Normal insulin sensitivity
and subsequently efficient metabolism of glucose are essential for healthy
reproduction in the female. Conditions of hyperinsulinemia can interfere with
normal ovarian cell function or be indirectly associated with other hormonal
conditions detrimental to optimal fertility [122–124]. Also,
exposure to high levels of glucose can have a deleterious effect on the oocyte [125, 126]. By normalising peripheral insulin signalling, PPARG
activation can circumvent many of these adverse effects of hyperinsulinemia, as
well as those detrimental outcome associated with persistently elevated blood
glucose levels.The genetic studies
detailed above, and the pharmacokinetics of TZD treatment improving insulin
sensitivity are both consistent with a direct role for PPARG in the regulation
of cellular insulin utilization. Despite this, it remains to be determined
exactly how TZD treatment and subsequent PPARG activation impacts gene
expression directly related to insulin signalling and glucose uptake (through
genes such as the insulin receptor (IR), IR-substrates, and glucose
transporters), as a range of conflicting results have emerged. Suggested
mechanisms include increases in glucosetransport protein 4 (GLUT4),
stimulation of phosphatidyl-3-kinase andmodified phosphorylation of
insulin receptor substrates [127–133]. In addition, it is well accepted that activation of PPARG does
improve not only basal hepatic glucose secretion, but also peripheral
insulin-stimulated glucose uptake, potentially indirectly via reduction of FFA,
TNFα, plasminogen activator inhibitor-1, and other
autocrine/endocrine signalling molecules which otherwise interfere with
efficient insulin signalling (reviewed in [134]). In this way, PPARG activation may improve female infertility
exacerbated by obesity and insulin resistance [25, 135–141].New reports are also
describing some of the first investigations into the ovarian-specific responses
to TZD that facilitate insulin sensitivity in this tissue. The work of
Seto-Young et al. [103] has shown that ovarian cells directly respond to TZDs to increase
transcription of insulin signalling components including IR alpha and beta
subunits and IRS-1, which would subsequently provide more efficient signal
transduction and cellular response to insulin.


## 4.2. Adipokines: Leptin and Adiponectin

Produced primarily by adipose tissue,
leptin and adiponectin are “adipokines” with contrasting actions on insulin
sensitivity. Whilst other adipokines such as visfatin and retinol-binding protein
4 (RBP-4) are also linked with
insulin sensitivity [142, 143] and the incidence of PCOS [144, 145], leptin and adiponectin are of
particular interest to those investigating female reproduction as it is known their presence can be detected by
ovarian cells which express leptin and adiponectin receptors. In addition,
although only the adiponectin promoter has been shown to contain a PPRE [146], transcriptional activity of both
leptin and adiponectin genes is
known to be decreased and increased, respectively, in the presence of
PPARG-activating ligands [147–151]. In this way, they can operate as
secondary messengers of signals initiated by PPARG activation.Leptin receptors are present in the granulosa
and thecal layers of the ovary [152, 153], and have been shown to be cyclically
regulated [154]. Leptin appears to influence ovarian
gonadotropin and steroid secretion [152, 153, 155], and affect oocyte quality and
developmental potential [156, 157].Adiponectin receptors AdipoR1 and
AdipoR2 are also both expressed by ovarian tissue [158] and adiponectin itself has been
identified within the follicular fluid of developed follicles in similar
concentrations to that observed in the serum [159]. Adiponectin appears to be involved in
many processes including those essential for ovulation, such as induction of COX-2 and prostaglandin E synthase expression in ovarian
granulosa cells [159].As the entire range of leptin and
adiponectin effects on ovarian cellular functions, including the outcomes of
PPARG activation (including enhancement of insulin sensitivity), are gradually
established, it is likely we will find that the improvements to reproductive
profiles and ovarian function of sub-fertile or infertile women treated with TZDs are mediated, at
least in part, through modulation of these two adipokines.


## 4.3. Lipid Uptake: CD36 and SCARB1

PPARG has a critical role in the regulation of adipocyte differentiation [94]. Among the best
characterised PPARG target genes are those involved in lipid metabolism,
including phosphoenolpyruvate carboxykinase [160], lipoprotein
lipase [161], fatty acid
binding protein [162, 163], and CD36 and
SCARB1 [164, 165]. CD36 and SCARB1,
class B scavenger receptors that mediate the endocytosis or selective
cholesterol uptake from oxLDL and HDL lipoproteins, are also both strongly
expressed by the ovary. The CD36 antigenis highly expressed by
granulosa cells of preantral and earlyantral follicles, with
moderatestaining also evident in the vascular thecal layers [166].In
this context, CD36 has been reported as a facilitator of thrombospondins-1 and
-2 activities [166, 167], influencing cell adhesion, wound healing, and angiogenesis [168, 169]; important components of the tissue and cellular changes that
occur during the ovarian cycle. CD36 is upregulated following
activation of PPARG in macrophages [164, 170], and a summary of
PPARG control of gene expression [171] suggested  this might act as a
positive feedback mechanism, such that more potential PPARG ligands can be
imported, enhancing expression of both PPARG and CD36.Ovarian SCARB1 expression appears to be strongly associated with HDL-cholesterol
ester requirement for production of androgen for aromatase-mediated conversion
to estradiol by the granulosa cells, and progesterone synthesis by luteal
cells. Thecal cells consistently express high levels of SCARB1 at all stages of
both healthy and atretic follicle development [172], and high
expression is also found within luteal structures [173].In these respects, PPARG activation may have profound influence on ovarian
function through the regulation of these genes or others regulating lipid
metabolism, by affecting availability of substrate for hormone synthesis, and
the remodelling of tissue structures required for oocyte release, luteinization,
and luteolysis.


## 4.4. Suppression of Chronic Inflammation

An important role for PPARG is the
suppression of immune cell synthesis and secretion of proinflammatory mediators
[174–182] (reviewed [183–185]). The role of the
immune system in female fertility is critical, both systemically, and locally
at the ovarian level.In addition, there are also interesting correlations between the development of adiposity, insulin
resistance and, chronic inflammation. Increased serum concentrations of TNF, NO,
and IL-6 are strongly associated with obesity [186, 187], and proinflammatory cytokines sourced from adipose tissue
including TNF, and IL-6 are among several important factors that participate in
the development of insulin resistance and type 2 diabetes mellitus [188–191]. Interestingly, together with central adiposity and insulin
resistance, we also find aspects of systemic inflammation independently
associated with impaired female fertility and PCOS [192, 193]. PPARG is implicated in improvements to the systemic inflammation
observed in obese and insulin resistant individuals treated with TZDs. These
studies describe reductions in serum C-reactive protein, IL-6, and soluble TNF
receptor 2 [194–198]. Other studies investigating the chronically inflamed profile
of PCOS patients support these findings, reporting that in addition to
restoring menstrual cyclicity and improving markers of hyperandrogensism, TZD
treatment is able to lower circulating C-reactive protein levels and the number
of circulating leukocytes [75, 85].


## 4.5. Ovarian Macrophages

Macrophages, dendritic cells, lymphocytes, and neutrophils have unique roles in the context
of ovarian physiology, and are essential for the normal regulation of ovulation
and control of the reproductive cycle [5, 199–201].
Macrophage distribution and numbers within the ovary varies across the cycle,
influenced by gonadotrophins and ovarian steroidogenic hormones. Resident
macrophages are present in the theca and stroma of the ovary during the late
stages of folliculogenesis [202].
Once the LH surge commences prior to ovulation, there is a massive recruitment
of new leukocytes from the circulation into the theca of the preovulatory
follicle [202, 203],
where they function to release proinflammatory cytokines and mediators assisting
the breakdown of the ovarian epithelium at ovulation. Their presence persists
until after ovulation, further increasing in number in the developing and
regressing corpus luteum [204].Ovarian macrophages maintain high levels of PPARG transcript expression until a
significant reduction in response to the proovulatory LH surge [1]. Immediately
following ovulation, expression is restored to high preovulatory levels [1]. In vitro
treatment of purified ovarian macrophages with the TZD troglitazone has been
shown to significantly alter proinflammatory gene expression [1]. Specifically,
these cells respond to TZD exposure by significantly suppressing mRNA
production of NOS2 (or inducible Nitric Oxide Synthase, iNOS), the enzyme that
catalyses the reaction producing the potent vasodilator, nitric oxide (NO). In
the human, NO seems to direct follicular selection and maturation [205], and application
of this NO property to IVF patients, deemed “poor responders”, has been found
to increase the number of oocytes retrieved [206]. This is an indication
that recruited and specialized ovarian macrophages can potentially respond
directly to TZDs administered systemically, and can regulate the availability
of ovulatory mediators. Such responses parallel the anti-inflammatory effects
of PPARG activation in nonovarian-activated macrophages [171], but was here
found to be specific to macrophages closely associated with the ovarian
environment (distinct to those located in the peritoneal cavity for instance).
This illustrates the unique influence of the ovarian milieu on normal PPARG
function and effects.


## 5. Conclusions

Many diverse endocrine and metabolic
components profoundly influence female fertility, including hormone production
as well as the development and ovulation of healthy oocytes. The role of PPARG in these events is
two-fold. PPARG activation of transcription has outcomes operating both
directly within the ovarian structure itself, and also indirectly through
influences on other tissue systems such as the adipose tissue and immune cells
(Figure3). In this way, PPARG controls key signals regulating the capacity for
normal reproduction. As PPARG is able, and required, to regulate many of
these actions, it is important that the roles of PPARG be carefully considered
as new concepts develop regarding the effects of dietary supplements such as
PUFAs, which are PPARG ligands, and the consequences of increased immunological
activation, such as occurs during obesity. As the
health crisis surrounding the obesity epidemic widens to include the damaging
effects on female fertility, it is important to remember the systemic
implications of metabolism and immune regulation on female fertility, and to
consider the role of PPARG in coordinating these contributions.
Tremendous opportunity exists for those interested in elucidating further the
exciting interactions between PPARG and female fertility. Publication of the
most extensive list to date of all genes containing potential PPREs in their
promoter regions [207] will provide a
valuable tool for such research, as many identified genes have known functions
within the context of ovarian physiology and pathology, in addition to
characterized roles in other tissues, including macrophages and adipose tissue.Figure 3
Schematic summarising the developing concept of PPARG influence on ovarian function and
female fertility. PPARG is able to strongly influence the activity of ovarian
cells directly, in particular steroidogenesis and tissue remodelling. In
addition, PPARG can further influence ovarian function via regulation of
external metabolic signals and immune cell contributions.



---

*Source: 243791-2008-01-03.xml*