M I N I - R E V I E W

Physiological and Pathological Androgen Actions

in the Ovary

Olga Astapova,1 Briaunna M. N. Minor,1 and Stephen R. Hammes1

1
Department of Medicine, Division of Endocrinology and Metabolism, University of Rochester School of
Medicine and Dentistry, Rochester, New York 14642

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ORCiD numbers: 0000-0002-7367-276X (S. R. Hammes).

Androgens, although traditionally thought to be male sex steroids, play important roles in female
reproduction, both in healthy and pathological states. This mini-review focuses on recent advances in
our knowledge of the role of androgens in the ovary. Androgen receptor (AR) is expressed in oocytes,
granulosa cells, and theca cells, and is temporally regulated during follicular development. Mouse
knockout studies have shown that AR expression in granulosa cells is critical for normal follicular
development and subsequent ovulation. In addition, androgens are involved in regulating dynamic
changes in ovarian steroidogenesis that are critical for normal cycling. Androgen effects on follicle
development have been incorporated into clinical practice in women with diminished ovarian reserve,
albeit with limited success in available literature. At the other extreme, androgen excess leads to
disordered follicle development and anovulatory infertility known as polycystic ovary syndrome
(PCOS), with studies suggesting that theca cell AR may mediate many of these negative effects. Finally,
both prenatal and postnatal animal models of androgen excess have been developed and are being
used to study the pathophysiology of PCOS both within the ovary and with regard to overall metabolic
health. Taken together, current scientific consensus is that a careful balance of androgen activity in the
ovary is necessary for reproductive health in women. (Endocrinology 160: 1166–1174, 2019)

ex steroids are crucial regulators of reproductive
S function. Although it was believed for a long time
that androgens are strictly male hormones and estrogens
are female hormones, we now know that both types of
sex steroids are necessary for normal development and
function of every human. Studies in androgen receptor
(AR) knockout (ARKO) mice have shown that in the
ovary, folliculogenesis does not reach its full potential in
the absence of androgens. At the other extreme, follicle
development is dysregulated in states of androgen excess
and can lead to polycystic ovary syndrome (PCOS), a
condition of hyperandrogenism and anovulatory in-
fertility. Although much of the literature suggests that
PCOS is caused by central defects in gonadotropin se-
cretion, other studies point to a direct action of andro-
gens in the ovary to alter folliculogenesis and ovulation.
This mini-review focuses on recent advances in our
knowledge of the role of androgens in cells that make
up ovarian follicles, biological models of androgen de-
ficiency and excess in the ovary, and the factors that
regulate ovarian responsiveness to androgens.
Ovarian Cell-Specific AR Deletion Studies
Much of what we know about androgen actions in the
ovary stems from mouse models lacking the AR gene.
Before the gene was identified, its locus on the X chro-
mosome was termed testicular feminization (Tfm) for its
effects in males. Men and male animal models with
absence of the Tfm locus were noted to have a female
appearance and infertility due to lack of responsiveness
to androgens. The infertility created a challenge in the
development of a female homozygous Tfm mutant model.
In the 1970s, Ohno et al. (1) managed to use chimeric

ISSN Online 1945-7170 Abbreviations: AR, androgen receptor; ARKO, androgen receptor knockout; Bmp15,
Copyright © 2019 Endocrine Society bone morphogenic protein 15; DHEA, dehydroepiandrosterone; DOR, diminished ovarian
Received 6 February 2019. Accepted 20 March 2019. reserve; Ezh2, enhancer of zeste homolog 2; Gdf9, growth differentiation factor 9; Hgf,
First Published Online 26 March 2019 hepatocyte growth factor; Kitl, kit ligand; PCOS, polycystic ovary syndrome; PNA,
prenatal dihydrotestosterone; Runx1, Runt-related transcription factor-1; Tfm, tes-
ticular feminization.

1166 https://academic.oup.com/endo Endocrinology, May 2019, 160(5):1166–1174 doi: 10.1210/en.2019-00101

doi: 10.1210/en.2019-00101
Tfm male mice and XO female mice, which are fertile, to
generate homozygous Tfm/O mutant females and were
the first to describe their phenotype. These females
appeared normal and were fully fertile shortly after
puberty; however, their reproductive ability declined
quickly and completely ceased much sooner than ex-
pected. Their ovarian morphology and follicle counts
were normal at 36 days of age, but at day 56 and on-
ward there was a reduction in the number of primordial
follicles and premature degeneration of oocytes in early
follicles with diffuse luteinization. XO mice have
functioning ovaries but develop the same abnormalities
starting much later, at day 210. This was the first piece
of evidence that androgen action is necessary for
maintaining the reproductive lifespan of a mouse ovary.
Without it, the ovaries failed prematurely.
Later that decade, Lyon and Glenister (2) described
the reproductive phenotype of homozygous Tfm/Tfm
mutant females, which confirmed the initial observa-
tions in Tfm/O mice. The Tfm/Tfm females initially had
normal fecundity but soon after started producing fewer
pups per litter and stopped reproducing sooner than
controls, resulting in an overall reduction of progeny
numbers by approximately half. Their ovarian mor-
phology findings were similar to those described by Ohno
et al. (1), suggesting that androgens prevent atresia of
early follicles.
With the advent of more sophisticated genetic
technologies, the AR gene was identified and AR de-
letion studies were carried out beginning in the early
2000s. Several ARKO female mouse models were de-
scribed and corroborated the findings of earlier Tfm
mouse studies. Two global ARKO mouse models were
created using Cre recombinase expression driven by the
actin promoter (3, 4) or the cytomegalovirus promoter
(5). Although the ARKO males had a femalelike ap-
pearance and were sterile due to arrest of spermato-
genesis, the females were phenotypically normal and
produced offspring. At a cursory glance, this may
suggest that androgen is dispensable in females. How-
ever, as seen with the Tfm animals, over the course of
their lifetimes, the AR-deficient female mice produced
significantly smaller numbers of litters and fewer pups
per litter, resulting in an overall reduction in fecundity of
up to 70% compared with their littermates. This was due
to premature depletion of follicles and early cessation of
fertility, a phenotype resembling diminished ovarian
reserve (DOR) in women. The ovaries of ARKO mice
appeared normal at 4 weeks of age, but their response
to gonadotropin stimulation in superovulation studies
was already diminished at that time. By 8 weeks, the
ovaries contained fewer corpora lutea and more atretic
follicles than controls (3–5). Follicles were completely
https://academic.oup.com/endo 1167
depleted by 40 weeks of age, resulting in infertility (5).
Heterozygous AR-deficient mice produced slightly fewer
pups than controls but were considerably more fertile
than their homozygous ARKO counterparts. These
studies proved that androgen action is not essential for
survival and reproduction of female mice, but their re-
productive potential is not fully realized in the absence of
androgen.
The work of Hu et al. (3) showed that the loss of
fecundity in female ARKO mice is due mostly to the
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reduced number of ovulated oocytes, with diminished
endometrial thickness likely also contributing. Although
AR is expressed at all levels of the reproductive hormone
axis, the subnormal response to gonadotropin stimula-
tion suggests that the primary defect in ARKO mice lies
within the ovary. AR has been specifically knocked out in
all three major ovarian cell types, and we have learned
that androgens play very different roles in each of them.
In 2010, Sen and Hammes (6) described the phenotypes
of oocyte-specific and granulosa cell–specific ARKO
mice. Although AR is expressed in the oocyte, its selective
deletion by Cre-recombinase expressed from the growth
differentiation factor 9 (Gdf9) gene promoter produced
no effect on fertility or fecundity, and follicle counts at 8
to 9 weeks of age were the same as in heterozygous mice.
On the contrary, granulosa cell–specific ARKO mice,
generated by anti-Müllerian hormone receptor II–driven
Cre expression, recapitulated the phenotype of the global
ARKO. These mice had longer estrous cycles and reduced
fertility, with premature ovarian failure. By 8 to 9 weeks
of age, the knockout mice already were producing only
one-quarter the number of offspring compared with
wild-type controls, and this declined further to ;10% by
24 to 25 weeks. Soon after this, FSH levels rose and the
mice developed premature ovarian failure. Progression of
small follicles to the antral stage was significantly re-
duced and apoptosis was increased. Interestingly, in this
study the fecundity of heterozygous ARKO mice was
unaffected, whereas global haploinsufficient mice had
mildly reduced fecundity, suggesting that another site of
full androgen activity, possibly the endometrium or
centrally in the pituitary or hypothalamus, is required for
optimal fertility. Still, this study was the first to prove that
the main site of androgen activity in the ovary is gran-
ulosa cells, where AR is necessary for normal preantral
follicle development. These findings were confirmed by
Walters et al. (7), who further demonstrated that not only
were the numbers of ovulated oocytes reduced when
AR was deleted in anti-Müllerian hormone–expressing
granulosa cells of preantral and antral follicles, but fewer
of those ovulated oocytes were fertilized and survived to
the two-cell stage, suggesting diminished oocyte quality
in these mice.
1168 Astapova et al Androgen Actions in the Ovary
Finally, a theca cell–specific ARKO mouse model was
described by Ma et al. (8) in 2017, in which AR was
deleted in Cyp17-expressing cells of the ovary. These
mice were phenotypically normal, with histologically
normal ovaries, and showed no reproductive deficit
under standard conditions. However, when treated with
prenatal dihydrotestosterone (PNA) to induce anovu-
lation mimicking the PCOS phenotype (described in
more detail below), mice lacking AR specifically in theca
cells were protected from the reproductive deficits as-
sociated with the condition. Although PNA mice
were completely acyclic and infertile, theca cell–specific
ARKO-PNA mice maintained some cyclicity and pro-
duced approximately half the number of offspring
compared with untreated controls. Thus, although the
mechanisms of PCOS induction by PNA treatment re-
main obscure, it is now clear that androgen actions in
theca cells are necessary at least in part in this disease
model.
The above studies of mouse ARKO models have shed
light on the pathogenesis of disorders related to androgen
imbalance in women. Both excess and deficiency of
androgens can cause female infertility. The proper bal-
ance of androgens in granulosa cells is necessary for
optimal follicle progression and evading apoptosis,
whereas an excess of androgen results in follicle arrest
and anovulation mainly through its actions in theca cells.
Regulation of AR Gene Expression
and Activity
As discussed above, ovarian AR mediates physiological
and pathogenic effects of androgens on female fertility.
AR expression levels in the ovary have been associated
with ovarian dysfunction, namely PCOS, but the factors
that regulate its expression in the ovary are not well
known. Much of what we do know about the regulation
of AR gene expression and activity comes from studies in
the male, particularly in prostate cancer–derived cell
lines. Factors at many levels of the gene expression
paradigm have been implicated in AR gene regulation,
including polymorphisms, splice variants, and cis- and
trans-acting transcription regulators, including andro-
gens themselves.
The human AR gene contains a poly-glutamine (CAG)
repeat region within the N-terminal transcription acti-
vation region. Most people have between 8 and 35 re-
peats (9), and the shorter length is associated with
increased activity of the receptor (10). Increased number
of CAG repeats, conversely, is associated with reduced
AR activity and has been linked to fertility problems in
men (11). A number of studies have focused on the
question of AR CAG repeat polymorphisms in women
Endocrinology, May 2019, 160(5):1166–1174
with PCOS, with the hypothesis that the shorter and
more active form of AR could lead to hyperandrogenism,
and the results have been variable. Some case-control
studies support this association whereas others find no
association (9, 12), possibly reflecting population dif-
ferences in the pathogenesis of this complex and het-
erogeneous syndrome. In ovulatory women with longer
CAG repeat length, intrafollicular fluid testosterone
concentration was lower and aromatase expression was
higher in one study (13), suggesting that AR may neg-
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atively regulate aromatase expression and reduce the
conversion of testosterone to estradiol. However, serum
testosterone measured in a different population was
associated with longer CAG repeat length (14), so other
factors are likely playing a role in this relationship.
Some studies, mostly in prostate cancer cells, suggest
that androgen can induce or suppress AR gene expres-
sion. For instance, an enhancer element in the second
intron of the AR gene binds AR and can either stimulate
or repress AR gene transcription depending on the
presence or absence of androgen (15). With androgen
present, lysine-specific demethylase 1 is recruited to this
location leading to transcription silencing. In the absence
of androgen, this site recruits transcription coactivators
FOXA1, OCT1, and GATA2 leading to H3K4 methyl-
ation and Pol II recruitment. In primates, but not in lower
species, a nonconsensus androgen response element is
found in the 50 UTR of the AR gene and suppresses its
transcription by approximately half when bound by AR
with physiologically relevant (10 nM) concentration of
DHT (16). However, there is no evidence to date that
these regulatory sites are active in the ovary. In one study,
3- to 10-day testosterone treatment induced AR mRNA
expression in granulosa cells of small and medium fol-
licles of rhesus monkeys (17), but the treatment resulted
in circulating testosterone levels that were more than
100-fold above normal, so the physiological significance
of this finding is unclear. Overall, most evidence suggests
that androgens can have a moderate suppressive effect on
AR mRNA levels in the prostate but no substantial effect
in the ovary, perhaps due to a difference in available
transcription factors in these tissues.
In a recent study, Liu et al. (18) reported that AR
mRNA splice variation in granulosa cells of patients with
PCOS results in the presence of variant forms of AR
(insertion-AR and deletion-AR), compared with ovula-
tory women. Both of these splice variants fail to trans-
locate into the nucleus upon DHT stimulation due to
altered interaction with cytoplasmic proteins HSP90 and
importin-a. Although it is not clear whether these vari-
ants contribute to the pathogenesis of PCOS, it is
intriguing to propose that extranuclear AR signaling
pathways may be involved. Lastly, AR phosphorylation
doi: 10.1210/en.2019-00101
at multiple serine residues was shown by immunostaining
in granulosa and theca cells of the marmoset ovary (19).
This was not affected by treatment with testosterone or
GnRH antagonist, and the physiological significance of
these phosphorylation sites is presently unknown.
Molecular Actions of Androgens in
the Ovary
As expected from ARKO studies in mice, multiple lines of
evidence from cell and follicle culture show that an-
drogens act via AR in the ovary to promote follicle
growth and reduce granulosa cell apoptosis (Table 1).
Microarray analysis of mRNA from global ARKO
mouse ovaries revealed that many genes involved in
folliculogenesis, including kit ligand (Kitl), bone mor-
phogenic protein 15 (Bmp15), Gdf9, and hepatocyte
growth factor (Hgf), are under control of AR (5). Still, the
cell-specific ovarian transcriptome directly regulated by
AR remains unknown. In addition, there are likely
nongenomic effects of AR that are important for ovarian
function and have yet to be elucidated.
Preantral follicles isolated from rat ovaries grow faster
in culture when DHT is present in the media in con-
centrations as low as 1 nM (20, 30), which is ;10-fold
higher than the reported EC50 of DHT for human AR
(21). In addition, preantral follicles from rats that are
chronically treated with DHT (83 mg continuous daily
release) also grow faster in culture (20), and the same has
been found in short-term DHT-treated monkeys (145
mg/kg/day for 5 days) (22). Thus, DHT has a trophic
effect on preantral follicle growth both in vitro and in
vivo. AR is most highly expressed in the granulosa cells of
small follicles, and its abundance decreases as the follicles
progress through development in rats (31) and in pri-
mates (32). Androgen actions in small follicles are likely
Table 1. Summary of Available Evidence From Studies of Androgen Actions in the Ovary
Experimental Species
Observed Effect and Design
Increased preantral follicle growth Rat
Monkey
Increased FSH receptor mRNA expression Rat
Monkey
Increased FSH receptor protein expression Mouse, human
Increased granulosa cell proliferation Rat
Pig
Reduced apoptosis of follicles Mouse
Increased expression of steroidogenic enzymes Rat
StAR, P450scc, and 3bHSD in mature granulosa
cells
Increased expression of cyclooxygenase-2 and Mouse
amphiregulin in periovulatory granulosa cells Human
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mediated by enhanced FSH and growth factor signaling
in granulosa cells. In fact, mRNA expression of FSH
receptor, the major trophic factor in granulosa cells, was
shown to be induced by androgen treatment of cultured
rat follicles (20), as well as monkey granulosa cells (23) in
vivo (0.4 or 4 mg/kg of testosterone for 3 days). In the
latter study, AR and FSH receptor were also found to
colocalize. Granulosa cell proliferation in response to
stimulation with FSH, IGF-1, and the oocyte-secreted
growth factor GDF-9, as well as intracellular messengers
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of these growth factors, are all augmented in the presence
of androgens in nanomolar concentrations (25–27)
(Fig. 1), and these effects seem more pronounced in
smaller preantral follicles. Coincidentally, apoptosis of
cultured follicles is suppressed by androgens (DHT,
25 nM) (24, 28) through mechanisms that may involve
granulosa-specific miRNA miR-125b (Fig. 1). Thus,
androgens appear most important in small follicles where
they promote follicle development and growth through
multiple pathways.
Nongenomic pathways involved in the proliferative
effects of androgens in the ovary have also been de-
scribed, though these are better characterized in prostate
cancer cells, where membrane-associated AR enhances
epidermal growth factor signaling through a fast-onset,
transcription-independent mechanism (34). In mouse
primary granulosa cells, as well as in the immortalized
granulosalike KGN cells, extranuclear AR induces FSH
receptor protein expression independently of gene
transcription, through an MAPK-dependent pathway
(24) (Fig. 1). Wartalski et al. (35) found that extranuclear
AR in porcine granulosa cells is modulated by environ-
mental fungicide vinclozolin and activates ERK1/2 and
AKT. In croaker granulosa cells, a membrane zinc
transporter family protein identified as ZIP9, unrelated
structurally to AR, is liganded by androgens and signals
DHT Dose Reference
In vitro 1 nM and higher (20, 21)
In vivo 83 mg continuous daily-release pellet (21)
In vivo 145 mg/kg/d for 5 d (22)
In vitro 1 nM and higher (20)
In vivo 0.4 or 4 mg/kg of testosterone for 3 d (23)
In vitro 25 nM (24)
In vitro 100 nM (25)
In vitro 500 nM (26, 27)
In vitro 25 nM (24, 28)
In vitro 100 nM (25)
In vivo 5 mg/g 4 h prior to RNA isolation (29)
In vitro 100 nM
1170 Astapova et al Androgen Actions in the Ovary Endocrinology, May 2019, 160(5):1166–1174

significantly induced by DHT treat-

ment of small follicles but suppressed
by the same treatment of large follicles.
Although there are differences between
studies, which may be a function of
transcription coregulators present in
granulosa cells at different stages of
development, it is clear that supra-
physiological levels of testosterone
can shift the steroid hormone balance

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within the ovary.
There are limited data suggesting that
androgens may directly affect ovulation
in certain circumstances. Ovulation-
related genes cyclooxygenase-2 and
amphiregulin are acutely induced by
DHT (5 mg/g) in periovulatory gran-
ulosa cells in mice (29) and in KGN
Figure 1. AR actions in granulosa cells. AR works through genomic (dashed lines) and
nongenomic (solid lines) pathways to promote growth and differentiation of granulosa cells, cells. Androgens can also promote oo-
suppress apoptosis and, in dominant follicles, increase steroid synthesis. The effects of cyte meiotic competence and matura-
granulosa growth factors IGF1, GDF9, and FSH are all enhanced in the presence of tion in some species, although there are
androgens through extranuclear activity of AR. At the gene level, AR induces the expression
of antiapoptotic miRNA miR125b, multiple steroidogenic enzymes, GDF9, and FSH receptor, likely redundant pathways for this in the
and regulates the activity of DNA methyltransferase Ezh2 through modulation of Ezh2 absence of androgens (41, 42). Con-
phosphorylation as well as transcriptional regulation of the miRNA miR101 (24–28, 33). versely, chronic excess of androgens is
detrimental to ovulation due to disor-
via Gsa and MAPK (36, 37). These pathways may act in dered follicular development, described in more detail
concert with nuclear AR to stimulate granulosa cell below. Overall it appears that androgens play a larger role
proliferation. early in follicular development, and ovulatory dysfunction
Some studies suggest that androgens modulate ovar- can result from defects in preantral follicles due to either
ian steroidogenesis downstream of FSH signaling. For insufficient or excessive androgen levels.
instance, the expression of key steroidogenic enzymes
StAR, P450scc, and 3bHSD are amplified by 100 nM Clinical Use of Androgens in
DHT treatment of cultured rat granulosa cells (25), Female Infertility
resulting in increased progesterone synthesis (Fig. 1).
This effect was not observed in cumulus granulosa cells The preponderance of evidence from animal studies has
(26), highlighting the specialization of granulosa cells shown that insufficient androgen activity in the female
throughout follicular development. Similarly, DHT has leads to subfertility that very closely resembles the clinical
variable effects on the expression levels of aromatase and presentation of DOR in women. Consequently, cautious
estrogen synthesis. In rat granulosa cells, testosterone use of dehydroepiandrosterone (DHEA), a less potent
stimulates aromatase gene expression. LRH-1 is a direct androgen than DHT, has been applied to women with
target gene of AR in this context and mediates the in- DOR undergoing fertility treatments. Empirical evidence
duction of aromatase and P450scc by testosterone (38). suggests there is little if any benefit of DHEA to fertility
However, these findings were not substantiated in cul- treatment success, but there is also no evidence of harm.
tured human granulosa cells, which are typically ob- DOR often presents as a time-sensitive infertility situa-
tained from later-stage follicles. Yang et al. (39) obtained tion when the desire is to try every available agent to
granulosa cells from women without PCOS and then maximize chances for success, and DHEA may be added
treated them in culture with testosterone at levels mea- to the treatment strategy. For similar reasons, recruitment
sured in the intrafollicular fluid of women with PCOS. In of subjects for placebo-controlled randomized trials of
this study, aromatase gene expression and protein DHEA in DOR is likely challenging and only small
abundance was downregulated by testosterone via AR. studies are available.
Developmental stage differences in follicular response to In a 2015 meta-analysis, Li et al. (43) evaluated eight
androgens were highlighted in a study of the marmoset studies, half of which were randomized clinical trials or
ovary by Harlow et al. (40): aromatase activity was case-control trials, and concluded that DHEA (75 mg
doi: 10.1210/en.2019-00101
daily in one or three divided doses for 3 to 4 months)
modestly improves clinical pregnancy rates in women
with DOR undergoing treatment with assisted reproductive
techniques. There was no difference in the number of
oocytes retrieved, contradicting the expected effect of
androgens in improving follicle recruitment. Overall,
the effect was minor and was lost in smaller studies.
In a subsequent randomized clinical trial, DHEA pre-
treatment in in vitro fertilization cycles (25 mg three times
daily for 8 weeks) did not improve ovarian response to
gonadotropin stimulation or clinical pregnancy rates in
women with DOR, though AR and FSH receptor ex-
pression were increased in granulosa cells (44). It is
possible that ovarian stimulation with supraphysiological
levels of gonadotropins typically used in IVF protocols
overcomes any additional effects of androgen treatment.
To speculate, as personalized medicine advances, there
may be a place for androgen supplementation in women
with DOR early in their reproductive lives with the goal of
decelerating follicle depletion before attempting to have
children.
Hyperandrogenism in PCOS
Both androgen insufficiency and androgen excess cause
ovarian dysfunction. Although lack of androgen activity
in the ovary, particularly in granulosa cells, leads to
ovarian insufficiency as described above, excess andro-
gen is linked to PCOS. In the latter, follicles are in-
creasingly recruited to the preantral and antral stage but
fail to progress to the Graafian stage and ovulate, and can
give the ovaries a characteristic polycystic appearance on
imaging. The hypothalamic and pituitary state in PCOS is
marked by increased pulse frequency of the GnRH and
high LH secretion. This leads to increased testosterone
production by the hypertrophic theca cell population,
adding to the hyperandrogenic state. This syndrome is in
some ways the opposite of ovarian insufficiency, but both
are frequent causes of anovulatory infertility. In addition,
PCOS is associated with metabolic syndrome.
Excess androgen appears to be both a cause and a
consequence of PCOS in a vicious cycle. For example,
chronic exposure to exogenous androgens is sufficient to
induce polyfollicular anovulatory infertility in mice (45)
and rats (46). Chronic DHT exposure of these females
through a pellet inserted prepuberty (27.5 mg in mice or
83 mg in rats daily continuous release for 90 days) results
in cessation of estrous cycles and the appearance of
multiple cystic, atretic follicles. The mice also develop
increased body fat and glucose intolerance, closely
mimicking PCOS in women. Unlike classic PCOS,
chronic exogenous androgen exposure in rodents is not
associated with increased LH levels or hyperthecosis and
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their ovarian morphology is distinct with many atretic
but few antral follicles. However, in other animal models
the syndrome can be programmed through transient
hormone disruption in utero, leading to hyperactive LH
tone and hyperplastic follicles that in turn secrete ab-
normally high amounts of testosterone.
A very well-validated method to induce a PCOS-like
phenotype in animal models is prenatal exposure to
excess androgens. In many mammals, including rodents
(47–50), sheep (51), and primates (52–54), in utero
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treatment with excess androgens during critical de-
velopmental windows produces remarkably consistent
results in female offspring. Invariably, the females de-
velop ovulatory dysfunction early during their re-
productive life, characterized by polyfollicular ovaries,
hyperandrogenemia, and LH hypersecretion. For in-
stance, injection of pregnant rhesus monkeys with 10 mg
of testosterone propionate daily during early (beginning
days 40 to 44) or late (beginning days 100 to 115)
gestation with the target circulating testosterone levels in
the male range resulted in the PCOS phenotype in female
offspring of both treatment groups (52). These females
had ;50% fewer menstrual cycles than controls, basal
testosterone levels 50% above controls, and poly-
follicular ovaries in 40% of treated animals compared
with 14% of controls. In this model, the early exposure
window coincided with the differentiation of the ovary
and appearance of oogonia, whereas the late exposure
occurred during ovarian maturation when follicles ap-
pear and begin to produce steroids and gain gonado-
tropin responsiveness. In some sheep and primate models
of PCOS, particularly those exposed to androgens earlier
in fetal development, increased follicle recruitment is
associated with early follicle depletion and reduced
ovarian reserve (55, 56). Studies using the AR antagonist
flutamide and the nonaromatizable androgen DHT have
demonstrated that prenatal testosterone in each of these
animal models indeed acts via AR to program re-
productive dysfunction. As mentioned above, AR in the
theca cells may be specifically responsible for much of
this effect (8).
It should be noted that, although prenatal androgen
treatment is the most effective method to induce a PCOS-
like phenotype in animal models, there are some im-
portant differences in treatment outcomes between
experimental species. For example, PNA mice (typically
injected with 250 mg DHT on days 16, 17, and 18 of
gestation) develop oligocyclicity and glucose intolerance
similar to women with PCOS (49), but lack the char-
acteristic polyfollicular ovarian morphology, which in
humans and primates is due to many persistent antral
follicles. This is due to the inherent differences between
the menstrual cycle in humans and the estrous cycle in
1172 Astapova et al Androgen Actions in the Ovary
rodents. In PNA mouse ovaries, a stark absence of
corpora lutea compared with untreated controls signifies
anovulation (57).
In addition to the reproductive defects, all prenatally
androgenized animal models develop insulin resistance,
impaired glucose tolerance, and increased visceral
adiposity, similar to the metabolic syndrome observed
with high frequency in women with PCOS (58, 59).
Depending on the timing of prenatal androgen exposure,
additional effects can include genital virilization and
behavioral masculinization (52). Although the etiology of
PCOS is still debated, the developmental origins hy-
pothesis is clearly in the lead (60), though the precise
mechanisms by which prenatal androgen excess pro-
grams this syndrome in animal models are still unclear.
In-depth characterization of the hypothalamic-
pituitary-ovarian axis in prenatally androgenized mice
and sheep has revealed hyperactive firing of GnRH
neurons programmed by the treatment (47, 49), leading
to increased LH secretion as well as reduced responsiveness
to both negative and positive feedback of estradiol
(61–63). These studies suggest that the pathogenesis of
PCOS by prenatal androgens may be central in origin.
However, intrinsic ovarian defects are evident in pre-
natally androgenized sheep as early as fetal days 90 to
140 (gestation length is 147 days). Although there is no
effect on ovarian morphology or germ cell volume, the
expression of steroidogenic enzymes and LH receptor is
significantly reduced (64), and the expression of AR, but
not estrogen or progesterone receptors, is selectively
increased (65).
As with other models of developmental programming
of adult disease, a proposed mechanism through which
these acute cellular changes during tissue development
can translate into dysfunction much later in life is
through long-lasting epigenetic changes. In support of
this, multiple studies have identified differences in pro-
moter methylation of ovarian genes that regulate re-
production in prenatally androgenized rodents (66–68)
and zebrafish (69) using genomic techniques. Genes with
significant promoter hypomethylation include AR and
steroidogenic enzymes Cyp11 and Cyp17 (67), Gata6
and StAR (68), whereas apoptosis-related genes Bcl2l1
and Scr5a1 are hypermethylated (66). It is not certain
that these specific epigenetic alterations affect gene ex-
pression levels, but overall epigenetic modifications may
be an important factor in the programming of ovarian
dysfunction by prenatal androgen excess. As an example
of androgen-induced changes in DNA methylation, acute
treatment of mouse granulosa cells with DHT results in
inactivation of the histone methyltransferase Polycomb
group protein enhancer of zeste homolog 2 (Ezh2) by
both rapid and long-acting mechanisms (33) (Fig. 1).
Endocrinology, May 2019, 160(5):1166–1174
Within minutes, DHT causes phosphorylation of Ezh2
via nonclassic extranuclear AR actions involving the
PI3K/AKT pathway. Additionally, after 48 hours of
DHT treatment, Ezh2 gene expression is silenced via the
miRNA miR-101. This results in hypomethylation of
the Runt-related transcription factor-1 (Runx1) gene, an
LH-responsive factor important in ovulation, leading
to its increased expression, and interruptions in this
androgen-stimulated pathway can suppress ovulation in
mice. Similar androgen-induced epigenetic changes may
Downloaded from https://academic.oup.com/endo/article-abstract/160/5/1166/5419221 by guest on 02 May 2020
underlie the defects programmed by prenatal androgen
excess.
Conclusions
To summarize, the proper balance of androgen activity
is necessary to achieve and maintain normal ovarian
function throughout the female reproductive lifespan.
The mechanisms of androgen actions in the ovary are
complex and dynamic, starting in the fetus and extending
into adulthood, and require the cooperation of many cell-
specific and follicle stage–specific factors, not all of which
have been identified. Both excess and lack of androgens
lead to ovarian dysfunction and are associated with the
most common causes of infertility in women. With more
studies, it is likely that clinicians will be able to better
target disorders of androgen actions in women to im-
prove their fertility.
Acknowledgments
Financial Support: O.A. was supported by National Institutes
of Health Grant F32HD097939-01.
Correspondence: Stephen R. Hammes, MD, PhD, Box
693, 601 Elmwood Avenue, Rochester, New York 14642.
Email: Stephen_Hammes@URMC.Rochester.edu.
Disclosure Summary: The authors have nothing to
disclose.
References
1. Ohno S, Christian L, Attardi B. Role of testosterone in normal
female function. Nat New Biol. 1973;243(125):119–120.
2. Lyon MF, Glenister PH. Reduced reproductive performance in
androgen-resistant Tfm/Tfm female mice. Proc R Soc Lond B Biol
Sci. 1980;208(1170):1–12.
3. Hu YC, Wang PH, Yeh S, Wang RS, Xie C, Xu Q, Zhou X, Chao
HT, Tsai MY, Chang C. Subfertility and defective folliculogenesis
in female mice lacking androgen receptor. Proc Natl Acad Sci USA.
2004;101(31):11209–11214.
4. Yeh S, Tsai MY, Xu Q, Mu XM, Lardy H, Huang KE, Lin H, Yeh
SD, Altuwaijri S, Zhou X, Xing L, Boyce BF, Hung MC, Zhang S,
Gan L, Chang C. Generation and characterization of androgen
receptor knockout (ARKO) mice: an in vivo model for the study of
androgen functions in selective tissues [published correction ap-
pears in Proc Natl Acad Sci USA. 2002;99(23):15245]. Proc Natl
Acad Sci USA. 2002;99(21):13498–13503.
doi: 10.1210/en.2019-00101
5. Shiina H, Matsumoto T, Sato T, Igarashi K, Miyamoto J,
Takemasa S, Sakari M, Takada I, Nakamura T, Metzger D,
Chambon P, Kanno J, Yoshikawa H, Kato S. Premature ovarian
failure in androgen receptor-deficient mice. Proc Natl Acad Sci
USA. 2006;103(1):224–229.
6. Sen A, Hammes SR. Granulosa cell-specific androgen receptors are
critical regulators of ovarian development and function. Mol
Endocrinol. 2010;24(7):1393–1403.
7. Walters KA, Middleton LJ, Joseph SR, Hazra R, Jimenez M,
Simanainen U, Allan CM, Handelsman DJ. Targeted loss of an-
drogen receptor signaling in murine granulosa cells of preantral
and antral follicles causes female subfertility. Biol Reprod. 2012;
87(6):151.
8. Ma Y, Andrisse S, Chen Y, Childress S, Xue P, Wang Z, Jones D,
Ko C, Divall S, Wu S. Androgen receptor in the ovary theca cells
plays a critical role in androgen-induced reproductive dysfunction.
Endocrinology. 2017;158(1):98–108.
9. Baculescu N. The role of androgen receptor activity mediated by
the CAG repeat polymorphism in the pathogenesis of PCOS. J Med
Life. 2013;6(1):18–25.
10. Chamberlain NL, Driver ED, Miesfeld RL. The length and location
of CAG trinucleotide repeats in the androgen receptor N-terminal
domain affect transactivation function. Nucleic Acids Res. 1994;
22(15):3181–3186.
11. Tut TG, Ghadessy FJ, Trifiro MA, Pinsky L, Yong EL. Long
polyglutamine tracts in the androgen receptor are associated with
reduced trans-activation, impaired sperm production, and male
infertility. J Clin Endocrinol Metab. 1997;82(11):3777–3782.
12. Zhang T, Liang W, Fang M, Yu J, Ni Y, Li Z. Association of the
CAG repeat polymorphisms in androgen receptor gene with
polycystic ovary syndrome: a systemic review and meta-analysis.
Gene. 2013;524(2):161–167.
13. Borgbo T, Macek M Sr, Chrudimska J, Jeppesen JV, Hansen LL,
Andersen CY. Size matters: associations between the androgen
receptor CAG repeat length and the intrafollicular hormone milieu.
Mol Cell Endocrinol. 2016;419:12–17.
14. Skrgatic L, Baldani DP, Cerne JZ, Ferk P, Gersak K. CAG repeat
polymorphism in androgen receptor gene is not directly associated
with polycystic ovary syndrome but influences serum testosterone
levels. J Steroid Biochem Mol Biol. 2012;128(3-5):107–112.
15. Cai C, He HH, Chen S, Coleman I, Wang H, Fang Z, Chen S,
Nelson PS, Liu XS, Brown M, Balk SP. Androgen receptor gene
expression in prostate cancer is directly suppressed by the androgen
receptor through recruitment of lysine-specific demethylase 1.
Cancer Cell. 2011;20(4):457–471.
16. Hay CW, Watt K, Hunter I, Lavery DN, MacKenzie A, McEwan IJ.
Negative regulation of the androgen receptor gene through a
primate-specific androgen response element present in the 50 UTR.
Horm Cancer. 2014;5(5):299–311.
17. Weil SJ, Vendola K, Zhou J, Adesanya OO, Wang J, Okafor J,
Bondy CA. Androgen receptor gene expression in the primate
ovary: cellular localization, regulation, and functional correlations.
J Clin Endocrinol Metab. 1998;83(7):2479–2485.
18. Liu Y, Wang Y, Wang F, Pan J, Xu J, Li J, Zhou C, Ding G, Wu Y,
Liu X, Sheng J, Huang H. Mechanism underlying the retarded
nuclear translocation of androgen receptor splice variants. Sci
China Life Sci. 2019;62(2):257–267.
19. McEwan IJ, McGuinness D, Hay CW, Millar RP, Saunders PT,
Fraser HM. Identification of androgen receptor phosphorylation in
the primate ovary in vivo. Reproduction. 2010;140(1):93–104.
20. Xue K, Liu JY, Murphy BD, Tsang BK. Orphan nuclear receptor
NR4A1 is a negative regulator of DHT-induced rat preantral
follicular growth. Mol Endocrinol. 2012;26(12):2004–2015.
21. Sonneveld E, Jansen HJ, Riteco JA, Brouwer A, van der Burg B.
Development of androgen- and estrogen-responsive bioassays,
members of a panel of human cell line-based highly selective
steroid-responsive bioassays. Toxicol Sci. 2005;83(1):136–148.
https://academic.oup.com/endo 1173
22. Vendola KA, Zhou J, Adesanya OO, Weil SJ, Bondy CA. An-
drogens stimulate early stages of follicular growth in the primate
ovary. J Clin Invest. 1998;101(12):2622–2629.
23. Weil S, Vendola K, Zhou J, Bondy CA. Androgen and follicle-
stimulating hormone interactions in primate ovarian follicle de-
velopment. J Clin Endocrinol Metab. 1999;84(8):2951–2956.
24. Sen A, Prizant H, Light A, Biswas A, Hayes E, Lee HJ, Barad D,
Gleicher N, Hammes SR. Androgens regulate ovarian follicular
development by increasing follicle stimulating hormone receptor
and microRNA-125b expression. Proc Natl Acad Sci USA. 2014;
111(8):3008–3013.
25. Hasegawa T, Kamada Y, Hosoya T, Fujita S, Nishiyama Y, Iwata
N, Hiramatsu Y, Otsuka F. A regulatory role of androgen in
Downloaded from https://academic.oup.com/endo/article-abstract/160/5/1166/5419221 by guest on 02 May 2020
ovarian steroidogenesis by rat granulosa cells. J Steroid Biochem
Mol Biol. 2017;172:160–165.
26. Hickey TE, Marrocco DL, Gilchrist RB, Norman RJ, Armstrong
DT. Interactions between androgen and growth factors in gran-
ulosa cell subtypes of porcine antral follicles. Biol Reprod. 2004;
71(1):45–52.
27. Hickey TE, Marrocco DL, Amato F, Ritter LJ, Norman RJ,
Gilchrist RB, Armstrong DT. Androgens augment the mitogenic
effects of oocyte-secreted factors and growth differentiation factor
9 on porcine granulosa cells. Biol Reprod. 2005;73(4):825–832.
28. Otala M, Mäkinen S, Tuuri T, Sjöberg J, Pentikäinen V, Matikainen
T, Dunkel L. Effects of testosterone, dihydrotestosterone, and
17beta-estradiol on human ovarian tissue survival in culture. Fertil
Steril. 2004;82(Suppl 3):1077–1085.
29. Yazawa T, Kawabe S, Kanno M, Mizutani T, Imamichi Y, Ju Y,
Matsumura T, Yamazaki Y, Usami Y, Kuribayashi M, Shimada M,
Kitano T, Umezawa A, Miyamoto K. Androgen/androgen receptor
pathway regulates expression of the genes for cyclooxygenase-2
and amphiregulin in periovulatory granulosa cells. Mol Cell
Endocrinol. 2013;369(1-2):42–51.
30. Laird M, Thomson K, Fenwick M, Mora J, Franks S, Hardy K.
Androgen stimulates growth of mouse preantral follicles in vitro:
interaction with follicle-stimulating hormone and with growth
factors of the TGFb superfamily. Endocrinology. 2017;158(4):
920–935.
31. Tetsuka M, Whitelaw PF, Bremner WJ, Millar MR, Smyth CD,
Hillier SG. Developmental regulation of androgen receptor in rat
ovary. J Endocrinol. 1995;145(3):535–543.
32. Hillier SG, Tetsuka M, Fraser HM. Location and developmental
regulation of androgen receptor in primate ovary. Hum Reprod.
1997;12(1):107–111.
33. Ma X, Hayes E, Biswas A, Seger C, Prizant H, Hammes SR, Sen A.
Androgens regulate ovarian gene expression through modulation
of Ezh2 expression and activity. Endocrinology. 2017;158(9):
2944–2954.
34. Sen A, O’Malley K, Wang Z, Raj GV, Defranco DB, Hammes SR.
Paxillin regulates androgen- and epidermal growth factor-induced
MAPK signaling and cell proliferation in prostate cancer cells.
J Biol Chem. 2010;285(37):28787–28795.
35. Wartalski K, Knet-Seweryn M, Hoja-Lukowicz D, Tabarowski Z,
Duda M. Androgen receptor-mediated non-genomic effects of
vinclozolin on porcine ovarian follicles and isolated granulosa cells:
vinclozolin and non-genomic effects in porcine ovarian follicles.
Acta Histochem. 2016;118(4):377–386.
36. Converse A, Zhang C, Thomas P. Membrane androgen receptor
ZIP9 induces croaker ovarian cell apoptosis via stimulatory G
protein alpha subunit and MAP kinase signaling. Endocrinology.
2017;158(9):3015–3029.
37. Thomas P, Converse A, Berg HA. ZIP9, a novel membrane an-
drogen receptor and zinc transporter protein. Gen Comp Endo-
crinol. 2018;257:130–136.
38. Wu YG, Bennett J, Talla D, Stocco C. Testosterone, not 5a-
dihydrotestosterone, stimulates LRH-1 leading to FSH-independent
expression of Cyp19 and P450scc in granulosa cells. Mol Endo-
crinol. 2011;25(4):656–668.
1174 Astapova et al Androgen Actions in the Ovary
39. Yang F, Ruan YC, Yang YJ, Wang K, Liang SS, Han YB, Teng XM,
Yang JZ. Follicular hyperandrogenism downregulates aromatase
in luteinized granulosa cells in polycystic ovary syndrome women.
Reproduction. 2015;150(4):289–296.
40. Harlow CR, Shaw HJ, Hillier SG, Hodges JK. Factors influencing
follicle-stimulating hormone-responsive steroidogenesis in marmoset
granulosa cells: effects of androgens and the stage of follicular
maturity. Endocrinology. 1988;122(6):2780–2787.
41. Makita M, Miyano T. Androgens promote the acquisition of
maturation competence in bovine oocytes. J Reprod Dev. 2015;
61(3):211–217.
42. Miedlich SU, Taya M, Young MR, Hammes SR. Paxillin and
embryonic polyadenylation binding protein (ePABP) engage to
regulate androgen-dependent xenopus laevis oocyte maturation - a
model of kinase-dependent regulation of protein expression. Mol
Cell Endocrinol. 2017;448:87–97.
43. Li J, Yuan H, Chen Y, Wu H, Wu H, Li L. A meta-analysis of
dehydroepiandrosterone supplementation among women with
diminished ovarian reserve undergoing in vitro fertilization or
intracytoplasmic sperm injection. Int J Gynaecol Obstet. 2015;
131(3):240–245.
44. Hu Q, Hong L, Nie M, Wang Q, Fang Y, Dai Y, Zhai Y, Wang S,
Yin C, Yang X. The effect of dehydroepiandrosterone supple-
mentation on ovarian response is associated with androgen re-
ceptor in diminished ovarian reserve women. J Ovarian Res. 2017;
10(1):32.
45. van Houten EL, Kramer P, McLuskey A, Karels B, Themmen AP,
Visser JA. Reproductive and metabolic phenotype of a mouse
model of PCOS. Endocrinology. 2012;153(6):2861–2869.
46. Mannerås L, Cajander S, Holmäng A, Seleskovic Z, Lystig T, Lönn
M, Stener-Victorin E. A new rat model exhibiting both ovarian and
metabolic characteristics of polycystic ovary syndrome. Endocri-
nology. 2007;148(8):3781–3791.
47. Sullivan SD, Moenter SM. Prenatal androgens alter GABAergic
drive to gonadotropin-releasing hormone neurons: implications
for a common fertility disorder. Proc Natl Acad Sci USA. 2004;
101(18):7129–7134.
48. Foecking EM, Szabo M, Schwartz NB, Levine JE. Neuroendocrine
consequences of prenatal androgen exposure in the female rat:
absence of luteinizing hormone surges, suppression of progesterone
receptor gene expression, and acceleration of the gonadotropin-
releasing hormone pulse generator. Biol Reprod. 2005;72(6):
1475–1483.
49. Roland AV, Moenter SM. Prenatal androgenization of female mice
programs an increase in firing activity of gonadotropin-releasing
hormone (GnRH) neurons that is reversed by metformin treatment
in adulthood. Endocrinology. 2011;152(2):618–628.
50. Tehrani FR, Noroozzadeh M, Zahediasl S, Piryaei A, Azizi F.
Introducing a rat model of prenatal androgen-induced polycystic
ovary syndrome in adulthood. Exp Physiol. 2014;99(5):792–801.
51. Padmanabhan V, Manikkam M, Recabarren S, Foster D. Prenatal
testosterone excess programs reproductive and metabolic dys-
function in the female. Mol Cell Endocrinol. 2006;246(1-2):
165–174.
52. Abbott DH, Barnett DK, Bruns CM, Dumesic DA. Androgen
excess fetal programming of female reproduction: a developmental
aetiology for polycystic ovary syndrome? Hum Reprod Update.
2005;11(4):357–374.
53. Abbott DH, Dumesic DA, Eisner JR, Colman RJ, Kemnitz JW.
Insights into the development of polycystic ovary syndrome
Endocrinology, May 2019, 160(5):1166–1174
(PCOS) from studies of prenatally androgenized female rhesus
monkeys. Trends Endocrinol Metab. 1998;9(2):62–67.
54. Dumesic DA, Schramm RD, Abbott DH. Early origins of polycystic
ovary syndrome. Reprod Fertil Dev. 2005;17(3):349–360.
55. Steckler T, Wang J, Bartol FF, Roy SK, Padmanabhan V. Fetal
programming: prenatal testosterone treatment causes intrauterine
growth retardation, reduces ovarian reserve and increases ovarian
follicular recruitment. Endocrinology. 2005;146(7):3185–3193.
56. Dumesic DA, Patankar MS, Barnett DK, Lesnick TG, Hutcherson
BA, Abbott DH. Early prenatal androgenization results in di-
minished ovarian reserve in adult female rhesus monkeys. Hum
Reprod. 2009;24(12):3188–3195.
57. Silva MS, Prescott M, Campbell RE. Ontogeny and reversal of
Downloaded from https://academic.oup.com/endo/article-abstract/160/5/1166/5419221 by guest on 02 May 2020
brain circuit abnormalities in a preclinical model of PCOS. JCI
Insight. 2018;3(7):99405.
58. Eisner JR, Dumesic DA, Kemnitz JW, Colman RJ, Abbott DH.
Increased adiposity in female rhesus monkeys exposed to androgen
excess during early gestation. Obes Res. 2003;11(2):279–286.
59. Roland AV, Nunemaker CS, Keller SR, Moenter SM. Prenatal
androgen exposure programs metabolic dysfunction in female
mice. J Endocrinol. 2010;207(2):213–223.
60. Franks S, Berga SL. Does PCOS have developmental origins? Fertil
Steril. 2012;97(1):2–6.
61. Sarma HN, Manikkam M, Herkimer C, Dell’Orco J, Welch KB,
Foster DL, Padmanabhan V. Fetal programming: excess prenatal
testosterone reduces postnatal luteinizing hormone, but not follicle-
stimulating hormone responsiveness, to estradiol negative feedback
in the female. Endocrinology. 2005;146(10):4281–4291.
62. Moore AM, Prescott M, Campbell RE. Estradiol negative and
positive feedback in a prenatal androgen-induced mouse model of
polycystic ovarian syndrome. Endocrinology. 2013;154(2):796–
806.
63. Unsworth WP, Taylor JA, Robinson JE. Prenatal programming of
reproductive neuroendocrine function: the effect of prenatal an-
drogens on the development of estrogen positive feedback and
ovarian cycles in the ewe. Biol Reprod. 2005;72(3):619–627.
64. Hogg K, McNeilly AS, Duncan WC. Prenatal androgen exposure
leads to alterations in gene and protein expression in the ovine fetal
ovary. Endocrinology. 2011;152(5):2048–2059.
65. Ortega HH, Salvetti NR, Padmanabhan V. Developmental pro-
gramming: prenatal androgen excess disrupts ovarian steroid re-
ceptor balance. Reproduction. 2009;137(5):865–877.
66. Zhang D, Cong J, Shen H, Wu Q, Wu X. Genome-wide identifi-
cation of aberrantly methylated promoters in ovarian tissue of
prenatally androgenized rats. Fertil Steril. 2014;102(5):1458–
1467.
67. Xia Y, Shen S, Zhang X, Deng Z, Xiang Z, Wang H, Yi L, Gao Q,
Wang Y. Epigenetic pattern changes in prenatal female Sprague-
Dawley rats following exposure to androgen [published online
ahead of print 31 March 2015]. Reprod Fertil Dev. doi: 10.1071/
RD14292.
68. Salehi Jahromi M, Hill JW, Ramezani Tehrani F, Zadeh-Vakili A.
Hypomethylation of specific CpG sites in the promoter region of
steroidogeneic genes (GATA6 and StAR) in prenatally androgen-
ized rats. Life Sci. 2018;207:105–109.
69. Xu N, Chua AK, Jiang H, Liu NA, Goodarzi MO. Early em-
bryonic androgen exposure induces transgenerational epigenetic
and metabolic changes. Mol Endocrinol. 2014;28(8):1329–
1336.