REPRODUCTION

REVIEW

Role of angiotensin in ovarian follicular development and

ovulation in mammals: a review of recent advances
Paulo Bayard Goncalves, Rogerio Ferreira, Bernardo Gasperin and Joao Francisco Oliveira
Laboratory of Biotechnology and Animal Reproduction BioRep, Departamento de Clnica de Grandes Animais,
Hospital Veterinario, Federal University of Santa Maria, Santa Maria, RS 97105-900, Brazil
Correspondence should be addressed to P B Goncalves; Email: bayard@ufsm.br

Abstract
Angiotensin (Ang) II is widely known for its role in the control of systemic blood vessels. Moreover, Ang II acts on the vascular control of
ovarian function, corpus luteum formation, and luteolysis. Over the past 10 years, our research group has been studying the new concept
of the reninangiotensin system (RAS) as an autocrine/paracrine factor regulating steroidogenesis and promoting different cellular
responses in the ovary, beyond vascular function. We have developed and used different in vivo and in vitro experimental models to study
the role of RAS in the ovary and a brief overview of our findings is presented here. It is widely accepted that there are marked species
differences in RAS function in follicle development. Examples of species-specific functions of the RAS in the ovary include the
involvement of Ang II in the regulation of follicle atresia in rats vs the requirement of this peptide for the dominant follicle development
and ovulation in rabbits and cattle. More recently, Ang-(17), its receptor, and enzymes for its synthesis (ACE2, NEP, and PEP) were
identified in bovine follicles, implying that Ang-(17) has an ovarian function. Other novel RAS components (e.g. (pro)renin receptor and
renin-binding protein) recently identified in the bovine ovary show that ovarian RAS is poorly understood and more complex than
previously thought. In the present review, we have highlighted the progress toward understanding the paracrine and autocrine control of
ovarian antral follicle development and ovulation by ovarian tissue RAS, focusing on in vivo studies using cattle as a model.
Reproduction (2012) 143 1120

Introduction development, the RAS peptides, especially Ang II and to

In the classical definition, the reninangiotensin system
(RAS) is considered as an endocrine system. The
vasoconstriction mediated by angiotensin (Ang) II on
vascular smooth muscle is an example of the systemic
action of RAS. However, the novel concept of a local or
tissue RAS has been established more recently. These
two distinct pathways (endocrine and local RAS) may be
present in the control of a single physiological event. In
the ovary, RAS underlies important ovarian blood
pressure control (Brannstrom et al. 1998, Acosta et al.
2000) and autocrine/paracrine signaling regulating
gene expression (Acosta et al. 1999, Portela et al. 2008,
Ferreira et al. 2011a). A detailed discussion on the local
RAS physiology has been presented by Paul et al. (2006).
Ovarian function in mammals is primarily orches-
trated by endocrine factors, mainly gonadotropins (FSH
and LH), their receptors (FSHR and LHR), and ovarian
steroids. It is well established that follicle growth occurs
in waves and that the follicular cohort development is
stimulated by a transient increase in FSH; however,
selection of a dominant follicle is poorly understood.
Among the autocrine and paracrine factors that are
known to be involved in regulating ovarian antral follicle
q 2012 Society for Reproduction and Fertility
ISSN 14701626 (paper) 17417899 (online)
a lesser extent Ang-(17), have recently emerged as key
factors in the regulation of follicle deviation (Ferreira
et al. 2011b). Follicle deviation is characterized by
reduction or cessation of growth of the subordinate
follicles and continuous growth of the future dominant
follicle (largest follicle) (Ginther et al. 1996). In cattle,
the dominant follicle acquires ovulatory capacity when
it reaches a diameter of around 12 mm (Sartori et al.
2001). At the end of the follicular phase, a complex series
of events culminates in ovulation, with the release of a
competent egg and the initiation of luteal development.
Ang II has been demonstrated to be essential in the
ovulatory process (Ferreira et al. 2007) and seems to be
an important mediator of LH action in the early increase
in ADAM activity that induces the cascade of events
leading to ovulation (Portela et al. 2011). However, the
physiological function of Ang II in the ovary is not yet
well established and the wide species variation makes
its understanding even more challenging (Bottari et al.
1993, Nielsen et al. 1995, Yoshimura 1997, Mow et al.
1999). In the present review, the focus is to highlight
the progress toward understanding the paracrine and
autocrine control of the ovarian antral follicle deve-
lopment and ovulation by ovarian tissue RAS.
DOI: 10.1530/REP-11-0192
Online version via www.reproduction-online.org
12 P B Goncalves and others
The bovine model
Cattle provide an excellent model for studying the role of
local factors in the control of follicle development. First,
cattle are a monovulatory species and the antral follicle
development and deviation can be monitored on a day-
to-day basis. Secondly, the timing of follicle deviation
and dominance, the LH surge, and ovulation can be
controlled. Finally, the follicular environment can be
easily modified by ultrasound-guided i.f. injection (Kot
et al. 1995, Ginther et al. 2004, Ferreira et al. 2007). Our
investigations were based on two in vitro and three in
vivo models. For the in vitro models, we used two serum-
free culture systems of granulosa cells without spon-
taneous luteinization (Gutierrez et al. 1997). In one
culture system, granulosa cells from medium-sized
follicles (48 mm) were used. In the other system,
granulosa cells of large follicles, in which the expression
of genes in the ovulatory cascade is responsive to LH,
were cultured (Portela et al. 2011). In one in vivo model,
we could predict the day before, during, and after
follicular deviation (Evans & Fortune 1997, Rivera et al.
2001) to evaluate RAS regulation. In another model, the
largest follicle was intrafollicularly injected with
competitive inhibitor, or the second largest follicle was
injected with an agonist to induce codominant follicles
at the time of deviation (Gastal et al. 1995, Ginther et al.
2004). In the other model, follicles were intrafollicularly
injected with a competitive inhibitor when they reached
the size of 12 mm and the cows were challenged with a
GNRH agonist to study ovulation (Ferreira et al. 2007,
Barreta et al. 2008).
Systemic RAS
Angiotensinogen, the precursor of RAS, is encoded by a
single gene, which is expressed mainly in the liver,
although its mRNA expression was detected in other
tissues, including the ovary (Ohkubo et al. 1986). Its
cleavage to form Ang I depends on kidney-secreted
renin. Catalytic activation of renin occurs after cleavage
of a segment of prorenin comprising 43 amino acids (Do
et al. 1987) and seems to occur only in kidneys because
renin disappears from blood after bilateral nephrectomy
(Sealey et al. 1977). However, noncatalytic activation of
prorenin has been described and will be further
discussed in the ovarian RAS section below.
Ang I is a decapeptide derived from angiotensinogen
cleavage by renin at the leucinevaline bond at the
N-terminal region in human or at the leucineleucine
bond in other species. Angiotensin converting enzyme
(ACE) is the major enzyme capable of converting the
decapeptide Ang I into octapeptide Ang II by specific
cleavage at Phe8His9 bond, releasing the dipeptide
HisLeu (Skeggs et al. 1955, 1956). Early studies showed
that only Ang II was capable of raising blood pressure.
Nevertheless, an i.v. injection of Ang I had the same
Reproduction (2012) 143 1120
hypertensive effect, indicating abundant ACE plasma
activity (Skeggs et al. 1954).
The RAS is a phylogenetically old system that is
present throughout the vertebrates. In most mammalian
species, the fifth amino acid of Ang II sequence is an
isoleucine (Ile), while cattle and nonmammalian tetra-
pods have a valine present at this position (named [Val5]-
Ang II). The systemic RAS is not the focus of the present
review and has been widely reviewed in Yoshimura
(1997), Campbell (2003), and Paul et al. (2006).
Ovarian RAS
More recently, a local RAS concept has emerged, and
novel kidney-independent enzymes capable of forming
Ang II have been identified. The evidence that supports
the existence of an ovarian RAS includes the following:
i) the concentrations of Ang II in the follicular fluid were
found to be higher than those in the plasma after human
chorionic gonadotropin (hCG) treatment (Husain et al.
1987), ii) high follicular fluid levels of Ang II were found
after bilateral nephrectomy (Husain et al. 1987), and
iii) in vitro-perfused rabbit ovaries produced Ang II when
exposed to hCG (Yoshimura et al. 1994). However, renin
is produced exclusively by kidneys and disappears from
the plasma after bilateral nephrectomy (Do et al. 1987).
Therefore, alternative pathways must be involved in the
local synthesis and regulation of Ang II concentration.
The contribution of this local system on ovarian
physiology will be discussed.
Prorenin is produced and secreted mainly by kidneys,
but extrarenal sources have also been described. Prorenin
concentration in follicular fluid is 12 times higher than
that found in the systemic circulation and is immunolo-
gically identical to kidney and plasma prorenin (Glorioso
et al. 1986). In the former studies, prorenin was not
identified as a catalytic enzyme; however, in the early
1990s, the ovarian concentration of prorenin was found
to be associated with follicular atresia (Schultze et al.
1989, Mukhopadhyay et al. 1991). Controversially, renin
secretion seems to be regulated by gonadotropins.
An increase in both plasma renin (Itskovitz et al. 1987)
and follicular fluid renin activity (Yoshimura et al.
1994) has been observed after hCG treatment.
For many years, prorenin was considered to be
the only inactive precursor of renin (Do et al. 1987).
However, the nonproteolytic activation of prorenin has
now been identified (Suzuki et al. 2003). The renin
and prorenin receptor (named (pro)renin receptor) is
able to not only bind renin and prorenin but also activate
prorenin by inducing a conformational change in protein
folding (Nabi et al. 2006). In vivo, (pro)renin receptor
binds mainly to prorenin due to its higher affinity to
prorenin than to renin and its high concentration in
tissues that produce it locally, i.e. in the ovary (Batenburg
et al. 2007). Moreover, prorenin also binds to
(pro)renin receptor and induces signaling-promoting
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angiotensin-like effects, independent of Ang II synthesis
(Kaneshiro et al. 2007). Recently, we have demonstrated
the presence of mRNA-encoding (pro)renin receptor in the
ovary and its differential mRNA expression in dominant
and subordinate follicles (Ferreira et al. 2011b), suggesting
a role for this receptor either by prorenin nonproteolytic
activation or by mediating Ang II-independent effects.
The role of ACE in ovarian physiology is not fully
understood. Captopril (an ACE inhibitor) did not inhibit
ovulation in rats (Daud et al. 1990). In contrast, the Ang II
type 2 receptor (AGTR2) blocker inhibited ovulation in
the rabbit and bovine (Yoshimura et al. 1992, Ferreira
et al. 2007), suggesting the presence of an alternative
enzymatic route to the synthesis of ovarian Ang II. The
members of plasminogen activator (PA) family, cathepsin
D and chymase, are enzyme candidates to convert Ang I
into the octapeptide Ang II (for review see Paul et al.
(2006)). Yoshimura et al. (1996a) used streptokinase, an
exogenous PA, to stimulate the intrafollicular Ang II
content and follicular growth in the rabbit. The same
authors have shown using an in vitro-perfused rabbit
ovary model that IGF1 stimulates both follicular growth
and PA activity in the follicular fluid. Recently, we
have demonstrated that the expression of ACE mRNA
in bovine granulosa cells is negatively correlated with
follicular growth (Ferreira et al. 2011b). Therefore,
additional studies are required to characterize the
enzyme responsible for cleaving Ang I into Ang II in
the ovary.
Ang II and follicle development
Ang II receptors in follicular cells
The role of Ang II in the regulation of follicular
development has mainly been studied in antral follicles.
However, the Ang II type 1 and 2 receptors (AGTR1 and
AGTR2) have been identified in porcine granulosa cells
of earlier stages of follicle development (Shuttleworth
et al. 2002b). A positive action of Ang II in estradiol
synthesis and growth of preantral follicles in culture
were also reported in swine (Shuttleworth et al. 2002a).
The hypothesis that RAS is involved in early follicle
development was recently reinforced. Pountain et al.
(2010) detected both AGTR1 and AGTR2 proteins in the
granulosa cells of primordial, primary, and secondary
follicles of pig ovaries from fetuses of at least 45 days of
gestation. They also detected mRNA-encoding prorenin
and angiotensinogen in preantral follicles, suggesting
Ang II synthesis during early follicle growth.
During antral follicle development, there are consider-
able differences between species regarding Ang II
receptor functions. In rats, AGTR2 was found to be
involved in follicle atresia through apoptosis (Tanaka
et al. 1995, Kotani et al. 1999). In fact, AGTR1 receptor
has been localized in healthy follicles, while the mRNA
expression of AGTR2 receptor has been identified
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Angiotensin and ovarian follicle 13
in small- to medium-sized atretic follicles in rats (de
Gooyer et al. 2004). Moreover, AGTR2 protein
expression has been found to be inhibited by FSH
(Pucell et al. 1988), and FSH-induced estradiol pro-
duction was inhibited by Ang II and that effect was
reversed by an Ang II receptor antagonist (PD123,319)
(Kotani et al. 1999).
In monovular species, the expression of AGTR2
receptor was first identified in theca cells from bovine
dominant follicles (Schauser et al. 2001). These data
were further confirmed by Berisha et al. (2002) who
demonstrated mRNA-encoding AGTR1 and AGTR2
receptors in both theca and granulosa cells from bovine
and observed a slight increase in AGTR2 mRNA in theca
cells from highly estrogenic follicles (O180 ng/ml) in
comparison with less estrogenic follicles (20180 ng/ml).
Recently, our group showed that the expression of
AGTR2 mRNA in granulosa cells was significantly higher
in healthy than in atretic follicles (Portela et al. 2008).
Furthermore, we also found that FSH, IGF1, and bone
morphogenetic protein-7, which augmented estradiol
secretion, increased AGTR2 expression at both mRNA
and protein levels (Portela et al. 2008). However,
fibroblast growth factors-7 and -10, which decreased
estradiol secretion, reduced AGTR2 protein levels in
primary cell cultures of granulosa cells (Portela et al.
2008). Collectively, these data suggest that rodent and
bovine models are different regarding Ang II signaling
during antral follicle development. The detection of high
AGTR2 expression in bovine dominant follicles suggests
a role for Ang II signaling during follicle deviation and
selection of dominant follicle in monovular species.
Ang II signaling: studies in vivo and in vitro
In cattle, i.f. injection of saralasin (an octapeptide
competitive antagonist of Ang II) inhibited dominant
follicle growth, decreased estradiol concentration in
follicular fluid, and downregulated CYP19A1 mRNA
expression in granulosa cells (Ferreira et al. 2011a).
However, the follicle regression induced by saralasin
was reversed with systemic FSH and the dominant
follicle reached ovulation (Fig. 1).
Analysis of follicular cells isolated from saralasin-
treated follicles revealed that the expression of AGTR2
was downregulated in theca cells, and mRNAs encoding
CYP19A1, LHR, cyclin D2, and 3bHSD were severely
downregulated in granulosa cells (Ferreira et al. 2011a;
Fig. 1). These are key genes for dominant follicle
development. There is strong evidence that Ang II is
required for and plays an important role in upregulating
essential genes to stimulate high levels of estradiol
secretion for maintaining follicle development when
FSH levels decline during follicular deviation (Adams
et al. 1992).
In another experiment conducted by our group, i.f.
injection of Ang II peptide or AGTR2 receptor-specific
Reproduction (2012) 143 1120
14 P B Goncalves and others
Saline or
Ang II or
Sar+FSH
DF
Ovulation
TC: AGTR2
GC: CYP19A1
3bHSD
Sar
CYCLIN D2
FF: E2
DF Atresia
Ang II or
CGP
SLF
Temporary
growth (24 h)
Figure 1 Effect of angiotensin II (Ang II), Ang II receptor antagonist
(saralasin Sar), or Ang II type 2 receptor (AGTR2)-specific agonist
(CGP42112A) on dominant (DF) and second largest follicle (SLF)
development in cattle. I.f. injections were performed in the largest
growing follicle when it reached 78 mm or in the SLF when the
difference between the largest and the second largest follicle was about
1 mm (w6 mm). After treatments, follicle growth was evaluated at 24 h
intervals until ovulation or atresia. Control follicles (saline) and those
injected with Ang II or Sar plus systemic (i.m.) FSH continued to grow
and ovulated after GNRH analog challenge. Sar-treated follicles
stopped growing and underwent atresia. Sar treatment significantly
reduced estradiol (E2) levels in follicular fluid, AGTR2 mRNA in theca
cells, and cyclin D2, 3bHSD, and CYP19A1 mRNA expression in
granulosa cells. I.f. injection of Ang II peptide or CGP42112A in the
SLF at deviation temporarily prevented its expected regression. GC,
granulosa cells; TC, theca cells; FF, follicular fluid.
agonist (CGP42112A) temporarily prevented the
expected regression of the second largest follicle
(subordinate follicle) at deviation (Ferreira et al. 2011a;
Fig. 1). In isolated perfused rabbit ovaries, IGF1
stimulated ovarian Ang II production (Yoshimura et al.
1996a), and in cattle, IGF1 induced an increase in
AGTR2 mRNA and protein (Portela et al. 2008). This
interaction between Ang II and IGF1 suggests that Ang II
is required for antral follicle development and may
explain the temporary rescue of the larger subordinate
follicle from atresia.
On the other hand, a series of independent studies
conducted with rodents have demonstrated that Ang II
is involved in the regulation of follicle atresia caused
by apoptosis of granulosa cells (Pucell et al. 1988, Daud
et al. 1990, Tanaka et al. 1995, Kotani et al. 1999,
de Gooyer et al. 2004). Transgenic rats that overexpress
renin and angiotensin in extrarenal tissues, including
ovarian cells, had a reduced number of large antral
follicles and small litter size (de Gooyer et al. 2004). In
the same study, similar negative effects on follicle
development were observed after Ang II infusion in
wild-type females. In swine, the concentrations of Ang II
Reproduction (2012) 143 1120
in follicular fluid were found to be negatively correlated
with follicular diameter (Li et al. 2004). These studies
indicate the existence of marked species differences of
the RAS function in follicle development and atresia.
Regulation of RAS members during follicle development
Estrus synchronization and follicular dynamics followed
by ovariectomy as described previously by Rivera et al.
(2001) allowed the characterization of novel RAS
components near follicular deviation. We recently
demonstrated that RAS is regulated and involved in
follicular deviation in monovular species (Ferreira et al.
2011b). Renin-binding protein (RnBP, an inhibitor of
renin activity) mRNA was found to be upregulated in
bovine subordinate follicles at the expected moment of
follicular deviation (day 3 of the follicular wave
emergence) and the day after deviation (day 4). This
correlation between increased RnBP mRNA expression
and atresia was further confirmed by i.f. injection of
fulvestrant (an estradiol receptor antagonist) (Ferreira
et al. 2011b). Prorenin activity was previously observed
to be negatively correlated with estradiol levels in bovine
follicular fluid, leading to atresia of large follicles
(Schultze et al. 1989, Mukhopadhyay et al. 1991).
Nevertheless, we observed that Ang II levels do not
increase in the subordinate follicle after deviation.
Therefore, we can hypothesize that factors other than
RAS peptides are regulating prorenin and renin activities
in the dominant and subordinate follicles. In contrast to
the data obtained in cattle, renin activity and estradiol
levels were positively correlated in the follicular fluid
from women who underwent ovarian stimulation
(Fernandez et al. 1985). However, there are not enough
studies to support the participation of RnBP in follicle
development and atresia.
Ang II production in follicular cells seems to be
regulated by gonadotropins. The concentrations of Ang II
in the follicular fluid of hCG-treated rodents after
bilateral nephrectomy were higher than those in the
plasma (Husain et al. 1987). Near ovulation, the levels of
Ang II also increased in the ovary after treating rabbits
with hCG (Yoshimura et al. 1994), cattle with LH (Acosta
et al. 2000, Shimizu et al. 2007), and women with
human menopausal gonadotrophin (hMG) and hCG
(Lightman et al. 1987). Similarly, Ang II and Ang III were
found to be positively correlated with estradiol levels
and follicle diameter in women stimulated with hMG
(Jarry et al. 1988).
Despite the evidence that Ang II synthesis is regulated
by gonadotropins, the pattern of Ang II levels in the
follicular fluid around the time of follicle deviation was
unknown until recently. One of the most recent findings
regarding the involvement of RAS in follicular develop-
ment is the increase in Ang II levels in the follicular fluid
of the dominant follicle at the expected moment of
deviation (day 3), which coincides with the acute
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increase in CYP19A1 mRNA expression and estradiol
levels (Ferreira et al. 2011a, b). Also, granulosa cells
treated with Ang II showed a positive correlation with
CYP19A1 mRNA expression in vitro. However, the inter-
regulation between Ang II and steroidogenesis in the
follicular cells is still poorly understood.
Ang II and ovulation
Ang II signaling and receptors near ovulation
Several studies on cattle demonstrated that Ang II levels
increase in follicular fluid after administration of an
ovulatory dose of gonadotropin (Acosta et al. 2000,
Shimizu et al. 2007). Schauser et al. (2001) observed
intense AGTR2 receptor binding in cow periovulatory
follicles, mainly in the theca cells. Using an in vivo
model, our group demonstrated that i.m. injection of
GNRH regulates the mRNA-encoding AGTR2 receptor
in theca cells from follicles R12 mm in diameter.
Ang II receptor antagonists inhibit ovulation and have
been used to study the role of RAS during the ovulatory
process in rodents, rabbits, and cattle (Pellicer et al.
1988, Kuji et al. 1996, Ferreira et al. 2007). I.p. injection
of saralasin blocked gonadotropin-induced ovulation
in immature rats (Pellicer et al. 1988). However, a
selective nonpeptide antagonist for AGTR2 receptors
(PD123,319) had no effects on the ovulation rate in
in vitro-perfused rat ovaries (Mikuni et al. 1998).
Furthermore, the administration of PD123,319 dimin-
ished but did not completely block ovulation in rats
(Mitsube et al. 2003). Taken together, these results
indicate that Ang II is essential for ovulation in rats, but
this effect is not exclusively regulated via the AGTR2.
In rabbit ovaries perfused in vitro, it was demonstrated
that Ang II signals through AGTR2 receptor subtype (Kuji
et al. 1996). Furthermore, ovulation induced by Ang II
and prostaglandins (PGE and PGF2a) synthesis induced
by hCG were inhibited by PD123,319 (Yoshimura et al.
1996b). We recently demonstrated that the ovulation is
inhibited when AGTR2 receptor antagonists (saralasin or
PD123,319) are intrafollicularly injected at the moment
of GNRH injection in cows (Ferreira et al. 2007).
Moreover, the LH-induced prostaglandin synthase 2
(PTGS2) mRNA expression is increased by Ang II and
inhibited by PD123,319 in granulosa cell cultures from
large dominant bovine follicles (Portela et al. 2011). In
both bovine and rabbit models, the AGTR1 blockade did
not affect gonadotropin-induced ovulation (Kuji et al.
1996, Ferreira et al. 2007). As discussed above, these in
vitro and in vivo studies suggest that Ang II plays an
important role in the early mechanism of ovulation via
the AGTR2 receptor subtype in rabbits and cattle.
Ang II is capable of inducing ovulation in rabbits even
in the absence of gonadotropins (Kuo et al. 1991,
Yoshimura et al. 1992). The effect of Ang II during
ovulation is mediated, at least in part, by PGs. In rabbit
ovaries perfused in vitro, Ang II stimulated the synthesis of
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Angiotensin and ovarian follicle 15
PGE2 and PGF2a in the absence of gonadotropins
(Yoshimura et al. 1993). The same authors inhibited Ang
II-induced PGs synthesis and ovulation using indometha-
cin (an inhibitor of cyclooxygenases). The fact that PGs
mediate Ang II functions in the periovulatory period was
further confirmed in other studies assessing meiotic
resumption of bovine oocytes (Barreta et al. 2008).
Factors from the EGF family, known as EGF-like
growth factors, are immediately upregulated by LH
surge in vivo and also after LH treatment in follicular cell
cultures (Park et al. 2004, Li et al. 2009). Amphiregulin
(AREG) and epiregulin (EREG) are two of the LH-induced
genes mediating the gonadotropin-induced PTGS2
expression (Park et al. 2004, Andric & Ascoli 2008, Li
et al. 2009). The hypothesis that Ang II is a mediator of
LH action in the cascade of events leading to ovulation
was confirmed by our group using bovine granulosa cell
cultures derived from O10 mm follicles. Using this
in vitro system, Ang II significantly amplified the positive
effect of LH on PTGS2 (mRNA and protein expression),
AREG, EREG, and plasminogen activators (Portela et al.
2011). A dramatic upregulation of ADAM17, an essential
factor for the initial ovulatory process (Yamashita et al.
2007), was also observed 1 h post-treatment with Ang II.
This early effect of Ang II preceded the induction of
LH-induced genes, such as AREG, EREG, and PTGS2 by
25 h. Galardin, a broad-spectrum matrix metallopro-
teinase inhibitor, completely blocked the effects of LHC
Ang II, demonstrating that sheddase activity is the direct
target of Ang II. Collectively, the results obtained in vivo
using Ang II receptors blockade and in vitro with
granulosa cell cultures suggest that Ang II is a mediator
of LH surge during the early mechanism of bovine
ovulation (Fig. 2).
Ang-(17) and follicle development and ovulation
Studies regarding Ang-(17) functions in ovarian physi-
ology are more recent and scarcer than those regarding
Ang II. Ang-(17) was first described in rat ovaries and
was shown to be regulated during the estrous cycle
(Costa et al. 2003). Recently, Ang-(17), MAS receptor,
and ACE2 were identified in all the stages of follicular
development in humans (Reis et al. 2011). Ang-(17)
g-protein-coupled receptor (MAS) and ACE2, an enzyme
capable of converting Ang I or Ang II into Ang-(17), are
expressed in the rat ovary (Pereira et al. 2009). Many
enzymes present in several tissues are involved in
Ang-(17) synthesis from either Ang I or Ang II. Neutral
endopeptidase (NEP) converts Ang I into Ang-(17).
Prolyl endopeptidase (PEP) and ACE2 are involved in the
conversion of Ang I or Ang II into Ang-(17). ACE and
aminopeptidases (AMPs) inactivate Ang-(17) through its
cleavage into smaller fragments. The pathways of Ang II
and Ang-(17) synthesis, their receptors, and interactions
between RAS components were recently revised by
Santos et al. (2008).
Reproduction (2012) 143 1120
16 P B Goncalves and others
ADAM17
Active
precursor
Active AREG/EREG
LHR AGTR2 ADAM17
Metalloproteinase Pro-EGFLP EGFR
activation
ADAM17 mRNA
AREG mRNA
EREG mRNA
PTGS2 mRNA
Figure 2 Proposed model of the reninangiotensin system in the
regulatory mechanism of ovulation. Angiotensin II (Ang II) amplified
the positive effect of LH on PTGS2, EREG, AREG, and ADAM17 mRNA
expression. The metalloproteinase inhibitor Galardin blocked the effect
of Ang II plus LH on AREG, EREG, and PTGS2 mRNA expression. These
findings, taken together with the data of the literature that ADAM17 is
the major sheddase in granulosa cells, suggest that epidermal growth
factor-like peptide precursors (Pro-EGFLP) are proteolytically cleaved
by ADAM17, resulting in amphiregulin (AREG) or epiregulin (ERG),
which binds to EGF receptor (EGFR). In short, Ang II seems to be a
mediator of LH action in promoting the activation/expression of
disintegrin and metalloproteinase (ADAM)-17 in the induction of
events leading to ovulation in cattle.
Functional studies evidenced a role for the RAS branch
composed of ACE2, MAS receptor, and Ang-(17) in
follicle development in the rodent model. In ovarian
homogenates from immature rats, Ang-(17) was shown
to increase during proestrus and estrus and after equine
chorionic gonadotropin (eCG) treatment (Costa et al.
2003). Immunolocalization studies demonstrated that
eCG-induced Ang-(17) synthesis occurs mainly in the
theca and interstitial cells (Pereira et al. 2009).
Interestingly, MAS receptor immunostaining, which
was not observed in immature ovaries, appeared in the
theca and interstitial cells from antral and preovulatory
follicles after eCG treatment (Pereira et al. 2009). A
positive effect of gonadotropins on Ang-(17) concen-
tration was also observed in the plasma from women
who underwent ovarian stimulation (Reis et al. 2011).
Perfusion of Ang-(17) or Ang II in immature rat
ovaries induced an increase in estradiol and pro-
gesterone synthesis (Costa et al. 2003). The positive
effect of Ang-(17) on steroidogenesis was inhibited by
the specific MAS receptor antagonist A-779. In contrast,
A-779 disrupted Ang II effect on progesterone, but not
estradiol synthesis, suggesting that Ang II effect on
progesterone may be a result of its conversion to Ang-
(17) (Costa et al. 2003, Simoes et al. 2004).
Ang-(17) protein is also present in rat oocytes, while
MAS and Ang-(17) are absent in rat granulosa cells
(Pereira et al. 2009). ACE2 was observed in stroma and
granulosa cells from immature ovaries and in all
follicular compartments, including the oocyte, from
Reproduction (2012) 143 1120
eCG-treated ovaries (Pereira et al. 2009). In accordance
with the protein levels and mRNA expression, MAS
receptor and ACE2 increase after eCG treatment in rat
ovaries homogenates (Pereira et al. 2009). In cattle,
ACE2 mRNA expression was upregulated in the
dominant follicle at the expected time of follicular
deviation, coinciding with an acute increase in intra-
follicular estradiol levels (MH Barreta, R Ferreira & PB
Goncalves 2011, unpublished data). The regulation of
ACE2 by gonadotropins and its differential mRNA
expression in dominant and subordinate follicles
reinforce the theory of local Ang-(17) production.
Regarding the enzymatic activity, ACE and NEP
activities in the rat ovaries were reduced after eCG
treatment (Pereira et al. 2009). In the bovine model, ACE
mRNA was more expressed in the subordinate follicles
entering atresia and was severely suppressed in the
estrogenic dominant follicle (Ferreira et al. 2011b).
Collectively, these data suggest that mRNA expression
and activity of ACE are negatively correlated with the
estradiol levels and moreover indicate a reduction in
Ang-(17) cleavage when estradiol levels are elevated.
The increased availability of Ang-(17) may positively
regulate estradiol synthesis as demonstrated previously
in rat ovaries perfused in vitro (Costa et al. 2003).
Regarding PEP enzyme, conflicting results were
observed in different models. In rodents, PEP activity
seems to be positively correlated with the estradiol levels
(Ohta et al. 1992, Pereira et al. 2009). On the other hand,
our preliminary results demonstrate that PEP mRNA
expression is upregulated during subordinate follicle
atresia in cattle.
In the ovulation process, Ang-(17) enhanced the
ovulatory efficiency in in vitro-perfused rabbit ovaries,
which was antagonized by A-779 (Reis et al. 2009).
Using a bovine model in which cows are synchronized
to obtain preovulatory follicles of at least 12 mm, Ang-
(17) levels increased in the follicular fluid between 12
and 24 h after GNRH injection in vivo (Fig. 3). The
increase in Ang-(17) just before ovulation is probably a
consequence of ACE2, NEP, and PEP activities, since
mRNAs that encode these three enzymes are upregu-
lated concomitantly to Ang-(17) increase (Santos et al.
2011). Collectively, these results and the fact that A-779
reduces hCG-induced ovulation in rabbits (Reis et al.
2009) suggest that Ang-(17) is a mediator of gonado-
tropin functions in the ovulatory cascade.
Summary and conclusion
It is increasingly evident that RAS is involved in the local
control of ovarian physiology. However, the intracellular
mechanism of action triggered by angiotensin is not yet
known in the ovary. New enzymes and receptors have
been described and seem to modulate the local synthesis
of angiotensin. It has been recently demonstrated that
prorenin, which is thought to be an inactive peptide, can
www.reproduction-online.org

GNRH injecti
Hou
rs after on
12
PEP
3 NEP
ACE2
AGTR2 Ang II peak
Expected Ang II
Ang-(17) peak
LH surge
E2
peak
0 24
Saralasin
or PD
I.f.
injection
6
Hou
rs after GNRH injection
bind to (pro)renin receptor and trigger local action
independently of Ang II. The RAS is extremely complex
and difficult to study, given that most of the active
peptides are post-translationally processed. For many
years, it was thought that Ang II was the only active
peptide of the RAS; however, it is now known that Ang-
(17), Ang III, and Ang IV play important roles in various
tissues. Based on recent findings, we propose that these
peptides are acting in the ovary. However, additional
studies are still necessary to better characterize the cell
and molecular pathways.
There is consensus that RAS functions in the ovary are
species specific. We have focused our research in the
cow model because it is a monovulatory species in
which we could monitor the follicle deviation, accu-
rately predict ovulation time, and modify the follicular
environment. We have shown cattle to be a reliable in
vivo animal model to study the RAS in ovarian function.
Moreover, a serum-free culture system of granulosa cells
without spontaneous luteinization and a culture system
of large follicles were used to further investigate RAS on
specific regulatory factors of follicular cells. Based on
these models, we found that i) Ang II concentration
increases in the follicular fluid of the dominant follicle
during and after deviation; ii) follicular development was
completely blocked with i.f. injection of a competitive
antagonist of Ang II or a selective antagonist for AGTR2
www.reproduction-online.org
Angiotensin and ovarian follicle 17
Figure 3 Time course of regulation of RAS
components in the bovine ovulatory follicle
(O12 mm) at 0, 3, 6, 12, and 24 h after GNRH
agonist challenge in vivo (upper middle part of the
figure). The LH surge is expected to occur between 0
and 3 h after GNRH injection. Estradiol levels
increased progressively in the first 3 h and declined
until 24 h after GNRH, validating the model.
Angiotensin II (Ang II) follicular fluid levels and
theca cell AGTR2 mRNA expression were
Ovulation
blockade upregulated during the first 6 h after GNRH
treatment. Ang-(17), its receptor MAS, and
enzymes responsible for Ang-(17) synthesis
(PEP, NEP, and ACE2) were upregulated between 6
and 24 h after GNRH. The lower middle part depicts
the blockade of ovulation following ultrasound-
mediated i.f. injection of an Ang II receptor
antagonist (saralasin) at 0 or 6 h or an Ang II
type 2 receptor (AGTR2)-specific antagonist
(PD123,319) at 0 h after GNRH agonist challenge.
The i.f. injection of saralasin at 12 h after GNRH did
not inhibit ovulation.
receptors; iii) i.f. injection of Ang II or AGTR2 agonist
prevented the expected regression of the second largest
follicle at deviation; iv) Ang II antagonist downregulated
the mRNA expression of genes involved in granulosa cell
proliferation and differentiation (LHR, CYP19A1, and
HSD3b) during follicular deviation; v) i.f. injection of a
competitive antagonist of Ang II decreased ovulation rate
in cows induced with a GNRH agonist; and finally vi)
Ang II acts via the AGTR2 receptor, increasing the
expression/activity of ADAM17 and resulting in
the upregulation of mRNA-encoding genes involved
in the ovulation cascade, such as AREG, EREG, and
PTGS2. Based on these findings and studies by other
groups, we conclude that RAS plays fundamental roles in
follicular development, deviation, atresia, and ovulation
in a species-specific manner.
Declaration of interest
The authors declare that there is no conflict of interest that
could be perceived as prejudicing the impartiality of the
research reported.
Funding
The research was funded by CNPq and CAPES.
Reproduction (2012) 143 1120
18 P B Goncalves and others
Acknowledgements
We thank the following collaborators: Dr Christopher A Price
(Universite de Montreal, Canada), Dr Adelina Martha dos Reis
(Universidade Federal de Minas Gerais, Brazil), Dr Jose
Buratini Junior (Universidade Estadual Paulista, Brazil), and
Dr Valerio M Portela (Universidade Federal de Santa Catarina,
Brazil). The authors also thank to Dr Vilceu Bordignon (McGill
University, Canada) for critical reading of the manuscript.
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oocyte maturation in rabbit ovaries via the AT2 receptor subtype.
Endocrinology 137 12041211. (doi:10.1210/en.137.4.1204)
Received 2 June 2011
First decision 19 July 2011
Revised manuscript received 19 October 2011
Accepted 1 November 2011
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