**2. RAS in the reproductive system**

### **2.1. RAS and ovary and follicular development**

Prorenin is produced by the ovarian follicular cells at different stages in oocyte matura‐ tion. As the ovarian follicle undergoes maturation, the prorenin concentration increases and remains elevated until the end of the luteal phase, near the start of menstruation, where it falls in parallel with progesterone levels [14]. Prorenin secretion in the ovary is regulated by gonadotropins, and thus, the rise in plasma‐luteinizing hormone (LH) levels shortly precedes the elevation of plasma prorenin, secreted into circulation mainly by the ovary [15, 16]. Of note, concentrations of prorenin, the inactive precursor of renin, are typically higher in the reproductive system than those of renin and it was originally postulated that it was locally activated by an unknown process. As such, studies demonstrating the expression of cathep‐ sin B, a potential activator of prorenin, in the maturating oocyte suggest that the increase in prorenin expression in the ovary can contribute to the rise in renin levels in the follicular fluid. Moreover, prorenin can activate the prorenin/renin receptor ((P)RR) and thus become active as well as stimulate Ang‐II‐independent pathways, which are associated to this receptor [17]. For instance, binding of prorenin to the (P)RR can promote cell growth and oocyte maturation [18]. More specifically, the (P)RR has recently been suggested to induce resumption of meiosis in oocytes [19].

Similarly to prorenin, local ovarian renin activity has been shown to be increased following the LH surge in rats, rabbits and human [20–22]. Moreover, increased renin mRNA expression has been measured in rat and primate following follicle‐stimulating hormone (FSH), estradiol or human chorionic gonadotropin (hCG) stimulation [23], suggesting that prorenin could be activated locally in the ovary and could contribute to the stimulation of the local RAS [15].

The ovarian expression of angiotensinogen (Agt) has been studied in rats and humans and has been shown to vary between species. In rat, Agt expression is found in ovaries, more spe‐ cifically during the mid‐ and late‐maturation of follicles (not during maturation of early‐pri‐ mary or primary follicles) [24]. The timing of Agt expression in maturing follicles matches the expression of gonadotropins. As such, given that Agt expression has been shown to be stimu‐ lated by estradiol in rat liver, it has been suggested that Agt expression in maturating follicles could be driven by gonadotropin‐stimulated‐estradiol local production. In humans, Agt has been measured in the follicular fluid and its levels are comparable or lower to circulating Agt [7]. However, there is no evidence of local ovarian Agt mRNA expression, suggesting that local ovarian Agt protein levels are derived from the circulation [15].

In contrast to the other RAS components mentioned above, the angiotensin‐converting enzyme (ACE) expression in the ovary does not follow gonadotropin‐stimulated cyclic expression pattern during the oestrous cycle since high ACE levels are found in the early stages of follicle maturation and in atretic follicles with very low levels in preovulatory follicles. This suggests that ACE has a role in early maturation of the follicles as well as their atresia [15].

Angiotensin II (Ang II) has been found to be produced and secreted by rabbit and rat ovaries in response to hCG elevation [21]. Since renin activity is stimulated by gonadotropins dur‐ ing preovulation, this increased renin activity probably drives the production of local Ang II. Similar observations have been made in women with natural or gonadotropin‐stimulated cycles [25].

Ang II mediates its actions in the ovary through both AT1R and AT2R. However, each recep‐ tor has different functions within the reproductive system. Indeed, AT1R has been reported to be mainly involved in the maintenance of ovarian vasculature which supplies nutrients to the developing follicles [26], whereas AT2R would be implicated in both the follicular develop‐ ment as well as in the regression of the luteal vasculature towards the end of the ovarian cycle. However, the timing of AT2R expression during oocyte maturation is uncertain and varies between species. Indeed, a study using autoradiography and gene expression measurements reported the expression of AT2R in granulosa cells of rat atretic follicles while it is almost absent in healthy follicles [27]. In contrast, studies in bovine ovaries demonstrate that AT2R expression is increased during follicular growth and maturation [15]. As such, it is very dif‐ ficult to conclude on a clear role of the ATRs in the ovary. In addition, the signalling pathways involved in AT2R modulation of follicular growth and maturation have not yet been studied. However, neuronal studies of AT2R signalling demonstrate that the MAPK pathway and acti‐ vation of nitric oxide promotes cell differentiation and could be putative pathways involved in follicular maturation in the ovary [28]. On the other hand, studies in rabbits have shown that ovarian RAS activation leads to estradiol production through AT2R stimulation. Based on the fact that gonadotropins stimulate the expression of many components of the RAS cas‐ cade, an intra‐ovarian paracrine or autocrine loop would exist between Ang II and estradiol [15]. However, the mechanisms responsible for the control of the autocrine loop are not well understood and more data are needed to confirm its activity in other species such as rodents and humans.

### **2.2. RAS during ovulation**

the reproductive system RAS has been shown to be implicated in different aspects of repro‐ duction, from fertility to embryo implantation and later through pregnancy [9, 10]. Important modulations of the RAS are observed from the very beginning of pregnancy and aberrant changes in RAS component expression can cause gestational problems such as preeclampsia [11–13]. The implication of the RAS in both normal and pathological pregnancy will be dis‐

Prorenin is produced by the ovarian follicular cells at different stages in oocyte matura‐ tion. As the ovarian follicle undergoes maturation, the prorenin concentration increases and remains elevated until the end of the luteal phase, near the start of menstruation, where it falls in parallel with progesterone levels [14]. Prorenin secretion in the ovary is regulated by gonadotropins, and thus, the rise in plasma‐luteinizing hormone (LH) levels shortly precedes the elevation of plasma prorenin, secreted into circulation mainly by the ovary [15, 16]. Of note, concentrations of prorenin, the inactive precursor of renin, are typically higher in the reproductive system than those of renin and it was originally postulated that it was locally activated by an unknown process. As such, studies demonstrating the expression of cathep‐ sin B, a potential activator of prorenin, in the maturating oocyte suggest that the increase in prorenin expression in the ovary can contribute to the rise in renin levels in the follicular fluid. Moreover, prorenin can activate the prorenin/renin receptor ((P)RR) and thus become active as well as stimulate Ang‐II‐independent pathways, which are associated to this receptor [17]. For instance, binding of prorenin to the (P)RR can promote cell growth and oocyte maturation [18]. More specifically, the (P)RR has recently been suggested to induce resumption of meiosis

Similarly to prorenin, local ovarian renin activity has been shown to be increased following the LH surge in rats, rabbits and human [20–22]. Moreover, increased renin mRNA expression has been measured in rat and primate following follicle‐stimulating hormone (FSH), estradiol or human chorionic gonadotropin (hCG) stimulation [23], suggesting that prorenin could be activated locally in the ovary and could contribute to the stimulation of the local RAS [15].

The ovarian expression of angiotensinogen (Agt) has been studied in rats and humans and has been shown to vary between species. In rat, Agt expression is found in ovaries, more spe‐ cifically during the mid‐ and late‐maturation of follicles (not during maturation of early‐pri‐ mary or primary follicles) [24]. The timing of Agt expression in maturing follicles matches the expression of gonadotropins. As such, given that Agt expression has been shown to be stimu‐ lated by estradiol in rat liver, it has been suggested that Agt expression in maturating follicles could be driven by gonadotropin‐stimulated‐estradiol local production. In humans, Agt has been measured in the follicular fluid and its levels are comparable or lower to circulating Agt [7]. However, there is no evidence of local ovarian Agt mRNA expression, suggesting that

local ovarian Agt protein levels are derived from the circulation [15].

cussed in this book chapter.

86 Renin-Angiotensin System - Past, Present and Future

in oocytes [19].

**2. RAS in the reproductive system**

**2.1. RAS and ovary and follicular development**

The process of ovulation depends on different signalling cascades involving cAMP release, steroids, prostaglandins and other chemical mediators [29, 30]. Several *in vitro* and *in vivo* studies have demonstrated that the RAS, especially through AT2R stimulation, has a role to play in ovulation. In particular, studies using *in vitro* perfused ovaries have demonstrated a dose‐dependent effect of Ang II on estradiol and prostaglandin secretion, correlating with the initiation of ovulation [31]. Therefore, the use of ACE inhibitors (which would lead to a decrease in Ang II production) for the treatment of hypertension in women who want to become pregnant may not be recommended. Of note, insulin‐like growth factor 1 (IGF‐1), through the activation of the plasminogen activator (PA), has been proposed to increase Ang II production, leading to the production of prostaglandins necessary for the rupture of the follicular wall and ovulation [32]. Hence, this could be a mechanism by which the IGF‐1 pro‐ duces its important effects on ovarian physiology and follicle development [33].

Studies on human follicular fluid samples collected from *in vitro* fertilization samples suggest that RAS activity correlates with follicular development. In particular, prorenin activity in follicular fluid is associated with the development, maturity and viability of the oocytes [18]. Indeed, low levels of follicular prorenin are associated with immature follicles while high pro‐ renin levels are correlated with atretic follicles, the latter being characterized by high levels of testosterone and low levels of estradiol. Intermediate levels of prorenin would therefore be necessary for normal ovulation to proceed. Interestingly, in our recently characterized model of preeclampsia superimposed on chronic hypertension, mice that overexpress both human renin and angiotensinogen (R+ A+ ), we observed that these mice have reduced litter size [34]. Given that this is not associated with increased foetal or neonatal mortality, this suggests that hypertension or the overexpression of the RAS in the reproductive system may decrease fer‐ tility by modulating ovulation or embryo implantation.

### **2.3. Corpus luteum**

Following ovulation, the remaining follicular cells undergo rapid remodelling and capil‐ lary invasion. Studies have shown that microvascular endothelial (MVE) cells in the corpus luteum express ACE and can convert Ang I to Ang II [26]. Both AT1R and AT2R have been detected in MVE cells with different levels of expression throughout the ovarian cycle: AT1R expression levels seem unchanged, whereas AT2R expression is lowest during the mid‐luteal phase and highest during the late luteal phase [26]. The regulation of angiogenic processes is a crucial step to ensure the constant flow of growth, maturation and demise of the corpus luteum. This angiogenic step requires the secretion of angiogenic factors such as the basic fibroblast growth factor (bFGF). Ang II would be one of the drivers of this rapid capillary invasion through AT1R‐dependent stimulation of bFGF expression. Hence, in luteal cells, the surge in LH that precedes ovulation would lead to increased Ang II production and enhanced AT1R stimulation which would drive the expression of bFGF. This would then promote angiogenesis and appropriate maintenance of the corpus luteum [35]. In contrast, the regression of the luteal vasculature would be attributed to the Ang II‐AT2R axis of the RAS [36].

### **2.4. Atresia**

At the beginning of each ovarian cycle, several primordial (immature) follicles undergo mat‐ uration. Due to the inefficient nature of folliculogenesis, most of those primordial follicles will not reach the final stage of maturation, and in humans, only one follicle will undergo ovulation. The remaining follicles degenerate through a process known as atresia. Atretic fol‐ licles are characterized by abnormally high prorenin levels associated with a low estradiol/ progesterone ratio [37]. These follicles have a thin layer of degenerated granulosa cells and the remaining active theca cells secrete prorenin [38]. In atretic granulosa cells, the Ang II receptor isoform that is most expressed is AT2R, which has been shown to drive apoptosis [27]. In follicles, FSH acts as a mild repressor of AT2R expression, so apoptosis cannot be trig‐ gered during the maturation phase of follicular development. However, in the luteal phase, FSH levels are reduced which relieves the inhibition on AT2R expression. As such, given the high Ang II level, AT2 stimulation increases granulosa cells apoptosis, promoting the atresia of immature follicles.

### **2.5. RAS and the placenta**

II production, leading to the production of prostaglandins necessary for the rupture of the follicular wall and ovulation [32]. Hence, this could be a mechanism by which the IGF‐1 pro‐

Studies on human follicular fluid samples collected from *in vitro* fertilization samples suggest that RAS activity correlates with follicular development. In particular, prorenin activity in follicular fluid is associated with the development, maturity and viability of the oocytes [18]. Indeed, low levels of follicular prorenin are associated with immature follicles while high pro‐ renin levels are correlated with atretic follicles, the latter being characterized by high levels of testosterone and low levels of estradiol. Intermediate levels of prorenin would therefore be necessary for normal ovulation to proceed. Interestingly, in our recently characterized model of preeclampsia superimposed on chronic hypertension, mice that overexpress both human

Given that this is not associated with increased foetal or neonatal mortality, this suggests that hypertension or the overexpression of the RAS in the reproductive system may decrease fer‐

Following ovulation, the remaining follicular cells undergo rapid remodelling and capil‐ lary invasion. Studies have shown that microvascular endothelial (MVE) cells in the corpus luteum express ACE and can convert Ang I to Ang II [26]. Both AT1R and AT2R have been detected in MVE cells with different levels of expression throughout the ovarian cycle: AT1R expression levels seem unchanged, whereas AT2R expression is lowest during the mid‐luteal phase and highest during the late luteal phase [26]. The regulation of angiogenic processes is a crucial step to ensure the constant flow of growth, maturation and demise of the corpus luteum. This angiogenic step requires the secretion of angiogenic factors such as the basic fibroblast growth factor (bFGF). Ang II would be one of the drivers of this rapid capillary invasion through AT1R‐dependent stimulation of bFGF expression. Hence, in luteal cells, the surge in LH that precedes ovulation would lead to increased Ang II production and enhanced AT1R stimulation which would drive the expression of bFGF. This would then promote angiogenesis and appropriate maintenance of the corpus luteum [35]. In contrast, the regression of the luteal vasculature would be attributed to the Ang II‐AT2R axis of the

At the beginning of each ovarian cycle, several primordial (immature) follicles undergo mat‐ uration. Due to the inefficient nature of folliculogenesis, most of those primordial follicles will not reach the final stage of maturation, and in humans, only one follicle will undergo ovulation. The remaining follicles degenerate through a process known as atresia. Atretic fol‐ licles are characterized by abnormally high prorenin levels associated with a low estradiol/ progesterone ratio [37]. These follicles have a thin layer of degenerated granulosa cells and the remaining active theca cells secrete prorenin [38]. In atretic granulosa cells, the Ang II receptor isoform that is most expressed is AT2R, which has been shown to drive apoptosis

), we observed that these mice have reduced litter size [34].

duces its important effects on ovarian physiology and follicle development [33].

A+

tility by modulating ovulation or embryo implantation.

renin and angiotensinogen (R+

88 Renin-Angiotensin System - Past, Present and Future

**2.3. Corpus luteum**

RAS [36].

**2.4. Atresia**

The placenta is an organ that provides nutrients and oxygen to the developing foetus and removes toxic waste products from the foetal circulation [39]. The formation of the pla‐ centa starts with the implantation of the embryo (at this developmental stage, the blas‐ tocyst) in the endometrium (known as the decidua during pregnancy). The blastocyst is composed of an inner cell mass (which will give rise to the foetus and the amniotic cavity) and the trophoblastic cells (a 'sticky' layer of cells forming the outer layer of the blastocyst). Implantation is initiated when the trophoblastic cells adhere to the surface of the decidua. This stimulates the proliferation of the trophoblastic cells, which divide into two cell types: the syncytial trophoblasts and cellular trophoblasts (also known as the chorion). The syn‐ cytial trophoblastic cells are multinucleated cells which are highly invasive. They secrete proteolytic enzymes that are responsible for the destruction of the decidua which creates cavities (known as endometrial lacunae). Simultaneously, the proliferating trophoblastic cells form protrusions, known as the chorionic villi, which become highly branched as well as vascularised by ramifications of the umbilical vein and artery. The endometrial lacu‐ nae will then be invaded by the branching chorionic villi, allowing the blastocyst to pen‐ etrate into the decidua and establishing the interface between the maternal and foetal blood where nutrients, blood gas and wastes will be exchanged. By the end of the first trimester, the uteroplacental circulation is fully established [40]. Maintaining optimal placental blood osmotic pressure and flow is crucial for the production of a viable offspring. Placental RAS is a key player in the regulation of maternal‐foetal blood flow during pregnancy [41]. Since many components of the RAS have been shown to be expressed in whole human placental extracts, human placental cell lines (human umbilical venous endothelial cells (HUVEC)), and in isolated primary placental cell fractions (primary trophoblastic cells fraction, pri‐ mary macrophage‐rich fraction and primary villous endothelial cells) [42–44], the RAS is believed to have a considerable influence in this organ [11, 45–48]. However, functional data of the placental RAS are very rare. RAS proteins have different level of expression in various areas of the placenta. Agt, renin, Ang I, Ang II, ACE, AT1R, and AT2R have been localized to the human and rat maternal decidua [49, 50], whereas Ang II and ACE have also been found in pericytes of endometrial spiral arteries. RAS components such as Agt and renin have also been detected in foetal capillaries [51] and AT1R has been found in cytotrophoblastic and syncytiotrophoblastic cells as well as in foetal capillaries. Many stud‐ ies have suggested the implication of the placental RAS in promoting trophoblastic cell migration, proliferation of the foetal vascular endothelium and vasodilation of the maternal vasculature [52, 53]. Hence, changes in placental RAS potentially contribute to alterations in uteroplacental perfusion, which are associated with gestational complications such as preeclampsia [54].

### **2.6. RAS and the uterus/endometrium**

Most components of the RAS can be found in both myometrium and endometrium of the uterus. However, the role of the RAS in the non‐pregnant uterus is still unknown [55]. Elevated expression and secretion of prorenin in stromal cells have been associated with decidualisation of the endometrium in early to mid‐proliferative phase [56]. Activation of the (P)RR by prorenin has been shown to promote vascular endothelial growth factor (VEGF) expression and could thus increase vascularity of the decidua to ensure an adequate blood flow to the placenta [56]. In addition, Ang II as well as AT1R and AT2R show a cyclical pattern of expression depending on the phase of the uterine cycle. First, AT2R is expressed at higher levels compared to AT1R, although both receptors show a similar expression pattern. Their expression gradually increases during the proliferative phase, reaching a maximum in late proliferative and early secretory phases, followed by a gradual decrease in expression through the rest of the secretory phase [57]. In comparison, plasma Ang II levels gradually increase through the menstrual cycle, reach‐ ing a peak in the late secretory phase [58]. Moreover, in the early to mid‐proliferative phase, endometrial Ang II levels and ATRs expression are mostly localized to the glandular and stro‐ mal cells of the endometrium, which could highlight a role for the RAS in modulating decidu‐ alisation and neovascularisation of the endometrium. Alternatively, in late secretory phase, they are localized mostly around blood vessels, where Ang II could contribute to the vasoconstriction of spiral arterioles which is necessary for the induction of menstruation [57]. In addition, angio‐ tensin‐(1‐7) (Ang‐(1‐7), a heptapeptide generated from Ang II cleavage by the enzyme ACE 2) and its receptor MAS (MAS‐R) have been shown to be expressed in the endometrium. While MAS‐R expression is localized to the epithelial and stromal cells and does not change through‐ out the menstrual cycle, Ang‐(1‐7) concentrations are highest in the glandular epithelium and in the stroma of the endometrium in mid‐ to late‐secretory phase [59]. Although the function of the Ang‐(1‐7)—Mas‐R axis is not well understood in the endometrium, by its vasodilatory, antiangiogenic and antimitotic properties, Ang‐(1‐7) could counterbalance Ang II actions and, possibly regulate endometrial regenerating processes according to homeostatic needs.

### **3. Pregnancy and RAS**

Pregnancy is characterized by an elevation in the levels of maternal circulating estrogen. Consequently, maternal circulating prorenin and renin are also increased during pregnancy. Prorenin reaches a peak within 20 days after conception and remains high until parturition while plasma‐renin activity rises during the first few weeks of pregnancy [60]. ACE is the only RAS component that decreases during pregnancy [61] while plasma Agt and Ang II levels are particularly elevated during the last trimester of normal gestation [62]. The elevated Ang II levels could be attributed in part to the stimulatory effect of estrogen on Agt expression but also to the elevated renin levels [63]. In addition, increased urinary and plasma aldosterone levels are observed during pregnancy which produces the increased plasma volume required for the growing placenta and foetus [64].

The increase in RAS in pregnant women should normally be associated with an increase in blood pressure. However, elevated blood pressure in not typically observed during normal pregnancy. On the contrary, due to the vasodilating effect of progesterone, a decrease in blood pressure is typically seen in the first and second trimesters, returning to baseline by deliv‐ ery [65]. Indeed, although Ang II levels are increased during pregnancy, normotensive preg‐ nant women are actually refractory to its vasopressor effects. Studies have reported a twofold increase in plasma Ang II levels concomitantly with a twofold decrease in the sensitivity to Ang II vasoconstrictive effects [66, 67]. Moreover, studies in pregnant women and animals have demonstrated that the elevation of plasma Ang‐(1‐7) would contribute to the reduction in blood pressure during pregnancy by counterbalancing the vasoconstrictor actions of elevated Ang II [68–70]. It was also demonstrated in rats, that arteries were more responsive to the vasodilatory effects of Ang‐(1‐7) during pregnancy [71]. The capacity of Ang‐(1‐7) to stimulate the release of the vasodilatory molecules prostaglandins would potentiate its own vasodilatory actions and would oppose Ang II effects [72]. A balance of the two biologically active peptides of the RAS, Ang II, a vasoconstrictor and angiogenic molecule, and Ang‐(1‐7), a vasodilator and anti‐angio‐ genic molecule, may therefore be essential for the maintenance of normal pregnancy [11, 73].

Trophoblasts are rich in AT1Rs and are thus responsive to the changes in Ang II concentra‐ tions that occur during pregnancy [74]. Recent studies demonstrate that multiple genes are regulated by AT1R signalling and include those encoding secreted proteins associated with trophoblast invasion (e.g., plasminogen activator inhibitor‐1, PAI‐I) and angiogenesis (soluble fms‐like tyrosine receptor‐1, sFlt‐1) which could promote endometrium decidualisation. Ang II signalling also activates NF‐kappa B and stimulates NADPH‐oxidase synthesis by tropho‐ blasts which would promote trophoblastic proliferation and invasiveness [75].
