**2. Role in follicular microenvironment and ovulatory capacity**

Oocyte and follicle development start during the fetal stage. Initially, the primordial germ cells migrate from the endoderm of the embryonic yolk sac to the gonadal ridge, where during migration the cells undergo mitotic divisions. In the gonadal ridge, cells are internalized and cease mitotic division. After being enclosed in ovigerous cords, the cells become referred to as oogonia. The oogonia present the onset of meiosis but are interrupted in prophase I, the moment that the chromosomes are decondensed and contained in the germinal vesicle. One layer of flattened epithelial cells (pre-granulosa cells) is formed around the oogonia, becoming a primordial follicle, and when the pre-granulosa cells become cuboidal granulosa cells (primary follicle), follicular growth begins, with the proliferation of granulosa cells turning into a secondary follicle (two to six layers), and later an antral follicle (more than six layers) (reviewed for [1, 2]).

The presence of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, and ANG II and ANG II receptors (AT1 and AT2 receptors) in the ovary is suggestive of a functional ovarian RAS. In cattle, the expression of ANG II is greatest in large follicles, suggesting that it is important during follicular growth and maturation [9]. Within the ovarian follicle, there appear to be species differences in the precise pattern of localization of AGTR2 protein. Infusion of ANG II induced ovulation in rabbits and the use of ANG II antagonists inhibited ovulation in rabbits, rats, and cattle [3–7].

ANG II acts through two distinct transmembrane receptors, namely AT1 (encoded by the AGTR1 gene) and AT2 (encoded by the AGTR2 gene [8]). In rabbits, the receptors are mostly AT2 receptors and are expressed in the granulosa cells of preovulatory follicles, consistent with the role of ANG II in ovulation. A similar role has been suggested in cattle by [7], who observed that AGTR2 mRNA in bovine granulosa cells was more abundant in healthy compared with atretic follicles.

Regarding the effects on oocyte maturation, Giometti et al. [9] investigated the role of ANG II in bovine oocyte nuclear maturation and suggested a role of ANG II in blocking the inhibitory effect of theca cells on nuclear maturation of bovine oocytes. Moreover, Barreta et al. [10] found strong evidence that ANG II mediates the resumption of meiosis induced by a luteinizing hormone (LH) surge in bovine oocytes, probably through the effects of prostaglandins produced by follicular cells. Recently, Siqueira et al. [11, 12] suggested that progesterone is also involved in oocyte meiotic resumption induced by the gonadotropin surge in cattle.

Furthermore, some reproductive biotechnologies, such as ovarian hyperstimulation, seem to affect ANG II in the ovaries. Numerous treatment protocols to induce multiple ovulations in cattle, using different gonadotropins, doses, routes of administration, and various hormone combinations and schedules, have been proposed in an attempt to improve embryo yield [13–16]. Recently, Barros et al. [17] showed higher levels of AGTR2 mRNA in cows submitted to ovarian hyperstimulation using FSH. These findings substantiate those of [7], who observed increases in AT2 receptor mRNA and protein levels after adding FSH to granulosa cell culture.

cells *in vivo* and *in vitro*. Moreover, the presence of this system in bovine, caprine and procine fetal ovaries ovaries suggests a role in preantral follicle development. In addition, in female germ cells, ANG II plays a key role in the oocytes during in vitro maturation in porcine and Cattle. In male reproduction as an important role in spermatogenesis to guarantee fertilization. Aiming to clarify the localization, role, and practical implications of the renin-angiotensin system (RAS) in male and female reproductive biology, this chapter highlights the roles of RAS in mammalian reproductive physiology, specifically, in the ovaries, testes, oviducts, and

Oocyte and follicle development start during the fetal stage. Initially, the primordial germ cells migrate from the endoderm of the embryonic yolk sac to the gonadal ridge, where during migration the cells undergo mitotic divisions. In the gonadal ridge, cells are internalized and cease mitotic division. After being enclosed in ovigerous cords, the cells become referred to as oogonia. The oogonia present the onset of meiosis but are interrupted in prophase I, the moment that the chromosomes are decondensed and contained in the germinal vesicle. One layer of flattened epithelial cells (pre-granulosa cells) is formed around the oogonia, becoming a primordial follicle, and when the pre-granulosa cells become cuboidal granulosa cells (primary follicle), follicular growth begins, with the proliferation of granulosa cells turning into a secondary follicle (two to six layers), and later an antral follicle (more than six layers)

The presence of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, and ANG II and ANG II receptors (AT1 and AT2 receptors) in the ovary is suggestive of a functional ovarian RAS. In cattle, the expression of ANG II is greatest in large follicles, suggesting that it is important during follicular growth and maturation [9]. Within the ovarian follicle, there appear to be species differences in the precise pattern of localization of AGTR2 protein. Infusion of ANG II induced ovulation in rabbits and the use of ANG II antagonists inhibited

ANG II acts through two distinct transmembrane receptors, namely AT1 (encoded by the AGTR1 gene) and AT2 (encoded by the AGTR2 gene [8]). In rabbits, the receptors are mostly AT2 receptors and are expressed in the granulosa cells of preovulatory follicles, consistent with the role of ANG II in ovulation. A similar role has been suggested in cattle by [7], who observed that AGTR2 mRNA in bovine granulosa cells was more abundant in healthy compared with atretic follicles. Regarding the effects on oocyte maturation, Giometti et al. [9] investigated the role of ANG II in bovine oocyte nuclear maturation and suggested a role of ANG II in blocking the inhibitory effect of theca cells on nuclear maturation of bovine oocytes. Moreover, Barreta et al. [10] found strong evidence that ANG II mediates the resumption of meiosis induced by a luteinizing hormone (LH) surge in bovine oocytes, probably through the effects of prostaglandins produced by follicular cells. Recently, Siqueira et al. [11, 12] suggested that progesterone is also involved in oocyte meiotic resumption induced by the gonadotropin surge in cattle.

**2. Role in follicular microenvironment and ovulatory capacity**

other reproductive tissues.

72 Renin-Angiotensin System - Past, Present and Future

(reviewed for [1, 2]).

ovulation in rabbits, rats, and cattle [3–7].

Although the focus of RAS is on antral follicle development and oocyte competence, RAS has also been detected in fetal ovaries; however, not much is known about the regulation of development of pre-antral follicles. ANG II could be one of the factors that activate oogonia and oocytes. Embryologically, ovarian development in mammals originates from the nephrogenic ridge, as well as fetal kidney [18], where ANG II plays a role in kidney development [19]. Renin was identified in pig and mouse mesonephros [18]. It is believed that mesonephric cells are the precursors of granulosa cells (reviewed for [1]).

ANG II is produced in fetal porcine ovaries, as well as other components of RAS (prorenin, angiotensin, AT1, and AT2 receptors) required for the production and action of ANG II. These components are present at about 45 days of gestation. The abundance of mRNA prorenin increases until day 90 and then stabilizes [20].

In early gestation in porcines, there is the presence of AT2 mRNA but the abundance decreases with the progress of gestation, unlike AT1 mRNA that remains stable during gestation. In addition, the protein of the receptors appears alternately; AT1 receptor is present throughout gestation but the amount decreases during evolution while AT2 appears steadily [20].

Still in porcines, proteins of the receptors are present in the epithelial surface with a predominance of AT1 receptors, in primordial germ cells [20, 21], granulosa cells of primordial, primary, and secondary follicles, and also in oocytes (except for oocytes of secondary follicles). However, proteins of the receptors are not present in theca or stroma cells [20].

In cattle, there is only one study confirming the presence of AT2 in fetal ovaries. Protein was detected in the cytoplasm of oogonia up to 60 days of gestation, becoming weak and unstable from day 75 of gestation. The AT2 protein appears again from day 210 in granulosa cells of primary and secondary follicles, and granulosa and theca cells of antral follicles. The mRNA AT2 abundance does not change throughout gestation [22].

The difference in the expression pattern of the protein and AT2 mRNA mentioned above is easily explained by the differences in follicular development between the species. The primary follicles appear earlier in bovines than in porcines, but in bovines the gestation is approximately three times longer than in porcines [22]. In caprine pre-antral ovaries, a high expression of AT1 and AT2 has been demonstrated in primordial follicles. It was also expressed in secondary follicles, but at a lower level [23].

Despite the presence of RAS components in fetal ovaries, the function of the system is not well understood. In pre-antral follicles, ANG II is associated with conserving follicular viability through binding to its receptors [23], and seems to be related to cellular atresia by binding to AT2 [20]. Furthermore, when porcine pre-antral follicles are cultured in a long-term culture system with the addition of ANG II, it seems to stimulate the division of granulosa cells and steroid synthesis [24].
