Angiotensin-(1-7), Angiotensin-Converting Enzyme 2 and Mas Receptor in Rat Polycystic Ovaries.

BACKGROUND: Hyperandrogenism is a pivotal mediator in the pathogenesis of the polycystic ovary syndrome (PCOS), but the mechanisms of androgen excess in this condition are not fully understood. Angiotensin (Ang)-(1-7) is an active peptide of the renin-angiotensin system (RAS) that stimulates ovarian follicular growth and testosterone release in vitro. OBJECTIVE: To investigate whether Ang-(1-7), its receptor Mas and angiotensin-converting enzyme 2 (ACE2), the enzyme that converts Ang II into Ang-(1-7), are expressed in rat polycystic ovaries (PCO) and thus if this peptide system might be associated with excess androgen production in PCO. METHODS: A rat model that shares some features of PCOS such as disruption of folliculogenesis and multiple ovarian cyst formation was used in the study. RESULTS: We found reduced levels of Ang-(1-7) and Mas receptor in PCO compared to normal ovaries. Also, ACE2 mRNA expression was reduced in PCO compared to ovaries of control rats (p < 0.05). PCO had high levels of estrogen and testosterone and increased mRNA for upstream enzymes of the steroidogenic cascade, but not of P450 aromatase. CONCLUSION: These findings suggest that the ovarian ACE2-Ang-(1-7)-Mas receptor axis is inhibited and therefore may not be a co-factor of excess testosterone production in rat PCO.

Localization of angiotensin-(1-7) and Mas receptor in the rat ovary throughout the estrous cycle.

We have previously demonstrated the presence of Angiotensin (Ang)-(1-7) in rat ovary homogenates and its stimulatory effect on estradiol and progesterone production. The present study was undertaken to identify the cellular localization of Ang-(1-7) and its receptor Mas in the rat ovary in the different phases of the estrous cycle. Ang-(1-7) and Mas were localized by immunohistochemistry and Mas mRNA expression was assessed by RT-PCR. Immunostaining for both Ang-(1-7) and Mas was found in all phases of the estrous cycle, particularly in the thecal and interstitial cells, as well as in regressing corpora lutea. However, granulosa cells were positive only in antral and preovulatory follicles at proestrus and estrus phases. This pattern contrasted with the distribution of the octapeptide Ang II, which was abundant in granulosa but not in theca cells. In addition, the expression of Mas mRNA was demonstrated in all estrous cycle phases. Angiotensin-converting enzyme activity did not vary between estrous cycle phases, whereas prolyl endopeptidase activity was significantly higher in diestrus and neutral endopeptidase activity was significantly higher in metestrus. These data provide the first evidence that new RAS components are dynamically expressed in the ovary across the rat estrous cycle. Further functional studies should clarify the role of Ang-(1-7) signaling through Mas receptor in the regulation of ovarian physiology.

Angiotensin peptides in the non-gravid uterus: Paracrine actions beyond circulation.

The renin-angiotensin system (RAS) involves a complex network of precursors, peptides, enzymes and receptors comprising a systemic (endocrine) and a local (paracrine/autocrine) system. The local RAS plays important roles in tissue modulation and may operate independently of or in close interaction with the circulatory RAS, acting in a complementary fashion. Angiotensin (Ang) II, its receptor AT1 and Ang-(1-7) expression in the endometrium vary with menstrual cycle, and stromal cell decidualization in vitro is accompanied by local synthesis of angiotensinogen and prorenin. Mas receptor is unlikely to undergo marked changes accompanying the cyclic ovarian steroid hormone fluctuations. Studies investigating the functional relevance of the RAS in the non-gravid uterus show a number of paracrine effects beyond circulation and suggest that RAS peptides may be involved in the pathophysiology of proliferative and fibrotic diseases. Endometrial cancer is associated with increased expression of Ang II, Ang-converting enzyme 1 and AT1 in the tumoral tissue compared to neighboring non-neoplastic endometrium, and also with a gene polymorphism that enhances AT1 signal. Ang II induces human endometrial cells to transdifferentiate into cells with myofibroblast phenotype and to synthetize extracellular matrix components that might contribute to endometrial fibrosis. Altogether, these findings point to a fully operating RAS within the uterus, but since many concepts rely on preliminary evidence further studies are needed to clarify the role of the local RAS in uterine physiology and pathophysiology.

Downregulation of natriuretic peptide system and increased steroidogenesis in rat polycystic ovary.

Atrial natriuretic peptide (ANP) is known to regulate ovarian functions, such as follicular growth and steroid hormone production. The aim of the present study was to investigate the natriuretic peptide system in a rat model of chronic anovulation, the rat polycystic ovary. Adult female Wistar rats received a single subcutaneous injection of 2mg estradiol valerate to induce polycystic ovaries, while the control group received vehicle injection. Two months later, their ovaries were quickly removed and analyzed. Polycystic ovaries exhibited marked elevation of testosterone and estradiol levels compared to control ovaries. The levels of ANP and the expression of ANP mRNA were highly reduced in the polycystic ovaries compared to controls. By immunohistochemistry, polycystic ovaries showed weaker ANP staining in stroma, theca cells and oocytes compared to controls. Polycystic ovaries also had increased activity of neutral endopeptidase, the main proteolytic enzyme that degrades natriuretic peptides. ANP receptor C mRNA was reduced and ANP binding to this receptor was absent in polycystic ovaries. Collectively, these results indicate a downregulation of the natriuretic peptide system in rat polycystic ovary, an established experimental model of anovulation with high ovarian testosterone and estradiol levels. Together with previous evidence demonstrating that ANP inhibits ovarian steroidogenesis, these findings suggest that low ovarian ANP levels may contribute to the abnormal steroid hormone balance in polycystic ovaries.

Evidence that angiotensin-(1-7) is an intermediate of gonadotrophin-induced oocyte maturation in the rat preovulatory follicle.

Several studies have shown the presence of components of the renin-angiotensin system in mammalian ovaries and their involvement in ovarian physiology. We have previously shown the presence of angiotensin-(1-7) [Ang-(1-7)], an important biologically active component of the renin-angiotensin system, and its receptor, Mas, in rat, rabbit and human ovaries. We have also shown the involvement of Ang-(1-7) in the rabbit ovulatory process in vitro. In the present study, we observed that Ang-(1-7) stimulated the resumption of meiosis in oocytes of rat preovulatory follicles, reaching more than 30% of oocytes with germinal vesicle breakdown. The specific antagonist of the Mas receptor, A-779, inhibited the germinal vesicle breakdown induced by Ang-(1-7) and reduced the oocyte maturation stimulated by luteinizing hormone (LH). Immunohistochemistry showed that LH increased both Ang-(1-7) and angiotensin-converting enzyme 2 (ACE2) staining in preovulatory follicles. The effect of gonadotrophins on mRNA expression of Mas and ACE2 in ovaries of immature equine chorionic gonadotrophin-primed rats was analysed by real-time PCR after 6 h of human chorionic gonadotrophin (hCG) injection, which exhibits LH-like effects. After hCG treatment, ACE2 mRNA expression was higher in the ovaries of treated rats than in the ovaries of control rats, whereas Mas mRNA levels were unchanged. A-779 changed the steroidogenesis stimulated by LH. An increased testosterone concentration and decreased progesterone levels were measured in the follicle medium. In conclusion, our results suggest that LH upregulates the ACE2-Ang-(1-7)-Mas axis and that Ang-(1-7) promotes meiotic resumption, possibly as a gonadotrophin intermediate.

Angiotensin-(1-7) induces ovulation and steroidogenesis in perfused rabbit ovaries.

A local renin-angiotensin system has been described in several organs, including the ovary; however, data indicating a role for angiotensin II in the induction of ovulation are controversial. We have previously shown the presence of a novel peptide, angiotensin-(1-7) [Ang-(1-7)], in the rat ovary and its effect on steroidogenesis. The objective of the present study was to determine whether Ang-(1-7) plays a role in ovulation. We first determined the presence and distribution of Ang-(1-7) and the receptor Mas in rabbit ovaries by immunohistochemistry. Angiotensin-(1-7) and Mas immunoreactivity were observed in interstitial cells and oocytes of immature ovaries. Immunoreactivity for Ang-(1-7) and Mas was also observed in theca and granulosa cells of preovulatory follicles in ovaries of gonadotrophin-stimulated rabbits. To verify the effect of Ang-(1-7) in ovulation and steroidogenesis, we used isolated ovaries from immature rabbits pretreated with equine chorionic gonadotrophin (50 i.u., 48 h before the experiment) and then perfused in vitro. The ovulatory efficiency was determined by the number of oocytes compared with the number of preovulatory follicles present in the ovary. Angiotensin-(1-7) stimulated oestradiol production and enhanced ovulatory efficiency, which was blocked by the specific Ang-(1-7) antagonist, A-779. Ovulation induced by human chorionic gonadotrophin was also antagonized by A-779. These results show, for the first time, the involvement of a novel regulatory peptide system, Ang-(1-7) and Mas, in the ovulatory process. More importantly, because A-779 antagonized hCG-induced ovulation, it may be inferred that Ang-(1-7) plays an important role in ovulation, possibly as a mediator of gonadotrophin action.

Angiotensin-(1-7), its receptor Mas, and the angiotensin-converting enzyme type 2 are expressed in the human ovary.

OBJECTIVE: To investigate whether angiotensin (Ang)-(1-7), its receptor Mas, and angiotensin-converting enzyme type 2 (ACE2) are present in human ovary. DESIGN: Cross-sectional study. SETTING: Academic hospital. PATIENT(S): Twelve reproductive-age women and five postmenopausal women undergoing oophorectomy for nonovarian diseases and seven women having controlled ovarian hyperstimulation for IVF. INTERVENTION(S): Ovarian tissue was obtained from the reproductive-age women and postmenopausal women undergoing oophorectomy for nonovarian diseases. Follicular fluid (FF) samples were obtained from the women having controlled ovarian hyperstimulation for IVF. MAIN OUTCOME MEASURE(S): Localization of Ang-(1-7) and Mas by immunohistochemistry; measurement of Ang-(1-7) in ovarian FF by RIA; detection of messenger RNAs encoding Mas and ACE2 with use of real-time polymerase chain reaction; assessment of 125I-labeled Ang-(1-7) binding to ovarian sections with use of autoradiographic binding assay. RESULT(S): Angiotensin-(1-7) and the receptor Mas were localized to primordial, primary, secondary, and antral follicles, stroma, and corpora lutea of reproductive-age ovaries. Postmenopausal women expressed both the peptide and its receptor in the ovarian stroma. Angiotensin-(1-7) was detectable in FF (mean±SE: 191±54 pg/mL). Both Mas and ACE2 messenger RNAs were expressed in ovarian tissue, as revealed by real-time polymerase chain reaction, and ovarian binding sites for 125I-labeled Ang-(1-7) were identified by autoradiography. CONCLUSION(S): Angiotensin-(1-7), its receptor Mas, and ACE2 are expressed in the human ovary. The peptide is present in several ovarian compartments and can be quantified in FF.

Angiotensin (1-7) and its receptor Mas are expressed in the human testis: implications for male infertility.

The presence of classical components of the renin-angiotensin system has been demonstrated in the male reproductive tract, mainly in the testes and epididymis. The objective of this study was to verify the localization of angiotensin (Ang)-(1-7) and its receptor Mas in human testis. The study included 12 men with previously proven fertility submitted to orchiectomy for prostate cancer and 20 infertile men submitted to testicular biopsy for infertility work-up, comprising a subgroup with obstructive azoospermia/normal spermatogenesis (n = 8) and another with non-obstructive azoospermia and severely impaired spermatogenesis (n = 12). Testicular tissue samples were processed by immunohistochemistry and real time polymerase chain reaction. Ang-(1-7) was strongly expressed in the interstitial compartment, mainly in Leydig cells, with similar intensity in all groups evaluated. The peptide was also detected in the seminiferous tubules, but with much less intensity compared to interstitial cells. The receptor Mas was equally distributed between interstitial and tubular compartments and was found in all layers of the normal seminiferous epithelium. However, neither Ang-(1-7) nor Mas were detected in the seminiferous tubules of samples with impaired spermatogenesis. The testicular samples of infertile men with impaired spermatogenesis (non-obstructive azoospermia) expressed Mas and ACE2 mRNA at lower concentrations (fold change = 0.06 and 0.04, respectively, P < 0.05) than samples with full spermatogenesis (obstructive azoospermia). This shows, for the first time, the immunolocalization of Ang-(1-7) and its receptor Mas in testes of fertile and infertile men, and suggests that this system may be altered when spermatogenesis is severely impaired.

Gonadotropin stimulation increases the expression of angiotensin-(1--7) and MAS receptor in the rat ovary.

We have previously shown the presence of immunoreactive angiotensin-(1-7) [Ang-(1-7)] in rat ovary homogenate and its stimulatory effect on estradiol and progesterone production in vitro. In the current study, we investigated the presence and cellular distribution of Ang-(1-7) and the Mas receptor, the expression of Mas and angiotensin-converting enzyme 2 (ACE2) messenger RNA (mRNA), and the enzymatic activity in the rat ovary following gonadotropin stimulation in vivo. Immature female Wistar rats (25 days old) were injected subcutaneously (SC) with equine chorionic gonadotropin (eCG, 20 IU in 0.2 mL) or vehicle 48 hours before euthanasia. Tissue distributions of Ang-(1-7), Mas receptor, and ACE2 were evaluated by immunohistochemistry, along with angiotensin II (Ang II) localization, while the mRNA expression levels of Mas receptor and ACE2 were evaluated by real-time polymerase chain reaction (PCR). In addition, we determined the activity of neutral endopeptidase (NEP), prolyl endopeptidase (PEP), and ACE by fluorometric assays. After eCG treatment, we found strong immunoreactivity for Ang-(1-7) and Mas primarily in the theca-interstitial cells, while Ang II appeared in the granulosa but not in the thecal layer. Equine chorionic gonadotropin treatment increased Mas and ACE2 mRNA expression compared with control animals (3.3- and 2.1-fold increase, respectively; P < .05). Angiotensin-converting enzyme and NEP activities were lower, while PEP activity was higher in the eCG-treated rats (P < .05). These data show gonadotropin-induced changes in the ovarian expression of Ang-(1-7), Mas receptor, and ACE2. These findings suggest that the renin-angiotensin system (RAS) branch formed by ACE2/Ang-(1-7)/Mas, fully expressed in the rat ovary and regulated by gonadotropic hormones, could play a role in the ovarian physiology.

Prolactin inhibits oocyte release after gonadotropin stimulation in the rat: putative mechanism involving ovarian production of beta-endorphin and prostaglandin.

OBJECTIVE: To evaluate whether prolactin (PRL) is able to inhibit ovulation induced with exogenous gonadotropins in the rat and whether this effect could be mediated by the ovarian production of beta-endorphin, prostaglandin, and nitric oxide (NO). DESIGN: Controlled in vivo and in vitro experiments. SETTING: Academic research laboratories. ANIMAL(S): Immature female rats undergoing ovulation induction with equine gonadotropins and hCG. INTERVENTION(S): Prolactin (100 or 200 microg), PRL + the opioid antagonist naloxone (200 microg each), or placebo were injected SC 4 hours after hCG administration for ovulation induction. In the in vitro experiments, isolated preovulatory ovaries were incubated with or without PRL in a final concentration of 100 or 200 ng/mL. MAIN OUTCOME MEASURES(S): Number of oocytes ovulated in vivo, ovarian beta-endorphin, PGE(2) and NO(2)(-)/NO(3)(-) release, and NO synthase activity in vitro. RESULT(S): Prolactin reduced significantly the number of oocytes ovulated at the doses of 100 and 200 microg, and this effect was partially reversed by naloxone administration together with 200 mug PRL. PRL also induced a twofold increase in the ovarian release of beta-endorphin and a threefold decrease in the ovarian production of PGE(2). Ovarian NO synthase activity and the concentrations of NO(2)(-)/NO(3)(-) in the incubation medium were not modified by PRL. CONCLUSION(S): Prolactin is able to reduce the number of oocytes released and modulate ovarian beta-endorphin and PGE(2) release, which may account for its peripheral anovulatory effects. This local effect of PRL could interfere in the process of ovulation induction by exogenous gonadotropins.

Effect of the pretreatment with prolactin on the distribution of immunoreactive beta-endorphin through different ovarian compartments in immature, superovulated rats.

Beta-endorphin and prolactin (PRL) are natural inhibitors of ovulation via central and peripheral mechanisms, but their possible interactions within the ovary are still unknown. The aims of the present study were to determine the gene expression and the topographic distribution of beta-endorphin, and the possible changes evoked by the pretreatment with PRL on the ovarian beta-endorphin localization in immature, superovulated rats. Prepuberal female Wistar rats weighing 60-70 g were superovulated with 20 IU equine gonadotrophins and, 48 h later, 20 IU human chorionic gonalotropin (hCG). Four hours after the hCG injection, the rats received either 200 microg rat PRL .i.p. (n = 12) or saline vehicle (n = 10). In the following morning the rats were killed and their ovaries were quickly removed. Beta-endorphin localization was assessed by immunohistochemistry and proopiomelanocortin (POMC) mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Beta-endorphin was expressed mostly in the corpora lutea and perivascular stroma, but a weak to moderate immunostaining was also present in the theca cells and some granulosa cells of tertiary/antral and preovulatory follicles. The main differences observed in the distribution of ovarian beta-endorphin between the two groups were a more intense immunostaining in the granulosa cells of antral follicles, corpus luteum and stroma of PRL-treated rats. POMC gene transcripts were detected in 2/5 samples from the control group and in 3/7 samples from the PRL-treated group. Thus, the expression of beta-endorphin in tertiary/antral follicles is enhanced by PRL treatment in immature, superovulated rats, providing a putative mechanism by which PRL could inhibit the ovarian response to induced ovulation.

Angiotensin-(1-7): a novel peptide in the ovary.

The present study was undertaken to investigate the presence of angiotensin-(1-7) [Ang-(1-7)] in the ovary and a possible role for it. Cycling female rats were killed in each phase of the estrous cycle, and ovarian Ang II and Ang-(1-7) were separated by HPLC and measured by RIA. The mean levels of Ang-(1-7) in proestrus and estrus were significantly higher than those in metestrus and diestrus (P < 0.05). Ang-(1-7) was also significantly higher in equine chorionic gonadotropin (eCG)-treated immature rats. Ang-(1-7) induced a significant increase in estradiol and progesterone production (P < 0.05) in the ovary of immature rats (24-25 d old) pretreated with eCG and perfused in a closed circuit system. This effect was blocked by A-779, a specific Ang-(1-7) antagonist (P < 0.05). The present data demonstrate the presence and physiological role of a novel renin-Ang system peptide in the ovary. The higher level of Ang-(1-7) in proestrus and estrus as well as in eCG-treated rats suggests the involvement of this renin-Ang system peptide in pre- and postovulatory events.