Virginia M. Pereira

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.

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) 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.

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.

Regulation of steroidogenesis by atrial natriuretic peptide (ANP) in the rat testis: Differential involvement of GC-A and C receptors

Previous studies have established a stimulatory effect of natriuretic peptides (NP) on testosterone production in mouse Leydig cells as intense as that of LH. Chronic administration of ANP in mice, on the other side, reduced testosterone levels. So, the understanding of the role of ANP on testicular steroidogenesis has been impaired by discrepant findings. The aim of the present study was to clarify the physiological role of ANP in the rat testis steroidogenesis using a model that preserves the interactions between testis cells and a medium devoid of any circulating factors that could interfere with testosterone production. First, ANP was immunolocalized in the interstitial compartment of the rat testis, mainly in Leydig cells. We also determined the presence of ANP and both GC-A (guanylyl cyclase A) and C receptors by real-time PCR in testis. Perfusion in vitro of testis with ANP (1 and 3x10(-7)M) stimulated testosterone production in a time- and dose-dependent manner. On the other side, testosterone secretion induced by LH was blunted by ANP. Similar effect was obtained using the specific C receptor ligand, cANF, indicating the involvement of C receptor in such response. In conclusion, ANP stimulated testosterone production in the rat testis perfused in vitro but decreased testosterone production LH-induced, effect that seems to involve C receptor. To this extent, our results suggest the existence of a local and complex peptidergic system in the rat testis, involving ANP and its receptors that could importantly modulate the androgen biosynthesis.

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.

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 μg 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.