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.

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.

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.

Cancer is one of the leading causes of death worldwide. Although several drugs are used clinically, some tumors either do not respond or are resistant to the existing pharmacotherapy, thus justifying the search for new drugs. Ursolic acid (UA) is a triterpene found in different plant species that has been shown to possess significant antitumor activity. However, UA presents a low solubility in aqueous medium, which presents a barrier to its biological applications. In this context, the use of liposomes presents a promising strategy to deliver UA and allow for its intravenous administration. In this work, long-circulating and pH-sensitive liposomes containing UA (SpHL-UA) were developed, and their chemical and physicochemical properties were evaluated. SpHL-UA presented adequate properties, including a mean diameter of 191.1 ± 6.4 nm, a zeta potential of 1.2 ± 1.4 mV, and a UA entrapment of 0.77 ± 0.01 mg/mL. Moreover, this formulation showed a good stability after having been stored for 2 months at 4°C. The viability studies on breast (MDA-MB-231) and prostate (LNCaP) cancer cell lines demonstrated that SpHL-UA treatment significantly inhibited cancer cell proliferation. Therefore, the results of the present work suggest the applicability of SpHL-UA as a new and promising anticancer formulation.

Endurance exercise is a remarkable intervention for the treatment of many diseases. Mitochondrial changes on skeletal muscle are likely important for many of the benefits provided by exercise. In this study, we aimed to evaluate the effects that a regular physical activity (swimming without workload) has on mitochondrial morphological alterations and glucometabolic parameters induced by a high-sugar diet (HSD). Weaned male Wistar rats fed with a standard diet or a HSD (68% carbohydrate) were subjected to 60 minutes of regular physical activity by swimming (without workload) for four- (20 sessions) or eight-week (40 sessions) periods. After training, animals were euthanized and the sera, adipose tissues, and skeletal muscles were collected for further analysis. The HSD increased body weight after an 8-week period; it also increased the fat pads and the adipose index, resulting in glucose intolerance and insulin resistance (IR). Transmission electron microscopy showed an increase in alterations of mitochondrial ultrastructure in the gastrocnemius muscle, as well as a decrease in superoxide dismutase (SOD) activity, and an increase in protein carbonylation. Regular physical activity partially reverted these alterations in rats fed a HSD, preventing mitochondrial morphological alterations and IR. Moreover, we observed a decrease in Pgc1α expression (qPCR analysis) in STD-EXE group and a less pronounced reduction in HSD-EXE group after an 8-week period. Thus, regular physical activity (swimming without workload) in rats fed a HSD can prevent mitochondrial dysfunction and IR, highlighting the crucial role for physical activity on metabolic homeostasis.