17-ß-Estradiol increases macrophage activity through activation of the G-protein-coupled estrogen receptor and improves the response of female mice to Cryptococcus gattii.

Cryptococcus gattii (Cg) is one of the agents of cryptococcosis, a severe systemic mycosis with a higher prevalence in men than women, but the influence of the female sex hormone, 17-ß-estradiol (E2), on cryptococcosis remains unclear. Our study shows that female mice presented delayed mortality, increased neutrophil recruitment in bronchoalveolar lavage fluid, and reduced fungal load after 24 hr of infection compared to male and ovariectomised female mice (OVX). E2 replacement restored OVX female survival. Female macrophages have more efficient fungicidal activity, which was increased by E2 and reversed by the antagonist of G-protein-coupled oestrogen receptor (GPER), which negatively modulates PI3K activation. Furthermore, E2 induces a reduction in Cg cell diameter, cell charge, and antioxidant peroxidase activity. In conclusion, female mice present improved control of Cg infection, and GPER is important for E2 modulation of the female response.

Angiotensin-converting enzyme 2 (ACE2), angiotensin-(1-7) and Mas receptor in gonadal and reproductive functions.

Angiotensin (Ang)-(1-7) is an active peptide formed from Ang I or Ang-(1-9) by multiple proteolytic steps involving angiotensin-converting enzyme (ACE) 1 and other peptidases, or by a single cleavage of Ang II catalyzed chiefly by ACE2. The effects of Ang-(1-7) are mediated by the G protein-coupled receptor Mas (or Mas1), encoded by the protooncogene MAS. The reproductive system expresses ACE2 quite abundantly and therefore is able to generate Ang-(1-7) using precursor peptides produced locally or taken from circulation. In several mammalian species, Ang-(1-7) stimulates ovarian follicle growth, oocyte maturation and ovulation. The peptide is found in human endometrium, mostly during the secretory phase of menstrual cycle when the uterus is receptive to embryo implantation. Rat models and human observational studies suggest that Ang-(1-7) is part of the maternal adaptive response to pregnancy and its deficiency is associated with poor circulation in the placental bed. Knockout mice revealed a relevant participation of Mas-mediated stimulus to the maintenance of normal spermatogenesis, even though the animal can still reproduce without it. In addition, the vasorelaxant effect of Ang-(1-7) participates in the physiological mechanism of corpus cavernosum blood influx and penile erection. We conclude that preclinical evidence encourages the pursuit of treatments for female and male reproductive dysfunctions based on Mas agonists, starting with its natural ligand Ang-(1-7).

Genetic deletion of the alamandine receptor MRGD leads to dilated cardiomyopathy in mice.

We have recently described a new peptide of the renin-angiotensin system, alamandine, a derivative of angiotensin-(1-7). Mas-related G protein-coupled receptor member D (MrgD) was identified as its receptor. Although similar cardioprotective effects of alamandine to those of angiotensin-(1-7) have been described, the significance of this peptide in heart function is still elusive. We aimed to evaluate the functional role of the alamandine receptor MrgD in the heart using MrgD-deficient mice. MrgD was localized in cardiomyocytes by immunofluorescence using confocal microscopy. High-resolution echocardiography was performed in wild-type and MrgD-deficient mice (2 and 12 wk old) under isoflurane anesthesia. Standard B-mode images were obtained in the right and left parasternal long and short axes for morphological and functional assessment and evaluation of cardiac deformation. Additional heart function evaluation was performed using Langendorff isolated heart preparations and inotropic measurements of isolated cardiomyocytes. Immunofluorescence indicated that the MrgD receptor is expressed in cardiomyocytes, mainly in the membrane and perinuclear and nuclear regions. Echocardiography showed left ventricular remodeling and severe dysfunction in MrgD-deficient mice. Strikingly, MrgD-deficient mice presented a pronounced dilated cardiomyopathy with a marked decrease in systolic function. Echocardiographic changes were supported by the data obtained in isolated hearts and inotropic measurements in cardiomyocytes. Our data add new evidence for a major role for alamandine/MrgD in the heart. Furthermore, our results indicate that we have identified a new gene implicated in dilated cardiomyopathy, unveiling a new target for translational approaches aimed to treat heart diseases. NEW & NOTEWORTHY The renin-angiotensin system is a key target for cardiovascular therapy. We have recently identified a new vasodepressor/cardioprotective angiotensin, alamandine. Here, we unmasked a key role for its receptor, Mas-related G protein-coupled receptor member D (MrgD), in heart function. The severe dilated cardiomyopathy observed in MrgD-deficient mice warrants clinical and preclinical studies to unveil its potential use in cardiovascular therapy.

Neonatal cardiomyocyte hypertrophy induced by endothelin-1 is blocked by estradiol acting on GPER.

Estradiol (E2) prevents cardiac hypertrophy, and these protective actions are mediated by estrogen receptor (ER)α and ERß. The G protein-coupled estrogen receptor (GPER) mediates many estrogenic effects, and its activation in the heart has been observed in ischemia and reperfusion injury or hypertension models; however, the underlying mechanisms need to be fully elucidated. Herein, we investigated whether the protective effect of E2 against cardiomyocyte hypertrophy induced by endothelin-1 (ET-1) is mediated by GPER and the signaling pathways involved. Isolated neonatal female rat cardiomyocytes were treated with ET-1 (100 nmol/l) for 48 h in the presence or absence of E2 (10 nmol/l) or GPER agonist G-1 (10 nmol/l) and GPER antagonist G-15 (10 nmol/l). ET-1 increased the surface area of cardiomyocytes, and this was associated with increased expression of atrial and brain natriuretic peptides. Additionally, ET-1 increased the phosphorylation of extracellular signal-related protein kinases-1/2 (ERK1/2). Notably, E2 or G-1 abolished the hypertrophic actions of ET-1, and that was reversed by G-15. Likewise, E2 reversed the ET-1-mediated increase of ERK1/2 phosphorylation as well as the decrease of phosphorylated Akt and its upstream activator 3-phosphoinositide-dependent protein kinase-1 (PDK1). These effects were inhibited by G-15, indicating that they are GPER dependent. Confirming the participation of GPER, siRNA silencing of GPER inhibited the antihypertrophic effect of E2. In conclusion, E2 plays a key role in antagonizing ET-1-induced hypertrophy in cultured neonatal cardiomyocytes through GPER signaling by a mechanism involving activation of the PDK1 pathway, which would prevent the increase of ERK1/2 activity and consequently the development of hypertrophy.

C-type natriuretic peptide: a link between hyperandrogenism and anovulation in a mouse model of polycystic ovary syndrome.

The polycystic ovary (PCO) syndrome (PCOS) is the most common cause of anovulatory infertility in women and is associated with several clinical disorders. Despite the great amount of research in the area, mechanisms involved in the genesis of this syndrome remain poorly understood. In a recent issue of Clinical Science (vol. 132, issue 7, 759-776), Wang and colleagues, highlight the important role of overactivated C-type natriuretic peptide (CNP) and natriuretic peptide receptor 2 (CNP/NPR2) system in preventing oocyte maturation and ovulation in PCOS mice model induced by androgen. Dehydroepiandrosterone (DHEA) treatment caused anovulation, high levels of androgen and estrogen receptors (AR and ER) in the ovary, high expression of CNP and natriuretic peptide receptor 2 (NPR2) in granulosa cells (GC), and an increase in testosterone and estradiol (E2) levels in sera. The high level of CNP/NPR2 was associated with oocyte meiotic arrest and very low ovulation rate. Treatment with human chorionic gonadotropin (hCG) or inhibitors of AR or ER reduced the level of CNP/NPR2, which resulted in meiotic resumption and ovulation. The article provided important information for understanding the effect of ovarian steroids on control of oocyte maturation and fertility and highlighted CNP/NPR2 as a specific pathway that is potentially involved in the ovulatory disruption in PCOS.

Angiotensin-(1-7) in human follicular fluid correlates with oocyte maturation.

STUDY QUESTION: Do angiotensin (Ang)-(1-7) levels in human ovarian follicular fluid (FF) correlate with the number and proportion of mature oocytes obtained for IVF? SUMMARY ANSWER: The present study shows for the first time that Ang-(1-7) levels in human FF correlate with the proportion of mature oocytes collected upon ovarian stimulation for IVF. WHAT IS KNOWN ALREADY: Ang-(1-7) is an active peptide of the renin-angiotensin system that stimulates oocyte maturation in isolated rabbit and rat ovaries. However, its role in human ovulation remains unexplored. STUDY DESIGN, SIZE, DURATION: This was a prospective cohort study including 64 participants from a single IVF center. Sample size was calculated to achieve a statistical power of 80% in detecting 20% differences in the proportion of mature oocytes between groups. The participants were enrolled in the study during six consecutive months. PARTICIPANTS/MATERIALS, SETTING, METHODS: Plasma samples were obtained from all subjects at Day 21 of the last menstrual cycle before starting pituitary blockade and controlled ovarian stimulation (COS). Plasma and FF samples were quickly mixed with a protease inhibitor cocktail and stored at -80°C. Ang-(1-7) was quantified in plasma and FF samples by a highly sensitive and specific radioimmunoassay, which was preceded by solid phase extraction, speed vacuum concentration and sample reconstitution in assay buffer. FF Ang-(1-7) levels were stratified into tertiles and the patients of each tertile were compared for COS/IVF outcomes using Kruskal-Wallis ANOVA. Multiple regression analysis was used to adjust correlations for potential confounders. The mRNA encoding for Mas, a receptor for Ang-(1-7), was investigated by real-time PCR in luteinized granulosa cells purified from the FF. MAIN RESULTS AND THE ROLE OF CHANCE: There was a four-fold increase in plasma Ang-(1-7) after ovulation induction (median 160.9 vs 41.4 pg/ml, P < 0.0001). FF Ang-(1-7) levels were similar to (169.9 pg/ml) but did not correlate with plasma Ang-(1-7) levels (r = -0.05, P = 0.665). Patients at the highest FF Ang-(1-7) tertile had a higher proportion of mature oocytes compared to patients at the lower FF Ang-(1-7) tertile (median 100% vs 70%, P < 0.01). There was a linear correlation between FF Ang-(1-7) and the proportion of mature oocytes (r = 0.380, P < 0.01), which remained significant after adjustment for age and duration of infertility (r = 0.447, P < 0.001). The luteinized granulosa cells expressed Mas receptor mRNA, which was positively correlated to the number of mature oocytes in women with more than three mature oocytes retrieved (r = 0.42, P < 0.01). LIMITATIONS, REASONS FOR CAUTION: This is an observational study, therefore, no causal relationship can be established between Ang-(1-7) and human oocyte maturation. Mas protein expression was not quantified due to limited availability of granulosa cells. WIDER IMPLICATIONS OF THE FINDINGS: Since this peptide promotes oocyte maturation in other species, it deserves further investigation as a potential maturation factor to human oocytes. STUDY FUNDING AND COMPETING INTEREST(S): Research supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). The authors have nothing to disclose.

Gender differences in normal left ventricle of adult FVB/N mice due to variation in interleukins and natriuretic peptides expression levels.

This study examined the sex differences for physical, morphological, histological, mRNA, and protein expression levels changes for interleukins and natriuretic peptides in left ventricle (LV) of two groups of adult FVB/N mice; males (WM) and females (WF). LV morphological, histological, reverse transcription and quantitative real-time PCR (RT-PCR), and immunohistochemical (IHC) alterations were determined in FVB/N mice at 34-35 weeks on gender basis. Confirming the gender dimorphism, FVB/N males (WM) illustrated a significant reduction in ANP and IL1-A levels as well as significantly increased body weight (BW (gm)), tibia length (TL (mm)), heart weight (HW (mg)), heart weight-to-body weight (HW/BW (mg/gm)) ratio, heart weight-to-tibia length (HW/TL (mg/mm)) ratio, left ventricle weight (LV (mg)), left ventricle-to-body weight (LV/BW (mg/gm)) ratio, and left ventricle-to-tibia length (LV/TL (mg/mm)) ratio, left ventricular (LV) cardiomyocyte diameter, high BNP, NPRA, IL-1B, and IL1R1 expression in comparison with FVB/N females (WF). Gender differences in relation to left ventricle (LV) may be due to differences in the interleukins and natriuretic peptides levels as an outcome of sex related hormones.

Natriuretic peptide clearance receptor ligand (C-ANP4-23 ) attenuates angiogenesis in a murine sponge implant model.

Natriuretic peptide receptor-C activation by the synthetic ligand C-ANP-4-23, a specific agonist for this receptor, has been shown to inhibit key events of the angiogenic cascade, such as migration, proliferation and vascular endothelial growth factor (VEGF) production. In the present study we investigated whether C-ANP4-23 could also inhibit angiogenesis in the sponge model in vivo. To this end, we evaluated the effects of C-ANP4-23 on inflammatory and angiogenic components of the fibrovascular tissue induced by polyether polyurethane sponge implants in mice. Measurements of the haemoglobin content (µg/mg wet tissue) and blood flow (laser Doppler perfusion imaging) of the implants, used as an index of vascularization, revealed that single (200 ng) or multiple (200 ng/day, 5 days) doses of C-ANP4-23 reduced angiogenesis in the implants relative to the phosphate-buffered saline-treated group. The peptide exerted an inhibitory effect on nitric oxide production (nitrite levels) and had a dual effect on VEGF levels, depending on the number of doses (i.e. stimulation at 4 days after one dose; inhibition at 7 days after five doses). Histological analysis corroborated the biochemical and functional parameters indicative of inhibition of neovascularization (decreased vessel number) by C-ANP4-23 . The peptide failed to modulate inflammation in our system. The inhibitory effect of C-ANP4-23 on the angiogenic component of the fibrovascular tissue induced by the synthetic matrix extends the range of the its actions and may indicate its therapeutic potential in controlling angiogenesis in fibroproliferative diseases.

Preovulatory changes in the angiotensin II system in bovine follicles.

The present study evaluated whether the gonadotrophin surge modulates components of the renin-angiotensin system and whether angiotensin II (Ang II) plays a role in the production of hormones by follicular cells during the ovulatory process. In Experiment 1, cows were ovariectomised at various times (0, 3, 6, 12 and 24 h) after GnRH injection to obtain preovulatory follicles. The concentration of Ang II in follicular fluid increased after GnRH and reached a peak at 24 h, concomitant with the peak of angiotensinogen (AGT) mRNA expression in granulosa cells. AGT mRNA was not expressed in theca cells. Ang II receptor type 2 and angiotensin-converting enzyme mRNA levels were transiently upregulated in theca cells. In Experiment 2, an in vitro culture was used to determine whether Ang II could modulate hormone production by healthy dominant follicles. In the absence of LH, Ang II did not alter hormonal production by either theca or granulosa cells. Ang II plus LH increased progesterone and prostaglandin secretion by granulosa cells. In summary, the renin-angiotensin system is actively controlled during the preovulatory period and Ang II amplifies the stimulatory effects of LH on the secretion of progesterone and prostaglandins by granulosa cells.

RFamide-related Peptide 3 Signaling via Neuropeptide FF Receptor Stimulates Prolactin Secretion in Female Rats.

The RF-amide peptides comprise a family of neuropeptides that includes the kisspeptin (Kp), the natural ligand of kisspeptin receptor (Kiss1r), and the RFamide-related peptide 3 (RFRP-3) that binds preferentially to the neuropeptide FF receptor 1 (Npffr1). Kp stimulates prolactin (PRL) secretion through the inhibition of tuberoinfundibular dopaminergic (TIDA) neurons. Because Kp also has affinity to Npffr1, we investigated the role of Npffr1 in the control of PRL secretion by Kp and RFRP-3. Intracerebroventricular (ICV) injection of Kp increased PRL and LH secretion in ovariectomized, estradiol-treated rats. The unselective Npffr1 antagonist RF9 prevented these responses, whereas the selective antagonist GJ14 altered PRL but not LH levels. The ICV injection of RFRP-3 in ovariectomized, estradiol-treated rats increased PRL secretion, which was associated with a rise in the dopaminergic activity in the median eminence, but had no effect on LH levels. The RFRP-3-induced increase in PRL secretion was prevented by GJ14. Moreover, the estradiol-induced PRL surge in female rats was blunted by GJ14, along with an amplification of the LH surge. Nevertheless, whole-cell patch clamp recordings showed no effect of RFRP-3 on the electrical activity of TIDA neurons in dopamine transporter-Cre recombinase transgenic female mice. We provide evidence that RFRP-3 binds to Npffr1 to stimulate PRL release, which plays a role in the estradiol-induced PRL surge. This effect of RFRP-3 is apparently not mediated by a reduction in the inhibitory tone of TIDA neurons but possibly involves the activation of a hypothalamic PRL-releasing factor.

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.

Norepinephrine modulation of heat dissipation in female rats lacking estrogen.

Postmenopausal hot flushes are caused by lack of estradiol (E2) but their neuroendocrine basis is still poorly understood. Here, we investigated the interrelationship between norepinephrine and hypothalamic neurons, with emphasis on kisspeptin neurons in the arcuate nucleus (ARC), as a regulatory pathway in the vasomotor effects of E2. Ovariectomized (OVX) rats displayed increased tail skin temperature (TST), and this increase was prevented in OVX rats treated with E2 (OVX + E2). Expression of Fos in the hypothalamus and the number of ARC kisspeptin neurons coexpressing Fos were increased in OVX rats. Likewise, brainstem norepinephrine neurons of OVX rats displayed higher Fos immunoreactivity associated with the increase in TST. In the ARC, the density of dopamine-ß-hydroxylase (DBH)-immunoreactive (ir) fibers was not altered by E2 but, importantly, DBH-ir terminals were found in close apposition to kisspeptin cells, revealing norepinephrine inputs to ARC kisspeptin neurons. Intracerebroventricular injection of the α2-adrenergic agonist clonidine (CLO) was used to reduce central norepinephrine release, confirmed by the decreased 3-methoxy-4-hydroxyphenylglycol/norepinephrine ratio in the preoptic area and ARC. Accordingly, CLO treatment in OVX rats reduced ARC Kiss1 mRNA levels and TST to the values of OVX + E2 rats. Conversely, CLO stimulated Kiss1 expression in the anteroventral periventricular nucleus (AVPV) and increased luteinizing hormone secretion. These findings provide evidence that augmented heat dissipation in OVX rats involves the increase in central norepinephrine that modulates hypothalamic areas related to thermoregulation, including ARC kisspeptin neurons. This neuronal network is suppressed by E2 and its imbalance may be implicated in the vasomotor symptoms of postmenopausal hot flushes.

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), 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.

Reduced dopaminergic tone during lactation is permissive to the hypothalamic stimulus for suckling-induced prolactin release.

Dopamine from tuberoinfundibular dopaminergic (TIDA) neurones tonically inhibits prolactin (PRL) secretion. Lactational hyperprolactinaemia is associated with a reduced activity of TIDA neurones. However, it remains controversial whether the suckling-induced PRL surge is driven by an additional decrease in dopamine release or by stimulation from a PRL-releasing factor. In the present study, we further investigated the role of dopamine in the PRL response to suckling. Non-lactating (N-Lac), lactating 4 hour apart from pups (Lac), Lac with pups return and suckling (Lac+S), and post-lactating (P-Lac) rats were evaluated. PRL levels were elevated in Lac rats and increased linearly within 30 minutes of suckling in Lac+S rats. During the rise in PRL levels, dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the median eminence (ME) and neurointermediate lobe of the pituitary did not differ between Lac+S and Lac rats. However, dopamine and DOPAC were equally decreased in Lac and Lac+S compared to N-Lac and P-Lac rats. Suckling, in turn, reduced phosphorylation of tyrosine hydroxylase in the ME of Lac+S. Domperidone and bromocriptine were used to block and activate pituitary dopamine D2 receptors, respectively. Domperidone increased PRL secretion in both N-Lac and Lac rats, and suckling elicited a robust surge of PRL over the high basal levels in domperidone-treated Lac+S rats. Conversely, bromocriptine blocked the PRL response to suckling. The findings obtained in the present study provide evidence that dopamine synthesis and release are tonically reduced during lactation, whereas dopamine is still functional with respect to inhibiting PRL secretion. However, there appears to be no further reduction in dopamine release associated with the suckling-induced rise in PRL. Instead, the lower dopaminergic tone during lactation appears to be required to sensitise the pituitary to a suckling-induced PRL-releasing factor.

Estradiol Potentiates But Is Not Essential for Prolactin-Induced Suppression of Luteinizing Hormone Pulses in Female Rats.

Hyperprolactinemia causes infertility by suppressing gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion. Because effects of prolactin (PRL) on the hypothalamus usually require estradiol (E2), we investigated the role of E2 in PRL-induced suppression of LH pulses. Ovariectomized (OVX) rats treated with oil or E2 (OVX + E2) received a subcutaneous injection of ovine PRL (oPRL) 30 minutes before serial measurement of LH in the tail blood by enzyme-linked immunosorbent assay. E2 reduced pulsatile LH secretion. oPRL at 1.5 mg/kg further reduced LH pulse frequency in OVX + E2 but had no effect in OVX rats. The higher dose of 6-mg/kg oPRL decreased LH pulse frequency in both OVX and OVX + E2 rats, whereas pulse amplitude and mean LH levels were lowered only in OVX + E2 rats. Kisspeptin immunoreactivity and Kiss1 messenger ribonucleic acid (mRNA) levels were decreased in the arcuate nucleus (ARC) of OVX + E2 rats. oPRL decreased both kisspeptin peptide and gene expression in the ARC of OVX rats but did not alter the already low levels in OVX + E2 rats. In the anteroventral periventricular nucleus, oPRL did not change kisspeptin immunoreactivity and, paradoxically, increased Kiss1 mRNA only in OVX + E2 rats. Moreover, oPRL effectively reduced Gnrh expression regardless of E2 treatment. In this study we used tail-tip blood sampling to determine the acute effect of PRL on LH pulsatility in female rats. Our findings characterize the role of E2 in the PRL modulation of hypothalamic components of the gonadal axis and LH release, demonstrating that E2 potentiates but is not essential for the suppression of pulsatile LH secretion caused by hyperprolactinemia.

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.

Correlation between BNP levels and Doppler echocardiographic parameters of left ventricle filling pressure in patients with Chagasic cardiomyopathy.

UNLABELLED: Cardiomyopathy is the most important manifestation of Chagas' disease. Brain natriuretic peptide (BNP) level and Doppler echocardiographic parameters for diastolic dysfunction have shown correlation with left ventricle (LV) filling pressures. OBJECTIVES: The purpose of this study is to compare BNP levels with Doppler echocardiographic parameters in patients with chagasic cardiomyopathy. METHODS: Forty-three patients (69.8% men; mean age 41.0 +/- 10.4 years) were submitted to an echocardiographic study and 39 had their BNP levels measured. RESULTS: BNP levels increased with the deterioration of the diastolic function (P=0.025). Pulmonary venous flow parameters were correlated with BNP levels, but E/E'ratio (E'measured at the inferior mitral annulus) was the only diastolic parameter that remained an independent predictor of elevated BNP levels in the multivariate analysis. The area under the receiver-operating curve for BNP to detect E/E' >15 was 0.875. A BNP value of 280.4 pg/ml had a sensitivity of 96% and a specificity of 75% for predicting E/E' >15. CONCLUSIONS: In a group of patients with chagasic cardiomyopathy, BNP levels correlated with diastolic function patterns regardless of systolic function. The E/E'ratio (inferior wall) was the only isolated parameter of diastolic function that was independently associated with BNP levels.

C-type natriuretic peptide signaling in human follicular environment and its relation with oocyte maturation.

Studies in mice have shown that C-type natriuretic peptide (CNP) is produced by granulosa cells and contributes to ovarian follicle growth and oocyte meiotic arrest until the preovulatory LH surge. In humans, the relationship between intraovarian CNP levels and oocyte meiotic resumption is unknown. The aim of this study was to investigate whether CNP and its receptor NPR2 are expressed in human ovarian follicles and if their levels change according to the meiotic phase of oocytes. We collected follicular fluid (FF) and luteinized granulosa cells (LGC) from follicle pools (n = 47), and FF, LGC and cumulus cells (CC) from individual follicles (n = 96) during oocyte pickup for in vitro fertilization. There was a positive linear correlation between CNP levels in FF pools and basal antral follicle counting (rs = 0.458; p = 0.002), number of preovulatory follicles >16 mm (rs = 0.361; p = 0.016) and number of oocytes retrieved (rs = 0,378; p = 0.011) and a negative correlation between CNP levels in FF pools and the percentage of mature (MII) oocytes retrieved (rs = -0.39; p = 0.033). FF CNP levels in follicles containing MII oocytes were significantly lower than in follicles containing immature (MI) oocytes (median = 0.44 vs. 0.57 ng/mL, p < 0.05). Accordingly, the CNP precursor gene NPPC was 50% less expressed in LGC from follicles containing MII oocytes than in follicles containing MI oocytes (p < 0.01). In addition, NPR2 mRNA was down-regulated in CC surrounding MII oocytes (60% reduction, p < 0.01). CNP signaling is downregulated in human ovarian follicles containing mature oocytes. Further studies should clarify whether CNP signaling is essential to keep oocyte meiotic arrest in humans.

Impairment of the natriuretic peptide system in follitropin receptor knockout mice and reversal by estradiol: implications for obesity-associated hypertension in menopause.

Estrogen is considered a major regulator of adipose tissue in females. Estrogen increases circulating levels of atrial natriuretic peptide (ANP), a hormone with renal and cardiovascular effects. The aim of this study was to determine the status of the natriuretic peptide system in female follitropin-receptor knockout (FORKO) mice that could be associated with obesity and hypertension observed in these mutants. Furthermore, estradiol treatment was used to reverse alterations observed. FORKO and wild-type (WT) mice received daily injections of estradiol for 4 d. On the fifth day, blood was collected for determination of plasma ANP levels, and selected tissues were collected for determination of ANP, natriuretic peptide receptor type-A (NPR-A) and type-C (NPR-C) gene expression by RT-PCR and binding of [(125)I]ANP by autoradiography. At 5 months of age, FORKO mice were heavier and had more adipose tissue than WT mice. FORKO mice had lower plasma ANP levels and atrial ANP gene expression and higher renal and adipocyte NPR-C gene expression than WT mice. Estradiol treatment reduced weight gain and increased atrial ANP synthesis as well as decreased ANP clearance NPR-C receptors, resulting in elevation of circulating ANP level. In conclusion, this study shows that FORKO females have an impaired natriuretic peptide system, which may contribute to the susceptibility of FORKO mice to developing age-related hypertension previously shown in these animals. This study establishes a relation between estrogen, adipose tissue, and ANP, which may have important implications in menopausal women.