João Francisco Coelho de Oliveira

The role of angiotensin II in the early stages of bovine ovulation

There is evidence that the renin-angiotensin system plays an important role in ovulation in cattle. Using an in vivo model, we investigated the role of angiotensin (Ang) II in bovine ovulation by injecting Ang II receptor antagonists into ovulatory follicles. Animals (n = 102) were pre-synchronized and, when the follicles reached 12 mm, they were given the respective treatment and the cows received GnRH agonist (i.m.) to induce ovulation. The ovulation rate was significantly lower when 100 mu M saralasin (Ang II receptor antagonist) was intrafollicularly injected (14.3%) in comparison with saline solution (83.3%). Based on these results, a second experiment was carried out to determine the timing of Ang IIs critical role in ovulation. Saralasin inhibited ovulation only when applied at 0 and 6 h (16.7 and 42.9% ovulation rate in the 0- and 6-h groups respectively), but not at 12 h (100%) following GnRH agonist treatment. To investigate the subtypes of Ang II receptors implicated in the LH-induced ovulation, losartan (LO; AT(1)-Ang II receptor antagonist), PD123 319 (AT(2)-Ang II receptor antagonist), LO+PD123 319, or saline were intrafollicularly injected when the cows were challenged with GnRH agonist. Ovulation was inhibited by PD123 319 and LO+PD123 319 (50.0 and 33.3% on ovulation rate respectively), but not by LO or saline solution (100% ovulation in both groups). From these results, we suggest that Ang II plays a pivotal role in the early mechanism of bovine ovulation via the AT(2) receptor subtype.

Endocrine Delivery of Interferon Tau Protects the Corpus Luteum from Prostaglandin F2 Alpha-Induced Luteolysis in Ewes

ABSTRACT Paracrine release of ovine interferon tau (oIFNT) from the conceptus alters release of endometrial prostaglandin F2 alpha (PGF) and prevents luteolysis. Endocrine release of oIFNT into the uterine vein occurs by Day 15 of pregnancy and may impart resistance of the corpus luteum (CL) to PGF. It was hypothesized that infusion of recombinant oIFNT (roIFNT) into the uterine or jugular veins on Day 10 of the estrous cycle would protect the CL against exogenous PGF-induced luteolysis. Osmotic pumps were surgically installed in 24 ewes to deliver bovine serum albumin (BSA; n = 12) or roIFNT (200 μg/day; n = 12) for 24 h into the uterine vein. Six ewes in each treatment group received a single injection of PGF (4 mg/58 kg body weight) 12 h after pump installation. In a second experiment, BSA or roIFNT was delivered at 20 or 200 μg/day into the uterine vein or 200 μg/day into the jugular vein for 72 h in 30 ewes. One half of these ewes received an injection of PGF 24 h after pump installation. Concentrations of progesterone in serum declined in BSA-treated ewes injected with PGF, but were sustained in all ewes infused with 20 μg/day of roIFNT into the uterine vein and 200 μg of roIFNT into the jugular vein followed 24 h later with injection of PGF. All concentrations of roIFNT and modes of delivery (uterine or jugular vein) increased luteal concentrations of IFN-stimulated gene (i.e., ISG15) mRNA. Infusion of 200 μg of IFNT over 24 h induced greater mRNA concentrations for cell survival genes, such as BCL2-like 1 (BCL2L1 or Bcl-xL), serine/threonine kinase (AKT), and X-linked inhibitor of apoptosis (XIAP) and decreased prostaglandin F receptor (PTGFR) mRNA concentrations, when compared to controls. It is concluded that endocrine delivery of roIFNT, regardless of route (uterine or jugular vein), effectively protects CL from the luteolytic actions of PGF by mechanisms that involve ISGs and stabilization of cell survival genes.

Angiotensin II, progesterone, and prostaglandins are sequential steps in the pathway to bovine oocyte nuclear maturation.

Oocyte meiotic resumption is triggered by the ovulatory gonadotropin surge; in cattle, angiotensin II (AngII) and prostaglandins (PG) are key mediators of this gonadotropin-induced event. Here, we tested the hypothesis that progesterone (P(4)) is also involved in oocyte meiotic resumption induced by the gonadotropin surge. In Experiment I, P(4) induced nuclear maturation in a dose-dependent manner using a coculture of follicular hemisections and cumulus-oocyte complexes. In the second experiment, using an in vivo model, an injection of mifepristone (MIFE; P(4) receptor antagonist) at the antrum of preovulatory follicles prevented GnRH-induced oocyte meiotic resumption in vivo. In Experiment III (coculture system similar to that of Experiment I), MIFE prevented stimulatory effects of AngII on resumption of meiosis, but saralasin (AngII receptor antagonist) did not inhibit P(4) actions. In Experiments IV and V, fibroblast growth Factor 10 (FGF10; known to suppress steroidogenesis in granulosa cells), blocked AngII-but not P(4)-induced oocyte meiotic resumption. Therefore, we inferred that AngII is upstream to P(4) in a cascade to induce meiotic resumption. Previously, we had reported that AngII acted throughout the PGs pathway to modulate nuclear progression. In Experiment V, indomethacin inhibited resumption of meiosis induced by P(4), providing further support to the AngII-P(4) sequential effect on meiotic resumption. In conclusion, we inferred that AngII, P(4) and PGs are sequential steps in the same pathway that culminates with bovine oocyte maturation.

Molecular characterization and regulation of the angiotensin-converting enzyme type 2/Angiotensin-(1-7)/MAS receptor axis during the ovulation process in cattle:

The objective of this study was to characterize the profiles of Ang-(1-7), MAS receptor, ACE2, NEP and PEP during the ovulatory process in cattle. For this study, 40 synchronized cows with follicular diameter ≥ 12 mm were ovariectomized at different time-points (0, 3, 6, 12 and 24 h) after i.m. application of gonadotropin-releasing hormone (GnRH) to induce a luteinizing hormone surge. Follicular fluid was collected for measuring Ang-(1-7) by radioimmunoassay. Theca and granulosa cells were isolated from the preovulatory follicles to evaluate the gene expression of MAS receptor, ACE2, NEP and PEP by qRT-PCR assay. Cross-contamination between theca and granulosa cells was tested by RT-PCR to detect cytochrome P450 aromatase (CYP19A1) and 17α-hydroxylase (CYP17A1) mRNA. Ang-(1-7) levels were constant until 12 h and then increased (p < 0.05) at 24 h after GnRH. Messenger RNA expression of MAS, ACE2, NEP and PEP was detected in theca and granulosa cells at all time-points after GnRH. In granulosa cells, ACE2, NEP and PEP were differentially expressed after GnRH treatment (p < 0.05). In conclusion, the Ang-(1-7), MAS receptor, ACE2, NEP and PEP profiles in preovulatory follicles indicate that Ang-(1-7) plays a role in the regulation of the ovulatory process in cattle.

Role of Angiotensin II in the Periovulatory Epidermal Growth Factor-Like Cascade in Bovine Granulosa Cells In Vitro

ABSTRACT Angiotensin II (AGT-2) induces follicular prostaglandin release in a number of species and ovulation in rabbits. Conversely, AGT-2 antagonists block ovulation in cattle. To determine the mechanism of action of AGT-2, we used a bovine granulosa cell model in which luteinizing hormone (LH) increased the expression of genes essential for ovulation in a time- and dose-dependent manner. The addition of AGT-2 to LH-stimulated cells significantly increased abundance of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA and protein, whereas AGT-2 alone had no effect. Upstream of PTGS2, AGT-2 increased abundance of mRNA encoding the epidermal growth factor-like ligands amphiregulin (AREG) and epiregulin (EREG) at 6 h posttreatment and abundance of a disintegrin and metalloprotease 17 (ADAM17), a sheddase, within 3 h of treatment. Inhibiting sheddase activity abolished the stimulatory effect of AGT-2 on AREG, EREG, and PTGS2 mRNA. The addition of selective AGT-2 antagonists to cells stimulated with LH plus AGT-2 demonstrated that AGT-2 did not act through the type 1 receptor and did not increase mitogen-activated protein kinase 3/1 phosphorylation. Combined with previous data from studies in vitro, we conclude that AGT-2 is an essential cofactor for LH in the early increase of ADAM expression/activity that induces the cascade of events leading to ovulation.

Role of angiotensin in ovarian follicular development and ovulation in mammals: a review of recent advances

Angiotensin (Ang) II is widely known for its role in the control of systemic blood vessels. Moreover, Ang II acts on the vascular control of ovarian function, corpus luteum formation, and luteolysis. Over the past 10 years, our research group has been studying the new concept of the renin-angiotensin system (RAS) as an autocrine/paracrine factor regulating steroidogenesis and promoting different cellular responses in the ovary, beyond vascular function. We have developed and used different in vivo and in vitro experimental models to study the role of RAS in the ovary and a brief overview of our findings is presented here. It is widely accepted that there are marked species differences in RAS function in follicle development. Examples of species-specific functions of the RAS in the ovary include the involvement of Ang II in the regulation of follicle atresia in rats vs the requirement of this peptide for the dominant follicle development and ovulation in rabbits and cattle. More recently, Ang-(1-7), its receptor, and enzymes for its synthesis (ACE2, NEP, and PEP) were identified in bovine follicles, implying that Ang-(1-7) has an ovarian function. Other novel RAS components (e.g. (pro)renin receptor and renin-binding protein) recently identified in the bovine ovary show that ovarian RAS is poorly understood and more complex than previously thought. In the present review, we have highlighted the progress toward understanding the paracrine and autocrine control of ovarian antral follicle development and ovulation by ovarian tissue RAS, focusing on in vivo studies using cattle as a model.

Angiotensin II profile and mRNA encoding RAS proteins during bovine follicular wave

Angiotensin II (AngII) has a role in ovarian follicle development, ovulation, and oocyte meiotic resumption. The objective of the present study was to characterise the AngII profile and the mRNA encoding RAS proteins in a bovine follicular wave. Cows were ovariectomised when the size between the largest (F1) and the second largest follicle (F2) was not statistically different (day 2), slightly different (day 3), or markedly different (day 4). AngII was measured in the follicular fluid and the mRNA abundance of genes encoding angiotensin-converting enzyme (ACE), (pro)renin receptor, and renin-binding protein (RnBP) was evaluated in the follicular cells from F1 and F2. The AngII levels increased at the expected time of the follicular deviation in F1 but did not change in F2. However, the expression of the genes encoding ACE, (pro)renin receptor, and RnBP was not regulated in F1 but was upregulated during or after the follicular deviation in F2. Moreover, RnBP gene expression increased when the F1 was treated with the oestrogen receptor-antagonist in vivo. In conclusion, the AngII concentration increased in the follicular fluid of the dominant follicle during and after deviation and further supports our finding that RAS is present in the ovary regulating follicular dominance.

Regulation of inducible nitric oxide synthase expression in bovine ovarian granulosa cells.

Nitric oxide (NO) is a potential regulator of ovarian follicle growth, and ovarian granulosa cells reportedly generate NO in response to gonadotrophins, suggesting that the regulated form of nitric oxide synthase (iNOS) is present. The objectives of the present study were to gain insight into the expression and role of iNOS in the follicle. Messenger RNA encoding iNOS was detected in granulosa cells, and abundance was higher in growing dominant follicles compared to subordinate follicles (P<0.01). FSH (P<0.05) and IGF1 (P<0.01) stimulated oestradiol secretion and iNOS mRNA abundance in granulosa cells in vitro, whereas FGF2 (P<0.05) and EGF (P<0.01) decreased oestradiol secretion and iNOS expression. The addition of an anti-oestrogen prevented FSH-induced iNOS mRNA accumulation. Inhibition of endogenous NO production did not affect steroidogenesis in granulosa cells, but increased FasL mRNA abundance, caspase-3 activation and the incidence of apoptotic cell death (P<0.05). These results demonstrate that iNOS is expressed in ruminant granulosa cells and is regulated by gonadotrophins and oestradiol. Physiological levels of NO may contribute to the survival of granulosa cells.

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.

Leukemia inhibitory factor stimulates the transition of primordial to primary follicle and supports the goat primordial follicle viability in vitro.

The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control - FC) and the remaining fragments were cultured in supplemented minimum essential medium (MEM) without (cultured control - CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for 1 or 7 days, at 39°C in air with 5% CO2. Fresh control, CC and treated ovarian fragments were processed for histological and fluorescence analysis. The percentage of histological normal preantral follicles cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than in the CC (32.6%; p < 0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC (p < 0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle viability of goat ovarian tissues cultured for 7 days.