Joabel Tonellotto dos Santos

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.

Angiotensin II Signaling Promotes Follicle Growth and Dominance in Cattle

It is generally understood that angiotensin II (AngII) promotes follicle atresia in rats, although recent data suggested that this may not be true in cattle. In this study, we aimed to determine in vivo whether AngII alters follicle development in cattle, using intrafollicular injection of AngII or antagonist into the growing dominant follicle or the second largest subordinate follicle. Injection of saralasin, an AngII antagonist, into the growing dominant follicle inhibited follicular growth, and this inhibitory effect was overcome by systemic FSH supplementation. Injection of AngII into the dominant follicle did not affect follicular growth, whereas injection of AngII into the second largest follicle prevented the expected atresia of this subordinate follicle, and the treated follicle grew at the same rate as the dominant follicle for the next 24 h. Inhibition of AngII action in the dominant follicle decreased estradiol concentrations in follicular fluid and the abundance of mRNA encoding aromatase, 3β-hydroxysteroid dehydrogenase, LH receptor, and cyclinD2 in granulosa cells, with minimal effects on theca cells. The effect of AngII on aromatase mRNA levels was confirmed using an in vitro granulosa cell culture system. In conclusion, these data suggest that AngII signaling promotes follicle growth in cattle and does so by regulating genes involved in estradiol secretion and granulosa cell proliferation and differentiation.

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.

FGF10 inhibits dominant follicle growth and estradiol secretion in vivo in cattle.

Fibroblast growth factors (FGFs) are involved in paracrine control of follicle development. It was previously demonstrated that FGF10 decreases estradiol (E(2)) secretion in granulosa cell culture and that theca cell FGF10 mRNA expression is decreased in healthy follicles from abattoir ovaries. The main objectives of this study were to evaluate FGF10 and FGFR2b mRNA expression during follicular development in vivo, to evaluate the effect of FGF10 on follicle growth using Bos taurus taurus cows as a model, and to gain more insight into the mechanisms through which FGF10 inhibits steroidogenesis. Messenger RNA encoding both FGF10 and FGFR2b (main FGF10 receptor) was significantly more expressed in subordinate follicles (SFs) than in dominant follicles (DFs). The intrafollicular injection of FGF10 into the largest growing follicle at 7-8 mm in diameter interrupted the DF growth in a dose-dependent manner (11±0.4, 8.3±1 and 5.9±0.3 mm for 0, 0.1, and 1 μg/ml FGF10, respectively, at 72 h after treatment; P<0.05). In a third experiment, follicles were obtained 24 h after FGF10 (1 μg/ml) or PBS treatment through ovariectomy. In theca cells, FGF10 treatment did not affect mRNA encoding steroidogenic enzymes, LHCGR and IGFBPs, but significantly upregulated FGF10 mRNA expression. The expression of CYP19A1 mRNA in granulosa cells was downregulated by FGF10 treatment, which was accompanied by a 50-fold decrease in E(2) production, and decreased cyclin D2 mRNA. These results have shown that FGF10 and its receptor FGFR2b are more expressed in SFs and provide solid in vivo evidence that FGF10 acts as an important regulator of follicular growth in cattle.

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.

The effect of equine chorionic gonadotropin on follicular size, luteal volume, circulating progesterone concentrations, and pregnancy rates in anestrous beef cows treated with a novel fixed-time artificial insemination protocol.

The objective was to determine the effects of eCG given on the day of, or 2 days before removal of an intravaginal progestin device, on ovarian follicle diameter, luteal volume, serum progesterone (P4) concentrations, and pregnancy per insemination in a fixed-time AI (FTAI) protocol. Lactating, anestrous, multiparous Bos taurus cross beef cows, 40 to 60 days postpartum, were given estradiol benzoate (2 mg im) and a progestin intravaginal device containing 250 mg of medroxyprogesterone acetate on Day 0 and cloprostenol (0.265 mg) on Day 6. Intravaginal devices were removed on Day 8 and GnRH (100 μg im) was given on Day 9, with timed AI 16 hours later. In experiment 1, cows were randomly assigned to receive 400 IU im eCG on Day 6 (eCG6; N = 8) or Day 8 (eCG8; N = 8), or to not receive eCG (control; N = 8). Dominant follicle diameter on Day 9 in the eCG6 group (10.0 ± 0.5 mm) was larger (P < 0.05) than in the eCG8 (8.6 ± 0.2 mm) or control (8.5 ± 0.4 mm) groups. Corpora lutea (CL) in all cows in the control group underwent premature luteolysis within 10 days after ovulation. Luteal volumes and P4 concentrations 10 and 15 days after ovulation were higher (P < 0.05) in the eCG6 group than in the eCG8 group. In experiment 2, the eCG6 (N = 121) and eCG8 (N = 125) protocols were compared in lactating anestrous cows that underwent FTAI. Pregnancy rate was higher (P < 0.05) in the cows that received eCG on Day 6 (27.3%; 33/121) than on Day 8 (16.0%; 20/125). Furthermore, CL volumes and P4 concentrations were higher (P < 0.05) in the eCG6 group (5784.0 ± 857.3 mm(3) and 8.1 ± 1.3 ng/mL, respectively) than in the eCG8 group (3220.9 ± 505.1 mm(3) and 4.5 ± 0.7 ng/mL, respectively). We concluded that eCG given 2 days before progestin removal in this FTAI protocol for anestrous beef cows increased diameter of the dominant follicle, luteal volume, serum P4 concentrations, and pregnancy rates.

Oil-Free Culture System for in Vitro Bovine Embryo Production

The use of oil to avoid water evaporation from cell culture has several disadvantages, amongst which there is the migration of compounds from media to oil and from oil to media. The aim of this study was to evaluate the osmolality of a culture system using fourwell plates with water in the central hole as an alternative to in vitro bovine embryo production (IVP). In addition, the osmolality changes of the oocyte washing medium were assessed in 35 mm dishes with or without 2 mL of silicon oil overlay. Osmolality of oocyte washing medium changed a great deal over time after 60 minutes on a 39°C heated plate (291 mOsm kg−1), which was not detected when the medium was overlaid with silicon oil (280 mOsm kg−1; P<0.05). During the maturation period, the presence of water in the central hole of four-well plates maintained the osmolality in the same pattern as oil overlay (293±0 vs 294±1.8 mOsm kg−1; P>0.05). Blastocyst rates were higher when embryos were cultured in presence of water or oil (29.7 and 29.9% for water and 33% in oil conventional microdrop system), except in the group that oocytes were washed in hyperosmotic washing medium (15.1%; P<0.05). Groups cultured in absence of water in the central hole had lower blastocyst rates (P<0.05) independently of exposure (15.5%) or not (16.2 and 16.8%) to hyperosmotic washing medium. In conclusion, fourwell plates with water in the central hole can be an alternative to replace oil overlay for bovine IVP, maintaining stable osmolality and embryo development rates.

Evaluation of P2X7 receptor expression in peripheral lymphocytes and immune profile from patients with indeterminate form of Chagas disease

Chagas disease (CD) is caused by Trypanosoma cruzi, an intracellular protozoan which is a potent stimulator of cell-mediated immunity. In the indeterminate form of CD (IFCD) a modulation between pro- and anti-inflammatory responses establishes a host-parasite adaptation. It was previously demonstrated that purinergic ecto-enzymes regulates extracellular ATP and adenosine levels, influencing immune and inflammatory processes during IFCD. In inflammatory sites ATP, as well as its degradation product, adenosine, function as signaling molecules and immunoregulators through the activation of purinergic receptors. In this work, it was analyzed the gene and protein expression of P2X7 purinergic receptor in peripheral lymphocytes and serum immunoregulatory cytokines from IFCD patients. Gene and protein expression of P2X7 receptor (P2X7R), and serum cytokines (IL-2, IL-10, IL-17 and IFN-γ) were unaltered. However, IFCD group showed significantly higher IL-4 and IL-6 levels while TNF-α was significantly decreased. These results indicate that imune profile of IFCD patients displays anti-inflammatory characteristics, consistent with the establishment of an immunomodulatory response. Further study about the molecular knowledge of P2X7R in IFCD is useful to clarify the participation of purinergic system in the regulatory mechanism which avoid the progression of CD.

Characterization of the kallikrein-kinin system during the bovine ovulation process.

The kallikrein-kinin system (KKS) has been described as an important mediator of physiologic processes. Kallikreins use kininogen (KNG) as substrate to generate bradykinin, the main active peptide of the KKS that acts through two types of receptors, the B(1)R and the B(2)R. The goal of this study was to characterize some components of the KKS in different compartments of the ovary during the bovine ovulation process. The KNG, B(1)R and B(2)R mRNA expression patterns were assessed in theca and granulosa cells, as well as the bradykinin concentration and kallikrein-like activity in follicular fluid of bovine periovulatory follicles. To obtain a periovulatory follicle (≥12 mm), twenty-seven cows were submitted to estrus synchronization protocol and ovariectomized by colpotomy at 0, 3, 6, 12 or 24h after a GnRH-analog injection (gonadorelin; 100 μg, IM). Follicular fluid was aspirated for enzymatic assays while granulosa and theca cells were harvested for mRNA analysis. The mRNA expressions in follicular cells were evaluated by real-time RT-PCR and data representation related to the cyclophilin housekeeping gene. The bradykinin concentration and kallikrein-like activity were measured in follicular fluid by enzymatic immunoassay and selective substrate cleavage, respectively. The B(2)R expression in theca cells and B(1)R expression in theca and granulosa cells showed different profiles during the periovulatory period (P<0.05). The bradykinin concentration and kallikrein-like activity in the follicular fluid were different (P<0.05) due to the time during the ovulation process. KNG mRNA expression was similar for both follicular cell types (P>0.05). Taken together, these results provide an important characterization of the presence and possible KKS regulation during the bovine ovulation.