Intravaginal devices impregnated with medroxyprogesterone acetate avoid early parturition and synchronize farrowing in sows.

Two experiments were performed to evaluate the use of an intravaginal device (IVD) impregnated with medroxyprogesterone acetate (MPA) to avoid early parturition and synchronize farrowing in sows. In both experiments with IVDs, the gestation length, stillbirth rate, birth weight, colostrum yield, lactational litter performance, and subsequent reproductive performance of sows were assessed. In Experiment 1 (Exp. 1; n = 91), sows were assigned to four treatments to evaluate the minimum required MPA dose: without IVD (CONT; control), 400 mg (MPA400), 600 mg (MPA600), and 800 mg (MPA800) of MPA in the IVD. The IVD was inserted on day 110 of gestation and removed on day 115. No sows farrowed during IVD treatment. Gestation length was increased in treatments with MPA (116.4 days) compared to the control (CONT; 114.9 days; P < 0.01), without effects on piglet birth weight (P = 0.98). A lower percentage of deaths around the farrow (P = 0.02) was observed in the CONT (1.8%) compared to MPA treatments (6.8%). The dose of 400 mg of MPA, validated in Exp. 1, was used in Experiment 2 (Exp. 2; n = 84) to evaluate the performance of sows and piglets in a sow farrowing synchronization protocol. Sows were treated with MPA from days 110-114 of gestation with or without 0.168 mg of cloprostenol sodium (PGF2α), for luteolysis induction, at IVD removal. Thus, four treatments were considered: CONT - without MPA or luteolysis induction (no interventions); PGF2α - luteolysis induction on day 114 of gestation without MPA; MPA114 - MPA treatment till 114 days of gestation without luteolysis induction; MPA114 + PGF2α - MPA treatment and luteolysis induction on day 114 of gestation. The gestation length in treatments with IVDs was longer (P < 0.01) than CONT without a difference for PGF2α treatment (P = 0.46). No impact of IVD use on piglet birth weight (P = 0.67) and deaths around the farrow (P = 0.50) were observed. The colostrum yield (P = 0.65), immunocrit (P = 0.72), piglet performance during lactation (P = 0.81), and weaning-to-estrus interval (P = 0.21) were similar among treatments. In conclusion, the use of IVDs impregnated with 400 mg of MPA between days 110 and 114 of gestation prevented early parturition with no implications for piglet survival at birth, colostrum yield, or litter performance.

The influence of prorenin/(pro)renin receptor on progesterone secretion by the bovine corpus luteum.

The aim of this study was to evaluate the role of prorenin/(pro)renin receptor activation on luteal progesterone (P4) secretion. Our hypothesis was that the nonproteolytic activation of (pro)renin receptor [P(RR)] is part of the regulatory mechanism responsible for corpus luteum (CL) function. In the first three experiments, prorenin was found to stimulate the production of P4, which is not inhibited by an angiotensin receptor antagonist (saralasin), but rather by a renin/prorenin inhibitor (aliskiren), a MAPK1/3 inhibitor (PD325901) or an EGFR inhibitor (AG1478), which are evidence of nonproteolytic activation of prorenin. Moreover, prorenin induced phosphorylation of MAPK1/3 in luteal cells. Following these in vitro experiments, a sequence of in vivo experiments was performed demonstrating that the intrafollicular injection of aliskiren in preovulatory follicles impaired P4 secretion in cows that ovulated. Furthermore, all profibrotic genes studied were present in the CL and TGFB1 and FN1 mRNA were upregulated from day 5-10 post-ovulation. During luteolysis, REN was downregulated at 48 h, whereas TGFB1 and SERPINE1 were dramatically upregulated in luteal tissue at 12 h after PGF. In summary, these data are evidence that nonproteolytic activation of (P)RR is involved in luteal function.

Insulin treatment does not affect follicular development but alters granulosa cell gene expression in dairy cows.

The use of strategies to stimulate follicular growth are important, especially for use in timed artificial insemination (TAI) protocols, aiming to increase dairy cow's fertility. The aim of this study was to investigate the effect of insulin on follicular growth, steroid production and expression of genes related to follicular development. For this, cows were submitted to a progesterone (P4) and estradiol (E2) based synchronization protocol. In study 1, eleven primiparous lactating Holstein cows, received a single s.c. application of 0.25 IU/kg human insulin or no treatment (control) on D8 of the protocol. Blood samples were collected, and the dominant follicle diameter was assessed daily via transrectal ultrasonography, from D8 to D12. In study 2, eight multiparous non-pregnant and non-lactating Jersey cows, received a single s.c. application of 0.25 IU/kg human insulin, whereas cows from the control group received a single s.c. injection (1 mL) of saline solution (NaCl 0.9%). Blood samples were collected, and the dominant follicle diameter was assessed daily via transrectal ultrasonography from D6 to D9 of the protocol. Sixteen hours after insulin injection, follicular aspiration was performed. In study 1, insulin treatment decreased systemic glucose levels, but did not affect follicular growth. In study 2, the glucose decrease induced by insulin treatment was accompanied by a tendency of decreased progesterone levels in follicular fluid, along with a decrease in steroidogenic acute regulatory protein (STAR) and insulin like growth factor binding protein 2 (IGFBP2) mRNA abundance in granulosa cells. In conclusion, insulin treatment does not increase follicle growth and estradiol secretion in dairy cows, but decreases IGFBP2 and tends to increase pappalysin (PAPPA) mRNA abundance in granulosa cells, suggesting a positive effect on follicle development.

Oxidative stress and biochemical markers in prenatally androgenized sheep after neonatal treatment with GnRH agonist.

BACKGROUND: Disruption of the balance between the production of ROS and their removal through enzymatic and non-enzymatic (antioxidant) processes has been proposed as a new mechanism in the pathology of polycystic ovary syndrome (PCOS). Evidence from animal models of PCOS (prenatally androgenized sheep) has suggested that treatment with insulin sensitizers, but not antiandrogens, can reduce increases in ROS. MATERIALS AND METHODS: In the present study, we investigated the effects of neonatal treatment with a gonadotropin-releasing hormone (GnRH) agonist (leuprolide acetate) on prenatally androgenized sheep with testosterone propionate to determine its impact on oxidative stress molecules (ferric reducing antioxidant power [FRAP], advanced oxidation protein product [AOPP], nitric oxide [NOx], albumin) at 8, 12, and 18 months of age. RESULTS: Androgenized ewes (but not leuprolide-treated ewes) showed reduced total cholesterol levels associated with a decrease in the ratio of visceral to subcutaneous adiposity (adjusted to abdominal area) as determined by computed tomography. In androgenized ewes at 12 months of age, an increase in subcutaneous fat and relative decrease in the visceral fat compartment did not affect the expression of REDOX markers. At 18 months of age, however, the levels of NOx metabolites decreased in androgenized animals, but remained close to normal in ewes subjected to neonatal treatment with leuprolide acetate. Other oxidative stress parameters (FRAP, AOPP, albumin) did not vary among groups. CONCLUSION: Our results demonstrate that the GnRH agonist leuprolide (as a single dose after birth) had weak effects on markers of the oxidative stress balance.

Prenatal Androgenization of Ewes as a Model of Hirsutism in Polycystic Ovary Syndrome.

The main clinical feature associated with hyperandrogenism in polycystic ovary syndrome (PCOS) in humans is hirsutism, where hair increases its length, pigmentation, and particularly its diameter. Currently, it is not known whether PCOS animal models also exhibit changes in the hair. Therefore, the aim of this study was to explore the wool characteristics in sheep prenatally androgenized (PA) with testosterone propionate. After 4 and 13 months of life, wool was collected from the top of the shoulder of both females and males (both androgenized and controls). The offspring sheep were followed for up to 19 months of life to evaluate testosterone and androstenedione serum levels by ultra-high-performance liquid chromatography-tandem mass spectrometry, determine insulin and glucose response to intravenous glucose tolerance test, and address estrus cyclicity during the second breeding season. PA male animals showed a reduction in wool fiber diameter at 4 months of age compared with controls (P = 0.02) but not at 13 months, whereas PA females showed increased hair diameter at 13 months (P = 0.002), with no difference at 4 months. No substantial changes in other hair parameters (length, color, and medullation) were identified. In addition, increased levels of serum testosterone were observed in PA female sheep compared with controls at 12 months (P = 0.03). Our results indicate for the first time, to our knowledge, that changes in wool fiber diameter observed in PA ewes replicate, at the translational level, the increase in hair diameter in hirsute women with PCOS.

Luteinizing hormone upregulates NPPC and downregulates NPR3 mRNA abundance in bovine granulosa cells through activation of the EGF receptor.

During folliculogenesis, the luteinizing hormone (LH) surge triggers dynamic events in granulosa cells that culminate with ovulation. The aim of this study was to evaluate if the epidermal growth factor receptor (EGFR) is required for ovulation in cattle, and if it regulates the expression of the natriuretic peptide (NP) system in granulosa cells after gonadotropin-releasing hormone (GnRH)/LH stimulation. It was observed that GnRH induces amphiregulin (AREG) and epiregulin (EREG) mRNA at 3 and 6 h after in vivo treatment, but the expression of these genes was not regulated by atrial (ANP) and C-type (CNP) NPs in granulosa cells cultured in vitro. The abundance of mRNA encoding the NP receptors (NPR1, 2 and 3) was not altered by LH supplementation and/or EGFR inhibition (AG1478; AG) in granulosa cells after 6 h of in vitro culture. However, in the same conditions, mRNA encoding the natriuretic peptide precursor C (NPPC) was upregulated by LH, whereas AG (0.5 and 5 µM) inhibited the LH effect. In order to confirm those results, 5 µM AG or saline were intrafollicularly injected in preovulatory follicles and cows were simultaneously treated with GnRH intramuscularly. Granulosa cells harvested at 6 h after GnRH injection revealed higher NPR3 and lower NPPC mRNA levels in AG-treated, compared to control cows. However, intrafollicular injection of AG did not inhibit GnRH-induced ovulation. In granulosa cells cultured in vitro, ANP associated with LH increased prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA abundance. In conclusion, we inferred that LH modulated NPPC and NPR3 mRNA abundance through EGFR in bovine granulosa cells, but ovulation in cattle did not seem to depend on EGFR activation.

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.

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.

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.

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, ACE(2), 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, ACE(2), 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, ACE(2), NEP and PEP was detected in theca and granulosa cells at all time-points after GnRH. In granulosa cells, ACE(2), NEP and PEP were differentially expressed after GnRH treatment (p < 0.05). In conclusion, the Ang-(1-7), MAS receptor, ACE(2), NEP and PEP profiles in preovulatory follicles indicate that Ang-(1-7) plays a role in the regulation of the ovulatory process in cattle.

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.