Cellular localization of androgen synthesis in equine granulosa-theca cell tumors: Immunohistochemical expression of 17α-hydroxylase/17,20-lyase cytochrome P450

Elevated blood testosterone concentrations, often accompanied by male-typical behaviors, is a common signalment of mares with granulosa-theca cell tumors (GCTCs), but no definitive information exists regarding the cellular differentiation of tumors associated with androgen secretion. This study was conducted to localize and thereby define the cellular expression of 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17), the enzyme most directly responsible for androgen synthesis, in 30 GTCTs and control tissues (gonads and adrenal glands) using immuno-histochemistry (IHC). Immuno-reactivity for P450c17 was evident in approximately half of 30 specimens examined, was most consistent in the interstitial cells surrounding existing or developing cysts, and was less intense in cells within cysts in the smaller proportion of specimens where this was observed. In control tissues, the expression of P450c17 was localized primarily in theca interna of normal ovarian follicles, in theca-lutein cells of some corpora lutea, but not in granulosa-lutein cells. Testicular interstitial cells and islands of adreno-cortical cells located in the adrenal medulla of the adrenal cortex further established the specificity of the antisera used. These data provided the first substantive evidence that polyhedral cells identified previously in GTCTs by histopathology have the potential to synthesize and secrete androgens, similar to theca interna and theca lutein cells in normal equine ovaries. © 2010 Elsevier Inc.

Female sex hormones mediate the allergic lung reaction by regulating the release of inflammatory mediators and the expression of lung E-selectin in rats

Background: Fluctuations of estradiol and progesterone levels caused by the menstrual cycle worsen asthma symptoms. Conflicting data are reported in literature regarding pro and anti-inflammatory properties of estradiol and progesterone.Methods: Female Wistar rats were ovalbumin (OVA) sensitized 1 day after resection of the ovaries (OVx). Control group consisted of sensitized-rats with intact ovaries (Sham-OVx). Allergic challenge was performed by aerosol (OVA 1%, 15 min) two weeks later. Twenty four hours after challenge, BAL, bone marrow and total blood cells were counted. Lung tissues were used as explants, for expontaneous cytokine secretion in vitro or for immunostaining of E-selectin.Results: We observed an exacerbated cell recruitment into the lungs of OVx rats, reduced blood leukocytes counting and increased the number of bone marrow cells. Estradiol-treated OVx allergic rats reduced, and those treated with progesterone increased, respectively, the number of cells in the BAL and bone marrow. Lungs of OVx allergic rats significantly increased the E-selectin expression, an effect prevented by estradiol but not by progesterone treatment. Systemically, estradiol treatment increased the number of peripheral blood leukocytes in OVx allergic rats when compared to non treated-OVx allergic rats. Cultured-BAL cells of OVx allergic rats released elevated amounts of LTB4 and nitrites while bone marrow cells increased the release of TNF-α and nitrites. Estradiol treatment of OVx allergic rats was associated with a decreased release of TNF-α, IL-10, LTB4 and nitrites by bone marrow cells incubates. In contrast, estradiol caused an increase in IL-10 and NO release by cultured-BAL cells. Progesterone significantly increased TNF- α by cultured BAL cells and bone marrow cells.Conclusions: Data presented here suggest that upon hormonal oscillations the immune sensitization might trigger an allergic lung inflammation whose phenotype is under control of estradiol. Our data could contribute to the understanding of the protective role of estradiol in some cases of asthma symptoms in fertile ans post-menopausal women clinically observed. © 2010 de Oliveira et al; licensee BioMed Central Ltd.

Evaluation of tissue reaction, cell viability and cytokine production induced by Sealapex Plus

The aim of this study was to investigate the effects of mineral trioxide aggregate (MTA), Sealapex, and a combination of Sealapex and MTA (Sealapex Plus) on the reaction of subcutaneous connective tissue of rats, and on cell viability and cytokine production in mouse fibroblasts. The tissue reaction was carried out with dentin tubes containing the materials implanted in the dorsal connective tissue of rats. The histological analysis was performed after 7 and 30 days. Millipore culture plate inserts with polyethylene tubes filled with materials were placed into 24-well cell culture plates with mouse fibroblasts to evaluate the cell viability by MTT assay. ELISA assays were also performed after 24 h of exposure of the mouse fibroblasts to set material disks. Histopathologic examination showed Von Kossa-positive granules that were birefringent to polarized light for all the studied materials at the tube openings. No material inhibited the cell viability in the in vitro test. It was detected IL-6 production in all root-end filling materials. MTA and Sealapex Plus induced a slight raise of mean levels of IL-1β. The results suggest that Sealapex Plus is biocompatible and stimulates the mineralization of the tissue.

Cimetidine-induced vascular cell apoptosis impairs testicular microvasculature in adult rats

Cimetidine, an H2 receptor antagonist used for treatment of gastric ulcers, exerts antiandrogenic and antiangiogenic effects. In the testes cimetidine impairs spermatogenesis, Sertoli cells and peritubular tissue, inducing apoptosis in the myoid cells. Regarding the importance of histamine and androgens for vascular maintenance, the effect of cimetidine on the structural integrity of the testicular vasculature was evaluated. Adult male rats received cimetidine (CMTG) and saline (CG) for 50 days. The testes were fixed in buffered 4% formaldehyde and embedded in historesin and paraffin. In the PAS-stained sections, the microvascular density (MVD) and the vascular luminal area (VLA) were obtained. TUNEL method was performed for detection of cell death. Testicular fragments embedded in Araldite were analyzed under transmission electron microscopy. A significant decrease in the MVD and VLA and a high number of collapsed blood vessel profiles were observed in CMTG. Endothelial cells and vascular muscle cells were TUNEL-positive and showed ultrastructural features of apoptosis. These results indicate that cimetidine induces apoptosis in vascular cells, leading to testicular vascular atrophy. A possible antagonist effect of cimetidine on the H2 receptors and/or androgen receptors in the vascular cells may be responsible for the impairment of the testicular microvasculature.

Effect of superstimulatory treatments on the expression of genes related to ovulatory capacity, oocyte competence and embryo development in cattle.

Multiple ovulation (superovulation) and embryo transfer has been used extensively in cattle. In the past decade, superstimulatory treatment protocols that synchronise follicle growth and ovulation, allowing for improved donor management and fixed-time AI (FTAI), have been developed for zebu (Bos indicus) and European (Bos taurus) breeds of cattle. There is evidence that additional stimulus with LH (through the administration of exogenous LH or equine chorionic gonadotrophin (eCG)) on the last day of the superstimulatory treatment protocol, called the 'P-36 protocol' for FTAI, can increase embryo yield compared with conventional protocols that are based on the detection of oestrus. However, inconsistent results with the use of hormones that stimulate LH receptors (LHR) have prompted further studies on the roles of LH and its receptors in ovulatory capacity (acquisition of LHR in granulosa cells), oocyte competence and embryo quality in superstimulated cattle. Recent experiments have shown that superstimulation with FSH increases mRNA expression of LHR and angiotensin AT(2) receptors in granulosa cells of follicles >8 mm in diameter. In addition, FSH decreases mRNA expression of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) in oocytes, but increases the expression of both in cumulus cells, without diminishing the capacity of cumulus-oocyte complexes to generate blastocysts. Although these results indicate that superstimulation with FSH is not detrimental to oocyte competence, supplementary studies are warranted to investigate the effects of superstimulation on embryo quality and viability. In addition, experiments comparing the cellular and/or molecular effects of adding eCG to the P-36 treatment protocol are being conducted to elucidate the effects of superstimulatory protocols on the yield of viable embryos.

Spermatogenesis of riffle bugs, Rhagovelia whitei and Rhagovelia sp (Veliidae), and backswimmers Martarega sp (Notonectidae).

We examined the course of spermatogenesis and the meiotic chromosome complements in aquatic species of true bugs, Heteroptera. The chromosome complement of the Veliidae species was 2n = 39 (38A + X0) and 23 (22A + X0) in Rhagovelia whitei and Rhagovelia sp, respectively, and in the species of the Notonectidae (Martarega sp) it was 26 (22A + 2m + XY); all collected from the region of São José do Rio Preto, SP, Brazil. An impressive characteristic of the first analysis was the size of the cells belonging to Martarega sp, which were six times larger than the same cells in Pentatomidae and twice as large as the cells in aquatic Heteroptera (Gerridae). Regarding spermatogenesis, all the species analyzed showed the same pattern: holocentric chromosomes and elongated spermatids with the chromatin distributed evenly along the head. The family Veliidae showed several bodies impregnated with silver nitrate at prophase, while the family Notonectidae displayed only one. The cells of Notonectidae also showed an evident and round body until the end of prophase I and in the family Veliidae the silver-impregnated bodies were disorganized, where the only region visualized was possibly that of the NOR. In metaphase, silver-stained regions were found at the periphery of all chromosomes in Veliidae and at the periphery of some chromosomes in Notonectidae. The spermatids of Veliidae showed a less silver-impregnated vesicle, while Notonectidae showed silver staining only in part of the nuclear membrane. Therefore, families of Heteroptera have some differences and features that can help identify and classify these species.

Control of breathing and blood pressure by parafacial neurons in conscious rats

New Findings: • What is the central question of this study? The main purpose of the present manuscript was to investigate the cardiorespiratory responses to hypoxia or hypercapnia in conscious rats submitted to neuronal blockade of the parafacial region. We clearly showed that the integrity of parafacial region is important for the respiratory responses elicited by peripheral and central chemoreflex activation in freely behavior rats. • What is the main finding and its importance? Since the parafacial region is part of the respiratory rhythm generator, they are essential for postnatal survival, which is probably due to their contribution to chemoreception in conscious rats. The retrotrapezoid nucleus (RTN), located in the parafacial region, contains glutamatergic neurons that express the transcriptor factor Phox2b and that are suggested to be central respiratory chemoreceptors. Studies in anaesthetized animals or in vitro have suggested that RTN neurons are important in the control of breathing by influencing respiratory rate, inspiratory amplitude and active expiration. However, the contribution of these neurons to cardiorespiratory control in conscious rats is not clear. Male Holtzman rats (280-300 g, n= 6-8) with bilateral stainless-steel cannulae implanted into the RTN were used. In conscious rats, the microinjection of the ionotropic glutamatergic agonist NMDA (5 pmol in 50 nl) into the RTN increased respiratory frequency (by 42%), tidal volume (by 21%), ventilation (by 68%), peak expiratory flow (by 24%) and mean arterial pressure (MAP, increased by 16 ± 4, versus saline, 3 ± 2 mmHg). Bilateral inhibition of the RTN neurons with the GABAA agonist muscimol (100 pmol in 50 nl) reduced resting ventilation (52 ± 34, versus saline, 250 ± 56 ml min-1 kg-1 with absolute values) and attenuated the respiratory response to hypercapnia and hypoxia. Muscimol injected into the RTN slightly reduced resting MAP (decreased by 13 ± 7, versus saline, increased by 3 ± 2 mmHg), without changing the effects of hypercapnia or hypoxia on MAP and heart rate. The results suggest that RTN neurons activate facilitatory mechanisms important to the control of ventilation in resting, hypoxic or hypercapnic conditions in conscious rats. © 2012 The Authors. Experimental Physiology © 2012 The Physiological Society.

Antiprotozoal activity of quinonemethide triterpenes from Maytenus ilicifolia (Celastraceae)

The present study describes the leishmanicidal and trypanocidal activities of two quinonemethide triterpenes, maytenin (1) and pristimerin (2), isolated from Maytenus ilicifolia root barks (Celastraceae). The compounds were effective against the Trypanosomatidae Leishmania amazonensis and Leishmania chagasi and Trypanosoma cruzi, etiologic agents of leishmaniasis and Chagas' disease, respectively. The quinonemethide triterpenes 1 and 2 exhibited a marked in vitro leishmanicidal activity against promastigotes and amastigotes with 50% inhibitory concentration (IC50) values of less than 0.88 nM. Both compounds showed IC50 lower than 0.3 nM against Trypanosoma cruzi epimastigotes. The selectivity indexes (SI) based on BALB/c macrophages for L. amazonensis and L. chagasi were 243.65 and 46.61 for (1) and 193.63 and 23.85 for (2) indicating that both compounds presented high selectivity for Leishmania sp. The data here presented suggests that these compounds should be considered in the development of new and more potent drugs for the treatment of leishmaniasis and Chagas' disease. © 2013 by the authors.

Effects of heat stress on development, quality and survival of Bos indicus and Bos taurus embryos produced in vitro

Heat stress is an important cause of poor development and low survival rates in bovine embryos. Experiments were conducted to test the hypothesis that Bos indicus embryos are more resistant to heat stress than are Bos taurus embryos. In experiment 1, Nelore and Jersey embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 6 hours), developmental ratios were assessed at Day 7 (Day 0 = day of fertilization), and blastocysts were frozen for RNA extraction. Experiment 2 evaluated expression of COX2, CDX2, HSF1, and PLAC8 in previously frozen blastocysts. In experiment 3, Nellore and Angus embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 12 hours) and transferred to recipients on Day 7. In experiment 4, embryos developed as in experiment 3 were fixed for Terminal deoxynucleotidyl transferase dUTP nick end labeling labeling and total cell counting. In experiment 1, heat stress decreased the percentage of Jersey oocytes that became blastocysts, but had no effect on Nellore embryos (34.6%, 25.0%, 39.5%, and 33.0% for Jersey control, Jersey heat-stressed, Nellore control, and Nellore heat-stressed oocytes, respectively; P < 0.05). In experiment 2, heat stress decreased (P < 0.05) expression of CDX2 and PLAC8, with higher expression of these genes in Nellore embryos than in Jersey embryos. Heat stress also decreased (P < 0.05) expression of COX2 in Jersey embryos, but had no effect on Nellore embryos. Expression of HSF1 was decreased (P < 0.05) by heat stress in both breeds, with a greater effect in Nellore embryos. In experiment 3, heat stress tended (P = 0.1) to decrease the percentage of pregnancies among cows (Day 30 to 35) that received Angus embryos. In experiment 4, heat stress increased (P < 0.05) the percentage of apoptotic blastomeres, but had no breed-specific effects. In addition, Nellore embryos had fewer (P < 0.05) Terminal deoxynucleotidyl transferase dUTP nick end labeling- positive blastomeres than did Angus embryos. We concluded that the detrimental effects of heat stress were dependent upon embryo breed and were more evident in Bos taurus embryos than in Bos indicus embryos. © 2013 Elsevier Inc.

Supplementation with the histone deacetylase inhibitor trichostatin A during in vitro culture of bovine embryos

Summary Trichostatin A (TSA) is a histone deacetylase inhibitor that induces histone hyperacetylation and increases gene expression levels. The aim of the present study was to establish a suitable condition for the use of TSA in in vitro cultures of bovine embryos, and to determine whether TSA would increase blastocyst rates by improvement of chromatin remodelling during embryonic genome activation and by increasing the expression of crucial genes during early development. To test this hypothesis, 8-cell embryos were exposed to four concentrations of TSA for different periods of time to establish adequate protocols. In a second experiment, three experimental groups were selected for the evaluation of embryo quality based on the following parameters: apoptosis, total cell number and blastocyst hatching. TSA promoted embryonic arrest and degeneration at concentrations of 15, 25 and 50 nM. All treated groups presented lower blastocyst rates. Exposure of embryos to 5 nM for 144 h and to 15 nM for 48 h decreased blastocyst hatching. However, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay revealed similar apoptosis rates and total cell numbers in all groups studied. Although, in the present study, TSA treatment did not improve the parameters studied, the results provided background information on TSA supplementation during in vitro culture of bovine embryos and showed that embryo quality was apparently not affected, despite a decrease in blastocyst rate after exposure to TSA. © Cambridge University Press 2011.

Hindbrain mineralocorticoid mechanisms on sodium appetite

Aldosterone acting on the brain stimulates sodium appetite and sympathetic activity by mechanisms that are still not completely clear. In the present study, we investigated the effects of chronic infusion of aldosterone and acute injection of the mineralocorticoid receptor (MR) antagonist RU 28318 into the fourth ventricle (4th V) on sodium appetite. Male Wistar rats (280-350 g) with a stainless-steel cannula in either the 4th V or lateral ventricle (LV) were used. Daily intake of 0.3 M NaCl increased to 46 ± 15 and 130 ± 6 ml/24 h after 6 days of infusion of 10 and 100 ng/h of aldosterone into the 4th V (intake with vehicle infusion: 2 ± 1 ml/24 h). Water intake fell slightly and not consistently, and food intake was not affected by aldosterone. Sodium appetite induced by diuretic (furosemide) combined with 24 h of a low-sodium diet fell from 12 ± 1.7 ml/2 h to 5.6 ± 0.8 ml/2 h after injection of the MR antagonist RU 28318 (100 ng/2 μl) into the 4th V. RU 28318 also reduced the intake of 0.3 M NaCl induced by 9 days of a low-sodium diet from 9.5 ± 2.6 ml/2 h to 1.2 ± 0.6 ml/2 h. Infusion of 100 or 500 ng/h of aldosterone into the LV did not affect daily intake of 0.3 M NaCl. The results are functional evidence that aldosterone acting on MR in the hindbrain activates a powerful mechanism involved in the control of sodium appetite. © 2013 the American Physiological Society.

High concentrations of glucose reduce the oxidative metabolism of dog neutrophils in vitro

Background: Dogs are commonly affected by hyperglycemic conditions. Hyperglycemia compromises the immune response and favors bacterial infections; however, reports on the effects of glucose on neutrophil oxidative metabolism and apoptosis are conflicting in humans and rare in dogs. Considering the many complex factors that affect neutrophil oxidative metabolism in vivo, we investigated in vitro the specific effect of high concentrations of glucose on superoxide production and apoptosis rate in neutrophils from healthy dogs.Results: The capacity of the neutrophils to reduce tetrazolium nitroblue decreased significantly in the higher concentration of glucose (15.13 ± 9.73% (8 mmol/L) versus 8.93 ± 5.71% (16 mmol/L)). However, there were no changes in tetrazolium nitroblue reduction at different glucose concentrations when the neutrophils were first activated with phorbol myristate acetate. High concentrations of glucose did not affect the viability and apoptosis rate of canine neutrophils either with or without prior camptothecin stimulation. This study provides the first evidence that high concentrations of glucose inhibit the oxidative metabolism of canine neutrophils in vitro in a manner similar to that which occurs in humans, and that the decrease in superoxide production did not increase the apoptosis rate.Conclusions: A high concentration of glucose reduces the oxidative metabolism of canine neutrophils in vitro. It is likely that glucose at high concentrations rapidly affects membrane receptors responsible for the activation of NADPH oxidase in neutrophils; therefore, the nonspecific immune response can be compromised in dogs with acute and chronic hyperglycemic conditions. © 2013 Bosco et al.; licensee BioMed Central Ltd.

Photodynamic and Antibiotic Therapy Impair the Pathogenesis of Enterococcus faecium in a Whole Animal Insect Model

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments.

Guided implant surgery: What is the influence of this new technique on bone cell viability?

Purpose: To evaluate the effect of implant osteotomy on immediate bone cell viability, comparing guided surgery for implant placement with the classic drilling procedure. Materials and Methods: For this study, 20 rabbits were used. The animals were divided into a guided surgery group (GG) and a control group (CG) and were then divided into 4 subgroups - subgroups 1, 2, 3, and 4 - corresponding to drills used 10, 20, 30, and 40 times, respectively. All animals received 5 osteotomies in each tibia, by use of the classic drilling procedure in one tibia and guided surgery in the other tibia. The osteotomized areas were removed and processed immunohistochemically for detection of osteocalcin, receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), and caspase 3. Results: Immunohistochemical analysis showed that osteocalcin expression was initially higher in the CG and remained constant after drill reutilization. Although the expressions of RANKL and OPG were not statistically different for the GG and CG, the RANKL/OPG ratio tended to be higher for the GG. Moreover, caspase 3 expression was elevated in the GG, proportionally to the number of osteotomies, indicating an increase in the apoptosis index in the GG. Conclusions: The classic drilling procedure is more favorable to cell viability than guided surgery.© 2013 American Association of Oral and Maxillofacial Surgeons.

Physiology and endocrinology symposium: Influence of cattle genotype (Bos indicus vs. Bos taurus) on oocyte and preimplantation embryo resistance to increased temperature

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)