Translocation of Oocytic HES1 into Surrounding Cumulus Cells in Bovine: Mechanism of Cellular Interaction during IVM?

HES1 (hairy and enhancer of split-1, effector of the NOTCH pathway) plays a role in oocyte maturation and has been detected so far mainly in somatic follicular cells. In this study, we aimed to investigate whether HES1 is present in both compartments of bovine cumulus oocyte complexes (COCs) and whether in vitro maturation itself has an effect on its distribution. We investigated the abundance of HES1 mRNA and protein in bovine COCs characterized by Brilliant-Cresyl-Blue (BCB) stainability by RT-PCR and immunofluorescence before and after in vitro maturation (IVM).

Influence of long-term thermal stress on the maturation on embryo development and Heat Shock Protein abundance in zebu cattle.

The objective of this study was to investigate the influence of long-term temperature stress during the maturation (IVM) of oocytes on the embryo production (IVP) and the abundance of HSP70 and HSP90 in zebu cattle. Viable cumulus-oocyte complexes (COCs) were incubated for 24 h at 37 °C, 38.5 °C, or 40 °C for the low-, physiological, and high-temperature stress treatments, respectively.

Oocyte quality and heat shock proteins in oocytes from bovine breeds adapted to the tropics under different conditions of environmental thermal stress.

In order to evaluate the influence of thermal stress on physiological parameters, and the oocyte quality of Girolando (n = 12) and adapted Pantaneira (n = 12) cattle, twelve sessions of ultrasound guided follicular aspiration (OPU) were performed, between January and November 2014 (during dry (May-September) and rainy season (October-April) in Brazil). The recovered cumulus-oocyte complexes (COCs) were selected and classified, according to quality, immediately after OPU. The oocytes were then stored in 3% paraformaldehyd before conducting immunofluorescence analysis under confocal microscopy to identify HSP70 and 90 proteins.

A Natural Mutation in Helix 5 of the Ligand Binding Domain of Glucocorticoid Receptor Enhances Receptor-Ligand Interaction.

The glucocorticoid receptor (GR) is a central player in the neuroendocrine stress response; it mediates feedback regulation of the hypothalamus-pituitary-adrenal (HPA) axis and physiological actions of glucocorticoids in the periphery. Despite intensive investigations of GR in the context of receptor-ligand interaction, only recently the first naturally occurring gain-of-function substitution, Ala610Val, of the ligand binding domain was identified in mammals. We showed that this mutation underlies a major quantitative trait locus for HPA axis activity in pigs, reducing cortisol production by about 40-50 percent.

The margin of safety of a single application of transdermal fentanyl solution when administered at multiples of the therapeutic dose to laboratory dogs.

Previous studies have demonstrated that a single, topical application of a novel, long-acting transdermal fentanyl solution provides analgesic fentanyl concentrations for at least 4 days. The objective of this study was to describe the margin of safety following application at multiples of the therapeutic dose. Twenty-four laboratory dogs were administered a single placebo or 1×, 3×, or 5× multiple of the dose of 2.

The ketone body β-hydroxybutyric acid influences agouti-related peptide expression via AMP-activated protein kinase in hypothalamic GT1-7 cells.

β-Hydroxybutyric acid (BHBA) acts in the brain to influence feeding behaviour, but the underlying molecular mechanisms are unclear. GT1-7 hypothalamic cells expressing orexigenic agouti-related peptide (AGRP) were used to study the AMP-activated protein kinase (AMPK) pathway known to integrate dietary and hormonal signals for food intake regulation. In a 25 mM glucose culture medium, BHBA increased intracellular calcium concentrations and the expression of monocarboxylate transporter 1 (MCT1 (SLC16A1)).

Studies on mechanisms of interferon-gamma action in pancreatic cancer using a data-driven and model-based approach.

Background: Interferon-gamma (IFNγ) is a multifunctional cytokine with antifibrotic and antiproliferative efficiency. We previously found that pancreatic stellate cells (PSC), the main effector cells in cancer-associated fibrosis, are targets of IFNγ action in the pancreas. Applying a combined experimental and computational approach, we have demonstrated a pivotal role of STAT1 in IFNγ signaling in PSC.

The mycotoxins alternariol and alternariol methyl ether negatively affect progesterone synthesis in porcine granulosa cells in vitro.

Mycotoxins as contaminants of animal food can impair fertility in farm animals. In the regulation of female fertility the ovarian steroid hormone progesterone (P(4)) plays an important role. In the present study we have investigated the influence of the mycotoxins alternariol (AOH), alternariol mono-methyl ether (AME), and tenuazonic acid (TeA) on cell viability, P(4) synthesis, abundance of the key enzymes of P(4) synthesis, P450 cholesterol side-chain cleavage enzyme (P450SCC) and 3-beta-hydroxysteroid dehydrogenase (3-beta-HSD), and of the corresponding Cyp11a1 and Hsd3b transcripts in cultured pig granulosa cells.

Galectin-1 induced activation of the mitochondrial apoptotic pathway: evidence for a connection between death-receptor and mitochondrial pathways in human Jurkat T lymphocytes.

Galectin-1 (gal-1) triggers T cell death by several distinct intracellular pathways including the activation of the death-receptor pathway. The aim of this study was to investigate whether gal-1 induced activation of the death-receptor pathway in Jurkat T lymphocytes mediates apoptosis via the mitochondrial pathway linked by truncated Bid (tBid). We demonstrate that gal-1 induced proteolytic cleavage of the death agonist Bid, a member of the Bcl-2/Bcl-xL family and a substrate of activated caspase-8, was inhibited by caspase-8 inhibitor II (Z-IETD-FMK).

The effect of feeding a diet naturally contaminated with deoxynivalenol (DON) and zearalenone (ZON) on the spleen and liver of sow and fetus from day 35 to 70 of gestation.

Pregnant sows were fed a control diet (CON, 0.15 mg deoxynivalenol (DON) and 0.0035 mg zearalenone (ZON) per kg diet) or diet containing 15% of Fusarium toxin contaminated triticale (MYCO, 4.

Changes in the spleen and liver of pregnant sows and full-term piglets after feeding diets naturally contaminated with deoxynivalenol and zearalenone.

Wheat contaminated naturally with the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZON) was fed to pregnant Landrace sows for 35days. On day 110, caesarean section was carried out, the offspring were killed immediately after birth, and their livers and spleens examined. At necropsy there were no macroscopic lesions observed in any organ of either sows or piglets.

Influence of diets with cereal grains contaminated by graded levels of two Fusarium toxins on selected enzymatic and histological parameters of liver in gilts.

Feeding experiments with diets containing Fusarium toxin-contaminated wheat were conducted to clarify the pathogenesis of enzymatic and histopathological effects of Fusarium toxins on porcine liver cells. A total of 36 prepuberal gilts were divided into four groups and fed diets with increasing proportions of Fusarium toxin-contaminated wheat at a total wheat proportion of 40% over a period of 35 days. The concentrations of the indicator toxins deoxynivalenol (DON) and zearalenone (ZON) which were analyzed by HPLC methods were 210/4, 3070/88, 6100/235, and 9570/358 microg/kg in the diets fed to groups I-IV, respectively.

Effects of diets with cereal grains contaminated by graded levels of two Fusarium toxins on selected immunological and histological measurements in the spleen of gilts.

Feeding experiments with diets containing Fusarium toxin-contaminated wheat were conducted to clarify the pathogenesis of immunological effects of Fusarium toxins to porcine spleen cells. Contaminated diets were fed to 36 Landrace prepubertal gilts for 35 d. Concentrations (as-fed basis) of the indicator toxins deoxynivalenol (DON) and zearalenone (ZON), respectively, were 210 and 4 (control--group I), 3,070 and 88 (group II), 6,100 and 235 (group III), and 9,570 and 358 microg/kg (group IV).

Influence of ovarian steroid hormones or platelet-activating factor on mRNA of platelet-activating factor receptor in endometrial explant perfusion cultures from ovariectomized bovine.

Platelet-activating factor (PAF) and its receptors are involved in inflammatory-like processes of the uterus associated with increased vascular permeability. PAF is supposed to be influenced by ovarian steroid hormones. The present study was undertaken to examine whether progesterone (P(4)), estradiol (E(2)) or PAF influence the PAF receptor gene expression in perfused endometrial explants derived from ovariectomized bovine.

Promoter-2-derived Cyp19 expression in bovine granulosa cells coincides with gene-specific DNA hypo-methylation.

Cyp19, the key gene of oestrogen biosynthesis, is expressed at very different concentrations and from different promoters in bovine granulosa cells (GCs) and in pregnant corpora lutea (CL), respectively. The present study was aimed to investigate if DNA methylation and thus epigenetic mechanisms might play a potential role in the regulation of Cyp19 expression and promoter-specific activity in GCs of cycling versus CL of pregnant cows. It was demonstrated that GCs express high concentrations of promoter-2-derived Cyp19 transcripts whereas CL samples isolated before and after implantation, and at the end of the first trimester, showed very low Cyp19 transcript concentrations, all of them derived from promoter 1.

Effects of galectin-1 on regulation of progesterone production in granulosa cells from pig ovaries in vitro.

The detection of galectin-1 (gal-1) in pig granulosa cell lysates by immunoblotting and its cytosolic as well as membrane-associated localization prompted us to study its effects on cell proliferation and regulation of progesterone synthesis. The lectin stimulated the proliferation of granulosa cells from pig ovaries cultured in serum-free medium. Gal-1 inhibited the FSH-stimulated progesterone synthesis of granulosa cells.

Mitochondrial aggregation patterns and activity in porcine oocytes and apoptosis in surrounding cumulus cells depends on the stage of pre-ovulatory maturation.

In this study, we evaluated the distribution and oxidative activity of mitochondria in ex vivo pre-ovulatory porcine oocytes using the fluorescence probe MitoTracker CMTM Ros Orange. Cumulus-oocyte complexes (COCs) were classified according to cumulus morphology and time from hCG administration. The meiotic configuration of the oocytes and the degree of apoptosis in the surrounding cumulus cells were also evaluated.

Expression of the bovine aromatase cytochrome P450 gene (Cyp19) is primarily regulated by promoter 2 in bovine follicles and by promoter 1.1 in corpora lutea.

Follicular development and differentiation are closely associated with increasing steroidogenesis. During the present study transcript concentration of Cyp19, Cyp11A1, and 3beta-hydroxysteroid dehydrogenase delta (3beta-HSD) encoding the steroidogenic enzymes P450(arom), P450(SCC), and 3beta-HSD were determined by real-time PCR in bovine granulosa cells (GC) as potential markers for follicular differentiation. Ovaries were collected from a local abattoir (experiment 1) and from synchronized animals at day 4 of estrus cycle (experiment 2).

Forskolin-induced cyclic AMP signaling in single adherent bovine oviductal cells: effect of dichlorodiphenyltrichloroethane (DDT) and tris(4-chlorophenyl)methanol (TCPM).

The influence of tris(4-chlorophenyl)methanol (TCPM) and dichlorodiphenyltrichloroethane (o,p'DDT) on forskolin induced cAMP signalling in single adherent bovine oviductal cells was investigated. An increase in the intracellular cAMP levels was measured indirectly by an increase in the 520/580 nm fluorescence emission ratio of the protein kinase A fluorosensor (FICRhR). FICRhR was microinjected into single cells, and the 520/580 nm fluorescence emission ratio was monitored by image cytometry with an image analysis system as a measure of intracellular cAMP concentration ([cAMP](i)).

Superovulation in cattle: practical aspects of gonadotropin treatment and insemination.

Embryo transfer (ET) in cattle has been used for the realisation of breeding programmes world-wide for more than 20 years. The efficiency of breeding technology, i.e.

Influence of tris(4-chlorophenyl)methanol (TCPM) on gap junction-mediated intercellular communication of cultured bovine granulosa cells.

Tris(4-chlorophenyl)methanol (TCPM) is a by-product in the manufacture of technical grade DDT, which is known to alter properties and functions of the female reproductive system. We investigated whether in vitro TCPM has an influence on the function of gap junction-mediated intercellular communication (GJIC) and gap junction protein expression of connexin 43 (Cx43) in cultured bovine granulosa cells. GJIC was assessed by fluorescent dye microinjection (dye-coupling).

Fluorometric detection of platelet activating factor receptor in cultured oviductal epithelial and stromal cells and endometrial stromal cells from bovine at different stages of the oestrous cycle and early pregnancy.

During the oestrous cycle and early pregnancy, the oviduct and uterus undergo a variety of morphological and physiological modifications in which the platelet activating factor receptor (PAF-R) plays an important role. PAF-R levels were quantified in bovine oviductal epithelial and stromal cells and endometrial stromal cells at days 2 to 4, 12, and 20 of the estrous cycle and during early pregnancy. Cells were grown in vitro and their intracellular PAF-R concentration was measured by flow cytometry using a polyclonal anti-PAF-R antibody system.

Platelet-activating factor (PAF)-like activity, localization of PAF receptor (PAF-R) and PAF-acetylhydrolase (PAF-AH) activity in bovine endometrium at different stages of the estrous cycle and early pregnancy.

PAF-like activity in the endometrium increased from days 2-4 to day 12 and day 20 in both cyclic and pregnant cows. There was an increase in platelet aggregation induced by PAF-like activity in the endometrium of pregnant animals on day 20 as compared to cyclic animals at the same point in time. Two major bands of PAF-R protein at 67 kDa and 97 kDa were detected by Western blot analysis.

Influence of inhibitors on increase in intracellular free calcium and proliferation induced by platelet-activating factor in bovine oviductal cells.

Oviductal endosalpingeal cells were isolated mechanically from heifers and cultured until there was 100% confluency. The cells were loaded with the Ca(2+)-sensitive fluorochrome, fura-2/acetoxymethylester, and cytosolic free calcium ([Ca2+]i) was monitored by spectrofluorimetry. Platelet-activating factor, at a concentration of 30 nmol l-1, induced an intracellular Ca2+ increase in cultured bovine oviductal cells, mainly via influx from the extracellular space.