Torsten Viergutz

Influence of 50 Hz electromagnetic fields in combination with a tumour promoting phorbol ester on protein kinase C and cell cycle in human cells

It still is an unsolved issue whether exposure to power-line frequency electromagnetic fields (EMF) may promote carcinogenesis and if so whether it does so by influencing the proliferation, the survival, and the differentiation of cells. Since the family of protein kinases C (PKC) takes part in these processes by interacting with signal transduction pathways at several levels including the activation of transcription factors, we evaluated in the present study the effects of exposure of human amniotic fluid cells (AFC) to 50Hz, 1 mT electromagnetic fields (EMF) alone and in combination with the tumour promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on the subcellular localization of PKC protein, on PKC enzyme activity, and on the cell cycle distribution.Quantitative analyses of the PKC expression pattern demonstrated the translocation of PKC from the cytosolic to the membrane fraction after exposure to 10, 50, 100nM, and 1 μM TPA. EMF exposure alone showed no effect on PKC translocation. Co-exposure to 10, 50, and 100nM TPA and 1 mT EMF revealed a significant additive effect (25 ± 50, 66 ± 29, 22 ± 50%, respectively) with the most prominent increase at the concentration of 50nM TPA. At the highest concentration of TPA used (1 μM) no additive effect of EMF could be observed. Data on enzymatic activity indicate that EMF modulate the PKC activity, showing a significant increase of 10 ± 16% in total PKC activity after co-exposure to 50nM TPA and 1 mT EMF when compared to 50nM TPA alone. Flow cytometric analyses showed a transient cell cycle arrest in G0/G1-phase followed by a delayed transit through S-phase in response to TPA, which was, however, not enhanced by co-exposure with EMF. We conclude that in AFC cells TPA at lower concentrations (≤ 100nM) induces a less than maximum effect on the PKC pathway, which can be enhanced by the applied EMF.

Effects of Dietary Isoflavones on Proliferation and DNA Integrity of Myoblasts Derived from Newborn Piglets

Soy-based formulas are consumed by growing numbers of infants and used as regular food supplements in livestock production. Moreover, constituent dietary phytoestrogens may compete with endogenous estrogens and affect individual growth. This study aimed to investigate the in vitro effects of isoflavones in comparison with estrogens on the proliferation of porcine satellite cells derived from neonatal muscle. After 7 h of exposure in serum-free medium, 17β-estradiol (1 nM, 1 μM), estrone (1 μM), and daidzein (1, 100 μM) slightly decreased whereas 100 μM genistein substantially lowered DNA synthesis. Declines in DNA amount were observed with genistein (1, 100 μM) and daidzein (100 μM). After 26 h of exposure, 100 μM genistein reduced DNA synthesis, whereas it was increased by 10 μM genistein and 10 and 100 μM daidzein. In the case of 10 μM genistein and 100 μM daidzein, these increases apparently resulted from the repair of damaged DNA. Genistein and daidzein (100 μM) reduced protein synthesis, caused a G2/M phase block, and decreased DNA amount in association with higher rates of cell death partially resulting from apoptosis. Conclusively, isoflavones at concentrations of greater than 1 μM act as inhibitors of porcine skeletal muscle cell proliferation.

N-acetylcysteine impairs survival of luteal cells through mitochondrial dysfunction.

N‐acetylcysteine (NAC) is known as an antioxidant and used for mucus viscosity reduction. However, this drug prevents or induces cell death depending on the cell type. The response of steroidogenic luteal cells to NAC is unknown. Our data shows that NAC can behave as an antioxidant or prooxidant in dependency on the concentration and mitochondrial energization. NAC elevated the flowcytometric‐measured portion of hypodiploid (dying) cells. This rise was completely abolished by aurintricarboxylic acid, an inhibitor of topoisomerase II. NAC increased the secretion of nitric oxide and cellular nitrotyrosine. An image analysis indicated that cells pretreated with NAC and loaded with DHR showed a fluorescent structure probably elicited by the oxidative product of DHR, rhodamine 123 that sequesters mitochondrially. Pretreating luteal cells with NAC or adding NAC directly to mitochondrial fractions followed by assessing the mitochondrial transmembrane potential difference (Δψ) by the JC‐1 technique demonstrated a marked decrease in Δψ. A protonophore restored Δψ and rotenone (an inhibitor of respiratory chain complex I) inhibited mitochondrial recovering. Thus, in steroidogenic luteal cells from healthy mature corpus luteum, NAC impairs cellular survival by interfering with mitochondrial metabolism. The protonophore‐induced recovering of NAC‐provoked decrease in Δψ indicates that an ATP synthase‐favored route of H+ re‐entry to the matrix is essentially switched off by NAC while other respiratory chain complexes remain intact. These data may be important for therapeutic timing of treatments with NAC.

Proteome and radioimmunoassay analyses of pituitary hormones and proteins in response to feed restriction of dairy cows

The hypothalamic‐pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2‐DE followed by MALDI‐TOF‐MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non‐esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine‐rich element‐binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co‐transcribed homolog, peroxiredoxin III, β‐tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood.

Oleic acid induces specific alterations in the morphology, gene expression and steroid hormone production of cultured bovine granulosa cells

After parturition, one of the major problems related to nutritional management that is faced by the majority of dairy cows is negative energy balance (NEB). During NEB, excessive lipid mobilization takes place and hence the levels of free fatty acids, among them oleic acid, increase in the blood, but also in the follicular fluid. This accumulation can be associated with serious metabolic and reproductive disorders. In the present study, we analyzed the effects of physiological concentrations of oleic acid on cell morphology, apoptosis, necrosis, proliferation and steroid production, and on the abundance of selected transcripts in cultured bovine granulosa cells. Increasing oleic acid concentrations induced intracellular lipid droplet accumulation, thus resulting in a foam cell-like morphology, but had no effects on apoptosis, necrosis or proliferation. Oleic acid also significantly reduced the transcript abundance of the gonadotropin hormone receptors, FSHR and LHCGR, steroidogenic genes STAR, CYP11A1, HSD3B1 and CYP19A1, the cell cycle regulator CCND2, but not of the proliferation marker PCNA. In addition, treatment increased the transcript levels of the fatty acid transporters CD36 and SLC27A1, and decreased the production of 17-beta-estradiol and progesterone. From these data it can be concluded that oleic acid specifically affects morphological and physiological features and gene expression levels thus altering the functionality of granulosa cells. Suggestively, these effects might be partly due to the reduced expression of FSHR and thus the reduced responsiveness to FSH stimulation.

Anti-Muellerian hormone levels in plasma of Holstein-Friesian heifers as a predictive parameter for ovum pick-up and embryo production outcomes

The aim of this study was to investigate whether plasma anti-Muellerian hormone (AMH) levels of Holstein-Friesian heifers could be used to predict ovum pick-up (OPU) and embryo production outcomes. Plasma samples and data were collected from 64 heifers, which underwent repeated OPU with subsequent in vitro embryo production followed by embryo flushing after superovulation. AMH levels were significantly positively correlated with the number of follicles aspirated per OPU session (r = 0.45), recovered oocytes per OPU (r =0.43) and in vitro produced embryos per OPU (r = 0.28). No significant correlations between AMH and in vivo produced embryos were ascertained. Our results suggest that correlations between AMH and outcomes of an OPU-IVF program are too low to use AMH as a precise predictive parameter for the success of a particular OPU procedure in Holstein-Friesian heifers. However, AMH can help to identify groups of very good or very poor oocyte donors.

LOX-1 regulates estrogenesis via intracellular calcium release from bovine granulosa cells.

Estradiol produced by ovarian granulosa cells triggers the luteinizing hormone surge which in turn initiates ovulation in female mammals. Disturbances in estradiol production from granulosa cells are a major reason for reproductive dysfunctions in dairy cows. Endogenous estradiol production might be altered by reactive oxygen species (ROS) such as oxidized low‐density lipoprotein (ox‐LDL). Inhibition of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), a receptor of ox‐LDL, leads to increased estrogenesis in granulosa cells. This activity is mediated by calcium release from endoplasmic reticulum (ER)‐dependent and ER‐independent calcium pools. Inhibition of the LOX‐1 signal transduction pathway is followed by mitochondrial alterations. The membrane potential ΔΨ increases and the ROS production decreases in mitochondria after blocking LOX‐1. Our data indicate that blocking the LOX‐1 receptor signal pathway might be a promising way to improve steroid hormone concentrations in metabolically highly active female mammals and, therefore, to defend against reproductive dysfunctions in humans and animals.

Postpartum levels of 8-iso-prostaglandin F2α in plasma and milk phospholipid fractions as biomarker of oxidative stress in first-lactating dairy cows

F2-isoprostanes such as 8-iso-prostaglandin F2 (8-iso-PGF2α) are formed by free radical-catalyzed mechanisms from membrane phospholipids and from low density lipoproteins through peroxidation of arachidonic acid. Esterified 8-iso-PGF2α is cleaved by phospholipases, circulates in blood and is excreted as putatively harmful oxidatively modified lipid via the kidney into urine. In this study we demonstrate that 8-iso-PGF2α concentrations in plasma samples from heifers are higher (p<0.005) compared to those from first-lactating dairy cows at 71 days postpartum. Furthermore, plasma 8-iso-PGF2α concentrations vary with ovarian activity and differ in response to luteolytic initiation as well as activation of the hypothalamic-pituitary-gonadal axis between heifers and first-lactating cows. Sustainable concentrations of 8-iso-PGF2α (50-150 pg/ml) are detectable in the phospholipid fraction of milk, suggesting milk as an additional excretion route for 8-isoprostanes. Plasma levels largely paralleled levels in milk (p<0.001). Plasma phospholipid 8-iso-PGF2α concentrations in cyclic cows decreased (p<0.05) from day 38 to day 71 postpartum, whereas milk phospholipid 8-iso-PGF2α rather increased (p<0.05). Cyclic cows tend to have higher 8-isoprostane levels compared to acyclic animals. In contrast to lipohydroperoxides, concentration of 8-iso-PGF2α were not correlated with milk yield (p>0.05). Our data indicate 8-iso-PGF2α may be a novel biomarker of oxidative stress in dairy cow, which is detectable in blood as well as in milk.

Modulation of vH+-ATPase is part of the functional adaptation of sheep rumen epithelium to high-energy diet.

Ruminal vacuolar H(+)-ATPase (vH(+)-ATPase) activity is regulated by metabolic signals. Thus, we tested whether its localization, expression, and activity were changed by different feeding. Young male sheep (n = 12) were either fed hay ad libitum (h) or hay ad libitum plus additional concentrate (h/c) for 2 wk. The vH(+)-ATPase B subunit signal was predominantly found in the cell membrane and cytosol of rumen epithelial cells (REC) with basal/parabasal phenotype. The elevated number (threefold) of these cells in rumen mucosa of h/c-fed sheep reflects a high proliferative capacity and, explains the 2.3-fold increase of the total number of vH(+)-ATPase-expressing REC. However, in accordance with a 58% reduction of the vH(+)-ATPase B subunit mRNA expression in h/c-fed sheep, its protein amount per single REC was decreased. Using the fluorescent probe BCECF and selective inhibitors (foliomycin, amiloride), the contribution of vH(+)-ATPase and Na(+)/H(+) exchanger to intracellular pH (pH(i)) regulation was investigated. REC isolated from h/c-fed sheep keep their pH(i) at a significantly higher level (6.91 ± 0.03 vs. 6.74 ± 0.05 in h-fed sheep). Foliomycin or amiloride decreased pH(i) by 0.16 ± 0.02 and 0.57 ± 0.04 pH units when applied to REC from h-fed sheep, but the effects were markedly reduced (-88 and -33%) after concentrate feeding. Nevertheless, we found that REC proliferation rate and [cAMP](i) were reduced after foliomycin-induced vH(+)-ATPase inhibition. Our results provide the first evidence for a role of vH(+)-ATPase in regulation of REC proliferation, most probably by linking metabolically induced pH(i) changes to signaling pathways regulating this process.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) activity decreases estrogenesis in ovarian granulosa cells

THE reproductive cycle in mammalian females is initiated by a surge of luteinizing hormone (LH). It is secreted from the pituitary gland in response to hypothalamic gonadotropin releasing hormone (GnRH). An increase in the activity of GnRH neurons predominantly results from rising secretion of estrogens from granulosa cells of the maturing ovarian follicle. The transient exposure to intense LH amplitudes instigates changes in the follicular expression of genes which are essential for the release or expulsion of the oocyte (ovulation). Prerequisite for its fertilization is a rupture of the apical follicle wall. The LH surge ceases (dependent on the species almost or completely) the proliferation and estrogenesis of mural granulosa cells while activates pathways also evident in challenging inflammatory response, such as the expression of cyclooxygenase-2 and the expression of the genomic progesterone receptor. It acts as a transcription factor that stimulates the transcription of genes which encode proteases capable of hydrolyzing follicular wall matrix, including cathepsin L and metalloproteinases such as ADAMTS. The transcription factor is also involved in the expression of the epidermal growth factor superfamily signaling for expansion of the granulosal cumulus cells (1). Recently, receptors known to play a role in the innate immunity, such as Toll-like 4 (2), and in an inflammatory process, including LOX-1 (3), have been identified in subpopulations of mural granulosa cells. Toll-like 4 receptor belongs to pattern-recognition receptor which have the capacity to recognize conserved motifs in complexes of lipids, proteins, and nucleic acids in contrast to selective antigen recognition by cells of the adaptive immunity. Expression and functionality of LOX-1 have been extensively studied in endothelial cells. They respond to LOX-1 activators, such as oxidized low-density lipoprotein (oxLDL), with raising the formation of reactive oxygen metabolites (ROM). The increase amplifies oxLDL binding and LOX-1 expression. An expression of LOX-1 has been recently detected in human ovarian follicular granulosa cells and LOX-1 has been reported to be involved in the granulosal reparative autophagy (4). Our analyses confirmed the LOX-1 expression using mural granulosa cells from bovine follicles. They were induced by a GnRH analogue after regressing the corpus luteum as described elsewhere (5). The concentration of transcripts was quantified by RT-PCR using the I-script-I-cycler technique (Biorad, Hamburg, Germany). LOX-1 protein was detected by immunochemistry and immunocytofluorimetry using a selective rabbit antibody against LOX-1 (LOX-1 AB) (BioVision, San Francisco, CA, USA), an anti-rabbit Alexa 488-conjugated antibody (Invitrogen, Darmstadt, Germany), and single cell analysis (Gallios, Beckman-Coulter, Krefeld, Germany) on the analogy of a described approach (6,7). We found an increase in LOX-1 mRNA in response to LH (5 ng/ml) following a culture (4 h) of granulosa cells in serum-free Mega cell medium (Sigma, Deisenhofen, Germany) and a correlation between the mRNA level and the LOX-1 protein (r 5 0.8, P < 0.05). A decline in mRNA for LOX-1 was observed in granulosa cells freshly prepared 20 h post GnRH (late ovulatory phase) in comparison with cells from follicles sampled 5 h post GnRH. These data are indicative of gonadotropin-dependent LOX-1 expression. To test the functionality of LOX-1, the granulosa cells from the late ovulatory phase were exposed to the selective rabbit antibody to LOX-1 (1.5 or 3.0 lg/ml). In comparison with an unspecific rabbit antibody (control), the mRNA for