Estrous cycle-dependent changes of Fas expression in the bovine corpus luteum: influence of keratin 8/18 intermediate filaments and cytokines

Background Fas expression and Fas-induced apoptosis are mechanisms attributed to the selective destruction of cells of the corpus luteum (CL) during luteal regression. In certain cell-types, sensitivity to these death-inducing mechanisms is due to the loss or cleavage of keratin-containing intermediate filaments. Specifically, keratin 8/18 (K8/K18) filaments are hypothesized to influence cell death in part by regulating Fas expression at the cell surface. Methods Here, Fas expression on bovine luteal cells was quantified by flow cytometry during the early (Day 5, postovulation) and late stages (Days 16–18, postovulation) of CL function, and the relationship between Fas expression, K8/K18 filament expression and cytokine-induced cell death in vitro was evaluated. Results Both total and cell surface expression of Fas on luteal cells was greater for early versus late stage bovine CL (89% vs. 44% of cells for total Fas; 65% vs.18% of cells for cell surface Fas; respectively, P0.05, n=4 CL/stage), despite evidence these conditions increased Fas expression on HepG2 cells (P0.05) or stage of CL (P\u3e0.05, n= 4 CL/stage) on this outcome. Conclusion In conclusion, we rejected our null hypothesis that the cell surface expression of Fas does not differ between luteal cells of early and late stage CL. The results also did not support the idea that K8/K18 filaments influence the expression of Fas on the surface of bovine luteal cells. Potential downstream effects of these filaments on death signaling, however, remain a possibility. Importantly, the elevated expression of Fas observed on cells of early stage bovine CL compared to late stage bovine CL raises a provocative question concerning the physiological role(s) of Fas in the corpus luteum, particularly during early luteal development

Evaluating the impact of the hexosamine biosynthesis pathway and O-GlcNAcylation on glucose metabolism in bovine granulosa cells

Granulosa cells (GCs) of ovarian follicles prefer glucose as a metabolic substrate for growth and maturation. Disruption of glucose utilization via the hexosamine biosynthesis pathway (HBP) impairs O-linked N-acetylglucosaminylation (O-GlcNAcylation) and inhibits proliferation of bovine GCs of both small (3–5 mm) and large (\u3e8.5 mm) antral follicles. Knowing that 2–5% of all glucose in cells is utilized via the HBP, the aim of this study was to characterize glucose metabolism in bovine GCs and determine the impact of the HBP and O-GlcNAcylation on metabolic activity. The GCs were initially cultured in serum-containing medium to confluency and then sub-cultured in serum-free medium in 96 well plates (n = 10 ovary pairs). The cells were exposed to vehicle and inhibitors of the HBP and O-GlcNAcylation for 24 h. Extracellular acidification rate (ECAR; an indicator of glycolysis) and oxygen consumption rate (OCR; an indicator of oxidative phosphorylation) of the GCs were measured using a Seahorse xFe96 Analyzer, including the implementation of glycolytic and mitochondrial stress tests. GCs from small antral follicles exhibited overall greater metabolic activity than GCs from large antral follicles as evidenced by increased ECAR and OCR. Inhibition of the HBP and O-GlcNAcylation had no effect on the metabolic activity of GCs from either type of follicle. The glycolytic stress test indicated that GCs from both types of follicles possessed additional glycolytic capacity; but again, inhibition of the HBP and O-GlcNAcylation did not affect this. Interestingly, inhibition of cellular respiration by 2-Deoxy-D-glucose impaired OCR only in GCs from small antral follicles, but exposure to the mitochondrial stress test had no effect. Conversely, in GCs from large antral follicles, oxidative metabolism was impaired by the mitochondrial stress test and was accompanied by a concomitant increase in glycolytic metabolism. Immunodetection of glycolytic enzymes revealed that phosphofructokinase expression is increased in GCs of small antral follicles compared to large follicles. Inhibition of O-GlcNAcylation impaired the expression of hexokinase only in GCs of small antral follicles. Inhibition of O-GlcNAcylation also impaired the expression of phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase in GCs of both types of follicles, but had no effect on the expression of lactate dehydrogenase. The results indicate that GCs of small antral follicles possess greater aerobic glycolytic capacity than GCs from large antral follicles; but disruption of the HBP and O-GlcNAcylation has little to no impact on metabolic activity

Chemokines in the corpus luteum: Implications of leukocyte chemotaxis

Chemokines are small molecular weight peptides responsible for adhesion, activation, and recruitment of leukocytes into tissues. Leukocytes are thought to influence follicular atresia, ovulation, and luteal function. Many studies in recent years have focused attention on the characterization of leukocyte populations within the ovary, the importance of leukocyte-ovarian cell interactions, and more recently, the mechanisms of ovarian leukocyte recruitment. Information about the role of chemokines and leukocyte trafficking (chemotaxis) during ovarian function is important to understanding paracrine-autocrine relationships shared between reproductive and immune systems. Recent advances regarding chemokine expression and leukocyte accumulation within the ovulatory follicle and the corpus luteum are the subject of this mini-review

Molecular manipulation of keratin 8/18 intermediate filaments: modulators of FAS-mediated death signaling in human ovarian granulosa tumor cells

Background: Granulosa cell tumors (GCT) are a rare ovarian neoplasm but prognosis is poor following recurrence. Keratin intermediate filaments expressed in these tumors are a diagnostic marker, yet paradoxically, may also constitute a target for therapeutic intervention. In the current study, we evaluated keratin 8/18 (K8/18) filament expression as a mechanism of resistance to apoptosis in GCT, specifically focusing on regulation of the cell surface death receptor, Fas (FAS). Methods: The GCT cell line, KGN, was transiently transfected with siRNA to KRT8 and KRT18 to reduce K8/18 filament expression. Expression of K8/18, FAS, and apoptotic proteins (PARP, cleaved PARP) were evaluated by fluorescence microscopy, flow cytometric analysis, and immunoblotting, respectively. The incidence of FAS-mediated apoptosis in KGN cells was measured by caspase 3/7 activity. All experiments were performed independently three to six times, using a fresh aliquot of KGN cells for each experiment. Quantitative data were analyzed by one- or two-way analysis of variance (ANOVA), followed by a Tukey’s post-test for multiple comparisons; differences among means were considered statistically significant at P \u3c 0.05. Results: Control cultures of KGN cells exhibited abundant K8/18 filament expression (~90 % of cells), and minimal expression of FAS (\u3c25 % of cells). These cells were resistant to FAS-activating antibody (FasAb)-induced apoptosis, as determined by detection of cleaved PARP and measurement of caspase 3/7 activity. Conversely, siRNA-mediated knock-down of K8/18 filament expression enhanced FAS expression (\u3e 70 % of cells) and facilitated FasAb-induced apoptosis, evident by increased caspase 3/7 activity (P \u3c 0.05). Additional experiments revealed that inhibition of protein synthesis, but not MEK1/2 or PI3K signaling, also prompted FasAb-induced apoptosis. Conclusions: The results demonstrated that K8/18 filaments provide resistance to apoptosis in GCT by impairing FAS expression. The abundance of keratin filaments in these cells and their role in apoptotic resistance provides a greater mechanistic understanding of ovarian tumorgenicity, specifically GCT, as well as a clinically-relevant target for potential therapeutic intervention

Nitroimidazole Action in Entamoeba histolytica: A Central Role for Thioredoxin Reductase

Metronidazole, a 5-nitroimidazole drug, has been the gold standard for several decades in the treatment of infections with microaerophilic protist parasites, including Entamoeba histolytica. For activation, the drug must be chemically reduced, but little is known about the targets of the active metabolites. Applying two-dimensional gel electrophoresis and mass spectrometry, we searched for protein targets in E. histolytica. Of all proteins visualized, only five were found to form adducts with metronidazole metabolites: thioredoxin, thioredoxin reductase, superoxide dismutase, purine nucleoside phosphorylase, and a previously unknown protein. Recombinant thioredoxin reductase carrying the modification displayed reduced enzymatic activity. In treated cells, essential non-protein thiols such as free cysteine were also affected by covalent adduct formation, their levels being drastically reduced. Accordingly, addition of cysteine allowed E. histolytica to survive in the presence of otherwise lethal metronidazole concentrations and reduced protein adduct formation. Finally, we discovered that thioredoxin reductase reduces metronidazole and other nitro compounds, suggesting a new model of metronidazole activation in E. histolytica with a central role for thioredoxin reductase. By reducing metronidazole, the enzyme renders itself and associated thiol-containing proteins vulnerable to adduct formation. Because thioredoxin reductase is a ubiquitous enzyme, similar processes could occur in other eukaryotic or prokaryotic organisms

Anti-filarial Activity of Antibiotic Therapy Is Due to Extensive Apoptosis after Wolbachia Depletion from Filarial Nematodes

Filarial nematodes maintain a mutualistic relationship with the endosymbiont Wolbachia. Depletion of Wolbachia produces profound defects in nematode development, fertility and viability and thus has great promise as a novel approach for treating filarial diseases. However, little is known concerning the basis for this mutualistic relationship. Here we demonstrate using whole mount confocal microscopy that an immediate response to Wolbachia depletion is extensive apoptosis in the adult germline, and in the somatic cells of the embryos, microfilariae and fourth-stage larvae (L4). Surprisingly, apoptosis occurs in the majority of embryonic cells that had not been infected prior to antibiotic treatment. In addition, no apoptosis occurs in the hypodermal chords, which are populated with large numbers of Wolbachia, although disruption of the hypodermal cytoskeleton occurs following their depletion. Thus, the induction of apoptosis upon Wolbachia depletion is non-cell autonomous and suggests the involvement of factors originating from Wolbachia in the hypodermal chords. The pattern of apoptosis correlates closely with the nematode tissues and processes initially perturbed following depletion of Wolbachia, embryogenesis and long-term sterilization, which are sustained for several months until the premature death of the adult worms. Our observations provide a cellular mechanism to account for the sustained reductions in microfilarial loads and interruption of transmission that occurs prior to macrofilaricidal activity following antibiotic therapy of filarial nematodes

Contributions of Mamu-A*01 Status and TRIM5 Allele Expression, But Not CCL3L Copy Number Variation, to the Control of SIVmac251 Replication in Indian-Origin Rhesus Monkeys

CCL3 is a ligand for the HIV-1 co-receptor CCR5. There have recently been conflicting reports in the literature concerning whether CCL3-like gene (CCL3L) copy number variation (CNV) is associated with resistance to HIV-1 acquisition and with both viral load and disease progression following infection with HIV-1. An association has also been reported between CCL3L CNV and clinical sequelae of the simian immunodeficiency virus (SIV) infection in vivo in rhesus monkeys. The present study was initiated to explore the possibility of an association of CCL3L CNV with the control of virus replication and AIDS progression in a carefully defined cohort of SIVmac251-infected, Indian-origin rhesus monkeys. Although we demonstrated extensive variation in copy number of CCL3L in this cohort of monkeys, CCL3L CNV was not significantly associated with either peak or set-point plasma SIV RNA levels in these monkeys when MHC class I allele Mamu-A*01 was included in the models or progression to AIDS in these monkeys. With 66 monkeys in the study, there was adequate power for these tests if the correlation of CCL3L and either peak or set-point plasma SIV RNA levels was 0.34 or 0.36, respectively. These findings call into question the premise that CCL3L CNV is important in HIV/SIV pathogenesis

All Our Babies Cohort Study: recruitment of a cohort to predict women at risk of preterm birth through the examination of gene expression profiles and the environment

<p>Abstract</p> <p>Background</p> <p>Preterm birth is the leading cause of perinatal morbidity and mortality. Risk factors for preterm birth include a personal or familial history of preterm delivery, ethnicity and low socioeconomic status yet the ability to predict preterm delivery before the onset of preterm labour evades clinical practice. Evidence suggests that genetics may play a role in the multi-factorial pathophysiology of preterm birth. The All Our Babies Study is an on-going community based longitudinal cohort study that was designed to establish a cohort of women to investigate how a women's genetics and environment contribute to the pathophysiology of preterm birth. Specifically this study will examine the predictive potential of maternal leukocytes for predicting preterm birth in non-labouring women through the examination of gene expression profiles and gene-environment interactions.</p> <p>Methods/Design</p> <p>Collaborations have been established between clinical lab services, the provincial health service provider and researchers to create an interdisciplinary study design for the All Our Babies Study. A birth cohort of 2000 women has been established to address this research question. Women provide informed consent for blood sample collection, linkage to medical records and complete questionnaires related to prenatal health, service utilization, social support, emotional and physical health, demographics, and breast and infant feeding. Maternal blood samples are collected in PAXgene™ RNA tubes between 18-22 and 28-32 weeks gestation for transcriptomic analyses.</p> <p>Discussion</p> <p>The All Our Babies Study is an example of how investment in clinical-academic-community partnerships can improve research efficiency and accelerate the recruitment and data collection phases of a study. Establishing these partnerships during the study design phase and maintaining these relationships through the duration of the study provides the unique opportunity to investigate the multi-causal factors of preterm birth. The overall All Our Babies Study results can potentially lead to healthier pregnancies, mothers, infants and children.</p