We found that incubation with either LPS or IL-1 β elicited a dose-dependent increase in both TIC viability and androgen production. Therefore, we evaluated the effects of the inflammatory agents lipopolysaccharide (LPS) and IL-1 β on rat theca-interstitial cells (TICs). However, the mechanistic basis for this linkage is unknown. An increasing body of evidence has pointed to a close association between PCOS and low-grade chronic systemic inflammation. MagMAX CORE Lysis Solution 1 50 mL 275 mL 1530C (room temperature) MagMAX CORE Binding Solution 45 mL 220 mL MagMAX CORE Wash Solution 1 60 mL 300 mL MagMAX CORE Wash Solution 2 60 mL 300 mL MagMAX CORE Elution Buffer 12 mL 55 mL MagMAX CORE Magnetic Beads 2.2 mL 11 mL MagMAX CORE Proteinase K (20 mg/mL) 1.25 mL 5 mLPolycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder characterized by theca cell hyperplasia and excessive androgen production.Gene ontology and pathway analysis revealed that both LPS and IL-1 β regulated genes highly enriched for many common functions, including the immune response and apoptosis. This provides a molecular explanation for the mechanism of action of inflammatory agents leading to increased androgen production. Among the genes upregulated by both LPS and IL-1 β were key regulatory genes involved in the cholesterol and androgen biosynthesis pathways, including Cyp17a1, Cyp11a1, Hsd3b, and Hmgcr. Using a stringent statistical cutoff, LPS and IL-1 β elicited differential expression of 52 genes, respectively.Le catalogue shogunmoto de piece AIE 275 MAGMAX comprend aux meilleurs prix toutes les pieces ncessaires son entretien.Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among women of reproductive age however, its underlying pathophysiology remains poorly understood. Pices et accessoires pour AIE 275 MAGMAX : pour accder votre catalogue de pice AIE 275 MAGMAX, slectionnez son anne dans le moteur de recherche. The system combines the CleanNA technology with our specially formulated.Equipement motard & Goodies. Together, these findings highlight the potential molecular mechanisms through which chronic low-grade inflammation contributes to the pathogenesis of androgen excess in PCOS.Biosystems MagMAX, Qiagen BioSprint, and other liquid handling instruments).
A leading model explaining PCOS is thecal cell dysfunction, the source of androgen biosynthesis in the human ovary ( 7). In most women with PCOS, androgen excess is the underlying culprit of the symptoms, including irregular ovulation, hirsutism, and acne ( 3– 6). The key features of PCOS include androgen excess, oligo-ovulation or anovulation, and the appearance of polycystic ovaries on ultrasound imaging ( 2, 3).FAST TOYS, S.M.C., QUADZILLA, AIE Atv Bike RAM 170 R 2003 - 002 RAM 170 R Stinger E 2003 - 002 RAM 250 R 2003 - 002 RAM 250 R Stinger E 2003 - 002 300 Quadzilla 2004 - 2006 179 FGR Street Bike V6. Installation id wont work for microsoft office 2016 macFor example, several key markers and mediators of systemic inflammation—including C-reactive protein ( 26, 27), TNF- α ( 28) and IL-6 ( 29)—are elevated in women with PCOS compared with body mass index-matched control subjects. Second, although PCOS has been associated with known proinflammatory metabolic disorders such as obesity, insulin resistance, and diabetes, the association of PCOS with inflammation has persisted in the absence of these confounders ( 24, 25). The converse is unlikely to be the case, because the administration of androgens, including nonaromatizable 5 α-DHT, have been found to reduce, rather than increase, the inflammatory responses ( 20– 23). First, several clinical trials have revealed that agents with anti-inflammatory properties, such as statins ( 13– 18) and resveratrol ( 19), improve PCOS symptoms and reduce androgen levels ( 15, 17– 19). Several lines of evidence have supported this possibility. Although excessive ovarian androgen production via theca cells contributes to many of the phenotypic manifestations of PCOS ( 3– 6, 10), the underlying cause of androgen excess has yet to be determined.Chronic low-grade inflammation has also been associated with PCOS, increasing the possibility that inflammation might contribute to the androgen excess in PCOS ( 11, 12). This leads to a plethora of responses, including upregulation of the inflammasomes ( 38) and activation of the MAPK and AKT pathways, with consequent effects on cellular proliferation and apoptosis ( 39, 40). LPS activates cells by binding to Toll-like receptor 4 (TLR4). It induces inflammation primarily via activation of macrophages and intracellular pathways in other cells, leading to the production of proinflammatory cytokines. LPS is a key pathogenic component of the cell wall of gram-negative bacteria. However, low doses of these substances mimic subclinical endotoxemia by failing to induce negative regulators of inflammatory pathways, allowing chronic low-grade inflammation to persist ( 37, 47). The ovarian IL-1 β system plays a major role in ovulatory processes ( 44, 45) thus, dysregulation of these inflammatory mediators might disrupt this tightly regulated process contributing to ovulatory disruption and anovulation ( 46).When administered at high concentrations, both LPS and IL-1 β cause sepsis-like symptoms and can lead to death. IL-1 β is first synthesized as an inactive precursor protein, pro-IL-1 β, and cleaved by caspase-1 into active IL-1 β as a result of inflammasome activation ( 38). LPS and IL-1 β regulated hundreds of the same genes but also regulated distinct sets of genes, consistent with their known overlapping, but distinctive, functions ( 38– 42, 47). These proinflammatory agents also had surprisingly widespread effects on gene expression in TICs, which included not only androgen biosynthesis pathways but also many other biochemical pathways, as revealed by RNA sequencing (RNAseq) analysis. In agreement with our hypothesis, we found that low doses of LPS and IL-1 β elicited increased expression of androgen biosynthesis genes in theca-interstitial cells (TICs) and increased androgen production in these cells. The potential sources of low-grade bacterial endotoxins in women with PCOS include altered gut microbiota, resulting in increased intestinal permeability ( 48, 49) or the persistence of low-grade chronic infection ( 50– 53). The ovaries were collected in ice-cold M199 isolation medium (Millipore Sigma) containing 2-mM l-glutamine, 25 mM HEPES, 1% antibiotic and antimycotic, and 0.1% BSA (pH 7.25). At 24 hours after the last injection, the rats were anesthetized using ketamine (Ketaset Zoetic, Kalamazoo, MI) and xylazine intraperitoneally (VETone, Boise, ID) and euthanized by intracardiac perfusion using 0.9% saline. Louis, MO) for 3 days to stimulate ovarian development and growth of antral follicles, as described previously ( 54). Starting at 27 days of age, the rats were injected subcutaneously with 17 β-estradiol (1 mg in 0.3 mL of sesame oil Millipore Sigma, St. Materials and Methods Isolation of rat TICsFemale Sprague-Dawley rats, aged 24 days, were obtained from Envigo (Placentia, CA). Aie Magmax 275 Free Of OviductsThe purified TICs were then washed twice with ice-cold M199 isolation medium, and the pellet was resuspended in a known volume of the ice-cold serum-free McCoy’s 5A culture medium (Gibco, Life Technologies, Carlsbad, CA) supplemented with 1% antibiotic/antimycotic mix, 0.1% BSA, and 2 mM glutamine (Gibco, Life Technologies). After 60 minutes of collagenase-DNase digestion, TICs were purified using discontinuous Percoll (Millipore Sigma) gradient centrifugation. The remaining ovaries were washed twice with ice-cold M199 isolation medium (Millipore Sigma) and then were minced and digested in 5 mL of collagenase-DNase solution containing 21.1 mg of collagenase (Worthington, Lakewood, NJ), 1.5 mg of DNase (Worthington), and 50 mg of BSA in M199 isolation medium at 37☌ for 60 minutes. The ovaries were gently punctured with a 26-gauge needle in ice-cold M199 isolation medium. In brief, the ovaries were removed from the rats and dissected free of oviducts and fat under a dissecting microscope.
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