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Schube U.,University of Leipzig | Nowicki M.,University of Leipzig | Jogschies P.,Clinic for Reproductive Medicine and Gynecological Endocrinology | Blumenauer V.,Clinic for Reproductive Medicine and Gynecological Endocrinology | And 2 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2014

Context: Obese women suffer from anovulation and infertility, which are driven by oxidative stress caused by increased levels of lipid peroxides and circulating oxidized low-density lipoprotein (ox-LDL). OxLDL binds to lectin-like oxLDL receptor 1 (LOX-1), cluster of differentiation 36 (CD36), and toll-like receptor 4 (TLR4) and causes cell death in human granulosa cells (GCs). Objective: Our objective was to reveal whether treatment with antioxidants resveratrol (RES) and/or desferoxamine (DFO) protect GCs from oxLDL-induced damage. Design and Setting: This basic research study was performed at the Institute of Anatomy and the Clinic of Reproductive Medicine. Patients: Patients were women undergoing in vitro fertilization therapy. Main Outcome Measures: GC cultures were treated with oxLDL alone or with RES or DFO under serum-free conditions for up to 36 hours. Dead cells were determined by propidium iodide uptake, cleaved caspase-3 expression, and electron microscopy. Mitosis was detected by Ki-67 immunostaining. LOX-1, TLR4, CD36, and heat-shock protein 60 were examined by Western blot. Measurement of oxidative stress markers (8-iso-prostaglandin F2α, advanced glycation end products, and protein carbonyl content) was conducted with ELISA kits. Results: Different subtypes of human GCs exposed to RES or DFO were protected as evidenced by the lack of cell death, enhanced mitosis, induction of protective autophagy, reduction of oxidative stress markers, and reduced expression of LOX-1, TLR4, CD36, and heat-shock protein 60. Importantly, RES could restore steroid biosynthesis in cytokeratin-positive GCs, which exhibited significant induction of steroidogenic acute regulatory protein. Conclusions: RES and DFO exert a protective effect on human GCs. Thus, RES and DFO may help improve the treatment of obese women or polycystic ovarian syndrome patients undergoing in vitro fertilization therapy. (J Clin Endocrinol Metab 99: 229-239, 2014). © Copyright 2014 by The Endocrine Society.

Serke H.,University of Leipzig | Bausenwein J.,University of Leipzig | Hirrlinger J.,The Interdisciplinary Center | Nowicki M.,University of Leipzig | And 5 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2010

Context: The oxidized low-density lipoprotein (oxLDL) and its lectin-like oxLDL receptor-1 (LOX-1) are found in the follicular fluid and in granulosa cells. Lipoprotein receptors and antioxidant enzymes could differ in granulosa cell subtypes. Objective: Our aim was to reveal cell-specific responses under oxLDL treatment. Design and Setting: We conducted basic research at the Institute of Anatomy and the Clinic of Reproductive Medicine. Patients: Women undergoing in vitro fertilization therapy participated in the study. Main Outcome Measures: Cultures of cytokeratin-positive/negative (CK +/CK-) granulosa cells and of cumulus cells were treated with 150 μg/ml oxLDL or native LDL under serum-free conditions for up to 36 h. Dead cells were determined by uptake of propidium iodide. LOX-1, toll-like receptor 4, and cluster of differentiation 36 (CD36) were examined in lysates by Western blots. The enzyme activities were determined in lysates and in supernatants. Results: Under oxLDL treatment, predominantly CK+ cells underwent nonapoptotic cell death. Receptors showed a cell-specific pattern of up-regulation: toll-like receptor 4 in CK+ cells, LOX-1 in CK - cells, and CD36 in cumulus cells. An antioxidant ranking occurred: superoxide dismutase activity in CK+ cells, total glutathione in CK- cells, and catalase activity in cumulus cells. The supernatants of oxLDL-treated CK+ cell cultures contained more catalase activity than in controls, whereas a moderate increase was noted for glutathione peroxidase (GPx) in supernatants of CK- and cumulus cells. Conclusions: Catalase/GPx activity in the supernatants may be due to cell death or to secretion. Oxidative stress could be sensed by CK+ cells and indicated by changes in catalase/GPx activity in the follicular fluid during ovarian disorders. Copyright © 2010 by The Endocrine Society.

Vilser C.,University of Leipzig | Hueller H.,University of Leipzig | Nowicki M.,University of Leipzig | Hmeidan F.A.,Clinic for Reproductive Medicine and Gynecological Endocrinology | And 2 more authors.
Fertility and Sterility | Year: 2010

Objective: To extend our recent observations on lectin-like oxidized low-density lipoprotein receptor (LOX-1) expression in human granulosa cell cultures with freshly harvested granulosa cells. Design: Clinical research. Setting: Institute of Anatomy and Clinic for Reproductive Medicine. Patient(s): Women undergoing IVF therapy were classified by total FSH dose, age, and body mass index. Main Outcome Measure(s): Purified granulosa cells were studied by Western blot and morphology for the presence of LOX-1, microtubule-associated light-chain protein 3 (LC3) and autophagosomes, which are both autophagic markers, cleaved caspase-3 for apoptosis, and apoptosis-inducing factor (AIF) for caspase-independent apoptosis. Intervention(s): None. Results: Active LOX-1 was found in all samples, being at its maximum in the younger obese group with a total FSH dose <2,000 IU. The LC3 II/LC3 I ratio, indicative of reparative autophagy, was at its maximum in younger normal-weight patients and increased under total FSH dose >2,000 IU. Autophagosomes in ultrathin sections were indicative of reparative autophagy. Cleaved caspase-3 was absent in all groups. The apoptotic AIF form was up-regulated in older patients. Unpurified granulosa cells consisted of ∼20% dead cells in the younger normal-weight group compared with up to 50% in the older obese group. Conclusion(s): The regulation of LOX-1 and of cell death in granulosa cells depends on oxidative stress. It becomes excessive during aging and obesity, because the power of reparative autophagy fades and antioxidant efficiency declines. © 2010 American Society for Reproductive Medicine.

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