TY - JOUR
AU - Serke,, H.
AB - 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 (oxLDL). 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. Obesity leads to irregular ovarian cycles and anovulation in women of reproductive age and is associated with systemic oxidative stress (1), but the mechanisms linking obesity and fertility are not fully understood. Highly augmented serum levels of triglycerides, free fatty acids, oxidized low-density lipoproteins (oxLDL), insulin, and glucose are metabolic consequences of obesity (2–4). Furthermore, the excessive lipid storage in the ovarian tissue is responsible for mitochondrial dysfunction with the augmented release of reactive oxygen species (ROS) leading to oxidative stress and ovarian damage (5). The ROS production recognized by indirect parameters responding to oxidative stress is probably involved in the increased rate of follicular atresia. Lectin-like oxLDL receptor 1 (LOX-1), toll-like receptor 4 (TLR4), and cluster of differentiation 36 (CD36) are receptors for oxLDL (5–8). We have previously demonstrated their existence on granulosa cells (GCs) from human preovulatory follicles. Moreover, we found that the oxLDL-dependent activation of these receptors can induce survival autophagy and, at higher doses, apoptosis in human GCs (9, 10), thus providing a potential mechanism of infertility in obesity. In this study, we tested whether oxLDL-induced follicle cell damage can be prevented by resveratrol (RES) or desferoxamine (DFO). Both compounds were found to be effective in a wide range of diseases including inflammation, cancers, heart failure, neurodegeneration, and ischemia (11, 12). However, little is known regarding their potential roles in regulation of human reproduction and ovarian physiology. RES is a plant phytoalexin of the large group of polyphenols bearing anti-inflammatory and antioxidative properties (13, 14). The RES antioxidative properties are based on their phenolic hydroxyl groups, which possess a high redox potential and therefore can act as highly effective scavengers. RES has the ability to stimulate the body's own antioxidant enzyme systems such as superoxide dismutase and catalase (13). It has been also reported that RES inhibits LDL oxidation and decreases cholesterol levels (14). In rats, RES inhibits luteinization and ovarian theca-interstitial cell proliferation and induces apoptosis; therefore, it may be effective in treating patients with polycystic ovarian syndrome (PCOS) (15). DFO, an iron chelator and antioxidant, is currently the first choice for the treatment of thalassemia. In cultured spinal ganglion cells, we have previously demonstrated protective effects of DFO caused by direct reduction of oxidative stress and induction of antioxidative molecules (11). Recently, Zafon et al (16) have demonstrated a link between obesity, body iron deposits, and oxidative stress; obesity seems to be associated with relative iron deficiency due to impaired intestinal uptake and inadequate iron bioavailability because of inflammation and oxidative stress. We have previously established protocols to isolate and culture 3 human GC types (cytokeratin [CK]+, CK−, and cumulus [Cum] cells) obtained from follicular aspirations of patients receiving in vitro fertilization (IVF) therapy (9, 10). We found oxLDL to be increased by approximately 2-fold in obese women, and high oxLDL levels in the preovulatory follicles correlates negatively with IVF outcomes (4). OxLDL-induced cell death and survival autophagy was dependent on the activation of the oxLDL-binding receptors LOX-1, TLR4, and CD36 (9, 10). Leptin-deficient (ob/ob) mice revealed excessive lipid storage in follicle cells together with advanced follicular atresia and defective steroidogenesis, which may well underlie their subfertility (5). Because all these findings point to oxidative stress as a key player in obesity-related infertility, we tested in this study whether oxLDL-induced damage of human GC subtypes can be prevented by RES and DFO. To this end, their effects on apoptosis, proliferation, oxLDL-binding receptors, and ROS generation have been studied. We demonstrate highly protective effects of both compounds, rendering them potential drugs in IVF therapy. Materials and Methods Primary cell cultures and oxLDL, RES, and DFO treatment GCs were obtained from patients undergoing IVF therapy. The Committee for Ethics at the University of Leipzig approved the experimental design (no. 068-12-05032012), and the patients provided written consent. The isolation, purification, and cultivation of GCs were performed as described previously (9, 10). For the oxLDL, oxLDL/RES or oxLDL/DFO treatments, confluent cultures of CK+, CK−, and Cum GCs, grown in 12-well cluster plates with serum-containing medium, were treated with 150 μg/mL oxLDL (Biomedical Technologies Inc) alone or with 30μM RES (Sigma-Aldrich) or 30μM DFO (Sigma) under endotoxin and serum-free conditions for 36 hours. The concentration of oxLDL indicated a mildly oxidized lipoprotein treatment (9, 10). Immunofluorescence localization of CK filaments Confluent cultures were developed on round coverslips, fixed with 4% buffered paraformaldehyde (PFA) plus 0.1% Triton X-100 (at room temperature for 10 minutes) and stained for CK as described by Serke et al (9). Cells were incubated with a primary mouse monoclonal antibody against pancytokeratin Lu5 (1:1000; Calbiochem) at 4°C overnight. For detection, the secondary antibody Cy3-goat antimouse (1:1000; Dianova) was used. Uptake of propidium iodide (n = 3) To determine cell death, cultures were washed with cold PBS and incubated with propidium iodide (PI) (12.5 μg/mL; Biochemica) at room temperature for 5 minutes. After a PBS rinse, cells were fixed in 4% PFA for 10 minutes. After another PBS rinse, coverslips were mounted upside down in Dako-Glycergel (DakoCytomation) containing 4′-6′-diamidino-2-phenylindole. The number of blue and pink nuclei (overlay of 4′-6′-diamidino-2-phenylindole in blue and PI in red) was counted in roughly 800 cells of each experiment. Ki-67 immunofluorescence staining (n = 3) To determine cell proliferation, cultures were fixed in 4% PFA for 10 minutes. Cells were incubated with a primary mouse monoclonal antibody against Ki-67 (1:500, DakoCytomation) at 4°C overnight. For detection, the secondary antibody Cy3-goat antimouse (1:1000; Dianova) was used. The number of blue and red nuclei was counted in roughly 600 cells of each experiment. Transmission electron microscopy (n = 3) To monitor signs of cell death or autophagy at the ultrastructural level, cultures were grown to confluence on Thermanox coverslips (Nunc, Inc,) in 4-well culture plates, fixed in 2.5% PBS-buffered glutaraldehyde at 4°C for 1 hour, and postfixed in 1% OsO4 for another hour. Monolayers were washed in 2.4% NaCl and then 0.2M sodium acetate buffer (pH 5.0). Ultrathin sections were prepared as described previously (9, 10). Western blot analysis (n = 6) Details of the technique were published by Serke et al (9, 10). The primary and secondary antibodies with the dilutions are listed in Supplemental Table 1 (published on The Endocrine Society's Journals Online website at http://jcem.endojournals.org) The peroxidase activity was visualized with the enhanced chemiluminescence kit (Amersham Pharmacia) and arbitrary units of the immunoreactive protein bands were determined according to Serke et al (9, 10). Protein loading was evaluated with housekeeping gene GAPDH (Fitzgerald). Positive controls are listed in Supplemental Table 2. Measurement of lipid peroxidation (n = 6) The F2α-isoprostanes are a recently described class of prostaglandins formed by free radical-mediated lipid peroxidation (17). Thus, the OXi-Select 8-iso-prostaglandin F2α (PGF2α) kit is a competitive ELISA for rapid detection and quantification of 8-iso-PGF2α. The quantity of 8-iso-PGF2α in samples is determined by comparing its absorbance (450 nm) with that of a known 8-iso-PGF2α standard curve. The assay and samples were processed according to the manufacturer's instructions. Measurement of advanced glycation end products (n = 6) The OXi-Select advanced glycation end product (AGE) kit is an ELISA for quantitative determination of the level of AGE protein adducts in which the quantity of this AGE adduct in protein samples is determined by comparing its absorbance (450 nm) with that of the a known AGE-BSA standard curve. The assay and samples were processed according to manufacturer's instructions. Measurement of protein oxidation (n = 6) The OXi-Select protein carbonyl ELISA kit provides a sensitive methodology for rapid detection and quantification of proteins modified by ROS and other reactive species. The quantity of protein carbonyls in protein samples is determined by comparing its absorbance (450 nm) with that of a known reduced/oxidized BSA standard curve. The assay and protein samples were processed according to the manufacturer's instructions. Photodocumentation and statistics Digitized pictures were taken with an Axioplan 2 light microscope (Zeiss) equipped with a Progress camera and otherwise used for epifluorescence illumination together with an axiovision camera (Zeiss). The mean ± SEM from 3 to 6 independent experiments was calculated. Statistics were calculated by ANOVA using the Bonferroni method (SigmaStat; Jandel Scientific). Results RES and DFO prevent oxLDL-dependent follicle cell death and increased cell proliferation The different follicle cell subtypes cultured under serum conditions were characterized by phase-contrast microscopy and CK filament immunostaining (Figure 1A, I–AIII) as described previously (10). Cell death was evaluated using a PI assay. A total of 62.6% ± 22.7% of dying cells were counted in oxLDL-treated CK+ cell cultures, whereas the number of dead cells was significantly reduced to 3.5% ± 1.4% under RES and 3.3% ± 1.2% under DFO treatment (Figure 1, A, b–d, and B). As described previously (9, 10), CK− cultures did not show oxLDL-induced degeneration (Figure 1Af) and remained intact also upon treatment with RES and DFO, respectively (Figure 1A, g and h). Untreated Cum cells exhibited a low degeneration of 7.6% ± 12.6% PI-positive cells (Figure 1Ai), which was elevated to 18.8% ± 15.4% upon exposure to oxLDL (Figure 1Aj). Both degeneration in controls and oxLDL-induced damage were prevented by RES or DFO (Figure 1, A, k and l, and B). Thus, oxLDL-induced degeneration is prevented by RES and DFO. Figure 1. Open in new tabDownload slide RES and DFO prevent oxLDL-induced cell death in human GCs. Cultures of human CK+, CK−, and Cum GCs were characterized by phase-contrast microscopy, immunofluorescence staining with the pancytokeratin antibody Lu5 under serum-free conditions. A, Dead cells were detected by PI uptake. B, Statistical evaluation of the number of dead cells. Cultures were treated under serum-free conditions with 150 μg/mL oxLDL alone or with 30μM RES or 30μM DFO for 36 hours. AI, CK+ cells were epithelioid and expressed high levels of CK. AII, The fibroblast-like CK− cells lacked detectable amounts of CK filaments. AIII, Cum cells exhibited a fibroblast-like morphology and consisted of CK+ as well as CK− cells. A, a–d, A total of 62.6% ± 22.7% of dying cells were counted in oxLDL-treated CK+ cell cultures, whereas the number of dead cells was significantly reduced under RES and DFO treatment. A, e–h, CK− cultures did not show oxLDL-induced degeneration and remained intact also upon treatment with RES and DFO, respectively. A, i–l, Untreated Cum cells exhibited a low rate of degeneration, which was elevated upon exposure to oxLDL. Both degeneration in controls and oxLDL-induced damage were prevented by RES or DFO. Values of 3 independent experiments represent means ± SEM. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05; **, P < .01; ***, P < .001. Scale bars, 100 μm (phase-contrast microscopy) and 50 μm (CK and PI staining). Figure 1. Open in new tabDownload slide RES and DFO prevent oxLDL-induced cell death in human GCs. Cultures of human CK+, CK−, and Cum GCs were characterized by phase-contrast microscopy, immunofluorescence staining with the pancytokeratin antibody Lu5 under serum-free conditions. A, Dead cells were detected by PI uptake. B, Statistical evaluation of the number of dead cells. Cultures were treated under serum-free conditions with 150 μg/mL oxLDL alone or with 30μM RES or 30μM DFO for 36 hours. AI, CK+ cells were epithelioid and expressed high levels of CK. AII, The fibroblast-like CK− cells lacked detectable amounts of CK filaments. AIII, Cum cells exhibited a fibroblast-like morphology and consisted of CK+ as well as CK− cells. A, a–d, A total of 62.6% ± 22.7% of dying cells were counted in oxLDL-treated CK+ cell cultures, whereas the number of dead cells was significantly reduced under RES and DFO treatment. A, e–h, CK− cultures did not show oxLDL-induced degeneration and remained intact also upon treatment with RES and DFO, respectively. A, i–l, Untreated Cum cells exhibited a low rate of degeneration, which was elevated upon exposure to oxLDL. Both degeneration in controls and oxLDL-induced damage were prevented by RES or DFO. Values of 3 independent experiments represent means ± SEM. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05; **, P < .01; ***, P < .001. Scale bars, 100 μm (phase-contrast microscopy) and 50 μm (CK and PI staining). To determine proliferation, immunostaining for Ki-67 was performed. Ki-67 protein is present during all active phases of the cell cycle except G0 (18). Proliferation was significantly decreased in oxLDL-treated CK+ cultures and restored by RES and DFO (Figure 2, A, a–d, and B). An unchanged proliferation rate was seen in all treatments of CK− cultures (Figure 2, A, e–h, and B). Untreated Cum cells exhibited a lower proliferation rate compared with untreated CK− and CK+ cells, which showed a trend to even lower levels upon treatment with oxLDL. The number of proliferating cells was significantly increased to 18.1% ± 4.2% under RES and 16.8% ± 2.6% under DFO treatment comparing to 10.1% ± 1.8% upon exposure to oxLDL (Figure 2, A, j–l, and B). Thus, oxLDL-induced inhibition of proliferation is restored by RES and DFO treatment. Figure 2. Open in new tabDownload slide RES and DFO induce proliferation of oxLDL-arrested human GCs. GC cultures were treated under serum-free conditions with 150 μg/mL oxLDL alone or with 30μM RES or 30μM DFO for 36 hours. Ki-67 immunostaining was performed. B, Statistical evaluation of proliferating cells is given in B. A, a–d, In oxLDL-treated CK+ cultures, proliferation was significantly decreased and restored by RES and DFO. A, e–h, An unchanged proliferation rate was seen in all treatments of CK− cultures. A, i–l, Untreated Cum cells exhibited a reduced proliferation compared with untreated CK− and CK+ cells. The number of proliferating cells was significantly increased under RES and DFO treatment compared with exposure to oxLDL. Values of 3 independent experiments represent means ± SEM. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05; **, P < .01; ***, P < .001. Scale bars, 50 μm. Figure 2. Open in new tabDownload slide RES and DFO induce proliferation of oxLDL-arrested human GCs. GC cultures were treated under serum-free conditions with 150 μg/mL oxLDL alone or with 30μM RES or 30μM DFO for 36 hours. Ki-67 immunostaining was performed. B, Statistical evaluation of proliferating cells is given in B. A, a–d, In oxLDL-treated CK+ cultures, proliferation was significantly decreased and restored by RES and DFO. A, e–h, An unchanged proliferation rate was seen in all treatments of CK− cultures. A, i–l, Untreated Cum cells exhibited a reduced proliferation compared with untreated CK− and CK+ cells. The number of proliferating cells was significantly increased under RES and DFO treatment compared with exposure to oxLDL. Values of 3 independent experiments represent means ± SEM. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05; **, P < .01; ***, P < .001. Scale bars, 50 μm. To determine (the mode of) cell death, the ultrastructure of GCs was examined under oxLDL, oxLDL/RES, or oxLDL/DFO conditions. Neither DNA condensation nor apoptotic bodies as morphological hallmarks of apoptosis were detected in CK+ and Cum cells upon exposure to oxLDL. Conversely, excessive cytoplasmic vacuolization, membrane-like remnants, and cytoplasmic and nuclear chromatin lysis were observed in oxLDL-treated CK+ and Cum cells as features of the necrotic/autophagic cell death (Figure 3I, b and j). In oxLDL/RES or oxLDL/DFO-treated CK+ as well as Cum cells, such signs of nonapoptic degeneration were found only occasionally. Interestingly, in untreated Cum cells, multilamellar bodies were considered as late lysosomal organelles after defective lipids/cholesterol had been enclosed by autophagosomes (Figure 3Ii). Additionally, we determined the presence of autophagosomes in untreated and oxLDL/RES- or oxLDL/DFO-treated CK+ as well as Cum cells (Figure 3I, c, d, k, and l). CK− cultures did not show oxLDL-induced degeneration and remained intact also upon treatment with RES and DFO, respectively. Here, early autophagosomes with the characteristic double membrane, autophagolysosomes, and intact mitochondria were observed (Figure 3I, e–h). Figure 3. Open in new tabDownload slide RES and DFO protect CK+ (a–d), CK− (e–h), and Cum (i–l) GCs from degeneration. GC cultures were treated under serum-free conditions with 150 μg/mL oxLDL alone or with 30 μM RES or 30 μM DFO for 36 hours. I, Ultrastructure of different GCs. II, Representative Western blots and semiquantitative evaluations of cleaved caspase-3 and LC3. I, A–D, The oxLDL-treated CK+ cells showed occasional membrane remnants (black arrows), excessive cytoplasm, and nuclear chromatin lysis (asterisk), whereas the oxLDL/RES- and oxLDL/DFO-treated CK+ cells were intact. Signs of autophagy are shown by double-membrane autophagosomes (white arrows). I, E–H, All treated CK− cells contained double-membrane autophagosomes (white arrows); mitochondria were intact. I, I–L, Some of the oxLDL-treated Cum cells showed excessive vacuolization and a nuclear chromatolysis (asterisk), whereas the oxLDL/RES- and oxLDL/DFO-treated cells were intact. Signs of autophagy are shown by autophagosomes (white arrows). Untreated Cum cells exhibited multilamellar bodies. Scale bars, 1 μm. IIA, A representative blot shows no expression of cleaved caspase-3 in all groups. IIB, A representative Western blot and semiquantitative evaluation display a significant increase in LC3-II/LC3-I ratio in CK− GCs, which was abolished in the presence of both antioxidants, corresponded to basal levels under serum-free conditions (control). Values of 6 independent experiments represent means ± SEM percentage of GAPDH control. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05; **, P < .01; ***, P < .001. Figure 3. Open in new tabDownload slide RES and DFO protect CK+ (a–d), CK− (e–h), and Cum (i–l) GCs from degeneration. GC cultures were treated under serum-free conditions with 150 μg/mL oxLDL alone or with 30 μM RES or 30 μM DFO for 36 hours. I, Ultrastructure of different GCs. II, Representative Western blots and semiquantitative evaluations of cleaved caspase-3 and LC3. I, A–D, The oxLDL-treated CK+ cells showed occasional membrane remnants (black arrows), excessive cytoplasm, and nuclear chromatin lysis (asterisk), whereas the oxLDL/RES- and oxLDL/DFO-treated CK+ cells were intact. Signs of autophagy are shown by double-membrane autophagosomes (white arrows). I, E–H, All treated CK− cells contained double-membrane autophagosomes (white arrows); mitochondria were intact. I, I–L, Some of the oxLDL-treated Cum cells showed excessive vacuolization and a nuclear chromatolysis (asterisk), whereas the oxLDL/RES- and oxLDL/DFO-treated cells were intact. Signs of autophagy are shown by autophagosomes (white arrows). Untreated Cum cells exhibited multilamellar bodies. Scale bars, 1 μm. IIA, A representative blot shows no expression of cleaved caspase-3 in all groups. IIB, A representative Western blot and semiquantitative evaluation display a significant increase in LC3-II/LC3-I ratio in CK− GCs, which was abolished in the presence of both antioxidants, corresponded to basal levels under serum-free conditions (control). Values of 6 independent experiments represent means ± SEM percentage of GAPDH control. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05; **, P < .01; ***, P < .001. Cleaved caspase-3 is not involved in follicle cell death and not activated under RES or DFO treatment Because programmed cell death does not necessarily cause all hallmarks of apoptosis such as membrane blebbing, shrinking, and nuclear fragmentation (19), and signs of necrotic-like cell death were evident after treatment with oxLDL in CK+ and Cum cells (Figures 1 and 3I), we tested whether caspase-3 was activated in the human GC subtypes using Western blot analysis (Figure 3IIa). Cleaved caspase-3 was absent under all treatments in the 3 GC types. Next, we wanted to study a potential shift from the cytosolic microtubule-associated protein1 light chain 3 (LC3-I) (18-kDa) protein to the membranous LC3-II (16-kDa) form as an established indicator of survival autophagy (20). As described previously (5, 9, 10), a high LC3-II/LC3-I ratio indeed indicates high amounts of autophagosomes. After treatment with oxLDL, only CK− GCs responded by a significant LC3-II/LC3-I shift, which was not observed when cells were also treated with RES and DFO (Fig. 3IIb), which is in keeping with their high resistance to oxLDL-induced degeneration (Figures 1 and 3I). RES- and DFO-reduced expressions of the oxLDL-binding receptors LOX-1, TLR4, and CD36 OxLDL-binding receptors are differentially regulated in GC subtypes (9, 10). In CK− GCs, LOX-1 protein expression was significantly up-regulated upon exposure to oxLDL. Notably, this effect was completely abolished in the presence of both antioxidants. The LOX-1 expression corresponded to basal level of controls (Figure 4A). No changes in LOX-1 synthesis were noted in CK+ or Cum cells (Figure 4A). In all experimental groups of CK+ cells, the TLR4 expression levels appeared to be higher compared with the other GC subtypes (Figure 4B). Furthermore, in all oxLDL-treated GCs, CD36 was significantly up-regulated. This up-regulation was abolished under RES and DFO treatments (Figure 4C). Thus, oxLDL-induced up-regulation of its receptors is prevented by RES and DFO. Figure 4. Open in new tabDownload slide A–D, The RES and DFO effect on expression of oxLDL-binding receptors LOX-1 (A), TLR4 (B), CD36 (C), and Hsp60 (D) examined by Western blot analysis in human GC subtypes under different conditions. GCs were treated with 150 μg/mL oxLDL alone or with 30μM RES or 30μM DFO for 36 hours. A, A representative blot and semiquantitative evaluation show significantly increased LOX-1 expression under oxLDL treatment in CK− GCs, which was decreased in the presence of both antioxidants. An unchanged LOX-1 protein level was evaluated in CK+ and Cum cells. B, No significant changes in TLR4 expression were noted in all treated GCs. C, Significant up-regulation of the CD36 protein is decreased upon exposure to RES and DFO in all cell subtypes. C, A representative Western blot for Hsp60 in GCs. The semiquantitative evaluation displays strong Hsp60 expression in oxLDL-treated CK− cells. This expression was decreased under treatments with RES or DFO. No significant regulation of Hsp60 was found in CK+ and Cum cells. Values of 6 independent experiments represent means ± SEM percentage of GAPDH control. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05; **, P < .01; *** P < .001. Figure 4. Open in new tabDownload slide A–D, The RES and DFO effect on expression of oxLDL-binding receptors LOX-1 (A), TLR4 (B), CD36 (C), and Hsp60 (D) examined by Western blot analysis in human GC subtypes under different conditions. GCs were treated with 150 μg/mL oxLDL alone or with 30μM RES or 30μM DFO for 36 hours. A, A representative blot and semiquantitative evaluation show significantly increased LOX-1 expression under oxLDL treatment in CK− GCs, which was decreased in the presence of both antioxidants. An unchanged LOX-1 protein level was evaluated in CK+ and Cum cells. B, No significant changes in TLR4 expression were noted in all treated GCs. C, Significant up-regulation of the CD36 protein is decreased upon exposure to RES and DFO in all cell subtypes. C, A representative Western blot for Hsp60 in GCs. The semiquantitative evaluation displays strong Hsp60 expression in oxLDL-treated CK− cells. This expression was decreased under treatments with RES or DFO. No significant regulation of Hsp60 was found in CK+ and Cum cells. Values of 6 independent experiments represent means ± SEM percentage of GAPDH control. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05; **, P < .01; *** P < .001. RES and DFO reduce expression of the Hsp60 The heat-shock protein 60 (Hsp60) is involved in stress responses and necessary for cellular survival under toxic or stressful circumstances (21, 22). Therefore, we have investigated Hsp60 expression in GCs under oxidative conditions. Interestingly, Hsp60 expression was significantly up-regulated only in oxLDL-treated CK− GCs (Figure 4D), which is in line with their relative resistance to oxidative stress (Figures 12–3). RES and DFO down-regulated Hsp60 to control levels in CK− but not in CK+ and Cum cells (Figure 4D). RES and DFO reduce ROS generation in oxLDL-treated follicle cells Obesity leads to mitochondrial dysfunction by enhanced ROS generation (2, 23), and we previously described enhanced ROS levels after 36 hours of oxLDL treatment in CK+ cells (9). To analyze the effect of both antioxidants on ROS generation, we determined the levels of 8-iso-PGF2α, AGEs, and protein carbonyl content as indicators of oxidative stress. A significant up-regulation of 8-iso-PGF2α level was detected in CK+ and CK− GCs under oxLDL treatment. This effect was completely abolished in the presence of both antioxidants. In Cum cells, the 8-iso-PGF2α level was reduced under RES treatment compared with the oxLDL-treated group (Figure 5A). Figure 5. Open in new tabDownload slide RES and DFO reduce levels of 8-iso-PGF2α (A), AGEs (B), and protein carbonyl (C) examined by ELISA in human GC subtypes under different conditions. GCs were treated with 150 μg/mL oxLDL alone or with 30μM RES or 30μM DFO for 36 hours. A, Semiquantitative evaluation displays a significant increase of 8-iso-PGF2α in oxLDL-treated CK+ and CK− cells and decrease upon exposure to both antioxidants. There was no significant increase in oxLDL-treated Cum cells, but RES and DFO led to a decrease of 8-iso-PGF2α. B, Semiquantitative evaluation shows a significant decrease of a high AGE level from both antioxidants in all treated GCs. C, Semiquantitative evaluation demonstrates a significant increased protein carbonyl level in all oxLDL-treated GCs, which was abolished by both antioxidants. Values of 6 independent experiments represent means ± SEM. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05, **, P < .01; ***, P < .001. Figure 5. Open in new tabDownload slide RES and DFO reduce levels of 8-iso-PGF2α (A), AGEs (B), and protein carbonyl (C) examined by ELISA in human GC subtypes under different conditions. GCs were treated with 150 μg/mL oxLDL alone or with 30μM RES or 30μM DFO for 36 hours. A, Semiquantitative evaluation displays a significant increase of 8-iso-PGF2α in oxLDL-treated CK+ and CK− cells and decrease upon exposure to both antioxidants. There was no significant increase in oxLDL-treated Cum cells, but RES and DFO led to a decrease of 8-iso-PGF2α. B, Semiquantitative evaluation shows a significant decrease of a high AGE level from both antioxidants in all treated GCs. C, Semiquantitative evaluation demonstrates a significant increased protein carbonyl level in all oxLDL-treated GCs, which was abolished by both antioxidants. Values of 6 independent experiments represent means ± SEM. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05, **, P < .01; ***, P < .001. AGEs are highly reactive molecules that are produced by nonenzymatic glycation and oxidation of proteins, lipids, and nucleic acids (24). High levels of AGEs were detected in all oxLDL-treated GCs and both antioxidants inhibited oxLDL-dependent AGE formation in all GC subtypes (Figure 5B). Protein carbonyl is the most commonly used marker of protein oxidation. In fact, accumulation of protein carbonyls has been observed in several human diseases such as diabetes and obesity (25). Strikingly, oxLDL treatment significantly augmented protein carbonyl levels in all GC types, and both RES and DFO abolished this increase (Figure 5C). Thus, 3 established parameters uniformly revealed enhanced oxidative stress upon oxLDL treatment and protection by both RES and DFO. RES increased the expression of StAR in CK+ cells To analyze the impact of RES and DFO on steroid biogenesis, the expression of steroidogenic acute regulatory protein (StAR) was investigated. StAR transfers cholesterol from the outer to the inner mitochondrial membrane and thus plays a key role in the production of steroid hormones (26). As described previously, untreated CK+ cells exhibit a reduced StAR expression compared with untreated CK− and CK+ cells (10). OxLDL-treated CK− cells showed an increased expression level of this protein, which was abolished in the presence of RES, and StAR expression corresponded to the basal level. Interestingly, in CK+ cells, a significant increased StAR synthesis was detected upon exposure to RES and DFO (Figure 6). Figure 6. Open in new tabDownload slide RES induces StAR expression, examined by Western blot analysis in human GC subtypes under different conditions. GCs were treated with 150 μg/mL oxLDL alone or with 30μM RES or 30μM DFO for 36 hours. The representative Western blot and the semiquantitative evaluation reveals a reduced StAR expression level of untreated CK+ cells compared with untreated CK− and Cum cells, but the treatment with RES led to increased StAR synthesis in CK+ cells. OxLDL-treated CK− cells exhibit an increased expression level of StAR protein, which was abolished in the presence of DFO. No significant regulation of StAR was found in treated Cum cells. Values of 6 independent experiments represent means ± SEM percentage of GAPDH control. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05; **, P < .01; ***, P < .001. Figure 6. Open in new tabDownload slide RES induces StAR expression, examined by Western blot analysis in human GC subtypes under different conditions. GCs were treated with 150 μg/mL oxLDL alone or with 30μM RES or 30μM DFO for 36 hours. The representative Western blot and the semiquantitative evaluation reveals a reduced StAR expression level of untreated CK+ cells compared with untreated CK− and Cum cells, but the treatment with RES led to increased StAR synthesis in CK+ cells. OxLDL-treated CK− cells exhibit an increased expression level of StAR protein, which was abolished in the presence of DFO. No significant regulation of StAR was found in treated Cum cells. Values of 6 independent experiments represent means ± SEM percentage of GAPDH control. A significantly decreased/increased level was observed with RES and DFO treatment: *, P < .05; **, P < .01; ***, P < .001. Discussion Recently, RES and DFO have been the focus of many in vitro and in vivo studies because of their pleiotropic and often beneficial biological activities (11, 14, 27–30). However, little is known regarding their potential roles in the regulation of reproduction and ovarian physiology. Obesity leads to irregular ovarian cycles, anovulation, and infertility in women of reproductive age and correlates with excessive lipid storage in ovarian tissue due to increased mitochondrial ROS production by adipose tissue (2). When ROS is not compensated by local antioxidant systems, oxidative stress causes harmful effects on female reproductive functions such as endometriosis and PCOS (31). It can also cause complications during pregnancy including spontaneous abortion, recurrent pregnancy loss, preeclampsia, and intrauterine growth restriction (32, 33). In human ovarian tissue, we have recently shown that enhanced ROS generation, and thereby oxLDL-induced oxidative stress, leads to follicle cell death (4, 5, 9, 10). To the best of our knowledge, until now, the role of RES and DFO in oxLDL-induced follicle cell death has not been clarified in the human system. In the present study, we show that RES and DFO have a protective effect against oxLDL-dependent follicle cell degeneration. Using PI uptake and transmission electron microscopy, we found RES and DFO to effectively abolish the oxLDL-induced follicle cell death. In addition, numerous autophagosomes in the RES- and DFO-treated cells suggested survival autophagy. Contrary to these results, a proapoptotic effect of RES has been described in human breast cancer cells and ovarian cancer cell lines (28, 34). RES has promoted apoptosis also in rat ovarian theca-interstitial cells due to an increase of caspase-3/7 activity (15). Furthermore, a high dose of RES and DFO had an antiproliferative activity in a wide variety of human cancer cell lines (28, 35). Ortega et al (29) have demonstrated RES to decrease the number of viable rat ovarian theca cells. In contrast, our results reveal a significant increase of GC viability. Under oxidative stress, the CK+ and Cum cells exhibit low proliferation rates, whereas proliferation increases under treatment with both RES and DFO. Taken together, the findings suggest RES and DFO have distinct effects on cell mortality and viability in the theca vs granulosa compartments. For this reason, the survival-promoting effect of RES and DFO on GCs and the antiproliferative effect on theca-interstitial cells are promising properties of these compounds as potential drugs for the treatment of obesity-induced infertility and PCOS (15). Additionally, it is well documented that RES has beneficial effects on dyslipidemia and obesity. This antioxidant corrects hyperglycemia by reducing blood glucose, improving insulin sensitivity, and weight loss (27, 36), which are metabolic features of obese women and PCOS patients. At the cellular level, we found RES and DFO to effectively decrease oxidative stress markers and oxLDL-binding receptor expression. As a well-known marker of oxidative cell damage, 8-iso-PGF2α is an important indicator of lipid peroxidation (17). Other markers for oxidative stress are AGEs and protein carbonyl levels. It has been reported that AGEs induce oxidative stress, and conversely, oxidative stress stimulates AGE formation (24). In GCs of PCOS patients, high levels of AGEs and their receptor (RAGE) have been found compared with GCs of healthy women (37). Indeed, both RES and DFO suppress ROS generation by decreasing 8-iso-PGF2α, AGEs, and protein carbonyl levels. Antioxidant mechanisms have been localized in granulosa and theca cells of the growing follicle (4, 29). Notably, the bidirectional cross-talk between the oocyte and its surrounding GCs (both Cum and mural layers) is crucial for normal follicle development (38). In obese women, GCs die early during folliculogenesis (4). Consequently, inadequate protection from oxidative stress is a potential trigger for follicular atresia (5). Our results strongly suggest increased oxidative stress (reflected by the above-mentioned markers) to interfere with folliculogenesis. It is unclear at present how exactly oocytes undergo damage under these conditions. The presented data, however, are in support of the role of oxidative stress in ovulatory dysfunction (39). Generally, it is known that oxLDL initiates oxidative stress by the up-regulation of its oxLDL-binding receptors such as LOX-1, TLR4, and CD36 via an internal feedback (10). Our results demonstrate that their up-regulation is also attenuated by both antioxidant treatments. Additionally, we show an up-regulation of Hsp60 expression under oxidative circumstances in CK− cells. Notably, this GC subpopulation is able to survive under oxLDL conditions. Hsp60 is necessary for protection of cells under toxic or stressful circumstances (21, 22). Our results reveal that RES and DFO decreases Hsp60 synthesis. Taken together, all these findings demonstrate that RES and DFO effectively decreased the oxidative stress in human follicle cells. Finally, we show that RES also restored steroid biosynthesis in CK+ GCs. Surprisingly, in CK− cells, StAR expression was strongly increased under oxLDL treatment. StAR is essential for the development and maintenance of corpora lutea and governs the translocation of cholesterol from the outer to the inner mitochondrial membrane, the rate-limiting step in steroidogenesis (26). Cholesterol can be derived from external sources, such as LDL via the LDL-binding receptors and high-density lipoproteins via the class B type I scavenger receptor (7). Notably, expression of CD36 was increased in all subtypes of human GCs. In the light of these results, we propose that GCs absorb more lipoprotein-derived cholesterol. In CK− GCs, this leads to increased activity of StAR and, thereby, to enhanced steroidogenesis. Thus, CK− GCs appear to compensate the oxLDL-induced oxidative stress and, thus, survive. In contrast, StAR expression remains unchanged in oxLDL-treated CK+ GCs, and they die. Interestingly, RES enhanced StAR synthesis in oxLDL-treated CK+ GCs, supporting their viability. Morita et al (40) have demonstrated RES to be an important player in the activation of luteinization and the terminal differentiation of GCs and that this effect is due to enhanced expression of SIRT1, StAR, LHR, and P450arom in rat ovarian GCs. These data strongly suggest to a crucial role of steroidogenesis activation in survival under oxidative circumstances. In conclusion, the present in vitro study shows that RES and DFO exert a protective effect on human GCs. Furthermore, RES as well as DFO decrease oxidative stress caused by oxLDL and therefore, they also prevent the different human GCs from oxLDL-dependent degeneration. Currently, we are developing an obesity-induced-infertile-mouse model in which we test the effects of RES and DFO on folliculogenesis, pregnancy and the F1-generation. This in vivo study may support our hypothesis that RES and DFO may help improving the treatment of obese women or PCOS patients undergoing IVF-therapy. 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TI - Resveratrol and Desferoxamine Protect Human OxLDL-Treated Granulosa Cell Subtypes From Degeneration
JF - Journal of Clinical Endocrinology and Metabolism
DO - 10.1210/jc.2013-2692
DA - 2014-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/resveratrol-and-desferoxamine-protect-human-oxldl-treated-granulosa-zljHLj7dWf
SP - 229
VL - 99
IS - 1
DP - DeepDyve
ER -