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Objective: Multilamellar bodies associated with an organized endoplasmic reticulum (ER) arise in various somatic cell types, and a subtype called multivesicular bodies is described in oocytes. Both entities, so far undetermined in significance, may occur in oocytes of follicles under oxidative stress. In preovulatory follicles, oxidative stress appears to be caused by oxidized low-density lipoprotein (ox-LDL)., Method: Cultures of preantral mouse follicles were treated with 100 µg/ml ox-LDL or normal LDL (n-LDL) for 12-48 h or for 12 days during antral follicle growth followed by in vitro ovulation and harvest of cumulus oophorus complexes (COCs) with metaphase II (MII) oocytes on day 13. Preantral follicles, COCs, or MII oocytes were immunostained with anti-tubulin antibody or stained with actin-binding phalloidin for confocal microscopy. Ultrathin sections were prepared for electron microscopy., Results: Preantral follicles exposed to n-LDL or ox-LDL developed normally, and MII oocytes in COCs possessed normal spindles with well-aligned chromosomes. In contrast, treated cumulus cells underwent apoptosis. Only the ox-LDL-treated preantral follicle oocytes showed ER-derived multilamellar bodies (EMBs) of type I, consisting of rough ER membranes for the envelope. The MII oocytes of COCs showed type II EMBs consisting of smooth/vesicular ER and were more prominent after ox-LDL than after n-LDL exposure. Degenerating mitochondria were prominent in oocytes of the ox-LDL group and judged as a sign of oxidative stress., Conclusion: Oxidative stress presumably induces damage of proteins and organelles in the oocytes. The EMBs might sequester the damaged structures for oocyte survival. Thus, EMBs could represent a novel form of autophagy., (Copyright © 2012 S. Karger AG, Basel.)

Oxidized low-density lipoprotein (oxLDL) induces apoptosis or autophagy in dependence on the cell type. We here investigated the effect of oxLDL on the B104 neuroblastoma and RN22 schwannoma cells being popular in neuroscience research. Cells were cultivated with and without oxLDL. To generate oxLDL, we added 50 μg/ml nLDL and 50 μM CuSO(4) into the culture medium. After a 24-h-long treatment, oxLDL was detectable in media from both cell culture types and its concentration was approximately 16 μg/ml. In the oxLDL-treated B104 neuroblastoma cell cultures 75% cells died after the 24-h exposure. The intact cells showed impaired mitochondria at the ultrastructural level. Western blot analysis revealed the increased expression of AIF 57 kDa (AIF(57)) protein, as a sign of caspase-independent cell death. In RN22 schwannoma cell cultures, oxLDL did not have any effect on cleaved caspase-3 and AIF(57) protein levels indicating absence of cell death. Treated RN22 schwannoma cells underwent survival autophagy by forming conspicuous autophagosomes and by processing LC3-I into LC3-II protein. Collectively, oxLDL induces AIF-dependent cell death in B104 neuroblastoma cells whereas in RN22 schwannoma cells enhanced signs of survival autophagy are noted., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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 microg/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.

DRG cells have been found to undergo apoptosis and necrosis after oxidized low-density lipoprotein (oxLDL) stimulation in vitro. However, the mechanism of oxLDL-induced DRG cell death is unclear. For this reason, we studied the expression of two potential oxLDL receptors: lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and toll-like receptor-4 (TLR4) in dorsal root ganglion (DRG) cell cultures from postnatal rats. Cells were cultivated with and without oxLDL. In oxLDL-treated DRG cell cultures, the increase of cleaved caspase-3 protein was observed as a sign of enhanced apoptosis. Untreated and oxLDL-treated DRG cell cultures expressed LOX-1 and TLR4 at similar levels. The LOX-1 expression remained unchanged after receptor blockade. However, the inhibition of LOX-1 caused a significant increase of cleaved caspase-3 and a decrease of TLR4 levels. The TLR4-inhibited DRG cell cultures lacked changes in LOX-1 expression for all experimental groups. The inhibition of TLR4 caused activation of jun N-terminal kinase (JNK) and a significant decrease of cleaved caspase-3 but did not change the TLR4 level. We conclude that LOX-1 and TLR4 are expressed in cultivated rat DRG cells and that the oxLDL-induced cell death in DRG cell cultures does not depend on the LOX-1 but on the TLR4., (2009 Wiley-Liss, Inc.)

The intrafollicular levels of oxidized low-density lipoprotein (oxLDL) and of enzyme antioxidants might contribute to reproductive disorders in obese and infertile women. Relevant data are missing. Eighty-four patients were grouped according to obese versus non-obese status and whether they had polycystic ovary syndrome (PCOS). The concentrations of oxLDL and the activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR) in the serum and follicular fluid were measured. Obese women with and without PCOS had significantly greater amounts of oxLDL in the follicular fluid as compared with non-obese women. The level of oxLDL in the follicular fluid was 1000 times lower than in serum. Obese women with and without PCOS had significantly higher catalase activity in the follicular fluid as compared with non-obese women. No differences were found for the SOD activity in the follicular fluid. The GPx and GR activities were up-regulated in obese patients without and with PCOS, yet not in respect to each serum and follicular fluid sample. We conclude that elevated levels of oxLDL in the follicular fluid of obese women are associated with higher catalase activity; both parameters are independent of PCOS. The levels of oxLDL and catalase activity appear to indicate different degrees of oxidative stress.

Autophagic cell death has been observed in granulosa cell cultures via the oxLDL-dependent activation of lectin-like oxidized low density lipoprotein receptor 1 (LOX-1). This activation might differ for cytokeratin-positive (CK(+)) and CK(-) granulosa cells. In particular, LOX-1 and toll-like receptor 4 (TLR4), one of the pattern recognition receptors of innate immunity, might be diversely regulated. Granulosa cell subtype cultures were established from the follicle harvests of patients undergoing in vitro fertilization (IVF) therapy. In response to oxLDL treatment, the fibroblast-like CK(-) cells upregulated LOX-1 and exhibited reparative autophagy, which could be blocked with anti-LOX-1 antibody. The epithelioid-like CK(+) cells did not regulate LOX-1 expression upon oxLDL application, but the expression of TLR4 and CD14 increased between 0 and 36 h of oxLDL/nDL treatment. This upregulation was associated with nonapoptotic cell death based on the absence of cleaved caspase-3. Reactive oxygen species (ROS) increased with 12 h oxLDL application and steroidogenic acute regulatory (StAR) protein expression was negligible. In CK(-) cells, the inhibition of TLR4 downregulated LOX-1 and induced apoptosis. We concluded that CK(-) granulosa cells are protected against oxLDL-dependent apoptosis by TLR4, whereas, in CK(+) cells, oxLDL-induced TLR4 activation triggers nonapoptotic cell death. The CK(+) cells might represent immune-like granulosa cells involved in ovarian remodeling processes.

The oxidized low-density lipoprotein (oxLDL)-dependent activation of the lectin-like oxLDL receptor-1 (LOX-1) triggers apoptosis in vascular cells and appears to be involved in atherosclerosis. Autophagy might be an alternate to apoptosis in endothelial cells. The EA.hy926 endothelial cell line has been reported to undergo necrosis under oxLDL stimulation. For this reason, we studied the expression of LOX-1 and its oxLDL-dependent function in EA.hy926 cells under serum starvation. Untreated and oxLDL-treated cells expressed the LOX-1 protein at similar levels 6h after starvation. After 24h without oxLDL and with native LDL (nLDL), statistically significant higher levels were found in LOX-1 than in the oxLDL-treated probes. The oxLDL cultures with low LOX-1 expression displayed stronger features of autophagy than those with nLDL as there were remodelling of actin filaments, disrupture of adherens junctions (immunofluorescence staining), and autophagosomes with the characteristic double membrane at the ultrastructural level. For the advanced oxLDL exposure times (18 and 24 h), autophagic vacuoles/autophagolysosomes were morphologically identified accompanied by a decrease in lysosomes. The autophagosome marker protein MAP LC3-II (Western blotting) was significantly augmented 6 and 18 h after oxLDL treatment compared with cultures treated with nLDL and medium alone. Signs of apoptosis were undetectable in cultures under oxLDL exposure, yet present under staurosporin (apoptosis inducer), i.e. presence of apoptotic bodies and cleaved caspase 3. We conclude that serum starvation upregulates LOX-1 in EA.hy926 cells, whereas the additional oxLDL treatment downregulates the receptor and intensifies autophagy probably by increase in oxidative stress.

The LOX-1 receptor, identified on endothelial cells, mediates the uptake of oxidized low-density lipoprotein (oxLDL). The oxLDL-dependent LOX-1 activation causes endothelial cell apoptosis. We here investigated the presence of LOX-1 in granulosa cells from patients under in vitro fertilization therapy. We were interested in the oxLDL-dependent LOX-1 receptor biology, in particular in the induction of apoptosis. In the human ovary, LOX-1 was localized in regressing antral follicles. In granulosa cell cultures, oxLDL-induced mRNA expression of LOX-1 in a time- and dose-dependent manner. The LOX-1 inhibitors (anti-LOX-1 antibody and kappa-carrageenan) abrogated the up-regulation of LOX-1. The oxLDL (100 microg/ml) treatment caused the autophagy form of programmed cell death: 1) reorganization of the actin cytoskeleton at the 6-h time point; 2) uptake of YO-PRO, a marker for the early step of programmed cell death, before propidium iodide staining to signify necrosis; 3) absence of apoptotic bodies and cleaved caspase-3; 4) abundant vacuole formation at the ultrastructural level; and 5) decrease of the autophagosome marker protein MAP LC3-I at the 6-h time point indicative of autophagosome formation. We conclude that follicular atresia is not under the exclusive control of apoptosis. The LOX-1-dependent autophagy represents an alternate form of programmed cell death. Obese women with high blood levels of oxLDL may display an increased rate of autophagic granulosa cell death.