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Endocrinology Vol. 147, No. 8 3851-3860
Copyright © 2006 by The Endocrine Society

Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1-Mediated Autophagy in Human Granulosa Cells as an Alternative of Programmed Cell Death

Nicole Duerrschmidt, Olga Zabirnyk1, Marcin Nowicki, Albert Ricken, Fayez A. Hmeidan, Verona Blumenauer, Jürgen Borlak2 and Katharina Spanel-Borowski

Institute of Anatomy (N.D., O.Z., M.N., A.R., K.S.-B.), University of Leipzig, D-04103 Leipzig, Germany; Centre for Reproductive Medicine (F.A.H., V.B.), Leipzig, Germany; and Fraunhofer Institute of Toxicology and Experimental Medicine (J.B.), Center for Drug Research and Medical Biotechnology, D-30625 Hannover, Germany

Address all correspondence and requests for reprints to: Katharina Spanel-Borowski, Institute of Anatomy, University of Leipzig, Liebigstrasse 13, D-04103 Leipzig, Germany. E-mail: spanelb{at}medizin.uni-leipzig.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 µg/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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OBESITY IS A serious problem in Western countries with a plethora of health effects. It is considered as main risk factor for diseases of the cardiovascular and the endocrine system, and obese women are often infertile (1, 2). There is strong evidence for obesity to be associated with oxidative stress, which then leads to augmented production of reactive oxygen species (ROS) (3). In fact, the native low-density lipoprotein (nLDL) can be oxidized to the modified form (oxLDL) by ROS.

In 1998 a new receptor was identified in bovine endothelial cells, which selectively binds and mediates the uptake of oxLDL (4). The receptor was named LOX-1 (lectin-like oxLDL receptor-1). It belongs to the C-type lectin family and has highest homology to natural killer cell receptors, which are implicated in tumor growth and cell recognition. The LOX-1 protein has no similarity with any known scavenger/LDL receptors (5, 6). LOX-1 was subsequently found in human endothelial cells, macrophages, vascular smooth muscle cells, and platelets (7, 8). A very recent study describes the LOX-1 presence in syncytiotrophoblasts of the human placenta, thus extending the receptor distribution to other organs than the vascular system (9). The oxLDL-dependent LOX-1 activation elicits the immediate production of ROS. Increased oxLDL formation leads to endothelial dysfunction and arteriosclerosis by a vicious cycle (10, 11). OxLDL up-regulates its own LOX-1 receptor and induces generation of more ROS molecules by a yet unknown mechanism. The more oxLDL is generated by oxidation, the more uptakes by LOX-1 are being observed (12, 13, 14). The physiological function of LOX-1 is involved to induce apoptosis in endothelial cells as documented by DNA fragmentation, activation of caspases-3 and -9 as well as the proapoptotic factor Bax. Additionally, LOX-1 inhibits the expression of the antiapoptotic factor Bcl-2 (12, 14, 15, 16).

Programmed cell death plays an important role in cell homoeostasis of the mammalian ovary. In each ovarian cycle, among growing antral follicles the preovulatory follicle is selected. Follicles, which do not ovulate, undergo follicular atresia (17). Disturbance of the atretic process leads to follicular cysts; 99.9% of all oocytes in different stages of follicular development and maturation die by atresia. In preantral follicles, the oocyte undergoes apoptosis, whereas the granulosa cells remain intact. In contrast, large antral follicles preferentially show apoptosis of granulosa cells (18, 19). The current knowledge on the molecular signaling in apoptotic granulosa cells points to the Fas-ligand pathway (20, 21, 22, 23). The susceptibility to Fas-ligand of preovulatory granulosa cells depends on the LH. Cells go into apoptosis only when harvested before the hormone surge (24). Whether apoptosis is induced in granulosa cells by other factors such as oxLDL is not known so far.

Until now, follicular atresia is judged as an exclusive process of apoptosis (24). However, in the goose and quail ovary, two other forms of programmed cell death, termed autophagy and necrosis-like form, coexist in addition to the apoptotic form (25, 26). These reports are of considerable importance because autophagy and the necrosis-like form have emerged as alternate nonapoptotic forms of programmed cell death. Autophagy is characterized by the degradation of proteins and damaged organelles, which occurs during tissue remodeling and starvation (27, 28). Autophagy is considered an evolutionary program for cell survival under stress. Stressed cells ultimately die when all internal resources of survival are exhausted. Autophagy is associated with the formation of large cytoplasmic vacuoles, which arise by isolated membranes, each surrounding cellular components under degradation and thus giving way to autophagosomes (29). The first step of autophagosome formation is independent on lysosome fusion. Notably, autophagy represents a caspase-3 and -9-independent signaling pathway without DNA fragmentation. Insights into the molecular machinery of autophagy and the necrosis-like form are just emerging (30).

We hypothesized that oxLDL-dependent LOX-1 receptor activation plays an essential role in the induction of human granulosa cell death. We report strong evidence for the autophagic program in mature human granulosa cells. The outcome of our study opens new frontiers to deepen our knowledge about follicular atresia and infertility in obese women.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Ovaries (n = 5; fixed with 4% formaldehyde).
These were collected from the archives of the Institute of Pathology, University of Leipzig, as described previously (31). According to the records, the ovaries embedded in paraffin wax were derived from women of reproductive age who had undergone oophorectomy for gynecological reasons between 1985 and 1995.

Cell culture of primary granulosa.
Granulosa cells were taken from patients under in vitro fertilization (IVF) therapy. The Committee for Ethical Approval at the Medical Faculty approved the experimental procedure and the use of the cells, and the patients gave their written consent. Bloodless aspirates from different follicles of one patient were pooled, centrifuged (1200 rpm for 3 min), the resuspended pellet plated into 50-ml plastic flasks, and cultured at 37 C in a humidified air with 5% CO2. The culture medium was a mixture of one part endothelial cell growth medium MV (PromoCell, Heidelberg, Germany) and four parts DMEM:F12 medium (1:1; Life Technologies, Inc., Karlsruhe, Germany) containing 15 mM HEPES, 22 mm NaHCO3, 5% (vol/vol) fetal bovine serum (PromoCell), and antibiotics. When conspicuous colonies of macrophages were noted in confluent cultures at the phase microscopical level (32), they were discarded. Single macrophages were damaged by shock freezing enzymatically dislodged granulosa cells in liquid nitrogen. Absence of leukocytes in the replated cultures was verified by immunostaining for CD11/18-positive macrophages.

The human granulosa cell line SVOG-4o.
This cell line was kindly provided by Nelly Auersperg (33) and cultured as described above. The intention was to have no cell limitation for mRNA and protein analysis. SVOG-4o cells turned out to decrease in cell proliferation at higher passages.

Preparation of oxLDL and nLDL and cell culture treatment
nLDL was isolated from human plasma by sequential gradient ultracentrifugation as described (14). oxLDL was prepared by incubation of nLDL with 5 µmol/liter CuSO4 for 24 h at 37 C. Oxidation was monitored using the thiobarbituric acid-reactive substances assay with tetraethoxypropane as an internal standard.

Granulosa cells were grown to confluence in serum-containing medium (see above) in either 50-ml flasks for Western blot analysis or 24-well-cluster plates for the morphological studies. Round glass coverslips had been mounted into each well. When not otherwise indicated, 100 µg/ml oxLDL or 100 µg/ml nLDL (Sigma, Taufkirchen, Germany) were applied in DMEM-F12 medium under serum-free conditions. Cultures with medium alone were also established. oxLDL-stimulated cultures could be simultaneously treated with 250 µg/ml {kappa}-carageenan (Sigma), a specific inhibitor of LOX-1. Time points of treatment changed between 1 and 24 h, depending on the different experiments.

RT-PCR analysis for LOX-1
Cultures of primary granulosa and SVOG-4o cells were stimulated with oxLDL/nLDL in the range between 10 and 100 µg/ml for 1–24 h. To show the specificity of LOX-1 mRNA up-regulation, the polyclonal rabbit anti-LOX-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was applied at 5 µg/ml together with LOX-1. Total RNA from oxLDL treated and untreated primary granulosa cells and SVOG-4o cells were isolated by peqGOLD RNAPure (Peqlab, Erlangen, Germany) according to the manufacturer’s recommendation. The quality of isolated RNA was checked using 1% agarose gel. The RNA concentration was quantified with UV spectroscopy (Ultrospec 4000; Pharmacia Biotech, Cambridge, UK).

Total RNA (1 µg), 300 ng random primer, and 1 mM deoxynucleotide triphosphate (Invitrogen, Karlsruhe, Germany) were preheated at 65 C for 5 min, and then 5 x first-strand buffer, 10 mm dithiothreitol, 40 U RNaseOUT ribonuclease inhibitor, 200 U SuperScript II reverse transcriptase (Invitrogen), and Rnase-free water were added to a final volume of 20 µl. Reverse transcription was carried out at 42 C for 1 h and stopped by heating to 70 C for 15 min. PCRs were carried out in a T3 thermal cycler (Biometra, Göttingen, Germany) using LOX-1-specific primer (forward: 5'-TGGGAAAAGAGCCAAGAGAA-3'; reverse: 5'-TGCAGCCAGCTAAATGACAG-3'; NM002543; 554-1003 bp) and 18S rRNA-primer (forward: 5'-GTTGGTGGAGCGATTTGTCTGG-3'; reverse: 5'-AGGGCAGGGACTTAATCAACGC; U13369; 5001–5348). The following cycling conditions were applied: denaturizing at 94 C for 40 sec, annealing at 55 C for 1 min, and extension at 72 C for 1 min (38–40 cycles). The PCRs were separated using 1.5% agarose gel, stained with ethidium bromide, and photographed on a transilluminator. The PCR fragments were quantified by densitometry using Aida (Raytest, Straubenhardt, Germany). The LOX-1 mRNA expression of each sample was normalized using 18S rRNA expression. The unstimulated control was set to 100%.

Western blot analysis for LOX-1 after oxLDL/nLDL stimulation for 3 h
Cultures were lysed by buffer containing 20 mM Tris/HCl (pH 6.8), 0.7% (wt/vol) sodium dodecyl sulfate, and 3.3% (wt/vol) saccharose. After homogenization by ultrasonification proteins were denaturized by heating (95 C, 5 min) and concentrations determined by the BCA protein assay kit (Pierce, Rockford, IL). Thirty micrograms protein of each sample were mixed 1:4 with loading buffer [250 mm Tris/HCl (pH 6.8); 400 mM dithiothreitol; 140 mm sodium dodecyl sulfate; 60% (vol/vol) glycerine and 0.02% (wt/vol) bromophenol blue], separated by SDS-PAGE and transferred to a nitrocellulose membrane. The nonspecific binding sites were blocked with 5% (wt/vol) milk powder in Tris-buffered saline (TBS) buffer [10 mM Tris/HCl (pH 7.3); 500 mm NaCl; 0.2% (vol/vol) Tween 20], and then the membrane was treated with a polyclonal rabbit anti-LOX-1 antibody (1:1500; Santa Cruz Biotechnology) overnight at 4 C. After rinsing the primary antibody was detected with a peroxidase-labeled goat antirabbit IgG (Vector Laboratories, Burlingame, CA) diluted 1:6000 for 1 h and by ECL detection reagents (Amersham Biosciences, Buckinghamshire, UK). Negative controls were incubated with rabbit IgG and secondary antibody. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level was detected with monoclonal mouse anti-GAPDH antibody (Santa Cruz Biotechnology) to equalize the protein.

Immunohistology of human ovaries and immunocytology of granulosa cells for LOX-1
Seven-micrometer-thick ovarian sections were deparaffinized, and endogenous peroxidase activity was quenched with 3% H2O2 in 50 mM TBS containing 10% methanol for 10 min. Sections were washed in PBS and saturated with 1.5% normal goat serum (DakoCytomation, Glostrup, Denmark) for 30 min and then incubated with the polyclonal rabbit anti-LOX-1 antibody (Santa Cruz Biotechnology) diluted 1:50 in PBS containing 0.25% BSA (overnight at 4 C; Sigma). The antibody detected the amino acids near the intracellular N terminus of LOX-1. After rinsing with PBS containing 0.2% (vol/vol) Tween 20 and PBS only, sections were incubated with biotinylated goat antirabbit IgG (Vector Laboratories) diluted 1:200 in PBS containing 1% normal goat serum for 30 min. Negative controls were incubated with rabbit IgG and the secondary antibody, respectively. PBS-washed sections were treated with the tyramide signal amplification system (TSA biotin system; PerkinElmer Life Science, Boston, MA) for 10 min. Then sections were incubated with the avidin-biotin-horseradish peroxidase-labeled complex (Vectastain, ABC reagent; Vector Laboratories) for 30 min. Bound peroxidase was detected by 0.025% (wt/vol) 3'3'-diaminobenzidine (Sigma, Deisenhofen, Germany) in TBS with 0.03% H2O2 added before use. After counterstaining with hematoxylin, dehydrated sections were embedded in Canada balsam.

Three-hour stimulated granulosa cell cultures were fixed with 4% formaldehyde in PBS for 15 min and permeabilized with 0.05% Tween 20 in PBS. After saturation of nonspecific binding sites with normal goat serum, cells were incubated with the rabbit polyclonal anti-LOX-1 antibody (Santa Cruz Biotechnology) diluted 1:400 in PBS containing 0.25% BSA (overnight at 4 C). The binding site was detected with a fluorochrome Cy3-labeled second antibody (Dianova, Hamburg, Germany) diluted 1:100 in PBS (30 min). Coverslips were mounted upside down in Dako Glycergel (DakoCytomation) containing 25 µg/ml 1,4-diacabicyclo[2,2,2] octane (Sigma) and 10 µg/ml 4'-6-diamidino-2-phenylindole (DAPI; Serva, Heidelberg, Germany). Negative controls were incubated with rabbit IgG and the second antibody, respectively.

Staining of actin filaments 6, 9, and 12 h after oxLDL/nLDL treatment.
Actin filaments are reported to behave differently during apoptosis and autophagy (34). Confluent cultures were fixed as described for immunostaining and permeabilized with 0.1% Triton X-100 for 10 min. Background staining was quenched with 0.1 M glycine in PBS for 5 min. Then cells were incubated (30 min) with fluorescein isothiocyanate-phalloidin (Sigma) diluted 1:200 in PBS. After PBS rinse coverslips were mounted upside down in Dako Glycergel (see above).

Detection of cell death with YO-PRO-1 and propidium iodide (PI) staining 6, 9, and 15 h after oxLDL/nLDL treatment
The green fluorescent YO-PRO-1 dye passes the cell membrane at an early step of programmed cell death and enters the nucleus, whereas the red fluorescent dye PI is seen in dead cells. The application of both fluorochromes allows differentiating between apoptosis and necrosis according to the supplier (Molecular Probes, Eugene, OR). Granulosa cell cultures were washed with cold PBS and incubated with the fluorochromes YO-PRO-1 and PI (Vybrant apoptosis assay kit no. 4, Molecular Probes) each 1:1000 diluted in PBS and applied together for 30 min. Cells were postfixed with 4% Formalyde in PBS for 15 min and further handled as described for immunocytology.

Detection of autophagic vacuoles with transmission electron microscopy after 15 and 24 h of oxLDL/nLDL treatment
The ultrastructural presence of autophagic vacuoles is the method of choice to monitor autophagy (35). Cultures were grown on Thermanox coverslips (Nunc, Inc., Naperville, IL) in 24-well culture plates. Cells were washed with PBS and fixed for 1 h in 2.5% PBS-buffered glutaraldehyde at 4 C and postfixed in 1% OsO4 for another hour. Monolayers were washed in 2.4% NaCl and then in 0.2 M sodium acetate buffer (pH 5.0). Cultures were subsequently treated with 1% uranyl acetate in 0.2 m sodium acetate buffer for 30 min. After rinsing with 0.2 M sodium acetate buffer, dehydration was carried out in 50–90% ethanol and 2-hydroxy-propyl-methacrylate (Merck, Darmstadt, Germany), which is compatible with plastic dishes. Monolayers were infiltrated with 2-hydroxy-propyl-methacrylate-Epon 812 mixtures by increasing the resin concentration gradually. Then coverslips were transferred to silicon molds, 300 µl Epon 812 added to each mold, and the samples evacuated before polymerization (2 d at 60 C). Polymerized Epon casts were rapidly frozen so that the coverslips could be removed from the castings that contained the cells. Areas were trimmed under a stereomicroscope and mounted on an Epon block. Ultrathin sections were contrasted with uranyl acetate and lead citrate and examined in a Zeiss EM 10 (Zeiss, Jena, Germany).

Detection of autophagic vacuoles by immunostaining for microtubule-associated protein light chain 3 (MAP LC3) and Western blot analysis 6–12 h after oxLDL/nLDL treatment
The MAP LC3 molecule has been initially identified as autophagosome marker protein (29, 35). This 16- to 18-kDa protein is soluble as cytosolic LC3-I molecule in nonstarved cells and becomes an autophagosome membrane-associated LC3-II molecule during starvation. Cultures were fixed with 4% formaldehyde and immunostaining conducted to detect autophagic vacuoles by using a rabbit polyclonal antibody against MAP LC3 (Santa Cruz Biotechnology) at a 1:4000 dilution. The binding site was detected as described for immunocytology. Additionally, confluent cultures were subjected to Western blot analysis (see above) by using the 1:2000 diluted rabbit antiserum against LC3 (a kind gift from Professor Yoshimori, National Institute of Genetics, Mishima, Japan) (29). The antiserum recognized both the cytosolic and membrane-bound molecules, i.e. LC3-I and LC3-II.

Verification of apoptosis in granulosa cells after staurosporine treatment 6–12 h after oxLDL/nLDL treatment
Staurosporine, a protein kinase inhibitor, is reported to induce apoptosis in human granulosa cells (36). We wanted to know whether apoptotic bodies and cleaved caspase-3 can be detected in staurosporine-treated cells, whereas oxLDL-treated granulosa cells lack these apoptotic signs. Cells were treated with 100 µg/ml oxLDL or 1 µM staurosporine (Sigma-Aldrich, catalog no. 62996-74-1) under serum-free conditions. Then cells were fixed and nuclei stained by DAPI (see immunocytology). Otherwise the protein was extracted from cultures in 50-ml flasks and blotted for the presence of cleaved caspase-3 by use of a specific rabbit antibody (Cell Signaling Technology via New England Biolabs GmbH, Frankfurt, Germany). The antibody was incubated at a 1:1000 dilution at 4 C over night. As positive controls, the company supplied cell extracts from Jurkat cells with and without induced apoptosis.

Photodocumentation and statistics
Digitized pictures were taken with an Axioplan 2 light microscope (Zeiss) equipped with a Progress camera and otherwise used with epifluorescence illumination together with an axiovision camera (Zeiss). For the actin filament staining and detection of autophagic vacuoles, pictures were obtained with the laser-scanning microscope LSM 510 Meta (Zeiss). Ultrastructural pictures were taken with a Zeiss EM10.

The data of each experiment related to three to six independent measurements and are shown as mean ± SEM. Statistical analysis was performed with ANOVA procedure by Bonferroni method (SigmaStat, Jandel Scientific, San Rafael, CA). Differences were taken as statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LOX-1 is localized in granulosa cells of the human ovary
Ovarian sections contained follicles of preantral and antral stage. Some of them expressed LOX-1 in granulosa cells. These follicles appeared to undergo atresia. In particular, antral follicles with advanced stages of atresia such as a loosened granulosa layer of irregular height and a deformed oocyte depicted a positive response within the cumulus, interestingly in the absence of apoptotic bodies (Fig. 1Go). When the thecal layer showed hypertrophy, LOX-1-positive thecal cells were also seen. We used as positive internal control the finding that, in the same section, vascular smooth muscle cells and endothelial cells displayed a LOX-1-positive signal. No signal was obtained in the negative control section.


Figure 1
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  		FIG. 1. LOX-1 occurrence in an antral follicle of advanced atresia as depicted by indirect immunostaining with a polyclonal anti-LOX-1 antibody. A, Deformation of the oocyte and loosening of the cumulus oophorus is shown. B, At higher magnification, LOX-1-positive granulosa cells are noted in the cumulus, of note, in the absence of apoptotic bodies. C, The negative control is conducted with unspecific rabbit IgG. D, The smooth muscle cells in the media of medullary vessels represent the positive control. Bar, A, 100 µm; B and C, 25 µm, D, 50 µm

 
LOX-1 up-regulation in cultured granulosa cells is dependent on oxLDL
The presence of LOX-1 in the intact ovary encouraged us to thoroughly investigate the LOX-1 expression by using the human granulosa cell line SVOG-4o and granulosa cells from IVF patients. We first examined whether LOX-1 mRNA was up-regulated under oxLDL treatment in a time- and concentration-dependent way. There was a striking increase as early as 3 h after stimulation with both the SVOG 4o and granulosa cells from IVF patients (Fig. 2Go); 100 µg/ml oxLDL maximally induced LOX-1 mRNA in SVOG-4o (235 ± 47% of control; P < 0.05) and granulosa cells of the patients (189 ± 33% of control; P < 0.05). Adding anti-LOX-1 antibody blocked the statistically significant up-regulation. The LOX-1 mRNA expression showed rather basic levels after stimulation with nLDL. For further experiments the primary granulosa cells were preferentially used and treated with 100 µg/ml because this concentration had up-regulated the LOX-1 mRNA expression at a statistically significant level.


Figure 2
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  		FIG. 2. LOX-1 mRNA expression in SVOG-4o and primary granulosa cells from IVF patients after incubation in a time (A)- and concentration (B)-dependent manner with oxLDL or nLDL under serum-free conditions for 1–15 h. The LOX-1 mRNA expression is quantified by semiquantitative RT-PCR. A, The oxLDL concentration of 100 µg/ml causes a significant up-regulation of LOX-1 mRNA after 3 h, compared with the control. B, The anti-LOX-1 antibody (anti-LOX-1 ab; 5 µg/ml) inhibits the significant LOX-1 up-regulation. nLDL has minor effect on LOX-1 mRNA expression (n = 3–6; *, P < 0.05 vs. control).

 
The LOX-1 protein expression was noted after oxLDL exposure for 3 h. A seemingly higher amount of the mature (50 kDa) and precursor form (40 kDa) was detected when compared with nLDL-stimulated cells and cells treated with the competitive LOX-1 inhibitor {kappa}-carrageenan (Fig. 3AGo). Under oxLDL stimulation as well as treatment with {kappa}-carrageenan, the rapid up-regulation of LOX-1 was prevented. Note the precursor form was up-regulated under oxLDL exposure without and with {kappa}-carageenan but hardly found under nLDL treatment. Because the LOX-1 protein expression was seen to be up-regulated in oxLDL-treated cells by using immunostaining after cell membrane permeabilization (Fig. 3BGo), the polyclonal anti-LOX-1 antibody most likely detected the precursor form. A high amount of LOX-1 protein was localized in the perinuclear area, which was absent under nLDL stimulation. The intense perinuclear response may reflect the augmented precursor protein translation.


Figure 3
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  		FIG. 3. LOX-1 protein expression in granulosa cells of IVF patients incubated with oxLDL and nLDL (100 µg/ml) under serum-free conditions for 3 h. A, For Western blot analysis, total proteins are isolated and 30-µg proteins per lane are incubated with rabbit anti-LOX-1 antibody (1:1500). OxLDL appears to induce a higher amount of LOX-1 protein (50 kDa), compared with nLDL and the LOX-1 inhibitor {kappa}-carrageenan (250 µg/ml). The LOX-1 precursor form (40 kDa) is up-regulated after treatment with oxLDL or {kappa}-carrageenan. A neglectable amount of the precursor form is detected under nLDL exposure. GAPDH shows equal loading of protein. Data are representative of two independent experiments. B, For localization by immunofluorescence with rabbit anti-LOX-1 antibody, the protein of punctuate immunostaining is more evident in permeabilized granulosa cells in A, compared with nLDL-treated granulosa cells in B. Bar, 20 µm.

 
Nonapoptotic cell death in cultured granulosa cells is dependent on oxLDL
Because oxLDL is known to induce apoptosis in endothelial cells by the activation of LOX-1 (12, 14, 15, 16), we looked for signs of cell death in oxLDL-treated IVF granulosa cells.

We reached four conclusions. First, we found a declining transcription rate of rRNA in a time-dependent manner. After 15 h of oxLDL exposure, 18S rRNA and 28S rRNA were lower in amount, compared with the serum-free control. After 24 h rRNAs were no longer detectable (Fig. 4AGo). Semiquantitative measurements of the total RNA displayed decreasing values between 6, 15, and 24 h of oxLDL treatment. The low amount of total RNA at the later time points were judged as sign for dying cells (Fig. 4BGo).


Figure 4
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  		FIG. 4. Decrease in rRNA and total RNA concentration after oxLDL stimulation in SVOG-4o cells and primary granulosa cells of IVF patients incubated with 100 µg/ml oxLDL under serum-free conditions for 1–24 h. Serum-free controls remained untreated. A, RNA is isolated by peqGOLD RNAPure and 28S and 18S rRNAs shown by gel electrophoresis. B, Total RNA is quantified by UV spectroscopy (n = 3–6; *, P < 0.05 vs. control).

 
Second, degradation of actin filaments is reported for the apoptotic process, whereas primarily intact actin filaments are described for autophagy (34). Here we observed that oxLDL treatment first caused the reorganization of the actin cytoskeleton followed second by the depolymerization of actin filaments in a time-dependent fashion. A subplasmalemmal ring of actin filaments developed 6 h after oxLDL stimulation (Fig. 5Go). After 9 h the actin filaments strikingly decreased in amount and few actin-positive granule-like structures were apparent. The complete loss of any actin filaments was noted after 12 h. The LOX-1 inhibitor {kappa}-carrageenan prolongated the process of actin depolymerization because at the 6- and 9-h time points, more actin filaments were noted, compared with the oxLDL-treated cells. {kappa}-Carrageenan thus delayed actin degradation by 3 h. Untreated and nLDL-treated cells were rich in filamentous actin of stress-fiber-like appearance when maintained for 12 h under serum-free conditions.


Figure 5
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  		FIG. 5. Remodeling and depolymerization of the actin cytoskeleton after oxLDL stimulation of granulosa cells from IVF patients in a time-dependent manner. Cells are stimulated with oxLDL and nLDL (100 µg/ml) between 6 and 12 h and stained with fluorescein isothiocyanate-phalloidin. After oxLDL treatment a distinct ring of actin filaments occurs adjacent to the cell membrane (6 h), and a few granular-like F-actin forms are noted (9 h). The loss of any actin-positive structures is complete after 12 h. The treatment with {kappa}-carrageenan is able to prolongate the process of remodeling from 6 to 9 h. Controls (none, nLDL) are rich in F-actin of stress-fiber like appearance 12 h under serum-free conditions. DAPI-stained nuclei do not reveal condensation or fragmentation. Laser scanning microscopy; bar, 10 µm

 
Third, to verify oxLDL-induced apoptosis in granulosa cells, the rapid and convenient Vybrant apoptosis assay (see Materials and Methods) was applied. After 9 h of oxLDL incubation, altered cells were detected by nuclear uptake of the green fluorochrome YO-PRO-1, a marker for the early step of cell death. After 15 h granulosa cells began to die as indicated by the PI uptake and red fluorescent nuclei (Fig. 6Go). The treatment with {kappa}-carrageenan for 15 h impeded the uptake of PI and thus slowed down cell death. Note that DAPI-stained nuclei never showed nuclear condensation and fragmentation, respectively, at the advanced time of investigation. This feature spoke against the apoptotic form of cell death.


Figure 6
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  		FIG. 6. Cell death assay incubating granulosa cells of IVF patients with YO-PRO-1 and PI. Cells are stimulated with oxLDL and nLDL (100 µg/ml each) under serum-free conditions for 6–15 h. At each time point, cultures are simultaneously incubated with the green fluorochrome YO-PRO-1 and the red fluorochrome PI. DAPI is used for nucleus staining. After oxLDL stimulation for 6 h, neither green nor red fluorescence is noted indicating living cells. After 9 h the nuclei display only green fluorescence (apoptotic cells) and after 15 h oxLDL incubation the nuclei demonstrate green and red fluorescence (dead cells). The treatment with {kappa}-carrageenan for 15 h impedes the uptake of PI and thus slows down cell death. After nLDL stimulation for 15 h, no uptake of fluorochromes is seen. Bar, 20 µm

 
Finally, ultrastructural changes typical for apoptosis such as cell blebbing, DNA condensation, or apoptotic bodies were searched for in oxLDL-treated granulosa cells by transmission electron microscopy. Neither DNA condensation nor apoptotic bodies were detected in granulosa cells 15 and 24 h after oxLDL treatment. OxLDL incubation for 15 h caused vacuolization, compared with the serum-free control (Fig. 7AGo), and the nLDL control, respectively. The vacuoles could contain floccular material or membrane-including debris. Intact mitochondria were detected. At the 24-h time point, vacuoles became more conspicuous (Fig. 7BGo). Prominent autophagosomes with membrane-bound inclusions were depicted. The cytoplasm and the karyoplasm began to undergo lysis. {kappa}-Carageenan could not protect from the oxLDL-induced vacuole formation. In the controls (serum-free and nLDL), the organelles remained intact apart from the appearance of autophagosomes often of myelin-like structure as is well known to be caused by serum-deprivation. The nLDL-treated granulosa cells developed a dilated rough endoplasmic reticulum (Fig. 7CGo).


Figure 7
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  		FIG. 7. Autophagic vacuoles after oxLDL stimulation are depicted at the ultrastructural level in granulosa cells of IVF patients. Cells are stimulated with oxLDL (100 µg/ml) (A and B) and with nLDL (C) under serum-free condition. A, At 15 h of oxLDL treatment many vacuoles are found. Because a few of them display inclusions (arrow), vacuoles are judged as autophagosomes. Intact mitochondria are seen (open arrow). B, At 24 h of oxLDL stimulation autophagic vacuoles have become more prominent. A circumscribed dilatation of the perinuclear space (triangle) and lysis of the karyoplasma are apparent. C, At 24 h of nLDL treatment, a dilated rough endoplasmic reticulum (arrow), lipid droplets (open arrow), and autophagosomes are found. Cytoplasm and organelles are intact. Bar, 2.5 µm for A and C; 1.1 µm for B.

 
OxLDL causes autophagic cell death in granulosa cells
Because the ultrastructural finding with a high amount of vacuoles sometimes together with cytoplasmic inclusions spoke for the autophagy form of programmed cell death, we investigated the presence of the MAP LC3 molecule, the initially identified autophagosome marker (29, 35), in granulosa cells. Earlier time points of oxLDL treatment were considered. After 6 h roughly 30% cells showed MAP LC3-positive immunostaining of punctuate pattern (Fig. 8AGo). The finding was substantiated by Western blot analysis showing the disappearance of the soluble LC3-I molecule, whereas changes in expression were inapparent for the LC3-II, compared with the controls (serum starvation and nLDL). After 12 h, the LC3-I and LC3-II proteins were neglectable for oxLDL treatment with and without {kappa}-carageenan (Fig. 8BGo).


Figure 8
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  		FIG. 8. The presence of the autophagosome marker, i.e. of MAP LC3-positive autophagosomes, is verified in granulosa cells of IVF patients. Cells are treated with oxLDL (100 µg/ml) without and with {kappa}-carageenan, with nLDL, or with medium (control). A, Immunocytochemistry is conducted in cultures treated for 6 h. The granulosa cell shows LC3-positive structures of punctuate immunostaining in (A), whereas no positive response is obtained under nLDL in (B). Bar, 10 µm. B, Western blot analysis is carried out to detect changes in the expression of the autophagosome marker. After 6 h (left side), a decrease of the LC3-I molecule is noted under oxLDL treatment both without and with {kappa}-carageenan. After 12 h, LC3-I and LC3-II are almost undetectable. Data are representative of two independent experiments.

 
Apoptosis induced in granulosa cells by staurosporine
To exclude the possibility that apoptosis may have escaped our notice in granulosa cell cultures under oxLDL treatment, it was necessary to establish a positive control. This was achieved by staurosporine-induced apoptosis. After 6 and 12 h of treatment, apoptotic bodies were seen in DAPI-stained samples (Fig. 9AGo). Furthermore, cleaved caspase-3 was apparent in Western blot analysis. In comparison, oxLDL-treated granulosa cells remained free of apoptotic features (Fig. 9BGo).


Figure 9
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  		FIG. 9. Induced apoptosis in granulosa cells from IVF patients after treatment with staurosporine (1 µM) in comparison with oxLDL (100 µg/ml) both under serum-free conditions. Controls are run under serum deprivation for the same time points. A, After 12 h of staurosporine treatment, many apoptotic bodies (arrows) are noted in the DAPI-stained culture, whereas intact nuclei appear to be present in the oxLDL-treated cells. B, Induced apoptosis is accompanied by the presence of cleaved caspase-3 in cultures treated for 6 and 12 h by staurosporine as noted by Western blot analysis. No caspase-3 appears under oxLDL treatment. Note the positive and negative caspase-3 response in Jurkat cell extracts with and without induced apoptosis. Data are representative of two independent experiments. Stau, Staurosporine

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we aimed to identify the role of oxLDL and LOX-1 receptor activity in ovarian function and show for the first time that the LOX-1 receptor can be localized in regressing antral follicles of the human ovary. Intact follicles most likely display a basal LOX-1 expression, which is below the level of detection by immunostaining. Notably, oxLDL stimulated LOX-1 receptor activity is also demonstrated in granulosa cells derived from IVF patients. In culture, the LOX-1 protein together with its precursor protein is up-regulated in granulosa cells already 3 h after oxLDL exposure. This time point of protein expression correlates with the statistically significant mRNA induction of LOX-1 in these cells, compared with the 6 and 15 h value. The response upon treatment of cells with 100 µg/ml oxLDL appears to be more rapid in granulosa cells than that observed for endothelial cells in which the LOX-1 mRNA peak occurs between 12 and 24 h of mild (10–40 µg/ml) oxLDL treatment (5, 37). A higher dose of oxLDL (60 µg/ml) sustains LOX-1 protein expression in vascular smooth muscle cells up to 24 h of treatment. The rapid oxLDL-dependent up-regulation of LOX-1 mRNA and protein in granulosa cells is selectively mediated by LOX-1 itself because LOX-1 inhibitors like anti-LOX-1 antibody and {kappa}-carageenan have prevented the increased receptor response. With regard to nLDL stimulation, there was a slight increase in LOX-1 mRNA level at the 3-h time point (Fig. 2Go); however, it was not at a statistically significant level. In line with this statement are the findings that stress fibers are present in granulosa cells and that no signs of cell death occur 12–24 h after nLDL treatment (Figs. 5–7GoGoGo).

The LOX-1 receptor has been preferentially studied in the vascular system. By now it becomes evident that other organ systems also express LOX-1 receptor such as trophoblasts of the human placenta according to a very recent publication (9). Here we report human granulosa cells to express LOX-1 receptor. It attracts considerable scientific interest because its oxLDL-dependent activation causes ROS production leading to apoptosis in vascular cells (12, 14, 15, 16). Apoptosis in the developing rat cerebellum is accompanied by the degradation of both the 28S and 18S of rRNAs (38). Despite the degradation of the rRNAs in oxLDL-treated human granulosa cells observed here, apoptosis could not be confirmed. No nuclear fragmentation/formation of apoptotic bodies has been seen in the DAPI-stained cultures and no cleaved caspase-3 activation observed.

The ultrastructural study represents the golden standard for describing forms of programmed cell death. Additional biochemical parameters serve as confirmation. In this study, abundant vacuole formation at the ultrastructural level and the appearance of the MAP LC3 molecule as autophagosome marker protein belong to the still limited, yet approved specific features to monitor autophagy (39, 40). We give clear evidence that the autophagy form of programmed cell death occurs in granulosa cells under oxLDL treatment. Our diagnosis of autophagy relies on the presence of abundant vacuoles at the ultrastructural level, some of them with inclusions of organelles. The typical double membrane, which arises from the so-called isolation membrane around an organelle, has most likely disappeared 15 h after oxLDL stimulation. At that late time point, granulosa cell necrosis is going on as indicated by the disappearance of actin filaments and the presence of PI-positive nuclei. The process of autophagosome formation and their turnover into autophagolysosomes occurs at an early time point. Six hours of oxLDL treatment is judged as a crucial period because here actin filaments are still remodeled and a decrease in protein expression is seen for the soluble LC3-I molecule. That we are unable to detect changes for the membrane-bound LC3-II in granulosa cells is explained by a rapid turnover of autophagosomes, which takes just minutes according to the literature (29). Additional data can be obtained by quantitative electron microscopy, a tedious process to estimate the cytoplasmic volume fraction of autophagic vacuoles (35). Staining with monodansylcadaverine at the light microscopical level as we successfully did 6 and 12 h of oxLDL treatment (our unpublished observations) is thought to be indicative of acidic autolysosomes (35, 41) and thus less valid for autophagosomes.

Work in progress on the downstream signals of the LOX-1-dependent transduction cascade indicates that MAPKs are involved (16). The heterodimeric G proteins also appear to play a role in the control of autophagy, in particular the involvement of GTPase in vacuole formation in hepatocytes (43). Here we obtain strong, indirect evidence that the monomeric Ras proteins play an essential role in transmitting the effect of oxLDL on granulosa cells. Ras proteins are tethered to the cytoplasmic face of the plasma membrane. For activation, Ras exchanges GDP for GTP. Downstream protein kinases are Roc and Roc-associated kinase carrying the signal from the plasma membrane toward the nucleus. Their activation is linked to rearrangement of the actin cytoskeleton (44, 45). We show that granulosa cells undergo remodeling of actin filaments 6 h after oxLDL treatment that may be the response of the cytoskeleton to the rise in vacuole formation. In contrast to apoptosis, the beginning of autophagy appears to depend on the intact cytoskeleton (34, 46). The early oxLDL-dependent remodeling of the actin cytoskeleton and the complete depolymerization at 15 h of oxLDL exposure represents a time-dependent process as supported by other findings. Within 2 min of oxLDL stimulation, human endothelial cells display remodeling of the actin cytoskeleton and disrupture of intercellular gaps combined with increased cell permeability (47). Otherwise oxLDL treatment more than 24 h considerably decreases total actin levels in macrophages and vascular smooth muscle cells (48, 49). Because in this study {kappa}-carageenan, the competitive LOX-1 inhibitor, is able to delay the actin filament alteration in oxLDL-treated granulosa cells for 3 h, {kappa}-carageenan is judged as reagent with protective effects on the LOX-1 signaling pathway. {kappa}-Carageenan may become of therapeutic interest.

The YO-PRO stain, a green fluorochrome, enters apoptotic cells, whereas the red fluorescent PI appears in necrotic cells due to loss of plasma membrane integrity. The presently observed YO-PRO-positive cells 9 h after oxLDL treatment, however, do not signalize apoptosis because DNA condensation and/or fragmentation do not follow at later time points of oxLDL treatment. Fragmentation is apparent neither in DAPI stained nuclei at the 15-h time point when PI-positive granulosa cells are additionally seen nor at 24 h, the latest time point of the ultrastructural study. The YO-PRO stain thus seems to be a general marker of cells undergoing programmed cell death by either apoptosis or autophagy. More subtle staining methods are wanted in the future to differentiate between the two forms. Interestingly, the appearance of YO-PRO-positive and PI-positive nuclei in granulosa cells 15 h after oxLDL treatment is blocked by {kappa}-carageenan, which again underlines its protective effects on the LOX-1-dependent signaling cascade. The effect of {kappa}-carageenan also shows that autophagy can be judged as an active molecular process with an upstream side, which is still able to switch back to cell recovery, whereas the downstream portion is doomed to make the cell die (50).

Atresia of antral follicles is understood to be under the exclusive control of apoptosis (19). Apoptotic bodies are seen (18, 20) and the Fas-ligand pathway is confirmed for granulosa cells from antral and preovulatory follicles (21, 22, 23, 24). We report autophagy to be an alternative of programmed cell death in LOX-1-positive granulosa from IVF patients. To further unravel the molecular mechanism of autophagy in the ovary, the recent report on the ovarian-voltage-activated Na+ channel in human granulosa cells contains a potentially interesting aspect (51). The activation of the endocrine Na+ channel is associated with an increase in autophagosomes and a decrease in progesterone secretion. In the past, morphological signs of autophagy and necrosis-like programmed cell death have been depicted for antral follicles of goose and quails (25, 26). These forms occur in addition to apoptosis. Also important is the convincing study about the existence of two distinct pathways of cell death in bovine follicles (52). The middle zone of the granulosa layer undergoes apoptosis, and the zone closest to the antrum forms atretic/pyknotic bodies. This is explained as a process of terminal differentiation as in the epidermis. All of these reports (25, 26, 52) give evidence for autophagy among different species. Autophagy can therefore be considered as a highly conserved process. This signifies that large vacuoles in granulosa cells often defined as artifacts of fixation have to be carefully reconsidered in ultrathin sections under the novel aspect of autophagy in follicular atresia. The process may also affect thecal cells because endogenous oxLDL is found to affect thecal function (42). An increased incidence of autophagic cell death in follicles may explain anovulations and infertility of obese women with high levels of oxLDL (1, 2, 3). Clinical studies on oxLDL levels in follicular fluid are wanted for obese women to show that obesity severely affects reproductive function. Altogether, another form of programmed cell death has been recognized, and the causes of it, and its significance needs further examination.


   Acknowledgments
 
We are indebted to Professor Yoshimori (National Institute of Genetics, Mishima, Japan) for supplying the rabbit anti-LC3 antibody. We also appreciate the technical skills of Nicole Peukert and Judith Craatz.


   Footnotes
 
Disclosure Summary: N.D., O.Z., M.N., A.R., F.A.H., V.B., J.B., K.S.-B. have nothing to declare.

First Published Online May 11, 2006

1 O.Z. contributed equally as first author. Back

2 J.B. contributed equally as senior author. Back

Abbreviations: DAPI, 4'-6-Diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IVF, in vitro fertilization; LOX-1, lectin-like oxLDL receptor-1; MAP LC3, microtubule-associated protein light chain 3; nLDL, native low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; PI, propidium iodide; ROS, reactive oxygen species; TBS, Tris-buffered saline.

Received January 23, 2006.

Accepted for publication May 2, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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