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Endocrinology Vol. 140, No. 6 2494-2500
Copyright © 1999 by The Endocrine Society

ARTICLES

Scavenger Receptor Class B Type I in the Rat Ovary: Possible Role in High Density Lipoprotein Cholesterol Uptake and in the Recognition of Apoptotic Granulosa Cells1

Per-Arne Svensson, Magnus S. C. Johnson, Charlotte Ling, Lena M. S. Carlsson, Håkan Billig and Björn Carlsson

Research Centre for Endocrinology and Metabolism, Department of Internal Medicine (P.-A.S., M.S.C.J., L.M.S.C., B.C.), Department of Pharmacology and Physiology (C.L.), and Center for Reproductive Medicine, Department of Obstetrics and Gynecology (H.B.), Sahlgrenska University Hospital, S-413 45 Goteborg, Sweden

Address all correspondence and requests for reprints to: Dr. Björn Carlsson, Research Centre for Endocrinology and Metabolism, Department of Internal Medicine, Gröna Stråket 8, Sahlgrenska University Hospital, S-413 45 Goteborg, Sweden. E-mail: bjorn.carlsson{at}ss.gu.se


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Scavenger receptor class B type I (SR-BI) mediates the selective uptake of high density lipoprotein cholesterol. SR-BI is expressed at high levels in the ovary, indicating that it plays a role in the delivery of cholesterol as substrate for steroid hormone production. However, SR-BI also binds anionic phospholipids with high affinity and could therefore be involved in the recognition of apoptotic cells. In this study we have characterized the expression of SR-BI in rat ovarian follicles undergoing atresia. Atretic follicles with cells undergoing apoptosis were identified by in situ DNA end labeling, and SR-BI expression was determined by in situ hybridization and immunohistochemistry. SR-BI was expressed in thecal cells at all stages of follicular development, including atretic follicles, and in corpus luteum. Isolated apoptotic granulosa cells (but not viable granulosa cells) bound annexin V, indicating that they display anionic phospholipids on the cell surface. Transfection of COS-7 cells with an expression vector carrying the rat SR-BI complementary DNA resulted in increased binding to apoptotic granulosa cells (46 ± 2% of the SR-BI-expressing cells bound at least one granulosa cell compared with 24 ± 3% for the mock-transfected cells; P < 0.0001), whereas the binding to viable granulosa cells was unchanged. Apoptotic granulosa cells also bound to isolated thecal shells. We conclude that thecal cells of both nonatretic and atretic follicles express SR-BI. The location of SR-BI expression in the ovary supports a role of this receptor in the uptake of high density lipoprotein cholesterol. In addition, our data suggest that SR-BI mediates the recognition of apoptotic granulosa cells by the surrounding thecal cells and that it therefore may play a role in the remodeling of atretic follicles to secondary interstitial cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SCAVENGER receptor class B type I (SR-BI) complementary DNA (cDNA) was cloned based on its ability to bind acetylated low density lipoprotein (1). However, subsequent studies have demonstrated that SR-BI is a high affinity receptor for high density lipoprotein (HDL) and that it mediates the selective uptake of HDL cholesterol (2, 3, 4, 5, 6, 7). SR-BI is expressed at all major sites of selective cholesterol uptake, such as the adrenal gland, ovary, testis, and liver (8, 9). Mice deficient in SR-BI have increased plasma cholesterol levels and reduced cholesterol content in the adrenal gland (10, 11), whereas overexpression of SR-BI in the liver results in reduced plasma HDL levels and increased cholesterol secretion into the bile (12).

In addition to its ability to bind HDL, SR-BI binds different forms of low density lipoprotein (1) and anionic phospholipids (13). Together with other scavenger receptors, SR-BI has been proposed to be involved in the host defense against exogenous pathogens and in the recognition of damaged molecules and apoptotic cells (14). For example, mice deficient in macrophage type I and type II class A scavenger receptors (MSR-A, also known as SR-AI/II) are less prone to develop atherosclerosis and have increased susceptibility to infections (15). Macrophages isolated from such mice also display reduced binding to apoptotic thymocytes (16). Class B scavenger receptors, including SR-BI, CD-36, and a related Drosophila melanogaster receptor, can bind to apoptotic cells (4, 17, 18). The binding is believed to be mediated by anionic phospholipids that are exposed on the cell surface on cells undergoing apoptosis (19, 20).

The ovary consists of several hundred thousand follicles at birth. However, only a few of these primordial follicles reach the preovulatory state and ovulate, whereas the majority of follicles undergo atresia. Follicular atresia occurs in both pre- and postnatal life and at all stages of follicular development and includes apoptosis of granulosa cells. The early stages of follicular atresia are characterized by pyknotic granulosa cells and the presence of cell debris in the follicular antrum. In the later stages, the basal lamina disintegrates, the number of granulosa cells is reduced, and the follicle collapses. At this stage, apoptotic granulosa cells are believed to be removed from the atretic follicle by viable ovarian cells and macrophages (21, 22). It is unclear which ovarian cell types are involved in removal of the apoptotic granulosa cells. However, the theca interna cells become hypertrophied and accumulate lipid droplets, which suggests that they participate in this process (21).

In this study we demonstrate that SR-BI is expressed in the thecal cells of both atretic and healthy follicles, and that SR-BI expressed in viable cells can mediate the binding of apoptotic granulosa cells. These results indicate that SR-BI, in addition to its role in HDL cholesterol delivery, may participate in remodeling of the atretic follicles to secondary interstitial cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
This study was approved by the local ethics committee for animal care and use, University of Goteborg (Goteborg, Sweden). Female Sprague Dawley rats (Alab, Stockholm, Sweden) were housed under standardized conditions with constant temperature (24–26 C), humidity (50–60%), and artificial 14-h light, 10-h dark cycle. The animals had free access to water and pelleted food. Mature rats (70 days old) were killed by decapitation, and ovaries were removed, rapidly frozen in liquid nitrogen, and stored at -70 C. Ovarian sections (10 µm) were thaw-mounted onto SuperFrost Plus slides (Eire Scientific, Portsmouth, NH) and stored at -70 C. Granulosa cells were isolated from ovaries obtained from PMSG-stimulated (10 IU PMSG; Sigma Chemical Co., St. Louis, MO; sc injection 2 days before death) immature rats (27 days old) by incising the largest follicles under a stereomicroscope (23). Directly isolated granulosa cells were considered viable because of the absence of internucleosomal DNA fragmentation. Apoptosis was induced by overnight incubation of granulosa cells in Eagle’s MEM (Life Technologies, Paisley, UK) at 37 C under 7% CO2 (24). Thecal shells were isolated from PMSG-stimulated immature rats. In brief, preovulatory follicles were dissected under a stereomicroscope. The follicles were cut in half using microscissors, and the granulosa cells were scraped off the follicular wall with a needle (25, 26).

Probes
[{alpha}-33P]UTP-labeled antisense rat SR-BI RNA was generated by in vitro transcription with T3 polymerase (Promega Corp., Madison, VI) using HindIII-linearized plasmid pMJ5:101 (9). [{alpha}-33P]UTP-labeled sense rat SR-BI RNA was generated by in vitro transcription with T7 polymerase (Promega Corp.) using SpeI-linearized pMJ5:101 as template.

In situ hybridization
In situ hybridization was performed one as previously described (9, 27, 28). In brief, sections were fixed in 4% formaldehyde for 10 min, acetylated, dehydrated through graded ethanol solutions, and delipidated in chloroform. Two consecutive sections on each slide were hybridized overnight at 50 C in a humidified chamber with 33P-labeled SR-BI antisense or sense RNA (2 x 105 cpm) in buffer containing 50% formamide, 25 mM Tris (pH 7.5), 1 mM EDTA (pH 8.0), 0.4 M NaCl, 1 x Denhardt’s solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA), 10% dextran sulfate (mol wt, 500,000), single stranded testicular DNA (250 µg/ml), and yeast transfer RNA (250 µg/ml). Sections were washed in 2 x SSC (standard saline citrate; at room temperature) and twice in 2 x SSC-50% formamide (50 C, 15 min), rinsed briefly in 2 x SSC (37 C), and incubated in 2 x SSC containing ribonuclease A (100 µg/ml) at 37 C for 30 min. After ribonuclease A treatment, the sections were rinsed in 2 x SSC, washed three times in 2 x SSC-50% formamide (50 C, 15 min), twice in 2 x SSC (room temperature, 5 min), dehydrated through graded ethanol, and air-dried. Sections were exposed to autoradiographic emulsion (LM-1; Amersham, Little Chalfont, UK) for up to 3 days, counterstained with hematoxylin-eosin, and studied in light- and darkfield using a Nikon Microphot-FX microscope (Nikon, Tokyo, Japan) with a Nikon FX-35A camera.

Immunohistochemistry
Ovarian cryosections were fixed in 4% formaldehyde for 10 min and rinsed in PBS three times. Sections where blocked with a solution containing PBS, 0.1% Triton X-100, and 3% donkey serum and then incubated (12 h, 8 C) with rabbit antiserum (29) raised against the extracellular domain of mouse SR-BI (RED1; 1:1000 dilution in PBS, 0.1% Triton X-100 and 3% donkey serum). After overnight incubation, the sections were rinsed in PBS three times, incubated with fluorescein-conjugated F(ab')2 of donkey antirabbit IgG (1:100 dilution in PBS with 3% donkey serum; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 3 h at 8 C, rinsed in PBS three times, and mounted in fluorescent mounting medium (DAKO Corp., Carpinteria, CA). The SR-BI antiserum was omitted in the negative control.

Analysis of DNA fragmentation
DNA fragmentation in ovarian sections was analyzed by in situ DNA 3'-end labeling essentially as previously described (23). Analysis of DNA fragmentation in isolated apoptotic and viable granulosa cells was performed as previously described (23). In brief, DNA isolated from granulosa cells was labeled at the 3'-ends with [35S]dideoxy-ATP using terminal transferase (Boehringer Mannheim, Mannheim, Germany) and separated on an agarose gel to determine the absence or presence of internucleosomal DNA fragmentation. The gel was dried and exposed on Hyperfilm (Amersham).

Analysis of SR-BI isoforms by RT-PCR
Total RNA was isolated from rat and mouse ovaries essentially as described by Chomczynski and Sacchi (30). For first strand cDNA synthesis, 5 µg RNA were heat denatured and reversed transcribed using 1 µg random hexamers (Boehringer Mannheim) and 20 U AMV-reverse transcriptase (Promega Corp.) in AMV-reverse transcriptase buffer (Promega Corp.). PCR was performed in Taq Extender buffer (Stratagene, La Jolla, CA) with 5 U Taq polymerase (Boehringer Mannheim) and 2.5 U Pfu polymerase (Stratagene), 1 µM of the primers rSRBIz5 (5'-GGG AAG ATC GAG CCA GTA-3'; Kebo, Spanga, Sweden) and rSRBIz3'NotI (5'-GCG CGG CCG CGG GGA CAG TGT GAC ATC T-3'; Kebo), cDNA, and deoxy-NTP (0.2 mM each), using GeneAmp PCR system 9600 (Perkin Elmer, Foster City. CA). The thermocycler was programmed for a "hot start" at 94 C (3 min) followed by a step-down procedure with 20-sec denaturation at 94 C, 20-sec annealing at 55–45 C (annealing temperatures decreased by 2° every cycle), and 30-sec elongation at 72 C. The step-down procedure was followed by 20 cycles with annealing at 48 C and a final 5-min elongation at 72 C. PCR products and a DNA marker (1 kb DNA ladder, Life Technologies, Gaithersburg, MD) were separated on a 3% agarose gel containing ethidium bromide and visualized by UV light. The PCR products were cloned into the pCRII vector (Invitrogen, Carlsbad, CA) and verified by DNA sequencing using a dye terminator cycle sequencing kit (Amersham) and an PE Applied Biosystems 377 automatic sequencer (Perkin-Elmer).

Annexin V binding analysis
Apoptotic granulosa cells (106) and viable granulosa cells (106) were washed in PBS and stained with fluorescein-labeled annexin V (Boehringer Mannheim) according to the manufacturer’s instructions. Unlabeled viable granulosa cells (106) were used as a control. Annexin V binding was measured in a Becton Dickinson and Co. FACSort (Becton Dickinson and Co., San Jose, CA) using the FL1 detector.

Expression vector constructs
The rat SR-BI cDNA was amplified by PCR (9) essentially as described above with primers HDL5'XbaI (5'-GGT CTA GAA CAT GGG CGT CAG CTC CA-3'; Genset, Paris, France) and HDL3'PvuII (5'-CCC AGC TGC CCT ACA GCT TGG CTT CTT-3'; Genset) and subcloned into pCR2.1 (Invitrogen) generating pPAS5–17; the insert was subsequently transferred into the XbaI site of the expression vector pSI (Promega Corp.) generating pPAS5–33. The construct was verified by DNA sequencing.

SR-BI transfection and granulosa cell binding
All reagents for cell culture and transfection were obtained from Life Technologies. COS-7 cells were grown in DMEM with Glutamax-1, 10% FBS, 2 µg/ml fungizone, and 50 µg/ml gentamicin sulfate at 37 C under 7.0% CO2. COS-7 cells were transfected with 8 µg pPAS5–33 or 8 µg pSI vector (Promega Corp.) and 40 µl lipofectamine in a 100-mm cell culture dish according to the manufacturer’s instructions. One day after the transfection, 50,000 transfected COS-7 cells were seeded into 35-mm cell culture dishes (Costar, Cambridge, MA). Seventy-two hours after transfection, the COS-7 cells were washed three times with serum-free DMEM. Apoptotic or viable granulosa cells (500,000 cells) in 1 ml serum-free DMEM medium were added to the transfected COS-7 cells and incubated for 1 h at 37 C. After washing five times with PBS (pH 7.3) to remove unbound granulosa cells, the percentage of COS-7 cells binding one or more granulosa cells was determined visually on randomly taken photographs using a Nikon Diaphot 300 microscope (Nikon).

Granulosa cell binding to thecal shells
Apoptotic granulosa cells (3 x 106) and viable granulosa cells (3 x 106) were stained with 0.5 µg/ml Hoechst 33258 (bisBenzimide, Sigma Chemical Co.) for 30 min at 37 C, washed three times in serum-free DMEM to remove excess dye, and resuspended in 1 ml serum-free DMEM. Both labeled apoptotic and viable granulosa cells showed intense nuclear staining, determined by visual inspection and fluorescein-activated cell sorter analysis (data not shown). Granulosa cells and thecal shells were incubated together for 1 h at 37 C. Eight thecal shells from each group were washed five times in PBS, mounted, and analyzed using a Nikon Microphot-FX microscope (Nikon).

Flow cytometry
Cell surface expression of SR-BI was determined by flow cytometry. COS-7 cells transfected with the SR-BI expression vector or pSI vector were detached from the cell culture dish, and 106 cells were incubated (2 h, room temperature) with rabbit antiserum raised against the extracellular domain of mouse SR-BI (RED1; 1:1000 dilution in PBS with 2% BSA; Sigma Chemical Co.). The cells were washed with PBS and incubated (30 min, room temperature) with fluorescein-conjugated F(ab')2 of donkey antirabbit IgG (1:500 dilution in PBS with 2% BSA; Jackson ImmunoResearch Laboratories, Inc.). Flow cytometry was performed using the FL1 detector on a Becton Dickinson and Co. FACSort. Flow cytometry was performed on the Hoechst 33258-labeled granulosa cells using the FL1 detector on a FACSort.

Statistical analysis
Statistical analysis was performed with unpaired t test using StatView software (Abacus Concepts, Berkeley, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SR-BI gene expression in atretic follicles
The distribution of SR-BI messenger RNA (mRNA) in the rat ovary was determined by in situ hybridization. In line with previous reports (9, 31), SR-BI mRNA was detected in thecal cells at all stages of follicular development and in corpus luteum (Fig. 1Go, B and E). To analyze whether SR-BI is expressed in atretic follicles, consecutive sections were analyzed for the presence of apoptotic cells, using in situ end labeling of DNA. SR-BI was expressed in thecal cells of both healthy follicles and follicles undergoing atresia (Fig. 1Go, B and C, E and F). SR-BI immunoreactivity was present in thecal cells at all stages of follicular development, including atretic follicles (Fig. 2Go, A and B). The most intense staining was present in the corpus luteum (Fig. 2BGo).



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  		Figure 1. Distribution of SR-BI expression and apoptotic cells in the rat ovary. Shown are darkfield microscopy of rat ovaries subjected to in situ hybridization with an SR-BI sense RNA probe (A and D) or an SR-BI antisense RNA probe (B and E). Apoptotic cells were identified by in situ DNA end labeling on consecutive sections (C and F). Indicated are healthy follicles (f), atretic follicles (af), corpus luteum (cl), thecal cells (tc), and granulosa cells (gc). Bar, 250 µm.

 


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  		Figure 2. Distribution of SR-BI immunoreactivity in the rat ovary. Cryosections were incubated with antiserum against SR-BI. Shown are an atretic follicle (A), antral follicle and corpus luteum (B), and control section without antisera against SR-BI (C). Indicated are corpus luteum (cl), thecal cells (tc), and granulosa cells (gc). Bar, 100 µm.

 
Analysis of SR-BI isoforms in the ovary
An alternative isoform of SR-BI with a unique C-terminal intracellular domain is present in the mouse (32). This receptor isoform (SR-BII) is generated by alternative splicing, which occurs in a region that is highly conserved between species (Fig. 3AGo), and it is therefore possible that SR-BII also exists in the rat. Using RT-PCR, we detected both SR-BI (476 bp) and SR-BII (374 bp) in RNA extracted from mouse ovaries; however, only SR-BI was detected in RNA extracted from rat ovaries (Fig. 3BGo). The identities of the PCR products were confirmed by DNA sequencing.



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  		Figure 3. Analysis of expression of SR-BI and SR-BII isoforms in the mouse and rat ovary. A, Alignment of the 3'-end of rat, mouse, and human SR-BI cDNA encoding the C-terminal domain of SR-BI (2 9 45 ). The exon structure and splicing pattern generating SR-BI are from the report by Cao et al. (42 ). The putative rat SR-BII sequence, as indicated in the figure, is based on the sequence homology to the mouse SR-BII isoform described by Webb et al. (32 ). The exon parts of the putative splice donor and acceptor sites as well as the putative stop codons (shaded) are conserved between species. B, RT-PCR analysis of expression of SR-BI and SR-BII in rat and mouse ovary. Lane 1, Mouse ovary; lane 2, rat ovary; lane 3, nontemplate control; lane 4, 1-kb DNA ladder. Indicated are PCR products corresponding to SR-BI (476 bp) and SR-BII (347 bp).

 
Characterization of apoptotic granulosa cells
Class B scavenger receptors are believed to bind apoptotic cells through interaction with anionic phospholipids, such as phosphatidylserine (PS), that are exposed on the outer leaflet of the membrane of apoptotic cells. We, therefore, characterized apoptotic granulosa cells in this respect. Annexin V is a protein that binds to phospholipids, especially to PS (33, 34). Apoptotic granulosa cells, but not viable granulosa cells, bound annexin V (Fig. 4BGo). Apoptosis was verified by internucleosomal DNA fragmentation (Fig. 4AGo).



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  		Figure 4. Analysis of internucleosomal DNA fragmentation and annexin V binding to apoptotic and viable granulosa cells. A, Analysis of DNA fragmentation in viable and apoptotic granulosa cells. The apoptotic granulosa cells show the presence of the characteristic internucleosomal DNA fragmentation, but viable granulosa cells did not. B, Binding of fluorescein-labeled annexin V to apoptotic granulosa cells and viable granulosa cells analyzed by flow cytometry.

 
Binding of apoptotic granulosa cells to COS-7 cells expressing SR-BI
In the next experiment we analyzed the possible interaction between apoptotic granulosa cells and COS-7 cells expressing SR-BI. Cell surface expression of SR-BI was verified by flow cytometry using antiserum raised against the extracellular domain of mouse SR-BI (Fig. 5EGo). Apoptotic and viable granulosa cells (Fig. 4AGo) were added to COS-7 cells that expressed SR-BI and to mock-transfected COS-7 cells. Binding was calculated as the percentage of COS-7 cells binding one or more granulosa cells. COS-7 cells expressing SR-BI bound significantly more apoptotic granulosa cells than mock-transfected control cells (P < 0.0001; Fig. 5Go, A, B, and D). In contrast, there was no significant difference in binding of viable granulosa cells to SR-BI-expressing COS-7 cells or mock-transfected control cells (Fig. 5DGo).



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  		Figure 5. Binding of apoptotic and viable granulosa cells (GC) to cells expressing SR-BI. A and B, Binding of apoptotic granulosa cells to COS-7 cells transfected with an expression vector for rat SR-BI (A) or control vector (B). C, Apoptotic granulosa cells and COS-7 cells before repeated washes with PBS. Apoptotic granulosa cells are seen as small white spheres bound to the larger dark COS-7 cells. Binding was calculated as the percentage of COS-7 cells that bound one or more granulosa cells. Binding of granulosa cells to transfected COS-7 cells shown as a box plot (D). The five horizontal lines of the box plot display the 10th, 25th, 50th (median), 75th, and 90th percentiles (***, P < 0.0001). Flow cytometric analysis of cell surface expression of SR-BI in COS-7 cells transfected with an expression vector for SR-BI and a control vector (E).

 
Granulosa cell binding to thecal shells
Because thecal cells of both atretic and healthy follicles express SR-BI, we tested the hypothesis that thecal cells may bind apoptotic granulosa cells. Thecal shells were isolated and incubated with fluorescently labeled apoptotic and viable granulosa cells. Thecal shells (n = 8) bound large numbers of apoptotic granulosa cells (Fig. 6AGo). In contrast, thecal shells (n = 8) incubated with viable granulosa cells bound only a very small number of cells (Fig. 6BGo).



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  		Figure 6. Binding of granulosa cells to isolated thecal shells. A and B, Isolated thecal shells incubated with labeled apoptotic granulosa cells (A) or labeled viable granulosa cells (B). Apoptotic and viable granulosa cells were labeled with Hoechst 33258. The labeled granulosa cells appear white in the picture. Note the increased number of bound granulosa cells in A. Indicated are the outside (Out) and the inside (In) of the thecal shell wall (TSW).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Scavenger receptors are known to bind multiple ligands, such as modified lipoproteins, apoptotic cells, and anionic phospholipids in vitro. However, the physiological relevance of many of these interactions is largely unknown (35). Recent studies have identified SR-BI as a receptor for HDL (2, 10, 12) that mediates selective cholesterol uptake in the liver and steroid hormone-producing tissues. We have shown, in line with other reports, that SR-BI expression in the rat ovary is high in the thecal cell layers of follicles and the corpus luteum (8, 9, 31, 36). This indicates that SR-BI plays a role in the uptake of HDL cholesterol for steroid hormone production in these compartments. In this study we show that SR-BI expression is not restricted to thecal cells of healthy follicles, but is also expressed in thecal cells of atretic follicles. In addition, we demonstrate that SR-BI can mediate the binding of viable cells to apoptotic granulosa cells.

Atresia of the ovarian follicles is an extensively studied process involving granulosa cell apoptosis. However, the mechanisms involved in removal of apoptotic cells are less well understood. Macrophages are the principal phagocytes that remove apoptotic cells and bodies, but other cell types adjacent to the apoptotic cells are now known to participate in this process (20). The role of nonprofessional phagocytes in the removal of apoptotic cells is easily recognized in specific tissues, i.e. the brain and testis, where inflammatory cells do not have access to apoptotic cells. In these tissues, other cells, such as Sertoli cells in the testis, act as nonprofessional phagocytes and remove apoptotic cells (37). In ovaries of adult rats, antral follicles undergoing atresia contain few macrophages; instead, neighboring follicular cells recognize and remove apoptotic cells (21, 22). However, the ovarian cell types that are involved in the removal of the apoptotic granulosa cells have not been defined. It is only in the final conversion from atretic follicles to secondary interstitial cells that macrophages appear to be involved in the removal of apoptotic cells. In this study, we show that apoptotic granulosa cells bound to isolated thecal shells. This binding may be mediated by SR-BI, as thecal cells express SR-BI at high levels, and SR-BI expressed in COS-7 cells mediated binding of apoptotic granulosa cells. However, the binding of apoptotic granulosa cells to thecal cells may involve additional mechanisms. Furthermore, it remains to be shown that the binding of apoptotic granulosa cells to thecal cells subsequently leads to phagocytosis of the apoptotic cells.

Previous studies have shown that class B scavenger receptors, i.e. SR-BI and CD-36, bind anionic phospholipids such as PS and phosphatidylinositol with high affinity in vitro, and cell lines expressing hamster SR-BI or the human SR-BI homolog (CLA-1) bind apoptotic MKM cells and apoptotic thymocytes, respectively (4, 38). The binding of apoptotic thymocytes to CLA-1-expressing cells could be inhibited by PS and phosphatidylinositol liposomes (4). In this study we also demonstrated that apoptotic granulosa cells bound annexin V, indicating that PS is exposed on the outer leaflet of the plasma membrane (33, 34). Taken together with previous reports demonstrating binding of PS to SR-BI (4), these results suggest that PS may mediate the binding of apoptotic granulosa cells to SR-BI expressed on viable cells. Furthermore, expression of SR-BI or CD36 in cell lines gives these cells phagocytic capacity for apoptotic cells (18, 38). Phagocytosis of apoptotic spermatogenic cells by Sertoli cells also appears to be dependent on the exposure of anionic phospholipids, and it has been suggested that this may involve SR-BI or CD36 (37).

We have previously identified a putative peroxisomal targeting sequence type I (PTS1) in the C-terminal of SR-BI (9). The PTS1 motif is recognized by the PTS1 import receptor, which mediates uptake of proteins in the peroxisome (39, 40). The PTS1 motif may therefore be of importance for SR-BI action, as the peroxisome is an important subcellular site for cholesterol metabolism (41). Interestingly, an alternatively spliced isoform of SR-BI exists in the mouse (32). This isoform, SR-BII, has a unique C-terminal intracellular domain lacking the PTS1 motif, suggesting that the receptor isoforms may have distinct biological functions. SR-BII has been shown to mediate both cholesterol uptake and efflux, but with lower efficiency than SR-BI (29). The predicted C-terminal sequences of both isoforms are highly conserved between species, suggesting that both isoforms may be present in several species. However, using RT-PCR we were unable to detect the transcript-encoding SR-BII isoform in the rat ovary.

The promoter region of the human SR-BI homolog, CLA-1, contains a recognition sequence for the orphan nuclear receptor steroidogenic factor-1 (SF-1). SF-1 binds to the CLA-1 promoter region and is of importance for regulation of CLA-1 expression (42). In the human ovary, the expression of SF-1 is abundant in granulosa cells and thecal cells of both mature and atretic follicles and in corpus luteum (43). The location of SR-BI mRNA in the rat ovary is different from that of SF-1 mRNA in human ovary, i.e. SR-BI is only expressed at very low levels in rat granulosa cells. This indicates that other factors may be involved in the regulation of SR-BI expression (42).

Scavenger receptors are multiligand receptors that have persisted through evolution (14, 44). The hypothesis that these receptors may, in fact, serve as multifunctional receptors is supported by the multiple biological consequences of the lack of SR-AI/II (15). The facts that SR-BI recognizes apoptotic granulosa cells and that it is expressed in atretic follicles as well as in healthy follicles open the possibility that SR-BI may participate in the recognition and removal of apoptotic cells in vivo in addition to its role as a receptor for HDL.


   Acknowledgments
 
We thank Prof. Deneys R. van der Westhuyzen for the generous gift of SR-BI antiserum, and Ulrica Carlsson for excellent technical assistance with the flow cytometric analysis.


   Footnotes
 
1 This work was supported by grants from the Wilhelm och Martina Lundgrens Vetenskapsfond, the Kungliga och Hvitfeldtska Stipendiestiftelsen, the Stiftelsen fonden för studerande av läkarvetenskap vid Sahlgrenska sjukhuset, and the Goteborg Medical Association, and Swedish Medical Research Council Grants 10380 11134 11285, 11331, 11502, and 13141. Back

Received August 5, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Acton SL, Scherer PE, Lodish HF, Krieger M 1994 Expression cloning of SR-BI, a CD36-related class B scavenger receptor. J Biol Chem 269:21003–21009[Abstract/Free Full Text]
  2. Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M 1996 Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 271:518–520[Abstract]
  3. Calvo D, GomezCoronado D, Lasuncion MA, Vega MA 1997 CLA-1 is an 85-kD plasma membrane glycoprotein that acts as a high-affinity receptor for both native (HDL, LDL, and VLDL) and modified (OxLDL and AcLDL) lipoproteins. Arteriosclerosis Thromb Vasc Biol 17:2341–2349[Abstract/Free Full Text]
  4. Murao K, Terpstra V, Green SR, Kondratenko N, Steinberg D, Quehenberger O 1997 Characterization of CLA-1, a human homologue of rodent scavenger receptor BI, as a receptor for high density lipoprotein and apoptotic thymocytes. J Biol Chem 272:17551–17557[Abstract/Free Full Text]
  5. Temel RE, Trigatti B, DeMattos RB, Azhar S, Krieger M, Williams DL 1997 Scavenger receptor class B, type I (SR-BI) is the major route for the delivery of high density lipoprotein cholesterol to the steroidogenic pathway in cultured mouse adrenocortical cells. Proc Natl Acad Sci USA 94:13600–13605[Abstract/Free Full Text]
  6. Xu S, Laccotripe M, Huang X, Rigotti A, Zannis VI, Krieger M 1997 Apolipoproteins of HDL can directly mediate binding to the scavenger receptor SR-BI, an HDL receptor that mediates selective lipid uptake. J Lipid Res 38:1289–1298[Abstract]
  7. Fluiter K, van der Westhuijzen DR, van Berkel TJC 1998 In vivo regulation of scavenger receptor BI and the selective uptake of high density lipoprotein cholesteryl esters in rat liver parenchymal and Kupffer cells. J Biol Chem 273:8434–8438[Abstract/Free Full Text]
  8. Landschulz KT, Pathak RK, Rigotti A, Krieger M, Hobbs HH 1996 Regulation of scavenger receptor, class B, type I, a high density lipoprotein receptor, in liver and steroidogenic tissues of the rat. J Clin Invest 98:984–995[Medline]
  9. Johnson MSC, Svensson P-A, Helou K, Billig H, Levan G, Carlsson LMS, Carlsson B 1998 Characterization and chromosomal localization of rat scavenger receptor class B type I, a high density lipoprotein receptor with a putative leucine zipper domain and peroxisomal targeting sequence. Endocrinology 139:72–80[Abstract/Free Full Text]
  10. Rigotti A, Trigatti BL, Penman M, Rayburn H, Herz J, Krieger M 1997 A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism. Proc Natl Acad Sci USA 94:12610–12615[Abstract/Free Full Text]
  11. Varban M, Rinninger F, Wang N, Fairchild-Huntress V, Dunmore J, Fang Q, Gosselin M, Dixon K, Deeds J, Acton S, Tall A, Huszar D 1998 Targeted mutation reveals a central role for SR-BI in hepatic selective uptake of high density lipoprotein cholesterol. Proc Natl Acad Sci USA 95:4619–4624[Abstract/Free Full Text]
  12. Kozarsky KF, Donahee MH, Rigotti A, Iqbal SN, Edelman ER, Krieger M 1997 Overexpression of the HDL receptor SR-BI alters plasma HDL and bile cholesterol levels. Nature 387:414–417[CrossRef][Medline]
  13. Rigotti A, Acton SL, Krieger M 1995 The class B scavenger receptors SR-BI and CD36 are receptors for anionic phospholipids. J Biol Chem 270:16221–16224[Abstract/Free Full Text]
  14. Krieger M 1997 The other side of scavenger receptors: pattern recognition for host defense. Curr Opin Lipidol 8:275–280[Medline]
  15. Suzuki H, Kurihara Y, Takeya M, Kamada N, Kataoka M, Jishage K, Ueda O, Sakaguchi H, Higashi T, Suzuki T, Takashima Y, Kawabe Y, Cynshi O, Wada Y, Honda M, Kurihara H, Aburatani H, Doi T, Matsumoto A, Azuma S, Noda T, Toyoda Y, Itakura H, Yazaki Y, Horiuchi S, Takahashi K, Kar Kruijt J, van Berkel TJC, Steinbrecher UP, Ishibashi S, Maeda N, Gordon S, Kodama T 1997 A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature 386:292–296[CrossRef][Medline]
  16. Terpstra V, Kondratenko N, Steinberg D 1997 Macrophages lacking scavenger receptor A show a decrease in binding and uptake of acetylated low-density lipoprotein and of apoptotic thymocytes, but not of oxidatively damaged red blood cells. Proc Natl Acad Sci USA 94:8127–8131[Abstract/Free Full Text]
  17. Franc NC, Dimarcq JL, Lagueux M, Hoffmann J, Ezekowitz RA 1996 Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity 4:431–443[CrossRef][Medline]
  18. Ren Y, Silverstein RL, Allen J, Savill J 1995 CD36 gene transfer confers capacity for phagocytosis of cells undergoing apoptosis. J Exp Med 181:1857–1862[Abstract/Free Full Text]
  19. Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM 1992 Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148:2207–2216[Abstract]
  20. Savill J, Fadok V, Henson P, Haslett C 1993 Phagocyte recognition of cells undergoing apoptosis. Immunol Today 14:131–136[CrossRef][Medline]
  21. Hsueh AJ, Billig H, Tsafriri A 1994 Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr Rev 15:707–724[Abstract/Free Full Text]
  22. Gaytan F, Morales C, Bellido C, Aguilar E, SanchezCriado JE 1998 Ovarian follicle macrophages: is follicular atresia in the immature rat a macrophage-mediated event? Biol Reprod 58:52–59[Abstract/Free Full Text]
  23. Billig H, Furuta I, Hsueh AJ 1993 Estrogens inhibit and androgens enhance ovarian granulosa cell apoptosis. Endocrinology 133:2204–2212[Abstract/Free Full Text]
  24. Tilly JL, Billig H, Kowalski KI, Hsueh AJW 1992 Epidermal growth factor and basic fibroblast growth factor suppress the spontaneous onset of apoptosis in cultured rat ovarian granulosa cells and follicles by a tyrosine kinase-dependent mechanism. Mol Endocrinol 6:1942–1950[Abstract/Free Full Text]
  25. Bergh C, Carlsson B, Olsson JH, Selleskog U, Hillensjo T 1993 Regulation of androgen production in cultured human thecal cells by insulin-like growth factor I and insulin. Fertil Steril 59:323–331[Medline]
  26. Nordenstrom K, Hamberger L 1981 Influence of gonadotrophins on cAMP formation in theca cells isolated from pre-ovulatory rat follicles. Acta Endocrinol (Copenh) 96:534–540[Abstract/Free Full Text]
  27. Carlsson B, Nilsson A, Isaksson OG, Billig H 1993 Growth hormone-receptor messenger RNA in the rat ovary: regulation and localization. Mol Cell Endocrinol 95:59–66[CrossRef][Medline]
  28. Bennett PA, Levy A, Sophokleous S, Robinson IC, Lightman SL 1995 Hypothalamic GH receptor gene expression in the rat: effects of altered GH status. J Endocrinol 147:225–234[Abstract/Free Full Text]
  29. Webb NR, Connell PM, Graf GA, Smart EJ, de Villiers WJ, de Beer FC, van der Westhuyzen DR 1998 SR-BII, an isoform of the scavenger receptor BI containing an alternate cytoplasmic tail, mediates lipid transfer between high density lipoprotein and cells. J Biol Chem 273:15241–15248[Abstract/Free Full Text]
  30. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  31. Mizutani T, Sonoda Y, Minegishi T, Wakabayashi K, Miyamoto K 1997 Cloning, characterization, and cellular distribution of rat scavenger receptor class B type I (SRBI) in the ovary. Biochem Biophys Res Commun 234:499–505[CrossRef][Medline]
  32. Webb NR, de Villiers WJ, Connell PM, de Beer FC, van der Westhuyzen DR 1997 Alternative forms of the scavenger receptor BI (SR-BI). J Lipid Res 38:1490–1495[Abstract]
  33. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH 1994 Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84:1415–1420[Abstract/Free Full Text]
  34. Verhoven B, Schlegel RA, Williamson P 1995 Mechanisms of phosphatidylserine exposure, a phagocyte recognition signal, on apoptotic T lymphocytes. J Exp Med 182:1597–1601[Abstract/Free Full Text]
  35. Freeman MW 1997 Scavenger receptors in atherosclerosis. Curr Opin Hematol 4:41–47[Medline]
  36. Li X, Peegel H, Menon KM 1998 In situ hybridization of high density lipoprotein (scavenger, type 1) receptor messenger ribonucleic acid (mRNA) during folliculogenesis and luteinization: evidence for mRNA expression and induction by human chorionic gonadotropin specifically in cell types that use cholesterol for steroidogenesis. Endocrinology 139:3043–3049[Abstract/Free Full Text]
  37. Shiratsuchi A, Umeda M, Ohba Y, Nakanishi Y 1997 Recognition of phosphatidylserine on the surface of apoptotic spermatogenic cells and subsequent phagocytosis by Sertoli cells of the rat. J Biol Chem 272:2354–2358[Abstract/Free Full Text]
  38. Fukasawa M, Adachi H, Hirota K, Tsujimoto M, Arai H, Inoue K 1996 SRB1, a class B scavenger receptor, recognizes both negatively charged liposomes and apoptotic cells. Exp Cell Res 222:246–250[CrossRef][Medline]
  39. Dodt G, Braverman N, Wong C, Moser A, Moser HW, Watkins P, Valle D, Gould SJ 1995 Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of the peroxisome biogenesis disorders. Nat Genet 9:115–125[CrossRef][Medline]
  40. Fransen M, Brees C, Baumgart E, Vanhooren JC, Baes M, Mannaerts GP, Van Veldhoven PP 1995 Identification and characterization of the putative human peroxisomal C-terminal targeting signal import receptor. J Biol Chem 270:7731–7736[Abstract/Free Full Text]
  41. Lazarow PB, Moser HW 1995 Disorders in Peroxisome Biogenesis. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The Metabolic and Molecular Bases of Inherited disease. McGraw-Hill, New York, vol 2:2287–2324
  42. Cao GP, Garcia CK, Wyne KL, Schultz RA, Parker KL, Hobbs HH 1997 Structure and localization of the human gene encoding SR-BI/CLA-1: evidence for transcriptional control by steroidogenic factor 1. J Biol Chem 272:33068–33076[Abstract/Free Full Text]
  43. Ramayya MS, Zhou J, Kino T, Segars JH, Bondy CA, Chrousos GP 1997 Steroidogenic factor 1 messenger ribonucleic acid expression in steroidogenic and nonsteroidogenic human tissues: Northern blot and in situ hybridization studies. J Clin Endocrinol Metab 82:1799–1806[Abstract/Free Full Text]
  44. Steinberg D 1997 Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem 272:20963–20966[Free Full Text]
  45. Calvo D, Vega MA 1993 Identification, primary structure, and distribution of CLA-1, a novel member of the CD36/LIMPII gene family. J Biol Chem 268:18929–18935[Abstract/Free Full Text]



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