1 INSERM UMR 636, Université de Nice-Sophia Antipolis, Faculty of Sciences Parc Valrose, 06108 Nice Cedex 2, France
2 CCMA, Université de Nice-Sophia Antipolis, Faculty of Sciences Parc Valrose, 06108 Nice Cedex 2, France
3 Departments of Biochemistry and Howard Hughes Medical Institute, Box 357370, University of Washington, Seattle, WA 98195, USA
* Author for correspondence (e-mail: vidal{at}unice.fr)
Accepted 7 April 2005
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Summary |
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Key words: CD36, Spermatogenesis, Phagocytosis, Sertoli cells, Germ cells
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Introduction |
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Fatty acids (FAs) are used for a large number of biological functions, however most tissues have limited or no capacity for fatty acid synthesis. Biochemical and biophysical approaches have provided evidence that argues for the existence of two distinct processes in cellular FA uptake: passive diffusion through the lipid bilayer and protein-facilitated transport. One of the proteins involved in this active FA uptake is CD36, an integral membrane glycoprotein found on the surface of a variety of cells. The primary role described for CD36 varies with the cell type on which it is expressed. It is commonly referred to as a facilitator for membrane fatty acid transport by muscle and adipose tissue (Abumrad et al., 1993; Coburn et al., 2000
; Ibrahimi and Abumrad, 2002
). But CD36 is also shown to be a scavenger receptor on macrophages, important for the phagocytosis and for the internalization of oxidized low-density lipoproteins (Kunjathoor et al., 2002
).
Previous studies have shown that cells of the seminiferous epithelium contain relatively large amounts of polyunsaturated fatty acids (PUFAs), with a non-homogeneous distribution among the cell population. Sertoli cells are very active in PUFA elongation and desaturation, while isolated germ cells are inefficient in synthesizing 22:5n-6 and 22:6n-3 (Retterstol et al., 2001a; Retterstol et al., 2001b
). PUFAs are known to contribute to membrane fluidity and flexibility, which are essential for fertilization.
We focused our attention on the CD36 protein in the seminiferous epithelium for two main reasons. First, CD36 belongs to the class B scavenger receptor family, which includes the receptor for selective cholesteryl ester uptake (scavenger receptor class B type I, SR-BI), and lysosomal integral membrane protein II. It has been shown that, in the Sertoli cells, SR-BI is partly responsible for the phagocytosis of apoptotic germ cells and residual bodies via interactions with external phosphatidylserine (Shiratsuchi et al., 1997; Shiratsuchi et al., 1999
). Second, in adipocytes, CD36 co-localizes with caveolin proteins in plasma membrane microdomains known as caveola (Febbraio et al., 2001
). These cholesterol- and sphingolipid-enriched structures are assumed to play important role in signal transduction but also in lipid trafficking (Pohl et al., 2004
). Moreover, caveolin 2 protein has been shown to be present in monolayer membranes surrounding lipids droplets within the cytoplasm (Fujimoto et al., 2001
; Tauchi-Sato et al., 2002
). Regarding the phagocytic activity and the particular fatty acid content of Sertoli cells in the seminiferous epithelium, we examined the expression and the localization of CD36 during spermatogenesis. We show here that germ cells, as well as free fatty acids, induce CD36 translocation to the plasma membrane of Sertoli cells. During the phagocytosis process, CD36 and caveolin 2 proteins co-localize at the site of phagocytosis. Moreover, the phagocytosis process involving CD36 is impaired by addition of free fatty acids to the culture medium.
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Materials and Methods |
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Cell culture
The 15P-1 cell line was grown in Dulbecco modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal calf serum (Gibco). Before phagocytosis experiments, cells were kept for 12 hours in serum-free medium. In another set of experiments, cells were then exposed to 100 µM of C18:2 in serum-free culture medium for 4 or 15 hours.
Centrifugal elutriation of testicular cells
Fractionation of Sertoli cells and of germ cells by elutriation was performed as previously described (Vidal et al., 2001). The purity of each fraction was determined by DNA staining with DAPI and light microscopy analysis.
Immunolabelling
Immunofluorescence analysis
Cells (2x104 cells per well) in four-well Labtek chamber glass slides (Nunc Inc, Naperville, IL) were fixed in cold methanol for 10 minutes before a 30-minute blocking step in PBS containing 0.03% Triton X-100, 1% normal goat serum (NGS, G9023, Sigma). The primary anti-mouse CD36 antibody (ABM-5525, Cascade Bioscience, MA) was diluted 1000-fold in PBS containing 0.03% Triton X-100, 1% NGS and incubated 2 hours at room temperature with the cell preparation. After three washes of 5 minutes each in PBS containing 1% NGS, the secondary goat anti-mouse FITC antibody (sc-2O8O, TEBU) diluted 1/400 in PBS was added for 1 hour at room temperature. Three additional washes in PBS were performed and slides were mounted in Vectashield reagent with DAPI (Vector Laboratories, Inc., Burlingame).
Mouse testes were embedded in tissue-freezing medium (Leica) and then frozen on isopentane and dry ice. Cryosections, 9 µm thick, were mounted on poly-L-lysine-coated slides (Merck), fixed in 4% paraformaldehyde for 15 minutes and rinsed in PBS three times. Sections were blocked with a solution containing PBS, 0.01% Triton X-100 and 3% NGS and then incubated 2 hours at room temperature with the above murine anti-mouse CD36 antibody (same dilution) followed by the FITC-conjugated secondary antibody (1:200 dilution in PBS) for 2 hours at room temperature. After three washes in PBS, sections were mounted in Vectashield reagent containing DAPI. The CD36 anti-serum was omitted in the negative controls. Samples were observed by conventional (Axiophot, Zeiss) or by confocal fluorescence microscopy (Fluoview, Olympus) with optical slices of 0.5 µm thickness.
Immunogold analysis
Immunogold labelling was performed on two different types of samples: ultrathin sections of testis embedded in LRWhite and ultrathin cryo-sections of 15P-1cells co-cultured for 24 hours with total germ cells.
Cryo-ultramicrotomy was performed on fixed with 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer pH 7.5 for 1 hour at 4°C. Cells were then washed with phosphate buffer and PBS containing 50 mM NH4Cl, carefully scraped off with a rubber policeman and embedded in 10% gelatin. Small blocks were infiltrated with 2.3 M sucrose, frozen in liquid nitrogen and cut at 120°C. Ultrathin sections (70 nm) were subjected to immunochemistry and embedded in methylcellulose as described elsewhere (Liou et al., 1996).
Mouse testes were fixed with 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer pH 7.5 for 4 hours, initially at room temperature, and then at 4°C. Testes were then washed with phosphate buffer for 1 hour at 4°C and dehydrated in ethanol before embedding in LRWhite.
Ultrathin sections (80 nm) were cut on a dry diamond knife and collected on formvar-coated nickel grids and processed for immunochemistry. Grids were deposited face down on the top of a small drop of the following solutions: PBS 50 mM NH4Cl (10 minutes), PBS 1% BSA 1% normal goat serum (5 minutes); anti-CD36 1/50 diluted in PBS 1% BSA 1% NGS (3 hours); PBS 1% BSA (10 minutes); PBS 0.01% fish-skin gelatin (5 minutes), colloidal gold conjugated anti-mouse IgG antibodies (10 nm) diluted 1/15 in PBS 0.01% fish-skin gelatin (30 minutes); PBS (2x5 minutes); PBS 1% glutaraldehyde (5 minutes); distilled water (5 minutes).
Preparations were observed with a Philips CM12 electron microscope operating at 80 kV. Micrographs were systematically taken on labelling area to count the number of beads on the structure. Non-specific background was never observed in our experimental conditions. No signal was observed when the first antibody was omitted.
Protein extraction and western blot analysis
Cells were lysed for 15 minutes on ice in TNET solution (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100) containing a cocktail of protease inhibitors (100 µM PMSF, 1 µM leupeptine, 1 µM pepstatine A) and phosphatase inhibitors (5 mM NaF, Na 2 mM orthovanadate). The lysate was then centrifuged to remove cellular debris (15 minutes, 15,000 g, 4°C). Protein concentration was estimated by the BCA reaction (Pierce, Rockford, IL). 50 µg of proteins for the testis or 4 µg of proteins for isolated cell fractions were loaded in Laemmli buffer, separated in 10% acrylamide gel, and transferred onto nitrocellulose. The primary CD36 antibody was diluted at 1:1000 and detection of specific immune complexes were detected by enhanced chemiluminescence kit, following the procedure specified by the manufacturer (Amersham, Buckinghamshire, UK).
Morphological analysis
Deconvolution microscopy
15P-1 cells are labelled with orange-fluorescent tetramethylrhodamine (CellTracker Orange CMTMR, Molecular Probes) and germ cells with green-fluorescent fluorescein diacetate (CellTracker Green CMFDA, Molecular Probes) according to the supplier's instruction. Cell interactions were recorded during the second and third hour of co-culture. Image acquisition (Objective 40x/1.35, Uapo) and analysis was performed using the MetaMorph Imaging System. Deconvolution microscopy using the constrained iterative algoritham (Sofworx 2.5) was performed with the Applied Precision DeltaVision system (Applied Precision, Issaquah, WA) built on an Olympus IX-70 base.
Scanning electron microscopy
Co-culture of 15P-1 and germ cells attached to a coverslip were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 for 2 hours at 4°C. They were carefully washed in phosphate buffer, dehydrated in a series of increasing concentrations of ethanol and then treated with hexamethyldisilazane (HMDS) for 10 minutes. Excess HMDS was evaporated in an airflow cabinet. Samples were then gold coated in a sputter coating unit and observed with a Jeol T300 operating at 20 kV.
Analysis of phagocytosis events
Germ cells or latex beads were added, in the presence or absence of free fatty acids, to 15P-1 cell cultures previously maintained for 12 hours in serum-free DMEM. After 8 hours co-culture, cells were gently washed with PBS, fixed in 4% PFA and mounted in DAPI containing medium. The remaining germs cells or latex beads were counted. For each experiment, five independant fields (containing at least five nuclei of 15P-1 cells) were observed by conventional microscopy (Axiophot, Zeiss). The mean number of latex beads or germ cell nuclei observed per field is similar in the presence or in absence of fatty acids. Morphological analysis enabled us to distinguish between germ cells or latex beads that has been engulfed by 15P-1 cells, and germ cells or latex beads remaining outside. Results were expressed as the proportion (%) of phagocytosed germ cells or latex beads compared with total germ cells or latex beads counted in each field.
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Results |
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CD36 protein is specifically relocalized at the plasma membrane of 15P-1 cells during phagocytosis
Phagocytic activity of the 15P-1 cell line has been previously described to be similar to the activity of Sertoli primary cell culture in vitro (Grandjean et al., 1997). We have checked for the modification of CD36 expression in 15P-1 cell cultures. Germ cell fractions enriched in elongating spermatids and also in residual bodies were able to induce a dramatic relocation of the CD36 protein as observed by immunofluorescence and immunogold labelling of the protein (Fig. 4). The protein was localized at the membrane contact between germ cells or residual bodies and 15P-1 cells (Fig. 4A,B). Electronic microscopy analysis of CD36 shows the protein located at the plasma membrane of 15P-1 cells (Fig. 4C). After phagocytosis, 25 gold beads were observed in the area in close contact with the engulfed elongated spermatid (Fig. 4D).
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By western blotting analysis, three CD36 immunoreactive bands between 52 and 121 kDa were observed in Sertoli-derived cell extracts, along with a band between 121 and 180 kDa. Alternatively, using the lysate of germ cell fractions enriched in spermatids or in spermatocytes the only band detectable is the largest one (Fig. 4E).
Concomitant with the relocalization of CD36, the morphology of 15P-1 cells was modified by the formation of protruding cytoplasmic extensions wrapped around germ cells, as shown by electron microscopy (Fig. 5A) or in time lapse experiments on live cells (Fig. 5B)
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CD36 protein co-localizes with caveolin 2
Long chain fatty acid uptake into adipocytes has been shown to depend partly on lipid raft function, involving CD36 and caveolin proteins in plasma membrane microdomains known as caveola (Pohl et al., 2004). Labelling with anti-caveolin 1 antibodies resulted in a uniform fluorescence all along the plasma membrane of Sertoli cells regardless of the presence or absence of germ cells (Fig. 6A,B). On the contrary, caveolin 2 and CD36 proteins co-localized in the luminal part of the testis close to the phagocytosis sites (Fig. 6C,D). Immunogold labelling of the two proteins with two different bead sizes confirms the co-localization of CD36 and caveolin 2 at the plasma membrane level during phagocytotic events (Fig. 6D). CD36 alone is also present on the plasma membrane and in the residual body, as previously observed (Fig. 2).
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Discussion |
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To examine the phagocytic activity of Sertoli cells and the re-localization of CD36, we took advantage of a cell line derived from Sertoli cells, the 15P-1 cell line, which maintains most of the characteristics of Sertoli cells (Paquis-Flucklinger et al., 1993; Rassoulzadegan et al., 1993
) and, in particular, the phagocytic activity (Grandjean et al., 1997
).
While the presence of a phagocytic activity in primary Sertoli cells has previously been demonstrated in rat (Pineau et al., 1991), the use of primary Sertoli cells would never give the basal conditions (namely the conditions where CD36 is not present at the cytoplasmic membrane) because every contaminant germ cell and/or dying cell and membrane ghost would stimulate the Sertoli cells. The 15P-1 cell line has been extensively described and is typically used for analysis of phagocytosis (Grandjean et al., 1997
; Jabado et al., 2002
) and can be considered to be an accurate model.
In accordance with the findings reported by others (Arenas et al., 2004; Alessio et al., 1996
; Greenwalt et al., 1992
), our western blot results support the idea that, depending on cell type, CD36 displays different molecular masses probably corresponding to different glycoforms. In germ cell fractions, only one reactive high molecular mass form of CD36 is observed, interpreted as a nonspecific band by several authors (Arenas et al., 2004
), while in testis and the Sertoli-derived cell line several forms can be detected. By immunolabelling experiments, we detect CD36 protein only in Sertoli cells in vivo as well as in 15P-1 cells in vitro, thus confirming the absence of CD36 expression in germ cells. This observation is consistent with the fact that expression of mature CD36 protein (i.e. membrane-associated CD36) in the testis is not related to variation in number of transcripts (data not shown) but reflects protein degradation and/or stability. The glycosylation status of the protein can account for its localisation and its accessibility in immunolabelling experiments, and remains to be further analysed.
Cyclic variation in the content of lipid droplets within the Sertoli cells of the rat is a phenomenon first described a long time ago (Kerr and De Kretser, 1975). The temporal relationship between phagocytosis of residual bodies and the increase of the volume of lipids in the Sertoli cells cytoplasm suggests that lipid droplets may form from the breakdown products of phagocytosed material (Kerr et al., 1984
; Sasso-Cerri et al., 2001
). The increase of lipid content in Sertoli cells occurs at stage IX, when spermatids pass down the seminiferous tubule as free spermatozoa, casting off residual bodies. We show here that the localization of CD36 follows the cyclic variation of lipids during spermatogenesis. Indeed, CD36 labelling is associated with globular structures in the basal part of the cytoplasm of Sertoli cells and relocated to the apical Sertoli cell membranes at the stages where phagocytosis occurs. Following phagocytosis, we typically found the CD36 protein at the membrane in contact with residual bodies.
CD36 is a fatty acid transporter and its affinity to the long chain polyunsaturated fatty acids suggests that these FAs may trigger the recognition of residual bodies. After the internalization of residual bodies, the CD36 protein is found inside the cell associated with vesicles containing lipids. Similarly, the same localization is observed after the addition of fatty acids to Sertoli cell cultures in vitro. Treating Sertoli cells with C18:2 (the preferential CD36 ligand) induces the relocalization of CD36 at the plasma membrane and the appearance of small lipid droplets, labelled by the antibodies. C18:2 impairs the capacity of Sertoli cells to phagocytose germ cells and residual bodies, supporting the idea that CD36 is essential for this process. In vivo, the Sertoli cell is in close contact with many germ cells at different differentiation stages, however, its phagocytosis activity is mainly directed to residual bodies. This activity is essential for sperm production and involves, in part, an apoptotic signal recognition (Maeda et al., 2002).
The existence of fatty acid transport vesicles surrounded by a lipid monolayer, as well as the presence of calveolin 2 inside this hemi-membrane, have already been reported in the literature (Fujimoto et al., 2001). This protein is also found associated with monolayer phospholipid membranes found around lipid transport vesicles. Our results indicated that CD36 and calveolin 2 co-localized during the phagocytosis of the residual bodies by Sertoli cells, both in vitro and in vivo. Membrane lipids and protein-lipid domains that contribute to the regulation of the dynamic endocytic pathway have already been described (Gruenberg, 2003
). The competition we observed between phagocytosis events and lipid uptake could be a key to understanding what mechanisms orchestrate the cross-talk between lipid membrane domains and lipid-protein complexes during the phagocytosis processes.
Fatty acids are used for a large number of biological functions, however, most tissues have limited or no capacity for fatty acid synthesis. In muscle, as well as in adipocytes, cellular FA uptake is acutely regulated by translocation of CD36 from an intracellular store to the plasma membrane (Bonen et al., 1999; Brinkmann et al., 2002
) when Sertoli cells swallow up germ cells. Several authors have carried out studies of the lipid composition in the seminiferous epithelium (Retterstol et al., 2001). It has been shown that cells contain relatively large amount of PUFAs, and that their distribution is not homogeneous among cell populations. Moreover, a broad change in lipid repartition between the residual body and the spermatozoan cytoplasmic membrane has been described in the literature (Retterstol et al., 2000
; Lenzi et al., 2000
). Within the seminiferous epithelium, Sertoli cells are very active in metabolizing PUFAs while isolated germ cells are inefficient in synthesizing 22:5n-6 and 22:6n-3 (Hurtado de Catalfo and de Gomez Dumm, 2002
). This observation can lead to the conclusion that there is a permanent exchange of lipids between Sertoli and germ cells. CD36-mediated phagocytosis could be one of the processes involved in these exchanges. The simplest way for Sertoli cells to ensure that there is sufficient lipid availability required for proliferation of millions of germ cells per day would be to recycle the lipid content in the residual body. Aside from genetic regulation, we can hypothesise that the seminiferous epithelium also functions as a real recycling centre. Future studies will enable us to follow fatty acids and membrane lipids after the phagocytosis of residual bodies and to determine the localization, composition and fate of lipid droplets in Sertoli cells.
Lipid metabolism is essential for hormonal regulation as well as structural differentiation leading to the production of spermatozoa. It has been shown in Leydig and Sertoli cells that hypercholesterolemia can have a detrimental effect on the secretary functions and the overall sperm fertilizing capacity (Fofana et al., 2000). In apoE-deficient mice submitted to chow diet, CD36 mRNAs levels are upregulated by at least 100%. When these mice are fed with a high cholesterol diet, arrest of spermatogenesis and subsequent atrophy of seminiferous tubules is observed (Zibara et al., 2002
).
In human, CD36, as well as SR-BI and HSL (hormone-sensitive lipase), expression and localization are modified in testis tumours reflecting an essential role for lipids and lipid receptors (Arenas et al., 2004). In mice, a null mutation in CD36 does not seem to affect the fertility of mutant animals but reveals important roles in fatty acid and lipoprotein metabolism (Febbraio et al., 1999
; Febbraio et al., 2002
). However, these mice do not have a generalized defect in expression of all scavenger receptors and the remaining ones could account for production of spermatozoa. Conversely, the absence of the hormone-sensitive lipase in HSL knockout mice induces severe oligospermia. It is interesting to note that HSL plays an important role in the regulation of CD36 expression (Chung et al., 2001
). In conclusion, the results discussed in the present article increase our understanding of the role of fatty acids as signalling molecules in the testis (Hajri et al., 2002
; Silva et al., 2002
) and the involvement of CD36 in these phenomena. Precise analysis of spermatogenesis in CD36 null mice would help us to understand better the physiological role of CD36 in the testis.
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Acknowledgments |
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