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Département de Pathologie et Biologie Cellulaire, Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada
Published, JLR Papers in Press, July 21, 2006.
1 To whom correspondence should be addressed. e-mail: marc.pelletier{at}umontreal.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary key words scavenger receptor class B type I • Sertoli cell • autoimmune orchitis • cell clearance • Leydig cell
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The nonenzymatic factors potentially involved in cholesterol homeostasis include ABCA1, which transfers cholesterol to apolipoprotein 1 (7, 21), and CD36 (22), which mediates the selective uptake of HDL cholesteryl esters and binds oxidized low density lipoproteins. In addition, scavenger receptor class B type I (SR-BI) selectively removes cholesteryl esters from the HDL to which it binds (23, 24) and contributes to the HDL-mediated cellular cholesterol efflux (25). Moreover, SR-BI is highly expressed in tissues with a strong cholesterol demand for steroidogenesis, such as adrenals, ovaries, and testes (23, 24). Experimental deficiency of SR-BI is accompanied by abnormal structure, composition, and abundance of lipoproteins, which alters the development of female gametes and causes a significant proportion of ovulated oocytes to die soon after ovulation, resulting in sterility in SR-B gene-knockout female mice (26). SR-BII, a spliced variant of the SR-B gene that differs from SR-BI only in the C-terminal cytoplasmic domain, also binds HDL with high affinity (27). In contrast to SR-BI, SR-BII does not mobilize intracellular cholesteryl ester stores and is less efficient at promoting cellular cholesterol efflux (27), but it effluxes 2-fold more FC than SR-BI in macrophages (28). Nonetheless, SR-BI and SR-BII both colocalize with cholesterol-rich plasma membrane caveolae (28–31), which influence cholesterol homeostasis by acting as conduits for cellular flux (32) and concentrate signaling proteins for signal transduction (33).
Autoimmune orchitis (AIO) is characterized by an increase in serum anti-sperm antibody levels, inflammation of the testis, leukocyte infiltration, degeneration of the germinal epithelium, spermatogenic arrest, and infertility (34). Spontaneous AIO has been documented in human, dog, mink, and rat (35). The incidence of inflammation of the testis is high in infertile males, and testicular inflammation has been identified as a cause of infertility, although symptomatic orchitis caused by bacterial or viral infections is a rare occurrence in men (36). Immunological male infertility is associated with pathological features that resemble granulomatous orchitis, with inflammation of the testis and persistent infertility documented in infertile mink with spontaneous AIO (35). To assess whether testicular SR-BI, SR-BII, esterified cholesterol (EC), and FC were affected by an impaired clearance of apoptotic germ cells caused by AIO, measurements performed in mink with spontaneous AIO were compared with those in normal development and the annual reproductive cycle. The model provides a unique opportunity to highlight the relationship between apoptosis, germ cell removal, SR-BI expression, and cholesterol levels. This information is significant because injection of a part of the extracellular domain of rat SR-BI fused with human FC in the tubules was claimed to cause an increased numbers of apoptotic germ cells (37) and because phagocytosis of apoptotic germ cells by Sertoli cells was said to be mediated by SR-BI in rodents (31, 37–39).
This study shows that the time periods during the annual seasonal reproductive cycle characterized by high levels of SR-BI and SR-BII were accompanied by low EC levels in the tubules, whereas the periods characterized by the predominance of one of the two SR-BI isoforms were accompanied by high EC levels. These results show that tubules with AIO were characterized by a deregulation of cholesterol homeostasis and a concurrent deregulation of SR-BI and SR-BII expression. This study also shows that an increase in only one of the two SR-BI isoforms in the tubules with AIO was accompanied by increased EC levels but not by a significant increase in apoptosis levels, suggesting that the recognition and phagocytosis of apoptotic cells are distinct physiological events and that the engulfment of apoptotic cells by Sertoli cells is only partly dependent on SR-BI.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For studies on infertile male mink, 2–3 year old mink that successfully mated and sired five litters of no fewer than four kits the year before but that were sterile during the current year and were diagnosed with spontaneous AIO were selected.
Clinical criteria of fertility
The ejaculated semen recovered from vaginal smears was evaluated with a light microscope, and the morphology, motility, and number of spermatozoa were assessed for each male mink during the mating season in March. For studies in infertile mink, those that failed to sire a litter, whose semen contained low counts of spermatozoa, immobile spermatozoa, or no spermatozoa with high serum levels of anti-sperm antibodies, and whose pathological features showed leukocyte infiltration and destruction of the seminiferous epithelium at autopsy typical of spontaneous AIO were used. Ten adult mink with AIO for February, March, and July (totaling 30 mink) were used. The right testis was processed for histological studies and the left for enriched tissue fractionation. The protocol was approved by the Université de Montréal Animal Care Committee.
Quantification of serum anti-sperm antibodies by ELISA
The methods of Ing et al. (41) and Benet-Rubinat et al. (42) were used as follows. One hundred microliters of spermatozoa suspended in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4) at a concentration of 1 x 106 cells/ml was added to each well (1 x 105 cells/well) of polystyrene flat-bottom plates (Immulon 2; Dynex Technology, Chantilly, VA), and the plates were centrifuged at 2,000 rpm in a GS-6R Beckman centrifuge (Beckman, Mississauga, Ontario, Canada) equipped with a GH 3.8 rotor at 4°C for 10 min. The supernatant was removed and the plates were dried. Spermatozoa were fixed in –20°C methanol for 30 min, dried, incubated with 1% BSA in PBS at 4°C overnight to eliminate nonspecific labeling, and washed in PBS containing 0.05% Tween 20. Spermatozoa were incubated first with various dilutions of mink sera (higher dilutions were needed when sera of infertile mink were used). Serum dilutions were prepared in 1% BSA in PBS ranging from 1:100 to 1:1,000. Spermatozoa were incubated with serum dilutions at 37°C for 1 h, next with rabbit anti-mink IgG (1:2,000 dilution) at 37°C for 1 h, and then with HRP-conjugated anti-rabbit IgG (1:2,000 dilution) at 37°C for 1 h. The reaction was detected by adding 100 µl of substrate solution containing 0.4 mg/ml orthophenylene diamine and 0.015% hydrogen peroxide in citrate buffer, pH 5.0, and stopped by the addition of 25 µl/well of 2 N sulfuric acid. Optical density was determined at 492 nm by an ELISA autoreader (Dynatech, Mississauga, Ontario, Canada). All samples were assayed in duplicate, and the results are expressed as fold dilution of serum where the absorbance value was 0.5.
Isolation of seminiferous tubule- and interstitial tissue-enriched fractions
Testes were decapsulated and immersed in PBS with or without phosphatase and protease inhibitors: 2 mM PMSF, 1 mM EGTA, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 4 mM Na3VO4, 80 mM NaF, 20 mM Na4P2O7, and 10 µM potassium bisperoxol-(1,10-phenanthroline)oxovanadate. The seminiferous tubules were teased apart and individually detached from the interstitial tissue with fine tweezers. Next, the tubule-interstitial tissue mixture was placed in PBS with or without phosphatase inhibitors in a water shaker bath set at 80 cycles/min at 4°C for 1 h. The mixture was allowed to sediment for 1 h in 15 ml conical tubes on ice and centrifuged at 600 rpm for 20 min in a GS-6R Beckman centrifuge equipped with a GH 3.8 rotor at 4°C. The seminiferous tubule-enriched fraction (STf) occupied the pellet, and the interstitial tissue fraction (ITf) occupied the top. The fractions were washed in PBS or PBS containing proteases and phosphatase inhibitors, and their purity was evaluated by light microscopy. This represents a modification of a previously published isolation method in which mild enzymatic digestion with collagenase D and soybean trypsin inhibitor was used to separate tubules from the interstitial tissue (19). The protein content of the ITf and STf samples was measured (43).
Quantification of apoptosis by ELISA
Measurements were done using a cell death detection ELISA kit (Boehringer-Mannheim, Laval, Québec, Canada) in accordance with the manufacturer's instructions. The assay kit allows the quantification of mononucleosome- and oligonucleosome-bound DNA in the cytoplasmic fraction of a cell homogenate that is caused by DNA fragmentation during apoptosis. Briefly, STf homogenates were incubated with the lysis buffer at room temperature for 30 min and centrifuged at 15,000 rpm in a Beckman centrifuge (Palo Alto, CA) at 4°C for 30 min. The cytosolic fractions were collected, and 20 µg of proteins was loaded onto streptavidin-coated microtiter plates, exposed to anti-histone biotin and peroxidase-conjugated anti-DNA antibody at room temperature for 2 h, washed, and incubated with 2,2'-azino-di-[3-ethylbenzithiazoline sulfonate] diammonium salt substrate solution. The results are expressed in absorbance unit per milligram of protein STf (AU/mg). Samples were assayed in duplicate.
Epididymal spermatozoa
Epididymides from normal adult mink euthanized in February and March were diced in cold PBS with or without proteases and phosphatase inhibitors, filtered through a 74 µm mesh, and centrifuged at 2,000 rpm for 15 min in a GS-6R Beckman centrifuge at 4°C to recover spermatozoa. The gametes were resuspended in 10 mM Tris-HCl, pH 8, containing 1 mM EDTA for 5 min to lyse epithelial and blood cells (44), washed twice, and diluted 1:1 in cold PBS with or without proteases and phosphatase inhibitors. The cells were sonicated in a Fisher Sonic Dismembrator (model 300; Fisher, Farmington, NY) during three 30 s intervals.
Antibodies against SR-BI and SR-BII
Three distinct antibodies were used for the detection of SR-BI and its isoform SR-BII: 1) the polyclonal antibody to SR-BI, which recognizes only the SR-BI isoform, was raised in rabbit against a peptide antigen corresponding to residues 496–509 of mouse SR-BI; 2) the goat polyclonal antibody to SR-BI produced against a synthetic peptide made to the C-terminal region of murine SR-BI, corresponding to residues 400–509; and 3) the rabbit polyclonal antibody, which recognizes exclusively the SR-BII isoform, was produced against a peptide antigen corresponding to residues 491–506. In addition, the rabbit SR-BII antibodies were affinity-purified against the recombinant SR-BII coupled to a CNBr-activated Sepharose 4B column. Antibodies 1 and 3 above were purchased from Novus Biologicals (Littleton, CO). The goat polyclonal antibody to SR-BI was a generous gift from Novus Biologicals. The affinity-purified antibodies are specific for each isoform of SR-BI and do not cross-react with other proteins in Western blot analyses of tissue extracts. The specificity of SR-BI and SR-BII antibodies was tested using mouse tissue as a positive control. The primary or secondary antibody alone was used for negative controls.
Electrophoresis and Western blots
Twenty micrograms of total proteins for each STf and/or ITf sample was denatured in electrophoresis sample buffer and loaded on a 10% polyacrylamide minigel, subjected to electrophoresis, and electrotransferred onto nitrocellulose membranes. Membranes blocked for 60 min at 37°C with 5% skim milk in TBS (140 mM NaCl and 50 mM Tris-HCl, pH 7.4) were incubated with purified anti-SR-BI (1:2,000 dilution) or anti-SR-BII (1:1,000) overnight and mixed with the corresponding HRP-conjugated secondary antibody. Some membranes were reprobed with either a goat polyclonal antibody to human vimentin (Sigma, St. Louis, MO) (1:500 dilution) or a monoclonal anti-human clusterin (Novocastra Laboratories, Ltd., New Castle upon Tyne, UK) (1:50 dilution).
For studies of AIO, myosin light chain (MLC) was used as an internal loading control. MLC has a molecular mass of 20 kDa, and the intensity of the band did not change in the different experimental conditions. The membranes between 40 and 50 kDa were incubated with monoclonal MLC antibody (Sigma) and then with anti-IgM peroxidase-conjugated antibody. Antigen-antibody complexes were revealed using enhanced chemiluminescence (Lumi-light+; Roche, Laval, Québec, Canada).
Plasma and testicular cholesterol analysis
Duplicate samples of enriched tissue fraction homogenates (60–80 µl each) and serum (40–60 µl) were extracted with 5 ml of hexane-isopropanol (3:2) for 30 min at room temperature and centrifuged at 2,500 rpm (GS-6R Beckman centrifuge) for 5 min (45). The supernatant was removed, and the extraction was repeated three times on the pellet. The supernatant from each sample was pooled, separated into two equal volumes, and evaporated at 37°C with N-EVAP Nitrogen Evaporators (Organomation Associates, Inc., Berlin, MA). Total cholesterol (TC) and unesterified FC were measured in the dried pellet from the 2 volumes with an enzymatic kit (Wako Chemical USA, Inc., Richmond, VA). EC levels were obtained by substraction of FC from TC. EC and FC contents are expressed in µg cholesterol/mg total protein for ITf and STf or in mg cholesterol/dl serum. The values are means ± SEM from three independent experiments.
Immunolabeling
Testes were perfusion-fixed with Bouin's fixative (46). Endogenous peroxidase activity was inhibited with 0.3% hydrogen peroxide, and immunolabeling was performed (19). Nonspecific binding was blocked with 0.5–3.0% skim milk. Paraffin sections were incubated with rabbit anti-SR-BI (1:50 dilution), goat anti-SR-BI (1:100 dilution), or rabbit anti-SR-BII (1:25 dilution), then with biotinylated anti-rabbit F(ab')2 (1:10,000 dilution) or anti-goat IgG (1:2,000 dilution) and peroxidase-conjugated streptavidin (1:200 dilution) (20). Controls included either the first or the second antibody alone.
Fluorescence microscopy
Spermatozoa mounted on poly-L-lysine-coated cover slips were air-dried, fixed, and permeabilized with –20°C methanol for 10 min. Preparations were incubated with 3% skim milk in PBS to block nonspecific binding. Next, spermatozoa were incubated with purified anti-SR-BI (1:10 dilution) or anti-SR-BII (1:5 dilution) for 60–120 min at 37°C, rinsed with PBS, and incubated with FITC-conjugated anti-rabbit IgG (1:400 dilution) at 37°C for 1 h. Cover slips were mounted in PBS/glycerol (1:1) containing 5% 1,4-diazabiscyclo-[2,2,2]octane and visualized with a Carl Zeiss Axioskop II fluorescence microscope.
Measurements of serum testosterone levels
Testosterone concentration in mink sera was measured by ELISA using ALPCO diagnostics kits (American Laboratory Products Company, Salem, NH) (19). The sensitivity of the method was 0.07 ng/ml.
Data analysis and statistical analysis
The bands of five independent immunoblots of SR-BI and SR-BII obtained for each time interval studied in ITf and STf were scanned on an Astra 2000S laser scanner (Umax Data System, Inc., Hainchu, Taiwan). The relative SR-BI and SR-BII protein contents of scanned bands were estimated by densitometry (Scion Image Software, Scion Corp.). The values were normalized to 60 day values for the studies of development or to August values for the studies of adults. The data are presented as means ± SEM and were evaluated with Student's t-test.
Source of chemicals and antibodies
Poly-L-lysine and 1,4-diazabiscyclo-[2,2,2]octane were from Sigma-Aldrich (Oakville, Ontario, Canada), and PMSF, biotinylated anti-rabbit IgG, biotinylated anti-goat IgG, leupeptin, aprotinin, and the Lumi-light+ chemiluminescence detection kit were from Roche. Potassium bisperoxol-(1,10-phenanthroline)oxovanadate(V) was from Calbiochem (San Diego, CA). Biotinylated anti-rabbit affinity-purified F(ab')2 fragment and peroxidase-conjugated anti-rabbit IgG, peroxidase-conjugated anti-mouse IgG, and anti-goat IgG were purchased from Jackson Immunoresearch Laboratories (Mississauga, Ontario, Canada).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western blot analyses of SR-BI and SR-BII in mink and mouse tissue fractions
When anti-SR-BI was used, one immunoreactive band was detected in the ITf and one in the STf (Fig. 1
). Although the molecular mass was the same in both mink tissue fractions, the mass was 82 kDa in mink and 85 kDa in mouse (Fig. 1). Anti-SR-BII showed a molecular mass of 85 kDa in the two tissue fractions in both species (Fig. 1).
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During development, traces of SR-BII were detected from 60 to 120 days, but the levels increased significantly from 120 to 150 days, from 150 to 180 days, and from 210 to 240 days (Fig. 2E). In contrast to SR-BI (Fig. 2D), SR-BII levels were low from July to October but high from November to June (Fig. 2F). SR-BII levels increased significantly from October to November (Fig. 2F) but decreased from June to July (Fig. 2F).
Measurements of SR-BI and SR-BII levels in the STf
During development, SR-BI levels increased significantly from 120 to 150 days, when they peaked and reached values that neared twice the ITf values before decreasing until 270 days; the decrease was significant between 180 and 210 days (Fig. 3A
). During the annual reproductive cycle, SR-BI levels were increased by August, then they decreased significantly by September before increasing again to reach peak values until February (Fig. 3B). From February to March, there was a very significant decrease in SR-BI levels, and only traces of the receptor were detected from April to June. However, this was followed by a significant increase in SR-BI levels from June to July (Fig. 3B). SR-BI was detected in epididymal spermatozoa isolated from adult mink in February and March (Fig. 3B).
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The immunoblot membranes used to measure SR-BI expression in the STf were stripped and reprobed for vimentin (Fig. 3E, F) and clusterin (data not shown because clusterin and vimentin variations were similar). The vimentin levels showed a profile of variations (Fig. 3E, F) reminiscent of that of SR-BI (Fig. 3A, B) but not SR-BII (Fig. 3C, D), suggesting that SR-BI is associated partly, albeit not exclusively, with Sertoli cells. This observation adds further support to the contention that SR-BII is principally associated with germ cells, because vimentin and clusterin have been considered to be normally synthesized by Sertoli cells, not germ cells.
Immunohistochemical studies of SR-BI
SR-BI localized to germ cells and Sertoli cells in a stage-specific manner in adult mink testes harvested in February (Fig. 4A
–D). SR-BI localized at the surface of the thin Sertoli cell cytoplasmic processes surrounding the germ cells during stage I (Fig. 4A). By stages VII and VIII, intercellular Sertoli cell junctional membranes in the vicinity of spermatocytes near the basal third of the epithelium (Fig. 4B, C) and the "trunk" of the supporting cells were labeled (Fig. 4D). The cell surfaces in the intercellular clefts between zygotene spermatocytes and Sertoli cell as well as those between adjoining Sertoli cell processes were SR-BI-positive during stage X (Fig. 4D). Stage X is concomitant with the spermatocyte migration from the basal to the lumenal compartment in mink. SR-BI-positive material was detected at the germ cell's surface and within the cytoplasm, where it appeared as SR-BI-positive minute dots (Fig. 4A–D). Intracellular SR-BI-positive clumps were present in apoptotic germ cells (Fig. 4B). SR-BI localized to the Leydig cell surface (Fig. 4E).
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Immunohistochemical studies of SR-BII
Most SR-BII was detected intracellularly in the vicinity of the nucleus in germ cells (Fig. 5A
, B) and Sertoli cells (Fig. 5A) in adult mink testes harvested in February, but this distribution did not change with the stage of the cycle (Fig. 5A, B). In addition, intercellular clefts between Sertoli cells and germ cells were weakly immunostained during stage X (Fig. 5B). The endothelial cells of blood vessels and other interstitial cells, but not Leydig cells, were SR-BII-positive (Fig. 5C). During testicular regression, the Sertoli cell surface was very weakly SR-BII-immunoreactive, although the immunoreactivity in this location was increased by May and June (cf. Fig. 5D, E with Fig. 5F). In addition, the intracellular immunoreactivity of SR-BII was localized to germ cells, in particular round spermatids and cells that showed signs of apoptosis (Fig. 5A). The protein was not detected in the interstitial space during testicular regression (Fig. 5D–F).
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45 µg cholesterol/mg protein by 120 days until adulthood. Conversely, the increase in EC levels from 90 to 150 days was followed by a significant decrease (P < 0.01, 150 vs. 180 days old) by 180 days. In adult mink, FC levels were approximately three times the EC values. FC and EC levels were lower during the periods when spermatogenesis was completed (from December to March) than when it was not (the rest of the annual reproductive cycle). In the STf, FC values were three times the EC values. During development, FC levels showed no significant changes but EC levels decreased, although not significantly, until adulthood. In the adult, FC levels decreased from December to March. The profile of the EC levels in the STf was reminiscent of that in the ITf, except that the November-to-December decrease (P < 0.01) was significant and there was a significant increase from March to April (P < 0.02) followed by a significant decrease from April to May (P < 0.05). Serum FC and EC values were near each other and ranged between 80 and 90 mg cholesterol/dl serum and 90 and 104 mg cholesterol/dl, respectively, without showing significant differences.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70 kDa" in membrane subfractions of primary cultured, pubertal 20 day old rat Sertoli cells and liver (38).
Cholesterol and apoptosis levels
Our results show that measurements generated with the N-EVAP Nitrogen Evaporator reflect real FC and EC levels and substantiate the data we had obtained previously by TLC (20).
Apoptosis levels measured by ELISA in whole testis extracts (52) and in enriched STfs (our unpublished results) showed a 2-fold increase in March and July in normal mink. The phagocytosis of massive numbers of apoptotic cells entails a cholesterol buildup that results in increased EC levels by March and April, whereas the decreased EC levels in July reflect reduced cholesterol amounts originating from fewer apoptotic cells remaining during this period. The observation of infrequent apoptotic figures in February in mink testis sections despite the report that apoptosis-mediated germ cell death accounts for half of the cell loss in normal testis (17, 18) implies a rapid removal and disposal by Sertoli cells. Moreover, our observation that low EC levels in February prevailed suggests a contribution of SR-BI/SR-BII to the removal of cholesterol-containing germ cells from the tubules back to the liver for the reverse transport of peripheral cholesterol during periods of spermatogenic activity. In normal mink, EC levels were lowest when SR-BI and SR-BII expression were both high, but the levels increased when only one isoform was predominant in the tubules. This suggests that the maintenance of low EC levels requires both isoforms and that each one influences cholesterol in a distinct manner in normal tubules. However, impaired clearance of cholesterol-containing apoptotic cells by Sertoli cells deregulates cholesterol homeostasis and results in increased EC levels in mink tubules with AIO, in which a significant EC increase was accompanied by a decrease in SR-BII but not SR-BI in March. This observation is consistent with the report that experimental hepatic overexpression of SR-BI induces a significant diminution of HDL cholesterol (53).
We show here the first measurements of SR-BI and SR-BII in individual compartments rather than in whole testis extract. Regarding the tubular compartment, the results with vimentin, a marker for Sertoli cells, showed that the concentration of vimentin-containing Sertoli cells in STf samples was lowest during periods when spermatogenesis was near completion, although the total Sertoli cell number per testis remained constant regardless of spermatogenic activity through the annual reproductive cycle. In addition, the vimentin concentration profiles during the development and annual cycle were similar to those in SR-BI. Therefore, SR-BI variations are concomitant with those in the proportion of Sertoli cells relative to the germ cell population in the sample. This does not exclude the possibility that the profile of variations in vimentin or in SR-BI indicates a specific change in vimentin and SR-BI gene expression. The predominance of SR-BI from July to February and of SR-BII from January to June suggests that an increase in SR-BII causes a restriction in SR-BI expression in normal mink tubules. During AIO, however, the feedback mechanism responsible for the expression in tandem of each isoform is perturbed, as shown by the observations that SR-BI and SR-BII expression was concurrent and that SR-BI increased in February but SR-BII increased in July, opposite of the normal measurements.
SR-BI/SR-BII expression and apoptosis levels
Our results show that the peak in apoptosis levels reported in whole testis extracts (52) and tubule-enriched fractions (our unpublished data) in March corresponds to increased SR-BII levels, whereas the peak in apoptosis recorded in July corresponds to high SR-BI levels. This could suggest the involvement of a distinct isoform in the removal of distinct generations of germ cells in normal tubules. However, our observation that increased SR-BI and/or SR-BII levels were not accompanied by significant increases in apoptosis in mink with AIO in February and July agrees with the reports that the expression of individual receptors (54–56) and the genetic deletion of scavenger receptor A (57) only produce minimal effects on the uptake of apoptotic cells in culture. Our results suggest that other multiligands besides SR-BI/SR-BII are involved in apoptosis and that this process is not causally related to SR-BI expression in testis. However, our results contrast with the report that injection of rat SR-BI into seminiferous tubules causes an increase in the number of apoptotic cells identified with the Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) method in mice (37). The TUNEL measures apoptotic cells not engulfed and remaining in tubules (i.e., clearance by Sertoli cells), whereas ELISA measurements reflect real apoptosis levels. Other studies have reported an increase in in situ end labeling (ISEL)-positive apoptotic germ cells in the tubules during experimental AIO in rat (58). More importantly, our finding of increased numbers of apostain-labeled apoptotic germ cells in mink tubules in which apoptosis levels measured by ELISA were not increased is proof that cell clearance is impaired during spontaneous AIO in mink (our unpublished data). Apoptosis is characterized by the appearance of plasma membrane blebs that have been shown to express externalized phosphatidylserine and to constitute sites for C1q and C-reactive protein binding, both of which are events connected with the noninflammatory clearance of cellular debris (59). The finding that phagocytosis of apoptotic germ cells was inhibited by liposomes containing phosphatidylserine (60) and/or by anti-SR-BI antibody provides strong support for the conclusion that SR-BI acts as a phosphatidylserine receptor (38).
Here, we show deregulated SR-BI and SR-BII expression in mink testis with AIO, which could alter the bleb recognition of dying germ cells and lead to impaired clearance by Sertoli cells. A deficit in SR-BI-mediated recognition of the nuclear antigens that have been shown to redistribute in blebs and apoptotic bodies (61) could favor intolerance to self proteins. The observation here that an increase in SR-BI or SR-BII was accompanied by minimal effects on apoptosis levels supports the notion that the recognition and phagocytosis of apoptotic cells are distinct physiological events and that the engulfment of apoptotic cells by Sertoli cells is only partly SR-BI-dependent.
The distribution of SR-BI and SR-BII
The interstitial compartment
Our finding of SR-BI at the mink Leydig cell plasma membrane agrees with similar findings in rat (62, 63). However, the discrepancy between our observation of a strong rather than "a very weak SR-BI staining" and "barely detectable" (62) SR-BI levels likely stems from the fact that mink Leydig cells are rich (64), whereas rat Leydig cells are poor, in cholesteryl ester-containing lipid droplets (65) and in the use endogenous, not exogenous, blood-borne HDL or LDL cholesterol for the purpose of testosterone production (62).
The seminiferous tubules
Our observation of SR-BI localized to mink Sertoli cells agrees with the finding of the receptor protein and mRNA (39) and of SR-BI as a phagocytosis-inducing phospholipid phosphatidylserine receptor in rat Sertoli cells (37). In addition, our detection of SR-BI on testicular cell and spermatozoan surfaces and of SR-BII on the inner acrosome overlying the nucleus is consistent with the report of 70% of SR-BI on the cell surface and 80–90% of SR-BII around the Chinese hamster ovary cell nucleus (66). In addition, our localization of SR-BI to mink Sertoli cell junctional membranes, except when it accumulates in the trunk of the cell during stage X of the cycle of the seminiferous epithelium, entails factors modifying its location to adapt to the physiological activities of the cell. This view is supported by the report of decreased SR-BI mRNA expression during stages I–VI in rat (39). In addition, SR-BI was reported to modify the structure of the membrane (67, 68) and the distribution of cholesterol within the membrane (69, 70). We showed that cholesterol accumulates in germ cell membranes (71), at the tight junctions of Sertoli cells (72–74), and within testicular cells (74). SR-BI/SR-BII-mediated selective lipid uptake in testicular cell membrane segments could modify the membrane fluidity causing local concentration of signaling proteins for signal transduction, which would in turn influence cell-cell interactions. In particular, spermatocyte translocation from one side to the other of the blood-testis barrier during stage X requires Sertoli cell junction plasticity while the spermatids release into the lumen, and the engulfment by Sertoli cells of lipid-containing residual bodies involves the intracellular management of cholesterol. A change in the scavenger receptor location could influence both of these events, because the "selective uptake of lipids by SR-BI and SR-BII correlates with the surface expression of SR-BI and SR-BII rather than with the rate at which the two receptors accumulate HDL intracellularly" (66, 75). The lack of a C-terminal leucine amino acid decreased the surface expression of SR-BI in mouse hepatocytes, and the three C-terminal amino acids required for binding PDZK1 are lacking in SR-BII (76). Interestingly, hormone-sensitive lipase (20) and SR-BI expression are both upregulated in the tubules during stage X.
In conclusion, cholesterol homeostasis and SR-BI expression are deregulated, but apoptosis levels are unchanged, in tubules with AIO. These results suggest that SR-BI and SR-BII expression favors the maintenance of low EC levels and that the predominance of only one isoform is associated with the accumulation of EC but not with apoptosis in the tubules.
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ACKNOWLEDGMENTS |
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Manuscript received April 10, 2006 and in revised form July 7, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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