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SR-BI and CD36
Normal and Pathological Human Testes Express Hormone-Sensitive Lipase and the Lipid Receptors CLA-1/SR-BI and CD36 MARIA I. ARENAS, PHD, MARIA V. T. LOBO, MD, ENRIQUE CASO, MD, LIDIA HUERTA, BS, RICARDO PANIAGUA, PHD, AND MARTIN A. MARTI´N-HIDALGO
Numerous studies have demonstrated the important role of cholesterol and cholesteryl esters in tumor cell proliferation and progression of cancer. However, few studies have focused on the role of lipid transporters and lipases in cancer development and progression. The present study examined the expression of hormone-sensitive lipase (HSL) and the scavenger receptors CLA-1/SR-BI and CD36 in normal human testis and in nontumor and tumor testicular disorders by immunohistochemistry and Western blotting analysis. In normal young testes, immunoreaction to CLA-1/SR-BI was found in the spermatid acrosomic vesicle and on the surface of Sertoli and Leydig cells. HSL was detected in spermatogonia, the Golgi region of spermatocytes, the nucleus of spermatids, and the cytoplasm of both Sertoli and Leydig cells. Elderly testes and testes with hypospermatogenesis showed a similar staining pattern to that of normal young testes except for CD36, which was expressed in Sertoli cells. Cryptorchid testes demonstrated intense labeling to HSL and weak labeling to SR-BI in Sertoli cells (nucleus and cytoplasm) and Leydig cells (cytoplasm). Seminiferous tubules with intratubular germ cell neoplasia exhibited intense immunolabeling to the 3 lipid receptors in the surface of neoplastic cells and to HSL in the nucleus. In seminoma and spermatocytic seminoma, neoplastic cells labeled to HSL but failed to stain with antilipid receptors; in the seminiferous tu-
bules at the periphery of the tumour, Charcot-Bo¨ttcher crystalloids of Sertoli cells were strongly positive to CLA-1. Testes with mature teratoma showed a weak reaction to CD36 and SR-BI in some cells of enteric-type glands, and immature teratoma were exclusively immunolabeled with HSL. Western blotting analysis revealed that multiple bands were immunolabeled, with differences seen between normal and pathological testes. The results of this study indicate that the presence of lipid receptors (CLA-1/SR-BI) and hormone-sensitive lipase in Leydig cells suggests a role of these proteins in steroidogenesis. Also, these proteins seem to be involved in spermiogenesis, as their labeling in spermatids suggests. In nonmalignant and malignant pathologies, cholesterol metabolism is probably altered, and HSL labeling in neoplastic germ cell nuclei suggests a still-unknown function of this enzyme, probably related to cell cycle regulation. HUM PATHOL 35:34-42. © 2004 Elsevier Inc. All rights reserved. Key words: HSL, CLA-1/SR-BI, CD36, immunohistochemistry, human testis. Abbreviations: FFA, free fatty acid; HDL, high-density lipoprotein; HSL, hormone-sensitive lipase; LDL, low-density lipoprotein; PPAR, peroxisome proliferator activated receptor; TBS, Tris-buffered saline.
Experimental and clinical evidence has clearly demonstrated a link between lipid metabolism disturbances and cancer development and progression. The influence of dietary lipids and changes in systemic and cellular lipid metabolism on colon, prostate, and breast cancer has been the subject of in-depth study. However, the possibility that these changes could influence tumors of other origins has been only scarcely analyzed. Cellular cholesterol homeostasis is a tightly regulated system in which the amount of unesterified cholesterol within the cells is controlled by the rate of cholesterol uptake and synthesis, esterification, and hydrolysis of the resultant cholesteryl esters. The main extracellular cholesterol sources are the lipoproteins, whose cholesterol is internalized by active, transporter-
dependent uptake mechanisms. Among these, class B scavenger receptors SR-BI and the thrombospondine receptor CD36 have received special attention because of their selectivity toward high-density lipoproteins (HDLs) and low-density lipoproteins (LDLs), as well as their broad ligand-binding specificity. The lipoprotein receptor CLA-1 (CD36 and LIMPII analogue I), whose murine analogue is the scavenger receptor BI (SR-BI), is a glycoprotein member of the CD36 gene family. CLA-1 is expressed in liver, digestive tract, and steroidogenic tissues, where it binds HDL, mediates the selective uptake of HDL cholesterol esters, and can also bind LDL with high affinity. SR-BI/ CLA-1 also transfers free cholesterol and anionic phospholipids into cells, and can mediate the lipid efflux from cells to the extracellular space. SR-BI/CLA-1 and CD36 have similar structure and ligand specificity, but CD36 binds oxidized low-density lipoproteins with higher affinity than CLA-1/SR-BI does. Moreover, CD36 internalizes not only cholesterol, but also its associated protein component, and also transports longchain fatty acids. In addition, CD36 is thought to be a possible component of the src signal transduction pathway, because CD36 can form a complex with the protein-tyrosine kinases Fyn, Yes, and Lyn in platelets. Both CD36 and SR-BI/CLA-1 have been suggested as medi-
From the Department of Cell Biology and Genetics, University of Alcala´, Alcala´ de Henares, Madrid, Spain and the Laboratory of Applied Molecular Oncology, Medical Oncology Service and Department of Investigation, Ramo´n y Cajal Hospital, Madrid, Spain. Accepted for publication August 18, 2003. Address correspondence and reprint requests to M. I. Arenas, MD, Department of Cell Biology and Genetics, University of Alcala´, 28871 Alcala´ de Henares, Madrid, Spain. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2003.08.015
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ators for the phagocytosis of apoptotic cells by macrophages, and SR-BI/CLA-1 has also been related to the phagocytic capabilities of Sertoli cells in rat testes. Hormone-sensitive lipase (HSL) has also merged as a key regulator of lipid metabolism in multiple tissues. A multifunctional enzyme, HSL catalyses the hydrolysis of monoacyl, diacyl, and triacyl glycerol, cholesterol, and retinyl esters, and controls plasma free fatty acid (FFA) levels by regulating the lipid metabolism in the adipocytes. HSL seems to be especially important in tumor pathology, because FFAs are ligands for members of the nuclear hormonal receptor family, such as peroxisome proliferator activated receptors (PPARs), and many of these receptors occur as obligate heterodimers with members of the family of the retinoic acid receptors. In human breast carcinoma cells, a possible relationship among cholesterol transport mediated by SR-BI/CLA-1 and the hydrolysis of the internalized cholesteryl esters by HSL has been suggested. These and other data have provided clues to the link between lipid metabolism and cancer development and progression. HSL also has been detected in several nonadipose rodent tissues, including muscle, pancreatic beta cells, placenta, adrenal gland, breast, ovary, and testis. In contrast, only a few studies have analyzed the expression and the possible role of HSL in human tissues. Furthermore, rodent steroidogenic tissues have been the main sources of information about cholesterol transport and intracellular metabolism, but the mechanisms that control and regulate these processes in human testis and their implications in testicular pathology remain obscure. Most researchers have focused their attention on the rodent testis, where HSL has been demonstrated to play a crucial role in the spermatogenetic process, because HSL knockouts are infertile. The rodent testis expresses HSL as a 3.9-kb mRNA and several proteins of 28 and 72 kDa. In contrast, the human testis expresses 2 different mRNAs for HSL (3.3 and 3.9 kb) and several anti-HSL immunoreactive proteins of 84 and 120 kDa. Moreover, in the rodent testis, HSL is located only in seminiferous tubules (Sertoli and spermatogenic cells), whereas in the human testis, HSL is located in both seminiferous tubules and Leydig cells. HSL’s absence in rodent Leydig cells excludes this lipase from playing any role in testicular steroidogenesis. However, its presence in human Leydig cells suggests differences in testicular cholesterol metabolism between humans and rodents. There also are no data about the expression and tissue distribution of CD36 in the human testis, or about the expression of HSL, SR-BI/CLA-1, and CD36 in human testicular pathology. Therefore, the first step should be to determine the cell types that might express these proteins and their possible changes in pathological situations, to explore the role of lipid transports and HSL in human testicular physiology. In the present study we explored the expression and distribution of HSL, SR-BI/CLA-1, and CD36 in the normal human testis and in nontumor and tumor testicu-
lar pathologies by immunohistochemical and Western blotting analysis methods. MATERIALS AND METHODS Materials Orchidectomy specimens were obtained for therapeutic purposes and with informed consent from 10 men (age 65 to 80 years) with prostatic carcinoma who had received no previous hormone or drug treatments and had no testicular, endocrine, or related disorders. In addition, testicular biopsy specimens were obtained from 10 young adult men (age 28 to 34 years) consulting for infertility, and another 32 testes obtained from testicular diseases surgery were fixed and processed in the same way. These were diagnosed as normal testes (n ⫽ 7), cryptorchid testes (n ⫽ 5), testicular torsion (n ⫽ 2), infertile testes with lesions in the adluminal compartment of seminiferous tubules (n ⫽ 5), intratubular germ cell neoplasia (n ⫽ 4), seminoma (n ⫽ 3), spermatocytic seminoma (n ⫽ 2), and teratoma (n ⫽ 4). Each sample was longitudinally sectioned in two equal portions; one portion was immediately processed for immunohistochemistry, and the other portion was frozen in liquid nitrogen and maintained at ⫺80°C for Western blotting analysis. The primary antibodies used were rabbit polyclonal antibody 1336, reactive with human CLA-1 (courtesy of M. A. Vega, CSIC, Madrid, Spain); rabbit polyclonal antibody to SR-BI/SR-BII (Novus Biologicals, Littleton, CO); goat polyclonal antibody to CD36 (Santa Cruz Biotechnologies, Santa Cruz, CA); and chicken polyclonal antibody to HSL (anti-rat adipocyte HSL; courtesy of C. Holm, Department of Cell and Molecular Biology, Lund University, Sweden).
Immunohistochemistry For light microscopy immunohistochemistry, the specimens were fixed for 24 hours at room temperature in either 0.1 mol phosphate-buffered 10% formaldehyde or Bouin’s solution, dehydrated, and embedded in paraffin. Sections (5 m thick) were processed using the avidin-biotin-peroxidase complex method. After deparaffinization, sections were hydrated and incubated for 30 minutes in 0.3% H2O2 diluted in methanol, to reduce endogenous peroxidase activity. Then, to retrieve the antigen, the sections were incubated with 0.1 mol citrate buffer, pH 6, for 5 minutes in a conventional pressure cooker. After rinsing in Tris-buffered saline (TBS), the slides were incubated with normal donkey serum at a 1:5 dilution in TBS for 30 minutes, to prevent nonspecific binding of the first antibody. Thereafter, the primary antibodies were applied at a dilution of 1:1000 for HSL, 1:200 for CD36, and 1:300 for CLA-1/SR-BI, in TBS/normal donkey serum at 4°C overnight. The sections were then washed twice in TBS and incubated with goat anti-chicken, rabbit anti-goat, and swine anti-rabbit biotinylated immunoglobulins (all from Dako, Carpinteria, CA) at 1:500 dilutions. After 1 hour of incubation with the secondary antibody, the sections were incubated with a standard streptavidin-biotin complex (Dako) and developed with 3,3'-diaminobenzidine, using the glucose oxidase-diaminobenzidine-nickel intensification method.
Western Blotting For Western blotting analysis, each sample was homogenized in 0.5 mol Tris-HCl buffer, pH 7.4, containing 1 mmol EDTA, 12 mmol 2-mercaptoethanol, 1 mmol benzamidine,
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and 1 mmol phenylmethylsulphonyl fluoride, with the addition of a cocktail of protease inhibitors (10 mmol of iodoacetamide, 0.01 mg/mL of soybean trypsin inhibitor, and 1 l/mL of leupeptin) and phosphatase inhibitors (10 mmol sodium fluoride and 1 mmol sodium orthovanadate) in the presence of 0.5% Triton X-100. Homogenates were centrifuged for 10 minutes at 10,000 rpm. After boiling for 2 minutes at 98°C, aliquots of 20 g of protein were separated in sodium dodecyl sulfate-polyacrylamide (9% w/v) slab minigels. Separated proteins were transferred for 4 hours at 0.25 A to nitrocellulose membranes (0.2 m), and the nitrocellulose sheets were then blocked for 1 hour with 5% blotto in 0.05 mol Tris-HCl and incubated overnight with the primary antibodies, diluted at 1:10000 (HSL), 1:1000 (CD36), and 1:3000 (CLA-1/SR-BI) in TBS. After extensive washing with TBS/Tween 20, the membranes were incubated with a horseradish peroxidase–labeled secondary antibody diluted in TBS (goat anti-chicken for HSL, 1:10,000 dilution; rabbit anti-goat for CD36, 1:4000 dilution; and swine anti-rabbit for CLA-1/ SR-BI, 1:4000 dilution; all from Chemicon, Temecula, CA) for 1 hour. After an intensive wash, the filters were developed with an enhanced chemiluminescence kit, following the procedure specified by the manufacturer (Amersham, Buckinghamshire, UK).
FIGURE 1. An intense signal for streptavidin-Cy3 can be observed in both Leydig cell (L) and germ cell cytoplasm. ST, seminiferous tubule. Bar ⫽ 25 m.
showed an intense surface reaction to CLA-1 and SR-BI (Fig 2A)and HSL labeling in the cytoplasm. In the seminiferous tubules, the basal cytoplasm of Sertoli cells and residual bodies immunoreacted intensely to CLA-1. A diffuse and weak reaction to both SR-BI and HSL was observed in the Sertoli cell cytoplasm. Spermatogonia demonstrated a negative immunoreaction to the lipid receptors and a weakly positive reaction to HSL. The Golgi region of spermatocytes was labeled with HSL only. Round spermatids demonstrated a positive reaction to CLA-1 and SR-BI in the acrosomic vesicle, whereas the nuclei exhibited HSL labeling (Fig 2B). CD36 was unreactive. In testes from aging men, the labeling pattern for CLA-1, SR-BI, and HSL was the same as in young men, except for those areas containing Sertoli cell– only tubules, in which Sertoli cell immunoreaction to HSL was more intense than in normal young testes. In the Leydig cells adjacent to Sertoli cell– only tubules, labeling was lower than in the Leydig cells of zones with complete spermatogenesis. Elderly testes demonstrated a patchy positive reaction to CD36 in the Sertoli cell surfaces (Fig 2C). Testes with hypospermatogenesis associated with primary spermatocyte sloughing exhibited the same reactivity to CLA-1 and SR-BI as normal testes. CD36 was positive in the Sertoli cell surfaces and in the cytoplasm of Leydig cells underneath the plasma membrane (Fig 2D). Spermatogonia cytoplasm reacted to HSL (Fig 2E). Testes with Leydig cell hyperplasia exhibited intense labeling to HSL around the nucleus. Cryptorchid testes demonstrated atrophic seminiferous tubules lined by a single layer of Sertoli cells. A strong immunoreaction to HSL in the cytoplasm and, surprisingly, in the nucleus of these cells was observed (Fig 2F). In areas with nodules of hyperplastic Leydig cells in cryptorchid testes, an intense HSL reaction was observed around the nucleus of these cells. A weak reaction to SR-BI was observed in the surface of both Sertoli and Leydig cells. CLA-1 and CD36 did not react. In testes with intratubular germ cell neoplasia of
Controls The specificity of the immunohistochemical procedures was checked using negative and positive control sections. Negative controls were created by either omitting the primary antibody or using the antibody preabsorbed with an excess of purified antigens. For HSL positive controls, sections of human white adipose tissue were incubated with the same antibody. For CD36, CLA-1, and SR-BI antibodies, monocytes present in testicular samples were used as internal controls. To assess the status of Leydig cell function, the presence of endogenous biotin and biotin-dependent enzymes, which are regulatory enzymes in lipogenesis and gluconeogenesis, was evaluated. Biotin was detected using streptavidin conjugated with Cy3 (Sigma-Aldrich, Madrid, Spain). Briefly, after incubation with streptavidin-Cy3 at 1:300 dilution, sections were stained with 4',6-diamidino-2-phenylindole and mounted with Mowiol. Microscopic analyses were done using an epifluorescence microscope (Axiophot; Carl Zeiss, Oberkochen, Germany), and photographs were taken with Fuji Super G400 ASA color film (Fujifilm, Tokyo, Japan).
RESULTS Controls Negative controls incubated with the preimmune serum or with the antibody preabsorbed with an excess of purified antigens demonstrated no labeling. Immunostaining of white adipose tissue sections were always positive for HSL. Monocytes present in human testicular interstitium exhibited a positive reaction to CD36 and CLA-1/SR-BI. On fluorescence microscopy, a strong signal in the Leydig cell cytoplasm and in the germ cell cytoplasm was found in all samples studied (Fig 1). Immunohistochemistry Study Normal young testes with complete spermatogenesis immunoreacted as follows (Table 1). Leydig cells 36
LIPASE AND LIPID RECEPTORS IN TESTES (Arenas et al)
TABLE 1. Immunoreaction Pattern to Lipid Receptors and HSL in Normal Young Testes
CLA-1 SR-BI HSL CD36
Spermatogonia
Spermatocytes
Spermatids
Sertoli cells
Leydig Cells
— — Cytoplasm (weak) —
— — Golgi region —
Acrosomic vesicle Acrosomic vesicle Nucleus —
Basal cytoplasm Cytoplasm (weak) Cytoplasm (weak) —
Surface Surface Cytoplasm —
the unclassified type, the affected tubules consisted of a population of malignant germ cells and Sertoli cells; the latter appeared displaced luminally. Both cell types demonstrated immunostaining to lipid receptors in the surface and focal areas of the cytoplasm (Fig 2G) and to HSL in the nucleus (Fig 2H). Leydig cells were immunolabeled with the 4 antibodies, with reactions similar to those observed in normal testes. Neoplastic cells present in seminoma and spermatocytic seminoma were immunopositive to HSL (Fig 1I), but they failed to stain with lipid receptors (Fig 2J). In these tumor types, the seminiferous tubules at the periphery of the tumor demonstrated only Sertoli cells with vacuolated cytoplasm, absence of lumen, and normal lamina propria. The Sertoli cells of these tubules labeled intensely to HSL. Lipid receptors were discretely immunostained in the cytoplasm of these cells, and an intense reaction to CLA-1 was seen in CharcotBo¨ ttcher crystalloids (Fig 2K). The Leydig cells present in these tubules immunostained to HSL in the cytoplasm and to lipid receptors on their surface. The mature teratomas studied here consisted of various somatic-type tissues, including enteric-type glands and cartilage. Some cells of enteric-type glands exhibited a positive reaction to CD36 and SR-BI (Fig 2L), but were unstained with CLA-1 and HSL. In immature teratomas, immature tissues, such as neuroepithelium, were immunolabeled exclusively with HSL.
tected in spermatocytic seminoma (Fig 3B, lane 4) and intratubular germ cell neoplasia (Fig 3B, lane 5) of the unclassified type, with an additional band of 70 kDa observed in the former. Multiple CLA-1 immunoreactive bands between 60 and 70 kDa and between 130 and 240 kDa were observed in normal young testes (Fig 3C, lane 2). In cryptorchid testes, the reactivity of higher molecular weight bands was lower (Fig 3C, lane 1). This pattern was similar in elderly testes (Fig 3C, lane 3). In spermatocytic seminoma and intratubular germ cell neoplasia, the high molecular weight bands were absent (Fig 3C, lanes 4 and 5). The immunoreactive band pattern for SR-BI (Fig 3D) was similar to that observed with the CLA-1 antibody, but the 60-kDa band was more intense with the SR-BI antibody. An immunoreactive band of 85 kDa was detected only in elderly testes and intratubular germ cell neoplasias when SR-BI antibody was used (Fig 3D, lanes 3 and 5). DISCUSSION In this study we have demonstrated the presence of lipid receptors (CLA-1, SR-BI, and CD36) and HSL in human testes, observing variations in this expression within different pathologies. The presence of CD36 in human testes has not been previously reported. We observed a positive reaction to CD36 in the surface of both Sertoli cells and Leydig cells in aging and pathological testes. This staining might be related either to increased phagocytic activity of the Sertoli cells or to accumulation of lipids in the cytoplasm of both cell types. Several pathological conditions are known to lead to lipid droplet accumulation, which is related to impaired lipid metabolism and steroidogenesis in Leydig cells or to arrested spermatogenesis in Sertoli cells.30 By Western blotting analysis, we detected 4 immunoreactive bands (at 55, 75, 130, and 150 kDa) to CD36 antibody, along with an additional band of 33 kDa in elderly testes. The CD36 cDNA predicts a polypeptide of 53 kDa with 10 potential N-linked glycosylation sites.31 For some authors, the mature protein has a molecular weight of 94 kDa and the CD36 precursor 74 kDa.31 Nevertheless, in platelets, Rhinehart-Jones and Greenwalt32 detected a 113-kDa isoform. In U937 cells, CD36 coprecipitated with two proteins of 33 and 130 kDa;31 these bands were interpreted as a protein associated with the 74-kDa CD36 precursor and as an artifact of the immunoprecipitation method, respectively. In accordance with the findings reported by others,33
Western Blotting Analysis The results of Western blotting analysis are shown in Figure 3. In all samples analyzed, HSL antibody stained multiple protein bands at different molecular weights. In normal human testes in young and aging men (Fig 3A, lanes 2 and 3), HSL-immunoreactive proteins were observed at 60, 84, 102, 110, and 120 kDa. An additional band at 28 kDa was observed in normal young testes. A similar pattern was found in cryptorchid testes (Fig 3A, lane 1). Testes with seminoma and spermatocytic seminoma exhibited immunoreactive bands at 28, 32, 110, and 130 kDa (Fig 3A, lane 4). Testes with intratubular germ cell neoplasia of the unclassified type demonstrated a band only at 60 kDa (Fig 3A, lane 5). In normal young and elderly testes, CD36 immunoreactive bands between 55 and 75 kDa were observed, along with 2 bands of 130 and 150 kDa (Fig 3B, lane 2) and in elderly testes an additional 33-kDa band was observed (Fig 3B, lane 3). The same pattern was observed in cryptorchid testes, but the reactivity of the higher molecular weight bands was lower (Fig 3B, lane 1). Four bands of 55, 74, 130, and 150 kDa were de37
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lated forms of CLA-1, mainly the 70-kDa form. The two antibodies that we used detected a protein of 70 kDa, but was preferentially bound by CLA-1. Landschulz et al37 found CLA-1/SR-BI expression at high levels in both the adrenal gland and liver of humans; it seems likely that one important function of this is to supply substrate for steroidogenesis.5 Reaven et al39 observed that under normal physiological conditions, rat testes do not require exogenous lipoprotein cholesterol for Leydig cell testosterone production; therefore, SR-BI receptor expression is low or absent. However, in rats treated with human chorionic gonadotropin, the expression of this receptor is increased, indicating that the hormone-treated cells take up HDLderived cholesterol esters to synthesize testosterone. In our study we observed SR-BI expression in Leydig cells from most samples, except in those cases where the Leydig cell population was altered or suppressed (seminomas and teratomas). This immunostaining suggests an active cholesterol ester flux for testosterone production. SR-BI expression is coordinately regulated with cholesterol metabolism,40 and its expression in steroidogenic cells in vivo and in vitro is regulated by trophic hormones.41 It has been demonstrated that in Sertoli cells from rat testis, SR-BI is a phosphatidylserine receptor responsible for the phagocytosis of apoptotic spermatogenetic cells,11 and that the phagocytic activity of Sertoli cells varies at different spermatogenetic stages. We have observed a weak reaction to SR-BI in the Sertoli cell cytoplasm of normal and hypospermatogenetic testes; however, the reaction to this antibody was more intense in Sertoli cells from cryptorchid testes and in intratubular germ cell neoplasia. Therefore, SR-BI expression increases in those disorders in which germ cells are absent or have a malignant phenotype. In contrast with rodents, in humans our data indicate that CLA-1/SR-BI cannot be related to the phagocytic activity of normal human Sertoli cells. Charcot-Bo¨ ttcher crystalloids are usually present in the Sertoli cell cytoplasm. Their origin and chemical composition are unknown, although Hadley and Dym42 suggested that they might contain heparin sulfate. These crystalloids are increased in cases of impaired spermatogenesis.43 No previous studies have reported immunostaining for any known protein within these crystalloids. We detected an accumulation of CLA-1 immunoreactived proteins in Charcot-Bo¨ ttcher crystal-
our results support the idea that, depending on cell type, CD36 displays different molecular masses corresponding to different glycoforms. However, the significance of the 33-kDa band is not clear, because this band was observed only in samples from elderly men; it could be interpreted as the result of intracellular CD36 processing. Recently, a variant of SR-BI (termed SR-BII) has been found in mice and humans. The SR-BII transcript is relatively abundant in tissues known to express SR-BI and represents approximately 30% of total mRNA SR-BI values in mouse adrenal glands and about 80% of total SR-BI values in mouse testes.34,35 However, whether SR-BII is present in human testes remains unknown. In our study, we used the commercial antibody reactive to SR-BI/II that recognized different bands with a wide range of 60 to 160 kDa; however, we observed that the predominant forms of SR-BI/BII in the human testes are low molecular weight proteins. These multiple bands might be the result of differently glycosylated fractions; Babitt et al36 reported a huge heterogeneity in the structures of the N-linked chains on SR-BI protein, similar to that encountered on CD36.33 Landschulz et al,37 using a rabbit anti-murine SR-BI polyclonal antibody, described a protein with an apparent molecular mass of 82 kDa in murine tissues, along with a larger band of approximately twice this mass (164 kDa) in both the adrenal gland and ovary and a faint protein of 129 kDa visible in the heart. In LDL receptor-deficient CHO cell mutants, Babitt et al36 described the following forms of SR-BI: a 82-kDa, Nglycosylated mature form, a 54-kDa immature form, and a 64-kDa intermediate form. Cao et al,38 using an antibody that recognizes only several proteins generated from SR-BI.1, detected a prominent band of 80 kDa in human tissues and an additional band of 50 kDa in the prostate. In addition, Shiratsuchi et al,11 using an antibody that recognizes amino acid residues 110 to 132 of hamster SR-BI, observed an immunoreactive band for SR-BI of 70 kDa in Sertoli cell cultures from rats. Because the human long form of CLA-1 shares 81% sequence identity with hamster SR-BI,1 we used an antibody reactive to human CLA-1. CLA-1 has a predicted molecular weight of 56 kDa with 10 potential N-glycosylation sites originating an 80-kDa mature form.3 In human testes, the 1336 antibody that we used preferentially recognizes the immature, low-glycosy-
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ FIGURE 2. (A) Normal young testis showing intense immunolabeling to SR-BI on the surface of Leydig cells (L) and in the acrosomic vesicles of round spermatids (Sp). The cytoplasm of Sertoli cells were weakly immunoreactive (S). (B) A strong reaction to HSL antibody can be observed in the nuclei of spermatids (Sp). The cytoplasm of Sertoli cells (S) shows weak staining. (C) Seminiferous tubules from elderly testes showing a patchy reaction to CD36 in the Sertoli cells. (D) Germ and Leydig cells from testes with primary spermatocyte sloughing exhibiting a strong reaction to CD36. Sg, spermatogonium. (E) Testes with primary spermatocytes sloughing were more strongly immunoreactive to HSL than normal testes; compare with (B). (F) Cryptorchid testis showed a strong immunoreaction to HSL in both the cytoplasm and the nucleus of Sertoli cells. Leydig cells were also stained. (G) Focal staining to CD36 observed in the malignant germ cell cytoplasm in intratubular germ cell neoplasia of the unclassified type. (H) Intense labeling to HSL antibody in Leydig cells (L) and in both the nucleus and cytoplasm of neoplastic cells in intratubular germ cell neoplasia of the unclassified type. (I) Neoplastic cells of spermatocytic seminoma demonstrating intense immunoreaction to HSL. (J) Negative staining to SR-BI in neoplastic cells of seminoma. (K) Seminiferous tubules at the periphery of spermatocytic seminoma. Charcot-Bo ¨ ttcher crystalloids are intensely labeled with CLA-1 antibody (arrowheads). (L) Some cells of enteric-type of mature teratoma exhibiting a positive reaction to SR-BI. Bar ⫽ 50 m.
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FIGURE 3. Immunoblots of HSL and scavenger receptors in normal and pathological human testes. Lane 1, cryptorchid testis; lane 2, normal young testis; lane 3, elderly testis; lane 4, spermatocytic seminoma; lane 5, intratubular germ cell neoplasia of unclassified type. Molecular masses are represented on the left. Each blot is representative of its respective group.
loids, supporting the notion of a dysfunction in the lipid transport of the Sertoli cells in a tumor environment. This dysfunction might be one of the changes occurring in the infertility associated with germ cell tumors. We have observed intense immunoexpression of CLA-1 in the Sertoli cells and malignant germ cells of seminiferous tubules with intratubular germ cell neo-
plasia; this expression was lost when malignant cells have an invasive phenotype. A number of studies have reported that cancer cells and solid tumors take up LDL more effectively than normal tissues,44 suggesting a higher cholesterol demand in dividing cells. An interesting, and not previously reported, finding is the positive reaction to CLA-1/SR-BI observed in the acrosomic vesicle of spermatids. These receptors 40
LIPASE AND LIPID RECEPTORS IN TESTES (Arenas et al)
testes. The presence of HSL in human Leydig cells suggests a direct relationship with steroid hormone production. In summary, the presence of lipid receptors (CLA1/SR-BI) and HSL in Leydig cells of normal and pathological human testes suggests that these proteins play a key role in steroidogenesis through the supply of free cholesterol for testosterone synthesis. These proteins are also involved in spermiogenesis, as their labeling in spermatids suggests. In nonmalignant and malignant pathologies, cholesterol metabolism is probably altered, and HSL labeling in the nucleus of neoplastic germ cells suggests a still-unknown function of this enzyme, probably related to cell cycle regulation.
likely are implicated either in transport of the lipids needed for the formation of acrosomal membranes or in a generalized redistribution of membrane cholesterol in the acrosome formation. It has been reported that SR-BI expression accelerates the bidirectional flux of free cholesterol and induces the formation of novel membrane structures45,46 and that CLA-1 plays a role in selective uptake of cholesterol esters from HDL.47 Durham and Grogan25 reported two types of cholesterol ester hydrolases in the testis: temperature-stable and temperature-labile. HSL may be related to temperature-labile cholesteryl ester hydrolase, because both enzymes are more active at low temperature and their activity increases with sexual maturity. Mairal et al47 characterized two different HSL mRNAs of 3.3 and 3.9 kb present in equal amounts in the human testis, a short-form HSL expressed in spermatogonia, pachytene spermatocytes, Sertoli cells, Leydig cells, and peritubular cells and a long-form HSL transcribed exclusively in haploid germ cells. Human adipose tissue expresses a 2.8-kb mRNA that encodes a 88-kDa protein. The mRNA and protein species expressed in the testis are larger, 3.9 kb and 120 kDa, respectively.48 In mouse, Blaise et al49 identified an HSL testis-specific promoter that contains 4 regions binding testicular nuclear proteins. One of these regions binds Sox proteins expressed in postmeiotic germ cells. Our results support that HSL present different testicular isoforms. By Western blotting analysis, we obtained different bands with apparent molecular masses of aproximately 28, 60, 84, 102, 110, and 130 kDa. Different HSLimmunoreactive bands have been reported in mammalian testes; for example, in guinea pig testes, Kabbaj et al50 detected bands of 104, 110, and 120 kDa in seminiferous tubules and only a band of 104 kDa in the interstitial tissue. In rat testis, Kraemer et al51 observed HSL-like immunoreactive proteins of 84, 89, and 102 kDa in sexually immature rats and additional 113- and 127-kDa immunoreactive proteins in mature animals. This pattern expression suggests that several tissue-specific promoters control the different forms of HSL, because when a gene is expressed in somatic tissues and in germ cells, tissue-specific expression often results from alternate promoter use.52 The significance of the nuclear immunostaining observed by light microscopy is unknown, although HSL might hydrolyze retinyl esters;16 recently, metabolism of cholesteryl esters was detected in the cell nucleus of hepatocytes.53 Because the main targets of retinoic acid derivatives are located in the cell nucleus, HSL might be related to the nuclear turnover of this derivative and/or to the nuclear metabolism of lipids. HSL is essential for spermatogenesis; HSL⫺/⫺ mice are infertile, have many epithelial vacuolated cells in the seminiferous tubules, and demonstrate increased amounts of cholesterol esters in their testes.24 Moreover, HSL may be essential for membrane stabilization and integrity in the seminiferous epithelium54 and also may be involved in the metabolism of lipid droplets in Sertoli cells,23 as suggested by the intense reaction to HSL observed in tubules seminiferous of pathological
REFERENCES 1. Acton S, Rigotti A, Landschulz KT, et al: Identification of a scavenger receptor SR-BI as a high-density lipoprotein receptor. Science 271:518-520, 1996 2. Sparrow CP, Pittman RC: Cholesterol esters selectively taken up from high-density lipoproteins are hydrolysed extralysosomally. Biochim Biophys Acta 1043:203-210, 1990 3. Calvo D, Vega MA: Identification, primary structure, and distribution of CLA-1, a novel member of the CD36/LIMPII gene family. J Biol Chem 268:18929-18935, 1993 4. Calvo D, Dopazo J, Vega MA: The CD36, CLA-1 (CD36L1), and LIMPII (CD36L2) gene family: Cellular distribution, chromosomal localization and genetic evolution. Genomics 25:100-106, 1995 5. Murao K, Terpstra V, Green SR, et al: 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, 1997 6. Acton SL, Scherer PE, Lodish HF, et al: Expression cloning of SR-BI, a CD36-related class B scavenger receptor. J Biol Chem 269: 21003-21009, 1994 7. Nozaki S, Kashiwagi H, Yamashita S, et al: Reduced uptake of oxidized low-density lipoprotein in monocyte-derived macrophages from CD36-deficient subjects. J Clin Invest 96:1859-1865, 1995 8. Abumrad NA, El-Maghrabi MR, Amri EZ, et al: Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation. J Biol Chem 268:17665-17668, 1993 9. Alessio M, Greco NJ, Primo L, et al: Platelet activation and inhibition of malarial cytoadherence by the anti-CD36 IgM monoclonal antibody NL07. Blood 82:3637-3647, 1993 10. Bull HA, Brickell PM, Dowd PM: Src-related protein tyrosine kinases are physically associated with the surface antigen CD36 in human dermal microvascular endothelial cells. FEBS Lett 351:41-44, 1994 11. Shiratsuchi A, Kawasaki Y, Ikemoto M, et al: Role of class B scavenger receptor type I in phagocytosis of apoptotic rat spermatogenic cells by Sertoli cells. J Biol Chem 274:5901-5908, 1999 12. Vaughan M, Berger JE, Steinberg D: Hormone-sensitive lipase and monoglyceride lipase activities in adipose tissue. J Biol Chem 239:401-409, 1964 13. Fredrikson G, Stralfors P, Nilsson NO, et al: Hormone-sensitive lipase of rat adipose tissue: Purification and some properties. J Biol Chem 256:6311-6320, 1981 14. Østerlund T, Danielsson B, Degerman E, et al: Domainstructure analysis of recombinant rat hormone-sensitive lipase. Biochem J 319:411-420, 1996 15. Lee ET, Adams JB, Garton AJ, et al: Hormone-sensitive lipase is involved in the hydrolysis of lipoidal derivatives of estrogens and other steroid hormones. Biochim Biophys Acta 963:258-264, 1998 16. Wei S, Lai K, Patel S, et al: Retinyl ester hydrolysis and retinol efflux from BFC-1 adipocytes. J Biol Chem 272:14159-14165, 1997 17. Yeaman SJ: Hormone-sensitive lipase: A multipurpose enzyme in lipid metabolism. Biochim Biophys Acta 1052:128-132, 1990
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18. Dreyer C, Krey G, Keller H, et al: Control of the peroxisomal -oxidation pathway by a novel family of nuclear hormone receptors. Cell 68:879-887, 1992 19. Amri EZ, Bonino F, Ailhaud G, et al: Cloning of a protein that mediates transcriptional effects of fatty acids in preadipocytes. Homology to peroxisome proliferator-activated receptors. J Biol Chem 270:2367-2371, 1995 20. Pussinen PJ, Karten B, Winersperger A, et al: The human breast carcinoma cell line HBL-100 acquires exogenous cholesterol from high-density lipoprotein via CLA-1 (CD-36 and LIMPII analogous 1)-mediated selective cholesteryl ester uptake. Biochem J 349: 559-566, 2000 21. Holm C, Belfrage P, Fredrikson G: Immunological evidence for the presence of hormone-sensitive lipase in rat tissues other than adipose tissue. Biochem Biophys Res Commun 148:99-105, 1987 22. Cook KG, Colbran RJ, Snee J, et al: Cytosolic cholesterol ester hydrolase from bovine corpus luteum: Its purification, identification, and relationship to hormone-sensitive lipase. Biochim Biophys Acta 752:46-53, 1983 23. Stenson Holst L, Hoffmann AM, Mulder H, et al: Localization of hormone-sensitive lipase to rat Sertoli cells and its expression in developing and degenerating testis. FEBS Lett 355:125-130, 1994 24. Osuga J, Ishibashi S, Oka T, et al: Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity. Proc Natl Acad Sci U S A 97:787-792, 2000 25. Durham LA, Grogan WM: Characterization of multiple forms of cholesteryl ester hydrolase in the rat testis. J Biol Chem 259:7433-7438, 1984 26. Calvo D, Go´mez-Coronado D, Lasuncio´n MA, et al: CLA-1 is an 85-kDa plasma membrane glycoprotein that acts as a high-affinity receptor for both native (HDL, LDL, and VLDL) and modified (OxLDL and AcLDL) lipoproteins. Arterioscler Thromb Vasc Biol 17:2341-2349, 1997 27. Hsu SM, Raine L, Fanger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580, 1981 28. Norton AJ, Jordan S, Yeomans P: Brief, high-temperature heat denaturation (pressure cooking): A simple and effective method of antigen retrieval for routinely processed tissues. J Pathol 173:371379, 1994 29. Hsu SM, Soban E: Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry. J Histochem Cytochem 30:1079-1082, 1982 30. Chung KW, Hamilton JB: Testicular lipids in mice with testicular feminization. Cell Tissue Res 27:69-80, 1975 31. Alessio M, de Monte L, Scirea A, et al: Synthesis, processing, and intracellular transport of CD36 during monocytic differentiation. J Biol Chem 271:1770-1775, 1996 32. Rhinehart-Jones T, Greenwalt DE: A detergent-sensitive 113kDa conformer/complex of CD36 exists on the platelet surface. Arch Biochem Biophys 326:115-118, 1996 33. Greenwalt DE, Lipsky RH, Ockenhouse CF, et al: Membrane glycoprotein CD36: A review of its roles in adherence, signal transduction, and transfusion medicine. Blood 80:1105-1115, 1992 34. Webb NR, de Villiers WJ, Connell PR, et al: Alternative forms of the scavenger receptor BI (SR-BI). J Lipid Res 38:1490-1495, 1997 35. Webb NR, Connell PR, Graf FA, et al: 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, 1998
36. Babitt J, Trigatti B, Rigotti A, et al: Murine SR-BI, a highdensity lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae. J Biol Chem 272:13242-13249, 1997 37. Landschulz KT, Pathak RK, Rigotti A, et al: Regulation of scavenger receptor, class B, type I, a high-density lipoprotein receptor, in liver and steroidogenic tissues of the rat. J Clin Invest 98:984995, 1996 38. Cao G, Garcia CK, Wyne KL, et al: Structure and localization of the human gene encoding SR-BI/CLA-1. Evidence for transcriptional control by steroidogenic factor 1. J Biol Chem 272:3306833076, 1997 39. Reaven E, Zhan L, Nomot A, et al: Expression and microvillar localization of scavenger receptor class B, type I (SR-BI) and selective cholesteryl ester uptake in Leydig cells from rat testis. J Lipid Res 41:343-356, 2000 40. Krieger M: Scavenger receptor class B type I is a multiligand HDL receptor that influences diverse physiologic systems. J Clin Invest 108:793-797, 2001 41. Trigatti B, Rigotti A, Krieger M: The role of the high-density lipoprotein receptor SR-BI in cholesterol metabolism. Curr Opin Lipidol 11:123-131, 2000 42. Hadley MA, Dym M: Immunocytochemistry of extracellular matrix in the lamina propria of the rat testis: Electron microscopic localization. Biol Reprod 37:1283-1289, 1987 43. Schulze C: Sertoli cells and Leydig cells in man. Adv Anat Embryol Cell Biol 88:1-104, 1984 44. Firestone RA: Low-density lipoproteins as a vehicle for targeting antitumor compounds to cells. Bioconjugate Chem 5:105-113, 1994 45. Llera-Moya M, Rothblat GH, Connelly MA, et al: Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface. J Lipid Res 40:575-580, 1999 46. Silver DL, Tall AR: The cellular biology of scavenger receptor class B type I. Curr Opin Lipidol 12:497-504, 2001 47. Mairal A, Malanie N, Laurell H, et al: Characterization of a novel testicular form of human hormone-sensitive lipase. Biochem Biophys Res Commun 291:286-290, 2002 48. Stenson Holst L, Langin D, Mulder H, et al: Molecular cloning, genomic organization, and expression of a testicular isoform of hormone-sensitive lipase. Genomics 35:441-447, 1996 49. Blaise R, Grober J, Rouet P, et al: Testis expression of hormone-sensitive lipase is conferred by a specific promoter that contains four regions binding testicular nuclear proteins. J Biol Chem 274:9327-9334, 1999 50. Kabbaj O, Holm C, Vitale ML, et al: Expression, activity, and subcellular localization of testicular hormone-sensitive lipase during postnatal development in the guinea pig. Biol Reprod 65:601-612, 2001 51. Kraemer FB, Patel S, Singh-Bist A, et al: Detection of hormone-sensitive lipase in various tissues, II. Regulation in the rat testis by human chorionic gonadotropin. J Lipid Res 34:609-616, 1993 52. Sun Z, Sassone-Corsi P, Means AR: Calspermin gene transcription is regulated by two cyclic AMP response elements contained in an alternative promoter in the calmodulin kinase IV gene. Mol Cell Biol 15:561-571, 1995 53. Albi E, Magni MV: The presence and the role of chromatin cholesterol in rat liver regeneration. J Hepatol 36:395-400, 2002 54. Chung S, Wang SP, Pan L, et al: Infertility and testicular defects in hormone-sensitive lipase-deficient mice. Endocrinology 142:4272-4281, 2001
42
Numerous studies have demonstrated the important role of cholesterol and cholesteryl esters in tumor cell proliferation and progression of cancer. However, few studies have focused on the role of lipid transporters and lipases in cancer development and progression. The present study examined the expression of hormone-sensitive lipase (HSL) and the scavenger receptors CLA-1/SR-BI and CD36 in normal human testis and in nontumor and tumor testicular disorders by immunohistochemistry and Western blotting analysis. In normal young testes, immunoreaction to CLA-1/SR-BI was found in the spermatid acrosomic vesicle and on the surface of Sertoli and Leydig cells. HSL was detected in spermatogonia, the Golgi region of spermatocytes, the nucleus of spermatids, and the cytoplasm of both Sertoli and Leydig cells. Elderly testes and testes with hypospermatogenesis showed a similar staining pattern to that of normal young testes except for CD36, which was expressed in Sertoli cells. Cryptorchid testes demonstrated intense labeling to HSL and weak labeling to SR-BI in Sertoli cells (nucleus and cytoplasm) and Leydig cells (cytoplasm). Seminiferous tubules with intratubular germ cell neoplasia exhibited intense immunolabeling to the 3 lipid receptors in the surface of neoplastic cells and to HSL in the nucleus. In seminoma and spermatocytic seminoma, neoplastic cells labeled to HSL but failed to stain with antilipid receptors; in the seminiferous tu-
bules at the periphery of the tumour, Charcot-Bo¨ttcher crystalloids of Sertoli cells were strongly positive to CLA-1. Testes with mature teratoma showed a weak reaction to CD36 and SR-BI in some cells of enteric-type glands, and immature teratoma were exclusively immunolabeled with HSL. Western blotting analysis revealed that multiple bands were immunolabeled, with differences seen between normal and pathological testes. The results of this study indicate that the presence of lipid receptors (CLA-1/SR-BI) and hormone-sensitive lipase in Leydig cells suggests a role of these proteins in steroidogenesis. Also, these proteins seem to be involved in spermiogenesis, as their labeling in spermatids suggests. In nonmalignant and malignant pathologies, cholesterol metabolism is probably altered, and HSL labeling in neoplastic germ cell nuclei suggests a still-unknown function of this enzyme, probably related to cell cycle regulation. HUM PATHOL 35:34-42. © 2004 Elsevier Inc. All rights reserved. Key words: HSL, CLA-1/SR-BI, CD36, immunohistochemistry, human testis. Abbreviations: FFA, free fatty acid; HDL, high-density lipoprotein; HSL, hormone-sensitive lipase; LDL, low-density lipoprotein; PPAR, peroxisome proliferator activated receptor; TBS, Tris-buffered saline.
Experimental and clinical evidence has clearly demonstrated a link between lipid metabolism disturbances and cancer development and progression. The influence of dietary lipids and changes in systemic and cellular lipid metabolism on colon, prostate, and breast cancer has been the subject of in-depth study. However, the possibility that these changes could influence tumors of other origins has been only scarcely analyzed. Cellular cholesterol homeostasis is a tightly regulated system in which the amount of unesterified cholesterol within the cells is controlled by the rate of cholesterol uptake and synthesis, esterification, and hydrolysis of the resultant cholesteryl esters. The main extracellular cholesterol sources are the lipoproteins, whose cholesterol is internalized by active, transporter-
dependent uptake mechanisms. Among these, class B scavenger receptors SR-BI and the thrombospondine receptor CD36 have received special attention because of their selectivity toward high-density lipoproteins (HDLs) and low-density lipoproteins (LDLs), as well as their broad ligand-binding specificity. The lipoprotein receptor CLA-1 (CD36 and LIMPII analogue I), whose murine analogue is the scavenger receptor BI (SR-BI), is a glycoprotein member of the CD36 gene family. CLA-1 is expressed in liver, digestive tract, and steroidogenic tissues, where it binds HDL, mediates the selective uptake of HDL cholesterol esters, and can also bind LDL with high affinity. SR-BI/ CLA-1 also transfers free cholesterol and anionic phospholipids into cells, and can mediate the lipid efflux from cells to the extracellular space. SR-BI/CLA-1 and CD36 have similar structure and ligand specificity, but CD36 binds oxidized low-density lipoproteins with higher affinity than CLA-1/SR-BI does. Moreover, CD36 internalizes not only cholesterol, but also its associated protein component, and also transports longchain fatty acids. In addition, CD36 is thought to be a possible component of the src signal transduction pathway, because CD36 can form a complex with the protein-tyrosine kinases Fyn, Yes, and Lyn in platelets. Both CD36 and SR-BI/CLA-1 have been suggested as medi-
From the Department of Cell Biology and Genetics, University of Alcala´, Alcala´ de Henares, Madrid, Spain and the Laboratory of Applied Molecular Oncology, Medical Oncology Service and Department of Investigation, Ramo´n y Cajal Hospital, Madrid, Spain. Accepted for publication August 18, 2003. Address correspondence and reprint requests to M. I. Arenas, MD, Department of Cell Biology and Genetics, University of Alcala´, 28871 Alcala´ de Henares, Madrid, Spain. 0046-8177/$—see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2003.08.015
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LIPASE AND LIPID RECEPTORS IN TESTES (Arenas et al)
ators for the phagocytosis of apoptotic cells by macrophages, and SR-BI/CLA-1 has also been related to the phagocytic capabilities of Sertoli cells in rat testes. Hormone-sensitive lipase (HSL) has also merged as a key regulator of lipid metabolism in multiple tissues. A multifunctional enzyme, HSL catalyses the hydrolysis of monoacyl, diacyl, and triacyl glycerol, cholesterol, and retinyl esters, and controls plasma free fatty acid (FFA) levels by regulating the lipid metabolism in the adipocytes. HSL seems to be especially important in tumor pathology, because FFAs are ligands for members of the nuclear hormonal receptor family, such as peroxisome proliferator activated receptors (PPARs), and many of these receptors occur as obligate heterodimers with members of the family of the retinoic acid receptors. In human breast carcinoma cells, a possible relationship among cholesterol transport mediated by SR-BI/CLA-1 and the hydrolysis of the internalized cholesteryl esters by HSL has been suggested. These and other data have provided clues to the link between lipid metabolism and cancer development and progression. HSL also has been detected in several nonadipose rodent tissues, including muscle, pancreatic beta cells, placenta, adrenal gland, breast, ovary, and testis. In contrast, only a few studies have analyzed the expression and the possible role of HSL in human tissues. Furthermore, rodent steroidogenic tissues have been the main sources of information about cholesterol transport and intracellular metabolism, but the mechanisms that control and regulate these processes in human testis and their implications in testicular pathology remain obscure. Most researchers have focused their attention on the rodent testis, where HSL has been demonstrated to play a crucial role in the spermatogenetic process, because HSL knockouts are infertile. The rodent testis expresses HSL as a 3.9-kb mRNA and several proteins of 28 and 72 kDa. In contrast, the human testis expresses 2 different mRNAs for HSL (3.3 and 3.9 kb) and several anti-HSL immunoreactive proteins of 84 and 120 kDa. Moreover, in the rodent testis, HSL is located only in seminiferous tubules (Sertoli and spermatogenic cells), whereas in the human testis, HSL is located in both seminiferous tubules and Leydig cells. HSL’s absence in rodent Leydig cells excludes this lipase from playing any role in testicular steroidogenesis. However, its presence in human Leydig cells suggests differences in testicular cholesterol metabolism between humans and rodents. There also are no data about the expression and tissue distribution of CD36 in the human testis, or about the expression of HSL, SR-BI/CLA-1, and CD36 in human testicular pathology. Therefore, the first step should be to determine the cell types that might express these proteins and their possible changes in pathological situations, to explore the role of lipid transports and HSL in human testicular physiology. In the present study we explored the expression and distribution of HSL, SR-BI/CLA-1, and CD36 in the normal human testis and in nontumor and tumor testicu-
lar pathologies by immunohistochemical and Western blotting analysis methods. MATERIALS AND METHODS Materials Orchidectomy specimens were obtained for therapeutic purposes and with informed consent from 10 men (age 65 to 80 years) with prostatic carcinoma who had received no previous hormone or drug treatments and had no testicular, endocrine, or related disorders. In addition, testicular biopsy specimens were obtained from 10 young adult men (age 28 to 34 years) consulting for infertility, and another 32 testes obtained from testicular diseases surgery were fixed and processed in the same way. These were diagnosed as normal testes (n ⫽ 7), cryptorchid testes (n ⫽ 5), testicular torsion (n ⫽ 2), infertile testes with lesions in the adluminal compartment of seminiferous tubules (n ⫽ 5), intratubular germ cell neoplasia (n ⫽ 4), seminoma (n ⫽ 3), spermatocytic seminoma (n ⫽ 2), and teratoma (n ⫽ 4). Each sample was longitudinally sectioned in two equal portions; one portion was immediately processed for immunohistochemistry, and the other portion was frozen in liquid nitrogen and maintained at ⫺80°C for Western blotting analysis. The primary antibodies used were rabbit polyclonal antibody 1336, reactive with human CLA-1 (courtesy of M. A. Vega, CSIC, Madrid, Spain); rabbit polyclonal antibody to SR-BI/SR-BII (Novus Biologicals, Littleton, CO); goat polyclonal antibody to CD36 (Santa Cruz Biotechnologies, Santa Cruz, CA); and chicken polyclonal antibody to HSL (anti-rat adipocyte HSL; courtesy of C. Holm, Department of Cell and Molecular Biology, Lund University, Sweden).
Immunohistochemistry For light microscopy immunohistochemistry, the specimens were fixed for 24 hours at room temperature in either 0.1 mol phosphate-buffered 10% formaldehyde or Bouin’s solution, dehydrated, and embedded in paraffin. Sections (5 m thick) were processed using the avidin-biotin-peroxidase complex method. After deparaffinization, sections were hydrated and incubated for 30 minutes in 0.3% H2O2 diluted in methanol, to reduce endogenous peroxidase activity. Then, to retrieve the antigen, the sections were incubated with 0.1 mol citrate buffer, pH 6, for 5 minutes in a conventional pressure cooker. After rinsing in Tris-buffered saline (TBS), the slides were incubated with normal donkey serum at a 1:5 dilution in TBS for 30 minutes, to prevent nonspecific binding of the first antibody. Thereafter, the primary antibodies were applied at a dilution of 1:1000 for HSL, 1:200 for CD36, and 1:300 for CLA-1/SR-BI, in TBS/normal donkey serum at 4°C overnight. The sections were then washed twice in TBS and incubated with goat anti-chicken, rabbit anti-goat, and swine anti-rabbit biotinylated immunoglobulins (all from Dako, Carpinteria, CA) at 1:500 dilutions. After 1 hour of incubation with the secondary antibody, the sections were incubated with a standard streptavidin-biotin complex (Dako) and developed with 3,3'-diaminobenzidine, using the glucose oxidase-diaminobenzidine-nickel intensification method.
Western Blotting For Western blotting analysis, each sample was homogenized in 0.5 mol Tris-HCl buffer, pH 7.4, containing 1 mmol EDTA, 12 mmol 2-mercaptoethanol, 1 mmol benzamidine,
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and 1 mmol phenylmethylsulphonyl fluoride, with the addition of a cocktail of protease inhibitors (10 mmol of iodoacetamide, 0.01 mg/mL of soybean trypsin inhibitor, and 1 l/mL of leupeptin) and phosphatase inhibitors (10 mmol sodium fluoride and 1 mmol sodium orthovanadate) in the presence of 0.5% Triton X-100. Homogenates were centrifuged for 10 minutes at 10,000 rpm. After boiling for 2 minutes at 98°C, aliquots of 20 g of protein were separated in sodium dodecyl sulfate-polyacrylamide (9% w/v) slab minigels. Separated proteins were transferred for 4 hours at 0.25 A to nitrocellulose membranes (0.2 m), and the nitrocellulose sheets were then blocked for 1 hour with 5% blotto in 0.05 mol Tris-HCl and incubated overnight with the primary antibodies, diluted at 1:10000 (HSL), 1:1000 (CD36), and 1:3000 (CLA-1/SR-BI) in TBS. After extensive washing with TBS/Tween 20, the membranes were incubated with a horseradish peroxidase–labeled secondary antibody diluted in TBS (goat anti-chicken for HSL, 1:10,000 dilution; rabbit anti-goat for CD36, 1:4000 dilution; and swine anti-rabbit for CLA-1/ SR-BI, 1:4000 dilution; all from Chemicon, Temecula, CA) for 1 hour. After an intensive wash, the filters were developed with an enhanced chemiluminescence kit, following the procedure specified by the manufacturer (Amersham, Buckinghamshire, UK).
FIGURE 1. An intense signal for streptavidin-Cy3 can be observed in both Leydig cell (L) and germ cell cytoplasm. ST, seminiferous tubule. Bar ⫽ 25 m.
showed an intense surface reaction to CLA-1 and SR-BI (Fig 2A)and HSL labeling in the cytoplasm. In the seminiferous tubules, the basal cytoplasm of Sertoli cells and residual bodies immunoreacted intensely to CLA-1. A diffuse and weak reaction to both SR-BI and HSL was observed in the Sertoli cell cytoplasm. Spermatogonia demonstrated a negative immunoreaction to the lipid receptors and a weakly positive reaction to HSL. The Golgi region of spermatocytes was labeled with HSL only. Round spermatids demonstrated a positive reaction to CLA-1 and SR-BI in the acrosomic vesicle, whereas the nuclei exhibited HSL labeling (Fig 2B). CD36 was unreactive. In testes from aging men, the labeling pattern for CLA-1, SR-BI, and HSL was the same as in young men, except for those areas containing Sertoli cell– only tubules, in which Sertoli cell immunoreaction to HSL was more intense than in normal young testes. In the Leydig cells adjacent to Sertoli cell– only tubules, labeling was lower than in the Leydig cells of zones with complete spermatogenesis. Elderly testes demonstrated a patchy positive reaction to CD36 in the Sertoli cell surfaces (Fig 2C). Testes with hypospermatogenesis associated with primary spermatocyte sloughing exhibited the same reactivity to CLA-1 and SR-BI as normal testes. CD36 was positive in the Sertoli cell surfaces and in the cytoplasm of Leydig cells underneath the plasma membrane (Fig 2D). Spermatogonia cytoplasm reacted to HSL (Fig 2E). Testes with Leydig cell hyperplasia exhibited intense labeling to HSL around the nucleus. Cryptorchid testes demonstrated atrophic seminiferous tubules lined by a single layer of Sertoli cells. A strong immunoreaction to HSL in the cytoplasm and, surprisingly, in the nucleus of these cells was observed (Fig 2F). In areas with nodules of hyperplastic Leydig cells in cryptorchid testes, an intense HSL reaction was observed around the nucleus of these cells. A weak reaction to SR-BI was observed in the surface of both Sertoli and Leydig cells. CLA-1 and CD36 did not react. In testes with intratubular germ cell neoplasia of
Controls The specificity of the immunohistochemical procedures was checked using negative and positive control sections. Negative controls were created by either omitting the primary antibody or using the antibody preabsorbed with an excess of purified antigens. For HSL positive controls, sections of human white adipose tissue were incubated with the same antibody. For CD36, CLA-1, and SR-BI antibodies, monocytes present in testicular samples were used as internal controls. To assess the status of Leydig cell function, the presence of endogenous biotin and biotin-dependent enzymes, which are regulatory enzymes in lipogenesis and gluconeogenesis, was evaluated. Biotin was detected using streptavidin conjugated with Cy3 (Sigma-Aldrich, Madrid, Spain). Briefly, after incubation with streptavidin-Cy3 at 1:300 dilution, sections were stained with 4',6-diamidino-2-phenylindole and mounted with Mowiol. Microscopic analyses were done using an epifluorescence microscope (Axiophot; Carl Zeiss, Oberkochen, Germany), and photographs were taken with Fuji Super G400 ASA color film (Fujifilm, Tokyo, Japan).
RESULTS Controls Negative controls incubated with the preimmune serum or with the antibody preabsorbed with an excess of purified antigens demonstrated no labeling. Immunostaining of white adipose tissue sections were always positive for HSL. Monocytes present in human testicular interstitium exhibited a positive reaction to CD36 and CLA-1/SR-BI. On fluorescence microscopy, a strong signal in the Leydig cell cytoplasm and in the germ cell cytoplasm was found in all samples studied (Fig 1). Immunohistochemistry Study Normal young testes with complete spermatogenesis immunoreacted as follows (Table 1). Leydig cells 36
LIPASE AND LIPID RECEPTORS IN TESTES (Arenas et al)
TABLE 1. Immunoreaction Pattern to Lipid Receptors and HSL in Normal Young Testes
CLA-1 SR-BI HSL CD36
Spermatogonia
Spermatocytes
Spermatids
Sertoli cells
Leydig Cells
— — Cytoplasm (weak) —
— — Golgi region —
Acrosomic vesicle Acrosomic vesicle Nucleus —
Basal cytoplasm Cytoplasm (weak) Cytoplasm (weak) —
Surface Surface Cytoplasm —
the unclassified type, the affected tubules consisted of a population of malignant germ cells and Sertoli cells; the latter appeared displaced luminally. Both cell types demonstrated immunostaining to lipid receptors in the surface and focal areas of the cytoplasm (Fig 2G) and to HSL in the nucleus (Fig 2H). Leydig cells were immunolabeled with the 4 antibodies, with reactions similar to those observed in normal testes. Neoplastic cells present in seminoma and spermatocytic seminoma were immunopositive to HSL (Fig 1I), but they failed to stain with lipid receptors (Fig 2J). In these tumor types, the seminiferous tubules at the periphery of the tumor demonstrated only Sertoli cells with vacuolated cytoplasm, absence of lumen, and normal lamina propria. The Sertoli cells of these tubules labeled intensely to HSL. Lipid receptors were discretely immunostained in the cytoplasm of these cells, and an intense reaction to CLA-1 was seen in CharcotBo¨ ttcher crystalloids (Fig 2K). The Leydig cells present in these tubules immunostained to HSL in the cytoplasm and to lipid receptors on their surface. The mature teratomas studied here consisted of various somatic-type tissues, including enteric-type glands and cartilage. Some cells of enteric-type glands exhibited a positive reaction to CD36 and SR-BI (Fig 2L), but were unstained with CLA-1 and HSL. In immature teratomas, immature tissues, such as neuroepithelium, were immunolabeled exclusively with HSL.
tected in spermatocytic seminoma (Fig 3B, lane 4) and intratubular germ cell neoplasia (Fig 3B, lane 5) of the unclassified type, with an additional band of 70 kDa observed in the former. Multiple CLA-1 immunoreactive bands between 60 and 70 kDa and between 130 and 240 kDa were observed in normal young testes (Fig 3C, lane 2). In cryptorchid testes, the reactivity of higher molecular weight bands was lower (Fig 3C, lane 1). This pattern was similar in elderly testes (Fig 3C, lane 3). In spermatocytic seminoma and intratubular germ cell neoplasia, the high molecular weight bands were absent (Fig 3C, lanes 4 and 5). The immunoreactive band pattern for SR-BI (Fig 3D) was similar to that observed with the CLA-1 antibody, but the 60-kDa band was more intense with the SR-BI antibody. An immunoreactive band of 85 kDa was detected only in elderly testes and intratubular germ cell neoplasias when SR-BI antibody was used (Fig 3D, lanes 3 and 5). DISCUSSION In this study we have demonstrated the presence of lipid receptors (CLA-1, SR-BI, and CD36) and HSL in human testes, observing variations in this expression within different pathologies. The presence of CD36 in human testes has not been previously reported. We observed a positive reaction to CD36 in the surface of both Sertoli cells and Leydig cells in aging and pathological testes. This staining might be related either to increased phagocytic activity of the Sertoli cells or to accumulation of lipids in the cytoplasm of both cell types. Several pathological conditions are known to lead to lipid droplet accumulation, which is related to impaired lipid metabolism and steroidogenesis in Leydig cells or to arrested spermatogenesis in Sertoli cells.30 By Western blotting analysis, we detected 4 immunoreactive bands (at 55, 75, 130, and 150 kDa) to CD36 antibody, along with an additional band of 33 kDa in elderly testes. The CD36 cDNA predicts a polypeptide of 53 kDa with 10 potential N-linked glycosylation sites.31 For some authors, the mature protein has a molecular weight of 94 kDa and the CD36 precursor 74 kDa.31 Nevertheless, in platelets, Rhinehart-Jones and Greenwalt32 detected a 113-kDa isoform. In U937 cells, CD36 coprecipitated with two proteins of 33 and 130 kDa;31 these bands were interpreted as a protein associated with the 74-kDa CD36 precursor and as an artifact of the immunoprecipitation method, respectively. In accordance with the findings reported by others,33
Western Blotting Analysis The results of Western blotting analysis are shown in Figure 3. In all samples analyzed, HSL antibody stained multiple protein bands at different molecular weights. In normal human testes in young and aging men (Fig 3A, lanes 2 and 3), HSL-immunoreactive proteins were observed at 60, 84, 102, 110, and 120 kDa. An additional band at 28 kDa was observed in normal young testes. A similar pattern was found in cryptorchid testes (Fig 3A, lane 1). Testes with seminoma and spermatocytic seminoma exhibited immunoreactive bands at 28, 32, 110, and 130 kDa (Fig 3A, lane 4). Testes with intratubular germ cell neoplasia of the unclassified type demonstrated a band only at 60 kDa (Fig 3A, lane 5). In normal young and elderly testes, CD36 immunoreactive bands between 55 and 75 kDa were observed, along with 2 bands of 130 and 150 kDa (Fig 3B, lane 2) and in elderly testes an additional 33-kDa band was observed (Fig 3B, lane 3). The same pattern was observed in cryptorchid testes, but the reactivity of the higher molecular weight bands was lower (Fig 3B, lane 1). Four bands of 55, 74, 130, and 150 kDa were de37
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LIPASE AND LIPID RECEPTORS IN TESTES (Arenas et al)
lated forms of CLA-1, mainly the 70-kDa form. The two antibodies that we used detected a protein of 70 kDa, but was preferentially bound by CLA-1. Landschulz et al37 found CLA-1/SR-BI expression at high levels in both the adrenal gland and liver of humans; it seems likely that one important function of this is to supply substrate for steroidogenesis.5 Reaven et al39 observed that under normal physiological conditions, rat testes do not require exogenous lipoprotein cholesterol for Leydig cell testosterone production; therefore, SR-BI receptor expression is low or absent. However, in rats treated with human chorionic gonadotropin, the expression of this receptor is increased, indicating that the hormone-treated cells take up HDLderived cholesterol esters to synthesize testosterone. In our study we observed SR-BI expression in Leydig cells from most samples, except in those cases where the Leydig cell population was altered or suppressed (seminomas and teratomas). This immunostaining suggests an active cholesterol ester flux for testosterone production. SR-BI expression is coordinately regulated with cholesterol metabolism,40 and its expression in steroidogenic cells in vivo and in vitro is regulated by trophic hormones.41 It has been demonstrated that in Sertoli cells from rat testis, SR-BI is a phosphatidylserine receptor responsible for the phagocytosis of apoptotic spermatogenetic cells,11 and that the phagocytic activity of Sertoli cells varies at different spermatogenetic stages. We have observed a weak reaction to SR-BI in the Sertoli cell cytoplasm of normal and hypospermatogenetic testes; however, the reaction to this antibody was more intense in Sertoli cells from cryptorchid testes and in intratubular germ cell neoplasia. Therefore, SR-BI expression increases in those disorders in which germ cells are absent or have a malignant phenotype. In contrast with rodents, in humans our data indicate that CLA-1/SR-BI cannot be related to the phagocytic activity of normal human Sertoli cells. Charcot-Bo¨ ttcher crystalloids are usually present in the Sertoli cell cytoplasm. Their origin and chemical composition are unknown, although Hadley and Dym42 suggested that they might contain heparin sulfate. These crystalloids are increased in cases of impaired spermatogenesis.43 No previous studies have reported immunostaining for any known protein within these crystalloids. We detected an accumulation of CLA-1 immunoreactived proteins in Charcot-Bo¨ ttcher crystal-
our results support the idea that, depending on cell type, CD36 displays different molecular masses corresponding to different glycoforms. However, the significance of the 33-kDa band is not clear, because this band was observed only in samples from elderly men; it could be interpreted as the result of intracellular CD36 processing. Recently, a variant of SR-BI (termed SR-BII) has been found in mice and humans. The SR-BII transcript is relatively abundant in tissues known to express SR-BI and represents approximately 30% of total mRNA SR-BI values in mouse adrenal glands and about 80% of total SR-BI values in mouse testes.34,35 However, whether SR-BII is present in human testes remains unknown. In our study, we used the commercial antibody reactive to SR-BI/II that recognized different bands with a wide range of 60 to 160 kDa; however, we observed that the predominant forms of SR-BI/BII in the human testes are low molecular weight proteins. These multiple bands might be the result of differently glycosylated fractions; Babitt et al36 reported a huge heterogeneity in the structures of the N-linked chains on SR-BI protein, similar to that encountered on CD36.33 Landschulz et al,37 using a rabbit anti-murine SR-BI polyclonal antibody, described a protein with an apparent molecular mass of 82 kDa in murine tissues, along with a larger band of approximately twice this mass (164 kDa) in both the adrenal gland and ovary and a faint protein of 129 kDa visible in the heart. In LDL receptor-deficient CHO cell mutants, Babitt et al36 described the following forms of SR-BI: a 82-kDa, Nglycosylated mature form, a 54-kDa immature form, and a 64-kDa intermediate form. Cao et al,38 using an antibody that recognizes only several proteins generated from SR-BI.1, detected a prominent band of 80 kDa in human tissues and an additional band of 50 kDa in the prostate. In addition, Shiratsuchi et al,11 using an antibody that recognizes amino acid residues 110 to 132 of hamster SR-BI, observed an immunoreactive band for SR-BI of 70 kDa in Sertoli cell cultures from rats. Because the human long form of CLA-1 shares 81% sequence identity with hamster SR-BI,1 we used an antibody reactive to human CLA-1. CLA-1 has a predicted molecular weight of 56 kDa with 10 potential N-glycosylation sites originating an 80-kDa mature form.3 In human testes, the 1336 antibody that we used preferentially recognizes the immature, low-glycosy-
4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ FIGURE 2. (A) Normal young testis showing intense immunolabeling to SR-BI on the surface of Leydig cells (L) and in the acrosomic vesicles of round spermatids (Sp). The cytoplasm of Sertoli cells were weakly immunoreactive (S). (B) A strong reaction to HSL antibody can be observed in the nuclei of spermatids (Sp). The cytoplasm of Sertoli cells (S) shows weak staining. (C) Seminiferous tubules from elderly testes showing a patchy reaction to CD36 in the Sertoli cells. (D) Germ and Leydig cells from testes with primary spermatocyte sloughing exhibiting a strong reaction to CD36. Sg, spermatogonium. (E) Testes with primary spermatocytes sloughing were more strongly immunoreactive to HSL than normal testes; compare with (B). (F) Cryptorchid testis showed a strong immunoreaction to HSL in both the cytoplasm and the nucleus of Sertoli cells. Leydig cells were also stained. (G) Focal staining to CD36 observed in the malignant germ cell cytoplasm in intratubular germ cell neoplasia of the unclassified type. (H) Intense labeling to HSL antibody in Leydig cells (L) and in both the nucleus and cytoplasm of neoplastic cells in intratubular germ cell neoplasia of the unclassified type. (I) Neoplastic cells of spermatocytic seminoma demonstrating intense immunoreaction to HSL. (J) Negative staining to SR-BI in neoplastic cells of seminoma. (K) Seminiferous tubules at the periphery of spermatocytic seminoma. Charcot-Bo ¨ ttcher crystalloids are intensely labeled with CLA-1 antibody (arrowheads). (L) Some cells of enteric-type of mature teratoma exhibiting a positive reaction to SR-BI. Bar ⫽ 50 m.
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FIGURE 3. Immunoblots of HSL and scavenger receptors in normal and pathological human testes. Lane 1, cryptorchid testis; lane 2, normal young testis; lane 3, elderly testis; lane 4, spermatocytic seminoma; lane 5, intratubular germ cell neoplasia of unclassified type. Molecular masses are represented on the left. Each blot is representative of its respective group.
loids, supporting the notion of a dysfunction in the lipid transport of the Sertoli cells in a tumor environment. This dysfunction might be one of the changes occurring in the infertility associated with germ cell tumors. We have observed intense immunoexpression of CLA-1 in the Sertoli cells and malignant germ cells of seminiferous tubules with intratubular germ cell neo-
plasia; this expression was lost when malignant cells have an invasive phenotype. A number of studies have reported that cancer cells and solid tumors take up LDL more effectively than normal tissues,44 suggesting a higher cholesterol demand in dividing cells. An interesting, and not previously reported, finding is the positive reaction to CLA-1/SR-BI observed in the acrosomic vesicle of spermatids. These receptors 40
LIPASE AND LIPID RECEPTORS IN TESTES (Arenas et al)
testes. The presence of HSL in human Leydig cells suggests a direct relationship with steroid hormone production. In summary, the presence of lipid receptors (CLA1/SR-BI) and HSL in Leydig cells of normal and pathological human testes suggests that these proteins play a key role in steroidogenesis through the supply of free cholesterol for testosterone synthesis. These proteins are also involved in spermiogenesis, as their labeling in spermatids suggests. In nonmalignant and malignant pathologies, cholesterol metabolism is probably altered, and HSL labeling in the nucleus of neoplastic germ cells suggests a still-unknown function of this enzyme, probably related to cell cycle regulation.
likely are implicated either in transport of the lipids needed for the formation of acrosomal membranes or in a generalized redistribution of membrane cholesterol in the acrosome formation. It has been reported that SR-BI expression accelerates the bidirectional flux of free cholesterol and induces the formation of novel membrane structures45,46 and that CLA-1 plays a role in selective uptake of cholesterol esters from HDL.47 Durham and Grogan25 reported two types of cholesterol ester hydrolases in the testis: temperature-stable and temperature-labile. HSL may be related to temperature-labile cholesteryl ester hydrolase, because both enzymes are more active at low temperature and their activity increases with sexual maturity. Mairal et al47 characterized two different HSL mRNAs of 3.3 and 3.9 kb present in equal amounts in the human testis, a short-form HSL expressed in spermatogonia, pachytene spermatocytes, Sertoli cells, Leydig cells, and peritubular cells and a long-form HSL transcribed exclusively in haploid germ cells. Human adipose tissue expresses a 2.8-kb mRNA that encodes a 88-kDa protein. The mRNA and protein species expressed in the testis are larger, 3.9 kb and 120 kDa, respectively.48 In mouse, Blaise et al49 identified an HSL testis-specific promoter that contains 4 regions binding testicular nuclear proteins. One of these regions binds Sox proteins expressed in postmeiotic germ cells. Our results support that HSL present different testicular isoforms. By Western blotting analysis, we obtained different bands with apparent molecular masses of aproximately 28, 60, 84, 102, 110, and 130 kDa. Different HSLimmunoreactive bands have been reported in mammalian testes; for example, in guinea pig testes, Kabbaj et al50 detected bands of 104, 110, and 120 kDa in seminiferous tubules and only a band of 104 kDa in the interstitial tissue. In rat testis, Kraemer et al51 observed HSL-like immunoreactive proteins of 84, 89, and 102 kDa in sexually immature rats and additional 113- and 127-kDa immunoreactive proteins in mature animals. This pattern expression suggests that several tissue-specific promoters control the different forms of HSL, because when a gene is expressed in somatic tissues and in germ cells, tissue-specific expression often results from alternate promoter use.52 The significance of the nuclear immunostaining observed by light microscopy is unknown, although HSL might hydrolyze retinyl esters;16 recently, metabolism of cholesteryl esters was detected in the cell nucleus of hepatocytes.53 Because the main targets of retinoic acid derivatives are located in the cell nucleus, HSL might be related to the nuclear turnover of this derivative and/or to the nuclear metabolism of lipids. HSL is essential for spermatogenesis; HSL⫺/⫺ mice are infertile, have many epithelial vacuolated cells in the seminiferous tubules, and demonstrate increased amounts of cholesterol esters in their testes.24 Moreover, HSL may be essential for membrane stabilization and integrity in the seminiferous epithelium54 and also may be involved in the metabolism of lipid droplets in Sertoli cells,23 as suggested by the intense reaction to HSL observed in tubules seminiferous of pathological
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