11 Immunoelectron microscopic localization of 3-hydroxysteroid and type 5 17-hydroxysteroid dehydrogenase in the human prostate and mammary gland

G Pelletier, V Luu-The, M El-Alfy, S Li and F Labrie Oncology and Molecular Endocrinology Research Center, Laval University Hospital (CHUL), Quebec, G1V 4G2, Canada (Requests for offprints should be addressed to G Pelletier, Oncology and Molecular Endocrinology Research Center, Laval University Hospital (CHUL), 2705, Laurier Boulevard, Quebec, Quebec, G1V 4G2, Canada; Email: [email protected])

ABSTRACT The subcellular distribution of steroidogenic organelle could be observed, although some concen- has so far been studied mostly in classical tration of gold particles was occasionally found over endocrine glands and in the placenta. In the bundles of microfilaments. In mammary gland peripheral intracrine organs which synthesize sex sections immunolabelled for 3-HSD or type 5 there is no indication about the organelles 17-HSD localization, labelling was observed in the which contain the enzymes involved in cytoplasm of the secretory epithelial cells in both biosynthesis. We have thus investigated the sub- the acini and terminal ducts. Immunolabelling was cellular localization of two enzymes involved in also found in the endothelial cells as well as in the production of sex steroids, namely 3- fibroblasts in stroma and blood vessels. The gold hydroxysteroid dehydrogenase (3-HSD) and type particles were not detected over any organelles, 517-hydroxysteroid dehydrogenase (17-HSD). except with the occasional accumulation of gold Using specific antibodies to these enzymes, we particles over microfilaments. The present data on conducted immunoelectron microscopic studies in the localization of two steroidogenic enzymes two peripheral tissues, namely the human prostate leading to the synthesis of testosterone indicate that and mammary gland. In the prostate, immunolabel- these enzymes are located not only in epithelial cells ling for both 3-HSD and type 5 17-HSD was but also in stromal and endothelial cells in both detected in the basal cells of the tube-alveoli as well tissues studied. The absence of any association of as in fibroblasts and endothelial cells lining the the enzymes with membrane-bound organelles blood vessels. In all the labelled cell types, the gold appears as a common finding in the reactive cell particles were distributed throughout the cyto- types of two peripheral tissues. plasm. No obvious association with any specific Journal of Molecular Endocrinology (2001) 26, 11–19

INTRODUCTION et al. 1991). In addition to the gonadal tissues, 3-HSD activity has been demonstrated in a variety The 3-hydroxysteroid dehydrogenase of peripheral intracrine tissues, including the (3-HSD) type 1 which catalyses the conversion prostate and mammary gland (Lachance et al. of 5–3-hydroxysteroid precursors into 4- 1990). At the light microscopic level, immuno- ketosteroids in all steroidogenic tissues has been reactive type 1 3-HSD has been detected in the purified from human placenta microsomes and testis, ovary and adrenal cortex (Pelletier et al. 1992) mitochondria (Luu-The et al. 1989b, 1990). and in the prostate (El-Alfy et al. 1999). In bovine Molecular cloning studies then revealed two types and rat adrenal cortex, 3-HSD activity has been of human 3-HSD (types 1 and 2) (Luu-The et al. shown to be associated with both the mitochondrial 1989b, 1990, Lachance et al. 1990, 1991, Rhéaume and microsomal fractions (Cherradi et al. 1993,

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1994, Sauer et al. 1994). By immunoelectron reproductive tissues, namely the prostate and microscopy, 3-HSD immunoreactivity was found mammary gland. As a positive control, the to be associated only with the smooth endoplasmic localization of 3-HSD was also studied in the reticulum (Ishimura et al. 1988) in bovine adrenal interstitial Leydig cells of the human testis. cortex. The enzyme 17-hydroxysteroid dehydrogenase (17-HSD) controls the last step in the formation of all androgens and oestrogens. So far, six types of MATERIALS AND METHODS 17-HSD have been characterized in the human (Jarabak et al. 1962, Peltoketo et al. 1988, Luu-The Tissue preparation et al. 1989a,Wuet al. 1993, Geissler et al. 1994, The mammary gland tissue specimens were Adamski et al. 1995, Lin et al. 1997, Nokelainen et obtained from pre-menopausal patients (25–40 al. 1998, Dufort et al. 1999, Krazeisen et al. 1999). years of age) who had breast reduction, while the Type 1 17-HSD is a soluble protein originally prostatic specimens were obtained from patients purified from human placenta (Jarabak et al. 1962), (55–65 years of age) with benign prostatic hyper- while types 2 and 3 17-HSD are microsomal plasia undergoing transurethral prostatectomy. enzymes cloned from human prostate and testis Testes were obtained from patients (50–60 years of cDNA libraries respectively (Wu et al. 1993, age) who were castrated for prostate cancer Geissler et al. 1994). Type 4 17-HSD (Adamski treatment. et al. 1995) is a peroxisomal enzyme expressed in The specimens were fixed in 3% paraformalde- virtually all human tissues. The recently charac- hyde and 1% glutaraldehyde in 0·2 M phosphate terized human type 5 17-HSD (Lin et al. 1997, buffer (pH 7·4) within 15 min after they had been Dufort et al. 1999) is a soluble enzyme catalysing dissected out. After 12 h, the tissues were post-fixed the reduction of 4-dione to testosterone in periph- in 0·2or0·5% osmium tetroxide, embedded in eral intracrine tissues. This enzyme plays a role in Araldite and cut at 0·1 µm with an ultramicrotome. the formation of active androgens in periph- For each organ under study, five separate tissue eral tissues. Type 7 17-HSD first cloned in a rat specimens were studied and the results were shown corpus luteum catalyses the transformation of to be consistent. oestrone (OE1) to oestradiol (OE2) (Nokelainen et al. 1998). Human type 7 17-HSD is a 37 kDa protein which has been found in the ovary, breast, placenta, testis, prostate and (Krazeisen et al. Immunocytochemistry 1999). The ultrathin sections were immunostained using Most of the data on the subcellular localization the protein A–gold complex (10 nm; British Biocell of steroidogenic enzymes have been obtained International, Cardiff, UK), as described (Roth following subcellular fractionation studies (Pudney et al. 1978). The antiserum to 3-HSD and type 5 et al. 1985, Haniu et al. 1987, Luu-The et al. 17-HSD were both used at a 1:500 dilution. The 1990, Cherradi et al. 1993, 1994, Sauer et al. 3-HSD antiserum used was raised by immunizing 1994). In fact, the ultrastructural localization of rabbits with purified human placental type 1 steroidogenic enzymes obtained by immuno- 3-HSD (Luu-The et al. 1989b, 1990). This electron microscopy has not so far been extensively antiserum has been widely used to localize 3-HSD investigated and the few reports on the subject are in several species, including the human (Pelletier related to classical steroidogenic glands such as the et al. 1992). The antiserum to type 5 17-HSD was testis, ovary and adrenal cortex (Ishimura et al. raised by immunizing rabbits against a peptide 1988, Shinzawa et al. 1988, Whitnall et al. 1993). sequence located at positions 297 to 320 We have recently developed antibodies against of the human type 5 17-HSD. The characteristics type 5 17-HSD and shown by immunocyto- of the antiserum have been reported recently chemistry that this enzyme could be detected in (El-Alfy et al. 1999, Pelletier et al. 1999). This reproductive organs, including the prostate, uterus antiserum has been successfully used for immuno- and mammary gland (El-Alfy et al. 1999, Pelletier cytochemical localization in several human tissues, et al. 1999). So far, the ultrastructural localization including the testis, prostate, breast, ovary and of type 5 17-HSD has not been reported. The uterus (El-Alfy et al. 1999, Pelletier et al. 1999). purpose of the present study was to investigate the Control experiments were performed by substi- electron microscopic localization of two enzymes tuting non-immunized rabbit serum (1:500) or involved in sex steroid biosynthesis, 3-HSD and the antiserum (1:500) absorbed with an excess  type 5 17-HSD, in two intracrine human (10 6 M) of their respective antigen.

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 1. Part of an epithelial secretory cell of a mammary gland stained for 3-HSD localization. Immunogold labelling (<) can be detected throughout the cytoplasm. Some gold particles are located over microfilament (MF) bundles. M: mitochondria; MV: microvilli projecting to the acinar lumen.64 000.

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 2. Part of a perivascular fibroblast of a mammary gland stained for type 5 17-HSD. Several gold particles are distributed throughout the cytoplasm. Some particles are located over microfilament (MF) bundles. M: mitochondria; V: endocytotic vesicles; N: nucleus.64 000.

RESULTS epithelial cells, the association of gold particles with bundles of microfilaments was occasionally Mammary gland observed in the endothelial cells and fibroblasts. The normal human mammary gland consists of When the sections were immunolabelled for type 5 ducts, acini and surrounding connective tissue. The 17-HSD, similar results were obtained, no label- epithelium lining the acini and ducts is composed of ling being associated with specific organelles in the two layers: an inner columnal or cuboidal glandular three labelled cell types (Fig. 2). epithelial layer and an outer discontinuous layer of myoepithelial cells. The myoepithelial cells are Prostate surrounded by a basement membrane. The epi- thelial cells contain free ribosomes, cisternae of The stratified epithelium lining the tube-alveoli is rough endoplasmic reticulum as well as Golgi divided into two layers: the basal layer made of low complexes which are not associated with secretory cuboidal cells which are separated from the stroma vesicles when the gland is resting. The epithelial by a basement membrane and the layer of columnar cells show luminal microvilli. The contractile secretory cells (luminal cells). The secretory myoepithelial cells are characterized by abundant cells possess luminal microvilli, well developed plasmalemmal vesicles and bundles of actin micro- rough endoplasmic reticulum and numerous large filaments. The cells also contain poorly developed secretory granules. The basal cells are devoid of any cisternae of rough and smooth endoplasmic secretory granules. reticulum and small Golgi complexes. In the tubulo-alveoli of human prostate speci- In sections immunolabelled with 3-HSD anti- mens, immunolabelling for type 3-HSD was bodies, the gold particles were localized throughout almost exclusively observed in the basal cells, as the cytoplasm of the epithelial cells in both the acini recently found at the light microscopic level and terminal ducts. No organelles appeared to be (El-Alfy et al. 1999, Pelletier et al. 1999). specifically labelled (Fig. 1). A few gold particles Immunolabelling was rather diffuse and did not were occasionally observed over bundles of micro- appear to be restricted to specific organelles (Fig. 3). filaments. No significant labelling could be detected As in the mammary gland, immunolabelling was over the myoepithelial cells. As previously observed also detected in the endothelial cells and fibroblasts at the light microscopic level (Pelletier et al. 1999), of blood vessels as well as in the fibroblasts of immunolabelling was found in the endothelial cells stroma. The intracellular distribution of gold and fibroblasts lining blood vessels as well as in particles was rather diffuse throughout the cyto- stromal fibroblasts. In these two cell types, plasm. No obvious association with organelles was immunolabelling did not generally appear to be detected, except those bundles of microfilaments associated with any specific organelle. As in which occasionally appeared to be more labelled

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 3. Part of a basal cell of the prostate epithelium  4. Prostate section stained for type 5 17-HSD stained for 3-HSD localization. Numerous gold particles localization. Part of an endothelial cell of a capillary. are overlying the cytoplasm. No association of gold Gold particles are distributed throughout the particles with organelles is observed. M: mitochondria; cytoplasm. V: endocytotic vesicles; MF: microfilaments; N: nucleus; BM: basement membrane.64 000. BM: basement membrane; L: capillary lumen.64 000.

that the other cellular components. Very similar DISCUSSION results were obtained with the anti-type 5 17-HSD (Fig. 4). In the prostate, the ultrastructural localization of In the testis, as previously observed by light 3-HSD and type 5 17-HSD immunoreactivity in microscope studies (Pelletier et al. 1992), immuno- basal cells of tube-alveoli, endothelial cells and labelling for the membrane-bound 3-HSD was fibroblasts confirm recent studies conducted at the restricted to the interstitial Leydig cells. The gold light microscopic level (El-Alfy et al. 1999, Pelletier particles were predominantly detected over the et al. 1999). Since type 5 17-HSD and 3-HSD are numerous mitochondria and to a lesser extent over both expressed in basal cells, it may be suggested cisternae of the smooth endoplasmic reticulum (data that testosterone synthesized in basal cells from not shown). When the antisera were immuno- circulating dehydroepiandrosterone (DHEA) reaches absorbed with their respective antigens, no associ- the luminal cells to be ultimately transformed into ation of gold particles with any cell type could be dihydrotestosterone (DHT) in the luminal cells by observed. Only very few dispersed gold particles the action of 5-reductase (Levine et al. 1996, could be detected throughout the sections (Figs 5 Pelletier et al. 1998). DHT would then exert its and 6). androgenic action in the luminal cells which contain www.endocrinology.org Journal of Molecular Endocrinology (2001) 26, 11–19

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 5. Control mammary gland section. Part of an epithelial secretory cell. Immunoabsorption of the antiserum to type 5 17-HSD with the antigen has completely prevented any labelling. Only a very few dispersed gold particles can be detected.64 000.

androgen receptors (Ruizeveld de Winter et al. prostate, the enzyme was also detected in stromal 1991, El-Alfy et al. 1999). fibroblasts and in endothelial cells and fibroblasts of In the mammary gland, the present data blood vessels. The present results also confirm demonstrate for the very first time the localization previous data obtained at the light microscopic level of 3-HSD in the epithelial secretory cells in acini indicating the presence of immunoreactive type 5 as well as in intralobular ducts. As observed in the 17-HSD in the mammary cell types (Pelletier et al.

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 6. Control prostate section. Part of an endothelial cell of a capillary. Immunoabsorption of the antiserum to 3-HSD with the purified human 3-HSD has completely prevented any staining. Only a very few gold particles can be seen. BM: basement membrane; L: capillary lumen.64 000.

1999) which also contain 3-HSD. It can therefore In steroid-secreting glands and in the placenta, be suggested that these enzymes in the mammary 3-HSD activity has been found to be associated gland are involved in testosterone biosynthesis with microsomal and mitochondrial fractions following transformation of circulating DHEA into (Luu-The et al. 1989b, 1990, Rhéaume et al. 1991, androstenedione. These cell types have also been Cherradi et al. 1993, 1994, Sauer et al. 1994). At shown to contain androgen receptors (Ruizeveld de the electron microscopic level, association of the Winter et al. 1991, Kimura et al. 1993). Therefore, enzyme with the smooth endoplasmic reticulum in it may be suggested that the intracellularly the adrenal cortical cells has been confirmed synthesized androgens directly influence the activity (Ishimura et al. 1988). In peripheral organs where of the same cells (intracrine activity) (Labrie et al. there is no typical steroid-secreting cell, the 1988). subcellular distribution of 3-HSD has not so far The subcellular distribution of different been reported. It is noteworthy that immuno- steroidogenic enzymes has been almost exclusively labelling for 3-HSD was not associated with any studied by subcellular fractionation experiments organelles in the human prostate and mammary performed in steroid-secreting glands and placenta gland. The significance of the association of gold (Pudney et al. 1985, Haniu et al. 1987, Luu-The particles with microfilaments in many of the et al. 1989b, 1990, Rhéaume et al. 1991, Cherradi examined cells of the prostate and mammary gland et al. 1993, 1994, Sauer et al. 1994). So far, the remains to be clarified. subcellular localization of steroidogenic enzymes Type 5 17-HSD is a soluble enzyme which has in peripheral organs has not been reported. been purified from mouse liver (Deyashiki et al. The present data obtained by immunoelectron 1995). In both the human prostate and mammary microscopy clearly demonstrate that in two periph- gland, type 5 17-HSD immunoreactivity did not eral tissues, the prostate and mammary gland, show any specific association with organelles, except 3-HSD and type 5 17-HSD are not confined to with an occasional localization over bundles of specific intracellular compartments in epithelial microfilaments. In the rat central nervous system, it cells, fibroblasts of the stromal as well as endothelial has been shown by immunoelectron microscopy cells and fibroblasts of blood vessels. The detection that type 1 17-HSD, which is also a soluble of 3-HSD in mitochondria and smooth endo- enzyme (Jarabak et al. 1962), was exclusively plasmic reticulum of the Leydig cells indicate that located in astrocytes (Pelletier et al. 1995). In this membrane-bound enzyme is associated with agreement with the present results, the gold specific organelles in a steroid-secreting cell. particles were observed throughout the cytoplasm of www.endocrinology.org Journal of Molecular Endocrinology (2001) 26, 11–19

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glial cells without any specific association with or- 17-hydroxysteroid dehydrogenase 4. Biochemical Journal ganelles, such as smooth endoplasmic reticulum and 311 437–443. Cherradi N, Defaye G & Chambaz EM 1993 Dual subcellular mitochondria. In the mammary gland, it is note- localization of the 3-hydroxysteroid dehydrogenase worthy that the myoepithelial cells which contain isomerase: characterization of the mitochondrial enzyme in a large number of microfilaments do not seem to the bovine adrenal cortex. Journal of Steroid contain any immunoreactivity for either enzyme. and Molecular Biology 46 773–779. A finding of interest was the localization of Cherradi N, Defaye G & Chambaz EM 1994 Characterization of the 3-hydroxysteroid dehydrogenase activity associated both 3-HSD and type 5 17-HSD in endothelial with bovine adrenocortical mitochondria. Endocrinology 134 cells in both prostate and mammary gland tissues. 1358–1364. This confirms previous observations at the light Deyashiki Y, Ohshima K, Nakanishi M, Satao K, Matsuura K microscopic level indicating the presence of 3- & Hara A 1995 Molecular cloning and characterization of mouse estradiol 17-dehydrogenase (A-specific),amemberof HSD and type 5 17-HSD in endothelial cells in the aldo-keto reductase family. Journal of Biological the prostate (El-Alfy et al. 1999, Pelletier et al. Chemistry 270 10461–10467. 1999) and that of type 5 17-HSD in endothelial Dufort I, Rheault P, Huang XF, Soucy P & Luu-The V 1999 cells in the mammary gland, ovary and uterus Characteristics of a highly labile human type 5 17- (Pelletier et al. 1999). On the other hand, these hydroxysteroid dehydrogenase. Endocrinology 140 1–7. Dupont E, Simard J, LabrieF&Pelletier G 1994 Localization enzymes were not detected in several other tissues of 3-hydroxysteroid dehydrogenase in rat brain as studied including testis (Pelletier et al. 1999), brain (Dupont by in situ hybridization. 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