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Immunoelectron Microscopic Localization of 3Β-Hydroxysteroid Dehydrogenase and Type 5 17Β-Hydroxysteroid Dehydrogenase In

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Immunoelectron Microscopic Localization of 3Β-Hydroxysteroid Dehydrogenase and Type 5 17Β-Hydroxysteroid Dehydrogenase In 11 Immunoelectron microscopic localization of 3-hydroxysteroid dehydrogenase 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- enzymes 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 steroids there is no indication about the organelles 17-HSD localization, labelling was observed in the which contain the enzymes involved in steroid 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 enzyme 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, Journal of Molecular Endocrinology (2001) 26, 11–19 Online version via http://www.endocrinology.org 0952–5041/01/026–011 2001 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 10/02/2021 09:00:19AM via free access 12 and others · 3-HSD and type 5 17-HSD in human prostate and breast 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 liver (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 amino acid 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. Journal of Molecular Endocrinology (2001) 26, 11–19 www.endocrinology.org Downloaded from Bioscientifica.com at 10/02/2021 09:00:19AM via free access 3-HSD and type 5 17-HSD in human prostate and breast · and others 13 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. www.endocrinology.org Journal of Molecular Endocrinology (2001) 26, 11–19 Downloaded from Bioscientifica.com at 10/02/2021 09:00:19AM via free access 14 and others · 3-HSD and type 5 17-HSD in human prostate and breast 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.
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