ARTICLE |
Correspondence to: Georges Pelletier, Lab. of Molecular Endocrinology, CHUL Research Center, 2705 Laurier Blvd., Québec, QC G1V 4G2, Canada.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
17ß-hydroxysteroid dehydrogenase (17ß-HSD) controls the last step in the formation of all androgens and all estrogens. At least six 17ß-HSD isoenzymes have been identified. The recently cloned Type 5 17ß-HSD transforms 4-dione into testosterone. To gain a better understanding of the role of this enzyme in reproductive tissues, we immunocytochemically localized the enzyme in human male and female reproductive organs. In the ovary of adult premenopausal women (2540 years of age), immunostaining was found in corpus luteum cells. In the uterus, staining was detected only in the epithelial cells of the endometrium. Immunolabeling was also detected in the mammary gland, a positive reaction being detected in epithelial cells of acini and intralobular ducts as well as in the surrounding stromal cells. In the testis, strong staining was seen in the Leydig cells, and a weak but specific reaction was occasionally detected in Sertoli and germ cells. In the prostate, specific labeling was observed in alveoli and stromal fibroblasts. In alveoli, all the basal cells were generally labeled, whereas the luminal cells exhibited variations in immunoreactivity. In all the reproductive organs examined, specific staining was routinely detected in the walls of blood vessels, including the endothelial cells. These results indicate a cell-specific localization of Type 5 17ß-HSD in the different human reproductive organs, thus suggesting new mechanisms of local androgen and estrogen formation that may play an important physiological role. (J Histochem Cytochem 47:731737, 1999)
Key Words: androgens, prostate, ovary, testis, 17ß-HSD localization
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is well recognized that androgens and estrogens arise from two main sources: the steroidogenic organs testis and ovary, which release the hormones into the general circulation, and local synthesis from the adrenal precursors dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and androstenedione (4-dione) (
To obtain information about the precise cellular localization of Type 5 17ß-HSD and then a better knowledge of the role of this enzyme in reproductive tissues, we developed specific antibodies against human Type 5 17ß-HSD and then immunocytochemically localized the enzyme in male and female human reproductive organs.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The samples of reproductive tissues, i.e., testis, prostate, ovary, uterus, and breast, were all obtained at surgery. The specimens used in the present immunocytochemical studies had a volume of about 2.5 cm3, with the exception of prostate chippings, which were of smaller sizes (approximatively 0.4 cm3). They were fixed in 4% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4) within 15 min after they had been dissected out. The average fixation time was 12 hr. The tissues were then dehydrated through increasing concentrations of ethanol, cleared in toluene, and embedded in paraffin. Ovaries, uterus, and breast tissues were obtained from premenopausal patients (2540 years of age). Prostate tissue was obtained from patients with symptomatic benign prostatic hyperplasia undergoing transurethral prostatectomy. The age of male patients ranged from 55 to 70 years. In each case, a minimum of three separate tissues was studied and the results were shown to be consistent.
Preparation of a Human Type 5 17ß-HSD Antibody
The peptide sequence N-GLDRNLHYFNSDSFASHPNYPYS located at amino acid position 297320 of the human Type 5 17ß-HSD (manuscript in preparation) was synthesized by le Service de Séquence de Peptides de l'Est du Québec (SSPEQ) (Québec, Canada) and was purified on HPLC. New Zealand rabbits (2.5 kg) received an SC INJECTION OF 100 µg of the peptide solubilized in 1 ml PBS containing 50% of complete Freund's adjuvant. The animals received two booster injections at 1-month intervals with 50 µg of the peptide in 1 ml of incomplete Freund's adjuvant. Two weeks after the last injection, the rabbits were sacrificed and the blood was collected. The antiserum obtained by decantation followed by centrifugation was stored at -80C. The specificity of the antiserum was verified by immunoblot analysis.
Immunoblot Analysis
Human embryonal kidney cells (293) were transfected with CMV-neo vectors expressing human Type 5 17ß-HSD, 3-HSD Types 1 and 3, and 5
-reductase Types 1 and 2, respectively. Stable transfectants were selected by their resistance to 10-7 M G-418. Positive clones were further confirmed by their ability to efficiently transform the corresponding substrate (unpublished data). Forty µg of the homogenate of each cell line was electrophoresed on a 515% SDS-PAGE as described (
Immunocytochemistry
The tissues were serially cut at 7 µm and sections mounted on glass slides. The sections were deparaffinized, hydrated, and incubated overnight at 4C with the human Type 5 17ß-HSD antiserum diluted 1:1000 in Tris-saline, pH 7.6. The sections were then washed and incubated at room temperature for 4 hr with peroxidase-labeled goat anti-rabbit -globulin (Hyclone; Logan, UT) diluted 1:500 as previously described (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The immunoblot analysis demonstrated that the antiserum reacted only with Type 5 17ß-HSD (Figure 1). No crossreactivity was detected either with 3-HSD Types 1 and 3, which share approximately 80% identity with 17ß-HSD and are present in the testis, mammary gland, and ovary (
-reductase Types 1 and 2, two enzymes that are abundant in prostate tissue (
|
In the ovary, immunolabeling was detected only in corpora lutea. In these structures, most of the luteal cells were moderately labeled (Figure 2A). Both large cells originating from the granulosa layer and small cells originating from the theca interna were immunolabeled. As shown in Figure 2B, immunoabsorption with the antigen completely prevented the staining. No labeling could be detected in the different types of follicles, including the secondary and preovulatory follicles, whereas specific staining was routinely detected in the walls of blood vessels (Figure 3). In the uterus, specific immunolabeling was detected in the endometrium, the myometrium being completely unlabeled. Both epithelial cells lining the glands and those covering the surface were strongly reactive (Figure 4). In the glandular epithelium, both the secretory (functional) cells and the basal cells (when they could be identified) showed a positive reaction. The endometrial stroma was devoid of labeling.
|
In mammary glands from premenopausal women, strong specific staining could be detected in the epithelial cells of acini and intralobular ducts and in stromal fibroblasts (Figure 5). In both cell types, the labeling appeared mainly cytoplasmic. Staining was also detected in the walls of blood vessels, including endothelial cells. In interlobular (Figure 5) and lactiferous ducts, staining also was detected in the epithelial cells. When the antiserum was absorbed with the antigen, no labeling could be detected (not shown).
|
In the testis, strong staining was found in the Leydig cells (Figure 6). In approximatively 10% of tubules, weak labeling could be observed in the seminiferous epithelium (not shown). Sertoli as well as germ cells, especially spermatogonia and spermatocytes, showed immunolabeling. In control sections, no labeling could be detected in Leydig cells as well as seminiferous epithelium. In the prostate, immunoreactivity was detected in alveoli and stroma (Figure 7 and Figure 8). In alveoli, staining was consistently found in the basal cells of the epithelium, whereas the luminal secretory cells exhibited variation in immunoreactivity. Whereas in some alveoli no luminal cells were labeled (Figure 7), other alveoli contained a large number of positive luminal cells (Figure 8). In general, the staining of luminal cells was weaker than that observed in basal cells. In the stroma, labeling was observed in the fibroblasts, the unstained cells probably being smooth muscle cells. Specific immunolabeling was found in the endothelial cells of capillaries, veins, and arteries. In veins, all the fibroblasts in the walls were stained, whereas in arteries, the tunica adventitia, but not the tunica media, was immunolabeled. When the antiserum was immunoabsorbed with the antigen, no staining could be detected.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To precisely determine the structures and the cell types producing Type 5 17ß-HSD in human reproductive tissues, we have developed antibodies against a synthetic peptide designed on the sequence of the Type 5 17ß-HSD protein (aa 297320). The antibodies appear to be specific, as verified by immunoblot analysis as well as immunoabsorption studies with the antigen in immunocytochemical studies.
The present data clearly indicate that, in each of the reproductive tissues investigated, the enzyme expression occurs in specific cell types. It is now quite clear that Type 5 17ß-HSD and Type 2 3-HSD are the same enzyme (
-HSD activity might be present in the cells immunolabeled with antibodies to Type 5 17ß-HSD. On the other hand, because previous results from this laboratory have indicated that all the reproductive tissues from human and rhesus monkey have an androgenic 17ß-HSD activity (
In the testis, the presence of Type 5 17ß-HSD in the Leydig cells suggests that these cells are involved in the conversion of 4-dione to testosterone needed for the internal male reproductive structures (epididymis, seminal vesicles, and vas deferens) as well as all secondary sex organs. Because 3ß-HSD is also localized in Leydig cells (
In the prostate alveoli, immunoactive Type 5 17ß-HSD has been detected predominantly in the basal cells. A similar finding has also been observed using in situ hybridization to localize Type 5 17ß-HSD mRNA (-reductase Types 1 and 2 (
-reductase Types 1 and 2 (
In the ovary, Type 5 17ß-HSD immunoactivity has been found in the majority of luteal cells but not in the follicular cells. This is in contrast to the findings on 3ß-HSD localization, which demonstrated the presence of this enzyme not only in luteal cells but also in theca interna cells (
In the mammary glands, strong immunolabeling was detected in epithelial cells of both alveoli and ducts, as well as in stromal cells that probably represent fibroblasts. Because these cell types have been shown to contain androgen receptors (
In the uterus, Type 5 17ß-HSD could be detected only in the epithelial cells of the endometrial glands. In the absence of androgen receptors in the uterus (
Another interesting finding was the localization of Type 5 17ß-HSD in blood vessel walls, including the endothelial cells. This observation correlates very well with recent findings from this laboratory indicating the presence of 5-reductase Types 1 and 2 mRNA in blood vessel walls in human prostate and skin (
In conclusion, the results obtained using specific antibodies to human Type 5 17ß-HSD clearly indicate a cell-specific localization of the enzyme in human reproductive organs. In fact, endocrine steroid-secreting cells (Leydig and luteal), as well as epithelial secretory cells and stromal cells, express Type 5 17ß-HSD. Therefore, the present data suggest new mechanisms of local androgen formation that may play an important physiological role in the different reproductive organs.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams JB, Archibald L, Clarke C (1978) Adrenal dehydroepiandrosterone and human mammary cancer. Cancer Res 36:4102-4106[Abstract]
Adams JB, Wong MSF (1968) Paraendocrine behaviour of human breast carcinoma: in vitro transformation of steroids to physiologically active hormone. J Endocrinol 41:41-52
Akinola LA, Poutanen M, Vihko R (1996) Cloning of rat 17ß-hydroxysteroid dehydrogenase type 2 and characterization of tissue distribution and catalytic activity of rat type 1 and type 2 enzymes. Endocrinology 137:1572-1579[Abstract]
Anderson S, Russell DW (1990) Structural and biochemical properties of cloned and expressed human and rat steroid 5-reductases. Proc Natl Acad Sci USA 87:3640-3644[Abstract]
Andersson S, Geissler WM, Patel S, Wu L (1995) The molecular biology of androgenic 17ß-hydroxysteroid dehydrogenases. J Steroid Biochem Mol Biol 53:37-39[Medline]
Biswas MG, Russell DW (1997) Expression cloning and characterization of oxidative 17ß- and 3-hydroxysteroid dehydrogenase from rat and human prostate. J Biol Chem 272:15959-15966
Dufort I, Rheault P, Huang XF, Soucy P, LuuThe V (1999) Characteristics of a highly labile human type 5 17ß-hydroxysteroid dehydrogenase. Endocrinology 140:1-7
Dufort I, Soucy P, Labrie F, LuuThe V (1996) Molecular cloning of human type 3 3-hydroxysteroid dehydrogenase that differs from 20
-hydroxysteroid dehydrogenase by seven amino acids. Biochem Biophys Res Commun 228:474-479[Medline]
Dumont M, LuuThe V, de Launoit Y, Labrie F (1992) Expression of human 17ß-hydroxysteroid dehydrogenase in mammalian cells. J. Steroid Biochem Mol Biol 41:605-608[Medline]
Eicheler W, Tuohimaa P, Vilja P, Adermann K, Forssmann W-G, Augmuller G (1994) Immunocytochemical localization of human 5-reductase 2 with polyclonal antibodies in androgen target and non-target tissue. J Histochem Cytochem 42:667-675
El-Alfy M, Huang XF, Berger L, LuuThe V, Labrie F, Pelletier G (1999) Localization of type 5 17ß-hydroxysteroid dehydrogenase, type 1 3ß-hydroxysteroid dehydrogenase and androgen receptor in the human prostate by in situ hybridization and immunocytochemistry. Endocrinology 140:1841-1849
FranckLissbrant I, Haggstrom S, Damber JE, Bergh A (1998) Testosterone stimulates angiogenesis and vascular regrowth in the ventral prostate in castrated adult rats. Endocrinology 139:451-456
Geissler WM, Davis DL, Wu L, Bradshaw KD, Patel S, Mendoça BB, Elliston KO, Wilson JD, Russell DW, Andersson S (1994) Male pseudohermaphroditism caused by mutations of testicular 17ß-hydroxysteroid dehydrogenase 3. Nature Genet 7:34-39[Medline]
HildPetito S, West NB, Brenner RM, Stouffer RL (1991) Localization of androgen receptor in the follicle and corpus luteum of the primate ovary during the menstrual cycle. Biol Reprod 44:561-568[Abstract]
Iwamura M, Abrahamsson P-A, Benning CM, Cockett ATK, Di Santagnese PA (1994) Androgen receptor immunostaining and its tissue distribution in formalin-fixed, paraffin-embedded sections after microwave treatment. J Histochem Cytochem 42:783-788
Jensen EV, DeSombre ER (1972) Estrogen-receptor interaction. Annu Rev Biochem 4:203-230
Khanna M, Qin KN, Wang RW, Cheng KC (1995) Substrate specificity, gene structure, and tissue-specific distribution of multiple human 3-hydroxysteroid dehydrogenases. J Biol Chem 270:20162-20168
Kimura N, Mizokami A, Oonuma T, Sasano H, Nagura H (1993) Immunocytochemical localization of androgen receptor with polyclonal antibody in paraffin-embedded human tissues. J Histochem Cytochem 41:671-678
Labrie F (1991) Intracrinology. Mol Cell Endocrinol 78:C113-C118[Medline]
Labrie F, LuuThe V, Lin SX, Labrie C, Simard J, Breton R, Bélanger A (1997) The key role of 17ß-HSDs in sex steroid biology. Steroids 62:148-158[Medline]
Levine AC, Wang JP, Ren M, Eliashvili E, Russell DW, Kirschenbaum A (1996) Immunohistochemical localization of steroid 5-reductase 2 in the human male fetal reproductive tract and adult prostate. J Clin Endocrinol Metab 81:384-389[Abstract]
Levy H, Deane HW, Rubin BL (1959) Visualization of steroid 3ß-O1-dehydrogenase activity in tissues of intact and hypophysectomized rats. Endocrinology 65:932-939
Lin HK, Jezx JM, Schlegel BP, Peehl DM, Pachter JA, Penning TM (1997) Expression and characterization of recombinant type 2 3-hydroxysteroid dehydrogenase (HSD) from human prostate: demonstration of bifunctional 3
/17ß-HSD activity and cellular distribution. Mol Endocrinol 11:1971-1984
LuuThe V, Sugimoto Y, Puy L, Labrie Y, Lopez I, Singh M, Labrie F (1994) Characterization, expression and immunohistochemical localization of 5-reductase in human skin. J Invest Dermatol 102:221-226[Abstract]
Martel C, Rhéaume E, Takahashi M, Trudel C, Couet J, LuuThe V, Simard J, Labrie F (1992) Distribution of 17ß-hydroxysteroid dehydrogenase gene expression and activity in rat and and human tissues. J Steroid Biochem Mol Biol 41:597-603[Medline]
McNatly KP, Makois A, DeGrozia C, Osothanondh R, Ryan KJ (1979) The production of progesterone androgens and estrogens by granulosa cells, thecal tissue and stromal tissue from human ovaries in vitro. J Clin Endocrinol Metab 49:687-699[Abstract]
NegriCesi P, Poletti A, Colciago A, Magni P, Martini P, Motta M (1998) Presence of 5-reductase isozymes and aromatase in human prostate cancer cells and in benign prostate hyperplastic tissue. Prostate 34:283-291[Medline]
Ohara A, Mori T, Taii S (1987) Functional differentiation in steroidogenesis of two types of luteal cells isolated from mature human corpora lutea of menstrual cycle. J Clin Endocrinol Metab 65:1192-1200[Abstract]
Orlowski J, Clark AF (1991) Epithelial-stromal interactions in the regulation of rat ventral prostate function: identification and characterization of pathways for androgen metabolism in isolated cell types. Endocrinology 128:872-884[Abstract]
Pelletier G, Dupont E, Simard J, LuuThe V, Bélanger A, Labrie F (1992) Ontogeny and subcellular localization of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in the human and rat adrenal, ovary and testis. J Steroid Biochem Mol Biol 43:451-467[Medline]
Pelletier G, LuuThe V, Huang XF, Lapointe H, Labrie F (1998) Localization by in situ hybridization of steroid 5-reductase isozyme gene expression in the human prostate and preputial skin. J Urol 160:577-582[Medline]
Pelletier G, Luu-The V, Labrie F (1995) Immunocytochemical localization of type 1 17ß-hydroxysteroid dehydrogenase in the rat brain. Brain Res 704:233-239[Medline]
Penning TM (1997) Molecular endocrinology of hydroxysteroid dehydrogenases. Endocrine Rev 18:281-305
Ruizeveld de Winter JA, Trapman J, Vermey M, Mulder E, Zegers ND, Van Der Kwast TH (1991) Androgen receptor expression in human tissues: an immunohistochemical study. J Histochem Cytochem 39:927-936[Abstract]
Sar M, Lubahn DB, French FS, Wilson EM (1990) Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 127:3180-3186[Abstract]