Cellular Localization of mRNA Expression of Enzymes Involved in the Formation and Inactivation of Hormonal Steroids in the Mouse Prostate
Oncology and Molecular Endocrinology Research Center, Laval University Medical Center, Québec, Canada, and Laval University, Québec, Canada
Correspondence to: Dr. Georges Pelletier, Oncology and Molecular Endocrinology Research Center, Laval University Hospital (CHUL), 2705, Laurier Boulevard, Québec, G1V 4G2, Canada. E-mail: georges.pelletier{at}crchul.ulaval.ca
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Summary |
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(J Histochem Cytochem 52:13511356, 2004)
Key Words: steroidogenic enzymes prostate androgens estrogens in situ hybridization
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Introduction |
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In recent years, the existence of a local biosynthesis of sex steroids, or intracrinology (Labrie et al. 1988; Labrie 1991
) has been supported by demonstration of the expression of several steroidogenic enzymes in peripheral tissues, including the prostate (Martel et al. 1994
; Luu-The 2001
; Labrie et al. 2003
). We have also reported, using immunocytochemistry and in situ hybridization (ISH), the cellular localization of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) type 1, 17ß-HSD type 5, and 5
-reductase types 1 and 2, the enzymes involved in the conversion of circulating DHEA to DHT in human prostate (Pelletier et al. 1998
,1999
; El-Alfy et al. 1999
). So far, the localization of steroidogenic enzyme mRNAs in the mouse prostate has not been reported.
To define the cell types involved in the local synthesis and degradation of sex steroids in the rodent prostate, we have studied the localization of mRNAs expressing a series of steroidogenic enzymes in the mouse ventral prostate by ISH. For each enzyme, we also measured mRNA levels in mouse ventral prostate using quantitative real-time PCR (RT-PCR).
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Materials and Methods |
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Histological Procedures
All the animals were perfused transcardially with 50 ml 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The different tissues, namely the liver, kidney, lung, adrenal, ovary, testis, mammary gland, uterus, and ventral lobe of the prostate were excised and postfixed in the same fixative for 24 hr at 4C. All the samples were then placed in 15% sucrose in 0.1 M phosphate buffer before being quickly frozen in isopentane chilled in liquid nitrogen.
In Situ Hybridization
Frozen sections (10 µm thick) were serially cut at 20C and mounted onto gelatin- and poly-L-lysine-coated slides. The vector used for production of the cRNA probe was constructed by insertion into a pBSKSII+ vector (Stratagene, La Jolla, CA) of cDNA fragments. The characteristics of the fragments of mouse enzyme cDNAs that have been studied are listed in Table 1.
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After hybridization, coverslips were removed and slides were rinsed in 2x SSC at room temperature for 30 min. Sections were then digested by RNase A (20 µg/ml in 2x SSC) at 37C for 30 min, rinsed in decreasing concentrations of SSC (2x SSC and 1x SSC) for 30 min at room temperature, washed in 0.5x SSC for 30 min at 37C, followed by 90 min at room temperature in 0.5 x SSC, and finally for 30 min at room temperature in 0.1x SSC.
The sections were then dehydrated and coated with liquid photographic emulsion (Kodak-NTB2; diluted 1:1 with water). Slides were exposed for 745 days, developed in a Dektol developer (Kodak, Rochester, NY) for 2 min, and fixed in rapid fixer (Kodak) for 4 min. Thereafter, the sections were rinsed and stained with haematoxylin.
RNA Preparation and Quantification of mRNA Expression Levels
Total RNA was isolated from a pool of 10 prostates with Trizol (Invitrogen, Burlington, Ontario). Twenty µg of total RNA was converted to cDNA by incubation with 400 U SuperScript II reverse transcriptase (Invitrogen), T7-oligo-d (T)24 as primer (5'-GGC-CAG-TGA-GTA-ATA-CGA-CTC-ACT-ATA-GGG-AGG-CGG-(dT)24-3'), 1x first-strand buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol) and 0.5 mM deoxynucleotide triphosphate at 42C for 1 hr.
Quantification of mRNA levels was performed using a quantitative RT-PCR method that permits monitoring the efficiency of the PCR amplification process by calculating the efficiency coefficient of corresponding standard curves. The LightCycler Realtime PCR apparatus as well as reagents were from Hoffman-La Roche, Inc. (Nutley, NJ). The reaction was performed using the amount of cDNA corresponding to 30 pg of initial total RNA. Oligoprimer pairs that allow the amplification of 200 bp of indicated mRNA were designed by GeneTools software, and their specificity was checked by blasting in the GenBank database. To avoid errors due to RNA and cDNA preparation and loading, we normalized with a housekeeping gene Atp5o (subunit O of ATPase) at each assay. Atp5o has been shown to be a gene that has stable expression levels from embryonic life through adulthood in various tissues (Warrington et al. 2000
). The mRNA levels are expressed as number of copies/µg total RNA that are calculated using a standard results curve of Atp5o.
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Results |
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Discussion |
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One cannot conclude, however, that when a hybridization signal cannot be detected in the mouse prostatic tissue, the enzyme mRNA is not expressed. The results obtained by quantitative RT-PCR indicate that with a few exceptions, there is a good correlation between the number of mRNA copies/µg total RNA and the detection of ISH signal, which was absent when the number of copies was below 2000. Each probe that provided negative results in the mouse prostatic tissue was shown to give positive results in at least one other tissue, indicating the effectiveness of each probe in detecting specific mRNAs under our experimental conditions. Altogether, the present results indicate that the failure to obtain an ISH signal for some enzymes is likely due to low levels of expression of those enzyme mRNAs.
With the approach involving the use of frozen sections, we could not localize any enzyme in the walls of blood vessels. In previous studies performed in the paraffin-embedded human prostate, we could detect immunoreactive 17ß-HSD type 5 in endothelial cells of capillaries, veins, and arteries (El-Alfy et al. 1999). Other studies involving the use of specific antibodies raised against mouse steroidogenic enzymes for immunocytochemical localization should provide additional information on the cell types expressing those enzymes in the mouse prostate.
From the present results, it clearly appears that the enzymes involved in the biosynthesis (17ß-HSD types 1, 3, and 7; 5-reductase type 2; and 11ß-HSD type 1), as well as the inactivation (17ß-HSD types 2, 4, 8, 9, 10, and 11; 7
-hydroxylase; estrogen sulfotransferase type 1; 11ß-HSD type 2; and UGT1A6), of hormonal steroids are all expressed by the epithelial cells, and several of them are also detected in stromal cells.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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