Localization of Type 8 17ß-hydroxysteroid Dehydrogenase mRNA in Mouse Tissues as Studied by In Situ Hybridization
Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL) and Laval University, Québec, Canada
Correspondence to: Dr. Georges Pelletier, Oncology and Molecular Endocrinology Research Center, Laval University Hospital (CHUL), 2705 Laurier Boulevard, Québec, Québec, G1V 4G2, Canada. E-mail: georges.pelletier{at}crchul.ulaval.ca
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Summary |
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Key Words: type 8 17ß-hydroxysteroid dehydrogenase estradiol inactivation estrone mouse tissues in situ hybridization
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Introduction |
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By Northern blot analysis, the mouse type 8 17ß-HSD mRNA was found to be expressed in the ovary, testis, kidney, and liver (Fomitcheva et al. 1998). By immunofluorescence, the type 8 17ß-HSD protein has been localized to the cumulus cells surrounding the oocyte and endothelial cells in the mouse ovary (Fomitcheva et al. 1998
). So far, the identification of cells expressing type 8 17ß-HSD mRNA has not been reported. To learn more about the physiological role of the enzymes in gonads and extragonadal tissues, we studied the precise localization of type 8 17ß-HSD mRNA in several mouse tissues by in situ hybridization.
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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 liver, lung, jejunum, colon, pancreas, dorsal skin, adrenal, pituitary, testis, prostate, ovary, uterus, vagina, and mammary gland were excised and postfixed in the same fixative for 24 hr at 4C. The tissues were 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-coated and poly-L-lysinecoated slides. The vector used for the production of cRNA probe was constructed by insertion into a pBSKSII+vector (Stratagene, La Jolla, CA) of a cDNA fragment of 537 bp of mouse type 8 17ß-HSD (Genebank accession number NM-013543). The cDNA fragment located at position 276-812 downstream from the adenine thymine guanine start codon was obtained by amplification using PCR. In situ hybridization with the antisense and sense 35S-labeled cRNA probes was performed as previously described (Givalois et al. 1997). Briefly, the sections were prehybridized at room temperature in a humid chamber for 2 hr in 450 µl/slide of a prehybridization buffer containing 50% formamide, 5x SSPE (1x SSPE being 0.1 M NaCl, 10 mM NaH2PO4, pH 7.4, mM EDTA), 5x Denhart's buffer 200 mg/ml, denatured salmon testis DNA (Sigma) 200 µg/ml yeast tRNA, 2 µg/ml Poly A (Boehringer-Mannheim, Montreal, Canada), and 4% dextran sulfate. After prehybridization treatment, 100 µl hybridization mixture (prehybridization buffer containing, in addition 10 mM dithiothreitol and 35S-labeled cRNA probe at a concentration of 10 x 106 cpm/3 ml) was spotted on each slide, sealed under a cover slip, and incubated at 37C overnight (1520 hr) in a humid chamber.
After hybridization, cover slips were removed and slides were rinsed in 2x SSC at room temperature for 30 min. Sections were 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 0.5x SSC, at 60C in 0.1x SSC and finally for 30 min at room temperature in 0.1x SSC.
The sections where then dehydrated and exposed to Kodak Biomax Mr films for 38 d before being coated with liquid photographic emulsion (Kodak-NTB2; diluted 1:1 with water). Slides were exposed for 745 d, developed in Dektol developer (Kodak) for 2 min, and fixed in rapid fixer (Kodak) for 4 min. Thereafter, tissues were rinsed in running water for 30 min, counterstained with hematoxylin and rapidly dehydrated through graded concentrations of ethanol, cleared in toluene, and cover-slipped with Permount (Fisher Scientific, Montreal, Canada).
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Results |
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In the ovary, specific labeling could be observed in granulosa cells in growing follicles at all stages of development and in luteal cells of corpora lutea (Figure 1). In the uterus, the hybridization signal was detected on epithelial cells (both luminal and glandular) and stromal cells, whereas the myometrial smooth muscle cells were unlabeled (Figure 2). In the vagina, both epithelial and stromal cells exhibited mRNA expression (Figure 3). In the female mammary gland, specific labeling was seen in periductal stromal cells and in ductal epithelial cells (Figure 4). In the clitoral gland, type 8 17ß-HSD mRNA was detected in epithelial cells of both acini and small ducts. The large excreting ducts were devoid of any specific reaction (Figure 5).
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Discussion |
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By Northern blot analysis, Fomitcheva et al. (1998) have detected type 8 17ß-HSD mRNA in the ovary. By immunofluorescence, the same authors reported the presence of the type 8 17ß-HSD protein in the cumulus cells in growing follicles and endothelial cells in the mouse ovary. In the present study, we found that the enzyme mRNA was expressed in all the granulosa cells in growing follicles and in luteal cells. This discrepancy observed between our results and those previously obtained by immunofluorescence could be explained by a higher sensitivity of the in situ hybridization technique or alternatively by a low transcription of the protein in the majority of granulosa and luteal cells. The in situ hybridization findings suggest that both granulosa and luteal cells can convert E2 into E1. Two enzymes involved in the conversion of E1 into E2 types 1 and 7 17ß-HSD have been found to be expressed in both granulosa and luteal cells (Nokelainen et al. 1996
; Pelletier et al. 2004
,2005
). It may be then suggested that type 8 17ß-HSD of which the predominant role is the conversion of E2 to E1 (Luu-The 2001
) might contribute to regulate the local concentration of E2 in both follicles and corpora lutea.
We report for the first time that type 8 17ß-HSD mRNA is expressed in the oviduct and uterus, the hybridization signal being found in the epithelial and stromal cells, but not in the myometrium. The enzyme might contribute to the fine regulation of estrogen levels in the endometrium, which is very sensitive to estrogen variations during the ovarian cycle. In human, the loss of estrogen inactivating enzyme (e.g., types 2 and 8 17ß-HSD) expression has been proposed as a mechanism involved in the pathogenesis of endometriosis (Bulun et al. 2002). In the mammary gland, type 8 17ß-HSD mRNA, which is expressed in both epithelial and stromal cells is likely involved, as in other tissues, in the regulation of the intracellular concentration of estrogens, locally produced or originating from the general circulation. In human breast proliferative disorders, type 2 17ß-HSD that, as with type 8 17ß-HSD converts E2 to E1, has not been found (Mindnich et al. 2004
). Studies to detect type 8 17ß-HSD expression in human breast cancer is in progress in our laboratory. In clitoral glands, which are highly estrogen-dependent (unpublished data), type 8 17ß-HSD mRNA is restricted to epithelial cells. This appears to be the first report on the expression of type 8 17ß-HSD mRNA in this gland. Interestingly, type 7 17ß-HSD, the enzyme that catalyzes the conversion of E1 into E2, has also been localized in epithelial cells of clitoral glands (Pelletier et al. 2005
). These findings suggest that epithelial cells are the major sites of E2 metabolism.
In the testis, type 8 17ß-HSD mRNA was expressed in germ cells, but not in Leydig cells, thus suggesting that the enzyme may contribute to the local metabolism of E2 and then protect germinal cells against exposure to high levels of estrogens. By Northern blot analysis, type 8 17ß-HSD mRNA has already been detected in the mouse testis, but there was no indication about the cell types involved in the expression of the enzyme mRNA (Fomitcheva et al. 1998). In the prostate, type 8 17ß-HSD mRNA was localized in both epithelial cells of the acini and stroma cells. The role of type 8 17ß-HSD might be related to the downregulation of estrogen levels in the prostate. The role of estrogens, which are locally synthesized through the action of aromatase or type 1 17ß-HSD, is still unclear (Pelletier et al. 2004
). ERß has been reported to be expressed in prostate epithelial and stroma cells in several species, including the mouse (Couse and Korach 1999
; Pelletier 2000
; Pelletier and El-Alfy 2000
). In ER
or ERß knockout mice, no abnormality of the development of prostate has been found (Couse et al. 2000
; Dupont et al. 2000
). On the other hand, we have reported that E2 administration to castrated rats could induce moderate hypertrophy of prostate epithelial cells and an increase in androgen receptor expression in epithelial and stromal cells (Pelletier 2002
). In preputial glands, which are equivalent to clitoral glands, the enzyme mRNA was similarly localized to the epithelial cells. Type 8 17ß-HSD could contribute to downregulate the levels of E2 in the epithelial cells of the gland, which also express ER
and AR (unpublished data).
In the pituitary of animals of both sexes, the hybridization signal is diffusely located in the anterior and intermediate lobes, whereas in the posterior lobe, the labeling appears mostly associated with the pituicytes. In the female, type 8 17ß-HSD is likely involved in the local regulation of estrogens coming from the general circulation and locally produced estrogens. In the male, the enzyme might regulate the estrogens locally produced through the action of type 1 17ß-HSD and aromatase (Sharpe 1998; Pelletier et al. 2004
). In the adrenal cortex, type 8 17ß-HSD mRNA has been found in the three layers. The expression of the enzyme in the adrenal cortex suggests that estrogens originating form the general circulation or locally produced might be downregulated in the steroidogenic cells. There is evidence that adrenal cortex can secrete estradiol probably through the action of aromatase (Fang 1977
; Bouraima et al. 2003
).
In the kidney, we observed that the expression of type 8 17ß-HSD mRNA was confined to proximal tubules. This finding is in agreement with previous reports indicating that Ke 6 or type 7 17ß-HSD protein could be localized by immunofluorescence to the proximal tubules and collecting tubules (Aziz et al. 1996). This might indicate that these kidney structures are the sites of E2 metabolism in the kidney. The expression of other enzymes involved in the conversion of E2 to E1, namely 17ß-HSD types 2, 4, 9, 10, and 11 in the kidney has not been reported.
In the liver, it appears that all the hepatocytes express the enzyme mRNA and might be involved in the conversion of E2 to E1. Type 7 17ß-HSD, the enzyme that converts E1 to E2, has also been found to be expressed in all the hepatocytes (Pelletier et al. 2005). The relative role of the different enzymes involved in the formation and inactivation of estrogens at the liver level remains to be clarified. In the lung, epithelial cells of alveoli and bronchioles express type 8 17ß-HSD. It then appears that the lung, as with the liver and kidney, can be considered as an organ involved in estrogen inactivation. It is noteworthy that type 2 17ß-HSD, another enzyme involved in the conversion of E2 to E1, has been shown to expressed in human lung fibroblast cell lines (Provost et al. 2002
). In the dorsal skin, the hybridization signal was diffusely located throughout the stroma. Such a localization of the enzyme might indicate that E2 is metabolized into E1 by type 8 17ß-HSD in the stroma.
In summary, we report for the first time the histological identification of the cell types expressing type 8 17ß-HSD mRNA in several tissues including gonads in the adult mouse. In those tissues, the role of the enzyme is likely related to the local regulation of estradiol levels coming from the general circulation or locally produced.
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Footnotes |
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Literature Cited |
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