Sex-related Expression of 20-hydroxysteroid Dehydrogenase mRNA in the Adult Mouse
Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL) and Laval University, Québec, (PQ) Canada
Correspondence to: Dr. Georges Pelletier, Oncology and Molecular Endocrinology Research Center and Québec Génome Center, Laval University Hospital (CHUL), 2705 Laurier Boulevard. Québec, PQ G1V 4G2, Canada. E-mail: georges.pelletier{at}crchul.ulaval.ca
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Summary |
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Key Words: progesterone catabolism 20-hydroxysteroid progesterone 20
-hydroxysteroid dehydrogenase mRNA expression in situ hybridization mouse autoradiography
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Introduction |
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Thus far, the precise localization of 20-HSD at the cellular level has not been reported. We therefore used in situ hybridization (ISH) to localize the 20
-HSD mRNA in several tissues in adult mice of both sexes to gain information about the exact sites of 20
-HSD expression and to determine whether there is a gender-related influence on the expression of the enzyme.
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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, i.e., liver, kidney, jejunum, pancreas, 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 on gelatin- and poly-L-lysine-coated 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 232 bp of mouse 20-HSD (GenBank accession number AB059565). The cDNA fragment located at position 36288 downstream from the ATG start codon was obtained by amplification using the polymerase chain reaction (PCR). ISH 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 (RT) in a humid chamber for 2 hr in 450 µl/slide of a prehybridization buffer containing 50% formamide, 5 x SSPE (1 x SSPE being 0.1 M NaCl, 10 mM NaH2PO4, pH 7.4, mM EDTA), 5 x Denhart's buffer, 200 mg/ml denatured salmon testis DNA (Sigma), 200 µg/ml yeast tRNA, 2 µg/ml Poly A (BoeringherMannheim; Montreal, PQ, 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/ml) was spotted on each slide, sealed under a coverslip, and incubated at 37C overnight (1520 hr) in a humid chamber.
After hybridization, coverslips were removed and slides were rinsed in 2 x SSC at RT for 30 min. Sections were digested by RNase A (20 µg/ml in 2 x SSC) at 37C for 30 min, rinsed in decreasing concentrations of SSC (2 x SSC and 1 x SSC) for 30 min at RT, washed in 0.5 x SSC for 30 min at 37C, followed by 90 min at RT in 0.5 x SSC, at 60C in 0.1 x SSC, and finally for 30 min at RT in 0.1 x SSC.
The sections were then dehydrated and exposed to Kodak Biomax MR films for 38 days before being coated with liquid photographic emulsion (Kodak-NTB2; diluted 1:1 with water). Slides were exposed for 145 days, developed in Dektol developer (Kodak; Rochester, NY) 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 coverslipped with Permount (Fisher Scientific; Montreal, PQ, Canada).
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Results |
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Discussion |
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In the female reproductive tract, 20-HSD mRNA expression was detected only in the epithelial cells of the uterine cervix . The role of the enzyme might be related to the delivery process. It has been reported that, in mice lacking 5
-reductase type 1, an enzyme that is also involved in progesterone catabolism and is expressed in the cervix, there was a failure of cervical ripening, leading to abnormal delivery (Mahendroo et al. 1999
). Such data suggest that 20
-HSD activity does not play a major role in the catabolism of progesterone in the cervix, at least during parturition.
In the mammary gland of the female mouse, weak 20-HSD expression was detected only in stromal cells surrounding the ducts. Progesterone receptors have been found to be expressed in both epithelial cells of the ducts and stromal cells in the mouse mammary gland (Fenn et al. 1998
). It therefore appears that progesterone metabolism is mainly restricted to stromal cells in mammary glands of the cycling mouse.
In the mouse testis, the hybridization signal was observed only in Leydig cells. The testicular localization of the enzyme has already been reported in the bovine and human species (Warren et al. 1993; Zhang et al. 2000
). The low expression of 20
-HSD mRNA reported in the human testis (Zhang et al. 2000
) can be explained by the fact that the expression of the enzyme is restricted to Leydig cells, which represent only a small proportion of the total number of testicular cells. The role of 20
-HSD might be related to some protective effect from progesterone in testicular tissue.
In the adrenal glands, 20-HSD mRNA expression was restricted to the cells of the zona reticularis. The hybridization signal was very high in the female and very low in the male. From pig adrenal cytosol, Nakajin et al. (1989)
have purified two proteins exhibiting 20
-HSD activities, which they named 20
-HSD I and II. We have recently reported low expression of 20
-HSD mRNA in total adrenal glands in whole human adrenal glands (Zhang et al. 2000
). This low expression of 20
-HSD mRNA might be explained by the restricted expression of 20
-HSD mRNA to the zona reticularis, which consists of a few layers of cells and represents a very small proportion of the total number of adrenal cells. The physiological role of 20
-HSD in the adrenal cortex remains to be clarified.
In the skin, 20-HSD mRNA was strongly expressed in sebaceous glands, but no specific labeling could be detected in the hair follicle and stromal cells or in the epidermis. The presence of progesterone receptors has been reported in sebaceous glands in the human (Wallace and Smaller 1998
). The enzyme might therefore regulate the availability of circulating and locally produced progesterone for progesterone receptors and thus control the influence of progesterone on sebaceous gland cell activity. Because it has been shown that progesterone treatment suppresses estrogen receptors in the monkey sex skin (West et al. 1990
), one role of progesterone might be to regulate estrogen receptor levels that are expressed in sebaceous glands (Pelletier 2000
). The very low expression of 20
-HSD mRNA in the male mouse skin is indicative that 20
-HSD mRNA might be positively regulated by ovarian hormones. In fact, we have recently observed that ovariectomy induced a striking decrease in 20
-HSD mRNA levels in mouse skin (unpublished data).
In the kidney, a specific hybridization signal was found in the epithelial cells in the distal convoluted tubules. As in other tissues, the signal was more striking in the female than in the male. In the human kidney, the expression of 20-HSD has been reported (RumkeVogl et al. 2002
) but there was no indication about the cell types involved in the expression of the enzyme. Interestingly, progesterone receptors have been found in epithelial cells of distal convoluted tubules (RumkeVogl et al. 2002
). From the results obtained in the mouse and human kidney, it appears that the same epithelial cells can express both progesterone receptors and the enzyme that inactivates progesterone. 20
-HSD might be involved in the fine regulation of the action of progesterone, which has been shown to be a potent mineralocorticoid receptor antagonist (Myles and Kuner 1996
). In the liver, 20
-HSD mRNA expression was observed in the hepatocytes, the expression being also higher in the female. Because of the large number of hepatocytes, such data indicate that liver might be considered as a major site of progesterone catabolism.
The absence of any hybridization signal in the prostate, uterus, vagina, and pituitary might be explained by a low expression of 20-HSD that could not be detected by ISH in these tissues. We might also consider the possibility that, in these four tissues, a fine regulation of intracellular progesterone concentrations is not critical for hormonal activity. By RT-PCR, Zhang et al. (2000)
have shown low expression of 20
-HSD mRNA in human prostate and uterus, whereas in the present studies we were unable to detect any hybridization signal in the same mouse tissues. These apparent species differences might be explained by a lower sensitivity of ISH compared to that obtained with the highly sensitive RT-PCR technique.
In summary, the present results on the localization of 20-HSD mRNA in mouse tissues indicate that the enzyme is highly expressed in the male and female gonads and in the uterine cervix. In non-reproductive tissue, the expression of 20
-HSD mRNA is always higher in the female than in the male, thus suggesting that the enzyme plays a predominant role in the inactivation of locally produced or circulating progesterone, which is much higher in the female. Other studies involving ovariectomy and hormone replacement therapy are required to clarify the role of ovarian steroids in the regulation of 20
-HSD expression.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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