Localization of Type 8 17β-hydroxysteroid Dehydrogenase mRNA in Mouse Tissues as Studied by In Situ Hybridization

Abstract

The enzyme type 8 17β-hydroxysteroid dehydrogenase (17β-HSD) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To obtain detailed information on the sites of action of type 8 17β-HSD, we have studied the cellular localization of type 8 17β-HSD mRNA in mouse tissues using in situ hybridization. In the ovary, hybridization signal was detected in granulosa cells of growing follicles and luteal cells. In the uterus, type 8 17β-HSD mRNA was found in the epithelial (luminal and glandular) and stromal cells. In the female mammary gland, the enzyme mRNA was seen in ductal epithelial cells and stromal cells. In the testis, hybridization signal was observed in the seminiferous tubule. In the prostate, type 8 17β-HSD was detected in the epithelial cells of the acini and stromal cells. In the clitoral and preputial glands, labeling was detected in the epithelial cells of acini and small ducts. The three lobes of the pituitary gland were labeled. In the adrenal gland, hybridization signal was observed in the three zones of the cortex, the medulla being unlabeled. In the kidney, the enzyme mRNA was found to be expressed in the epithelial cells of proximal convoluted tubules. In the liver, all the hepatocytes exhibited a positive signal. In the lung, type 8 17β-HSD mRNA was detected in bronchial epithelial cells and walls of pulmonary arteries. The present data suggest that type 8 17β-HSD can exert its action to downregulate E2 levels in a large variety of tissues.

Keywords

type 8 17β-hydroxysteroid dehydrogenase estradiol inactivation estrone mouse tissues in situ hybridization

T he 17β-hydroxysteroid dehydrogenase (17β-HSD) are enzymes responsible for the formation and inactivation of active androgens and estrogens. Multiple types of 17β-HSDs (named types 1–12) have been cloned and have been shown to be expressed in several human and animal tissues (Peltoketo et al. 1999; Luu-The 2001; Mindnich et al. 2004). The type 8 17β-HSD has been recently identified. It is also known as the product of the Ke 6 gene, which is found in the human leukocyte antigen region (Kikuti et al. 1997). This area is known to contain genes encoding the human major histocompatibility complex. In the mouse, the complex is thought to be involved in polycystic kidney disease because aberrant gene expression has been found in two models of polycystic kidney disease (Aziz et al. 1993). Recently, Fomitcheva et al. (1998) have reported that the mouse protein overexpressed was a 17β-HSD that efficiently catalyzes the transformation of 17β-estradiol (E2) to estrone (E1). The transformation of testosterone (T) to 4-dione is ≃25% of that of E2 to E1. Peltoketo et al. (1999) suggests designing it as type 8 17β-HSD. Recently, using RT-PCR, we have isolated a DNA fragment corresponding to the coding region of the human Ke6 gene (Luu-The 2001). Transfection into HEK-293 cells showed that this enzyme transforms efficiently E2 into E1 (Luu-The 2001).

By Northern blot analysis, the mouse type 8 17β-HSD mRNA was found to be expressed in the ovary, testis, kidney, and liver (Fomitcheva et al. 1998). By immunofluorescence, the type 8 17β-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 17β-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 17β-HSD mRNA in several mouse tissues by in situ hybridization.

Figure 1

Section through an ovary. (A) After hybridization with the antisense probe, labeling can be detected over granulosa cells (GC) of growing follicles, as well as luteal cells in an adjacent corpus luteum (CL). (B) Adjacent section hybridized with the sense probe. Only diffuse labeling can be observed. X600.

Figure 2(A)

Section through the uterus hybridized with the antisense probe. Labeling can be seen on the luminal (L) and glandular (G) epithelial cells and on stromal cells (right arrow). (B) Consecutive section hybridized with the sense probe. Only a few dispersed silver grains are present. X600.

Figure 3(A)

Section through the vagina. Silver grains are detected over the epithelial (E) and stromal cells (S). (B) Control section hybridized with the sense probe. Only a few dispersed silver grains are present. X600.

Materials and Methods

Animals

Four adult male (26–30 g) and female (24–27 g) C57BL6 mice were housed under constant temperature (21 ± 1C) and light (lights on from 0600 to 2000 hr) regimen. The animals received Purina Chow (Ralston-Purina, St Louis, MO) and tap water ad libitum. The experiment was conducted in an animal facility approved by the Canadian Council on Animal Care and the Association for Assessment and Accreditation of Laboratory Animal Care. The study was performed in accordance with the Canadian Council on Animal Care Guide for Care and Use of Experimental Animals. The animals were all perfused between 9 and 10 hr for histological procedures as described in the following section. The females were on proestrus.

Figure 4(A)

Section through the mammary gland of a female mouse. Labeling can be observed in epithelial (E) cells of ducts as well as in stromal (S) cells at proximity of the ducts. (B) Control section hybridized with the sense probe. Only diffuse background is observed. X600.

Figure 5

Section through a clitoral gland. Strong labeling is present in the sebaceous cells of acini (A). (B) Control section hybridized with the sense probe. Only a few dispersed grains are observed. X600.

Figure 6(A)

Section through a testis. Accumulation of silver grains is observed over germ cells (G). The Leydig cells (L) being unlabeled. (B) In the central consecutive section hybridized with the sense probe, only dispersed silver grains can be seen. X600.

Figure 7(A)

Section through the prostate. Silver grains are found over the epithelial cells (E) bordering the lumen (L) as well as the stroma cells (S). (B) Adjacent section hybridized with the sense probe. Few dispersed silver grains can be observed. X600.

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-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 537 bp of mouse type 8 17β-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 (1 X 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 (15–20 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 0.5 X SSC for 30 min at 37C, followed by 90 min at room temperature in 0.5 X SSC, at 60C in 0.1 X SSC and finally for 30 min at room temperature in 0.1 X SSC.

The sections where then dehydrated and exposed to Kodak Biomax M _r films for 3–8 d before being coated with liquid photographic emulsion (Kodak-NTB2; diluted 1:1 with water). Slides were exposed for 7–45 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).

Results

After 3 to 8 days of exposure of the films, specific hybridization signal could be detected in the following tissues: ovary, oviduct, uterus, mammary gland, vagina, testis, prostate, clitoral gland, preputial gland, pituitary gland, adrenal gland, kidney, dorsal skin, and lung. No specific labeling have been observed in seminal vesicles, epididymis, jejunum, and colon. From photographic emulsion-coated sections, precise information could be obtained about the cell types expressing type 8 17β-HSD mRNA in the different tissues.

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 17β-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).

In the testis, a specific hybridization signal was observed over seminiferous tubules, Leydig cells being devoid of any specific labeling (Figure 6). Because we used frozen sections, a procedure that does not totally preserve the integrity of the tissues, it was not possible to identify the cell types expressing type 8 17β-HSD mRNA in the tubules. In the prostate, in situ hybridization signal was observed in both the epithelial cells bordering the acinar lumen and the stromal cells (Figure 7). In the preputial gland, which is homologous to the clitoral gland, specific labeling was similarly observed over the sebaceous cells forming the acini and the epithelium of the ducts.

In the pituitary gland, adrenal glands, kidney, liver, dorsal skin, and lung, the expression of type 8 17β-HSD was similar in both sexes. In the pituitary gland, specific autoradiographic reactions were detected over the cells of the three lobes (Figure 8). In the anterior and intermediate lobes, the vast majority of cells if not all the cells were labeled, whereas, in the posterior lobe, the silver grains were mostly detected over the pituicytes. In the adrenal glands, the three zonae of the cortex—namely glomerulosa, fasciculata, and reticularis zona—were specifically labeled (Figure 9). No specific reaction could be found in the adrenal medulla. In the kidney, hybridization signal was found in the epithelial cells of proximal convoluted tubules (Figure 10). In the liver, all the hepatocytes were expressing the enzyme mRNA (Figure 11). In the lung, type 8 17β-HSD mRNA was found to be expressed in bronchial epithelial cells and walls of pulmonary arteries (not shown). In the dorsal skin, specific labeling was detected in the stroma (not shown).

Figure 8(A)

Section through the pituitary gland of a female mouse. Silver grains are overlying the cells in the three lobes. (B) Control adjacent section. Very few grains can be detected. AL: anterior lobe; LL: intermediate lobe; PL: posterior lobe. X600.

Figure 9(A)

Section through the adrenal cortex of a male mouse. The hybridization signal is seen in cells in the three zonae of the adrenal cortex. G: glomerulosa; F: fasciculate; R: reticularis. (B) Consecutive section hybridized with the sense probe. A diffuse background can be observed. X650.

Figure 10(A)

Section through the kidney of a male mouse. Labeling is observed in the epithelial cells of proximal convoluted tubules (PCT). (B) Consecutive section hybridized with the sense probe. Only dispersed silver grains can be observed. G: glomerulus. X600.

In all the tissues expressing type 8 17β-HSD mRNA, hybridization with the radiolabeled sense probe generated only a light uniform background (Figures 1–11).

Discussion

The present data obtained by in situ hybridization clearly demonstrate that type 8 17β-HSD mRNA is expressed in reproduction organs and in several peripheral tissues in male and female mouse. This appears as the first report on the cellular localization of type 8 17β-HSD mRNA.

Figure 11(A)

Section through the liver of a female mouse. All the hepatocytes exhibit hybridization signal. (B) Control adjacent section hybridized with the sense probe. Only weak labeling can be detected. X600.

By Northern blot analysis, Fomitcheva et al. (1998) have detected type 8 17β-HSD mRNA in the ovary. By immunofluorescence, the same authors reported the presence of the type 8 17β-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 17β-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 17β-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 17β-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 17β-HSD) expression has been proposed as a mechanism involved in the pathogenesis of endometriosis (Bulun et al. 2002). In the mammary gland, type 8 17β-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 17β-HSD that, as with type 8 17β-HSD converts E2 to E1, has not been found (Mindnich et al. 2004). Studies to detect type 8 17β-HSD expression in human breast cancer is in progress in our laboratory. In clitoral glands, which are highly estrogen-dependent (unpublished data), type 8 17β-HSD mRNA is restricted to epithelial cells. This appears to be the first report on the expression of type 8 17β-HSD mRNA in this gland. Interestingly, type 7 17β-HSD, the enzyme that catalyzes the conversion of E 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 17β-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 17β-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 17β-HSD mRNA was localized in both epithelial cells of the acini and stroma cells. The role of type 8 17β-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 17β-HSD, is still unclear (Pelletier et al. 2004). ERβ 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 ERα or ERβ 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 17β-HSD could contribute to downregulate the levels of E2 in the epithelial cells of the gland, which also express ERα 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 17β-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 17β-HSD and aromatase (Sharpe 1998; Pelletier et al. 2004). In the adrenal cortex, type 8 17β-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 17β-HSD mRNA was confined to proximal tubules. This finding is in agreement with previous reports indicating that Ke 6 or type 7 17β-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 17β-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 17β-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 17β-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 17β-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 17β-HSD in the stroma.

In summary, we report for the first time the histological identification of the cell types expressing type 8 17β-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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