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Original Studies |
Department of Pathology (J.T., T.S., G.H., Y.M., H.N., H.S.) and Pediatrics (J.T., K.I.), Tohoku University School of Medicine, Sendai, Miyagi 980-8575; Research Institute of Life Science, Snow Brand Co. Ltd. (J.N., K.K., M.Y.), Tochigi 329-0512; and the Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University (N.H.), Toyoake, Aichi 470-1192, Japan; and the Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center (S.A.), Dallas, Texas 75235
Address all correspondence and requests for reprints to: Dr. Junji Takeyama, Department of Pathology, Tohoku University School of Medicine, 2–1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. E-mail: j-takeyama{at}patholo2.med.tohoku.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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17ß-Hydroxysteroid dehydrogenase (17ßHSD) isozymes regulate the
relative concentrations of potent 17ß-hydroxy and biologically weak
17ß-keto forms of estrogens and androgens locally at the target
tissue level. The type 1 isozyme of 17ßHSD (17ßHSD1), which is
associated with a high specificity for
C18-steroids, primarily converts the inactive
C18-steroid estrone (E1) to
the biologically active estrogen estradiol (E2)
(6, 7, 8). In contrast, attenuation of E2 action is
achieved by expression of the type 2 isozyme of 17ßHSD (17ßHSD2),
an enzyme that inactivates E2 to
E1 (8, 9, 10). 17ßHSD2 is also active on
androgens, and therefore, this enzyme is generally considered to play
important roles in the peripheral inactivation of androgens and
estrogens and in the maintenance of steady state levels of these
steroids in target tissues. In addition, 17ßHSD2 catalyzes the
20
-oxidation of the C21-steroid,
20-dihydroprogesterone, to the active progestin, progesterone
(9, 10, 11).
We have previously reported the immunolocalization of 17ßHSD1 and 17ßHSD2 protein in the human placenta (12). 17ßHSD1 immunoreactive protein is localized to the syncytiotrophoblast, where the active estrogen, E2, is produced and secreted into the maternal circulation. On the other hand, 17ßHSD2 protein is detected in the endothelial cells of capillaries and veins of the chorionic villi, where E2 is converted to E1 before entering the fetal circulation. Therefore, 17ßHSD2 in the human placenta can serve as a barrier or gatekeeper to the passage of excessive, maternally derived estrogens into the fetal circulation.
The interconversion of E1 and E2 has been demonstrated in rat and human adult tissues (13). Moreover, the cell type-specific expression of 17ßHSD2 immunoreactive protein has been detected in hepatocytes of the fetal liver (14). However, the enzyme activity and expression of 17ßHSD isozymes in the human fetus have not been examined systematically. In this study, we examined the tissue distribution of 17ßHSD activities using 14C-labeled E2 or E1 as a substrate in homogenates from various human fetal tissues.
We then studied the expression of messenger ribonucleic acid (mRNA) transcripts for 17ßHSD1 and 17ßHSD2 using Northern blot analysis. In addition, we examined these isozymes by RT-PCR using fluorescent dye-labeled primers. Furthermore, we used immunohistochemistry to study the cellular distribution of 17ßHSD2 protein in the human fetus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human fetal tissues (11–21 weeks gestational age) were obtained after elective termination in normal pregnant women at Tohoku University Hospital and Nagaike Maternal Clinic (Sendai, Japan). This research protocol was approved by the committee on the ethics of Tohoku University School of Medicine, and informed consent was obtained from these pregnant women before elective termination. The ages of the fetuses were estimated by the last menstrual date, body weight, or crown-rump length. Human fetal tissues of 26, 30, 36, and 38 weeks of age were obtained at the time of autopsy at Tohoku University Hospital. The time elapsed from fetal death to the removal of the tissues ranged from 0.5–1 h in the case of elective termination and from 2–6 h in autopsy cases. These samples did not have any significant histopathological abnormalities. The specimens for enzyme assays and RNA isolation were snap-frozen and stored at -80 C, and those for immunohistochemistry were fixed in 10% neutral formalin for 18 h at 4 C and embedded in paraffin. Due to the availability of the specimens, enzyme assay, Northern blotting, and RT-PCR analysis could not be performed in the same fetal specimen. Among the cases examined for enzyme assay, Northern blotting, and RT-PCR analysis, one of the three cases (21 weeks) in which enzyme assay was analyzed and one of the two cases (20 weeks) in which RT-PCR analysis was performed were also evaluated by immunohistochemistry.
Enzyme assay
The specimens used in this study were obtained from fetuses aged 20 weeks (male), 20 weeks (female), and 21 weeks (female). Human fetal tissues were homogenized at 4 C in phosphate buffer (100 mmol/L KCl, 10 mmol/L KH2PO4, 10 mmol/L Na2HPO4, and 1 mmol/L ethylenediamine tetraacetate, pH 7.5) using a Potter-Elvehjam glass-Teflon homogenizer and centrifuged for 15 min at 1000 x g. Aliquots of the supernatant were incubated for 30 min at 37 C in 0.5 ml phosphate buffer containing 1 µmol/L 14C-labeled E2 or E1 and 10 mmol/L NAD+, NADH, NADP+, or NADPH. Incubations were terminated and extracted with 2 ml diethyl ether. The organic phase was evaporated, and the residue was subjected to TLC; the developing solvent was cyclohexane-ethyl acetate (1:1, vol/vol). The chromatoplates were autoradiographed for 18 h at room temperature, and the radioactivity signals were detected and quantitated using a GS-250 Molecular Imager (Bio-Rad Laboratories, Inc., Hercules, CA). The protein content of the tissue homogenates was measured by the method of Lowry (15) using BSA as a standard. The differences in 17ßHSD activity among various fetal organs were statistically evaluated using Bonferroni’s test. The cofactor preference for the oxidative and reductive reactions of 17ßHSD was also studied and evaluated with Student’s t test. P < 0.01 was considered significant.
Northern blot analysis
The RNA used in this study was extracted from a female fetus (20 weeks gestation). The procedures employed in this study were previously described in detail by us (10). In brief, total RNA was purified from frozen tissue using the RNA STAT-60 System (Tel-Test, Friendswood, TX) according to the manufacturer’s instructions. Ten micrograms of total RNA were size fractionated by electrophoresis, transferred to a nylon membrane, and hybridized with complementary DNA (cDNA) probes under high stringency conditions as described.
Analysis of the mRNAs for 17ßHSD types 1 and 2 by RT-PCR with a fluorescent primer
RNA was extracted from fetuses aged 13 weeks (male) and 20 weeks
(female). Semiquantitative analysis of 17ßHSD1, 17ßHSD2, and
ß-actin mRNAs was carried out in the total RNA fractions from various
human fetal tissues by RT-PCR using a specific pair of fluorescent
dye-labeled primers and their standard RNAs as previously described
(16, 17). Oligonucleotide primers for RT-PCR were synthesized as shown
in Table 1
. The coding sequence between the
two PCR primer sites is located 5' upstream of the RT primer site in
each cDNA for 17ßHSD1, 17ßHSD2, and ß-actin. The standard RNAs
for 17ßHSD1, 17ßHSD2, and ß-actin were synthesized in
vitro with T7 RNA polymerase using their cDNA as templates,
purified, and then spectrophotometrically quantitated. Total RNA
(0.2–2 µg) and standard RNAs (0.02–2 amol) were subjected to RT
with Superscript II reverse transcript (Life Technologies, Inc., Grand Island, NY) and a specific antisense primer at 47 C
for 45 min. The resulting cDNAs were amplified by PCR using a specific
pair of fluorescent dye-labeled sense primers and antisense primers for
23–28 cycles. The fluorescent PCR products were analyzed on a 2%
agarose gel with a Gene Scanner 362 Fluorescent Fragment Analyzer
(Perkin-Elmer Corp., Foster City, CA). Relative amounts
for 17ßHSD1, 17ßHSD2, and ß-actin mRNAs in the tissue RNA were
calculated from the peak areas of the fluorescent PCR products of a
specific mRNA and its standard RNA.
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The monoclonal antibody, mAb-C2–12 (subclass IgG1/
), was
produced by immunizing mice with a synthetic carboxyl-terminal peptide
corresponding to amino acids 375–387 of 17ß-HSD2. The detailed
characterization of this monoclonal antibody has been previously
reported (14).
Immunohistochemistry
The gestational age of the fetus examined ranged from 11–38 weeks. Paraffin-embedded tissues (3.0-µm sections) were mounted onto clean silane-coated glass slides (Matsunami Co. Ltd., Tokyo, Japan). After deparaffinization, the sections were pretreated by hydrated autoclaving in 10 mmol/L sodium citrate buffer (pH 6.0) at 120 C for 5 min to retrieve the antigenicity (18). The slides were then immersed in methanol containing 0.3% hydrogen peroxidate for 30 min to block endogenous peroxidase activity. The immunohistochemical staining procedure employed in this study was the streptavidin-biotin amplified method. We used peroxidase-conjugated streptavidin from a Histofine Kit (Nichirei Co., Tokyo, Japan) and visualized the antigen-antibody complex with 3,3'-diaminobenzidine. Normal rabbit serum was incubated for 30 min at room temperature to decrease nonspecific staining. Sections were then incubated with the primary antibody for 17ß-HSD2 at a concentration of 1:5 for 18 h at 4 C. After washing, the sections were incubated with biotinylated rabbit antimouse Ig and then with peroxidase-conjugated streptavidin, followed by incubation in a solution containing 0.05% hydroxychloride (pH 7.6), 0.06 mmol/L 3,3'-diaminobenzidine, and 2 mmol/L hydrogen peroxide. Immunoreactive sections were counterstained with hematoxylin and mounted with a glycerol-gelatin water-soluble medium. As negative controls for immunostaining, the sections were incubated with normal mouse IgG instead of primary antibodies. No specific immunoreactivity was detected in these sections.
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Results |
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The levels of estrogenic 17ßHSD activity measured in various
human fetal tissues are demonstrated in Table 2. The rates of estrogenic 17ßHSD activity
detected in the placenta and liver were statistically higher than those
in other fetal tissues (P < 0.0001). 17ßHSD activity
in placenta was significantly higher than that in liver
(P < 0.0001). Low, but significant, levels of 17ßHSD
activity were also detected in the stomach, small intestine
(P < 0.01 compared to 17ßHSD activity in brain,
adrenal gland, heart, and lung), colon, and kidney. 17ßHSD activity
in the brain and adrenal gland was extremely low. The specimens from
heart and lung showed no detectable 17ßHSD activity under the
experimental conditions used. For cofactor preference, the enzyme
present in the liver, gastrointestinal tract, and kidney responsible
for 17ßHSD activity significantly preferred NAD(H) to NADP(H)
(P < 0.001).
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Expression of the 1.3-kb mRNA for 17ßHSD1 was detected only in
the placenta (Fig. 1). On the other hand, the
1.5-kb mRNA for 17ßHSD2 was markedly detected in the placenta, liver,
ileum, jejunum, and rectum (Fig. 1). In addition, relatively low levels
of 17ßHSD2 mRNA hybridization signals were detected in the stomach,
duodenum, sigmoid colon, uterus, kidney, and urinary bladder.
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Results of the semiquantitative analysis of 17ßHSD1 and
17ßHSD2 mRNAs are summarized in Table 3.
All quantitated data are expressed on the basis of ß-actin mRNA
levels. The greatest amount of 17ßHSD1 mRNA was detected in the
placenta, whereas the brain, lung, heart, and kidney expressed
relatively lower amounts of this gene. 17ßHSD1 expression in the
adrenal gland, liver, and gastrointestinal tract was extremely low. On
the other hand, a relatively large amount of 17ßHSD2 mRNA was
detected in the placenta, liver, kidney, and gastrointestinal tract.
The expression of the 17ßHSD1 gene was predominant in the placenta,
brain, heart, lung, and adrenal gland, whereas that of 17ßHSD2 was
much more dominant in the liver, gastrointestinal tract, and
kidney.
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Results are summarized in Table 4. In
small intestine and liver, immunoreactivity of 17ßHSD2 was marked
from early gestational weeks and throughout the course of gestation. In
the stomach, colon, and kidney, 17ßHSD2 immunoreactivity was not
detected in the early stages of gestation; it was detected and
subsequently increased in intensity from mid to late stages of
gestation. 17ßHSD2 immunoreactivity was detected in surface
epithelial cells of the stomach, but not in gastric glands (Fig. 2a). In the small intestine
and colon, 17ßHSD2 immunoreactivity was detected in absorptive
epithelial cells (Fig. 2, b and c). No immunoreactivity was seen in the
lamina propria, muscularis mucosae, or the submucosal layers of the
stomach and intestine. Hepatocytes in the fetal liver demonstrated
marked immunoreactivity for 17ßHSD2 (Fig. 2d). The immunolocalization
of 17ßHSD2 in the kidney was exclusively present in interstitial
cells surrounding the urinary tubules of the renal medulla (Fig. 2, e
and g), but not in the urinary tubules and glomerulus (Fig. 2f).
Immunoreactivity for 17ßHSD2 was analyzed in skin at 20 gestational
weeks and was detected only in sebaceous glands, not in the epidermis
(Fig. 2h).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we spectrophotometrically examined the mRNA for 17ßHSD1 and 17ßHSD2 in various human fetal tissues by RT-PCR using fluorescent dye-labeled primers for semiquantitation of the transcripts. We could not examine them in the same fetal tissues, but 17ßHSD1 and -2 transcripts that appeared weak or absent on Northern blot analysis were detected by RT-PCR. For instance, 17ßHSD1 mRNA, which could not be detected by Northern blot analysis, was observed in almost all tissues examined by RT-PCR. 17ßHSD1 was predominantly expressed in the brain, heart, lung, and adrenal. These results suggest that estrogens are locally activated in these tissues and possibly play crucial roles in the control of growth and/or differentiation. On the other hand, relatively high levels of 17ßHSD2 expression were detected in the liver, gastrointestinal tract, and kidney, which is consistent with the results from our Northern blot analysis. However, the number of cases examined in the mRNA study was limited, and further investigations are required for clarification.
We also characterized 17ßHSD2 localization in human fetal tissues using immunohistochemistry. Among gastrointestinal tract tissues in which 17ßHSD2 mRNA transcripts were detected, 17ßHSD2 immunoreactivity was detected in surface epithelial cells of the stomach and absorptive epithelial cells of the small intestine and colon. 17ßHSD2 enzyme in epithelial cells of the gastrointestinal tract has been suggested to be involved in the inactivation of E2 before its absorption (20). Intense 17ßHSD2 immunoreactive protein was detected in the small intestine in early stages of gestation. 17ßHSD2 in the small intestine may be involved in the inactivation of E2 in swallowed amniotic fluid. In the stomach and large intestine, relative 17ßHSD2 immunointensity increased from mid to late stages of gestation as the concentrations of estrogens in amniotic fluid increased (4). Hepatocytes in the liver from early stages of gestation demonstrated marked 17ßHSD2 immunoreactivity, which probably plays a major role in E2 inactivation from the fetal portal circulation. We previously reported that 17ßHSD2 was immunolocalized in the endothelial cells of capillaries in chorionic villi, which prevent excessive E2 from entering into the fetal circulation (12). Therefore, large quantities of E2 derived from the maternal circulation and amniotic fluid are inactivated by 17ßHSD2 present in the placenta, stomach, small intestine, colon, and liver before entering the fetal systemic circulation and targeting various fetal tissues.
In the kidney, the expression of 17ßHSD2 immunoreactive protein was present in interstitial and stromal cells surrounding the urinary tubules, but not in the tubules or glomerulus. The possible physiological role of 17ßHSD2 in the human fetal kidney may include the inactivation of E2, possibly leaking from the urinary tubules because of their immaturity. However, further investigations are required to clarify the biological significance of 17ßHSD2 in this tissue. In the skin, 17ßHSD2 was present only in sebaceous glands, not in the epidermis. Therefore, estrogens in amniotic fluid may enter the fetal circulation by diffusion through fetal skin. 17ßHSD2 is highly expressed in sebaceous glands of adult skin and regulates sebum production (21). The present results indicate that sebum production occurs in human fetal skin from as early as 20 weeks gestation.
At present, six isozymes of 17ßHSD have been identified. 17ßHSD
type 3 is predominantly expressed in the testis and converts the
inactive C19-steroid androstenedione to the
active androgen testosterone (22). 17ßHSD type 4, which is expressed
in many tissues, with the highest levels in liver, heart, prostate, and
testis, has been shown to convert E2 to
E1 and androstenediol to dehydroepiandrosterone
(23). 17ßHSD type 5 is an aldoketoreductase that preferentially
reduces androgens and estrogens using NADPH as a cofactor (24).
17ßHSD type 6, isolated from rat ventral prostate, preferentially
oxidizes 3-androstanediol to androsterone using
NAD+ as a cofactor and oxidizes testosterone and
estradiol (25). It has been previously reported that 17ßHSD type 1,
2, 4, and 5 isozymes were detected by Northern blots of whole mouse
embryos, and each of the enzymes showed a unique pattern of expression
(26). Further investigations of 17ßHSD isozymes are required to
clarify the regulation of various sex steroids in human fetal
tissues.
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Acknowledgments |
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Footnotes |
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Received July 6, 1999.
Revised October 6, 1999.
Accepted October 14, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxysteroid dehydrogenase activity. J Biol
Chem. 169:12964–12969.
-hydroxysteroid dehydrogenase activities
of human placental microsomes: kinetic evidence for two enzymes
differing in substrate specificity. Arch Biochem Biophys. 239:206–215.[CrossRef][Medline]
-hydroxysteroid
dehydrogenases from rat and human prostate. J Biol Chem. 272:15959–15966.This article has been cited by other articles:
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  Y. Miki, T. Nakata, T. Suzuki, A. D. Darnel, T. Moriya, C. Kaneko, K. Hidaka, Y. Shiotsu, H. Kusaka, and H. Sasano Systemic Distribution of Steroid Sulfatase and Estrogen Sulfotransferase in Human Adult and Fetal Tissues J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5760 - 5768. [Abstract] [Full Text] [PDF]
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J. Takeyama, T. Suzuki, S. Inoue, C. Kaneko, H. Nagura, N. Harada, and H. Sasano Expression and Cellular Localization of Estrogen Receptors {{alpha}} and {beta} in the Human Fetus J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2258 - 2262. [Abstract] [Full Text]
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