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Biocenter Oulu, World Health Organization Collaborating Center for Research in Human Reproduction (M.V.J.M., V.V.I., M.H.P., R.K.V., P.T.V.), and the Departments of Clinical Chemistry (M.V.J.M., V.V.I., M.H.P., R.K.V., P.T.V.), Obstetrics and Gynecology (T.V., J.T.), and Pathology (F.S.), University of Oulu, FIN-90220 Oulu, Finland
Address all correspondence and requests for reprints to: Dr. Pirkko T. Vihko, Department of Clinical Chemistry, University of Oulu, Kajaanintie 50, FIN-90220 Oulu, Finland. E-mail: pvihko{at}whoccr.oulu.fi
Abstract
According to the current hypothesis, 17ß-hydroxysteroid dehydrogenases (17HSDs) regulate the extent of estrogen influence in the endometrium by converting estradiol (E2) locally into a biologically less active sex steroid, estrone (E1), and vice versa. Recently, we have shown that both 17HSD type 1 and type 2 are expressed in the human endometrium, and in the present work, using in situ hybridization, we show that 17HSD type 2 is localized in the glandular epithelial cells as previously shown for the type 1 enzyme, but in contrast to type 1, the expression of type 2 is highest at the end of the cycle. Hence, we hypothesize that the differential expression of the two 17HSD enzymes, with opposite activities in same cell types, could modulate intracellular E2 concentrations during the end of the luteal phase of the menstrual cycle. We further analyzed the expression of 17HSD type 1 and type 2 mRNAs in term human placenta. Expression of 17HSD type 1 mRNA was detected in the syncytiotrophoblasts, and signals for type 2 mRNA were found inside the villi, corresponding to cytotrophoblasts. The expression of 17HSD type 2 in the placenta may serve to maintain the presence of inactive sex steroids and attenuate the formation of biologically potent androgens and estrogens.
THE PHYSIOLOGICAL activities of estrogens
and androgens are regulated by redox reactions at position C-17 by
17ß-hydroxysteroid dehydrogenases (17HSDs), which are responsible for
the interconversion of low activity sex steroids such as estrone
(E1), androstenedione, and 5
-androstanedione
to more potent forms: estradiol (E2),
testosterone (T), and 5-dihydrotestosterone. To date, six different
17HSDs have been characterized (1, 2, 3, 4, 5, 6). Type 1 and type 3 enzymes prefer
the reduction of 17-ketosteroids to 17-hydroxy forms, and the enzymes
have been shown to be associated with gonadal sex steroid
E2 and T production in the ovary and testis,
respectively (3, 7, 8, 9, 10). Type 2 and type 4 enzymes, present in various
classical and nonclassical steroid hormone target tissues, catalyze a
reaction opposite that catalyzed by the type 1 and type 3 enzymes,
thereby oxidating E2 to E1,
and T to androstenedione (2, 4, 10, 11, 12, 13). However, according to the
results of recent studies, the type 4 enzyme prefentially catalyzes the
dehydrogenase and hydratase reactions involved in ß-oxidation of
fatty acids and bile acid intermediates (12, 13). This together with
data showing that 17HSD type 2 is abundantly expressed in several
extragonadal tissues (10, 14) suggest that of all the 17HSD enzymes
characterized, 17HSD type 2 has the most substantial role in the
inactivation of female sex steroids.
Recently, it has been demonstrated that both 17HSD type 1 and type 2 are expressed in the human endometrium, and that the expression of the enzymes in endometrial epithelial cells is regulated in a progestin-dependent manner (10, 15, 16, 17). According to the current hypothesis, 17HSDs regulate the extent of estrogen influence in the endometrium by converting E2 locally into a biologically less active sex steroid, E1, and vice versa. In line with the expression of 17HSD type 1 and type 2 enzymes in the endometrium, 17HSD activity is present mainly in the glandular cells (18).
The aim of the present work was to investigate, using in situ hybridization, the expression and localization of 17HSD type 2 in human endometrial tissue at various times during the menstrual cycle. Furthermore, activity data suggest that both type 1 and type 2 enzymes are expressed in the human placenta (19), but until now only the localization of 17HSD type 1 is known in the placenta (15, 20). To reveal in which cell types 17HSD type 2 is expressed in the placenta, the expressions of type 1 and type 2 mRNAs were compared in term placental tissue.
Materials and Methods
Chemicals and reagents
Radiolabeled [-35S]deoxy-CTP (1300 Ci/mmol) was
purchased from DuPont-New England Nuclear (Boston, MA). T7 and SP6 RNA
polymerases were obtained from Promega (Madison, WI), and proteinase K
and transfer RNA were purchased from Boehringer Mannheim (Mannheim,
Germany). Other reagents not mentioned were either purchased from Sigma
Chemical Co. (St. Louis, MO) or Merck (Darmstadt, Germany) and were of
the highest purity grade available.
Tissue specimens
Twenty-one tissue samples were collected on different days of the menstrual cycle by curettage from regularly cycling women undergoing tubal sterilization. Three additional endometrial samples were obtained from hysterectomies. The phase of the menstrual cycle was estimated histologically by hematoxylin-eosin staining and measurement of serum progesterone (P) concentrations. Placental tissue specimens were obtained from healthy mothers at full-term delivery. Small intestine samples were obtained from patients operated upon because of intestinal carcinoma. They were examined microscopically to ensure that they did not contain any carcinoma tissue. Approval for the study was obtained from the ethical comittee of the Faculty of Medicine and the University Hospital of Oulu.
All tissue specimens were briefly washed twice with 1 x phosphate-buffered saline, fixed overnight at 4 C in 4% paraformaldehyde and phosphate-buffered saline, dehydrated, and embedded in paraffin (solidification point, 51–53 C; Merck). Thereafter, 7-µm sections were cut and collected on glass slides. The sections were dewaxed with xylene, and before hybridization, reactive aldehyde groups remaining after fixation were eliminated by 10-min treatment in 0.1 M glycine and 0.2 M Tris-HCl, pH 7.4.
In situ hybridization
A 376-bp fragment (nucleotides 1–376) of human 17HSD type 1
cDNA (1) and a 380-bp fragment (nucleotides 191–570) of human 17HSD
type 2 cDNA (2) were cloned in pGEM-4Z plasmids (Promega) and used as
templates for in vitro transcription. Sense and antisense
[-35S]CTP-labeled RNA probes were transcribed with T7
or SP6 RNA polymerases, using linearized plasmids as templates. The
in situ hybridization reactions were performed as previously
described by Chotteau-Lelievre et al. (21) and Mustonen
et al. (22, 23).
Results
Desquamative and proliferative endometrium
During the desquamative and early proliferative phase of the
endometrium (days 2–9), 17HSD type 2 was undetectable (Table 1
and Fig. 1A). However, in the late proliferative
phase (days 10–14), weak to moderate expression of type 2 mRNA was
detected in the epithelial cells of some glands (Table 1 and Fig. 1B).
The serum P concentrations in the patients during desquamative and
proliferative stages ranged from 0.2–1.5 nmol/L.
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During the early secretory phase (days 15–19), the expression of
17HSD type 2 mRNA increased in the glandular epithelial cells (Table 1
and Fig. 1C). In the midsecretory phase (days 22–25), the expression
of type 2 mRNA was strongly increased in the epithelium of all glands
(Table 1 and Fig. 1D). In the late secretory phase (days 26–29), 17HSD
type 2 mRNA expression in the epithelium of the glands remained at a
high level, slightly decreasing toward the end of the cycle (Table 1
and Fig. 1E). The serum P concentrations in the patients during
secretory phase ranged from 5–34 nmol/L.
Term placenta and small intestine
In line with previous immunohistochemical results (15), strong
expression of 17HSD type 1 mRNA was detected in the
syncytiotrophoblasts in term placenta (Fig. 2A). In contrast, 17HSD type 2 mRNA was
moderately expressed inside the villi and was localized to
cytotrophoblasts (Fig. 2B). Hence, the data indicate different cellular
localization of 17HSD type 1 and type 2 mRNAs in human placenta. In the
small intestine, which was used as a positive control for 17HSD type 2,
strong expression of type 2 mRNA was detected in the surface epithelium
(Fig. 2C). This is in line with data recently obtained using mouse
intestine (23). The spot-like pattern that was detected in inner layers
of the villi was also seen with the sense control probe (data not
shown). Human 17HSD type 1 was not detected in the small intestine
sections (data not shown).
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The present results show that the intensity of 17HSD type 2 mRNA expression varies in the endometrium with the stage of the menstrual cycle, and when present, the mRNA signal is detected in the epithelial cells of the glands, whereas the stroma is negative. In addition and differing slightly from previous results (17), the expression of 17HSD type 2 mRNA was not directly related to a rise in serum P concentrations, unlike 17HSD type 1 enzyme expression, which follows serum P concentrations (15). Hence, the expression of type 2 mRNA appeared in the late proliferative phase before type 1 enzyme could be detected in a previous study (15). However, the mRNA expression of 17HSD type 2 was further increased when E2-synthetizing type 1 enzyme expression vanished after the early/midsecretory phase (15). These data are in line with the results of previous studies showing that oxidative 17HSD activity, mainly present in the glandular endometrium, increases toward the end of the menstrual cycle (18, 24, 25, 26, 27). In addition, P increases E1 sulfotransferase expression and decreases the synthesis of estrogen receptors (ERs) in human endometrium (28, 29, 30, 31). Hence, we hypothesize that the differential expression of the two 17HSD enzymes, with opposite activities, modulates intracellular E2 concentrations during the luteal phase and thereby decreases the influence of E2 together with reduced ER concentrations and increased E1 sulfotransferase expression.
Previous activity measurements have shown that both reductive and oxidative 17HSD activities are present in the human placenta (19). Histochemical studies have, furthermore, shown that 17HSD activity is present in the trophoblasts and endoderm of the yolk sac (32, 33). Other steroidogenic enzymes, such as cytochrome P450 side-chain cleavage enzyme and 3ßHSD type 1 are also expressed in trophoblastic cells of the placenta (34, 35). In line with the results of previous immunohistochemical studies and Northern analyses (15, 20, 36), we showed that 17HSD type 1 is localized in the placental syncytiotrophoblasts. It has been assumed that the major endocrine functions of the placenta are restricted to the syncytial layer of trophoblasts (37, 38), but in this study we now show that the sex steroid-inactivating 17HSD type 2 enzyme is expressed in the cytotrophoblasts of human placenta. Our results are in line with those of previous RNAse protection assay studies (36), in which type 2 mRNA expression was only barely detected in fresly isolated cytotrophoblasts, and it vanished as the cells were further cultured. This could be due to the fact that during the maturation of cytotrophoblasts, cells lose mitotic activity and fuse, resulting in the formation of syncytiotrophoblasts, a terminally differentiated state of the trophoblast lineage (39). Further, this is the first study showing that 17HSD type 1 and type 2 are expressed in different cell types of the human placenta, but additional studies are needed to evaluate the role of enzymes during gestation.
It is well known that both T and 5-dihydrotestosterone are needed
for the normal growth and differentiation of the male genital tract.
These highly active male sex steroids are locally synthesized in the
fetal gonads by 17HSD type 3 and 5-reductase, respectively (3, 40, 41). Our preliminary results show that human 17HSD type 2 has a higher
efficiency toward androgens than estrogens in cultured cells
(unpublished data). Thus, the presence of 17HSD type 2 in the placenta
may serve to inactivate T in order to maintain low concentrations of
active androgens in the maternal circulation. In addition, at least
human 17HSD type 2 also has 20-hydroxysteroid dehydrogenase activity
both in vitro and in vivo (2), but the role of
this activity in the placenta and in the maintenance of normal
gestation as well as during the menstrual cycle remains to be studied
in more detail.
In line with the results of previous Northern analyses (10, 17), we detected no 17HSD type 1 expression in the small intestine. However, similar to that recently shown for the mouse enzyme (23), 17HSD type 2 was strongly expressed in the luminal surface epithelial cells of the small intestine. The expression of 17HSD type 2 in luminal epithelial cells of both mouse and human small intestine raises the possibility that the type 2 enzyme may have a role in the inactivation of sex steroids, and possibly steroid-like compounds, found in the digestive system. It is well known that orally administered E2 and T are inactivated rapidly and thereby do not enter the circulation in significant amounts. Hence, it is possible that 17HSD type 2 is one of the key enzymes involved in the rapid degradation and excretion of steroids in surface epithelial cells in the intestine. Estrogens are needed in the regulation of fatty acid-binding protein levels in the small intestine and thereby in the regulation of fatty acids to be absorbed (42). In addition, estrogens are involved in normal gastrointestinal motility (43). Furthermore, recent work has revealed the presence of sex steroid receptors in the stromal elements, but not in intestinal epithelial cells (44). Thus, additional studies are needed to characterize the roles of estrogens/ERs/17HSD type 2 in gastric physiology and pathology.
Acknowledgments
We thank Ms. Liisa Kaarela for her expert technical assistance with in situ hybridizations.
Footnotes
1 This work was mainly supported by the Research Council for Health of
the Academy of Finland (Project 3314) and the Sigrid Jusélius
Foundation (to J.T. and T.V.). M.V.J.M. was also supported by the
Finnish Cancer Society and the Research and Science Foundation of
Farmos. The Department of Clinical Chemistry is a WHO Collaborating
Center for Research in Human Reproduction supported by the Ministries
of Education, Social Affairs and Health, and Foreign Affairs,
Finland. 
2 Present address: Department of Physiology, Institute of
Biomedicine, University of Turku, Kiinamyllynkatu 10, FIN-90520 Turku,
Finland.
Received September 10, 1997.
Revised November 20, 1997.
Accepted January 22, 1998.
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