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Original Articles |
Department of Woman and Child Health, Division for Obstetrics and Gynecology (G.S., B.v.S.), Department of Plastic Surgery (M.W.), and Department of Pathology, Division for Clinical Cytology (L.S.), Karolinska Hospital, S-171 76 Stockholm, Sweden; and Biocenter Oulu and Department of Clinical Chemistry, University of Oulu (M.P., R.V.), FIN-90220 Oulu, Finland
Address all correspondence and requests for reprints to: Gunnar Söderqvist, M.D., Ph.D., Department of Woman and Child Health, Division for Obstetrics and Gynecology, Karolinska Hospital, S-171 76 Stockholm, Sweden.
Abstract
Our purpose was to assess 17ß-hydroxysteroid dehydrogenase (17HSD) type 1 protein expression in normal breast tissue during the menstrual cycle and hormonal contraception. We analyzed 17HSD type 1 protein expression by immunohistochemistry during the regular menstrual cycle (n = 12) and hormonal contraception (n = 7) in women undergoing reduction mammoplasty. 17HSD type 1 protein was detected in normal breast epithelial cells throughout the menstrual cycle and in all women using hormonal contraception. Mean 17HSD type 1 staining intensity was higher in alveolar epithelial cells in women using hormonal contraception (2.14) than in untreated women (1.25; P < 0.04). For ducts, this difference approached significance (2.29 vs. 1.41; P = 0.06). There was a negative correlation between serum estradiol (E2) levels and 17HSD type 1 protein expression for both alveolar (rs = -0.68; P = 0.004) and ductal (rs = -0.75; P = 0.002) breast epithelial cells.
Enhanced 17HSD type 1 protein expression might increase the conversion to E2 in normal breast tissue during hormonal contraception. The negative correlation between serum E2 levels and 17HSD type 1 suggests this enzyme to be one of the regulatory mechanisms of intratissue E2 concentration in normal breast tissue.
ESTROGEN is generally accepted to be a promoter of breast epithelial cell proliferation and to be involved in the development and growth of breast cancer (1, 2). Recent data have indicated that estrogen action can be strongly affected by hormone metabolism and biosynthesis in peripheral target tissues (3). Thus, plasma concentrations of estrogens do not totally reflect their biological potency at the target tissue level. One of the key enzymes regulating estrogen action is 17ß-hydroxysteroid dehydrogenase (17HSD), which catalyzes the interconversion between the low active estrone (E1) and the highly active estradiol (E2). In the endometrium, the reaction is mainly oxidative, favoring the inactivation of E2 to E1 (4). However, in the granulosa cells of the ovary, 17HSD activity is an essential step in E2 biosynthesis, thereby favoring the activation of E1 to E2. It was for a long time obscure why the enzyme was favoring E2 formation in some and E1 formation in other organs and conditions (5). Some clarification has been achieved by the recent characterization of four human 17HSD type 1–4 enzymes, each with specific properties (6, 7, 8). Current data suggest that from the characterized 17HSD enzymes, types 1 and 2 are most closely associated with estrogen metabolism (3). The human type 1 enzyme is cytosolic and prefers E1 as a substrate, whereas 17HSD type 2 is a membrane-bound enzyme capable of using both estrogens and androgens (6) and, to a lesser extent, progestogens as substrates. These two enzymes predominantly catalyze opposite reactions: type 1 converts E1 to E2, and type 2 converts E2 to E1 (6, 9).
In the present study, 17HSD type 1 protein expression was assessed in apparently normal breast tissue from women undergoing reduction mammoplasties during the menstrual cycle and hormonal contraception. In addition, estrogen (ER) and progesterone receptor (PR) content and sex steroid serum levels were analyzed.
Experimental Subjects
The clinical material comprised 19 premenopausal women (mean age, 34 yr; range, 21–50 yr) undergoing reduction mammoplasty for inconvenient breast size. Twelve of the women of fertile age (mean age, 38 yr; range, 22–50 yr) had regular menstrual cycles, and seven women (mean age, 26 yr; range, 21–38 yr) were subject to hormonal contraception. None of the normally cycling women had taken any sex steroid-containing drugs during the last 6 months preceding the study. In addition, three untreated healthy postmenopausal women, aged 52, 52, and 59 yr were investigated.
Venous blood samples and anamnestic menstrual cycle data were collected on the day of surgery. E2, progesterone, and PRL were analyzed by routine hospital methods. The study was approved by the local ethics committee, and all women gave informed consent.
Materials and Methods
Immediately after surgical removal, the tissue samples were washed with saline and frozen at -70 C until analyzed.
Immunohistochemistry
17HSD type 1. The breast specimens were sectioned to 5–7 µm at -20 C and thaw-mounted on poly-L-lysine-coated glass slides. Tissue sections were treated with 0.01 mol/L phosphate-buffered saline (PBS)-10% FCS (pH 7.4) for 30 min at room temperature to block nonspecific binding. The polyclonal antibody used in this study has been characterized in detail previously (10, 11, 12).
The antiserum raised against 17HSD type 1 (1:200 dilution in PBS-10% FCS) was added to the slides, and they were incubated overnight at 4 C. The slides were washed three times for 5 min each time with PBS. Thereafter, the sections were incubated at room temperature for 2 h with a 1:50 dilution of biotinylated goat antirabbit antibody (Dako Laboratories, Copenhagen, Denmark). The slides were washed three times for 5 min each time with PBS, and a 1:50 dilution of fluorescein isothiocyanate-conjugated streptavidin (Dako Laboratories, Copenhagen, Denmark) was added. After incubation for 1 h at room temperature, the sections were washed five times with PBS, 5 min each time, and mounted (Immu-Mount, Shandon, PA). Specific staining was controlled by staining the sections with preimmune serum or by preabsorbing the antiserum with highly purified 17HSD type 1 before staining.
Sex steroid receptors. Staining was performed on formalin-fixed, paraffin-embedded tissues. The basic staining procedure applies an avidin-biotin-peroxidase method modified for antigen retrieval from paraffin-embedded tissue (13). The ER and PR analyses were performed with reagents supplied by Abbott Laboratories (North Chicago, IL).
Quantification of immunohistochemical staining
Immunostained cells were quantified by cell counting in sections by an observer blinded to treatments. Epithelial cells lining the alveoli/terminal ducts and major ducts were considered separately. Labeled cell cytoplasm for 17HSD type 1, and nuclei for steroid receptors were graded as unlabeled (0), weakly labeled (1+), moderately labeled (2+), or intensely labeled (3+). At least 100 cells/slide were counted at 3 different sites for each combination of tissue site and stain type.
Statistical analysis
Differences were assessed by the Wilcoxon rank sum test for unpaired observations. For correlation analyzes, Spearman’s rank correlation coefficient was calculated.
Results
The summary of the results for 17HSD type 1, ER, and PR stainings
is given in Table 1
. The data showed that
in breast tissue, 17HSD type 1 was detected exclusively in epithelial
cells throughout the menstrual cycle, and that the staining intensity
was similar in alveoli and ducts, with no statistical differences
between the two structures. Typical stainings for the enzyme in the
normal breast are illustrated in Fig. 1.
In specimens taken during the follicular phase, weak to moderate 17HSD
type 1 staining was observed. Luteal phase staining varied from weak to
strong, and staining for the enzyme was also found in the late luteal
phase. Furthermore, staining for 17HSD type 1 was observed in two of
the three postmenopausal women.
|
|
). Alveolar 17HSD type 1 staining
intensity was significantly higher (mean, 2.14; median, 2; range, 1–3)
in women using contraceptives than in women with untreated cycles
(mean, 1.25; median, 1.5; range, 0–2; P < 0.04). For
ducts, this difference approached significance (mean, 2.29; median, 2;
range, 1–3 vs. mean, 1.41; median, 2; range, 0–3;
P = 0.06).
|
). For women with normal cycles, this
correlation reached significance for ducts (rs = -0.58;
P = 0.05), whereas the values for alveoli were not
significant (rs = -0.45; P = 0.13).
|
ER content was lower in the luteal phase in untreated premenopausal women. PR ranged from low to high in both phases. There was a significantly positive correlation between ductal 17HSD type 1 staining intensity and the percentage of ER-positive cells (rs = 0.59; P < 0.05) and a tendency for a positive correlation in alveoli (rs = 0.53; P = 0.09). No significant correlations were seen between 17HSD type 1 and PR levels.
There were significantly negative correlations between serum progesterone levels and the proportion of alveolar ER-positive cells in untreated premenopausal women (rs = -0.68; P = 0.03), all premenopausal women (rs = -0.48; P = 0.05), and the whole study group (rs = -0.61; P < 0.01). For ducts, this correlation was significant only for the whole study group (rs = -0.49; P = 0.03).
Discussion
17HSD type 1 protein was readily detected in normal breast epithelial cells, in ducts and alveoli, during both follicular and luteal phases of the menstrual cycle, and the staining was also observed in postmenopausal women. These data indicate that the regulation of 17HSD type 1 in normal breast is different from that previously shown in endometrial epithelial cells. In the endometrium, the enzyme is absent during the follicular phase. Maximal protein expression was observed during the early and midluteal phase of the cycle and gradually disappeared during the late luteal phase (11). In the endometrium, the increase in staining intensity for 17HSD type 1 was associated with increased serum progesterone levels.
A difference between the endometrium and breast was also suggested by the present results indicating a negative correlation between serum E2 levels and the enzyme protein expression. The present data indicate a greater tissue conversion from E1 to E2 in subjects with low endogenous E2 levels. However, it should be recalled that the regulation and expression of oxidative 17HSD type 2 also should be studied before conclusions about intratissue E2 concentrations are made (3, 5).
There was also a tendency for a negative correlation between serum progesterone levels and the expression of 17HSD type 1 in normal breast tissue of untreated women. Previously, in breast cancer tissue a positive correlation was found between 17HSD type 1 and serum progesterone levels (12). This may be a mechanism by which high intratissular E2 levels are retained in malignant breast tissue. In the T47D breast cancer cell line, which is rich in PR, treatment of the cells with the synthetic progestogen ORG 2058 increased the 1.3-kilobase 17HSD type 1 messenger ribonucleic acid (14).
In this study, 17HSD type 1 was detected in 9 of 12 (75%) premenopausal women with regular cycles and in 2 of 3 postmenopausal women. This is in line with previous findings in benign breast disease, where 71% of samples had positive 17HSD type 1 staining (14). The corresponding value in malignant breast tissue was 47%. Here, strong and moderate 17HSD type 1 staining was related to the presence of PR. In normal breast tissue we found no correlation between 17HSD type 1 staining intensity and PR. However, a significant positive correlation was found between 17HSD type 1 staining and ER. This is in line with the negative correlation between 17HSD type 1 staining intensity and serum E2 levels found in this study and with a negative correlation between the ER level and serum E2 levels found recently (13).
Previously, a decline in PR number was found in the epithelial cells of the endometrium during the luteal phase, whereas in the breast, the number of PR remained unchanged throughout the cycle (15).
As expected, there was a negative correlation between serum progesterone levels and ER content. Women using hormonal contraception showed a significantly higher 17HSD type 1 expression than untreated women. This up-regulation of the reductive enzyme indicates a potential for increased formation of highly potent E2 from E1 in hormonal contraceptive users. The present results indicate a difference between progestogens used for contraception and physiological progesterone with regard to 17HSD type 1 protein expression in normal breast tissue. Previously, discrepant effects were also found for sulfatase activity. Although there was a positive correlation between serum progesterone levels and sulfatase activity in untreated women, the conversion of E1 sulfate to E1 was suppressed during hormonal contraception (16).
In conclusion, the negative correlation between serum E2 levels and 17HSD type 1 protein expression indicates a potential for greater tissue conversion from E1 to E2 in subjects with low endogenous E2 serum levels. This may be important for the estrogenic stimulation of the breasts and could be one of the regulatory mechanisms of intratissue E2 concentration in normal breast tissue.
Acknowledgments
We gratefully acknowledge the efforts of Tuula Eklöf, Torsten Hägerström, Birgitta Byström, Eva Segersten, Siv Rydås, Ulrika Olsson, Mia Karlsson, and Liisa Karela.
Footnotes
1 This work was supported by the Swedish Medical Research Council
(Project 5982), Karolinska Institute Research Funds, the Swedish Cancer
Society, the Research Council for Health, the Academy of Finland
(Project 1051135), and the Ministries of Education, Social Affairs and
Health and of Foreign Affairs, Finland. 
Received March 21, 1997.
Revised November 21, 1997.
Accepted December 23, 1997.
References
-hydroxysteroid dehydrogenase activity. J Biol
Chem. 268:12964–12969.This article has been cited by other articles:
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  K. A Brown, K. Sayasith, N. Bouchard, J. G Lussier, and J. Sirois Molecular cloning of equine 17{beta}-hydroxysteroid dehydrogenase type 1 and its downregulation during follicular luteinization in vivo J. Mol. Endocrinol., January 1, 2007; 38(1): 67 - 78. [Abstract] [Full Text] [PDF]
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 Y. Nagayoshi, T. Ohba, H. Yamamoto, Y. Miyahara, H. Tashiro, H. Katabuchi, and H. Okamura Characterization of 17{beta}-hydroxysteroid dehydrogenase type 4 in human ovarian surface epithelial cells Mol. Hum. Reprod., September 1, 2005; 11(9): 615 - 621. [Abstract] [Full Text] [PDF]
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