Human Reproduction vol.7 no. 10 pp. 1461 -1466, 1992
Immunohistochemical localization of androgen receptor in the human endometrium, decidua, placenta and pathological conditions of the endometrium
Kiyoshige Horie1, Kenji Takakura, Kimitoshi Imai, Shutsung Liao2 and Takahide Mori Department of Gynaecology and Obstetrics, Faculty of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan and 2The Ben May Institute, University of Chicago, IL, USA 'To whom correspondence should be addressed
The immunohistochemical localization of the androgen receptor in the human endometrium at various stages of the menstrual cycle and post-menopausal period, in decidua and placenta of early pregnancy, and in several pathological conditions of the endometrium has been investigated. At any phase of the menstrual cycle, both endometrial glandular cells and endometrial stromal cells showed positive nuclear staining. Endometrial stromal cells of the functional layer showed stronger staining than those of the basal layer, but endometrial glandular cells of both layers showed the same staining intensity. There was little staining in myometrium. Even after menopause, endometrial glandular and stromal cells showed the same staining pattern as the basal layer of pre-menopausal endometrium and the staining intensity of endometrial stromal cells was weak. In decidua and placenta of early pregnancy, decidual and trophoblastic cells showed positive staining and there was no staining in the stromal cells of placenta. The expression of the androgen receptor was also detected in adenomyosis, endometriosis and endometrial carcinoma. Although the proliferation and differentiation of endometrium are mediated mainly by oestrogen and progesterone receptors, the androgen receptor may play some role in modulating these changes. These results suggest that it may be involved in both physiological and pathological changes of the endometrium.
Key words: androgen receptor/endometrium/human/ imrnunohistochemistry /placenta
Introduction Human uterine endometrium undergoes monthly cyclic changes during a woman's reproductive period and once pregnancy is established, is transformed into the gestational endometrium. These changes are mainly controlled by sex steroids, the activities of which are mediated by their receptors. Androgens have a great influence on many target tissues, including male and femalereproductiveorgans. Androgen activity is supposed to be the sum of a direct effect mediated by androgen receptor and an indirect effect of oestrogens produced by © Oxford University Press Downloaded from https://academic.oup.com/humrep/article-abstract/7/10/1461/664797 by Bournemouth University user on 04 June 2018
aromatization of androgens (Mooradian et al., 1987). In the female reproductive organs, androgens play an important role in ovarian follicular function, by androgen receptor-mediated activity (Hillier, 1987), and have a direct inhibitory effect on the growth of human endometrial cells in vitro (Neulen et al., 1987; Rose et al., 1988). By the ligand binding assay, human androgen receptor has been demonstrated in the endometrium (Tamaya et al., 1986; Muechler, 1987), placenta (McCormick et al., 1981), and pathological conditions of the endometrium (Tamaya et al., 1979; Kato and Seto, 1985). This assay provides quantitative data of tissue homogenates but cannot provide the precise cellular or subcellular localization of this receptor. Recently, the localization of steroid receptors has been studied immunohistochemically using specific monoclonal antibodies for these receptors. The expression of androgen receptor in various tissues of human and other species has been demonstrated (Takeda et al., 1990; Sar et al., 1990; Ruizeveld de Winter et al., 1991) and we reported its localization in the human ovary throughout the menstrual cycle (Horie etal., 1992). In the human endometrium, oestrogen and progesterone receptors can be examined immunohistochemically (Lessey et al., 1983) but there has been no report concerning the precise localization of the androgen receptor. In the present study, we examined its localization in the human endometrium during the menstrual cycle, in the decidua and placenta, and in endometrium suffering from several pathological conditions.
Materials and methods Subjects Human endometria and other tissues were obtained from 27 women, aged 34—65 years at laparotomies performed for medical indications. They had regular menstrual cycles (28—30 days) and the stage of their menstrual cycles was determined by endometrial dating (Noyes et al., 1950). Human placentae and deciduae were obtained from three women, aged 24—29 years, who had undergone legal abortions. Gestational age was calculated from the data of the last menstrual period and, if necessary, was adjusted by taking into account ultrasonic measurement of the gestational sac and fetal crown—rump length. Informed consent for experimental purposes was obtained from each woman. Antibodies The preparation of rat anti-human androgen receptor monoclonal antibody has been described previously (Chang et al., 1989). The antibody recognized the androgen receptor amino-terminal region in human, rat and monkey, and was diluted to 1:25 with 1461
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phosphate-buffered saline (PBS) containing 0.1 % bovine serum albumin (BSA) (Sigma Chemical Co., St Louis, MO, USA) and 0.1% NaN3. Immunohistochemical staining Fresh endometrial tissues were cut into small pieces, quickly frozen, and stored in OCT compound (Miles Inc., Elkhart, IN, USA) at -80°C until use. The immunostaining procedure was
performed on cryostat sections by the avidin-biotin-peroxidase complex (ABC) method (Vectastain ABC kit (PK^tOO4), Vector Laboratories, Burlingame, CA, USA). Briefly, 4 /tin cryostat sections were mounted on glass slides (coated with 3-aminopropyl-triethoxysilane), fixed in a freshly made 4% paraformaldehyde solution for 2 min, and rinsed in PBS for 30 min. Next they were incubated with normal goat serum, to minimize non-specific staining, and then incubated with rat anti-
Fig. 1. Sections of cyclically changed endometrium. Endometria at late proliferative phase (A,B,F), at mid-secretory phase (C,D) and at late secretory phase (E) are shown as well as functional (A,C,E,F) and basal layers (B,D) of endometria. Endometrial stromal cells of the functional layer showed stronger staining than those of the basal layer, but endometrial glandular cells of both layers showed the same staining intensity (A-D). The immunostaining was detected in the pre-decidualized stromal cells at late secretory phase (E); control section is shown (F). Magnification X125.
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Androgen receptor in human endometrium
androgen receptor monoclonal antibody with control rat IgG (ZYMED Laboratories, Inc., San Francisco, CA, USA) overnight at 4°C. The sections were incubated with biotinylated anti-rat IgG antibody for 30 min at room temperature, followed by incubation with ABC reagent for 30 min. Every incubation was followed by washing twice with PBS. Finally, immunoreaction was visualized by incubating the sections in a solution consisting of diaminobenzidine (1.2 mg/ml) in tris buffer solution containing 0.02% H2Q2 for 3 min. The staining appeared as brown-coloured grains in the cells. Counterstaining was carried out with 3% methylgreen, which was essential for identifying cellular nuclei. We could not recognize positive or negative staining without counterstaining, and minimized the influence of methylgreen on the photomicrogaphs by using a green filter. For a control, the sections were incubated with normal rat IgG (ZYMED Laboratories, Inc., San Franciso, CA, USA). For a positive control of anti-androgen receptor antibody, the sections of benign prostatic hypertrophy were used. Results Staining with anti-androgen receptor antibody was confirmed predominantly in the nuclei of prostatic epithelial cells used as positive controls (data not shown). No immunostaining was observed with normal rat IgG instead of primary anti-androgen receptor antibody. Background staining in figures included a weak nuclear counterstaining with methylgreen, which did not mask specific immunostaining. Endometrium throughout the menstrual cycle and after menopause Endometria of 14 women with normal menstrual cycles were examined. Among them, six were at proliferative phase and eight were at secretory phase. At proliferative phase, both endometrial glandular cells and endometrial stromal cells showed positive nuclear staining (Figure 1A,B). Endometrial stromal cells of the
functional layer showed stronger staining than those of the basal layer, but endometrial glandular cells of both layers showed the same staining intensity (Figure 1A,B). There was little staining in myometrium. At secretory phase, the expression of androgen receptor showed a similar result to that at proliferative phase (Figure 1C,D) and in the pre-decidualized stromal cells at late secretory phase, similar immunostaining was also detected (Figure IE). Endometria of three women after menopause were examined. Endometrial glandular and stromal cells showed the same staining pattern as the basal layer of pre-menopausal endometrium and the staining intensity of endometrial stromal cells was weak (Figure 2A,B). Immunostaining was detected in the cells of cystic atrophy (Figure 2B), which were characteristic of the post-menopausal endometrium (Ferenczy, 1987), and squamous metaplasia (Figure 2A). Sections observed under the microscope showed negative staining for androgen receptor in endometrial lymphoid cells. Decidua and placenta Deciduae and placentae of three women were examined, all of which were obtained from women at 9 - 1 2 weeks of pregnancy. Decidual and trophoblastic cells showed positive nuclear staining (Figure 3A,B). No staining was detected in the stromal cells of placenta (Figure 3B). Pathological conditions of the endometrium Adenomyosis of four women and external endometriosis of four women were examined, both of which contained ectopic endometrium. In these cases, endometrial glandular and stromal cells showed positive nuclear staining at any phase of the menstrua] cycle (Figure 4A—D). No immunostaining was detected in the myometrium and stromal cells around these lesions (Figure 4 A - D ) . Endometrial carcinoma of four women were examined, all of which were Grade II adenocarcinoma. The malignant cells of these cases showed positive staining (Figure 5A,B). Immuno-
Fig. 2. Sections of post-menopausal endometrium. Endometrial glandular and stromal cells showed the same staining pattern as the basal layer of pre-menopausal endometrium and the staining intensity of endometria] stromal cells was weak (A,B)- Immunostaining was detected in the cells of squamous metaplasia (A; arrow) and cystic atrophy (B; arrow) which was characteristic of the post-menopausal endometrium. Magnification X125.
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Fig. 3. Sections of decidua and placenta. At 12 weeks of pregnancy, decidual and trophoblastic cells showed positive nuclear staining (A,B) and no staining was detected in the stromal cells of placenta (B). Magnification x 125.
B
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endometrium within myometrium (A; arrow) showed positive nuclear staining (A,B)- 1° endometriosis of cul-de-sac (C) and ovary (ovarian endometriotic cyst) (D), endometrial glandular and stromal cells showed positive nuclear staining (C,D); control (B; inset). Magnification X50 (A); X125 ( B - D ) .
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Androgen receptor in human endometrium
Fig. 5. Sections of endometrial carcinoma. The malignant cells in both glandular (A) and solid (B) regions showed positive nuclear staining. Magnification X125.
staining was detected in both glandular and solid lesions (Figure 5A,B). Discussion Our current immunohistochemical data about androgen receptor localization confirmed results reported previously by ligand binding assay and visualized the exact localization of the androgen receptor without the need for tissue homogenates. In our study, endometrial stromal and glandular cells showed the same pattern of immunostaining at any phase of the menstrual cycle and stromal cells in the functional layer showed stronger staining than those in the basal layer. Although the expression of the androgen receptor seems to be constant throughout the menstrual cycle, the peripheral blood level of androgens and the activities of endometrial androgen-metabolizing enzymes show cyclical changes (Strauss and Gurpide, 1991). The androgenic activity in the endometrium may be influenced by these factors. The consistent receptor expression was quite different from the cycle-dependent expressions of oestrogen and progesterone receptors (Lessey et al., 1988). Oestrogen receptor levels reach a maximum at late proliferative phase and decline during secretory phase, reflecting down-regulation by progesterone. Progesterone receptor levels increase throughout proliferative phase, and progesterone receptors on endometrial glandular cells during the secretory phase markedly decline, while those on endometrial stromal cells are rather constant. In human breast cancer cell line, androgens induce downregulation of oestrogen receptor and suppress oestrogendependent induction of progesterone receptor, by androgen receptor-mediated activity (Poulin et al., 1989). Proliferation and differentiation of the endometrium are mediated mainly by oestrogen and progesterone receptors, and the consistent presence of androgen receptors suggests their possible modulation by androgen receptors. It has been suggested that oestrogen-induced proliferation of endometrial glandular cells may be influenced by underlying endometrial stromal cells (Cunha et al., 1983; Bigsby and Cunha,
1986) and that oestrogen may suppress production of a factor by endometrial stromal cells which restrains endometrial glandular cell replication (Chen et al., 1988; Tabibzadeh et al., 1989). High androgen receptor expression in endometrial stromal cells of the functional layer may reflect the stromal regulation of epithelial cell function. After menopause, the ratio of androgen to oestrogen is high (Yen, 1977) and steroidal effect on the endometrium is predominantly androgenic. The expression of androgen receptors was observed in both glandular and stromal cells of the endometrium. Furthermore, the cells of cystic atrophy, which are characteristics of the post-menopausal endometrium (Ferenczy, 1987), showed positive androgen receptor staining. These pathological changes of post-menopausal endometrium may thus be influenced by androgens mediated by androgen receptors. Androgen receptors were expressed in several pathological conditions of the endometrium: adenomyosis, endometriosis, and endometrial carcinoma. Danazol is an effective therapeutic agent in the treatment of adenomyosis and endometriosis. As one possible mechanism, it is considered that this drug binds to androgen receptors and exerts a direct inhibitory effect on endometrial tissues (Rose et al., 1988). Our detection of these receptors in adenomyosis and endometriosis provides further evidence for this effect. The demonstration of androgen receptors in endometrial carcinoma confirmed a previous study (Kato and Seto, 1985) by ligand binding assay. Androgens as well as progesterone have an anti-proliferative effect on endometrial cells (Rose etal., 1988) and may be clinically applied in the management of endometrial carcinoma. Androgen receptor expression in various tissues of human and other species has been reported immunohistochemically (Takeda et al., 1990; Ruizeveld de Winter et al., 1991; Sar et al., 1991; Horie et al., 1992). Ruizeveld de Winter et al. (1991) reported that no significant staining was observed with F 39.4, another monoclonal antibody for androgen receptor, in the human uteri of four women examined. This discrepancy might be due to a difference of monoclonal antibody and immunohistochemical procedure. Our immunohistochemical data provide a new strategy 1465
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K.Horie et at. to evaluate the inter-relationship between each steroid receptor and whether androgen receptors may be involved in both physiological and pathological changes of the endometrium. Acknowledgements This research was supported in part by a grant-in-akl from the Ministry of Education, Science, and Culture of Japan (no. 01570927, 02454380 and 02857227) and by US National Institute of Health Grant no. DK37694.
References
Ruizeveld de Winter J. A., Trapman.J., Vermey.M., Mulder.E., Zegers.N.D. and ver der Kwast.T.H. (1991) Androgen receptor expression in human tissues: an immunohistochemical study. J. Histochem. Cytochem., 39, 927-936. Sar,M., Lubahn.D.B., French.F.S. and Wilson,M.E. (1990) Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology, 127, 3180-3186. StraussJ.F,HI and Gurpide.E. (1991) The endometrium: regulation and dysfunction. In Yen,S.S.C. and Jaffe,R.B. (eds), Reproductive Endocrinology. W.B. Saunders Company, Philadelphia, p. 309. Tabibzadeh.S.S., Santhanam.U., Sehgal.P.B. and May.L.T. (1989) Cytokine-induced production of IFN-02/IL-6 by freshly explanted human endometrial stromal cells; modulation by estradiol-17/3. J. Immunol, 142, 3134-3139. Takeda.H., Chodak.G., Mutchnik.S., Nakamoto.T. and Chang,C. (1990) Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor. J. Endocrinol, 126, 17-25. Tamaya.T., Motoyama.T., Ohono.Y., Ide.N., Tsurusaki.T. and Okada.H. (1979) Steroid receptor levels and histology of endometriosis and adenomyosis. Fertil. SteriL, 31, 396-400. Tamaya.T., Murakami.T. and Okada,H. (1986) Concentrations of steroid receptors in normal human endometrium in relation to the day of the menstrual cycle. Acta Obstet. Gynecol. Scand., 65, 195-198. Yen.S.S. (1977) The biology of menopause. J. Reprod. Med., 18, 287-296.
Bigsby.R.M. and Cunha.G.R. (1986) Estrogen stimulation of deoxyribonucleic acid synthesis in uterine epithelial cells which lack estrogen receptors. Endocrinology, 119, 390—396. Chang.C, Whelan.C.T., Popovich.T.C, KokontisJ. and Liao.S. (1989) Fusion proteins containing androgen receptor sequences and their use in the production of poly- and monoclonal anti-androgen receptor antibodies. Endocrinology, 125, 1097-1099. Chen,L., Mory.Y., Zilberstein.A. and Revel.M. (1988) Growth inhibition of human breast carcinoma and leukemia/lymphoma cell lines by recombinant IFN-/32. Proc. Natl. Acad. Sci. USA, 85, 8037-8041. Cunha,G.R., Chung,L.W.K., Shannon,.!.M., Taguchi.O. and Fujii.H. (1983) Hormone-induced morphogenesis and growth: role of mesenchymal-epithelial interaction. Recent Prog. Horn. Res., 39, Received on June 25, 1992; accepted on September 1, 1992 559-598. Ferenczy.A. (1987) Anatomy and histology of the uterine corpus. In Kurman,R.J. (ed.), Blaustein 's Pathology of the Female Genital Tract. Springer-Verlag, New York, p. 257. Hillier.S.G. (1987) Intrafollicular paracrine function of ovarian androgen. J. Steroid Biochem., 27, 351-357. Horie.K., Takakura.K., Fujiwara,H., Suginami.H., Liao.S. and Mori,T. (1992) Immunohistochemical localization of androgen receptor in the human ovary throughout the menstrual cycle in relation to oestrogen and progesterone receptor expression. Hum. Reprod., 7, 184 — 190. KatoJ. and Seto,T. (1985) Correlation of androgen receptors with histological differentiation in human endometrial carcinomas. Acta Obstet. Gynecol. Scand., 64, 209-212. Lessey.B.A., Killam.A.P., Metzger.D.A., Haney,A.F., Greene.G.L. and McCarty,K.S.,Jr (1988) Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle. J. Clin. Endocrinol. Metab., 67, 334-340. McCormick.P.D., Razel.A.J., Spelsberg.T.C. and Coulam.C.B. (1981) Evidence for an androgen receptor in the human placenta. Am. J.
Obstet. Gynecol, 140, 8-13. Mooradian.A.D., Morley.J.E. and Korenman.S.G. (1987) Biological actions of androgens. Endocr. Rev., 8, 1-28. Muechler.E.K. (1987) The androgen receptor of the human endometrium. Endocr. Res., 13, 69—84. Neulen.J., Wagner.B., Runge.M. and Breckwoldt,M. (1987) Effect of progestins, androgens, estrogens and antiestrogens of 3H-thymidine uptake by human endometrial and endosalpinx cells in vitro. Arch. Gynecol, 240, 25-232. Noyes.R.W., Hertig.A.T. and Rock,J. (1950) Dating the endometrial biopsy. Fertil SteriL, 1, 3 - 2 5 . Poulin.R., SimardJ., Labrie.C, Petitclerc.L., Dumont,M., Lagace,L. and Labrie.F. (1989) Down-regulation of estrogen receptors by androgens in the ZR-75-1 human breast cancer cell line. Endocrinology, 125, 392-399. Rose.G.L., White,J.O., Dowsett.M., Jeffcoate,S.L. and MudgeJ.E. (1988) The inhibitory effects of danazol, danazol metabolites, gestrinone, and testosterone on the growth of human endometrial cells in vitro. Fertil SteriL, 49, 224-228. 1466 Downloaded from https://academic.oup.com/humrep/article-abstract/7/10/1461/664797 by Bournemouth University user on 04 June 2018
Immunohistochemical localization of androgen receptor in the human endometrium, decidua, placenta and pathological conditions of the endometrium
Kiyoshige Horie1, Kenji Takakura, Kimitoshi Imai, Shutsung Liao2 and Takahide Mori Department of Gynaecology and Obstetrics, Faculty of Medicine, Kyoto University, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan and 2The Ben May Institute, University of Chicago, IL, USA 'To whom correspondence should be addressed
The immunohistochemical localization of the androgen receptor in the human endometrium at various stages of the menstrual cycle and post-menopausal period, in decidua and placenta of early pregnancy, and in several pathological conditions of the endometrium has been investigated. At any phase of the menstrual cycle, both endometrial glandular cells and endometrial stromal cells showed positive nuclear staining. Endometrial stromal cells of the functional layer showed stronger staining than those of the basal layer, but endometrial glandular cells of both layers showed the same staining intensity. There was little staining in myometrium. Even after menopause, endometrial glandular and stromal cells showed the same staining pattern as the basal layer of pre-menopausal endometrium and the staining intensity of endometrial stromal cells was weak. In decidua and placenta of early pregnancy, decidual and trophoblastic cells showed positive staining and there was no staining in the stromal cells of placenta. The expression of the androgen receptor was also detected in adenomyosis, endometriosis and endometrial carcinoma. Although the proliferation and differentiation of endometrium are mediated mainly by oestrogen and progesterone receptors, the androgen receptor may play some role in modulating these changes. These results suggest that it may be involved in both physiological and pathological changes of the endometrium.
Key words: androgen receptor/endometrium/human/ imrnunohistochemistry /placenta
Introduction Human uterine endometrium undergoes monthly cyclic changes during a woman's reproductive period and once pregnancy is established, is transformed into the gestational endometrium. These changes are mainly controlled by sex steroids, the activities of which are mediated by their receptors. Androgens have a great influence on many target tissues, including male and femalereproductiveorgans. Androgen activity is supposed to be the sum of a direct effect mediated by androgen receptor and an indirect effect of oestrogens produced by © Oxford University Press Downloaded from https://academic.oup.com/humrep/article-abstract/7/10/1461/664797 by Bournemouth University user on 04 June 2018
aromatization of androgens (Mooradian et al., 1987). In the female reproductive organs, androgens play an important role in ovarian follicular function, by androgen receptor-mediated activity (Hillier, 1987), and have a direct inhibitory effect on the growth of human endometrial cells in vitro (Neulen et al., 1987; Rose et al., 1988). By the ligand binding assay, human androgen receptor has been demonstrated in the endometrium (Tamaya et al., 1986; Muechler, 1987), placenta (McCormick et al., 1981), and pathological conditions of the endometrium (Tamaya et al., 1979; Kato and Seto, 1985). This assay provides quantitative data of tissue homogenates but cannot provide the precise cellular or subcellular localization of this receptor. Recently, the localization of steroid receptors has been studied immunohistochemically using specific monoclonal antibodies for these receptors. The expression of androgen receptor in various tissues of human and other species has been demonstrated (Takeda et al., 1990; Sar et al., 1990; Ruizeveld de Winter et al., 1991) and we reported its localization in the human ovary throughout the menstrual cycle (Horie etal., 1992). In the human endometrium, oestrogen and progesterone receptors can be examined immunohistochemically (Lessey et al., 1983) but there has been no report concerning the precise localization of the androgen receptor. In the present study, we examined its localization in the human endometrium during the menstrual cycle, in the decidua and placenta, and in endometrium suffering from several pathological conditions.
Materials and methods Subjects Human endometria and other tissues were obtained from 27 women, aged 34—65 years at laparotomies performed for medical indications. They had regular menstrual cycles (28—30 days) and the stage of their menstrual cycles was determined by endometrial dating (Noyes et al., 1950). Human placentae and deciduae were obtained from three women, aged 24—29 years, who had undergone legal abortions. Gestational age was calculated from the data of the last menstrual period and, if necessary, was adjusted by taking into account ultrasonic measurement of the gestational sac and fetal crown—rump length. Informed consent for experimental purposes was obtained from each woman. Antibodies The preparation of rat anti-human androgen receptor monoclonal antibody has been described previously (Chang et al., 1989). The antibody recognized the androgen receptor amino-terminal region in human, rat and monkey, and was diluted to 1:25 with 1461
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phosphate-buffered saline (PBS) containing 0.1 % bovine serum albumin (BSA) (Sigma Chemical Co., St Louis, MO, USA) and 0.1% NaN3. Immunohistochemical staining Fresh endometrial tissues were cut into small pieces, quickly frozen, and stored in OCT compound (Miles Inc., Elkhart, IN, USA) at -80°C until use. The immunostaining procedure was
performed on cryostat sections by the avidin-biotin-peroxidase complex (ABC) method (Vectastain ABC kit (PK^tOO4), Vector Laboratories, Burlingame, CA, USA). Briefly, 4 /tin cryostat sections were mounted on glass slides (coated with 3-aminopropyl-triethoxysilane), fixed in a freshly made 4% paraformaldehyde solution for 2 min, and rinsed in PBS for 30 min. Next they were incubated with normal goat serum, to minimize non-specific staining, and then incubated with rat anti-
Fig. 1. Sections of cyclically changed endometrium. Endometria at late proliferative phase (A,B,F), at mid-secretory phase (C,D) and at late secretory phase (E) are shown as well as functional (A,C,E,F) and basal layers (B,D) of endometria. Endometrial stromal cells of the functional layer showed stronger staining than those of the basal layer, but endometrial glandular cells of both layers showed the same staining intensity (A-D). The immunostaining was detected in the pre-decidualized stromal cells at late secretory phase (E); control section is shown (F). Magnification X125.
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androgen receptor monoclonal antibody with control rat IgG (ZYMED Laboratories, Inc., San Francisco, CA, USA) overnight at 4°C. The sections were incubated with biotinylated anti-rat IgG antibody for 30 min at room temperature, followed by incubation with ABC reagent for 30 min. Every incubation was followed by washing twice with PBS. Finally, immunoreaction was visualized by incubating the sections in a solution consisting of diaminobenzidine (1.2 mg/ml) in tris buffer solution containing 0.02% H2Q2 for 3 min. The staining appeared as brown-coloured grains in the cells. Counterstaining was carried out with 3% methylgreen, which was essential for identifying cellular nuclei. We could not recognize positive or negative staining without counterstaining, and minimized the influence of methylgreen on the photomicrogaphs by using a green filter. For a control, the sections were incubated with normal rat IgG (ZYMED Laboratories, Inc., San Franciso, CA, USA). For a positive control of anti-androgen receptor antibody, the sections of benign prostatic hypertrophy were used. Results Staining with anti-androgen receptor antibody was confirmed predominantly in the nuclei of prostatic epithelial cells used as positive controls (data not shown). No immunostaining was observed with normal rat IgG instead of primary anti-androgen receptor antibody. Background staining in figures included a weak nuclear counterstaining with methylgreen, which did not mask specific immunostaining. Endometrium throughout the menstrual cycle and after menopause Endometria of 14 women with normal menstrual cycles were examined. Among them, six were at proliferative phase and eight were at secretory phase. At proliferative phase, both endometrial glandular cells and endometrial stromal cells showed positive nuclear staining (Figure 1A,B). Endometrial stromal cells of the
functional layer showed stronger staining than those of the basal layer, but endometrial glandular cells of both layers showed the same staining intensity (Figure 1A,B). There was little staining in myometrium. At secretory phase, the expression of androgen receptor showed a similar result to that at proliferative phase (Figure 1C,D) and in the pre-decidualized stromal cells at late secretory phase, similar immunostaining was also detected (Figure IE). Endometria of three women after menopause were examined. Endometrial glandular and stromal cells showed the same staining pattern as the basal layer of pre-menopausal endometrium and the staining intensity of endometrial stromal cells was weak (Figure 2A,B). Immunostaining was detected in the cells of cystic atrophy (Figure 2B), which were characteristic of the post-menopausal endometrium (Ferenczy, 1987), and squamous metaplasia (Figure 2A). Sections observed under the microscope showed negative staining for androgen receptor in endometrial lymphoid cells. Decidua and placenta Deciduae and placentae of three women were examined, all of which were obtained from women at 9 - 1 2 weeks of pregnancy. Decidual and trophoblastic cells showed positive nuclear staining (Figure 3A,B). No staining was detected in the stromal cells of placenta (Figure 3B). Pathological conditions of the endometrium Adenomyosis of four women and external endometriosis of four women were examined, both of which contained ectopic endometrium. In these cases, endometrial glandular and stromal cells showed positive nuclear staining at any phase of the menstrua] cycle (Figure 4A—D). No immunostaining was detected in the myometrium and stromal cells around these lesions (Figure 4 A - D ) . Endometrial carcinoma of four women were examined, all of which were Grade II adenocarcinoma. The malignant cells of these cases showed positive staining (Figure 5A,B). Immuno-
Fig. 2. Sections of post-menopausal endometrium. Endometrial glandular and stromal cells showed the same staining pattern as the basal layer of pre-menopausal endometrium and the staining intensity of endometria] stromal cells was weak (A,B)- Immunostaining was detected in the cells of squamous metaplasia (A; arrow) and cystic atrophy (B; arrow) which was characteristic of the post-menopausal endometrium. Magnification X125.
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Fig. 3. Sections of decidua and placenta. At 12 weeks of pregnancy, decidual and trophoblastic cells showed positive nuclear staining (A,B) and no staining was detected in the stromal cells of placenta (B). Magnification x 125.
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endometrium within myometrium (A; arrow) showed positive nuclear staining (A,B)- 1° endometriosis of cul-de-sac (C) and ovary (ovarian endometriotic cyst) (D), endometrial glandular and stromal cells showed positive nuclear staining (C,D); control (B; inset). Magnification X50 (A); X125 ( B - D ) .
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Androgen receptor in human endometrium
Fig. 5. Sections of endometrial carcinoma. The malignant cells in both glandular (A) and solid (B) regions showed positive nuclear staining. Magnification X125.
staining was detected in both glandular and solid lesions (Figure 5A,B). Discussion Our current immunohistochemical data about androgen receptor localization confirmed results reported previously by ligand binding assay and visualized the exact localization of the androgen receptor without the need for tissue homogenates. In our study, endometrial stromal and glandular cells showed the same pattern of immunostaining at any phase of the menstrual cycle and stromal cells in the functional layer showed stronger staining than those in the basal layer. Although the expression of the androgen receptor seems to be constant throughout the menstrual cycle, the peripheral blood level of androgens and the activities of endometrial androgen-metabolizing enzymes show cyclical changes (Strauss and Gurpide, 1991). The androgenic activity in the endometrium may be influenced by these factors. The consistent receptor expression was quite different from the cycle-dependent expressions of oestrogen and progesterone receptors (Lessey et al., 1988). Oestrogen receptor levels reach a maximum at late proliferative phase and decline during secretory phase, reflecting down-regulation by progesterone. Progesterone receptor levels increase throughout proliferative phase, and progesterone receptors on endometrial glandular cells during the secretory phase markedly decline, while those on endometrial stromal cells are rather constant. In human breast cancer cell line, androgens induce downregulation of oestrogen receptor and suppress oestrogendependent induction of progesterone receptor, by androgen receptor-mediated activity (Poulin et al., 1989). Proliferation and differentiation of the endometrium are mediated mainly by oestrogen and progesterone receptors, and the consistent presence of androgen receptors suggests their possible modulation by androgen receptors. It has been suggested that oestrogen-induced proliferation of endometrial glandular cells may be influenced by underlying endometrial stromal cells (Cunha et al., 1983; Bigsby and Cunha,
1986) and that oestrogen may suppress production of a factor by endometrial stromal cells which restrains endometrial glandular cell replication (Chen et al., 1988; Tabibzadeh et al., 1989). High androgen receptor expression in endometrial stromal cells of the functional layer may reflect the stromal regulation of epithelial cell function. After menopause, the ratio of androgen to oestrogen is high (Yen, 1977) and steroidal effect on the endometrium is predominantly androgenic. The expression of androgen receptors was observed in both glandular and stromal cells of the endometrium. Furthermore, the cells of cystic atrophy, which are characteristics of the post-menopausal endometrium (Ferenczy, 1987), showed positive androgen receptor staining. These pathological changes of post-menopausal endometrium may thus be influenced by androgens mediated by androgen receptors. Androgen receptors were expressed in several pathological conditions of the endometrium: adenomyosis, endometriosis, and endometrial carcinoma. Danazol is an effective therapeutic agent in the treatment of adenomyosis and endometriosis. As one possible mechanism, it is considered that this drug binds to androgen receptors and exerts a direct inhibitory effect on endometrial tissues (Rose et al., 1988). Our detection of these receptors in adenomyosis and endometriosis provides further evidence for this effect. The demonstration of androgen receptors in endometrial carcinoma confirmed a previous study (Kato and Seto, 1985) by ligand binding assay. Androgens as well as progesterone have an anti-proliferative effect on endometrial cells (Rose etal., 1988) and may be clinically applied in the management of endometrial carcinoma. Androgen receptor expression in various tissues of human and other species has been reported immunohistochemically (Takeda et al., 1990; Ruizeveld de Winter et al., 1991; Sar et al., 1991; Horie et al., 1992). Ruizeveld de Winter et al. (1991) reported that no significant staining was observed with F 39.4, another monoclonal antibody for androgen receptor, in the human uteri of four women examined. This discrepancy might be due to a difference of monoclonal antibody and immunohistochemical procedure. Our immunohistochemical data provide a new strategy 1465
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K.Horie et at. to evaluate the inter-relationship between each steroid receptor and whether androgen receptors may be involved in both physiological and pathological changes of the endometrium. Acknowledgements This research was supported in part by a grant-in-akl from the Ministry of Education, Science, and Culture of Japan (no. 01570927, 02454380 and 02857227) and by US National Institute of Health Grant no. DK37694.
References
Ruizeveld de Winter J. A., Trapman.J., Vermey.M., Mulder.E., Zegers.N.D. and ver der Kwast.T.H. (1991) Androgen receptor expression in human tissues: an immunohistochemical study. J. Histochem. Cytochem., 39, 927-936. Sar,M., Lubahn.D.B., French.F.S. and Wilson,M.E. (1990) Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology, 127, 3180-3186. StraussJ.F,HI and Gurpide.E. (1991) The endometrium: regulation and dysfunction. In Yen,S.S.C. and Jaffe,R.B. (eds), Reproductive Endocrinology. W.B. Saunders Company, Philadelphia, p. 309. Tabibzadeh.S.S., Santhanam.U., Sehgal.P.B. and May.L.T. (1989) Cytokine-induced production of IFN-02/IL-6 by freshly explanted human endometrial stromal cells; modulation by estradiol-17/3. J. Immunol, 142, 3134-3139. Takeda.H., Chodak.G., Mutchnik.S., Nakamoto.T. and Chang,C. (1990) Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor. J. Endocrinol, 126, 17-25. Tamaya.T., Motoyama.T., Ohono.Y., Ide.N., Tsurusaki.T. and Okada.H. (1979) Steroid receptor levels and histology of endometriosis and adenomyosis. Fertil. SteriL, 31, 396-400. Tamaya.T., Murakami.T. and Okada,H. (1986) Concentrations of steroid receptors in normal human endometrium in relation to the day of the menstrual cycle. Acta Obstet. Gynecol. Scand., 65, 195-198. Yen.S.S. (1977) The biology of menopause. J. Reprod. Med., 18, 287-296.
Bigsby.R.M. and Cunha.G.R. (1986) Estrogen stimulation of deoxyribonucleic acid synthesis in uterine epithelial cells which lack estrogen receptors. Endocrinology, 119, 390—396. Chang.C, Whelan.C.T., Popovich.T.C, KokontisJ. and Liao.S. (1989) Fusion proteins containing androgen receptor sequences and their use in the production of poly- and monoclonal anti-androgen receptor antibodies. Endocrinology, 125, 1097-1099. Chen,L., Mory.Y., Zilberstein.A. and Revel.M. (1988) Growth inhibition of human breast carcinoma and leukemia/lymphoma cell lines by recombinant IFN-/32. Proc. Natl. Acad. Sci. USA, 85, 8037-8041. Cunha,G.R., Chung,L.W.K., Shannon,.!.M., Taguchi.O. and Fujii.H. (1983) Hormone-induced morphogenesis and growth: role of mesenchymal-epithelial interaction. Recent Prog. Horn. Res., 39, Received on June 25, 1992; accepted on September 1, 1992 559-598. Ferenczy.A. (1987) Anatomy and histology of the uterine corpus. In Kurman,R.J. (ed.), Blaustein 's Pathology of the Female Genital Tract. Springer-Verlag, New York, p. 257. Hillier.S.G. (1987) Intrafollicular paracrine function of ovarian androgen. J. Steroid Biochem., 27, 351-357. Horie.K., Takakura.K., Fujiwara,H., Suginami.H., Liao.S. and Mori,T. (1992) Immunohistochemical localization of androgen receptor in the human ovary throughout the menstrual cycle in relation to oestrogen and progesterone receptor expression. Hum. Reprod., 7, 184 — 190. KatoJ. and Seto,T. (1985) Correlation of androgen receptors with histological differentiation in human endometrial carcinomas. Acta Obstet. Gynecol. Scand., 64, 209-212. Lessey.B.A., Killam.A.P., Metzger.D.A., Haney,A.F., Greene.G.L. and McCarty,K.S.,Jr (1988) Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle. J. Clin. Endocrinol. Metab., 67, 334-340. McCormick.P.D., Razel.A.J., Spelsberg.T.C. and Coulam.C.B. (1981) Evidence for an androgen receptor in the human placenta. Am. J.
Obstet. Gynecol, 140, 8-13. Mooradian.A.D., Morley.J.E. and Korenman.S.G. (1987) Biological actions of androgens. Endocr. Rev., 8, 1-28. Muechler.E.K. (1987) The androgen receptor of the human endometrium. Endocr. Res., 13, 69—84. Neulen.J., Wagner.B., Runge.M. and Breckwoldt,M. (1987) Effect of progestins, androgens, estrogens and antiestrogens of 3H-thymidine uptake by human endometrial and endosalpinx cells in vitro. Arch. Gynecol, 240, 25-232. Noyes.R.W., Hertig.A.T. and Rock,J. (1950) Dating the endometrial biopsy. Fertil SteriL, 1, 3 - 2 5 . Poulin.R., SimardJ., Labrie.C, Petitclerc.L., Dumont,M., Lagace,L. and Labrie.F. (1989) Down-regulation of estrogen receptors by androgens in the ZR-75-1 human breast cancer cell line. Endocrinology, 125, 392-399. Rose.G.L., White,J.O., Dowsett.M., Jeffcoate,S.L. and MudgeJ.E. (1988) The inhibitory effects of danazol, danazol metabolites, gestrinone, and testosterone on the growth of human endometrial cells in vitro. Fertil SteriL, 49, 224-228. 1466 Downloaded from https://academic.oup.com/humrep/article-abstract/7/10/1461/664797 by Bournemouth University user on 04 June 2018
