0003.9969,92 55.00 + 0.00 Copyright 0 1992 Pergamon Press Ltd
Arch.soral &al. Vol. 37, No. 6, pp. 51 l-514. 1992 Printed in Great Britain. All rights reserved
SHORT COMMUNICATION IMMUNOHISTOCHEMICAL DETECTION OF ANDROGEN RECEPTORS IN HUMAN ORAL MUCOSA A. OJANOTKO-HARRI,’ H. FORSSELL,‘.*M. LAINE,’ H. HUR-ITIA,’M. BLKVER* and P. TUOHNAA~ ‘Institute of Dentistry, University of Turku, SF-20520 Turku and *Department of Biomedical Sciences, University of Tampere, 33101 Tampere, Finland (Received 6 November 1991; accepted 4 February 1992) Summary-Oral mucosal biopsies from I1 healthy volunteers, 7 women and 4 men, were analyscd for the localization of androgen, oestrogen and progesterone receptors. The samples were dissected as quickly as possible and immediately frozen in liquid nitrogen to be stored at -70°C. Only androgen receptors could be detected by the methods used. These were mainly located in the nuclei of basal epithehal cells, and to some extent in the nuclei of fibroblasts and endothelial cells. Failure in the localization of oestrogen and progesterone receptors might have been due either to low numbers of receptors or to the insensitivity of antibodies used. The method is thus suitable only for detecting androgen receptors. Key words: oral mucosa, sex steroids, immunohistochemistry.
Gingival tissue and oral mucosa are known to be sensitive to changes in the hormonal balance, especially to changes in female sex steroids. Wellknown forms of gingivitis are associated with puberty and pregnancy (Liie, 1965; Sutcliffe, 1972). In addition, many women complain of a burning sensation and pain in the oral mucosa after onset of menopause (Pisanty, Rafaely and Bolischuk, 1975). The gingival tissue and oral mucosa of man and animals are capable of metabolizing steroid hormones (ElAttar and Hugoson, 1974; OjanotkoHart-i, 1985; Ojanotko-Harri, Hurttia and Harri, 1986). This metabolism is enhanced during gingival inflammation (ElAttar and Hugoson, 1974; 1985). Active metabolites of Ojanotko-Hart-i, testosterone (dihydrotestosterone) and oestrogens (17/l-oestradiol) are formed especially in the inflamed gingiva, whereas progesterone is metabolized to inactive metabolites. The selective action of steroid hormones (e.g. through their active metabolites) on the metabolism of their target tissues depends on the presence of high-affinity receptors. Binding of steroids with these receptors allows them to control the gene expression of their target cells via pathways such as their involvement in the synthesis of structural proteins and enzymes and the growth of the target tissue (Beato, 1989; Bechet, 1986; Carson-Jurica, Schrader and O’Malley, 1990). Sex steroid hormones have interactions in their target tissues and may control each other’s actions through effects such as
*To whom all correspondence
should be addressed.
regulation of the number of receptors (Pertschuk, 1990, p. 2). Only a few studies have demonstrated sex steroid hormone receptors in the gingiva and oral mucosa (Southern et al., 1978; Vittek et al., 1982 a-c). In these a ligand-binding method was used to measure the receptor content of human and rabbit gingiva, and these tissues were found to contain receptors for dihydrotestosterone, 17p-oestradiol and progesterone. The disadvantages of ligand binding are its inability to locate receptors and the large sample needed (100-200 mg for each hormone receptor), which has prevented simultaneous demonstration of all sex steroid receptors. Some attempts have also been made to localize these receptors by autoradiography (Mohamed, 1974; Aufdemorte and Sheridan, 1981; Vittek et af., 1985), but non-specific labelling has impeded interpretation of the results. Both these methods demonstrate only unoccupied hormone receptors and thus over- or underestimation may occur, depending on the hormonal status of the tissue (Pertschuk, 1990, p. 15). Immunohistochemical techniques require only small tissue samples, and simultaneous demonstration of all sex steroid receptors is possible. In addition, the antibodies used in these techniques can detect both occupied and unoccupied receptors. Our aim in this preliminary study was to demonstrate and localize all sex steroid receptors in healthy oral mucosa using immunohistochemical techniques. Our protocol, using gingival biopsies from healthy volunteers, was reviewed and approved by the Ethical Committee of the University of Turku, Finland. After informed consent, oral
511
512
A. OJANOTKO-HARRI et d.
mucosal biopsies (6 gingival, 5 buccal mucosal) were taken from 11 healthy volunteers, 7 women (ages 3, 23, 27, 30, 32, 71, 84yr) and 4 men (ages 7, 10, 27, 59 yr). The samples were dissected as quickly as possible and immediately frozen in liquid nitrogen to be stored for no more than I month at -70°C until further processing. The samples weighed about IO-20 mg. Primary antibodies
Polyclonal rabbit anti-human androgen receptor anti-body was kindly provided by Dr S. Liao from the University of Chicago, IL, U.S.A. Immunohistochemical staining of oestrogen and progesterone receptors was done with the Abbott ER-ICA kit and with the monoclonal antibody mPR1 from Transbio, respectively. Immunohistochemical procedures
Androgen receptors were visualized as described in detail by Blauer et al. (1991). In brief, to reduce non-specific binding, sections were incubated with non-fat milk powder and normal goat serum. The sections were thereafter incubated overnight with anti-human androgen receptor antibody and, after washing, processed using the immunoperoxidase technique. Biotin-labelled goat anti-rabbit IgG (Vector, Burlingame, CA, U.S.A.) was-added as the second antibody and, after washing, sections were incubated with avidin-biotin-horseradish peroxidase complex (Vectastain Elite ABC-kit, Vector, Burlingame, CA, U.S.A.), prepared according to the manufacturer’s instructions. Peroxidase activity was visualized using 0.02% 3’3-diaminobenzidine tetrahydrochloride (Sigma, St Louis, MO, U.S.A.) and IO mM imidazole and 0.01% H?O,. The sections were dehydrated and mounted in Entellan (Merck, Darmstadt, Germany). Oestrogen receptors were visualized using the ERICA kit according to kit instructions. For progesterone receptor staining, biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, U.S.A.) was used as the secondary antibody and normal horse serum was substituted for normal goat serum. Otherwise, the staining procedure was the same as that for androgen receptors. Ligand binding was assayed with the dextran-coated charcoal method according to Lovgren et al. (1978). With these immunohistochemical methods we detected androgen receptors in all oral mucosal tissues studied; no receptors for oestrogens and progesterone were detected. Figure 1 shows a sample
incubated with anti-human androgen receptor antibody. Positive staining is seen in the nuclei, especially in the basal epithelial cells, and also in fibroblasts and endothelial cells. Figure 2 shows a control section without added antibody and without any staining of the nuclei. Our negative results are not in agreement with several earlier studies in which oral mucosal and gingival tissues were found to be target tissues of all sex steroid hormones (Ojanotko-Harri, 1990, p. 23). Oral mucosa is not, however, as sensitive to female sex steroids as the classical target organs, e.g. uterus and vagina. The reactions of oral mucosa should rather be compared with those of vulva] epithelium, as many oral complaints related to the menopause are similar to the sym$oms of vulvitis, which often coincide with oral symptoms (Ojanotko-Hart-i, 1990, p. 19). The reasons for our non-detection of oestrogen and progesterone receptors may be methodological. Previous studies have used either ligand binding or autoradiographic methods for the detection of receptors (Mohamed, 1974; Vittek er al., 1982 a
Arch.soral &al. Vol. 37, No. 6, pp. 51 l-514. 1992 Printed in Great Britain. All rights reserved
SHORT COMMUNICATION IMMUNOHISTOCHEMICAL DETECTION OF ANDROGEN RECEPTORS IN HUMAN ORAL MUCOSA A. OJANOTKO-HARRI,’ H. FORSSELL,‘.*M. LAINE,’ H. HUR-ITIA,’M. BLKVER* and P. TUOHNAA~ ‘Institute of Dentistry, University of Turku, SF-20520 Turku and *Department of Biomedical Sciences, University of Tampere, 33101 Tampere, Finland (Received 6 November 1991; accepted 4 February 1992) Summary-Oral mucosal biopsies from I1 healthy volunteers, 7 women and 4 men, were analyscd for the localization of androgen, oestrogen and progesterone receptors. The samples were dissected as quickly as possible and immediately frozen in liquid nitrogen to be stored at -70°C. Only androgen receptors could be detected by the methods used. These were mainly located in the nuclei of basal epithehal cells, and to some extent in the nuclei of fibroblasts and endothelial cells. Failure in the localization of oestrogen and progesterone receptors might have been due either to low numbers of receptors or to the insensitivity of antibodies used. The method is thus suitable only for detecting androgen receptors. Key words: oral mucosa, sex steroids, immunohistochemistry.
Gingival tissue and oral mucosa are known to be sensitive to changes in the hormonal balance, especially to changes in female sex steroids. Wellknown forms of gingivitis are associated with puberty and pregnancy (Liie, 1965; Sutcliffe, 1972). In addition, many women complain of a burning sensation and pain in the oral mucosa after onset of menopause (Pisanty, Rafaely and Bolischuk, 1975). The gingival tissue and oral mucosa of man and animals are capable of metabolizing steroid hormones (ElAttar and Hugoson, 1974; OjanotkoHart-i, 1985; Ojanotko-Harri, Hurttia and Harri, 1986). This metabolism is enhanced during gingival inflammation (ElAttar and Hugoson, 1974; 1985). Active metabolites of Ojanotko-Hart-i, testosterone (dihydrotestosterone) and oestrogens (17/l-oestradiol) are formed especially in the inflamed gingiva, whereas progesterone is metabolized to inactive metabolites. The selective action of steroid hormones (e.g. through their active metabolites) on the metabolism of their target tissues depends on the presence of high-affinity receptors. Binding of steroids with these receptors allows them to control the gene expression of their target cells via pathways such as their involvement in the synthesis of structural proteins and enzymes and the growth of the target tissue (Beato, 1989; Bechet, 1986; Carson-Jurica, Schrader and O’Malley, 1990). Sex steroid hormones have interactions in their target tissues and may control each other’s actions through effects such as
*To whom all correspondence
should be addressed.
regulation of the number of receptors (Pertschuk, 1990, p. 2). Only a few studies have demonstrated sex steroid hormone receptors in the gingiva and oral mucosa (Southern et al., 1978; Vittek et al., 1982 a-c). In these a ligand-binding method was used to measure the receptor content of human and rabbit gingiva, and these tissues were found to contain receptors for dihydrotestosterone, 17p-oestradiol and progesterone. The disadvantages of ligand binding are its inability to locate receptors and the large sample needed (100-200 mg for each hormone receptor), which has prevented simultaneous demonstration of all sex steroid receptors. Some attempts have also been made to localize these receptors by autoradiography (Mohamed, 1974; Aufdemorte and Sheridan, 1981; Vittek et af., 1985), but non-specific labelling has impeded interpretation of the results. Both these methods demonstrate only unoccupied hormone receptors and thus over- or underestimation may occur, depending on the hormonal status of the tissue (Pertschuk, 1990, p. 15). Immunohistochemical techniques require only small tissue samples, and simultaneous demonstration of all sex steroid receptors is possible. In addition, the antibodies used in these techniques can detect both occupied and unoccupied receptors. Our aim in this preliminary study was to demonstrate and localize all sex steroid receptors in healthy oral mucosa using immunohistochemical techniques. Our protocol, using gingival biopsies from healthy volunteers, was reviewed and approved by the Ethical Committee of the University of Turku, Finland. After informed consent, oral
511
512
A. OJANOTKO-HARRI et d.
mucosal biopsies (6 gingival, 5 buccal mucosal) were taken from 11 healthy volunteers, 7 women (ages 3, 23, 27, 30, 32, 71, 84yr) and 4 men (ages 7, 10, 27, 59 yr). The samples were dissected as quickly as possible and immediately frozen in liquid nitrogen to be stored for no more than I month at -70°C until further processing. The samples weighed about IO-20 mg. Primary antibodies
Polyclonal rabbit anti-human androgen receptor anti-body was kindly provided by Dr S. Liao from the University of Chicago, IL, U.S.A. Immunohistochemical staining of oestrogen and progesterone receptors was done with the Abbott ER-ICA kit and with the monoclonal antibody mPR1 from Transbio, respectively. Immunohistochemical procedures
Androgen receptors were visualized as described in detail by Blauer et al. (1991). In brief, to reduce non-specific binding, sections were incubated with non-fat milk powder and normal goat serum. The sections were thereafter incubated overnight with anti-human androgen receptor antibody and, after washing, processed using the immunoperoxidase technique. Biotin-labelled goat anti-rabbit IgG (Vector, Burlingame, CA, U.S.A.) was-added as the second antibody and, after washing, sections were incubated with avidin-biotin-horseradish peroxidase complex (Vectastain Elite ABC-kit, Vector, Burlingame, CA, U.S.A.), prepared according to the manufacturer’s instructions. Peroxidase activity was visualized using 0.02% 3’3-diaminobenzidine tetrahydrochloride (Sigma, St Louis, MO, U.S.A.) and IO mM imidazole and 0.01% H?O,. The sections were dehydrated and mounted in Entellan (Merck, Darmstadt, Germany). Oestrogen receptors were visualized using the ERICA kit according to kit instructions. For progesterone receptor staining, biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, U.S.A.) was used as the secondary antibody and normal horse serum was substituted for normal goat serum. Otherwise, the staining procedure was the same as that for androgen receptors. Ligand binding was assayed with the dextran-coated charcoal method according to Lovgren et al. (1978). With these immunohistochemical methods we detected androgen receptors in all oral mucosal tissues studied; no receptors for oestrogens and progesterone were detected. Figure 1 shows a sample
incubated with anti-human androgen receptor antibody. Positive staining is seen in the nuclei, especially in the basal epithelial cells, and also in fibroblasts and endothelial cells. Figure 2 shows a control section without added antibody and without any staining of the nuclei. Our negative results are not in agreement with several earlier studies in which oral mucosal and gingival tissues were found to be target tissues of all sex steroid hormones (Ojanotko-Harri, 1990, p. 23). Oral mucosa is not, however, as sensitive to female sex steroids as the classical target organs, e.g. uterus and vagina. The reactions of oral mucosa should rather be compared with those of vulva] epithelium, as many oral complaints related to the menopause are similar to the sym$oms of vulvitis, which often coincide with oral symptoms (Ojanotko-Hart-i, 1990, p. 19). The reasons for our non-detection of oestrogen and progesterone receptors may be methodological. Previous studies have used either ligand binding or autoradiographic methods for the detection of receptors (Mohamed, 1974; Vittek er al., 1982 a
