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Testosterone and estradiol up-regulate androgen and estrogen receptors in immature and adult rat thyroid glands in vivo
Steroids 67 (2002) 1007–1014
Testosterone and estradiol up-regulate androgen and estrogen receptors in immature and adult rat thyroid glands in vivo Sakhila K. Banu∗ , P. Govindarajulu, Michael M. Aruldhas Department of Endocrinology, Dr. ALM. PG. Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600113, India Received 8 October 2001; received in revised form 16 April 2002; accepted 23 April 2002
Abstract Thyroid gland is one of the non-classical target organs for sex steroids. Presence of androgen and estrogen receptors in the neoplastic and non-neoplastic thyroid glands of mammalian species is well documented. The aim of the present study is to elucidate the changes in serum and thyroidal sex steroids, and their receptors in the thyroid gland of rats from immature to adult age under gonadectomized (GDX) and sex steroids replaced conditions. Normal Wistar male and female rats from immature to adult age (day 21, 30, 45, 60 and 160 post-partum (pp)) were used in the present study. One group (I) of rats was GDX at an early age (day 10 pp) and the other group (II) at the adult age (day 120 pp). Group I rats were sacrificed at different experimental periods such as 21, 30, 45 and 60 days pp, and group II rats were sacrificed at day 160 pp. Another group of GDX rats from group I and II were replaced with physiological doses of testosterone or estradiol. Serum and thyroidal concentrations of sex steroids were estimated by RIA method and the concentrations of receptors by radioreceptor assay. Gonadectomy significantly decreased serum and thyroidal testosterone and estradiol and concentrations of androgen receptor (AR) and estrogen receptor (ER) in the thyroid. Replacement of sex steroids to GDX rats restored the normal level of sex steroids, AR and ER. Therefore, it is suggested from the present study that (i) sex steroids up-regulate their own receptors in the thyroid, (ii) sex steroids may influence thyroid growth and the proliferation of thyrocytes by modulating their receptor concentrations in the thyroid. © 2002 Published by Elsevier Science Inc. Keywords: Gonadectomy; Thyroid; Sex steroid-receptor
1. Introduction The extreme female predominance of thyroid diseases suggests that sex hormones may have a pathogenic role in the thyroid [1,2]. A recent study described a divergent role for 17-beta estradiol and its metabolite 2-methoxy estradiol (2-ME) on human thyroid cell proliferation. While estradiol stimulated cell cycle progression early in G1 phase by induction of cyclin D1 gene expression in benign and malignant thyroid cell, 2-ME induced G2/M cycle arrest, apoptosis and disruption of thyroid follicles [1]. Androgen also has a direct influence on thyroid cell proliferation [3–5]. Presence of AR has been reported in thyroid tissue of rat, primate and human [4–8]. Early studies suggest that the gonadal steroids indirectly act through the hypothalamic–pituitary axis to modulate thyroid function [9–11]. However, our recent studies proved the direct effects of testosterone and estradiol on the proliferation of normal (rat) [3] and cancerous (human) [12] ∗
Corresponding author. Present address: Ontogenie et ReproductionCRBR, CHUL, 2705 Boul W. Laurier, Laval University, Ste-Foy, Que., Canada, GIV 4G2. Tel.: +1-418-656-4141x6125; fax: +1-418-654-2765. E-mail address: [email protected] (S.K. Banu). 0039-128X/02/$ – see front matter © 2002 Published by Elsevier Science Inc. PII: S 0 0 3 9 - 1 2 8 X ( 0 2 ) 0 0 0 6 3 - 6
thyroid cells in vitro. Even though sex steroids regulate thyroid growth in vivo [4,6] and in vitro [3,12], the mechanism underlying this phenomenon is unproven. The present study addresses the effect of sex steroids on the concentration of AR and ER in the thyroid of immature and adult Wistar rats of various periods of maturation.
2. Experimental 2.1. Animals Wistar rats (Rattus norvegicus) of day 1–160 pp were maintained under controlled temperature (25 ± 2 ◦ C) and lighting (12 h dark and 12 h light) conditions. Animals were provided with a standard pellet diet (Gold Mohur, Lipton India Ltd., India) and clean drinking water ad libitum. 2.2. Chemicals Triamcinolone acetonide, testosterone, 17-estradiol, hydroxylapatite slurry and phenyl methyl sulfonyl fluoride
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(PMSF) were purchased from Sigma, St. Louis. Pre-packed PD 10 and G-25 sephadex columns were purchased from Pharmacia Biotech, Sweden. 2,4,6,7-[3 H]-estradiol (85–110 Ci/mmol) and 1,2,6,7-[3 H]-testosterone (70–105 Ci/mmol) were procured from Amersham Int. Plc., Amersham, UK. 2.3. Blood and tissue collection Blood samples were collected from rats of day 21–160 pp and sera separated for hormone assays. Animals were sacrificed by decapitation under ether anesthesia and thyroid glands from all the experimental rats were dissected out and were stored in liquid nitrogen for AR and ER assays. 2.4. Experimental design Two major groups of male and female rats were used in the present study. Group I: Rats belonging to this group were gonadectomized (GDX) at the age of day 10 pp (peak mitotic index) and autopsied at the age of day 21 (immature-weaning), 30 (immature-pre-pubertal), 45 (peri-pubertal) and 60 pp (pubertal), and were subdivided into the following subgroups. Sub-group i: 21 days old rats GDX at day 10 pp and treated with the vehicle propylene glycol, i.p. from day 11 pp. Sub-group ii: 21 days old rats GDX at day 10 pp and treated with testosterone daily, i.p. from day 11 pp. Sub-group iii: 21 days old rats GDX at day 10 pp and treated with estradiol daily, i.p. from day 11 pp. Sub-group iv: 30 days old rats GDX at day 10 pp and treated with the vehicle propylene glycol, i.p. from day 11 pp. Sub-group v: 30 days old rats GDX at day 10 pp and treated with testosterone daily, i.p. from day 11 pp. Sub-group vi: 30 days old rats GDX at day 10 pp and treated with estradiol daily, i.p. from day 11 pp. Sub-group vii: 45 days old rats GDX at day 10 pp and treated with the vehicle propylene glycol daily, i.m. from day 11 pp. Sub-group viii: 45 days old rats GDX at day 10 pp and treated with testosterone daily, i.m. from day 11 pp. Sub-group ix: 45 days old rats GDX at day 10 pp and treated with estradiol daily, i.m. from day 11 pp.
Sub-group x: 60 days old rats GDX at day 10 pp and treated with the vehicle propylene glycol daily, i.m. from day 11 pp. Sub-group xi: 60 days old rats GDX at day 10 pp and treated with testosterone daily, i.m. from day 11 pp. Sub-group xii: 60 days old rats GDX at day 10 pp and treated with estradiol daily, i.m. from day 11 pp. Group II: These rats were GDX at the age of day 120 pp, and left for 10 days without any hormonal replacement, and supplemented with sex steroids daily from day 131 pp and autopsied at day 160 pp. Rats belonging to this group were sub-divided into the following sub-groups. Sub-group xiii: 160 days old rats GDX at day 120 pp and treated with the vehicle daily, i.m. from day 130 pp. Sub-group xiv: 160 days old rats GDX at day 120 pp and treated with testosterone daily, i.m. from day 130 pp. Sub-group xv: 160 days old rats GDX at day 120 pp and treated with estradiol daily, i.m. from day 130 pp. Replacement regimen of testosterone and estradiol to gonadectomized rats Age groups
Immature (day 11–30 pp) Pubertal (day 30–60 pp) Adult (day 130–160 pp)
Testosterone (g/100 g BW)
Estradiol (g/100 g BW)
Male
Female
Male
0.1
0.05
0.5
100
1.0
0.5
5.0
200
2.0
1.0
10.0
5.0
Female
2.5. Extraction of steroids from the thyroid tissue Sex steroids from thyroid tissue were extracted with 1:1 ethyl acetate and iso-octane solution as described in our previous paper [4]. 2.6. Radioimmunoassay of serum and tissue testosterone and estradiol Testosterone and estradiol were estimated according to the method of Sufi et al. [13], using specific antibodies. The percentage binding of the antigen to the antibody was 36% for testosterone and 37–40% for estradiol. Minimum detectable levels of testosterone and estradiol were 0.3 and 0.1 pg per tube, respectively. Testosterone concentration
S.K. Banu et al. / Steroids 67 (2002) 1007–1014
was expressed as ng/ml serum and ng/g tissue, and the concentration of estradiol in serum and thyroid tissue has been expressed as pg/ml and pg/g thyroid, respectively. The inter- and intraassay of variations and cross-reactivity for testosterone and estradiol were as mentioned in our previous paper [4].
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3. Results 3.1. Effect of gonadectomy on serum and intrathyroidal testosterone and estradiol 3.1.1. Testosterone Gonadectomy significantly decreased serum and intrathyroidal testosterone concentration in male and female rats of all the experimental groups. Supplementation of testosterone brought back normal titres of serum and thyroidal testosterone in all the groups. Estradiol treatment to GDX rats did not bring about any change in testosterone titres in rats from any of the group (Tables 1 and 2).
2.7. Radioreceptor assay AR and ER concentrations in the nuclear fraction of thyroid tissue were quantified by radioreceptor assay followed by the method of Marugo et al. [14]. The entire assay was carried out at 4 ◦ C. The assay details were elaborated in our previous paper [4]. Bmax and kd values were calculated by Scatchard plot analysis [15]. The concentration of the receptors for sex steroids were expressed as fmol/mg DNA.
3.1.2. Estradiol As in the case of testosterone, gonadectomy perceptibly decreased estradiol concentration in serum and thyroid tissue of male and female rats from all the experimental groups. Supplementation of estradiol brought back normality of serum and tissue estradiol titres in all the above groups.
2.8. Statistical analysis The data were subjected to ANOVA [16]. Table 1 Serum testosterone in rats (ng/ml)
Gonadectomized
GDX + T
GDX + E2
Age in days
Gonad intact
Male 21 30 45 60 160
0.47 0.75 2.80 4.40 5.30
± ± ± ± ±
0.03 0.10 0.13 0.20 0.21
0.05 0.15 0.20 0.25 0.20
± ± ± ± ±
0.01a 0.01a 0.02a 0.02a 0.01a
0.51 0.81 3.10 4.70 5.91
± ± ± ± ±
0.03b 0.05b 0.12b 0.20b 0.18b
0.04 0.20 0.20 0.30 0.25
± ± ± ± ±
0.01ac 0.01ac 0.02ac 0.02ac 0.02ac
Female 21 30 45 60 160
0.07 0.17 0.20 0.41 0.52
± ± ± ± ±
0.004 0.01 0.01 0.02 0.04
0.005 0.05 0.04 0.03 0.07
± ± ± ± ±
0.001a 0.005a 0.01a 0.01a 0.01a
0.06 0.19 0.23 0.42 0.57
± ± ± ± ±
0.01b 0.02b 0.02b 0.01b 0.04b
0.005 0.03 0.03 0.04 0.09
± ± ± ± ±
0.002ac 0.01ac 0.01ac 0.02ac 0.02ac
Each value is mean ± S.E.M. of 10 samples. Level of significance at P < 0.01. a: Gonad intact vs. others; b: GDX vs. others; c: GDX + testosterone vs. others. GDX: gonadectomized; T: testosterone; E2 : estradiol.
Table 2 Intrathyroidal testosterone in rats (ng/g thyroid) Gonadectomized
GDX + T
GDX + E2
Age in days
Gonad intact
Male 21 30 45 60 160
2.30 1.40 8.60 9.60 9.30
± ± ± ± ±
0.08 0.05 0.32 0.30 0.21
0.30 0.07 1.50 1.25 1.20
± ± ± ± ±
0.01a 0.01a 0.04a 0.04a 0.03a
2.50 1.50 9.10 11.10 10.20
± ± ± ± ±
0.15b 0.10b 0.32b 0.65b 0.48b
0.20 0.05 0.90 1.30 1.40
± ± ± ± ±
0.01ac 0.008ac 0.03ac 0.08ac 0.09ac
Female 21 30 45 60 160
0.59 0.68 0.71 0.75 0.58
± ± ± ± ±
0.02 0.02 0.02 0.02 0.02
0.05 0.05 0.09 0.10 0.10
± ± ± ± ±
0.01a 0.005a 0.01a 0.01a 0.01a
0.63 0.72 0.76 0.82 0.61
± ± ± ± ±
0.03b 0.03b 0.03b 0.04b 0.04b
0.05 0.06 0.03 0.09 0.09
± ± ± ± ±
01ac 0.01ac 0.01ac 0.02ac 0.01ac
Each value is mean ± S.E.M. of four samples pooled from 15 to 30 rats. Level of significance at P < 0.01. a: Gonad intact vs. others; b: GDX vs. others; c: GDX + testosterone vs. others. GDX: gonadectomized; T: testosterone; E2 : estradiol.
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Fig. 1. Effect of gonadectomy on androgen receptor concentration in male and female Wistar rats. Each value is mean ± S.E.M. of five replications, pooled from 30 to 50 rats. Level of significance at P < 0.05. (a) Gonad intact vs. others; (b) GDX vs. others; (c) GDX + testosterone vs. others.
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Fig. 2. Effect of gonadectomy on estrogen receptor concentration in male and female Wistar rats. Each value is mean ± S.E.M. of five replications, pooled from 30 to 50 rats. Level of significance at P < 0.05. (a) Gonad intact vs. others; (b) GDX vs. others; (c) GDX + testosterone vs. others.
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Table 3 Serum estradiol in rats (pg/ml) Age in days
Gonad intact
Gonadectomized
GDX + T
GDX + E2
Male 21 30 45 60 160
0.33 0.45 0.72 0.63 1.00
± ± ± ± ±
0.01 0.01 0.02 0.02 0.08
0.10 0.10 0.15 0.19 0.20
± ± ± ± ±
0.01a 0.01a 0.005a 0.02a 0.01a
0.15 0.19 0.28 0.30 0.30
± ± ± ± ±
0.007ab 0.009ab 0.012ab 0.01ab 0.01ab
0.30 0.49 0.79 0.71 1.20
± ± ± ± ±
0.01bc 0.01bc 0.03bc 0.02bc 0.09bc
Female 21 30 45 60 160
21.07 9.50 30.20 25.31 35.20
± ± ± ± ±
0.90 0.31 0.80 1.30 1.60
2.20 0.90 1.40 1.20 2.20
± ± ± ± ±
0.01a 0.03a 0.06a 0.05a 0.08a
5.8 2.60 4.20 5.00 6.40
± ± ± ± ±
0.20ab 0.10ab 0.13ab 0.12ab 0.16ab
19.50 8.90 33.0 28.10 33.80
± ± ± ± ±
1.00bc 0.25bc 1.60bc 1.00bc 1.30bc
Each value is mean ± S.E.M. of 10 samples. Level of significance at P < 0.01. a: Gonad intact vs. others; b: GDX vs. others; c: GDX + testosterone vs. others. GDX: gonadectomized; T: testosterone; E2 : estradiol. Table 4 Intrathyroidal estradiol in rats (pg/g thyroid) Age in days
Gonad intact
Gonadectomized
GDX + T 0.50 0.80 1.20 1.00 0.90
± ± ± ± ±
0.03ab 0.13ab 0.08ab 0.04ab 0.04ab
2.42 7.64 7.20 9.30 9.15
± ± ± ± ±
0.08bc 0.25bc 0.25bc 0.38bc 0.22bc
4.06 2.60 6.30 6.60 3.90
± ± ± ± ±
0.16ab 0.05ab 0.16ab 0.09ab 0.10ab
12.95 30.25 136.03 150.09 111.00
± ± ± ± ±
0.80bc 1.30bc 4.20bc 4.40bc 2.70bc
Male 21 30 45 60 160
2.10 7.40 6.80 8.80 8.60
± ± ± ± ±
0.12 0.10 0.12 0.28 0.21
ND ND ND ND ND
Female 21 30 45 60 160
13.2 28.1 140.0 147.0 118.0
± ± ± ± ±
0.90 1.30 5.60 4.20 4.60
0.90 0.60 1.90 2.00 1.20
± ± ± ± ±
0.06a 0.03a 0.05a 0.06a 0.08a
GDX + E2
Each value is mean ± S.E.M. of four samples pooled from 15 to 30 rats. Level of significance at P < 0.01. a: Gonad intact vs. others; b: GDX vs. others; c: GDX + testosterone vs. others. GDX: gonadectomized; T: testosterone; E2 : estradiol; ND: non-detectable.
Substitution of testosterone to GDX rats also increased the concentration of serum and intrathyroidal estradiol in female rats of all age groups, when compared with respective age-matched GDX controls. Males also showed a similar trend. However, in both the sexes, the level of serum estradiol did not reach the control levels in testosterone-supplemented rats when compared with normal controls (Tables 3 and 4). 3.1.3. Androgen receptors (AR) Gonadectomy markedly decreased the concentration of AR in male and female rats in all experimental groups. Replacement of testosterone to GDX male and female rats brought back the normal level of AR in the thyroid, while estradiol replacement did not alter the concentration of the same (Fig. 1). 3.1.4. Estrogen receptors (ER) Concentration of ER in male and female rats of different experimental groups decreased significantly due to gonadectomy. Normal level of ER was regained in GDX rats given estradiol supplementation in both the sexes. Though testos-
terone supplementation did not maintain normality of ER, it significantly elevated the concentration of ER in the thyroid of all the experimental groups compared to GDX rats, except in 30 days males (Fig. 2).
4. Discussion Gender specific modulatory effects of testosterone and estradiol on the proliferation of normal thyroid epithelial cells from immature and adult rats, and human thyroid carcinoma cell lines were reported from our laboratory recently [3,12]. Data from our in vitro study with isolated thyrocytes from immature and adult rats revealed a direct mitogenic effect of sex steroids (testosterone and estradiol) on thyrocytes which is gender specific [3]. Further, the inhibitory effect of tamoxifen/flutamide on the basal and estradiol/testosterone-induced thyrocyte proliferation confirmed the role of sex steroids on thyrocyte proliferation [3]. However, thyrotropin (TSH) has a better stimulatory effect on thyrocyte proliferation, which is independent of
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sex. Co-administration of TSH along with sex steroids resulted in the maximum proliferative response of thyrocytes, suggesting the potentiating effect of sex steroids on TSH-mediated thyrocyte proliferation [17]. Testosterone and estradiol modulate TSH-binding in the thyrocytes of Wistar rats, which is influenced by age and sex of the animals [18]. Therefore, we suggest that sex steroids have direct effect on thyroid growth apart from their potentiating effect on TSH-mediated growth events. Our recent study revealed a positive correlation between thyroid growth and sex steroids, and their receptor status in post-natal, prepubertal, pubertal and adult rats, in vivo [6]. Evidence for a direct stimulatory effect of estradiol on thyroid cell growth has recently been reported in FRTL-5 rat thyroid cell line that expresses functional ER [19], and human thyroid tumor cells [2,12]. Expression of ER-mRNA and protein in benign and malignant thyroid cells have been demonstrated recently, confirming the ER-mediated events in thyroid tumorigenesis [2]. The growth stimulatory effect of estradiol on benign and malignant thyroid cells was reported to be associated with an increased expression of cyclin D1, the protein that plays a key role in the regulation of G1/S transition in the cell cycle [2]. It is interesting that cyclin D1 gene possesses an estrogen-responsive regulatory region [20]. Over expression of cyclin D1 is reported in thyroid carcinoma tissues [21]. Estrogen activates transcription of oncogenes such as c-fos and, c-myc [22] and it also promotes thyroid tumor growth via MAP-kinase cytoplasmic signaling [2]. We have recently reported that gonadectomy of rats at the post-natal to adult age has decreased thyroid growth indices such as mitotic index, concentration of DNA and number of thyrocytes and supplementation of sex steroids restored them to normal level [23]. The above study also exhibited parallel changes in the concentration of serum TSH and its receptor. It is suggested that sex steroids may modulate thyroid growth through up- or down-regulation of their receptor numbers in the thyroid, apart from their effect on TSH and TSH-receptor. Therefore, the present study is attempted to evaluate the status of sex steroids and their receptors in vivo under GDX and sex steroid replaced conditions. Previous report on human subjects revealed that the concentration of AR is higher in the thyroid gland of males, while ER is higher in females [8]. A similar phenomenon is also observed in normal rats in the present study. Castration significantly decreased the incidence of thyroid tumors in rats treated with irradiation or carcinogen [9,10], whereas testosterone replacement was associated with increased tumor yield [24]. AR and ER are found in higher concentration in neoplastic human thyroid tissue when compared with the adjacent non-neoplastic tissue [9,25]. Estradiol up-regulates estrogen and progesterone receptor gene expression in ovine uterus [26]. Testosterone up-regulates AR gene expression in the testis of rats [27]. A similar pattern of regulation of AR and ER by androgen and estrogen, respectively, might
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have occurred in the thyroid. The present study shows the up-regulation of sex steroid receptors in the thyroid for the first time. Therefore, our study reveals that sex steroid receptors in the thyroid are under homologous regulation by their respective ligands. This may be deduced from the consistent reduction of AR and ER concentration in the thyroid of GDX rats, and the specific stimulation of AR by testosterone and ER by estradiol in these rats, irrespective of the age and sex of animals. A careful look at the data on nuclear steroid receptors may reveal that testosterone is capable of increasing ER concentration in GDX female rats to a small but significant level. In fact, serum and thyroidal concentrations of estradiol increased to a significant level in the female rats supplemented with testosterone after gonadectomy, though not at par with gonad intact control. Probably, this is the reason for the heterologous up-regulation of ER by testosterone. Since there is also an increase in circulating estradiol in these rats, it is possible that there occurs peripheral conversion of testosterone into estradiol in GDX females. This in turn should have resulted in increased thyroidal concentration of estradiol and thus, ER. Bioavailability of testosterone and estradiol in the thyroid tissue of male and female rats is a novel finding in our previous [4,6] and present study. Our recent studies revealed that changes in the sex steroid status between male and female rats have a definite influence on the pattern of thyroid growth [4,6]. Gonadectomy impaired thyroid histoarchitecture in immature and adult male and female rats (our unpublished data). Presence of androgen and ER in the thyroid glands of immature, pubertal and adult rats, and the regulation of AR and ER concentration by sex steroids under normal physiological conditions indicate that sex steroids might have an essential role not only in thyroid pathogenesis, but also in the normal development and growth of the thyroid. Moreover, the differences in the incidence of thyroid diseases between male and female sex may be due to the differences in the thyroidal concentration of sex steroids and their receptors, and difference in sex steroid-mediated signal transduction events. Future studies in this area might strengthen this proposal. Acknowledgments One of the authors (Sakhila K. Banu) gratefully acknowledges Council of Scientific and Industrial Research (CSIR), New Delhi, India, for sponsoring the senior research fellowship. References [1] Wang SH, Myc A, Koenig RJ, Bretz JD, Arscott PL, Baker JR. 2-Methoxy estradiol, an endogenous estrogen metabolite, induces thyroid cell apoptosis. Mol Cell Endocrinol 2000;165:163–72. [2] Manole D, Schildknecht B, Gosnell B, Adams E, Derwahl M. Estrogen promotes growth of human thyroid tumor cells by different molecular mechanisms. J Clin Endocrinol Metab 2001;86:1072–7.
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[15] Scatchard G. The attraction of proteins for small molecules and ions. Ann N Y Acad Sci 1949;31:660–72. [16] Zar JH. Biostatistical analysis. Englewood Cliffs, NJ: Prentice-Hall, 1984. p. 151–81. [17] Banu SK. Gender specific modulatory effects of testosterone and estradiol on the post-natal growth and development of the thyroid gland in Wistar rats. Ph.D. Thesis, University of Madras, Chennai, India. [18] Banu SK, Govindarajulu P, Aruldhas MM. Testosterone and estradiol modulate TSH-binding in the thyrocytes of Wistar rats: influence of age and sex. J Steroid Biochem Mol Biol 2001;78:329–42. [19] Furnaletto TW, Nguyen LQ, Jameson JL. Estradiol increases proliferation and down-regulates the sodium/iodide symporter gene in FRTL-5 cells. Endocrinology 1999;140:5705–11. [20] Altucci L, Addeo R, Cicatiello L, Dauvois S, Parker MG, Truss M, et al. 17beta-Estradiol induces cyclin D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested human breast cancer cells. Oncogene 1996;12:2315–24. [21] Farid NR. Molecular pathogenesis of thyroid cancer: the significance of oncogenes, tumour suppressor genes, and genomic instability. Exp Clin Endocrinol Diabetes 1996;104:1–12. [22] Schuchard M, Landers JP, Sandhu NP, Spelsberg TC. Steroid hormone regulation of nuclear proto-oncogenes. Endocrine Rev 1993;14:659–69. [23] Banu SK, Arosh JA, Govindarajulu P, Aruldhas MM. Testosterone and estradiol differentially regulate thyroid growth in Wistar rats from immature to adult age. Endocrine Res 2001;27:447–63. [24] Napalkov NP. On some new aspects of experimental carcinogenesis in the thyroid gland. Int J Cancer 1963;19:756–7. [25] Clark OH, Gerend PL, Davis M, Goretzki PE, Hoffman PG. Estrogen and thyroid stimulating hormone (TSH) receptors in neoplastic and non-neoplastic human thyroid tissue. J Surg Res 1985;38:89–96. [26] Ing NH, Tornesi MB. Estradiol up-regulates estrogen receptor and progesterone receptor gene expression in specific ovine uterine cells. Biol Reprod 1997;56:1205–15. [27] Turner KJ, Morley M, MacPherson S, Millar MR, Wilson JA, Sharpe RM, et al. Modulation of gene expression by androgen and estrogen in the testis and prostate of the adult rat following androgen withdrawal. Mol Cell Endocrinol 2001;178:73–87.
Testosterone and estradiol up-regulate androgen and estrogen receptors in immature and adult rat thyroid glands in vivo Sakhila K. Banu∗ , P. Govindarajulu, Michael M. Aruldhas Department of Endocrinology, Dr. ALM. PG. Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600113, India Received 8 October 2001; received in revised form 16 April 2002; accepted 23 April 2002
Abstract Thyroid gland is one of the non-classical target organs for sex steroids. Presence of androgen and estrogen receptors in the neoplastic and non-neoplastic thyroid glands of mammalian species is well documented. The aim of the present study is to elucidate the changes in serum and thyroidal sex steroids, and their receptors in the thyroid gland of rats from immature to adult age under gonadectomized (GDX) and sex steroids replaced conditions. Normal Wistar male and female rats from immature to adult age (day 21, 30, 45, 60 and 160 post-partum (pp)) were used in the present study. One group (I) of rats was GDX at an early age (day 10 pp) and the other group (II) at the adult age (day 120 pp). Group I rats were sacrificed at different experimental periods such as 21, 30, 45 and 60 days pp, and group II rats were sacrificed at day 160 pp. Another group of GDX rats from group I and II were replaced with physiological doses of testosterone or estradiol. Serum and thyroidal concentrations of sex steroids were estimated by RIA method and the concentrations of receptors by radioreceptor assay. Gonadectomy significantly decreased serum and thyroidal testosterone and estradiol and concentrations of androgen receptor (AR) and estrogen receptor (ER) in the thyroid. Replacement of sex steroids to GDX rats restored the normal level of sex steroids, AR and ER. Therefore, it is suggested from the present study that (i) sex steroids up-regulate their own receptors in the thyroid, (ii) sex steroids may influence thyroid growth and the proliferation of thyrocytes by modulating their receptor concentrations in the thyroid. © 2002 Published by Elsevier Science Inc. Keywords: Gonadectomy; Thyroid; Sex steroid-receptor
1. Introduction The extreme female predominance of thyroid diseases suggests that sex hormones may have a pathogenic role in the thyroid [1,2]. A recent study described a divergent role for 17-beta estradiol and its metabolite 2-methoxy estradiol (2-ME) on human thyroid cell proliferation. While estradiol stimulated cell cycle progression early in G1 phase by induction of cyclin D1 gene expression in benign and malignant thyroid cell, 2-ME induced G2/M cycle arrest, apoptosis and disruption of thyroid follicles [1]. Androgen also has a direct influence on thyroid cell proliferation [3–5]. Presence of AR has been reported in thyroid tissue of rat, primate and human [4–8]. Early studies suggest that the gonadal steroids indirectly act through the hypothalamic–pituitary axis to modulate thyroid function [9–11]. However, our recent studies proved the direct effects of testosterone and estradiol on the proliferation of normal (rat) [3] and cancerous (human) [12] ∗
Corresponding author. Present address: Ontogenie et ReproductionCRBR, CHUL, 2705 Boul W. Laurier, Laval University, Ste-Foy, Que., Canada, GIV 4G2. Tel.: +1-418-656-4141x6125; fax: +1-418-654-2765. E-mail address: [email protected] (S.K. Banu). 0039-128X/02/$ – see front matter © 2002 Published by Elsevier Science Inc. PII: S 0 0 3 9 - 1 2 8 X ( 0 2 ) 0 0 0 6 3 - 6
thyroid cells in vitro. Even though sex steroids regulate thyroid growth in vivo [4,6] and in vitro [3,12], the mechanism underlying this phenomenon is unproven. The present study addresses the effect of sex steroids on the concentration of AR and ER in the thyroid of immature and adult Wistar rats of various periods of maturation.
2. Experimental 2.1. Animals Wistar rats (Rattus norvegicus) of day 1–160 pp were maintained under controlled temperature (25 ± 2 ◦ C) and lighting (12 h dark and 12 h light) conditions. Animals were provided with a standard pellet diet (Gold Mohur, Lipton India Ltd., India) and clean drinking water ad libitum. 2.2. Chemicals Triamcinolone acetonide, testosterone, 17-estradiol, hydroxylapatite slurry and phenyl methyl sulfonyl fluoride
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(PMSF) were purchased from Sigma, St. Louis. Pre-packed PD 10 and G-25 sephadex columns were purchased from Pharmacia Biotech, Sweden. 2,4,6,7-[3 H]-estradiol (85–110 Ci/mmol) and 1,2,6,7-[3 H]-testosterone (70–105 Ci/mmol) were procured from Amersham Int. Plc., Amersham, UK. 2.3. Blood and tissue collection Blood samples were collected from rats of day 21–160 pp and sera separated for hormone assays. Animals were sacrificed by decapitation under ether anesthesia and thyroid glands from all the experimental rats were dissected out and were stored in liquid nitrogen for AR and ER assays. 2.4. Experimental design Two major groups of male and female rats were used in the present study. Group I: Rats belonging to this group were gonadectomized (GDX) at the age of day 10 pp (peak mitotic index) and autopsied at the age of day 21 (immature-weaning), 30 (immature-pre-pubertal), 45 (peri-pubertal) and 60 pp (pubertal), and were subdivided into the following subgroups. Sub-group i: 21 days old rats GDX at day 10 pp and treated with the vehicle propylene glycol, i.p. from day 11 pp. Sub-group ii: 21 days old rats GDX at day 10 pp and treated with testosterone daily, i.p. from day 11 pp. Sub-group iii: 21 days old rats GDX at day 10 pp and treated with estradiol daily, i.p. from day 11 pp. Sub-group iv: 30 days old rats GDX at day 10 pp and treated with the vehicle propylene glycol, i.p. from day 11 pp. Sub-group v: 30 days old rats GDX at day 10 pp and treated with testosterone daily, i.p. from day 11 pp. Sub-group vi: 30 days old rats GDX at day 10 pp and treated with estradiol daily, i.p. from day 11 pp. Sub-group vii: 45 days old rats GDX at day 10 pp and treated with the vehicle propylene glycol daily, i.m. from day 11 pp. Sub-group viii: 45 days old rats GDX at day 10 pp and treated with testosterone daily, i.m. from day 11 pp. Sub-group ix: 45 days old rats GDX at day 10 pp and treated with estradiol daily, i.m. from day 11 pp.
Sub-group x: 60 days old rats GDX at day 10 pp and treated with the vehicle propylene glycol daily, i.m. from day 11 pp. Sub-group xi: 60 days old rats GDX at day 10 pp and treated with testosterone daily, i.m. from day 11 pp. Sub-group xii: 60 days old rats GDX at day 10 pp and treated with estradiol daily, i.m. from day 11 pp. Group II: These rats were GDX at the age of day 120 pp, and left for 10 days without any hormonal replacement, and supplemented with sex steroids daily from day 131 pp and autopsied at day 160 pp. Rats belonging to this group were sub-divided into the following sub-groups. Sub-group xiii: 160 days old rats GDX at day 120 pp and treated with the vehicle daily, i.m. from day 130 pp. Sub-group xiv: 160 days old rats GDX at day 120 pp and treated with testosterone daily, i.m. from day 130 pp. Sub-group xv: 160 days old rats GDX at day 120 pp and treated with estradiol daily, i.m. from day 130 pp. Replacement regimen of testosterone and estradiol to gonadectomized rats Age groups
Immature (day 11–30 pp) Pubertal (day 30–60 pp) Adult (day 130–160 pp)
Testosterone (g/100 g BW)
Estradiol (g/100 g BW)
Male
Female
Male
0.1
0.05
0.5
100
1.0
0.5
5.0
200
2.0
1.0
10.0
5.0
Female
2.5. Extraction of steroids from the thyroid tissue Sex steroids from thyroid tissue were extracted with 1:1 ethyl acetate and iso-octane solution as described in our previous paper [4]. 2.6. Radioimmunoassay of serum and tissue testosterone and estradiol Testosterone and estradiol were estimated according to the method of Sufi et al. [13], using specific antibodies. The percentage binding of the antigen to the antibody was 36% for testosterone and 37–40% for estradiol. Minimum detectable levels of testosterone and estradiol were 0.3 and 0.1 pg per tube, respectively. Testosterone concentration
S.K. Banu et al. / Steroids 67 (2002) 1007–1014
was expressed as ng/ml serum and ng/g tissue, and the concentration of estradiol in serum and thyroid tissue has been expressed as pg/ml and pg/g thyroid, respectively. The inter- and intraassay of variations and cross-reactivity for testosterone and estradiol were as mentioned in our previous paper [4].
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3. Results 3.1. Effect of gonadectomy on serum and intrathyroidal testosterone and estradiol 3.1.1. Testosterone Gonadectomy significantly decreased serum and intrathyroidal testosterone concentration in male and female rats of all the experimental groups. Supplementation of testosterone brought back normal titres of serum and thyroidal testosterone in all the groups. Estradiol treatment to GDX rats did not bring about any change in testosterone titres in rats from any of the group (Tables 1 and 2).
2.7. Radioreceptor assay AR and ER concentrations in the nuclear fraction of thyroid tissue were quantified by radioreceptor assay followed by the method of Marugo et al. [14]. The entire assay was carried out at 4 ◦ C. The assay details were elaborated in our previous paper [4]. Bmax and kd values were calculated by Scatchard plot analysis [15]. The concentration of the receptors for sex steroids were expressed as fmol/mg DNA.
3.1.2. Estradiol As in the case of testosterone, gonadectomy perceptibly decreased estradiol concentration in serum and thyroid tissue of male and female rats from all the experimental groups. Supplementation of estradiol brought back normality of serum and tissue estradiol titres in all the above groups.
2.8. Statistical analysis The data were subjected to ANOVA [16]. Table 1 Serum testosterone in rats (ng/ml)
Gonadectomized
GDX + T
GDX + E2
Age in days
Gonad intact
Male 21 30 45 60 160
0.47 0.75 2.80 4.40 5.30
± ± ± ± ±
0.03 0.10 0.13 0.20 0.21
0.05 0.15 0.20 0.25 0.20
± ± ± ± ±
0.01a 0.01a 0.02a 0.02a 0.01a
0.51 0.81 3.10 4.70 5.91
± ± ± ± ±
0.03b 0.05b 0.12b 0.20b 0.18b
0.04 0.20 0.20 0.30 0.25
± ± ± ± ±
0.01ac 0.01ac 0.02ac 0.02ac 0.02ac
Female 21 30 45 60 160
0.07 0.17 0.20 0.41 0.52
± ± ± ± ±
0.004 0.01 0.01 0.02 0.04
0.005 0.05 0.04 0.03 0.07
± ± ± ± ±
0.001a 0.005a 0.01a 0.01a 0.01a
0.06 0.19 0.23 0.42 0.57
± ± ± ± ±
0.01b 0.02b 0.02b 0.01b 0.04b
0.005 0.03 0.03 0.04 0.09
± ± ± ± ±
0.002ac 0.01ac 0.01ac 0.02ac 0.02ac
Each value is mean ± S.E.M. of 10 samples. Level of significance at P < 0.01. a: Gonad intact vs. others; b: GDX vs. others; c: GDX + testosterone vs. others. GDX: gonadectomized; T: testosterone; E2 : estradiol.
Table 2 Intrathyroidal testosterone in rats (ng/g thyroid) Gonadectomized
GDX + T
GDX + E2
Age in days
Gonad intact
Male 21 30 45 60 160
2.30 1.40 8.60 9.60 9.30
± ± ± ± ±
0.08 0.05 0.32 0.30 0.21
0.30 0.07 1.50 1.25 1.20
± ± ± ± ±
0.01a 0.01a 0.04a 0.04a 0.03a
2.50 1.50 9.10 11.10 10.20
± ± ± ± ±
0.15b 0.10b 0.32b 0.65b 0.48b
0.20 0.05 0.90 1.30 1.40
± ± ± ± ±
0.01ac 0.008ac 0.03ac 0.08ac 0.09ac
Female 21 30 45 60 160
0.59 0.68 0.71 0.75 0.58
± ± ± ± ±
0.02 0.02 0.02 0.02 0.02
0.05 0.05 0.09 0.10 0.10
± ± ± ± ±
0.01a 0.005a 0.01a 0.01a 0.01a
0.63 0.72 0.76 0.82 0.61
± ± ± ± ±
0.03b 0.03b 0.03b 0.04b 0.04b
0.05 0.06 0.03 0.09 0.09
± ± ± ± ±
01ac 0.01ac 0.01ac 0.02ac 0.01ac
Each value is mean ± S.E.M. of four samples pooled from 15 to 30 rats. Level of significance at P < 0.01. a: Gonad intact vs. others; b: GDX vs. others; c: GDX + testosterone vs. others. GDX: gonadectomized; T: testosterone; E2 : estradiol.
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Fig. 1. Effect of gonadectomy on androgen receptor concentration in male and female Wistar rats. Each value is mean ± S.E.M. of five replications, pooled from 30 to 50 rats. Level of significance at P < 0.05. (a) Gonad intact vs. others; (b) GDX vs. others; (c) GDX + testosterone vs. others.
S.K. Banu et al. / Steroids 67 (2002) 1007–1014
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Fig. 2. Effect of gonadectomy on estrogen receptor concentration in male and female Wistar rats. Each value is mean ± S.E.M. of five replications, pooled from 30 to 50 rats. Level of significance at P < 0.05. (a) Gonad intact vs. others; (b) GDX vs. others; (c) GDX + testosterone vs. others.
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Table 3 Serum estradiol in rats (pg/ml) Age in days
Gonad intact
Gonadectomized
GDX + T
GDX + E2
Male 21 30 45 60 160
0.33 0.45 0.72 0.63 1.00
± ± ± ± ±
0.01 0.01 0.02 0.02 0.08
0.10 0.10 0.15 0.19 0.20
± ± ± ± ±
0.01a 0.01a 0.005a 0.02a 0.01a
0.15 0.19 0.28 0.30 0.30
± ± ± ± ±
0.007ab 0.009ab 0.012ab 0.01ab 0.01ab
0.30 0.49 0.79 0.71 1.20
± ± ± ± ±
0.01bc 0.01bc 0.03bc 0.02bc 0.09bc
Female 21 30 45 60 160
21.07 9.50 30.20 25.31 35.20
± ± ± ± ±
0.90 0.31 0.80 1.30 1.60
2.20 0.90 1.40 1.20 2.20
± ± ± ± ±
0.01a 0.03a 0.06a 0.05a 0.08a
5.8 2.60 4.20 5.00 6.40
± ± ± ± ±
0.20ab 0.10ab 0.13ab 0.12ab 0.16ab
19.50 8.90 33.0 28.10 33.80
± ± ± ± ±
1.00bc 0.25bc 1.60bc 1.00bc 1.30bc
Each value is mean ± S.E.M. of 10 samples. Level of significance at P < 0.01. a: Gonad intact vs. others; b: GDX vs. others; c: GDX + testosterone vs. others. GDX: gonadectomized; T: testosterone; E2 : estradiol. Table 4 Intrathyroidal estradiol in rats (pg/g thyroid) Age in days
Gonad intact
Gonadectomized
GDX + T 0.50 0.80 1.20 1.00 0.90
± ± ± ± ±
0.03ab 0.13ab 0.08ab 0.04ab 0.04ab
2.42 7.64 7.20 9.30 9.15
± ± ± ± ±
0.08bc 0.25bc 0.25bc 0.38bc 0.22bc
4.06 2.60 6.30 6.60 3.90
± ± ± ± ±
0.16ab 0.05ab 0.16ab 0.09ab 0.10ab
12.95 30.25 136.03 150.09 111.00
± ± ± ± ±
0.80bc 1.30bc 4.20bc 4.40bc 2.70bc
Male 21 30 45 60 160
2.10 7.40 6.80 8.80 8.60
± ± ± ± ±
0.12 0.10 0.12 0.28 0.21
ND ND ND ND ND
Female 21 30 45 60 160
13.2 28.1 140.0 147.0 118.0
± ± ± ± ±
0.90 1.30 5.60 4.20 4.60
0.90 0.60 1.90 2.00 1.20
± ± ± ± ±
0.06a 0.03a 0.05a 0.06a 0.08a
GDX + E2
Each value is mean ± S.E.M. of four samples pooled from 15 to 30 rats. Level of significance at P < 0.01. a: Gonad intact vs. others; b: GDX vs. others; c: GDX + testosterone vs. others. GDX: gonadectomized; T: testosterone; E2 : estradiol; ND: non-detectable.
Substitution of testosterone to GDX rats also increased the concentration of serum and intrathyroidal estradiol in female rats of all age groups, when compared with respective age-matched GDX controls. Males also showed a similar trend. However, in both the sexes, the level of serum estradiol did not reach the control levels in testosterone-supplemented rats when compared with normal controls (Tables 3 and 4). 3.1.3. Androgen receptors (AR) Gonadectomy markedly decreased the concentration of AR in male and female rats in all experimental groups. Replacement of testosterone to GDX male and female rats brought back the normal level of AR in the thyroid, while estradiol replacement did not alter the concentration of the same (Fig. 1). 3.1.4. Estrogen receptors (ER) Concentration of ER in male and female rats of different experimental groups decreased significantly due to gonadectomy. Normal level of ER was regained in GDX rats given estradiol supplementation in both the sexes. Though testos-
terone supplementation did not maintain normality of ER, it significantly elevated the concentration of ER in the thyroid of all the experimental groups compared to GDX rats, except in 30 days males (Fig. 2).
4. Discussion Gender specific modulatory effects of testosterone and estradiol on the proliferation of normal thyroid epithelial cells from immature and adult rats, and human thyroid carcinoma cell lines were reported from our laboratory recently [3,12]. Data from our in vitro study with isolated thyrocytes from immature and adult rats revealed a direct mitogenic effect of sex steroids (testosterone and estradiol) on thyrocytes which is gender specific [3]. Further, the inhibitory effect of tamoxifen/flutamide on the basal and estradiol/testosterone-induced thyrocyte proliferation confirmed the role of sex steroids on thyrocyte proliferation [3]. However, thyrotropin (TSH) has a better stimulatory effect on thyrocyte proliferation, which is independent of
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sex. Co-administration of TSH along with sex steroids resulted in the maximum proliferative response of thyrocytes, suggesting the potentiating effect of sex steroids on TSH-mediated thyrocyte proliferation [17]. Testosterone and estradiol modulate TSH-binding in the thyrocytes of Wistar rats, which is influenced by age and sex of the animals [18]. Therefore, we suggest that sex steroids have direct effect on thyroid growth apart from their potentiating effect on TSH-mediated growth events. Our recent study revealed a positive correlation between thyroid growth and sex steroids, and their receptor status in post-natal, prepubertal, pubertal and adult rats, in vivo [6]. Evidence for a direct stimulatory effect of estradiol on thyroid cell growth has recently been reported in FRTL-5 rat thyroid cell line that expresses functional ER [19], and human thyroid tumor cells [2,12]. Expression of ER-mRNA and protein in benign and malignant thyroid cells have been demonstrated recently, confirming the ER-mediated events in thyroid tumorigenesis [2]. The growth stimulatory effect of estradiol on benign and malignant thyroid cells was reported to be associated with an increased expression of cyclin D1, the protein that plays a key role in the regulation of G1/S transition in the cell cycle [2]. It is interesting that cyclin D1 gene possesses an estrogen-responsive regulatory region [20]. Over expression of cyclin D1 is reported in thyroid carcinoma tissues [21]. Estrogen activates transcription of oncogenes such as c-fos and, c-myc [22] and it also promotes thyroid tumor growth via MAP-kinase cytoplasmic signaling [2]. We have recently reported that gonadectomy of rats at the post-natal to adult age has decreased thyroid growth indices such as mitotic index, concentration of DNA and number of thyrocytes and supplementation of sex steroids restored them to normal level [23]. The above study also exhibited parallel changes in the concentration of serum TSH and its receptor. It is suggested that sex steroids may modulate thyroid growth through up- or down-regulation of their receptor numbers in the thyroid, apart from their effect on TSH and TSH-receptor. Therefore, the present study is attempted to evaluate the status of sex steroids and their receptors in vivo under GDX and sex steroid replaced conditions. Previous report on human subjects revealed that the concentration of AR is higher in the thyroid gland of males, while ER is higher in females [8]. A similar phenomenon is also observed in normal rats in the present study. Castration significantly decreased the incidence of thyroid tumors in rats treated with irradiation or carcinogen [9,10], whereas testosterone replacement was associated with increased tumor yield [24]. AR and ER are found in higher concentration in neoplastic human thyroid tissue when compared with the adjacent non-neoplastic tissue [9,25]. Estradiol up-regulates estrogen and progesterone receptor gene expression in ovine uterus [26]. Testosterone up-regulates AR gene expression in the testis of rats [27]. A similar pattern of regulation of AR and ER by androgen and estrogen, respectively, might
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have occurred in the thyroid. The present study shows the up-regulation of sex steroid receptors in the thyroid for the first time. Therefore, our study reveals that sex steroid receptors in the thyroid are under homologous regulation by their respective ligands. This may be deduced from the consistent reduction of AR and ER concentration in the thyroid of GDX rats, and the specific stimulation of AR by testosterone and ER by estradiol in these rats, irrespective of the age and sex of animals. A careful look at the data on nuclear steroid receptors may reveal that testosterone is capable of increasing ER concentration in GDX female rats to a small but significant level. In fact, serum and thyroidal concentrations of estradiol increased to a significant level in the female rats supplemented with testosterone after gonadectomy, though not at par with gonad intact control. Probably, this is the reason for the heterologous up-regulation of ER by testosterone. Since there is also an increase in circulating estradiol in these rats, it is possible that there occurs peripheral conversion of testosterone into estradiol in GDX females. This in turn should have resulted in increased thyroidal concentration of estradiol and thus, ER. Bioavailability of testosterone and estradiol in the thyroid tissue of male and female rats is a novel finding in our previous [4,6] and present study. Our recent studies revealed that changes in the sex steroid status between male and female rats have a definite influence on the pattern of thyroid growth [4,6]. Gonadectomy impaired thyroid histoarchitecture in immature and adult male and female rats (our unpublished data). Presence of androgen and ER in the thyroid glands of immature, pubertal and adult rats, and the regulation of AR and ER concentration by sex steroids under normal physiological conditions indicate that sex steroids might have an essential role not only in thyroid pathogenesis, but also in the normal development and growth of the thyroid. Moreover, the differences in the incidence of thyroid diseases between male and female sex may be due to the differences in the thyroidal concentration of sex steroids and their receptors, and difference in sex steroid-mediated signal transduction events. Future studies in this area might strengthen this proposal. Acknowledgments One of the authors (Sakhila K. Banu) gratefully acknowledges Council of Scientific and Industrial Research (CSIR), New Delhi, India, for sponsoring the senior research fellowship. References [1] Wang SH, Myc A, Koenig RJ, Bretz JD, Arscott PL, Baker JR. 2-Methoxy estradiol, an endogenous estrogen metabolite, induces thyroid cell apoptosis. Mol Cell Endocrinol 2000;165:163–72. [2] Manole D, Schildknecht B, Gosnell B, Adams E, Derwahl M. Estrogen promotes growth of human thyroid tumor cells by different molecular mechanisms. J Clin Endocrinol Metab 2001;86:1072–7.
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