Branko Filipović

Chronic Estradiol Exposure Modulates Thyroid Structure and Decreases T4 and T3 Serum Levels in Middle-Aged Female Rats

Objectives: In human medicine, estrogen is applied in prevention and treatment of health problems associated with the menopause. The aim of this study was to examine the effects of chronic estradiol dipropionate (EDP) treatment on thyroid gland structure and function in middle-aged female rats. Methods: At 14 months of age, Wistar rats received 0.625 mg EDP/kg b.w./day intraperitoneally for 2 weeks. The peripheral and central zones of the thyroid were stereologically analyzed and the following morphometric parameters determined: volume density of follicles, follicular epithelium, interstitium and colloid, epithelial height and the index of activation rate. Serum levels of TSH, T4 and T3 were determined by ELISA. Results: EDP treatment led to significant decreases in volume densities of follicles and follicular epithelium, epithelial height and index of activation rate (by 11%, p < 0.05; 23%, p < 0.005; 11%, p < 0.05 and 21%, p < 0.05, respectively) in comparison to control values. Hyperplasia of thyroid follicular cells was noticed in 25% of EDP-treated animals. Serum levels of T4 and T3 were decreased (by 33%, p < 0.005 and 28%, p < 0.001, respectively), but TSH concentration was not significantly different from that of the controls. Conclusion: Chronic estradiol treatment significantly decreased volume density and height of centrally located follicular epithelium, follicular activation index and serum level of total thyroid hormones in middle-aged rats.

Suppressive effects of genistein and daidzein on pituitary–thyroid axis in orchidectomized middle-aged rats

High intake of soybean phytoestrogens, isoflavones genistein (G) and daidzein (D), has been associated with health benefits. However, isoflavones were reported to affect adversely thyroid function in the presence of other goitrogenic factors. As the thyroid gland becomes functionally impaired with age, we examined whether supplementary doses of G or D would affect morphology and function of pituitary–thyroid axis in middle-aged male rats. Sixteen-month-old orchidectomized Wistar rats were treated with 10 mg/kg of either G or D, while the control sham-operated and orchidectomized group received just the vehicle for three weeks. The animals were fed soy-free diet with increased iodine content, and killed 24 h after the last treatment. Their pituitaries and thyroids were excised and prepared for further immunohistochemical and morphometric investigation. The concentrations of thyroid-stimulating hormone (TSH), total T4 and T3, in the serum were determined. In both isoflavone-treated groups, pituitary TSH-immunopositive cells had increased cellular volume and relative volume density (P < 0.05), as well as increased serum TSH levels (P < 0.05) in comparison to the controls; their thyroid tissue was characterized by increased volume of thyroglobulin-immunopositive epithelium (P < 0.05), epithelial height and index of activation rate (P < 0.05), while the volume of luminal colloid, and total serum T4 and T3 levels decreased (P < 0.05) in comparison to the controls. In conclusion, this study provides the first direct evidence that both G and D can induce microfollicular changes in the thyroid tissue and reduce the level of thyroid hormones in Orx middle-aged male rats, a model of andropause. This reduction consequently led to a feedback stimulation of pituitary TSH cells. The detected stimulatory effect was higher in the daidzein-treated rats.

Unbiased Stereological Estimation of the Rat Fetal Pituitary Volume and of the Total Number and Volume of TSH Cells After Maternal Dexamethasone Application

Glucocorticoids have an inhibitory influence on proliferation activity of the pituitary cells while stimulating apoptosis. Therefore, it was hypothesized that the synthetic glucocorticoid, dexamethasone (DX), has an inhibitory influence on the number of thyroid‐stimulating hormone (TSH) cells during fetal development. The effects of maternal administration of DX on stereological parameters of TSH cells, and TSH serum concentration were investigated in 21‐day‐old rat fetuses. On day 16 of pregnancy, the experimental dams received 1.0 mg DX/kg b.w. subcutaneously, followed by 0.5 mg DX/kg b.w./day on days 17 and 18 of gestation. The control gravid females received the same volume of saline vehicle. TSH cells were stained immunocytochemically by the peroxidase–antiperoxidase (PAP) method. The fetal pituitary volumes were estimated using Cavalieris principle. A physical disector counting technique in combination with the fractionator sampling method was used for estimation of pituitary TSH cell number. Cell and nuclear volumes were measured with a planar rotator. Maternal DX application was found to cause a significant decrease of pituitary volume and number of TSH cells per pituitary in 21‐day‐old fetuses in comparison with the control fetuses. TSH cell number expressed per body weight unit declined significantly after maternal DX administration. These results indicate an inhibitory DX influence on proliferative activity of precursors and likely differentiated TSH cells and increased apoptotic prevalence. The histological appearance, volume of TSH cells and TSH serum concentration suggest intensive synthetic activity in TSH cells of DX exposed fetuses. Microsc. Res. Tech. 73:1077–1085, 2010.

Genistein-induced histomorphometric and hormone secreting changes in the adrenal cortex in middle-aged rats.

The soybean phytoestrogen, genistein, is increasingly consumed as an alternative therapeutic for age-related diseases, namely cardiovascular conditions, cancer and osteoporosis. Besides estrogenic/antiestrogenic action, this isoflavone exerts a prominent inhibitory effect on tyrosine kinase and the steroidogenic enzyme families, thus affecting hormonal homeostasis. The aim of this study was to examine the effects of genistein on: histomorphometric features of the adrenal cortex, blood concentrations of aldosterone, corticosterone and dehydroepiandrosterone (DHEA) and adrenal tissue corticosterone content in orchidectomized middle-aged male rats. Sixteen-month-old Wistar rats were divided into sham-operated (SO), orchidectomized (Orx) and genistein-treated orchidectomized (Orx+G) groups. Genistein (30 mg/kg/day) was administered subcutaneously for three weeks, while the control groups received the vehicle alone. The adrenal cortex was analysed histologically and morphometrically. Circulating concentrations of aldosterone, corticosterone and DHEA, as well as adrenal tissue corticosterone levels, were determined by immunoassay. When compared to the SO group, orchidectomy decreased the ZG and ZR cell volume by 43% and 29%, respectively (P < 0.05). Serum concentrations of aldosterone and DHEA were markedly lower [13% and 41%, respectively (P < 0.05)], while serum and adrenal tissue levels of corticosterone did not change after orchidectomy. Orchidectomy followed by genistein treatment increased the ZG, ZF and ZR cell volume by 54%, 34% and 77%, respectively (P < 0.05), compared to the untreated orchidectomized group. Histological analysis revealed noticeable vacuolization of the ZG and ZF cells in the Orx+G group. Serum aldosterone and corticosterone concentrations together with adrenal tissue corticosterone were 47%, 31% and 44% lower, respectively (P < 0.05), whereas serum DHEA concentration was 342% higher (P < 0.05) in this group in comparison with the Orx group. This study shows that in orchidectomized middle-aged rats, genistein can cause the shunting of metabolic pathways in the adrenals, supporting DHEA secretion and inhibiting corticosterone and aldosterone secretion.

A BRIEF COMMUNICATION Subcutaneously Administrated Genistein and Daidzein Decrease Serum Cholesterol and Increase Triglyceride Levels in Male Middle-Aged Rats

Nutritional supplements containing soybean phytoestrogens, the isoflavones genistein (G) and daidzein (D), are increasingly used as alternative therapy for osteoporosis, cancer, and cardiovascular and other diseases with a frequency that increases with advancing age. In this study we examined the effects of subcutaneous administration of either G or D on serum lipid levels in orchidectomized (Orx) and intact (IA) middle-aged male rats, which are experimental models of andropause. Sixteen-month-old Wistar rats were treated with 10 mg/kg and 30 mg/kg of either G or D. The control groups received testosterone, estradiol, or vehicle for 3 weeks, after which the total serum cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), and total triglycerides (TT) were measured. Compared with the matching vehicle-treated controls, the higher doses of G and D and testosterone treatment significantly (P < 0.05) lowered the TC and lipoprotein cholesterol levels. The greatest effect was observed regarding LDL-C in both Orx and IA males after G and D treatments, in which LDL-C decreased by more than 30%. The lower isoflavone doses induced a significant cholesterol-lowering effect (P < 0.05) only in the Orx group. Like the estradiol treatment, the higher doses of G and D increased the TT levels in both rat models by more than 50% (P < 0.05). The lower doses of isoflavones increased TT only in the Orx group. In male middle-aged rats, injections of higher doses of G and D decreased the serum cholesterol levels, as did testosterone injection, and brought about an increase in serum triglycerides similar to that observed after estradiol treatment.Nutritional supplements containing soybean phytoestrogens, the isoflavones genistein (G) and daidzein (D), are increasingly used as alternative therapy for osteoporosis, cancer, and cardiovascular and other diseases with a frequency that increases with advancing age. In this study we examined the effects of subcutaneous administration of either G or D on serum lipid levels in orchidectomized (Orx) and intact (IA) middle-aged male rats, which are experimental models of andropause. Sixteen-month-old Wistar rats were treated with 10 mg/kg and 30mg/kg of either G or D. The control groups received testosterone, estradiol, or vehicle for 3 weeks, after which the total serum cholesterol (TC), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), and total triglycerides (TT) were measured. Compared with the matching vehicle-treated controls, the higher doses of G and D and testosterone treatment significantly (P < 0.05) lowered the TC and lipoprotein cholesterol levels. The greatest effect was observed regarding LDL-C in both Orx and IA males after G and D treatments, in which LDL-C decreased by more than 30%. The lower isoflavone doses induced a significant cholesterol-lowering effect (P < 0.05) only in the Orx group. Like the estradiol treatment, the higher doses of G and D increased the TT levels in both rat models by more than 50% (P < 0.05). The lower doses of isoflavones increased TT only in the Orx group. In male middle-aged rats, injections of higher doses of G and D decreased the serum cholesterol levels, as did testosterone injection, and brought about an increase in serum triglycerides similar to that observed after estradiol treatment.

Soy isoflavones interfere with thyroid hormone homeostasis in orchidectomized middle-aged rats

We previously reported that genistein (G) and daidzein (D) administered subcutaneously (10mg/kg) induce changes in the angio-follicular units of the thyroid gland, reduce concentration of total thyroid hormones (TH) and increase thyrotropin (TSH) in serum of orchidectomized middle-aged (16-month-old) rats. To further investigate these effects, we now examined expression levels of the thyroglobulin (Tg), thyroperoxidase (Tpo), vascular endothelial growth factor A (Vegfa) and deiodinase type 1 (Dio 1) genes in the thyroid; in the pituitary, genes involved in TH feedback control (Tsh β, Dio 1, Dio 2, Trh receptor); and in the liver and kidney, expression of T3-activated genes Dio 1 and Spot 14, as well as transthyretin (Ttr), by quantitative real-time PCR. We also analyzed TPO-immunopositivity and immunofluorescence of T4 bound to Tg, determined thyroid T4 levels and measured deiodinase enzyme activities in examined organs. Decreased expression of Tg and Tpo genes (p<0.05) correlated with immunohistochemical staining results, and together with decreased serum total T4 levels, indicates decreased Tg and TH synthesis following treatments with both isoflavones. However, expression of Spot 14 (p<0.05) gene in liver and kidney was up-regulated, and liver Dio 1 expression and activity (p<0.05) increased. At the level of pituitary, no significant change in gene expression levels, or Dio 1 and 2 enzyme activities was observed. In conclusion, both G and D impaired Tg and TH synthesis, but at the same time increased tissue availability of TH in peripheral tissues of Orx middle-aged rats.

Orchidectomy of Middle-Aged Rats Decreases Liver Deiodinase 1 and Pituitary Deiodinase 2 Activity

Endogenous androgens are involved in regulation of thyroid function and metabolism of thyroid hormones. As serum testosterone level progressively declines with age, this regulation may change. We tested how androgen deprivation, achieved by orchidectomy, affects thyroid homeostasis in middle-aged rats. Fifteen-month-old Wistar rats were orchidectomized (Orx) or sham-operated under ketamine anesthesia (15 mg/kg body weight). Five weeks after the surgery, animals were decapitated. Thyroids were used for histomorphometric and ultrastructural examinations and together with livers and pituitaries for real-time quantitative PCR and deiodinase (DIO) activity measurements. Serum testosterone, TSH, l-thyroxine (T(4)), and cholesterol (Chol) levels were determined. As expected, middle-aged control rats had lower (P<0.05) testosterone and T(4) compared with 3-month-old males. In the Orx middle-aged group, we detected diminished serum testosterone (P<0.05), no change in TSH and T(4) levels, and higher Chol level (P<0.05), in comparison with age-matched controls. Histomorphometric analysis of thyroid tissue revealed decreased relative volume densities of follicles and colloid (P<0.05). Relevant gene expressions and DIO1 enzyme activity were not changed in the thyroids of Orx rats. Liver Dio1 gene expression and DIO1 activity were decreased (P<0.05) in comparison with the control values. Pituitary levels of TSHβ, Dio1, and Dio2 mRNAs did not change, while DIO2 activity decreased (P<0.05). In conclusion, orchidectomy of middle-aged rats affected thyroid structure with no effect on serum T(4) and TSH. However, decreased liver DIO1 and pituitary DIO2 enzyme activities indicate compensatory-adaptive changes in local T(3) production.

Testosterone application decreases the capacity for ACTH and corticosterone secretion in a rat model of the andropause.

The culminating phase of ageing in males-andropause is characterized by enhanced activity of the hypothalamic-pituitary-adrenal axis and frequent glucocorticoid excess. In parallel, free testosterone deficiency provides the baseline hormonal milieu for the ageing male. The aim of this study was to illustrate (using diverse microscopic and biochemical methodologies) the effects of testosterone application on the capacity for adrenocorticotropic hormone (ACTH) and corticosterone secretion in a rat model of the andropause. Middle-aged Wistar rats were divided into sham-operated (SO; n=8), orchidectomized (Orx; n=8) and testosterone treated orchidectomized (Orx+T; n=8) groups. Testosterone propionate (5 mg/kg b.w./day) was administered for three weeks, while SO and Orx groups received the vehicle alone. ACTH cells and the adrenal cortex were stained using immuno-histochemical, immuno-fluorescent and histochemical procedures. Circulating concentrations of testosterone, estradiol, ACTH and corticosterone, as well as the adrenal tissue corticosterone levels were measured by immunoassays. Testosterone application led to increased (p<0.05) serum concentrations of sex steroids. Consequently, in Orx+T rats the ACTH cell nuclei volume increased (p<0.05) by 34%, while the volume density of ACTH cells and their relative intensity of fluorescence decreased (p<0.05) by 46% and 21%, respectively, in comparison with the corresponding parameters in the Orx group. Testosterone also induced vasodilatation in the adrenocortical zona fasciculata, and decreased (p<0.05) the ACTH concentrations and adrenal tissue corticosterone levels by 38% and 31%, respectively, compared to the Orx group. In conclusion, testosterone administration caused a decrease in the capacity for ACTH and corticosterone secretion in a rat model of the andropause.

Testosterone and estradiol treatments differently affect pituitary-thyroid axis and liver deiodinase 1 activity in orchidectomized middle-aged rats.

We previously reported that orchidectomy (Orx) of middle-aged rats (15-16-month-old; MA) slightly affected pituitary-thyroid axis, but decreased liver deiodinase (Dio) type 1 and pituitary Dio2 enzyme activities. At present, we examined the effects of subsequent testosterone-propionate treatment (5mg/kg; Orx+T), and compared the effects of testosterone with the effects of estradiol-dipropionate (0.06mg/kg; Orx+E) treatment. Hormones were subcutaneously administered, daily, for three weeks, while Orx and sham-operated (SO) controls received only the vehicle. The applied dose of T did not alter serum TSH, T4 and T3 concentrations in Orx- MA, though it increased TSH when administrated to Orx young adults (2.5-month-old; Orx-YA). However, pituitaries of Orx-MA+T rats had higher relative intensity of immunofluorescence (RIF) for TSHβ; in their thyroids we found increased volume and height of follicular epithelium, decreased volume of the colloid and higher RIF for T4-bound to thyroglobulin (Tg-T4). Liver Dio1 activity was increased. E-treatment did not affect serum hormone levels, pituitary RIF for TSHβ, or liver Dio1 activity in Orx-MA rats. Thyroids had decreased relative volume and height of follicular epithelium, increased relative volume of the colloid, decreased volume of sodium-iodide symporter-immunopositive epithelium and lower RIF for Tg-T4. Detected changes were statistically significant. In conclusion, androgenization enhanced pituitary TSHβ RIF, thyroid activation and liver Dio1 enzyme activity in Orx-MA, without elevating serum TSH as in Orx-YA rats. Estrogenization induced pituitary enlargement with no effect on pituitary TSHβ RIF, serum TSH or liver Dio1 activity. E also induced alterations in thyroid histology that indicate mild suppression of its functioning, and contributed to thyroid blood vessel enlargement in Orx-MA rats.

The effects of sex steroids on thyroid C cells and trabecular bone structure in the rat model of male osteoporosis

Androgen deficiency is one of the major factors leading to the development of osteoporosis in men. Since calcitonin (CT) is a potent antiresorptive agent, in the present study we investigated the effects of androgen deficiency and subsequent testosterone and estradiol treatment on CT‐producing thyroid C cells, skeletal and hormonal changes in middle‐aged orchidectomized (Orx) rats. Fifteen‐month‐old male Wistar rats were either Orx or sham‐operated (SO). One group of Orx rats received 5 mg kg−1 b.w. testosterone propionate (TP) subcutaneously, while another group was injected with 0.06 mg kg−1 b.w. estradiol dipropionate (EDP) once a day for 3 weeks. A peroxidase–antiperoxidase method was applied for localization of CT in the C cells. The studies included ultrastructural microscopic observation of these cells. The metaphyseal region of the proximal tibia was measured histomorphometrically using an imagej public domain image processing program. TP or EDP treatment significantly increased C cell volume (Vc), volume densities (Vv) and serum CT concentration compared with the Orx animals. Administration of both TP and EDP significantly enhanced cancellous bone area (B.Ar), trabecular thickness (Tb.Th) and trabecular number (Tb.N) and reduced trabecular separation (Tb.Sp). Serum osteocalcin (OC) and urinary Ca concentrations were significantly lower after these treatments in comparison with Orx rats. These data suggest that testosterone and estradiol treatment in Orx middle‐aged rats affect calcitonin‐producing thyroid C cells, which may contribute to the bone protective effects of sex hormones in the rat model of male osteoporosis.