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As the mediator between the mother and fetus, the placenta allows the most appropriate environment and optimal fetal growth. The placenta of one sex sometimes has a greater ability over the other to respond to and protect against possible maternal insults. Here, we characterized sex differences in the placenta’s morphological features and antioxidant status following dexamethasone (Dx) exposure. Pregnant rats were exposed to Dx or saline. The placenta was histologically and stereologically analyzed. The activity of the antioxidant enzymes, lipid peroxides (TBARS), superoxide anion and nitric oxide (NO) was measured. The decrease in placental zone volumes was more pronounced (p < 0.05) in female placentas. The volume density of PCNA-immunopositive nuclei was reduced (p < 0.05) in both sexes. The reduced (p < 0.05) antioxidant enzyme activities, enhanced TBARS and NO concentration indicate that Dx exposure triggered oxidative stress in the placenta of both fetal sexes, albeit stronger in the placenta of female fetuses. In conclusion, maternal Dx treatment reduced the size and volume of placental zones, altered placental histomorphology, decreased cell proliferation and triggered oxidative stress; however, the placentas of female fetuses exerted more significant responses to the treatment effects. The reduced placental size most probably reduced the transport of nutrients and oxygen, thus resulting in the reduced weight of fetuses, similar in both sexes. The lesser ability of the male placenta to detect and react to maternal exposure to environmental challenges may lead to long-standing health effects.

The aim of the present study was to determine and elaborate on all changes in old-aged (OA) versus young-aged (YA) rat thyroids by using stereological, ultrastructural, hormonal, and gene expression analyses. We used 4- and 24-month-old male Wistar rats in our evaluation, presenting all changes in comparison with YA rats. Results showed that the thyroid parenchyma was characterized by higher absolute volumes of the gland, colloid, epithelium, and interstitium by 135, 135, 140, and 142% (p < 0.05) respectively, while the relative volumes of colloid and glands were unchanged. Ultrastructural analysis revealed less active glands, with smaller amounts of lysosomes, thyroglobulin (Tg) granules, and microvilli in the luminal colloid. Optical density values for thyroid peroxidase (TPO), Tg, and vascular-endothelial growth factor immunostaining remained unchanged; however, TPO and Tg exhibited visually stronger expression in small active follicles. Thyroxine (T4)-Tg, the relative intensity of fluorescence (RIF), serum T4, and the sodium-iodide symporter immunohistochemical and gene expressions decreased by 20, 40, 29, and 31% (p < 0.05), respectively, in OA thyroids. Pituitary thyroid-stimulating hormone (TSH) RIF increased by 44% (p < 0.05), but the TSH serum concentration remained unchanged. In conclusion, the obtained results indicate depression of the thyroid gland synthetic and secretory capacity with advanced age.

The use of the antidepressant drug chlorpromazine (CPZ) is linked to the occurrence of oxidative stress in some brain structures. Thus, overcoming the side effects of CPZ is of great importance. Because agmatine (AGM) can act as a free radical scavenger, it is an interesting compound as an adjunct to CPZ therapy. The aim of our study was to investigate the enzymatic parameters of oxidative stress in the hippocampus and striatum of rats after CPZ treatment, and the potential protective effects of AGM. Rats were injected as follows with (i) 1 mL/kg b.w. saline; (ii) a single intraperitoneal (i.p.) dose of CPZ (38.7 mg/kg); (iii) CPZ (38.7 mg/kg) and AGM (75 mg/kg); (iv) AGM (75 mg/kg). CPZ induced an increase in superoxide anion radical (O2 •-) concentration, while the activities of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), were lowered in both the hippocampus and striatum. Cotreatment with CPZ and AGM protected the examined brain structures by reversing the antioxidant enzyme activities to the control values. Following CPZ treatment, the effects were more pronounced for SOD and GPx in the hippocampus, and for SOD, CAT and GPx in the striatum. The full effect of restored superoxide production was achieved in the striatum, which points to the role of CAT. The obtained results suggest that CPZ in combination with AGM may be considered as a new treatment strategy.

Thyroid C-cells secrete the hormone calcitonin (CT) which acts as an inhibitor of bone resorption. Our aim was to examine the age-related changes in the structure and function of CT-producing C-cells, using histomorphometric, ultrastructural, and biochemical analyses. We used young adult (3-months-old), middle-aged (16-months-old), and old (24-months-old) male rats. The peroxidase-antiperoxidase method was applied for localization of CT. Stereological analysis was performed using the newCAST stereological software package. Serum samples were analyzed for the determination of CT, testosterone (T), calcium (Ca2+), and phosphorus (P). We found a significant increase in the volume density (Vv) of C-cells in both older groups (p < 0.05). The percentage of smaller volume range C-cells increased (p < 0.0001), while the proportion of greater volume range C-cells decreased (p < 0.05) with ageing. Ultrastructural analysis revealed a larger number of secretory granules in older rats. Serum CT increased (p < 0.001), while serum T and P were reduced (p < 0.01) in older rats. Serum Ca2+ was lower (p < 0.0001) in middle-aged rats compared to young adults. We revealed a 20% incidence of C-cell hyperplasia in older rats and one case of medullary thyroid carcinoma in an old rat. Our findings indicate that the ageing process causes significant histomorphometric changes at the thyroid C-cell level.

Vitamin D plays an essential role in prevention and treatment of osteoporosis. Thyroid hormones, in addition to vitamin D, significantly contribute to regulation of bone remodeling cycle and health. There is currently no data about a possible connection between vitamin D treatment and the thyroid in the context of osteoporosis. Middle-aged Wistar rats were divided into: sham operated (SO), orchidectomized (Orx), and cholecalciferol-treated orchidectomized (Orx + Vit. D3; 5 µg/kg b.m./day during three weeks) groups (n = 6/group). Concentration of 25(OH)D in serum of the Orx + Vit. D3 group increased 4 and 3.2 times (p < 0.0001) respectively, compared to Orx and SO group. T4, TSH, and calcitonin in serum remained unaltered. Vit. D3 treatment induced changes in thyroid functional morphology that indicate increased utilization of stored colloid and release of thyroid hormones in comparison with hormone synthesis, to maintain hormonal balance. Increased expression of nuclear VDR (p < 0.05) points to direct, TSH independent action of Vit. D on thyrocytes. Strong CYP24A1 immunostaining in C cells suggests its prominent expression in response to Vit. D in this cell subpopulation in orchidectomized rat model of osteoporosis. The indirect effect of Vit. D on bone, through fine regulation of thyroid function, is small.

We examined whether isoflavones interfere with thyroid homeostasis, increase hepatic thyroid hormone concentrations and affect cholesterol metabolism in middle-aged (MA) male rats. Thirteen-month-old Wistar rats were injected subcutaneously with 35 mg/kg b.w./day of genistein, daidzein or vehicle (controls) for four weeks. Hepatic Dio1 gene expression was up-regulated by 70% (p < 0.001 for both) and Dio1 enzyme activity increased by 64% after genistein (p < 0.001) and 73% after daidzein treatment (p < 0.0001). Hepatic T3 was 75% higher (p < 0.05 for both), while T4 increased only after genistein treatment. Serum T4 concentrations were 31% lower in genistein- and 49% lower in dadzein-treated rats (p < 0.001 for both) compared with controls. Hepatic Cyp7a1 gene expression was up-regulated by 40% after genistein and 32% after daidzein treatment (p < 0.05 for both), in agreement with a 7α-hydroxycholesterol increase of 50% (p < 0.01) and 88% (p < 0.001), respectively. Serum 24- and 27-hydroxycholesterol were 30% lower (p < 0.05 for both), while only 24-hydroxycholesterol was decreased in the liver by 45% after genistein (p < 0.05) and 39% (p < 0.01) after dadzein treatment. Serum concentration of the cholesterol precursor desmosterol was 32% (p < 0.05) lower only after dadzein treatment alone, while both isoflavones elevated this parameter in the liver by 45% (p < 0.01). In conclusion, isoflavones increased T3 availability in the liver of MA males, despite decreasing serum T4. Hepatic increase of T3 possibly contributes to activation of the neutral pathway of cholesterol degradation into bile acids in the liver. While isoflavones obviously have the potential to trigger multiple mechanisms involved in cholesterol metabolism and oxysterol production, they failed to induce any hypocholesterolemic effect.

Estrogen signaling plays an important role in pituitary development and function. In sensitive rat or mice strains of both sexes, estrogen treatments promote lactotropic cell proliferation and induce the formation of pituitary adenomas (dominantly prolactin or growth-hormone-secreting ones). In male patients receiving estrogen, treatment does not necessarily result in pituitary hyperplasia, hyperprolactinemia or adenoma development. In this review, we comprehensively analyze the mechanisms of estrogen action upon their application in male animal models comparing it with available data in human subjects. Sex-specific molecular targets of estrogen action in lactotropic (PRL) cells are highlighted in the context of their proliferative and secretory activity. In addition, putative effects of estradiol on the cellular/tumor microenvironment and the contribution of postnatal pituitary progenitor/stem cells and transdifferentiation processes to prolactinoma development have been analyzed. Finally, estrogen-induced morphological and hormone-secreting changes in pituitary thyrotropic (TSH) and adrenocorticotropic (ACTH) cells are discussed, as well as the putative role of the thyroid and/or glucocorticoid hormones in prolactinoma development, based on the current scarce literature.

The neuroendocrine system can be modulated by a magnetic field and cerebral ischemia as external and internal stressors, respectively. This study deals with the separate or combined effects of an extremely low frequency (ELF) magnetic field (50 Hz, average magnetic field of 0.5 mT) for 7 days and global cerebral ischemia for 10 min on the morpho-functional features of pituitary adrenocorticotrophic (ACTH) and thyrotrophic (TSH) cells in 3-month-old gerbils. To determine the immediate and delayed effects of the applied stressors, measurements were made on the 7th and 14th days after the onset of the experiment. The ELF magnetic field and 10-min global cerebral ischemia, separately and particularly in combination, decreased (P < 0.05) the volume density of ACTH cells, while only in combination were intracellular ACTH content and plasma ACTH concentration increased (P < 0.05) on day 7. The ELF magnetic field elevated serum TSH concentration on day 7 and intracellular TSHβ content on day 14 (P < 0.05). Also, 10-min global cerebral ischemia alone increased serum TSH concentration (P < 0.05), while in combination with the ELF magnetic field it elevated (P < 0.05) intracellular TSHβ content on day 14. In conclusion, an ELF magnetic field and/or 10-min global cerebral ischemia can induce immediate and delayed stimulation of ACTH and TSH synthesis and secretion. This is the pre-peer reviewed version of the following article: Rauš Balind S, Manojlović-Stojanoski M, Šošić-Jurjević B, Selaković V, Milošević V, Petković B. An Extremely Low Frequency Magnetic Field and Global Cerebral Ischemia Affect Pituitary ACTH and TSH Cells in Gerbils. Bioelectromagnetics. 2020;41(2):91-103, which has been published in final form at [http://dx.doi.org/10.1002/bem.22237]. Related to: [http://ibiss-r.rcub.bg.ac.rs/handle/123456789/3764].

Vitamin D plays an essential role in prevention and treatment of osteoporosis. Thyroid hormones, in addition to vitamin D, significantly contribute to regulation of bone remodeling cycle and health. There is currently no data about a possible connection between vitamin D treatment and the thyroid in the context of osteoporosis. Middle-aged Wistar rats were divided into: sham operated (SO), orchidectomized (Orx), and cholecalciferol-treated orchidectomized (Orx + Vit. D3; 5 µg/kg b.m./day during three weeks) groups (n = 6/group). Concentration of 25(OH)D in serum of the Orx + Vit. D3 group increased 4 and 3.2 times (p < 0.0001) respectively, compared to Orx and SO group. T4, TSH, and calcitonin in serum remained unaltered. Vit. D3 treatment induced changes in thyroid functional morphology that indicate increased utilization of stored colloid and release of thyroid hormones in comparison with hormone synthesis, to maintain hormonal balance. Increased expression of nuclear VDR (p < 0.05) points to direct, TSH independent action of Vit. D on thyrocytes. Strong CYP24A1 immunostaining in C cells suggests its prominent expression in response to Vit. D in this cell subpopulation in orchidectomized rat model of osteoporosis. The indirect effect of Vit. D on bone, through fine regulation of thyroid function, is small.

The aim of the study was to examine the potential of the principal soy isoflavones, genistein and daidzein, or isoflavone rich soy extract to recover pituitary castration cells in orchidectomized adult male rats in comparison with the effects of estradiol. Two weeks post orchidectomy (Orx), animals received estradiol-dipropionate, genistein, daidzein or soy extract subcutaneously for 3 weeks. Control sham-operated (So) and Orx rats received just the vehicle. Changes in the volumes of pars distalis, of individual follicle-stimulating hormone (FSH) and luteinizing hormone (LH) containing cells, their volume, numerical density and number were determined by unbiased design-based stereology. The intracellular content of βFSH and βLH was estimated by relative intensity of fluorescence (RIF). Orchidectomy increased all examined stereological parameters and RIF. Compared to Orx, estradiol increased the volume of pars distalis, but reversed RIF and all morphometric parameters of gonadotropes to the level of So rats, except their number. Treatments with purified isoflavones and soy extract decreased RIF to the control So level, expressing an estradiol-like effect. However, the histological appearance and morphometrical features of gonadotropes did not follow this pattern. Genistein increased the volume of pars distalis, decreased the volume density of LH-labeled cells and raised the number of gonadotropes. Daidzein decreased the cell volume of gonadotropic cells but increased their number and numerical density. Soy extract induced an increase in number and numerical density of FSH-containing cells. Therefore, it can be concluded that soy phytoestrogens do not fully reverse the Orx-induced changes in pituitary castration cells. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Prenatal glucocorticoid overexposure could largely influence pituitary-adrenal activity and anxiety-like behavior in offspring. Our aim was to study the possible potentiating effect of moderate dose of fructose – common ingredient of today’s diet – on prenatal glucocorticoid treatment-induced hypothalamo-pituitary-adrenal (HPA) axis changes. Pregnant female rats were treated with multiple dexamethasone (Dx) doses (3 x 0.5 mg/kg/b.m. Dx; 16th-18th gestational day). Half of female offspring from control and Dx treated dams were supplemented with 10% fructose solution, from weaning till adulthood. Immunohistochemistry, unbiased stereological evaluation and hormonal analysis are used to provide the morpho-functional state of pituitary and adrenal gland. Anxiety-like behavior was assessed using the light/dark box test and the elevated plus maze test. Prenatally Dx exposed females, with or without fructose consumption, had markedly reduced adrenocortical volume (p < 0.05) comparing to controls. Increased basal plasma ACTH level in these females (p < 0.05) maintained corticosterone concentration at control level produced by smaller adrenal glands. In parallel, anxiety-like behavior was shown by both tests used. In conclusion, prenatal Dx exposure cause negative psychophysiological outcome reflected in increased HPA axis activity and anxiety behavior in female offspring, while moderately increased fructose consumption failed to evoke any alteration or to potentiate effects of prenatal Dx exposure. This is the peer reviewed version of the following article: Manojlović-Stojanoski M, Nestorović N, Petković B, Balind SR, Ristić N, Trifunović S, Ajdžanović V, Filipović B, Šošić-Jurjević B, Milošević V. The effects of prenatal dexamethasone exposure and fructose challenge on pituitary-adrenocortical activity and anxiety-like behavior in female offspring. Tissue Cell. 2020;62:101309. [https://doi.org/10.1016/j.tice.2019.101309]

Letter to the Editor

The aim of the present study was to examine the morphometric characteristics of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) immunoreactive cells in the pituitaries of orchidectomized (Orx) and Orx testosterone- or estradiol-treated rats. Adult male Orx Wistar rats, 2 weeks post operation, received estradiol dipropionate (E) or testosterone propionate (T) for 3 weeks. Both controls, sham-operated (So) and Orx rats, were injected with solvent, in the same regime. Changes in the volume of pars distalis , the volumes of individual FSH- and LH-labeled cells, their numerical density and number were determined by unbiased design-based stereology. The FSH and LH intracellular content was estimated by relative intensity of fluorescence (RIF). We observed that Orx caused hyperstimulation of gonadotropic cells. Their volume, volume density, number, numerical density and intracellular hormone content significantly increased in comparison to So controls. Compared to Orx controls, T caused a significant decrease in the volume and volume density of gonadotropic cells and immunoreactive FSH and LH content in their cytoplasm. The volume of the pars distalis , the numerical density and number of gonadotropic cells were not changed as compared to Orx controls. Estradiol treatment caused a significant increase in the volume of the pars distalis , decreases in cell volume, volume and numerical density of gonadotropic cells, and FSH and LH intracellular content in comparison to Orx controls. The number of FSH-labeled cells increased. In conclusion, both T and E reversed all of the examined parameters of gonadotropic cells of Orx rats to the level of So controls, except in number. DOI: 10.2298/ABS151201070N Key words: estradiol; gonadotropic cells; orchidectomy; stereology; testosterone Received: December 1, 2015; Revised: February 17, 2016; Accepted: February 18, 2016; Published online: August 17, 2016 How to cite this article: Nestorovic NM, Trifunovic SL, Jaric IM, Manojlovic-Stojanoski MN, Ristic NM, Filipovic BR, Sosic-Jurjevic BT, Milosevic VLj. Sex steroid application reverses changes in rat castration cells: Unbiased stereological analysis. Arch Biol Sci. 2016;68(4):821-8.

As a major phytoestrogen of soy, genistein effectively prevents bone loss in both humans and rat models of osteoporosis. However, although the bone‐sparing effects of genistein are achieved directly through estrogen receptors, its mode of action on bone by modulation of other endocrine functions is not entirely clear. Thus, thyroid hormones and calcitonin (CT) have an essential influence on bone metabolism. Besides its action on bones, in this study we examined the effect of genistein on the activity of two different endocrine cell populations, thyroid follicular and C‐cells. Fifteen‐month‐old Wistar rats were either bilaterally orchidectomized (Orx) or sham‐operated (SO). Two weeks after surgery, half of the Orx rats were treated chronically with 30 mg kg(−1) b.w. genistein (Orx + G) subcutaneously (s.c.) every day for 3 weeks, while the remaining Orx rats and the SO rats were given the same volume of sterile olive oil to serve as controls. For histomorphometrical analysis of the trabecular bone microarchitecture an imagej public domain image processing programme was used. Thyroid sections were analysed histologically and stereologically after visualization of follicular and C‐cells by immunohistochemical staining for thyroglobulin and CT. Thyroid follicular epithelium, interstitium, colloid and CT‐immunopositive C‐cells were examined morphometrically. Serum concentrations of osteocalcin (OC), triiodothyronine (T(3)), thyroxine (T(4)) and CT were determined as well as urinary calcium (Ca(2+)) concentrations. Genistein treatment significantly increased cancellous bone area (B.Ar), trabecular thickness (TbTh) and trabecular number (TbN) (P

Thyroid C-cells produce calcitonin (CT), a hypocalcemic hormone, that acts as an inhibitor of bone resorption. In this study, we investigated the effects of tamoxifen (TAM) as a selective estrogen receptor modulator on thyroid C-cells, trabecular bone and biochemical markers of bone metabolism in an animal model of androgen deficiency, represented by middle-aged orchidectomized (Orx) rats. Fifteen-month-old male Wistar rats were divided into: Orx and sham-operated (SO) groups. Rats from one Orx group were injected subcutaneously with TAM citrate (Orx + TAM; 0.3 mgkg(-1) b.w.), while the rats from SO and a second Orx group received vehicle alone, once a day for 3 weeks. The peroxidase-antiperoxidase method was applied for localization of CT in C-cells. Thyroid C-cells were morphometrically and ultrastructurally analyzed. An ImageJ image-processing program was used to measure bone histomorphometric parameters. Blood serum samples were analyzed for CT, osteocalcin (OC), calcium (Ca2+) and phosphorus (P). Urinary Ca2+ concentrations were measured. TAM treatment significantly increased thyroid C-cell volume (V-c) and serum CT when compared with vehicle-treated Orx rats. Analysis of trabecular microarchitecture of the tibia showed that administration of TAM significantly increased cancellous bone area, trabecular thickness and trabecular number, whereas trabecular separation was significantly decreased compared with vehicle-treated Orx rats. Serum OC and urinary Ca2+ concentrations were significantly lower in comparison with the control Orx group. These results indicate that in our rat model of androgen deficiency, TAM stimulated calcitonin-producing thyroid C-cells and increased trabecular bone mass. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173009]

In contrast to studies in women, male osteoporosis is poorly understood and strictly related to advancing age. Among the first antiresorptive substances used in the prevention and treatment of osteoporosis is calcitonin (CT), a hypocalcemic hormone that potently inhibits osteoclastic bone resorption. Natural CT is produced and secreted by thyroid C-cells. The other endocrine population of thyroid cells produces thyroid hormones (TH), which also affect bone turnover. The aim of this study was to evaluate the influence of salmon CT on trabecular bone microarchitecture with special reference to effects on the structure and function of both CT- and TH-producing thyroid cells in orchidectomized (Orx) middle-aged rats. Twenty-four male Wistar rats aged 15 months were randomly divided into Orx and sham-operated (SO) groups. One group of Orx animals received (s.c.) synthetic salmon CT (Orx + CT; 100 IU kg(-1) b.w.) subcutaneously every second day for 6 weeks. The second Orx group and SO rats were given the same volume of vehicle alone by the same schedule. Trabecular bone histomorphometrical parameters were: cancellous bone area (B.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular separation (Tb.Sp) were obtained with an ImageJ public-domain image-processing program. The peroxidase-antiperoxidase method was applied for localization of CT in C-cells. Anti-human CT antisera served as the primary antibodies. For immunohistochemical characterization of vascular endothelial growth factor (VEGF) in thyroid tissue, rabbit antisera against human VEGF, served as primary antibodies. CT-immunopositive thyroid C-cells, thyroid follicular epithelium, interstitium and colloid were evaluated morphometrically. Blood serum samples were analyzed for CT, osteocalcin (OC), and thyroxine (T4 ), and calcium (Ca(2+) ) concentration was determined in urine samples. Salmon CT application significantly increased B.Ar, TbTh and TbN, but markedly decreased Tb.Sp. Administration of exogenous CT significantly decreased mean volume (Vc) and relative volume density (Vv) of thyroid C-cells in relation to both SO and Orx groups. The Vv of the colloid was higher, whereas the VV of the follicular epithelium was lower after CT treatment compared with Orx alone. CT treatment markedly elevated serum CT, whereas serum OC, T4 and urinary Ca(2+) concentrations were lower than in the Orx group. These results indicate that salmon CT stimulates trabecular bone microarchitecture, strongly inhibits thyroid C-cells and changes the structure of the thyroid gland, indicating hypoactivity. This is the pre-peer reviewed version of the following article: Filipović B, Šošić-Jurjević B, Ajdžanović V, Živanović J, Ristić N, Trifunović S, Milošević V. Response of trabecular bone, thyroid C and follicular cells to synthetic salmon calcitonin in middle-aged orchidectomized male rats. J Anat. 2017, which has been published in final form at [http://doi.org/10.1111/joa.12600]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. Journal of Anatomy (2017)

The effects of estradiol (EDP) and human chorionic gonadotropin (HCG) on histological and stereological features of adrenocorticotropic (ACTH) cells in peripubertal female rats were examined. The first group of females received five injections of EDP (0.25 mg/kg b.w.), every second day from the 4th to 14th day after birth, and was killed at the peripubertal stage. The second group of females was given two injections of pregnyl-gonadotrophinum chorionicum (HCG; 50 IU/kg body weight) on the 36th and 37th days after birth, and it was killed 24 h after the last treatment. The controls were injected with an equivalent volume of the corresponding vehicle. ACTH cells were immunohistochemically labeled and stereologically evaluated. Stereological analysis showed that the volume of ACTH cells and their volume density in peripubertal females treated with EDP, were decreased by 15.6\% and 53.8\% (p < 0.05), respectively, compared to the controls. In HCG-treated animals, the observed parameters were increased by 39.2\% and 15.4\% (p < 0.05), respectively, in comparison with the control females. These findings suggest that the application of EDP or HCG exerted opposite effects on the stereological features of pituitary ACTH cells. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}ON 173009]

The long-term effects of somatostatin 14 (SST-14) on the pituitary-ovarian axis were examined. Female Wistar rats received 20 mu g/100 g b.w. doses subcutaneously twice daily for 5 consecutive days in the infantile (from 1lth to 15th day) or peripubertal (from 33rd to 37th day) period of life. Females treated as infants were killed in the peripubertal (38th day) or adult period of life (80th day), and those treated during peripuberty as adults (80th day). Pituitary follicle-stimulating (FSH), luteinizing (LH) and somatotropic (GH) cells, and ovaries were analyzed by stereology and morphometry. Serum FSH and LH concentrations were determined by RTA. FSH and LH cell volumes were significantly decreased in pituitaries of peripubertal females treated with SST-14 as infants, and in adult females treated during peripuberty. GH cell volume was decreased in all treated rats. In the ovaries, enlargement of the non-growing pool of follicles was detected in adult females treated during peripuberty. SST-14 applied to infant rats did not lead to changes in initial follicular recruitment, but it disturbed follicle growth and development at later stages. It can be concluded that SST-14 exerted long-term inhibitory effects on the pituitary-ovarian axis and GH cells in rats. Ministry for Science of Serbia {[}173009]

Summary Background: Breast cancer is the most common form of cancer in women. It arises from multiple genetic changes in oncogenes and tumor suppressor genes. Among so far studied oncogenes relatively few, including epdermal growth factor receptor (EGFR), cyclinD1 (CCND1)and cmyc, have been found to play an important role in progression of this type of human malignancy. The aim of this study was to examine the prognostic potential of CCND1, c-myc and EGFR amplification and their possible cooperation in breast carcinogenesis. Methods: Copy number analyses of CCND1 and c-myc genes were done by TaqMan based quantitative real time PCR. Am pli fication status of EGFR was determined by differential PCR. Results: Amplification of CCND1, c-myc and EGFR onco- gene has been found in 20.4%, 26.5% and 26.5% of breast cancer cases, respectively. Analysis showed that amplification of CCND1 oncogene was significantly associated with the stage II of disease while amplification of EGFR gene was sig- nificantly associated with overexpression of HER-2/neu. Tu- mour stage and expression of HER-2/neu appeared to be significant predictors of patient's outcome. Stage I patients lived significantly longer then stage III patients (p=0.04) while patients with FiER-2/neu overexpression had worse prognoses and lived significantly shorter (p=0.001). Finally, survival of patients who underwent hormone therapy only was significantly longer (p=0.001) then survival of the rest of patients. Conclusions: Amplification of CCND1 or EGFR oncogene is associated with the progression of breast cancer and bad prognosis. No co-ordination in amplification of CCND1, c- myc and EGFR oncogenes were established in this cohort of breast cancer patients.

Ageing in men is accompanied by an increased occurrence of osteoporosis, but traditional hormonal replacement therapy elevates the risk of developing endocrine cancer. Vitex agnus-castus L. (Vac) essential oil is commonly used as an alternative therapy for ageing symptoms in both men and women. It is known that calcitonin (CT), thyroid C cell hormone, inhibits bone resorption. The purpose of this experimental study was to investigate the influence of Vac essential oil administration on the immunohistomorphometric features of thyroid C cells and bone metabolism in 16-month-old male Wistar rats. The first group of animals (n=8) was treated subcutaneously (s.c.) with 60 mg/kg of Vac essential oil once a day for 3 weeks. Control animals (n=8) received sterile olive oil s.c. by the same schedule. After Vac treatment significant increases (p

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.

Vitex Agnus-CastusL. Essential Oil Increases Human Erythrocyte Membrane FluidityErythrocyte membrane fluidity is related to their rheologic behavior, the dynamic quality of erythrocytes, which is tempted in hypertension and atherosclerosis. An increased risk of these and other cardiovascular diseases occurs in ageing women. Menopause-related conditions are often treated with hormone replacement therapy that may increase the risk of malignancies.Vitex agnus-castusL. essential oil contains various organic compounds (monoterpenes, sesquiterpenes and terpenoids), and is increasingly used as an alternative therapy for menopausal symptoms. These components of the oil may be incorporated into cell membranes, thereby changing the membrane fluidity. The aim of this study was to determine the effects ofVitex agnus-castusessential oil on human erythrocyte membrane fluidity at graded depths. We used Electron Paramagnetic Resonance spectroscopy and fatty acid spin probes (5-doxyl stearic acid and 12-doxyl stearic acid), whose spectra depend on membrane fluidity. After treatment withVitex agnus-castusessential oil the erythrocytes had a significant (p=0.029) and reversible increase in membrane fluidity in the deeper hydrophobic membrane regions, with no change (p>0.05) in fluidity near the membrane's hydrophilic surface. These results document increased fluidity of the human erythrocyte membrane byVitex agnus-castusessential oil, and this action may be useful in patients with menopause-related hypertension and other cardiovascular conditions.

Andropause, the culminating phase of ageing in males is characterized by the decline of the growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis that is responsible for somatic development. Estrogens on the other hand, assume a relevant somatic role, as well as the one in the pituitary. They are particularly interesting because of their therapeutic uses in certain common ageing-associated diseases. The aim of the present study was to examine the effects of subcutaneous treatment with estradiol, dipropionate on the immunohistomorphometric features of GH cells, in an animal model of the andropause. Sixteen-month-old Wistar rats were divided into sham-operated (SO), orchidectomized (Orx) and estradiol dipropionate-treated orchidectomized (Orx+Edp) groups. Estradiol dipropionate (0.625 mg/kg/day) was administered subcutaneously for three weeks while the SO and Orx groups received the vehicle alone. GH cells were identified by the peroxidase-antiperoxidase (PAP) immunohistochemical procedure. In the Orx+Edp group, GH cell volumes as well as the relative volume densities were significantly (p

Morphofunctional Characteristics of Pituitary Adrenocorticotropes in An Animal Model of Heat StressAs a result of the global warming, the average ambient temperature during summertime has increased in regions with moderate continental cli mate. The effects of 24 h exposure to heat stress (35±1 °C) on the morphology and function of pituitary adrenocorticotropes were examined in adult male Wistar rats. Significant changes in the morphofunctional features of adrenocorticotropes were found after the heat stress, with no differences noted in the cell shape or localization, compared to controls. The adrenocorticotropes cell volume, as well as the volume density, were significantly decreased (p

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.

The maintenance of erythrocyte membrane fluidity at the physiological level is an important factor affecting the ability of erythrocytes to pass through blood vessels of small luminal diameter. Genistein and daidzein, which are used as alternative therapeutics in cardiovascular conditions, can be incorporated into the cell membrane and change its fluidity. The aim of this study was to examine the effects of genistein and daidzein on erythrocyte membrane fluidity at graded depths. We used electron paramagnetic resonance (EPR) spectroscopy and fatty acid spin probes (5-DS and 12-DS) where EPR spectra were dependent on fluidity. The results showed that genistein significantly (p < 0.05) decreased erythrocyte membrane fluidity near the hydrophilic surface, while daidzein significantly (p < 0.05) increased the same parameter in deeper regions of the membrane. These data suggest that the deep fluidizing effects of daidzein on erythrocyte membranes make it a better therapeutic choice than genistein in some cardiovascular conditions. Ministry for Science of Serbia [143007B, 143016B]

Ghrelin, a growth hormone secretagogue that exerts an important role in appetite and weight regulation, participates in the activation of the hypothalamo-pituitary-adrenal (HPA) axis. Male Wistar rats (5/group) received daily for 5 days, via an ICV (intracerebroventricular) cannula, 5 microl phosphate buffered saline with or without 1 microg of rat ghrelin. Two hours after the last injection, blood and adrenal glands were collected from decapitated rats for blood hormone analyses and histologic and morphometric processing. Ghrelin treatment resulted in increased (p

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. Exp Biol Med 234:148-156, 2009 Ministry of Science and Technological Development of Serbia [143007B]

The soybean phytoestrogen, genistein, is increasingly consumed as an alternative therapeutic for age-related diseases. The aim of this study was to examine the morphofunctional characteristics of adrenocorticotrophic (ACTH) cells and blood concentrations of ACTH in sham-operated, orchidectomized and genistein-treated orchidectomized, 16-month-old Wistar male rats. Genistein (10 mg/kg/day) was administered subcutaneously for three weeks, while the control groups received the vehicle alone. Orchidectomy and genistein treatment decreased the volume density of ACTH cells and reduced (p < 0.05) circulating ACTH concentrations in comparison with control groups. In conclusion, genistein modulated the morphofunctional features of ACTH cells and decreased blood ACTH levels. Ministry for Science of Serbia [143007B]

Elevated glucocorticoid levels in the gravid female circulation affect a number of endocrine functions in the fetuses and neonates. The aim of this study was to examine the effects of maternal dexamethasone (Dx) administration during late pregnancy on the ovaries of neonatal offspring. On the 16th day of pregnancy, experimental dams received subcutaneously 1.0 mg Dx/kg b.w., followed by 0.5 mg Dx/kg b.w./day on the 17th and 18th days of gestation. The control gravid females received the same volume of saline vehicle. Left ovaries from 5-day-old female pups were stereologically analyzed. The ovary volumes were estimated using Cavalieri's principle. The number of healthy and atretic primordial and primary follicles was estimated using a fractionator-physical disector method. The number of secondary follicles was determined by exact counts of every fourth section encompassing whole cross-sections of the ovary. The ovary volume was significantly decreased (by 44.4%; P < 0.05) in the group of female pups from Dx-treated mothers comparing to the controls. The numbers of healthy primordial and atretic follicles were 38.8% (P < 0.05) and 50.9% (P < 0.05), respectively, reduced in the ovaries of pups from the Dx-treated mothers, when compared with the control values. There were 53.4% (P < 0.05) fewer healthy primary and 41.8% (P < 0.05) fewer healthy secondary follicles as well. The numbers of atretic primary and secondary follicles were reduced by 60.0% (P < 0.05) and 61.7% (P < 0.05), respectively. It can be concluded that fetal exposure to glucocorticoids decreased the pool of non-growing follicles in the neonatal ovary, whereas the processes of folliculogenesis and atresia remained unaffected. Ministry for Science of Serbia [143007B]

Neonatal female rats (3-7 days old) were injected subcutaneously twice daily with 20 ?g of SRIH-14/100g b.w. for five consecutive days. The animals were sacrificed in the peripubertal (38 days old) or adult (80 days old) periods of life. Control rats were treated in the same way with identical volumes of saline. SRIH-14 led to significant reduction (p0.05) changes in the morphometric parameters of adult adrenal cortex after SRIH-14 treatment compared with control females. It can be concluded that subcutaneously applied SRIH-14 in the neonatal period inhibits growth of adrenal ZG cells in female rats only up to the peripubertal stage, while the adrenal gland cortex recovers before mature adulthood is reached.

Glucocorticoids and thyroid hormones control many aspects of fetal development. Using immunohistochemistry and stereology, in the present study we investigated the effects of dexamethasone (Dx) administration during pregnancy on pituitary TSH cells of 19-day-old fetuses. Doses of 1.0, 0.5, and 0.5 mg Dx/kg bw/day were given to the dams on three consecutive days starting on day 16 of gestation. Administration of Dx to pregnant rats induced a significant decline of fetal TSH cell number per unit of area and their volume density in comparison with the corresponding controls. Our results showed that maternal Dx administration inhibited multiplication of TSH cells in 19-day-old fetuses.

The effects of estradiol dipropionate (EDP) or calciumglucoheptonate (Ca) on the morphology and stereology of the PRLcellsin14-month-oldWistarfemaleratswerestudied.Theanimalsweretreated daily with EDP in the dose of 0.625 mg/kg b.w. or calciumglucoheptonate(Ca;11.4mg/kgb.w.)fortwoweeks.Thecontrolswereinjected with vehicle alone by the same schedule. Mammotrophs (PRLcells) were immunocytochemically localized by the PAP method. BloodPRL concentration was determined by Delfia procedure. In animalstreated with EDP the volume of both, PRL cells and their nuclei, as wellas the volume densities were significantly (p 0.05) decreased compared to control rats. Serum concentration ofPRL was significantly increased (p 0.05) changed by 2% compared to controls. Based on theseresults, it can be concluded that EDP expresses a strong stimulatoryeffect on the morphology and function of pituitary PRL cells.Keywords:PRL,mammotrophs,estradiol,calcium,middle-aged,female 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 I and pituitary Dio2 enzyme activities. At present, we examined the effects of subsequent testosterone-propionate treatment (5 mg/kg; Orx + T), and compared the effects of testosterone with the effects of estradiol-clipropionate (0.06 mg/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 (RIP) for TSHI3; in their thyroids we found increased volume and height of follicular epithelium, decreased volume of the colloid and higher RIF for T,{[}-bound to thyroglobulin (Tg-T4). Liver Diol activity was increased. E-treatment did not affect serum hormone levels, pituitary RIF for TS1-11i, or liver Diol 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 TSHr, RIF, thyroid activation and liver Diol enzyme activity in Orx-MA, without elevating serum TSH as in Orx-YA rats. Estrogenization induced pituitary enlargement with no effect on pituitary TSHI) RIF, serum TSH or liver Diol 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. (C) 2015 Elsevier Inc. All rights reserved. Ministry of Education and Science of the Republic of Serbia {[}173009]; Deutsche Forschungsgemeinschaft {[}DFG-GK 1208, TP3, RE3038/1-1]; Charite Universitatsmedizin Berlin; European Society for Endocrinology (ESE)

The structure and function of C cells of adult female rats after ovariectomy (Ovx) were investigated. Intact control and Ovx rats were i.p. treated with sterile olive oil for 4 weeks. Peroxidase-antiperoxidase (PAP) immunohistochemical procedure was applied to localize calcitonin (CT) in thyroid C cells while its serum content was determined by RIA method. Morphometric analyses of the C cells volume, that of their nuclei and relative volume density included stereological method with the multipurpose test system M42. Also, the average number of C cells number per mm2 was calculated. Ovx led to a significant increase in body weight (21%; p

Overexposure to glucocorticoids during the fetal period induces changes in developmental processes in various fetal tissues. The aim of this study was to investigate the effects of the synthetic glucocorticoid, dexamethasone (Dx), on pituitary volume and gonadotropic cells during a critical period of pituitary development. The effects of Dx on stereological parameters of the pituitary gland and FSH and LH cells were investigated in 19 and 21-day old 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. FSH and LH cells were stained immunohistochemically by the peroxidase-antiperoxidase method (PAP). In 19-day old fetuses, exposure to Dx caused a significant decrease of pituitary volume, estimated by Cavalieri's principle. Also, the total number of FSH and LH cells per pituitary, determined by physical fractionator counting technique, was significantly reduced. These changes persisted until fetal day 21. Volume densities and numerical densities of FSH and LH cells after exposure to Dx in 19 and 21-day old fetuses remained unaffected. Our results suggest that altered stereological parameters in pituitary gland after exposure to dexamethasone in fetal period could be long-lasting. (C) 2014 Elsevier GmbH. All rights reserved. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173009]

Somatopause, the complex aspect of andropause, is recognizable by reduced growth hormone - GH/insulin-like growth factor 1 axis function in the ageing male. Soy isoflavones (usually genistein and daidzein), which are known for their beneficial effects in the treatment of ageing symptoms, are active in the pituitary, as well. The immunohistomorphometric and -fluorescent characteristics of pituitary growth hormone secreting cells, in an animal model of andropause, were examined after a treatment with genistein or daidzein. Andropausal Wistar rats were divided into sham operated, orchidectomized and genistein or daidzein treated orchidectomized groups. Genistein or daidzein (30 mg/kg/day) were administered subcutaneously for three weeks, while sham operated and orchidectomized groups received the vehicle alone. Growth hormone secreting cells were identified by the peroxidase-antiperoxidase immuno-histochemical, and immuno-fluorescent procedure. The main characteristic of growth hormone secreting cells in soy isoflavones treated groups is a weaker immuno-histochemical staining and immuno-fluorescent signal compared to sham operated and orchidectomized groups. The growth hormone secreting cell volume in orchidectomized +genistein or +daidzein groups is by 13.8% and 11.9% (p

Thyroid hormones (TH) and glucocorticoids strongly contribute to the maturation of fetal tissues in the preparation for extrauterine life. Influence of maternal dexamethasone (Dx) administration on thyroid glands morpho-functional characteristics of near term rat fetuses was investigated applying unbiased stereology. On the 16th day of pregnancy dams received 1.0 mg/Dx/kg/b.w., followed by 0.5 mg/Dx/kg/b.w. on the 17th and 18th days of gestation. The control females received the same volume of saline. The volume of fetal thyroid was estimated using Cavalieri's principle; the physical/fractionator design was applied for the determination of absolute number of follicular cells in mitosis and immunohistochemically labeled C cells; C cell volume was measured using the planar rotator. The functional activity of thyroid tissue was provided from thyroglobulin (Tg) and thyroperoxidase (TPO) immunohistochemical staining. Applying these design-based modern stereological methods it was shown that Dx treatment of gravid females led to a significant decrease of fetal thyroid gland volume in 19- and 21-day-old fetuses, due to decreased proliferation of follicular cells. The Tg and TPO immunohistochemistry demonstrated that intensive TH production starts and continues during the examined period in control and Dx-exposed fetuses. Under the influence of Dx the absolute number of C cells was lower in both groups of near term fetuses, although unchanged relation between the two populations of endocrine cells, follicular and C cells suggesting that structural relationships within the gland are preserved. In conclusion maternal glucocorticoid administration at the thyroid gland level exerts growth-inhibitory and maturational promoting effects in near term rat fetuses. (C) 2014 Elsevier Inc. All rights reserved. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173009]

Numerous studies have established a link between estrogen levels and activity of the hypothalamic pituitary adrenal (HPA) system. Considering the ``weak estrogenic{''} effect of soy isoflavones, this study was designed to evaluate the influence of soy extract treatment on some morphofunctional parameters of rat pituitary corticotropes in vivo. Adult male orchidectomized Wistar rats received estradiol-dipropionate or soy extract in oil/ethanol solvent subcutaneously for 3 weeks. Both controls, sham-operated (So) and orchidectomized (Orx) rats, were injected with solvent, in the same regime. Changes in pituitary volume, total volume, total number and volume of individual corticotropes were evaluated stereologically, while ACTH levels were determined biochemically. In comparison with So rats, estradiol treatment provoked increases (p < 0.05) of ACTH level (166\%), pituitary weight (167\%) and volume (102\%), total volume (20\%) and total number of corticotropes (18\%). In comparison to Orx, following estradiol treatment elevation (p < 0.05) of ADE level (69\%), pituitary weight (131\%) and volume (82\%), total (30\%) and individual volume (29\%) of corticotropes was observed. Soy extract treatment led to enhancement (p < 0.05) of: ACTH level (28\%), total (25\%) and individual volume (20\%) of corticotropes. It can be concluded that soy extract acts in a similar way to estradiol, but the increased activity of corticotropes was weaker. (C) 2013 Elsevier GmbH. All rights reserved. Ministry of Education and Science of Serbia {[}173009]

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 (T4), and cholesterol (Chol) levels were determined. As expected, middle-aged control rats had lower (P4 compared with 3-month-old males. In the Orx middle-aged group, we detected diminished serum testosterone (P4 levels, and higher Chol level (PPDio1 gene expression and DIO1 activity were decreased (PTSHβ, Dio1, and Dio2 mRNAs did not change, while DIO2 activity decreased (P4 and TSH. However, decreased liver DIO1 and pituitary DIO2 enzyme activities indicate compensatory–adaptive changes in local T3 production.

Proper and balanced nutrition is very important in prevention and treatment of chronic diseases. Many individuals modify their diet and/or take different nutraceuticals expecting to attain optimum health, extend their lifespan and prevent diseases such as cardiovascular, cancer, osteoporosis, obesity, or diabetes type II. Based on „Japanese phenomenon“ (Adlercreutz, 1998), numerous advertisements suggest that soy-based diet, and its phytoestrogens (PE) in particular, provide protection against many chronic diseases and contribute to the long lifespan often observed in Asia. That is why soy and other phytoestrogen rich plants became increasingly popular in the U.S. and western countries in the past 30 years. Furthermore, in these countries, PEs are often consumed in its purified form, as nutritional supplements, “designed” for special medical purposes. These supplements are freely available in pharmacies, health food shops, grocery shops and are usually consumed without medical control. There is a lack of awareness that uncontrolled consumption of natural PEs may be potentially harmful to human health. Even more concerning is that some people consume supplements in excess of suggested daily dosage (Wuttke et al., 2007). The soybean (Glycine max), compared to other legumes, is richer in protein levels and quality, based on its digestibility and concentration of essential amino acids (Rand et al., 2003). It is also good source of fiber, certain vitamins and minerals, such as folate and potassium (Rochfort and Panozzo, 2007). It has very high antioxidant content, similar to fruits famous for their antioxidant activity (Galleano et al., 2010). Also, despite their high carbohydrate content, the glycemic load of soybeans is relatively low due to their low glycemic index. In addition, soy-food has high levels of iron in the form of ferritin (Lonnerdal et al., 2006). The concentration of calcium in soymilk is much lower than in cow milk, however, its absorption from soy milk is similar to that from cow milk (Reinwald and Weaver, 2010). Besides the favorable nutritional attributes, soybean contains a number of biologically active components (saponins and lunasin, phytic acids, phytosterols, trypsin inhibitors, and peptides) including isoflavones genistein (G), daidzein (D) and glycitein (Gy). As soybean phytoestrogens, isoflavones are considered the most important in prevention and treatment of hormone-dependent cancers, cardiovascular diseases, osteoporosis, menopausal symptoms and other age-related diseases. In addition, some studies suggest that soy and its isoflavones affect body weight homeostasis. Modern world is a controversy with ever-increasing obesity on one side, and a high percent of starving people around the globe, on the other side. Having that in mind, combined with

A decrease of erythrocyte membrane fluidity can contribute to the pathophysiology of hypertension. Soy products, which are used as alternative therapeutics in some cardiovascular conditions, contain various isoflavones (genistein, daidzein, and their glucosides, genistin and daidzin), which can incorporate cellular membrane and change its fluidity. The aim of this study was to examine the effects of soy extract (which generally corresponds to the soy products of isoflavone composition) on erythrocyte membrane fluidity at graded depths. We used electron paramagnetic resonance spectroscopy and fatty acid spin probes (5-DS and 12-DS), the spectra of which are dependent on membrane fluidity. After being treated with soy extract, erythrocytes showed a significant (P = 0.016) decrease of membrane fluidity near the hydrophilic surface, while there were no significant changes of fluidity in deeper hydrophobic membrane regions. These results suggest that soy products containing high levels of genistein and isoflavone glucosides may not be suitable for use in hypertension because they decrease erythrocyte membrane fluidity. Ministry of Science of the Republic of Serbia [143007B, 143016B]

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 Cavalieri's 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. (C) 2010 Wiley-Liss, Inc. Ministry for Science and Environmental Protection of Republic Serbia [143007B]

The effects of pregnancy and multiple dexamethasone (Dx) treatment on morphometric parameters of adenohypophyseal gonadotropic cells that produce follicle stimulating hormone (FSH) and luteinizing hormone (LH) were investigated in female Wistar rats. The rats in the experimental group received injections of 1.0, 0.5 and 0.5mg Dx/kg b.w. on days 16-18 of pregnancy, white the control group received equal volumes of saline. There was also an age-matched adult virgin control group. The experimental and control animals were sacrificed 24 and 72h after the last injection. Using the peroxidase-anti-peroxidase immunohistochemical labeling procedure, morphometric analyses showed that cell volume and volume density of FSH and LH cells on day 19 of pregnancy, as well as the number of LH cells, were significantly decreased compared to the virgin control values. On day 21 of gestation, the volume of FSH and LH cells remained smaller than in the virgin controls. Moreover, FSH and LH cell volume was significantly decreased 24h after multiple Dx treatment in comparison with the pregnant controls. Thus, during the last days of pregnancy, the morphometric parameters of gonadotropic cells decreased in comparison with the control virgin rats, but Dx treatment of pregnant rats had an inhibitory influence on FSH and LH cell size only 24h after the last dose. (c) 2007 Elsevier GmbH. All rights reserved. null

Different hormones and growth factors control the homeostasis of the anterior pituitary gland. We examined the morphological features of pituitary thyroid stimulating hormone (TSH)-producing cells in juvenile and peripubertal female rats after treatments with estradiol-dipropionate (EDP), human chorionic gonadotropin (hCG) and a combination of both hormones (EDP+hCG). TSH-producing cells were labelled using a peroxidase-antiperoxidase immunohistochemical procedure for binding of a rabbit anti-rat beta-thyrotropic polyclonal antisera. Morphometric differences in the cytoplasmic and nuclear volume densities in thyrotropes after the treatments were determined using a stereological method. The relative weights of the pituitary glands were significantly higher in the EDP- and EDP+hCG-treated juvenile and peripubertal rats than in untreated age-matched controls. Treatment with EDP promoted a decrease, and treatment with hCG an increase, of the cellular and nuclear volumes in TSH cells in both juvenile and peripubertal females in comparison with the respective controls. Treatment with a combination of EDP+hCG did not induce any significant changes. The cytoplasmic and nuclear volume densities in TSH cells in the EDP+hCG-treated group were significantly higher than in the EDP-treated, and significantly lower than in hCG-treated rats at both growth stages. These findings suggest that estradiol and hCG exerted opposite effects on pituitary TSH-immunoreactive cells. The observed effects on thyrotrope morphology were apparently independent of the stage of development. (C) 2006 Elsevier GmbH. All rights reserved. null