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Amphibians are useful bioindicators for monitoring aquatic health and the influence of xenobiotics such as endocrine disrupting chemicals. Because aquatic ecosystems experience the majority of global pollution, aquatic organisms are most exposed and vulnerable to endocrine disruptors. Furthermore, penetration of endocrine disruptors into aquatic organisms especially in amphibians is even easier because of more permeable skin, resulting in high bioavailability and bioaccumulation of chemicals. One of the most potent endocrine disruptors is thiourea, which chemically blocks the synthesis of thyroid hormones and prevents metamorphosis in am-phibians. We investigated the influence of thiourea on histomorphology of the thyroid gland in Triturus newts at the metamorphic stage, when thyroid hormone concentrations should reach their maximum level. Chronic exposure to thiourea induced hypertrophy and hyperplasia of follicular cells as well as a significant reduction of interstitial tissue. The intensity of the thyroglobulin immunostaining signal significantly decreases upon chronic exposure to thiourea. Successful cross-reactivity of human primary antibody in immunochemical detection of thyroglobulin in Urodela confirms potential homology in thyroglobulin structure throughout the vertebrates.

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.

In a series of our previous works, we revealed the beneficial effects of applied soy isoflavones (genistein or daidzein) on the wide context of corticosteroidogenesis in vivo, in a rat model of the andropause. Soy isoflavones decreased the circulating levels of pituitary adrenocorticotropic hormone, inhibited aldosterone secretion, as well as corticosterone production and secretion, but stimulated dehydroepiandrosterone secretion, all in andropausal rats. In vitro studies indicate that the mechanism underlying these hormonal changes relies on inhibition of the pituitary tyrosine kinase and adrenocortical 3β-hydroxysteroid dehydrogenase enzymes by soy isoflavones. Although the clinical studies are in their infancy, the opinion is that genistein and daidzein have therapeutic potential for the safe treatment of ageing-caused androgen deprivation and glucocorticoid excess with related metabolic/hemodynamic issues in males. Our accumulated experience and knowledge in the field of biomedical effects of plant polyphenols have provided a platform for potential recommending the agenda to organize and accelerate experimental research aimed at producing the optimal supplementation. We hypothesize that an in vivo approach should first be exploited in the sequence of investigative steps, followed by in vitro studies and synchronously conducted molecular docking analyses. In vivo research, besides establishing the margin of exposure safety or adjustment of the correct polyphenol dose, enables identification and quantification of the metabolites of applied polyphenols in the blood. Subsequent in vitro exploitation of the metabolites and related docking analyses provide clarification of the molecular mechanisms of action of applied polyphenols. Chemical modification of the polyphenol structure or coupling it with nanoparticles might be the next step in optimizing the design of supplementation. Selected, intact or chemically-modified polyphenol molecules should be included in preclinical studies on a more closely-related species, while clinical studies would finally assess the safety and effectiveness of a polyphenol-based remedial strategy. The final supplement represents a product of an appropriate technological process, conducted in accordance with the recommendations derived from the preceding research.

Soy isoflavone genistein (Gen) exerts beneficial effects against prostate cancer cells in vitro and in vivo. However, its use as a chemoprevention/therapeutic agent is largely limited due to its low bioavailability. In this study we synthesized two variants of a new delivery system, genistein–gold nanoparticles conjugates Gen@AuNPs1 and Gen@AuNPs2, by an environmentally friendly method, using a dual role of Gen to reduce Au3+ and stabilize the formed AuNPs, with no additional component. The formation of Gen@AuNPs was confirmed via UV–Vis spectroscopy, FTIR, and Raman spectra measurements. The spherical shape and uniform size of Gen@AuNPs1 and Gen@AuNPs2 (10 ± 2 and 23 ± 3 nm, respectively), were determined by transmission electron microscopy. The nano-conjugates also varied in hydrodynamic diameter (65.0 ± 1.7 and 153.0 ± 2.2 nm) but had similar negative zeta potential (−35.0 ± 2.5 and −37.0 ± 1.6 mV), as measured by dynamic light scattering. The Gen loading was estimated to be 46 and 48%, for Gen@AuNPs1 and Gen@AuNPs2, respectively. The antiproliferative activities of GenAuNPs were confirmed by MTT test in vitro on three malignant prostate carcinoma cell lines (PC3, DU 145, and LNCaP), while selectivity toward malignant phenotype was confirmed using non-cancerous MRC-5 cells. Flow cytometric analysis showed that the inhibition on cell proliferation of more potent Gen@AuNPs1 nano-conjugate is comparable with the effects of free Gen. In conclusion, the obtained results, including physicochemical characterization of newly synthesized AuNPs loaded with Gen, cytotoxicity, and IC50 assessments, indicate their stability and bioactivity as an antioxidant and anti-prostate cancer agent, with low toxicity against human primary cells. © 2021 Elsevier B.V.

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.

International audience; A growing population of elderly people consume citrus flavanones, naringenin, and hesperetin in the form of fruits or juices. Flavanones are bioactives with potent antioxidant properties and have potential in slowing down the aging process. Because flavanones exert controversial effects on pituitary-thyroid functioning, our study on the old-aged rat model aimed to elucidate the mechanism by which naringenin and hesperetin affect this axis. Naringenin and hesperetin increased the Sirt1 mRNA level by 91 and 71% (p < 0.05), which was followed by increased Sirt1 expression by 20 and 15% (p < 0.05), respectively. Only naringenin decreased thyroid-stimulating hormone expression by 20% (p < 0.05). Thyroid peroxidase protein expression was upregulated after naringenin or hesperetin by 62 and 43% (p < 0.05), respectively. Naringenin lowered mRNA levels of Tpo, Sod1, Sod2, Cat, and Nrf2 by 50, 32, 45, 35, and 42% (p < 0.05), respectively, and increased Gpx by 54% (p < 0.05), while hesperetin decreased Sod1 and Sod2 mRNA levels by 46 and 55% (p < 0.05), respectively. Naringenin increased the protein expressions of Nrf2 and SOD2 by 58 and 50% (p < 0.05), respectively, and decreased SOD1 expression by 48% (p < 0.05), while hesperetin protein decreased expressions of SOD1 and Nrf2 by 63 and 32% (p < 0.05), respectively. Altogether, our findings suggest that citrus flavanones contribute to restoring the impaired thyroid functioning in the old-aged rats.

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.

Citrus flavanones naringenin (NAR) and hesperetin (HES) are potent antioxidants that may contribute to maintenance of health at old age by improving cardiovascular and metabolic status. However, they may also affect thyroid hormone economy. Keeping in mind impaired thyroid function at older age, in this study we tested wheather NAR or HES administration potentiate this decline. NAR or HES were administrated orally (15 mg/kg) to male 24-month-old Wistar rats during 4 Weeks. Control groups received vehicle, sunflower oil. Qualitative and quantitative immunohistochemical and immunofluorescent expression of specific proteins and stereological analyses of thyroid tissue were performed. Thyroid stimulating hormone (TSH) and total thyroxine (T-4) concentrations were measured in serum. Thyroid parenchyma of both flavanone-treated groups was characterized by lower (p LT 0.05) absolute and relative volume of luminal colloid, accompanied by elevated (p LT 0.05) relative volume of stroma in comparison with the controls. No hypertrophy or absolute thyroid volume change was detected. Intensity of immunopositive signal for thyroglobulin (Tg) and T-4 bound to Tg (T-4-Tg) increased (p LT 0.05) in the colloid of thyroid follicles after both flavanone treatments. Serum TSH increased (p LT 0.05) after NAR, while T-4 remained unchanged after both treatments. In conclusion, NAR elevated serum TSH in old-aged males, thus being more potent than HES in altering pituitary-thyroid axis. However, changes in thyroid structure, namely moderate colloid depletion and higher Tg and T-4-Tg protein expressions after both treatments, indicate preserved capacity of the gland to compensate flavanone interfering, and maintain T-4 production in old-aged males. (C) 2017 Elsevier GmbH. All rights reserved.

The authors (Vladimir Ajdzanovic, Ivana Jaric, Dragan Milenkovic and Verica Milosevic) are participating in the COSTAction FA 1403 POSITIVe (Inter individual variation in response to consumption of plant food bioactives and determinants involved),supported by COST (European Cooperation in Science and Technology). We are grateful to Mr. Hinko Sauter (“Swiss-ConceptSciences” d.o.o., Belgrade, Serbia) for additional technical support and to Mrs. Maja R. Vojvodi´c for language correction of the manuscript; Diosgenin, a steroidal sapogenin of natural origin, has demonstrated benefits when it comes to the treatment of malignancies, cardiovascular issues and menopausal symptoms. In this study, we investigated the histological changes of the adrenal gland after diosgenin application in a rat model of the menopause. Middle-aged, acyclic female Wistar rats were divided into control (C; n=6) and diosgenin treated (D; n=6) groups. Diosgenin (100mg/kg b.w./day) was orally administered for four weeks, while C group received the vehicle alone. A histological approach included design-based stereology, histochemistry and immunohistochemistry. The adrenal cortex volume decreased in D females by 15% (p

Introduction and Aim Daidzein application may represent an effective and less harmful alternative to indicated, classical estrogenization of ageing men. The aim of this study was to perform structural and hormonal analysis of the adrenal cortex, after estradiol or daidzein supplementation in a rat model of the andropause. Material and methods Middle-aged Wistar rats were divided into sham operated (SO; n = 8), orchidectomized (Orx; n = 8), estradiol treated orchidectomized (Orx + E; n = 8) and daidzein treated orchidectomized (Orx + D; n = 8) groups. Estradiol (0.625 mg/kg b.m./day) or daidzein (30 mg/kg b.m./day) were administered subcutaneously for three weeks, while the SO and Orx groups received the vehicle alone. Set objectives were achieved using stereology, histochemistry/immunohistochemistry, immunoassays and ultrastructural analysis. Results Both estradiol and daidzein treatment significantly increased volumes of the zona glomerulosa cell and nuclei, but decreased circulating aldosterone levels. Estradiol markedly increased volumes of the zona fasciculata cell and nuclei in parallel with significant decrease of the adrenal tissue level of corticosterone, while daidzein significantly decreased both the adrenal and circulating levels of corticosterone. Serum DHEA level and volumes of the zona reticularis cell and nuclei significantly increased upon estradiol treatment, whereas daidzein even stronger increased the circulating level of DHEA. Shunting of the corticosteroidogenesis pathways towards adrenal androgens production, after the treatments, corresponded to the ultrastructural findings and zonal capillary network rearrangements. Conclusions Given the coherence of its effects and relative safety, daidzein could be the remedy of choice for the treatment of ageing-caused androgen deprivation and the hypothalamo-pituitary-adrenal axis hyperfunction/related metabolic issues in males.

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

In a rat model of the andropause we aimed to examine the influence of daidzein, soy isoflavone, on the structure and function of parathyroid glands (PTG) and the expression levels of some of the crucial regulators of Ca2+ and Pi homeostasis in the kidney, and to compare these effects with the effects of estradiol, serving as a positive control. Middle-aged (16-month-old) male Wistar rats were divided into the following groups: sham-operated (SO), orchidectomized (Orx), orchidectomized and estradiol-treated (Orx+E; 0.625mg/kg b.w./day, s.c.) as well as orchidectomized and daidzein-treated (Orx+D; 30mg/kg b.w./day, s.c.) group. Every treated group had a corresponding control group. PTH serum concentration was decreased in Orx+E and Orx+D groups by 10% and 21% (p

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

It is well known that stress changes levels of pituitary hormones in the bloodstream and in the pituitary itself. However, almost nothing is known about the impact of stress on histological and stereological parameters of the growth hormone producing cells (somatotrophs-GH cells). The aim of the present study was to investigate the effect of: acute and repeated immobilization; acute and repeated restraint on histological and morphofunctional parameters of somatotrophs in adult Wistar rats. Changes in the pituitary gland volume; the volume density and volume of somatotrophs following acute and repeated immobilization (IMO, R-IMO); acute and repeated restraint (R, R-R) were evaluated using a stereological system (newCAST), while growth hormone level within pituitary was determined by Western blot. Our results demonstrated the decrease (p < 0.05) of the pituitary volume (17%, 19%) in the IMO and R groups, respectively, and the increase in the R-R group. The volume density of GH cells decreased (p < 0.05) in the R-IMO (7%), R (26%) and R-R (18%) group in comparison to the control value. The pituitary GH content was increased (p < 0.05) after the IMO (2-fold), R (2.5-fold) and R-R (2.1-fold) as compared to the control group. These results point out that acute and repeated immobilization and/or restraint lead not only to changes in GH hormone concentration, but also modify the morphological aspects of GH cells within the rat pituitary.

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]

The isoflavone, daidzein is a biologically active, plant-derived compound that interacts with estrogen receptors. Data from previous studies have suggested that daidzein exerts beneficial effects in many diseases; however, as an endocrine disrupter, it may also alter the functioning of the endocrine system. Data regarding the effect of daidzein on the morphofunctional and histological parameters of the hypothalamic-pituitary-adrenal (HPA) system is still lacking. Therefore, using the newCAST stereological software, we investigated the effects of chronic (21 days) daidzein treatment on corticotropin-releasing hormone (CRH) neurons within the hypothalamus and corticotropes (ACTH cells) in the pituitary, while image analysis was employed to-examine the intensity of fluorescence of CRH in the median eminence (ME) and adrenocorticotropin hormone in the pituitary in adult orchidectomized (Ovx) rats. Circulating ACTH and corticosterone levels were also analyzed. This study showed that daidzein treatment decreased the volume density of CRH neurons within the paraventricular nucleus as well as CRH immunofluorescence in the ME. The total number of ACTH cells was decreased, while ACTH cell volume and the intensity of ACTH fluorescence were increased following daidzein treatment. Both ACTH and corticosterone blood levels were increased after daidzein administration. The results of performed experiments clearly demonstrate that volume density of CRH neurons; total number and volume of ACTH cells, as well as stress hormones levels are vulnerable to the effects of daidzein.

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.

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)

Soy-food and its isoflavones, genistein (G) and daidzein (D), were reported to exert mild cholesterol-lowering effect, but the underlying mechanism is still unclear. In this research, first we studied age-related alterations in hepatic cholesterol metabolism of acyclic middle-aged (MA) female rats. Then we tested if purified isoflavones may prevent or reverse these changes, and whether putative changes in hepatic thyroid hormone availability may be associated with this effect. Serum and hepatic total cholesterol (TChol), bile acid and cholesterol precursors, as well as serum TSH and T4 concentrations, hepatic deiodinase (Dio) 1 enzyme activity and MCT8 protein expression were determined by comparing data obtained for MA with young adult (YA) intact (IC) females. Effects of subcutaneously administered G or D (35 mg/kg) to MA rats were evaluated versus vehicle-treated MA females. MA IC females were characterized by: higher (p < 0.05) serum TChol, lower (p < 0.05) hepatic TChol and its biosynthetic precursors, lower (p < 0.05) hepatic 7α-hydroxycholesterol but elevated (p < 0.05) 27- and 24-hydroxycholesterol in comparison to YA IC. Both isoflavone treatments decreased (p < 0.05) hepatic 27-hydroxycholesterol, G being more effective than D, without affecting any other parameter of Chol metabolism. Only G elevated hepatic Dio1 activity (p < 0.05). In conclusion, age-related hypercholesteremia was associated with lower hepatic Chol synthesis and shift from main neutral (lower 7α-hydroxycholesterol) to alternative acidic pathway (higher 27-hydroxycholesterol) of Chol degradation to bile acid. Both isoflavones lowered hepatic 27-hydroxycholesterol, which may be considered beneficial. Only G treatment increased hepatic Dio1 activity, thus indicating local increase in thyroid hormones, obviously insufficient to induce prominent cholesterol-lowering effect. Experimental Gerontology (2017), 92: 74-81

Aging is a complex process characterized by a progressive decline in cellular function, alterations of the endocrine system and the increased incidence of thyroid diseases. Citrus flavanones naringenin (NAR) and hesperetin (HES) may contribute to maintenance of health at old age however they may also affect thyroid hormone economy. The aim of this study was to test weather NAR or HES administration alter thyroid functioning. NAR or HES were administrated orally (15 mg/kg) to male 24-month-old Wistar rats during 4 weeks. Control groups received vehicle, sunflower oil. Immunohistochemical staining of thyroglobulin (Tg), thyroxine (T4) bound to Tg (T4-Tg) and sodium iodide sysmporter (NIS) were performed. Immunopositivity of Tg or T4-Tg in thyroid follicles were measured as intensity of optical density (OD) or relative intensity of fluorescence (RIF). Thyroid stimulating hormone (TSH) and total T4 concentrations were measured in serum. After citrus flavanones treatments, thyroids showed Tg and T4-Tg immunopositivity mainly in the colloid of the follicles. The obtained results showed increased (P ! 0.05) intensity of OD for Tg and T4-Tg RIF after both flavanones treatments, while NIS expression did not change. Serum TSH increased (P ! 0.05) only after NAR, although T4 remained unchanged after both treatments. In conclusion, citrus flavanones induced stronger expression of thyroid-specific proteins in gland of old-aged males. However, only NAR elevated serum TSH, being more potent than HES. This alteration is in line with preserved capacity of the thyroid to compensate the mild interference of flavanones treatments with thyroid hormone production in old-aged males.

Calcitonin (CT) is a hypocalcemic hormone produced by thyroid C-cells that acts as an bone antiresorptive agent. The other endocrine cell population in thyroid, called follicular cells produce thyroid hormones (TH) which also affect bone turnover. In this study, we evaluated the effects of salmon CT administration on structure and function of both CT and TH producing thyroid cells as well as trabecular bone microarchitecture in orchidectomized (Orx) middle-aged rats. Fifteen-month-old male Wistar rats were either Orx or sham-operated (SO). One group of Orx animals were injected subcutaneously with synthetic salmon CT (Orx + CT; 100 IU/kg b.w.) every second day for 6 weeks. The rats from SO and second Orx group received the same volume of vehicle alone by the same schedule. The peroxidase–antiperoxidase method was applied for localization of CT in C-cells. CT-immunopositive thyroid C-cells, thyroid follicular epithelium, interstitium and colloid were evaluated morphometrically. An ImageJ public domain image processing program was used to measure bone histomorphometric parameters of the proximal tibial specimens. Blood serum samples were analyzed for CT, osteocalcin (OC) and thyroxine (T4), and urine samples fot calcium (Ca2+) concentration. We found a significant decrease in the Vc and Vv of thyroid C-cells after CT treatment compared to both SO and Orx. The Vv of the colloid was higher, while VV of the follicular epithelium was lower after CT treatment compared to Orx. Analysis of trabecular microarchitecture showed that salmon CT administration significantly increases of cancellous bone area (B.Ar), trabecular thickness (Tb.Th), and trabecular number (Tb.N) whereas trabecular separation (Tb.Sp) was significantly decreased. CT treatment markedly elevated serum CT, but serum OC, T4 and urinary Ca2+ concentrations were lower than in the Orx group. These findings indicate that administration of salmon CT inhibited calcitonin-producing thyroid C-cells and changes the structure of the thyroid gland indicating hypoactivity.

Light regulates numerous physiological functions and synchronizes them with the environment, in part by adjusting secretion of different hormones. We hypothesized that constant light (CL) would disturb pituitary-thyroid axis. Our aim was to determine morphological and functional changes in this endocrine system in such extreme conditions and, based on the obtained results, to propose the underlying mechanism(s). Starting from the thirtieth postnatal day, female Wistar rats were exposed to CL (600 lx) for the following 95 days. The controls were maintained under the regular laboratory lighting conditions. After decapitation, pituitaries and thyroids were prepared for further histomorphometric, immunohistochemical, and immunofluorescence examinations. Concentration of thyroid stimulating hormone (TSH), total T4 and T3 (TH) were determined. Thyroid tissue of light-treated rats was characterized by microfollicular structure. We detected no change in total thyroid volume, localization and accumulation of thyroglobulin, thyroid peroxidase, and sodium-iodide symporter in the follicular epithelium of CL rats. The volume of follicular epithelium and activation index were increased, while volume of the colloid and serum levels of TH decreased. In the pituitary, the relative intensity of TSH β-immunofluorescence signal within the cytoplasm of thyrotrophs increased, but their average cell volume and the relative volume density decreased. Serum TSH was unaltered. We conclude that exposure of female rats to CL induced alterations in pituitary-thyroid axis. Thyroid tissue was characterized by microfollicular structure. Serum TH levels were reduced without accompanying increase in serum TSH. We hypothesize that increased secretion and clearance of TH together with unchanged or even decreased hormonal synthesis, resulted in decreased serum TH levels in CL group. We assume this decrease consequently led to increased synthesis and/or accumulation of pituitary TSH. However, decreased average TSH cell volume and relative volume density, together with unchanged serum TSH, point to additional, negative regulation of thyrotrophs.

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

We previously reported that Vitex agnus-castus L. essential oil (VACEO), when administered to middle-aged males, exerts a bone-protective effect, induces silencing of locomotor activities and decreases pituitary prolactin immunopositivity. To further assess the putative endocrine effects of VACEO, we examined the pituitary-thyroid and -adrenocortical axes in our model. Sixteen-month-old Wistar rats were subcutaneously administered 60mg/kg of VACEO dissolved in sterile olive oil, while the control group received the same amount of vehicle alone for three weeks. Pituitaries, thyroids and adrenals were analyzed by qualitative and quantitative histological approaches. Concentration of thyroid stimulating hormone (TSH), total thyroxine and triiodothyronine (TH), adrenocorticotrophic hormone (ACTH), corticosterone in serum and in adrenal tissue were measured. In VACEO-treated rats, the relative volume density of pituitary thyrotrophs increased (p

This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia Grant No. 173009; This study aimed to investigate effects of citrus flavanones naringenin (NAR) and hesperetin (HES) on liver antioxidant status and membrane phospholipid composition in 24-month-old rats. NAR and HES (15 mg/kg) were administrated orally to male Wistar rats, once per day, for 4 weeks. Control group received either vehicle (sunflower oil) or remained intact. The results showed decreased (p < 0.05) activity of antioxidant enzymes (AOE), specifically catalase (CAT), superoxide dismutase (SOD) 1 and glutathione reductase (GR) in the liver of intact control old-aged rats in comparison to young intact controls. Flavanone administration to old-aged males increased (p < 0.05) examined AOE activities in comparison to vehicle-administered animals. Namely, NAR was more potent in comparison to HES regarding the increase (p < 0.05) in activities of examined antioxidant enzymes (SOD 1 and 2, glutathione peroxidase-GPx and GR) and the liver glutathione (GSH), while HES elevated (p < 0.05) only activity of CAT and GR. Both flavanones significantly decreased (p < 0.05) TBARS and improved (p < 0.05) membrane phospholipid composition in favor of n-3 PUFA and n-6/n-3 PUFA ratio. Both flavanones did not affect liver histology and reduced (p < 0.05) alanine aminotransferase and aspartate aminotransferase levels in serum. The results of this study indicate beneficial potential of citrus flavanones in the old-aged rat liver

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.

This study aimed to examine the influence of pregnancy on responsiveness of the pituitary–adrenal (PA) axis to dexamethasone (Dx) in rats. Dams and virgins were injected with Dx in three consecutive daily doses of 1.0, 0.5, 0.5 mg/kg/bw, starting from day 16 of pregnancy, and sacrificed 24 and 72 h after termination of treatment. Immunocytochemical, morphometrical and biochemical methods were used to assess pituitary and adrenal function. Three-way ANOVA revealed Dx inhibition of the PA axis in virgin and pregnant females after 24 and 72 h. Pregnant females demonstrated a significantly lower (p

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