Ivana Jarić

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

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

Effects of age and soybean isoflavones on hepatic cholesterol metabolism and thyroid hormone availability in acyclic female rats

ABSTRACT 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 7&agr;‐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 7&agr;‐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. HIGHLIGHTSAge‐related hypercholesteremia in middle‐aged acyclic female rats (13‐month‐old) is associated with:(i) lower hepatic cholesterol biosynthesis and(ii) shift from neutral to acydic pathway of cholesterol degradation to bile acid in the liver.(iii) unchanged TSH and total T4 in serum, as well as Dio1 enzyme activity and MCT8 protein expression in the liverSoy isoflavones reversed only hepatic 27‐hydroxycholesterol to the level of young adults.Only genistein elevated hepatic Dio1 activity, thus indicating local increase in thyroid hormones.

Diosgenin-caused changes of the adrenal gland histological parameters in a rat model of the menopause

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<0.05) while the volume of adrenal medulla increased (p<0.05) by 64%, compared to the same parameters in C group. Volume density of the zona glomerulosa (expressed per absolute adrenal gland volume) in D rats increased (p<0.05) by 22% in comparison with C animals. Diosgenin treatment decreased (p<0.05) the volume density of the zona fasciculata (expressed per volume of adrenal cortex) by 15% when compared to C females. Absolute volume of the zona reticularis in D group decreased (p<0.05) by 38% in comparison with the same parameter in C rats. Also, after diosgenin application, the volume density of the zona reticularis (expressed per volume of adrenal cortex) and the zona reticularis cell volume were decreased by 51% and 20% (p<0.05) respectively, compared to C animals. Our results, reflecting a decrease in many stereological parameters of the adrenal cortex, indicate that diosgenin took over the role of corticosteroid precursors and became incorporated into steroidogenesis.

Dexamethasone exposure affects paraventricular nucleus and pituitary corticotrophs in female rat fetuses: An unbiased stereological and immunohistochemical study

The hypothalamic paraventricular nucleus (PVN) drives the stress response by activating the hypothalamo-pituitary-adrenal (HPA) axis, particularly vulnerable to glucocorticoid exposure during development. To evaluate the effects of fetal dexamethasone (Dx) exposure on the stereological features of PVN and HPA axis activity in female rat fetuses, pregnant rats received 0.5mg Dx/kg/b.w./day on days 16, 17 and 18 of pregnancy and 21-day-old fetuses were obtained; controls received the same volume of saline. In an unbiased stereological approach, Cavalieris principle and an optical fractionator were used for estimating volume and total cell number of the PVN, respectively. The intensity of corticotropin-releasing hormone (CRH) immunoreactivity in the median eminence (ME) was determined by CRH optical density and the adrenocorticotropic hormone (ACTH) relative fluorescence signal intensity (RIF) in pituitary corticotrophs was measured using Image J. Significant reductions (p<0.05) in PVN volume and cell number were found in fetuses exposed to Dx. Additionally, CRH optical density in the ME and ACTH RIF (p<0.05) in the corticotrophs were decreased. The established results suggest that the reduced number of cells in the PVN after maternal Dx administration negatively affects the CRH content in the ME and the ACTH quantity in pituitary corticotrophs in near-term fetuses.

Lignin model compound in alginate hydrogel: a strong antimicrobial agent with high potential in wound treatment

Nowadays bacterial resistance to known antibiotics is a serious health problem. In order to achieve more efficient treatment, lately there is an effort to find new substances, such as certain biomaterials, that are non-toxic to humans with antibiotic potential. Lignins and lignin-derived compounds have been proposed to be good candidates for use in medicine and health maintenance. In this study, the antibacterial activity of the lignin model polymer dehydrogenate polymer (DHP) in alginate hydrogel (Alg) was studied. The obtained results show that DHP-Alg has strong antimicrobial activity against several bacterial strains and biofilms and does not have a toxic effect on human epithelial cells. These results strongly suggest its application as a wound healing agent or as an adjunct substance for wound treatments.

Functional morphology of pituitary -thyroid and -adrenocortical axes in middle-aged male rats treated with Vitex agnus castus essential oil.

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<0.001), while intensity of cytoplasmic TSHβ immunostaining decreased (p<0.001), consistent with elevated TSH in serum (p<0.01). The thyroid tissue was characterized by a micro-follicular structure, increased relative volume of follicular epithelium (p<0.05), decreased volume of luminal colloid (p<0.001) and increased basolateral expression of sodium-iodide symporter-immunopositivity (p<0.05). Serum TH also increased (p<0.01). The relative volume density of pituitary corticotrophs decreased (p<0.05), compatible with decline in circulating ACTH (p<0.05). Neither tissue nor serum corticosterone levels were affected by VACEO treatment. In conclusion, the observed changes in TSH and ACTH strongly indicate central endocrine effects of prolonged VACEO treatment. In this respect, production of ACTH decreased without impact on corticosterone production. Increase in serum concentration of both TH and TSH are not compatible with a negative feedback loop and suggest a major change in set-point regulation of the hypothalamic-pituitary-thyroid axis.

Daidzein upregulates anti-aging protein Klotho and NaPi 2a cotransporter in a rat model of the andropause

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<0.05) respectively, in comparison with the Orx. PTG volume was decreased in Orx+E group by 16% (p<0.05), when compared to the Orx. In Orx+E group expression of NaPi 2a was lower (p<0.05), while NaPi 2a abundance in Orx+D animals was increased (p<0.05), when compared to Orx. Expression of PTH1R was increased (p<0.05) in Orx+E group, while in Orx+D animals the same parameter was decreased (p<0.05), in comparison with Orx. Klotho expression was elevated (p<0.05) in Orx+D rats, in regard to Orx. Orx+D induced reduction in Ca2+/creatinine and Pi/creatinine ratio in urine by 32% and 16% (p<0.05) respectively, in comparison with Orx. In conclusion, presented results indicate the more coherent beneficial effects of daidzein compared to estradiol, on disturbed Ca2+ and Pi homeostasis, and presumably on bone health, in the aging male rats.

Genistein and daidzein treatments differently affect uterine homeostasis in the ovary-intact middle-aged rats.

&NA; This study aimed to investigate the effects of soy isoflavones, genistein (GEN) and daidzein, (DAI) on the uterine function in ovary‐intact middle‐aged rats. GEN and DAI (35 mg/kg) were subcutaneously administrated to acyclic (12‐month‐old) Wistar females, daily, for 4 weeks. Control group received either vehicle (olive oil and ethanol, 9:1) or remained intact. We found that GEN and DAI differently affect uterine morphophysiology. GEN significantly increased the uterine wet weight which was associated with hyperplastic changes, revealed by stereological and histomorphometrical analyses. Also, PCNA immunoexpression was increased, whereas expression of apoptotic marker (caspase‐3) was decreased. Protein and gene expressions of ER&agr; were down‐regulated, while PR and ER&bgr; were up‐regulated after GEN application. Also, GEN caused an increase of LAC and VEGF mRNA expression, together with an up‐regulation of Akt activity. In contrast, DAI did not change the uterine wet weight and stereological features of the main uterine compartments as well as LAC and VEGF gene expression. Absence of hyperplastic changes were illustrated by an increase in caspase‐3 immunoexpression, associated with reduced PCNA expression. DAI up‐regulated only the expression of ER&bgr;, while the expression levels of ER&agr; and PR remain unaffected. Also, DAI inhibited the activation of Akt due to down‐regulation of phosphorylated and total form of Akt protein expression. Compared to GEN, DAI did not promote events associated with the endometrial cell proliferation in the conducted study, figuring as the compound with a potential safety profile, which justifies further investigation. HighlightsDAI did not change the uterine wet weight and stereological features of the uterus.DAI up‐regulated the expression of ER&bgr;, while the expression levels of ER&agr; and PR remain unaffected.DAI inhibits the activation of Akt due to down‐regulation of phosphorylated form.Compared to GEN, DAI did not promote events associated with endometrial cell proliferation.

Soy Phytoestrogens Do Not Fully Reverse Changes in Rat Pituitary Castration Cells: Unbiased Stereological Study: PHYTOESTROGENS WEAKLY AFFECT CASTRATION CELLS

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.

The Effect of Moderate Heat on Rat Pituitary ACTH Cells: Histomorphometric, Immunofluorescent and Hormonal Study

Abstract In areas with moderate continental climate, increased average ambient temperature during the summer represents a stressogenic factor that affects the hypothalamo-pituitaryadrenocortical axis in mammals. Therefore, we wanted to examine the effects of 4 days of constant exposure to moderately elevated ambient temperature (35 ± 1oC) on the histomorphometric and immunofl uorescent characteristics, as well as on the hormonal secretion of pituitary corticotropes (ACTH) cells in adult male rats. In comparison with the controls kept at 20 ± 2oC, a signifi cant increase (p<0.05) of the absolute and relative pituitary weight (23.1% and 36.1%, respectively) was registered after exposure to heat. The localization, as well as the shape of the ACTH cells in the heat exposed group was not signifi cantly altered, but their immunopositivity was weaker. After 4 days of heat exposure, a weaker signal confi rmed the relative fl uorescence intensity of the ACTH cells (15.3%, p<0.05). In heat exposed rats, an increase of the cellular and nuclear volumes of immunolabelled ACTH cells and decrease of their volume density (6.9%, 14.3% and 20.0%, respectively; p<0.05) was registered. Observed histomorphometric and immunofl uorescent features of the pituitary ACTH cells were in accordance with the increased (p<0.05) value of plasma adrenocorticotropic hormone (ACTH) by 23.7% compared to the control rats. It can be concluded that the 4-day exposure to moderately elevated ambient temperature intensifi es pituitary ACTH secretion in adult male rats.