Nataša Ristić

Genistein affects ovarian folliculogenesis: A stereological study

The effects of short‐term genistein exposure on ovarian folliculogenesis in immature rats were examined stereologically. To determine whether genistein acts as an estrogen agonist or antagonist, the results were compared with the effects of 17α‐ethynylestradiol. Immature female rats received 50 mg/kg/bw of genistein in dimethyl sulfoxide subcutaneously daily for three consecutive days from 18 to 20 days. The second group was injected with 1 μg/kg/bw of 17α‐ethynylestradiol in olive oil in the same schedule. Each group had a corresponding control. Genistein increased ovary and ovarian stroma volumes by 18.50% (P < 0.05) and 53.40% (P < 0.05), respectively, and changed the parenchyma to stroma ratio in favor of stroma. Genistein induced decreases in the number of primordial (by 17.23%; P < 0.05), primary (16.62%; P < 0.05), and secondary follicles (12.29%: P < 0.05), whereas the number of atretic secondary follicles increased (5.10‐fold; P < 0.05). The number of healthy large follicles was raised by 27.3% (P < 0.05), accompanied by 35.64% more atretic large follicles (P < 0.05). Similarly to genistein, estradiol changed the parenchyma to stroma ratio in favor of stroma, and reduced the number of primordial follicles, but the number of primary follicles was elevated. There were more healthy and atretic small and large follicles. In conclusion, genistein acted as an estrogen antagonist and had an inhibitory effect on the initial phase of folliculogenesis. In the other phases, genistein acted as an estrogen agonist, stimulating transition from the preantral to antral stage of folliculogenesis, and altering the ratio of follicular parenchyma and ovarian stroma in favor of stroma. Microsc. Res. Tech. 2012.

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

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

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

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

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 genistein on gonadotropic cells in immature female rats

The effects of genistein on pituitary gonadotropic cells of immature female rats were examined and compared to actions of the synthetic estrogen, 17α-ethynylestradiol. Immature female rats received 50mg/kg/bw of genistein in dimethylsulfoxide (DMSO) subcutaneously (s.c.) daily for 3 days at 18, 19 and 20 days of age. A second group was injected with 1μg/kg of 17α-ethynylestradiol in olive oil in the same schedule. The genistein control group received DMSO only, while 17α-ethynylestradiol controls were given sterile olive oil only. Changes in cell number per mm(2), cell volume and volume density of follicle-stimulating (FSH) and luteinizing (LH) immunolabeled cells were evaluated by morphometry and stereology. Genistein induced significant increases in the number of FSH cells (by 21%) and LH cells (by 20%) per mm(2) compared to corresponding controls. Volumes of FSH and LH cells were significantly increased by 19.7% and 20% and their volume densities by 20% and 20.2%, respectively. Estradiol markedly affected gonadotropes in the same manner, but to a greater extent. It can be concluded that genistein acted as an estrogenic agonist in the pituitaries of immature female rats, and as such, stimulated gonadotropic cells.

Morphological and functional changes in pituitary-thyroid axis following prolonged exposure of female rats to constant light

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. J. Morphol. 275:1161–1172, 2014.

A BRIEF COMMUNICATION

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

Morpho-functional characteristics of rat fetal thyroid gland are affected by prenatal dexamethasone exposure.

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 1.0mg/Dx/kg/b.w., followed by 0.5mg/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 Cavalieris 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.

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.

Immunohistomorphometric Features of ACTH Cells in Juvenile Rats after Treatment with Estradiol or Human Chorionic Gonadotropin

Immunohistomorphometric Features of ACTH Cells in Juvenile Rats after Treatment with Estradiol or Human Chorionic Gonadotropin Estradiol and human chorionic gonadotropin (hCG) are very important in controlling the secretory activity of hormone producing cells in the female rat pituitary glands. The aim of the present study was to examine the morphometric parameters of immunohistochemically labeled ACTH cells in juvenile (16th day) female rat pituitaries after treatment with five doses of estradiol dipropionate (EDP) and two doses of hCG during the neonatal period of life. The controls were treated on the same schedule with an equivalent volume of vehicle. All animals were sacrificed 24 h after the last treatment. ACTH-producing cells were studied using the peroxidase-antiperoxidase immunohistochemical procedure. The absolute and relative pituitary weights were increased (p<0.05) only in the EDP treated group by 120.0% and by 121.1% respectively, in comparison with the controls. In this group, the volume of ACTH cells, volume of their nuclei and volume density were significantly decreased (p<0.05) by 6.4%, 33.3% and 46.2% respectively, compared to the corresponding controls. After treatment with hCG, there were no significant (p>0.05) changes neither in the volume of ACTH cells nor in the volume of their nuclei, in comparison with the controls. On the basis of the results obtained in our study, it can be concluded that EDP, injected into female rats during the neonatal period of life, has an inhibitory effect on the immunohistomorphometric parameters of ACTH cells, but such an effect is not clearly expressed after treatment with hCG. Imunohistomorfometrijske Odlike ACTH Ćelija u Juvenilnih Pacova Nakon Tretmana Estradiolom Ili Humanim Horionskim Gonadotropinom Estradiol i humani horionski gonadotropin (hCG) veoma su važni u kontroli sekretorne aktivnosti ćelija hipofize koje proizvode hormone. Cilj ove studije je bio ispitati morfometrijske parametre imunohistohemijski obeleženih ACTH ćelija hipofize ženki pacova, u juvenilnom periodu (16. dan), nakon tretmana sa pet doza estradiol-dipropionata (EDP) ili dve doze humanog horionskog gonadotropina (hCG). Kontrole su bile tretirane na istovetan način sa odgovarajućom količinom rastvarača. Sve životinje su bile žrtvovane 24 h nakon poslednjeg tretmana. ACTH ćelije su bile imunohistohemijski obeležene metodom peroksidaza-antiperoksidaza (PAP). Dobijeni rezultati ukazuju na značajno povećanje (p<0,05) apsolutne i relativne mase hipofize za 120% i 121,1% kod ženki tretiranih sa EDP u odnosu na kontrolu. U ovoj grupi ženki pacova volumen ACTH ćelija i njihovih jedara, kao i njihova volumenska gustina su bili značajno smanjeni (p<0,05) za 6,4%, 33,3% i 46,2% u poređenju sa kontrolom. Nakon tretmana hCG-om, volumen ACTH ćelija, kao i volumen njihovih jedara, nisu bili značajno (p>0,05) promenjeni u odnosu na kontrolu. Na osnovu dobijenih rezultata možemo zaključiti da EDP, injeciran ženkama pacova tokom neonatalnog perioda života, ima inhibitorni efekat na imunohistomorfometrijske parametre ACTH ćelija, što nije tako jasno izraženo nakon tretmana hCG-om.