Changes of growth hormone-releasing hormone and somatostatin neurons in the rat hypothalamus induced by genistein: a stereological study.

OBJECTIVES: Genistein is a plant-derived estrogenic isoflavone commonly found in dietary and therapeutic supplements, due to its potential health benefits. Growth hormone-releasing hormone (GHRH) and somatostatin (SS) are neurosecretory peptides synthesized in neurons of the hypothalamus and regulate the growth hormone secretion. Early reports indicate that estrogens have highly involved in the regulation of GHRH and SS secretions. Since little is known about the potential effects of genistein on GHRH and SS neurons, we exposed rats to genistein. METHODS: Genistein were administered to adult rats in dose of 30 mg/kg, for 3 weeks. The estradiol-dipropionate treatment was used as the adequate controls to genistein. Using applied stereology on histological sections of hypothalamus, we obtained the quantitative information on arcuate (Arc) and periventricular (Pe) nucleus volume and volume density of GHRH neurons and SS neurons. Image analyses were used to obtain GHRH and SS contents in the median eminence (ME). RESULTS: Administration of estradiol-dipropionate caused the increase of Arc and Pe nucleus volume, SS neuron volume density, GHRH and SS staining intensity in the ME, when compared with control. Genistein treatment increased: Arc nucleus volume and the volume density of GHRH neurons (by 26%) and SS neurons (1.5 fold), accompanied by higher GHRH and SS staining intensity in the ME, when compared to the orhidectomized group. DISCUSSION: These results suggest that genistein has a significant effect on hypothalamic region, involved in the regulation of somatotropic system function, and could contribute to the understanding of genistein as substance that alter the hormonal balance.

Effects of soy phytoestrogens on pituitary-ovarian function in middle-aged female rats.

The aim of this study was to assess the effects of genistein (G) and daidzein (D) on the histological, hormonal, and functional parameters of the pituitary-ovarian axis in middle-aged female rats, and to compare these effects with the effects of estradiol (E), commonly used in the prevention and treatment of menopausal symptoms. Middle-aged (12 month old) Wistar female rats subcutaneously received 35 mg/kg of G, or 35 mg/kg of D, or 0.625 mg/kg of E every day for 4 weeks. Each of the treated groups had a corresponding control group. An intact control group was also established. G and D did not change the intracellular protein content within gonadotropic and lactotropic cells, but vacuolization was observed in all the cell types. In contrast, E caused an inhibition of gonadotropic and stimulation of lactotropic cells. Also, ovaries of middle-aged female rats exposed to G or D have more healthy primordial and primary follicles and less atretic follicles. E treatment in the ovaries had a mostly negative effect, which is reflected by the increased number of atretic follicles in all tested classes. G and D provoked decrease in CuZnSOD and CAT activity, while E treatment increased MnSOD and decreased CuZnSOD and GSHPx activity. All the treatments increased serum estradiol and decreased testosterone levels, while D and E increased the serum progesterone level. In conclusion, soy phytoestrogens exhibited beneficial effects on pituitary-ovarian function in middle-aged female rats, as compared to estradiol.

Membrane steroid receptor-mediated action of soy isoflavones: tip of the iceberg.

Soy isoflavone's (genistein and daidzein in particular) biological significance has been thoroughly studied for decades, so we started from the premise that refreshed investigation approach in this field should consider identification of their new molecular targets. In addition to recently described epigenetic aspects of polyphenole action, the cell membrane constituents-mediated effects of soy isoflavones are worthy of special attention. Accordingly, the expanding concept of membrane steroid receptors and rapid signaling from the cell surface may include the prominent role of these steroid-like compounds. It was observed that daidzein strongly interacts with membrane estrogen receptors in adrenal medullary cells. At low doses, daidzein was found to stimulate catecholamine synthesis through extracellular signal-regulated kinase 1/2 or protein kinase A pathways, but at high doses, it inhibited catecholamine synthesis and secretion induced by acetylcholine. Keeping in mind that catecholamine excess can contribute to the cardiovascular pathologies and that catecholamine lack may lead to depression, daidzein application promises to have a wide range of therapeutic effects. On the other hand, it was shown in vitro that genistein inhibits LNCaP prostate cancer cells invasiveness by decreasing the membrane fluidity along with immobilization of the androgen receptor containing membrane lipid rafts, with down regulation of the androgen receptors and Akt signaling. These data are promising in development of the molecular pharmacotherapy pertinent to balanced soy isoflavone treatment of cardiovascular, psychiatric, and steroid-related malignant diseases.

Exposure to dexamethasone reduces pituitary volume and gonadotropic cell number in rat fetuses.

Overexposure to glucocorticoids during the fetal period induces changes in developmental processes in various fetal tissues. The aim of this study was to investigate the effects of the synthetic glucocorticoid, dexamethasone (Dx), on pituitary volume and gonadotropic cells during a critical period of pituitary development. The effects of Dx on stereological parameters of the pituitary gland and FSH and LH cells were investigated in 19 and 21-day old fetuses. On day 16 of pregnancy, the experimental dams received 1.0 mg Dx/kg b.w. subcutaneously, followed by 0. 5mg Dx/kg b.w./day on days 17 and 18 of gestation. The control gravid females received the same volume of saline. FSH and LH cells were stained immunohistochemically by the peroxidase-antiperoxidase method (PAP). In 19-day old fetuses, exposure to Dx caused a significant decrease of pituitary volume, estimated by Cavalieri's principle. Also, the total number of FSH and LH cells per pituitary, determined by physical fractionator counting technique, was significantly reduced. These changes persisted until fetal day 21. Volume densities and numerical densities of FSH and LH cells after exposure to Dx in 19 and 21-day old fetuses remained unaffected. Our results suggest that altered stereological parameters in pituitary gland after exposure to dexamethasone in fetal period could be long-lasting.

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 ß-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.

Effects of genistein on stereological and hormonal characteristics of the pituitary somatotrophs in rats.

The hypothalamic-pituitary somatotropic system plays a pivotal role in the regulation of physiological processes and metabolism, which is modulated by gonadal steroids. Considering that genistein belongs to the phytoestrogen family and acts via similar mechanisms to estrogens, the present study was designed to demonstrate whether genistein modulates the morphofunctional characteristic of somatotrophs [growth hormone (GH) cells] in adult rats in comparison with the effects of estradiol. In the study, the orchidectomized adult rats were used as an appropriate model system for testing the effects of this hormone-like substance. Changes in the pituitary somatotrophs were evaluated histologically and stereologically, while GH level was determined biochemically. Using immunolabelling and stereological methods, we showed that orchidectomy (Orx) provoked the decrease of GH cell volume density. After estradiol treatment of Orx rats, the most prominent change concerned the pituitary relative intensity of GH fluorescence and circulating GH level, which were elevated 77 % and 4.7-fold, respectively. Clearly, in contrast to orchidectomy, estradiol treatment enhanced the GH cells activity. Genistein treatment increased pituitary weight and volume, GH cell volume density, the total number of GH cells, and GH blood concentration (1.3-fold) in comparison to the Orx group. Although identical tendencies followed estradiol and genistein administration, the changes observed after genistein treatment were milder compared to estradiol treatment.

Diosgenin does not express estrogenic activity: a uterotrophic assay.

This study assessed the effects of diosgenin on estrogenic activity using a uterotrophic assay. Immature female rats received diosgenin orally at doses of 200, 100, or 20 mg/kg body mass; and 17α ethynylestradiol at doses of 1 or 0.3 µg/kg, daily, for 3 consecutive days from day 19 to day 21. Controls were distributed among 2 groups: an intact control group and a vehicle control group. Animals were sacrificed 24 h after the last application of diosgenin, estradiol, or vehicle (22nd day of life). Uterine wet weight, stereological and histomorphometrical changes, immunohistochemical expression of estrogen receptor alpha (ERα), progesterone receptor (PR), and the expression of lactoferrin (LF) were examined. Diosgenin did not affect the uterine wet weight, epithelium height, volume densities of endometrium, endometrial epithelia, number of endometrial glands, or histological appearance of vaginal epithelia. ERα, PR, and LF immunostaining intensity were not altered in the animals that received diosgenin. High-potency reference ER agonist 17α-ethynylestradiol induced a significant increase in all of the measured parameters, and as expected, decreased ERα immunostaining intensity. Based on these data, it can be concluded that diosgenin, at doses of 20-200 mg/kg, did not act as an estrogen agonist in the immature rat uterotrophic assay.

The influence of different radiopacifying agents on the radiopacity, compressive strength, setting time, and porosity of Portland cement.

OBJECTIVES: The aims of this study were to evaluate the radiopacity, compressive strength, setting time, and porosity of white Portland cement (PC) with the addition of bismuth oxide (Bi2O3), zirconium dioxide (ZrO2), and ytterbium trifluoride (YbF3) after immersion at 37 °C for 7 days in distilled water or phosphate buffer saline. MATERIALS AND METHODS: Specimens measuring 8 mm in diameter and 1 mm in thickness were fabricated from PC with the addition of 10, 20, and 30 wt% Bi2O3, ZrO2 or YbF3. ProRoot MTA (Dentsply, Tulsa, OK, USA) and pure PC were used as controls. For radiopacity assessments, specimens were radiographed alongside a tooth slices and an aluminum stepwedge on Extraspeed occlusal dental films (Insight Kodak, Rochester, New York). Mean optical density of each specimen was calculated and used to express radiopacity of the material as an equivalent thickness of aluminum. Compressive strength was measured by using 4-mm diameter and 6-mm high specimens and Universal testing machine. High-pressure mercury intrusion porosimeter (Carlo Erba Porosimeter 2000) was employed to measure the porosity of the specimens. The setting time was measured by using a needle of 100 g in weight. The morphology of specimens was evaluated using a scanning electron microscope (TESCAN Mira3 XMU, USA Inc.). Data were analyzed by one-way ANOVA and post hoc Tukey test (P < 0.05). RESULTS: The PC with the addition of at least 10 wt% Bi2O3 and 20 wt% ZrO2 or YbF3 demonstrated greater radiopacity value than the recommended 3 mmAl cut-off. ZrO2 and YbF3 increased the compressive strength of PC, but it was not statistically significant (P > 0.05), while Bi2O3 decreased it (P < 0.05). All radiopacifiers significantly increased the porosity of the experimental cements (P < 0.05). Bi2O3 extended the setting time of PC (P < 0.05), whilst ZrO2 and YbF3 did not significantly affect it (P > 0.05). CONCLUSIONS: ZrO2 and YbF3 may be used as a suitable alternative to replace Bi2O3 in MTA without influencing its physical properties.

Soy isoflavones and cellular mechanics.

Soy isoflavones are diphenolic compounds that are frequently used for alternative treatment of ageing symptoms in both genders. They operate at principally two hierarchical levels of functional organization - cellular and molecular, while these 'types' of action appear to have indefinite borders. Soy isoflavone action at the cellular level involves inter alia the effects on cell mechanics. This epigenetic and modular determinant of cell function and fate is defined by: the anchorage to extracellular matrix (ECM) and neighboring cells, cytoskeleton organization, membrane tension and vesicle trafficking. Soy isoflavones have been reported to: (i) generally fashion an inert cell phenotype in some cancers and enhance the cell anchorage in connective tissues, via the effects on ECM proteins, focal adhesion kinases-mediated events and matrix metalloproteinases inhibition; (ii) affect cytoskeleton integrity, the effects being related to Ca(2+) ions fluxes and involving cell retraction or differentiation/proliferation-related variations in mechanical status; (iii) increase, remain "silent" or decrease membrane tension/fluidity, which depends on polarity and a number and arrangement of functional groups in applied isoflavone; (iv) provoke inhibitory effects on vesicle trafficking and exo-/endocytosis, which are usually followed by changed cell morphology. Here we present and discuss the abundance of effects arising from cells' "encounter" with soy isoflavones, focusing on different morphofunctional definers of cell mechanics.

In vitro radiographic detection of cement overhangs on cement-retained implant restorations.

PURPOSE: The aim of this in vitro study was to investigate the potential of digital and conventional radiography to detect small amounts of residual dental luting cements around implant abutments. MATERIALS AND METHODS: Artificial cement and aluminum overhangs in varying thicknesses, heights, and depths were radiographed adjacent to implant restorations with a radiovisiography sensor. Five trained evaluators were asked to identify the smallest depth of overhang that could be detected on radiographs. RESULTS: For detection of cement overhangs adjacent to implant abutments, a luting agent must have greater radiopacity than what is recommended by relevant International Organization for Standardization directives. To detect a 0.1-mm-thick portion of excess cement adjacent to an implant abutment, the cement should have a radiopacity of at least 1.7 mm of aluminum for high-resolution digital radiographs and 2.2 mm of aluminum for film-based radiographs. Two-way analysis of variance revealed that the thickness of the specimens, type of imaging detector, and type of cement all affected the radiopacity threshold for artificial cement excess (P < .05). The height of the specimens and the surrounding structures were not statistically significant factors in cement detection. CONCLUSIONS: Digital radiography offers better possibilities for visualization of cement excess than conventional radiography.

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.

Genistein stimulates the hypothalamo-pituitary-adrenal axis in adult rats: morphological and hormonal study.

Genistein, the soy isoflavone structurally similar to estradiol, is widely consumed for putative beneficial health effects. However, there is a lack of data about the genisteins' effects in adult males, especially its effects on the hipothalamo-pituitary-adrenal (HPA) axis. Therefore, the present study was carried out to investigate the effects of genistein on the HPA axis in orchidectomized adult rats, and to create a parallel with those of estradiol. Changes in the hypothalamic corticotrophin-releasing hormone (CRH) neurons and pituitary corticotrophs (ACTH cells) were evaluated stereologically, while corticosterone and ACTH levels were determined biochemically. Orchidectomy (Orx) provoked the enlargement (p<0.05) of: hypothalamic paraventricular nucleus volume (60%), percentage of CRH neurons (23%), percentage of activated CRH neurons (45%); pituitary weight (15%) and ACTH level (57%). In comparison with Orx, estradiol treatment provoked the enlargement (p<0.05) of: percentage of CRH neurons (28%), percentage of activated CRH neurons (2.7-fold), pituitary weight (131%) and volume (82%), ACTH level (69%), the serum (103%) and adrenal tissue (4.8 fold) level of corticosterone. Clearly, Orx has induced the increase in HPA axis activity, which even augments after estradiol treatment. Also, compared to Orx, genistein treatment provoked the enhancement (p<0.05) of: percentage of activated CRH neurons (2.3-fold), pituitary weight (28%) and volume (21%), total number of ACTH cells (22%) ACTH level (45%), the serum (2.6-fold) and adrenal tissue (2.8 fold) level of corticosterone. It can be concluded that an identical tendency, concerning the HPA axis parameters, follows estradiol and genistein administration to the orchidectomized adult rats.

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.

The effect of chronic food restriction on immunopositive ACTH cells in peripubertal female rats.

In peripubertal female rats, we have previously found that 50% food restriction (FR) increases plasma IL-6, haptoglobin and both alanine transaminase (ALT) and alkaline phosphatase (AST) aminotransferases, indicating the existence of an inflammatory response. To study whether such FR influences the hypothalamic-pituitary-adrenal (HPA) axis, we examined by immunohistochemistry the morphofunctional features of pituitary adrenocorticotroppic (ACTH) cells. In FR rats the volume and volume density of ACTH cells as well as plasma ACTH levels were increased by 17.6%, 12.5% and 13.4%, respectively, in comparison with controls (p<0.05). We concluded that chronic FR is a systemic stressor in young females, capable to stimulate the HPA axis, probably as a result of IL-6 action.