Differences in Cholesterol Metabolism, Hepato-Intestinal Aging, and Hepatic Endocrine Milieu in Rats as Affected by the Sex and Age.

Age and sex influence serum cholesterol levels, but the underlying mechanisms remain unclear. To investigate further, we measured cholesterol, precursors (surrogate synthesis markers), degradation products (oxysterols and bile acid precursors) in serum, the liver, jejunum, and ileum, as well as serum plant sterols (intestinal absorption markers) in male and female Wistar rats (4 and 24 months old). The analysis of histomorphometric and oxidative stress parameters (superoxide dismutase, catalase, glutathione-related enzyme activities, lipid peroxide, and protein carbonyl concentrations) in the liver and jejunum offered further insights into the age- and sex-related differences. The hepatic gene expression analysis included AR, ERα, and sex-specific growth hormone-regulated (Cyp2c11 and Cyp2c12) and thyroid-responsive (Dio1, Tbg, and Spot 14) genes by qPCR. We observed age-related changes in both sexes, with greater prominence in females. Aged females had significantly higher serum cholesterol (p < 0.05), jejunum cholesterol (p < 0.05), and serum plant sterols (p < 0.05). They exhibited poorer hepato-intestinal health compared with males, which was characterized by mild liver dysfunction (hydropic degeneration, increased serum ALT, p < 0.05, and decreased activity of some antioxidant defense enzymes, p < 0.05), mononuclear inflammation in the jejunal lamina propria, and age-related decreases in jejunal catalase and glutathione peroxidase activity (p < 0.05). Aged females showed increased levels of 27-hydroxycholesterol (p < 0.05) and upregulated ERα gene expression (p < 0.05) in the liver. Our study suggests that the more significant age-related increase in serum cholesterol in females is associated with poorer hepato-intestinal health and increased jejunal cholesterol absorption. The local increase in 27-hydroxycholesterol during aging might reduce the hepatoprotective effects of endogenous estrogen in the female liver.

High Fat Diet Exaggerate Metabolic and Reproductive PCOS Features by Promoting Oxidative Stress: An Improved EV Model in Rats.

Background and Objectives: Polycystic ovary syndrome (PCOS) is a frequent multifactorial endocrinopathy affecting women in the reproductive period, often associated with infertility and metabolic disorders. The use of animal models helps to better understand etiopathogenesis, enabling the examination of the effects of certain drugs in order to discover the best possible therapeutic approach. We tried to investigate the additional effect of estradiol-valerate (EV) and high-fat diet (HFD) in female rats to explore PCOS-related alterations with special focus on oxidative stress. Materials and Methods: Animals were divided into three groups: control group (CTRL, n = 6), estradiol-valerate group (EV, n = 6), and estradiol-valerate group on HFD (EV + HFD, n = 6). PCOS was induced by single subcutaneous injection of long-acting EV in a dose of 4 mg/per rat. We tried to improve the metabolic characteristics of the PCOS animal model by adding HFD, so the CTRL and EV group had a regular diet, while the EV + HFD group had HFD during the induction period of 60 days. Results: We observed alterations of anthropometric parameters and hormonal disturbances, along with estrus cycle impairment reassembly to obese-type PCOS phenotype. Moreover, glucose metabolism was impaired after addition of HFD to EV protocol, contrary to EV administered alone. Histological analysis confirmed more numerous cystic follicles after the combination of EV and HFD protocol. The alterations of oxidative stress markers could be related to and serve as the mechanistic base for development of PCOS-related endocrine, reproductive, and metabolic properties. Conclusions: The additive effect of EV and HFD was obvious in the majority of the parameters observed. Our study strongly demonstrated metabolic as well as reproductive properties of PCOS in rats.

Age-Related Changes in Calcitonin-Producing Thyroid C-Cells of Male Wistar Rats.

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.

Antenatal Dexamethasone Treatment Induces Sex-dependent Upregulation of NTPDase1/CD39 and Ecto-5'-nucleotidase/CD73 in the Rat Fetal Brain.

Dexamethasone (DEX) is frequently used to treat women at risk of preterm delivery, but although indispensable for the completion of organ maturation in the fetus, antenatal DEX treatment may exert adverse sex-dimorphic neurodevelopmental effects. Literature findings implicated oxidative stress in adverse effects of DEX treatment. Purinergic signaling is involved in neurodevelopment and controlled by ectonucleotidases, among which in the brain the most abundant are ectonucleoside triphosphate diphosphohydrolase 1 (NTPDase1/CD39) and ecto-5'-nucleotidase (e5'NT/CD73), which jointly dephosphorylate ATP to adenosine. They are also involved in cell adhesion and migration, processes integral to brain development. Upregulation of CD39 and CD73 after DEX treatment was reported in adult rat hippocampus. We investigated the effects of maternal DEX treatment on CD39 and CD73 expression and enzymatic activity in the rat fetal brain of both sexes, in the context of oxidative status of the brain tissue. Fetuses were obtained at embryonic day (ED) 21, from Wistar rat dams treated with 0.5 mg DEX/kg/day, at ED 16, 17, and 18, and brains were processed and used for further analysis. Sex-specific increase in CD39 and CD73 expression and in the corresponding enzyme activities was induced in the brain of antenatally DEX-treated fetuses, more prominently in males. The oxidative stress induction after antenatal DEX treatment was confirmed in both sexes, although showing a slight bias in males. Due to the involvement of purinergic system in crucial neurodevelopmental processes, future investigations are needed to determine the role of these observed changes in the adverse effects of antenatal DEX treatment.

Normotensive rats with PCOS exhibit the hypertensive pattern: focus on oxidative stress.

Numerous evidence implies complex interrelations between polycystic ovary syndrome (PCOS) and hypertension (HT) in reproductive-age women. In this study, we aimed to investigate the potential strain differences in ovarian morphology, hemodynamic, and biochemical characteristics in an androgen-induced PCOS rat model. A total of 24 rats of 3 weeks old (12 Wistar Kyoto - WK and 12 spontaneously hypertensive rats - SHR) were divided into four groups: WK, WK PCOS, SHR, and SHR PCOS. PCOS was induced by daily s.c. injections of testosterone enanthate (1 mg/100 g body weight) administered for 5 weeks. PCOS induction led to estrus cyclicity cessation, cystic ovarian appearance, and sex hormones disturbances in both strains. The morphometric parameters in ovaries were altered in a manner of PCOS-related changes in both strains (higher number in preantral, atretic, and cystic follicles). Ultrasonographically, a significant decrease in ovarian volume (OV) was registered in PCOS groups but also in SHR compared to WK rats. All blood pressure parameters were higher in SHR compared to WK. PCOS modeling increased systolic, mean arterial, and pulse pressure in WK strain, while in SHR, only mean arterial and pulse pressure were higher. Alterations in oxidative stress parameters could provide a molecular basis for PCOS-related changes: in PCOS groups, thiobarbituric acid reactive substance and superoxide anion radical levels were higher in both strains, while superoxide dismutase and glutathione were significantly lowered.

The effects of prenatal dexamethasone exposure and fructose challenge on pituitary-adrenocortical activity and anxiety-like behavior in female offspring.

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.

The phytoestrogen genistein prevents trabecular bone loss and affects thyroid follicular cells in a male rat model of osteoporosis.

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 (T3 ), thyroxine (T4 ) and CT were determined as well as urinary calcium (Ca2+ ) concentrations. Genistein treatment significantly increased cancellous bone area (B.Ar), trabecular thickness (TbTh) and trabecular number (TbN) (P < 0.05), but trabecular separation (Tb.Sp) was decreased (P < 0.05) compared with control Orx rats. In the thyroid, genistein treatment significantly elevated the relative volume density (Vv) of the follicular cells (P < 0.05) compared with Orx, whereas Vv of the colloid was lower (P < 0.05) than in the Orx. Evaluation of the biochemical parameters showed significant reductions in serum OC, T3 , T4 and urinary Ca2+ concentrations (P < 0.05), compared with Orx rats. These data indicate that genistein treatment improves the trabecular microarchitecture of proximal tibia, induces histomorphometrical changes in thyroid glands, and decreases circulating thyroid hormone levels in orchidectomized rat model of male osteoporosis.

Histological and morphofunctional parameters of the hypothalamic-pituitary-adrenal system are sensitive to daidzein treatment in the adult rat.

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.

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

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 (35mg/kg) were subcutaneously administrated to acyclic (12-month-old) Wistar females, daily, for 4weeks. 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 ERα were down-regulated, while PR and ERß 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 ERß, while the expression levels of ERα 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.

Effects of Estradiol on Histological Parameters and Secretory Ability of Pituitary Mammotrophs in Ovariectomized Female Rats.

OBJECTIVES: Estrogen replacement therapy remains current as a therapeutic approach to treat menopausal symptoms and may significantly affect hormone-producing cells in the female pituitaries. The aim of this study was to examine the histological parameters of pituitary mammotrophs and prolactin secretion after chronic estradiol treatment in ovariectomized adult female rats, reflecting premature menopause. MATERIALS AND METHODS: In this experimental study, adult female Wistar rats were divided into non-ovariectomized (C), ovariectomized (OVX) and estradiol-treated ovariectomized (OVX+E) groups. Estradiol dipropionate [0.625 mg/kg body mass per day] was administered for four weeks, while the C and OVX groups received vehicle alone. Mammotrophs were identified by the peroxidase-antiperoxidase (PAP) immunohistochemical procedure, while prolactin concentrations were measured by the non-isotopic two-step assay (Delfia) method. Comparison of the differences between groups was performed using one-way analysis of variance (ANOVA) and Tukay (honest significant difference) HSD test. RESULTS: Ovariectomy caused significant (P<0.05) decreases in mammotroph optical density (OD), volume density (VV) and number per mm2 by 29, 27 and 34%, respectively, in comparison with the C females. In the OVX+E group, significant (P<0.05) increases in OD, cell volume, VV and number of mammotrophs per mm2 by 181, 15%, 5.8-fold and 5.2-fold, respectively, were observed when compared to OVX animals. The serum prolactin concentration in OVX females was significantly (P<0.05) decreased by 14% in comparison to the C group, while in OVX+E females, prolactin levels were significantly (P<0.05) increased by 53% compared to the OVX controls. CONCLUSIONS: Estradiol supplementation in ovariectomized females is followed by stimulatory histological and secretory changes of the mammotrophs. These results could serve as indicators of possible prolactinome development upon estradiol application in premature menopausal subjects.

Response of trabecular bone, thyroid C and follicular cells to synthetic salmon calcitonin in middle-aged orchidectomized male rats.

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 (Ca2+ ) 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 Ca2+ 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.

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.

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 TSHß 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.

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, Cavalieri's 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.

Effects of prolonged alcohol exposure on somatotrophs and corticotrophs in adult rats: Stereological and hormonal study.

Exposure to alcohol alters many physiological processes, including endocrine status. The present study examined whether prolonged alcohol (A) exposure could modulate selected stereological and hormonal aspects of pituitary somatotrophs (growth hormone-GH cells) and corticotrophs (adrenocorticotropic hormone-ACTH cells) in adult rats. Changes in pituitary gland volume; the volume density, total number and volume of GH and ACTH cells following alcohol exposure were evaluated using a stereological system (newCAST), while peripheral GH and ACTH levels were determined biochemically. Our results demonstrated the reduction (p<0.05) of the volume density (37%) and volume of GH cells (29%) in the group A. Also, there was a tendency for the total number of GH cells to be smaller in the group A. Serum GH level was significantly decreased (p<0.05; 70%) in the group A when compared to control values. Moreover, prolonged alcohol exposure induced declines (p<0.05) in volume density (24%) and volume of ACTH cells (29%). The total number of ACTH cells and ACTH level were higher (p<0.05; 42%) in the group A than in control rats. Collectively, these results indicate that prolonged alcohol exposure leads not only to changes in GH and ACTH hormone levels, but also to alterations of the morphological aspects of GH and ACTH cells within the pituitary.

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.

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.

Soy extract-dependent changes in morphofunctional parameters of the pituitary corticotropes in adult rats.

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