The isoflavones genistein and daidzein increase hepatic concentration of thyroid hormones and affect cholesterol metabolism in middle-aged male rats.

We examined whether isoflavones interfere with thyroid homeostasis, increase hepatic thyroid hormone concentrations and affect cholesterol metabolism in middle-aged (MA) male rats. Thirteen-month-old Wistar rats were injected subcutaneously with 35 mg/kg b.w./day of genistein, daidzein or vehicle (controls) for four weeks. Hepatic Dio1 gene expression was up-regulated by 70% (p < 0.001 for both) and Dio1 enzyme activity increased by 64% after genistein (p < 0.001) and 73% after daidzein treatment (p < 0.0001). Hepatic T3 was 75% higher (p < 0.05 for both), while T4 increased only after genistein treatment. Serum T4 concentrations were 31% lower in genistein- and 49% lower in dadzein-treated rats (p < 0.001 for both) compared with controls. Hepatic Cyp7a1 gene expression was up-regulated by 40% after genistein and 32% after daidzein treatment (p < 0.05 for both), in agreement with a 7α-hydroxycholesterol increase of 50% (p < 0.01) and 88% (p < 0.001), respectively. Serum 24- and 27-hydroxycholesterol were 30% lower (p < 0.05 for both), while only 24-hydroxycholesterol was decreased in the liver by 45% after genistein (p < 0.05) and 39% (p < 0.01) after dadzein treatment. Serum concentration of the cholesterol precursor desmosterol was 32% (p < 0.05) lower only after dadzein treatment alone, while both isoflavones elevated this parameter in the liver by 45% (p < 0.01). In conclusion, isoflavones increased T3 availability in the liver of MA males, despite decreasing serum T4. Hepatic increase of T3 possibly contributes to activation of the neutral pathway of cholesterol degradation into bile acids in the liver. While isoflavones obviously have the potential to trigger multiple mechanisms involved in cholesterol metabolism and oxysterol production, they failed to induce any hypocholesterolemic effect.

Testosterone and estradiol treatments differently affect pituitary-thyroid axis and liver deiodinase 1 activity in orchidectomized middle-aged rats.

We previously reported that orchidectomy (Orx) of middle-aged rats (15-16-month-old; MA) slightly affected pituitary-thyroid axis, but decreased liver deiodinase (Dio) type 1 and pituitary Dio2 enzyme activities. At present, we examined the effects of subsequent testosterone-propionate treatment (5mg/kg; Orx+T), and compared the effects of testosterone with the effects of estradiol-dipropionate (0.06mg/kg; Orx+E) treatment. Hormones were subcutaneously administered, daily, for three weeks, while Orx and sham-operated (SO) controls received only the vehicle. The applied dose of T did not alter serum TSH, T4 and T3 concentrations in Orx- MA, though it increased TSH when administrated to Orx young adults (2.5-month-old; Orx-YA). However, pituitaries of Orx-MA+T rats had higher relative intensity of immunofluorescence (RIF) for TSHß; in their thyroids we found increased volume and height of follicular epithelium, decreased volume of the colloid and higher RIF for T4-bound to thyroglobulin (Tg-T4). Liver Dio1 activity was increased. E-treatment did not affect serum hormone levels, pituitary RIF for TSHß, or liver Dio1 activity in Orx-MA rats. Thyroids had decreased relative volume and height of follicular epithelium, increased relative volume of the colloid, decreased volume of sodium-iodide symporter-immunopositive epithelium and lower RIF for Tg-T4. Detected changes were statistically significant. In conclusion, androgenization enhanced pituitary TSHß RIF, thyroid activation and liver Dio1 enzyme activity in Orx-MA, without elevating serum TSH as in Orx-YA rats. Estrogenization induced pituitary enlargement with no effect on pituitary TSHß RIF, serum TSH or liver Dio1 activity. E also induced alterations in thyroid histology that indicate mild suppression of its functioning, and contributed to thyroid blood vessel enlargement in Orx-MA rats.

Daidzein effects on ACTH cells: immunohistomorphometric and hormonal study in an animal model of the andropause.

Daidzein is a potential natural alternative to estradiol during therapy of some malignancies in men. Besides weak inhibition of tyrosine kinase activity, daidzein has a sizeable inhibitory effect on calcium channels. The aim of this study was to examine the effects of daidzein on the immunohistomorphometric features of pituitary adrenocorticotropes (ACTH cells) and circulating levels of ACTH and corticosterone, in comparison with estradiol, in an animal model of the andropause. Sixteen-month-old Wistar rats were divided into sham operated (SO), orchidectomized (Orx), estradiol treated orchidectomized (Orx+E) and daidzein treated orchidectomized (Orx+D) groups. Estradiol (0.625 mg/kg/day) and daidzein (30 mg/kg/day) were administered subcutaneously for three weeks, while the SO and Orx groups received the vehicle alone. ACTH cells were identified by the peroxidase-antiperoxidase (PAP) immunohistochemical procedure. Peripheral circulating concentrations of ACTH and corticosterone were measured by immunoassay. Orchidectomy reduced (p<0.05) the cell volume and volume density of adrenocorticotropes by 11% and 16%, respectively, in comparison to SO rats. In Orx+E rats, the volume density of ACTH cells decreased (p<0.05) by 25%, but the circulating level of ACTH increased (p<0.05) by 29%, compared to Orx rats. Daidzein treatment significantly decreased (p<0.05): volume density of ACTH cells, circulating ACTH and corticosterone by 24%, 48% and 33%, respectively, compared to the Orx group. In conclusion, this study revealed that daidzein negatively modulated the immunohistomorphometric features of ACTH cells and, unlike estradiol, decreased ACTH and corticosterone secretion, in an animal model of the andropause.

Somatostatin 14 affects the pituitary-ovarian axis in infant rats.

The effects of multiple somatostatin (SRIH-14) treatment on the pituitary-ovarian axis were examined in infant rats. Female Wistar rats received subcutaneously two daily 20 µg/100g b.w. doses for five consecutive days (from 11 to 15 days of age). Changes in cell volume, volume density and number per unit area (mm²) of follicle-stimulating (FSH), luteinizing (LH) and somatotropic (GH) immunolabeled cells were evaluated by stereology and morphometry. Serum FSH and LH concentrations were determined by RIA. Ovaries were analyzed by simple point counting of follicles. SRIH-14 treatment significantly reduced FSH and LH cell volume, while their volume density and number per unit area were unaltered. Serum concentrations of FSH and LH were significantly reduced. Volume and volume density of GH cells was significantly decreased after SRIH-14 treatment, while their number per unit area was unaltered. In the ovary, SRIH-14 induced a significant increase in the percentage of primordial follicles followed by a significant decrease in percentage of primary follicles. The number of healthy and atretic preantral follicles was unchanged. It can be concluded that SRIH-14 treatment during the infantile period markedly inhibits pituitary FSH, LH and GH cells. In the ovary, SRIH-14 acts by inhibiting initial folliculogenesis without affecting atretic processes.

Daidzein administration positively affects thyroid C cells and bone structure in orchidectomized middle-aged rats.

SUMMARY: Thyroid C cells hormone, calcitonine, inhibits bone resorption. We have demonstrated that daidzein treatment of orchidectomized rats (model for osteoporosis) stimulated C cells and increased trabecular bone mass. These results suggest that, besides direct action, daidzein may also affect bone structure indirectly through enhancement of thyroid C cell activity. INTRODUCTION: Thyroid C cells produce calcitonin (CT) which acts as an inhibitor of bone resorption. In this study, the influence of daidzein treatment on thyroid C cells, bone structure, and bone function in orchidectomized (Orx) middle-aged rats was investigated. METHODS: Sixteen-month-old Wistar rats were divided into Orx and sham-operated (SO) groups. Half the Orx rats were given subcutaneous injections of daidzein (30 mg/kg b.w./day) for 3 weeks. CT-immunopositive thyroid C cells were morphometrically analyzed. The metaphyseal region of the proximal tibia was measured histomorphometrically, and cancellous bone area (B.Ar), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were calculated. Serum samples were analyzed for CT and osteocalcin (OC), calcium (Ca) and phosphorus concentrations, and urine samples for Ca levels. RESULTS: Treatment of Orx animals with daidzein significantly increased volume of C cells compared to the Orx rats. Daidzein also enhanced B.Ar, Tb.Th, and Tb.N and reduced Tb.Sp. The serum OC and urinary Ca concentrations decreased significantly in comparison with the Orx group. CONCLUSIONS: These findings indicate that daidzein treatment stimulates thyroid C cells, increase trabecular bone mass, and decrease bone turnover in Orx middle-aged rats, which is the model of male osteoporosis.

Maternal dexamethasone treatment reduces ovarian follicle number in neonatal rat offspring.

Elevated glucocorticoid levels in the gravid female circulation affect a number of endocrine functions in the fetuses and neonates. The aim of this study was to examine the effects of maternal dexamethasone (Dx) administration during late pregnancy on the ovaries of neonatal offspring. On the 16th day of pregnancy, experimental dams received subcutaneously 1.0 mg Dx/kg b.w., followed by 0.5 mg Dx/kg b.w./day on the 17th and 18th days of gestation. The control gravid females received the same volume of saline vehicle. Left ovaries from 5-day-old female pups were stereologically analyzed. The ovary volumes were estimated using Cavalieri's principle. The number of healthy and atretic primordial and primary follicles was estimated using a fractionator-physical dissector method. The number of secondary follicles was determined by exact counts of every fourth section encompassing whole cross-sections of the ovary. The ovary volume was significantly decreased (by 44.4%; P < 0.05) in the group of female pups from Dx-treated mothers comparing to the controls. The numbers of healthy primordial and atretic follicles were 38.8% (P < 0.05) and 50.9% (P < 0.05), respectively, reduced in the ovaries of pups from the Dx-treated mothers, when compared with the control values. There were 53.4% (P < 0.05) fewer healthy primary and 41.8% (P < 0.05) fewer healthy secondary follicles as well. The numbers of atretic primary and secondary follicles were reduced by 60.0% (P < 0.05) and 61.7% (P < 0.05), respectively. It can be concluded that fetal exposure to glucocorticoids decreased the pool of non-growing follicles in the neonatal ovary, whereas the processes of folliculogenesis and atresia remained unaffected.

Multiple treatments with SRIH-14 or octreotide affect adrenal zona glomerulosa in adult male rats.

Somatostatin analogues are currently used to treat various disorders such as hypersecretion and different neuroendocrine tumors. In this study we examined the effects on the adrenal cortex of somatostatin (SRIH-14) and octreotide administered subcutaneously twice daily for 5 days to adult male rats. Control rats received saline under the same regime. After sacrifice, the adrenal glands were removed and examined morphometrically using the M(42) multipurpose test system. Blood samples were prepared for biochemical tests. Both SRIH-14 and octreotide induced morphofunctional changes in adrenal zona glomerulosa. We found significant decreases (p < 0.05) in the absolute cell and nuclear volumes of zona glomerulosa in both experimental groups in comparison to the control. The serum aldosterone level was 11% lower (p < 0.05) in the SRIH-14 and 13% (p < 0.05) lower in the octreotide-treated group in comparison with the control group. Morphometric parameters of zona fasciculata and zona reticulata and corticosterone levels were not altered significantly (p > 0.05) in either treated group. It may therefore be concluded that both SRIH-14 and octreotide affected zona glomerulosa in the same manner by decreasing morphofunctional characteristics.

Influence of maternal dexamethasone treatment on morphometric characteristics of pituitary GH cells and body weight in near-term rat fetuses.

Growth hormone (GH) and glucocorticoids have a powerful influence on controlling fetal growth, differentiation and maturation of numerous tissues. In the present study, the effect of maternal dexamethasone (Dx) treatment on GH cells and body weight in 19- and 21-day-old rat fetuses was investigated using immunocytochemical and morphometric methods. Pregnant female rats received daily injections of 1.0-0.5-0.5 mg Dx/kg b.w. on days 16-18 of pregnancy (experimental group), while the control group received an equal volume of saline. Dx treatment of pregnant rats enhanced immunostaining intensity and significantly increased (p<0.05) GH nuclear and cell volume, as well as volume density and number of GH cells per square millimeter in 19-day-old fetuses compared to the controls. In 21-day-old fetuses after maternal Dx administration, immunoreactivity, volume density and number of GH cells remained significantly increased (p<0.05). Dx treatment of pregnant rats resulted in marked body weight reduction of 21-day-old but not 19 days old fetuses in comparison with the corresponding controls. The presented results demonstrate that maternal Dx application has pronounced effect on morphometric parameters of GH cells of 19- and 21-day-old fetuses. Also, in near-term rat fetuses body weight was largely independent of pituitary GH cell activity.

Effects of ovariectomy and chronic estradiol administration on pituitary-thyroid axis in adult rats.

The effects of ovariectomy (Ovx) and of ovariectomy followed by chronic estradiol dipropionate administration (Ovx EDP) on the structure and function of the pituitary-thyroid axis were examined in the rat. Pituitary TSH cells and thyroid tissue were histologically, immunohistochemically and stereologically investigated. Serum TSH and T(4) levels were determined by RIA. Ovx did not affect pituitary weight, but subsequent treatment with EDP led to its more than two-fold increase (p<0.05). After ovariectomy, the cellular volume of pituitary TSH-immunoreactive cells increased by 28%, p<0.05 compared to sham-operated animals (SO). Treatment of Ovx rats with EDP partially reversed this change. However, the relative volume density of thyrotrophs decreased in comparison to the Ovx and SO groups (by 18% and 23%, p<0.05, respectively). No statistically significant differences in serum TSH levels were observed between the experimental groups. In thyroid tissue both peripheral and central follicles responded to Ovx and EDP treatments. Compared to SO rats, the relative volume densities of the follicles and colloid were increased (by 14% and 30%, p<0.05, respectively) in Ovx rats. Chronic EDP treatment of Ovx rats reversed these changes to the pre-ovariectomy state. Hyperplasia of thyroid follicular cells and a significant reduction (by 21%, p<0.05) of the serum level of T(4) were detected. In conclusion, estradiol deficiency and chronic treatment affected pituitary TSH cells and thyroid tissue. The sum effect of Ovx on the pituitary-thyroid axis was slightly stimulatory. Subsequent EDP treatment decreased thyroid functioning but at the same time preserved serum TSH at the control level.

Chronic exposure to constant light affects morphology and secretion of adrenal zona fasciculata cells in female rats.

The effect of chronic exposure to light of adult Wistar rats on growth and function of adrenal zona glomerulosa (ZG) and zona fasciculata (ZF) were examined. The females were exposed to continuous light of 600 lux for 95 days, starting on day 30 of age. The controls were kept under a 12:12 h light-dark cycle, at ambient temperature. The rats were sacrificed by decapitation and the left adrenal gland of each animal was dissected out and prepared for morphometric analyses. In animals exposed to chronic lighting, the absolute and relative volume of ZG were insignificantly increased by 5% (p>0.05) compared to controls. The volume of ZG cells and their nuclei were insignificantly changed by 1% (p>0.05) in comparison with corresponding controls. The absolute and relative volume of ZF were significantly increased (by 14 and 9%, respectively; p<0.05), as compared to controls. The volume of ZF cells and their nuclei were significantly increased (by 12 and 9%, respectively; p<0.05). Serum concentration of corticosterone was also significantly (p<0.05) increased by 13% in light-exposed group in comparison with control rats. These findings suggest that continuous exposure of female rats to constant light increased growth and secretory activity of ZF cells.

The effects of synthetic salmon calcitonin on thyroid C and follicular cells in adult female rats.

Structural and morphometric features of thyroid C and follicular cells were studied in adult rat females after treatment with synthetic salmon calcitonin (CT). The animals were chronically treated with either a low (10 IU/kg b.w) or a high (100 IU/kg b.w) dose of CT. A stereological method was applied to determine the volume density and the number of immunoreactive C cells. The height and volume density of follicular epithelium, colloid, interstitium and the follicles (epithelium plus colloid), as well as the index of activation rate were calculated. A significant decrease in body weight, as well as the volume density of immunoreactive C cells and the number of C cells per mm2, was observed in rats treated with both doses of CT. The height and volume density of follicular epithelium and follicles, as well as the index of activation rate were significantly increased in the animals given the high CT dose, while the volume densities of colloid and interstitium were reduced. No significant changes in the examined morphometric parameters were detected after treatment with the low CT dose. According to these results it can be concluded that the structural features of thyroid C and follicular cells were affected by the high dose CT treatment in the opposite manner, while the low dose CT treatment influenced only C cells.

Chronic somatostatin treatment affects pituitary gonadotrophs, ovaries and onset of puberty in rats.

The effects of chronic somatostatin (SRIH-14) treatment on the pituitary gonadotrophs (FSH and LH cells) and ovaries of female Wistar rats were examined. Females were given 20 microg/100 g b.w. twice per day from the immature (23rd day) till the adult period of life (71st day). The onset of puberty was determined by daily examination for vaginal opening. The peroxidase-antiperoxidase immunocytochemical procedure was used to study the gonadotrophs. Changes in the number per unit area (mm2), cell volume and volume densities of LH- and FSH-immunoreactive cells were evaluated by morphometry and stereology. Ovaries were analysed by simple point counting of follicles and corpora lutea (CL). Follicles were divided by size according to the classification of Gaytán and Osman. The mitotic indexes of granulosa and theca cells in the follicles were estimated at all stages of folliculogenesis. The number, volume and the volume density of FSH- and LH-immunoreactive cells decreased after chronic SRIH-14 treatment, particularly the latter. In the ovary, SRIH-14 treatment decreased the number of healthy follicles at all phases of folliculogenesis, lowered the mitotic indexes of granulosa and theca cells but increased the number of atretic follicles. Healthy CL were fewer in number, while regressive CL were increased. Vaginal opening occurred at a later age in treated females. It can be concluded that chronic SRIH-14 treatment markedly inhibited LH cells and to a lesser extent FSH cells. In the ovary SRIH-14 inhibited folliculogenesis, enhanced atretic processes and lowered proliferative activity of granulosa and theca cells. It also delayed puberty onset.

The thyroid C cells of ovariectomized rats treated with estradiol.

The structure and function of thyroid C cells were studied in ovariectomized (Ovx) adult female rats without and after chronic treatment with estradiol dipropionate (EDP). A peroxidase-antiperoxidase method was applied for localization of calcitonin (CT) in the C cells. Morphometric changes in their volume, nuclei, and relative volume density were evaluated in comparison with sham-operated control rats using a stereological method. The number of C cells was calculated. CT content in the sera was determined by radioimmunoassay. Ovariectomy (Ovx) led to a 21% increase in body weight ( P<0.005), while treatment of Ovx rats with EDP decreased body weight by 25% ( P<0.01). The immunoreactivity for CT in C cells of the Ovx rats was markedly increased. Significant decreases in the volume of C cells (by 13%; P<0.05) and serum CT (by 45%) were recorded, while the C cell number increased by 59% ( P<0.05) in relation to the corresponding controls. The treatment of Ovx rats with EDP caused conspicuous degranulation of the C cells. The cellular volume was increased by 11% and serum CT by 36% in comparison with Ovx animals. At the same time a decrease in C cell number by 29% ( P<0.05) was evident. It may be concluded that estradiol deficiency after Ovx reduced the synthesis and release of CT, while chronic treatment of these animals with EDP had a positive effect on the secretory activity of thyroid C cells.