Exogenous Nerve Growth Factor Increases the Activity of High-Affinity Choline Uptake and Choline Acetyltransferase in Brain of Fisher 344 Male Rats

The objective of this study was to determine the effect of age and chronic intracerebral administration of nerve growth factor (NGF) on the activity of the presynaptic cholinergic neuronal markers hemicholinium-sensitive high-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) in the brain of Fisher 344 male rats. In 24-month-old rats, a substantial decrease in ChAT activity (30%) was measured in striatum, and decreases in HACU were found in frontal cortex (28%) and hippocampus (23%) compared with 4-month-old controls. Cholinergic neurons in brain of both young adult and aged rats responded to administration of exogenous NGF by increased expression of both phenotypes. In 4-month-old animals, NGF treatment at 1.2 micron/day resulted in increased activities of both ChAT and HACU in striatum (175 and 170%, respectively), frontal cortex (133 and 125%), and hippocampus (137 and 125%) compared with untreated and vehicle-treated 4-month-old animals; vehicle treatment had no effect on the activity of either marker. In 24-month-old animals treated with NGF for 2 weeks, ChAT activity was increased in striatum (179%), frontal cortex (134%), and hippocampus (119%) compared with 24-month-old control animals. Synaptosomal HACU in 24-month-old rats was increased in striatum (151%) and frontal cortex (128%) after 2 weeks of NGF treatment, but hippocampal HACU was not significantly different from control values.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of estrogens on cholinergic neurons in the rat basal nucleus

Estrogen replacement in postmenopausal women may help prevent or delay development of Alzheimer's disease. Because loss of basal forebrain cholinergic neurons with reductions in choline acetyltransferase (ChAT) concentration are associated with Alzheimer's disease, we investigated the effect of estradiol (E(2)) and J 861, a non-feminizing estrogen, on cholinergic neurons in the basal forebrain. Ovariectomized rats received E(2), J 861 or vehicle, and basal forebrain sections through the substantia innominata, medial septum, and nucleus of the diagonal band were immunostained for ChAT. ChAT-immunoreactive cells in the basal forebrain were significantly reduced in the ovariectomized rats compared to intact rats, but those ovariectomized rats receiving estrogen replacement with E(2) and J 861 had near normal levels of ChAT-positive neurons. While retrograde tracing experiments with fluorogold injected into the prefrontal cortex showed no significant differences in the number of fluorogold-labeled cells among the groups, ChAT-immunoreactive cells and double-labeled cells were significantly lower in OVX rats than in intact and E(2) rats. Some substantia innominata cells in the J 861 rats were ChAT/estrogen receptor alpha-positive. These results suggest that E(2) and J 861 have positive effects on cholinergic neurons that project from the basal nucleus to the forebrain cortex.     

Regulation of duodenal insulin-like growth factor I and active calcium transport by ovariectomy in female rats.

There is a significant body of data that supports the concept that reproductive hormones in females have effects on duodenal calcium transport that are not mediated via altered circulating concentrations of 1,25-dihydroxyvitamin D (1,25(OH)2D). Previously, we have shown parallel alterations in duodenal Ca transport and longitudinal bone growth rate in sexually maturing female rats in response to ovariectomy and estradiol (E) treatment of ovariectomized (OVX) rats (OVX+E) without any change in circulating levels of 1,25(OH)2D or parathyroid hormone. Results are presented here from experiments designed to: (i) further explore the relationship between 1,25(OH)2D and ovarian status in the regulation of duodenal calcium transport, and (ii) determine whether OVX and E replacement alter circulating and duodenal levels of insulin-like growth factor I (IGF-I) that might be related to effects on Ca transport. Growth hormone, which has been shown to affect intestinal Ca absorption and vitamin D metabolism, is thought to act indirectly by stimulating IGF-I. Six-week-old female rats were OVX, given estradiol implants (OVX+E), and fed a diet containing either 0.5% or 0.1% Ca for 3 weeks. In both diet groups, the OVX animals exhibited a higher level of Ca transport, as measured by the everted gut sac method, than either the intact controls or the OVX+E group; there was no difference in calcium transport between the different diet groups. Although there was no difference in circulating levels of 1,25(OH)2D among the intact, OVX, and OVX+E groups fed either diet, animals fed the 0.1% Ca diet had higher circulating levels of 1,25(OH)2D than those fed the 0.5% Ca diet. There was no difference in duodenal levels of calbindin9K among intact, OVX, and OVX+E animals in either diet group, although the animals fed the 0.1% Ca diet had higher levels of calbindin9K than the animals fed the 0.5% Ca diet. In animals fed the 0.5% Ca diet, OVX resulted in elevated serum and duodenal levels of IGF-1, as compared with intact and OVX+E animals on the same diet. In animals fed the 0.1% Ca diet, there was no elevation of IGF-I in the OVX group relative to intact and OVX+E animals. These results lend additional support to the concept that alterations in duodenal active calcium transport that occur with alterations in ovarian hormones are not mediated by changes in serum levels of 1,25(OH)2D, but may be related to some factor related to growth, possibly IGF-I.     

针刺对去卵巢大鼠脑内胆碱乙酰转移酶基因表达的影响

本工作旨在探讨雌激素对脑内乙酰胆碱生成的影响和电针刺激“足三里”穴对去卵巢大鼠脑内乙酰胆碱生成的调整作用。实验选用成年Wistar雌性大鼠,将动物分为正常对照组(INT)、去卵巢组(OVX)和去卵巢针刺组(OVX AC)。用放射免疫分析方法测定血中雌二醇含量,采用RT-PCR方法获得大鼠脑内胆碱乙酰转移酶(ChAT)mRNA的逆转录表达产物——cDNA,用琼脂糖凝胶电泳方法检测,并通过原位杂交方法观察海马ChAT mRNA阳性神经元的表达,然后用计算机图像分析系统进行统计分析。实验结果显示：去卵巢组大鼠体内雌激素水平明显降低,脑内ChAT mRNA的RT-PCR产物和海马ChAT mRNA阳性表达产物的平均面积、平均积分光度值均明显减少,与对照组和针刺组比较有显著性差异;去卵巢针刺“足三里”穴组与去卵巢组相比,大鼠血中雌激素水平明显升高,脑内ChAT mRNA RT-PCR产物明显增多,海马的ChAT mRNA表达阳性神经元增多。以上结果提示：脑内ChAT基因表达与体内雌激素水平有密切关系,去卵巢后针刺“足三里”穴对ChAT的调节作用可能是针刺增强脑内乙酰胆碱含量的机制之一。     

Effect of quantity and quality of dietary protein on choline acetyltransferase and nerve growth factor, and their mRNAs in the cerebral cortex and hippocampus of rats

The brain protein synthesis is sensitive to the dietary protein; however, the role of dietary protein on biomarkers including choline acetyltransferase and nerve growth factor (NGF) for the function of cholinergic neurons remains unknown in young rats. The purpose of this study was to determine whether the quantity and quality of dietary protein affects the concentration of NGF and activity of choline acetyltransferase, and their mRNA levels in the brains of young rats. Experiments were carried out on five groups of young rats (4 weeks) given the diets containing 0, 5, 20% casein, 20% gluten or 20% gelatin for 10 days. The activity of choline acetyltransferase in the cerebral cortex and hippocampus declined gradually with a decrease in quantity and quality of dietary protein. The concentration of NGF in the cerebral cortex and the mRNA levels of choline acetyltransferase in the cerebral cortex and hippocampus did not differ among groups. However, the concentration and mRNA level of NGF in the hippocampus was significantly lower in rats fed with lower quantity of protein or lower quality of protein. In the hippocampus, the mRNA levels of NGF significantly correlated with the NGF concentration when the quantity (r = 0.704, P < 0.01) and quality (r = 0.682, P < 0.01) of dietary protein was manipulated. It was further found that a significant positive correlation existed between the NGF concentration and the activity of choline acetyltransferase in the hippocampus (dietary protein quantity, r = 0.632, P < 0.05; dietary protein quality, r = 0.623, P < 0.05). These results suggest that the ingestion of lower quantity and quality of dietary protein are likely to control the mRNA level and concentration of NGF, and cause a decline in the activity of choline acetyltransferase in the brains of young rats.     

Effect of daidzein, a soy isoflavone, on bone metabolism in Cd-treated ovariectomized rats

This study compared the ability of daidzein, a soy isoflavone, with that of 17beta-estradiol to prevent bone loss in cadmium (Cd)-exposed ovariectomized (OVX) rats during growth. Four week-old female Wistar rats were randomly assigned to five treatment groups of 9 rats each, either (1) sham-operated (SH); (2) OVX and placed on experimental diets (OVX); (3) OVX fed 50 ppm of CdCl2 (OVX-Cd); (4) OVX fed 50 ppm of CdCl2 and 10 microg of daidzein per kg of body mass (OVX-CD-D); or (5) OVX fed 50 ppm of CdCl2 and 10 microg of estrogen per kg of body mass (OVX-CD-E). All rats were given free access to AIN-76 modified diet and drinking water, with or without Cd, for 8 weeks. The OVX groups gained more (P < 0.05) body mass than the SH group. Femoral mass was increased by feeding daidzein and estradiol, whereas femoral length was not (P > 0.05) significantly different among groups. Femoral breaking force was not significantly different among groups, however, femoral BMD was significantly lower in OVX-Cd than in the SH and OVX groups. Morphologically proliferative cartilage and hypertrophic cells in femur showed normal distribution in OVX-Cd-D and OVX-Cd-E groups unlike those in OVX-Cd group. These findings suggest that Cd-OVX-induced osteopenia or osteoporosis probably results from an increase in bone turnover.     

Correlative Regulation of Nerve Growth Factor Level and Choline Acetyltransferase Activity by Thyroxine in Particular Regions of Infant Rat Brain

Abstract: Effects of thyroxine (T₄) on nerve growth factor (NGF) level and choline acetyltransferase (ChAT) activity of rat brains were investigated. Repetitive intraperitoneal administration of T₄ caused increases in both NGF level and ChAT activity in the frontal cortex, septum, hippocampus, and striatum and decreases in the cerebellum in 2-day-old rats. Only ChAT activity was elevated in the olfactory bulb, and the NGF level remained unchanged there. No changes were observed in the midbrain and pons/medulla. Furthermore, T₄ was effective on the post-natal rats only up to day 11. These results suggest that T₄ plays a role in the developmental regulation of NGF level and ChAT activity in rat brain in a region- and/or stage-specific manner. That (1) changes in NGF level and ChAT activity occurred in regions nearly identical to those that contained NGF-responding neurons, and (2) the change in NGF level in the hippocampus and frontal cortex was followed by the change of ChAT activity after a single injection of T₄ suggest that the effects of T₄ on cholinergic differentiation are, at least in part, mediated via NGF, which itself is quantitatively regulated by T₄.     

Dietary soy isoflavone-aglycone lowers food intake in female rats with and without ovariectomy

Objective: Estrogens downregulate eating behavior, and soy isoflavones are known to be estrogenic agents. We aimed to examine whether the estrogenic property of soy isoflavones can affect food intake and body weight. Methods and Procedures: Seven‐week‐old male, female, and ovariectomized (OVX) Sprague‐Dawley rats were given free access to a diet containing 100–300 mg total isoflavone/kg diet, or to a control diet, either with or without concurrent administration of estradiol by subcutaneous implantation. Results: Dietary soy isoflavone was shown to lower food intake in female rats, whether or not the animals had undergone ovariectomy. Administration of estradiol lowered the food intake in male rats and in OVX female rats. The decrease in weekly food intake in female rats led to a reduction in their weekly gain in body weight. Dietary soy isoflavone significantly increased the concentration of serum isoflavones, especially equol (a metabolite of daidzein), regardless of gender or ovariectomy. Dietary soy isoflavone did not affect either serum estradiol concentration or uterine and didymus weights, but estradiol administration improved the uterine atrophy in OVX rats, and decreased the didymus weight in male rats. Discussion: Soy isoflavone lowers the food intake in female rats, but not in the male animals. Contrary to the hypothesis currently in vogue, the reduction in food intake caused by soy isoflavone may not be a purely estrogenic effect. This follows from the finding that the effects of soy isoflavones on food intake and on the reproductive organs differ from the corresponding effects produced by estrogen.     

Basal forebrain cholinergic neurons are necessary for estrogen to enhance acquisition of a delayed matching-to-position T-maze task

Intraseptal injections of the selective cholinergic immunotoxin 192 IgG-saporin (SAP) were performed to determine whether basal forebrain cholinergic neurons are necessary for hormone-mediated enhancement of acquisition in a delayed matching-to-position (DMP) T-maze task. The DMP task is a simple spatial learning task. Studies have shown that continuous estradiol replacement enhances acquisition of the DMP task in young ovariectomized rats and that long-term treatment with either estradiol or estradiol + progesterone can prevent a deficit in DMP acquisition in old rats. In the present study, continuous estradiol replacement significantly enhanced acquisition of the DMP task by non-SAP-treated, ovariectomized rats. In contrast, neither continuous estradiol nor weekly administration of estradiol + progesterone significantly enhanced acquisition of the DMP task in rats that received intraseptal injections of either a high dose (1.0 microg) or a low dose (0.22 microg) of SAP. Animals that reached criterion were significantly impaired by rotating the maze 180 degrees regardless of treatment, suggesting that animals in all groups used extramaze cues to at least some degree to solve the task. SAP-treated animals were slightly more sensitive to increasing the intertrial delay than non-SAP-treated controls, suggesting that the SAP lesions produced a modest deficit in spatial working memory. Immunohistochemistry confirmed the loss of cholinergic neurons in specific regions of the basal forebrain of SAP-treated animals. In addition, DMP acquisition correlated significantly with ChAT activity in the hippocampus and frontal cortex. The data suggest that basal forebrain cholinergic projections are necessary for hormone-mediated enhancement of DMP acquisition.     

染料木素对卵巢切除大鼠学习记忆能力的影响

目的：探讨染料木素对卵巢切除大鼠学习记忆能力的影响及作用机制。方法：将40只SD雌性大鼠随机分为用假手术组、去卵巢对照组、染料木素高剂量、低剂量组、17β-雌二醇组,切除卵巢建立学习和记忆能力受损的模型。灌胃给药6周后Morris水迷宫测定各组大鼠学习记忆能力,免疫组化法观察大鼠海马微管相关蛋白（tau蛋白）阳性表达情况,测定大鼠脑组织中乙酰胆碱酯酶（AchE）、乙酰胆碱转移酶（ChaT）、超氧化物歧化酶（SOD）的活性及丙二醛（MDA）的含量,观察海马组织超微结构变化。结果：大鼠切除卵巢后Morris水迷宫测定的学习记忆能力显著下降,微管相关蛋白（tau蛋白）异常磷酸化阳性表达率增高,前脑皮质中超氧化物歧化酶（SOD）、乙酰胆碱转移酶（ChaT）活性降低,丙二醛（MDA）含量、乙酰胆碱酯酶（AchE）活性增高。低剂量的染料木素可以发挥类雌激素样作用,改善去卵巢大鼠的以上症状。结论：染料木素对卵巢切除导致的学习和记忆能力下降有改善作用,低剂量效果显著,其可能的机制是：抑制了脑内AchE的活性,使乙酰胆碱的降解减少;增强脑组织抗氧化能力;稳定微管相关蛋白（tau蛋白）,降低tau蛋白异常磷酸化水平。     

Short term effects on bone quality associated with consumption of soy protein isolate and other dietary protein sources in rapidly growing female rats

Beneficial effects of soy protein consumption on bone quality have been reported. The effects of other dietary protein sources such as whey protein hydrolysate (WPH) and rice protein isolate (RPI) on bone growth have been less well examined. The current study compared effects of feeding soy protein isolate (SPI), WPH and RPI for 14 d on tibial bone mineral density (BMD) and bone mineral content (BMC) in intact and ovariectomized (OVX) rapidly growing female rats relative to animals fed casein (CAS). The effects of estrogenic status on responses to SPI were also explored. Tibial peripheral quantitative computerized tomography (pQCT) showed all three protein sources had positive effects on either BMD or BMC relative to CAS (P < 0.05), but SPI had greater effects in both intact and OVX female rats. SPI and E2 had positive effects on BMD and BMC in OVX rats (P < 0.05). However, trabecular BMD was lower in a SPI + E2 group compared to a CAS + E2 group. In OVX rats, SPI increased serum bone formation markers, and serum from SPI-fed rats stimulated osteoblastogenesis in ex vivo. SPI also suppressed the bone resorption marker RatLaps (P < 0.05). Both SPI and E2 increased alkaline phosphatase gene expression in bone, but only SPI decreased receptor activator of nuclear factor-kappaB ligand (RANKL) and estrogen receptor gene expression (P < 0.05). These data suggest beneficial bone effects of a soy diet in rapidly growing animals and the potential for early soy consumption to increase peak bone mass.     

Effects of estradiol and estradiol sulfamate on the liver of ovariectomized or ovariectomized and hypophysectomized rats

This study was performed to evaluate and compare the effects of estradiol sulfamate (J995) and estradiol (E2) on the hepatic levels of the estrogen receptor (ER) and its mRNA, in ovariectomized (OVX) and OVX+hypophysectomized (OVXHX) female rats and to study the effects on the liver-derived serum compounds angiotensin I, triglycerides, high-density lipoprotein (HDL) and cholesterol. ER concentrations were determined using ligand-binding assay (LBA) and enzyme immuno assay (EIA), and the mRNA levels using solution hybridization.

The rats were treated orally (p.o.) or subcutaneously (s.c.) for 7 days, with treatments initiated 14 days after surgery.

No differences were found in ER mRNA levels between J995 and E2 treated rats.

The s.c. administered estrogens increased ER levels in OVX rats. Addition of GH+DEX to OVXHX rats restored the ER to levels above those seen in intact rats, whereas simultaneous oral treatment with E2 significantly decreased ER levels again. The s.c. treatment with either J995 or E2 limited the increase caused by addition of GH+DEX.

After oral treatment angiotensin I levels were increased by E2, but not by J995, while triglycerides, HDL and cholesterol levels were decreased by oral E2, J995 showing a similar pattern but was less effective.

In summary, these results on hepatic ER levels and estrogen dependent compounds produced by the liver showed that J995 has a lower impact on the normal liver functions after oral treatment than E2. Thus, J995 is a very promising substance for development of oral estrogen treatment with reduced hepatic side effects.     

Effects of 17β-estradiol and antioxidant administration on oxidative stress and insulin resistance in ovariectomized rats

The prevalence of insulin resistance syndrome increases during menopause with the overproduction of reactive oxygen species and impairment of the free radical scavenger function. Therefore, we investigated the effects of 17β-estradiol (E(2)) and vitamin E, as an antioxidant, on lipid peroxidation and antioxidant levels in the brain cortex and liver of ovariectomized rats as well as on insulin resistance in those rats. Forty female Sprague-Dawley rats, 3?months of age and weighing 231.5?± 9.4 g, were divided into 4 groups: sham, ovariectomized (OVX), OVX treated with E(2) (40 μg/kg subcutaneously), and OVX treated with E(2) and vitamin E (100?mg/kg intraperitoneally). The 4 groups received the appropriate treatment every day for 8?weeks. Levels of glutathione, glutathione peroxidase, superoxide dismutase , catalase, and malondialdehyde in the brain cortex and liver of ovariectomized rats were measured. Also, fasting plasma insulin, glucose, and homeostatis model assessment of insulin resistance (HOMA-IR) were determined. Malondialdehyde increased and antioxidants (glutathione, glutathione peroxidase, catalase, superoxide dismutase) decreased in the brain cortex and liver of OVX rats. Also, fasting glucose, insulin, and HOMA-IR increased in OVX rats. E(2) and E(2) plus vitamin E decreased malondialdehyde and increased antioxidants in the brain cortex and liver of OVX rats. Moreover, they decreased fasting glucose, insulin, and HOMA-IR in ovariectomized rats. This study demonstrates that E(2) and E(2) plus vitamin E supplementation to OVX rats may improve insulin resistance, strengthen the antioxidant system, and reduce lipid peroxidation.     

Effect of serum estradiol and leptin levels on thyroid function, food intake and body weight gain in female Wistar rats

We evaluated the interplay among estrogen, leptin and thyroid function in the regulation of body mass in female rats. Adult female rats were divided into four groups: control (C, sham-operated), ovariectomized (OVX), ovariectomized treated with estradiol benzoate (Eb) 0.7 or 14 μg/100 g bw per day, during 21 days. OVX led to an increase in body mass, food intake and food efficiency (change in body mass as function of the amount of food ingested) which were normalized by the lower Eb dose, and decreased significantly when the higher dose was given. Serum leptin levels were increased more than two-fold in all ovariectomized groups. Serum T4 levels of the Eb treated OVX were significantly lower than in the controls. Serum T3 and TSH were unaffected by OVX or by Eb treatment. Uterine type 2 iodothyronine deiodinase (D2) activity changed in parallel with serum estradiol: decreased after OVX, returned to control levels after the lower E2 treatment, and increased significantly after the high Eb dosage. The hypothalamic D2 activity was reduced around 30% in all castrated groups, treated or not with estrogen, whereas in the brown adipose tissue the enzyme was not changed. Interestingly, although estrogen-treated OVX rats had lower body weight, serum leptin was high, suggesting that estrogen increases leptin secretion. Our results show that estradiol is necessary for the hypothalamic action of leptin, since the increase in leptin levels observed in all ovariectomized rats was associated with a decrease in food intake and food efficiency only in the rats treated with estrogen.     

Influences of age and ovarian follicular reserve on estrous cycle patterns, ovulation, and hormone secretion in the Long-Evans rat

This study examined the influences of aging and reduced ovarian follicular reserve on estrous cyclicity, estradiol (E(2)) production, and gonadotropin secretion. Young virgin and middle-aged (MA) retired breeder female rats were unilaterally ovariectomized (ULO) or sham operated (control). Unilateral ovariectomy of young rats reduced the ovarian follicular reserve by one-half, to a level similar to that found in MA controls. Unilateral ovariectomy of MA females reduced the follicular pool further, to one half of MA controls. The incidence of regular cyclicity was significantly lower in MA ULO females than in young controls, with intermediate cycle frequency in young ULO and MA controls. Among cyclic rats, the magnitude of the proestrous LH surge was highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. Similarly, ovulation rates were highest in young controls, intermediate in young ULO rats and MA controls, and lowest in MA ULO females. While young ULO rats exhibited augmented secondary FSH surges on estrous morning, middle-aged ULO females displayed secondary FSH levels comparable to young controls. The effects of age and reduced follicle number on estrous cyclicity and gonadotropin secretion were not due to altered E(2) secretion, as preovulatory E(2) levels were similar among all groups. Thus, experimental reduction in the follicular reserve exerts acute effects on the preovulatory LH surge, ovulation rate, and estrous cyclicity in both young and MA rats. However, decreased follicle number increases FSH levels only in young rats, indicating aging-related alterations in the feedback regulation of FSH.     

Effects of estradiol and progesterone on body composition, protein synthesis, and lipoprotein lipase in rats

Prior studies suggest that estradiol and progesterone regulate body composition in growing female rats. Because these studies did not consider the confounding effect of changes in food intake, it remains unclear whether ovarian hormones regulate body composition independently of their effects on food intake. We utilized a pair-feeding paradigm to examine the effects of these hormones on body composition. In addition, skeletal muscle protein fractional synthesis rate and adipose tissue lipoprotein lipase activity were measured to examine pathways of substrate deposition into fat and fat-free tissue. Female Sprague-Dawley rats [pubertal: 7-8 wk old; 190 +/- 0.5 (SE) g] were separated into four groups: 1) sham-operated (S; n = 8), 2) ovariectomized plus placebo (OVX; n = 8), 3) ovariectomized plus estradiol (OVX+E; n = 8), and 4) ovariectomized plus progesterone (OVX+P; n = 8). All ovariectomized groups were pair-fed to the S group. Body composition was measured using total body electrical conductivity. The relative increase in fat-free mass was greater (P < 0.01) in the OVX group (31 +/- 2%) than in the S (17 +/- 2%), OVX+E (18 +/- 2%), and OVX+P (22 +/- 2%) groups. The fractional synthetic rates of gastrocnemius muscle protein paralleled changes in fat-free mass: OVX had a higher (P < 0.05) synthesis rate (21 +/- 3%/day) than S (12 +/- 2%/day), OVX+E (11 +/- 2%/day), and OVX+P (8 +/- 1%/day) groups. Body fat increased in the S group (31 +/- 7%; P < 0.01), whereas the OVX groups lost fat (OVX: -10 +/- 7%; OVX+E: -15 +/- 7%; OVX+P: -13 +/- 7%). No differences in lipoprotein lipase were found. Our results suggest that estradiol and progesterone may regulate the growth of fat and fat-free tissues in female rats. Moreover, ovarian hormones may influence skeletal muscle growth through their effects on skeletal muscle protein synthesis.     

Effect of dietary calcium: Phosphorus ratio on bone mineralization and intestinal calcium absorption in ovariectomized rats

We investigated the effect of dietary calcium:phosphorus (Ca:P) ratio on bone mineralization and intestinal Ca absorption in ovariectomized (OVX) rat models of osteoporosis and sham-operated rats. Thirty 12-wk-old female Wistar rats were divided into three groups of OVX rats and three groups of sham rats. Thirty days after the adaptation period, OVX rats and sham rats were fed a diet formulated Ca:P, 1:0.5, 1:1 or 1:2 (each diet containing 0.5% Ca), respectively for 42 d. In both sham and OVX rats, serum osteocalcin, a marker of bone turnover, was increased by decreasing Ca:P ratio (1:2). In contrast, rats fed the Ca:P = 1:0.5 diet (dietary P restriction) suppressed the increased serum parathyroid hormone, osteocalcin and urinary deoxypyridinoline, and increased Ca absorption in both sham and OVX rats compared to the Ca:P = 1:1 and 1:2 diets. Especially, in OVX rats, the decreased bone mineral density of the fifth lumbar was also suppressed when rats were fed the Ca:P = 1:0.5 diet. These results indicated that the elevation of dietary Ca:P ratio may inhibit bone loss and increase intestinal Ca absorption in OVX rats.     

Combinatorial effects of the phytoestrogen genistein and of estradiol in uterus and liver of female Wistar rats

The knowledge about safety of phytoestrogens on proliferative endpoints in the endometrium is rather limited, particularly when low amounts of estrogens are present like in postmenopausal women. Therefore, we now studied how genistein (GEN) exposure affects proliferative endpoints in the endometrium in estrogenized animals. We investigated the effects of GEN (10 mg/(kg day) BW) on uterine proliferation and on general uterine response markers in intact female rats and ovariectomized (OVX) female rats co-treated with different doses of estradiol (E2; 1 or 4 μg/(kg day) BW). In parallel we investigated generalized hepatic effects of GEN in this co-stimulatory protocol. In agreement to our previous results, GEN treatment of OVX animals for 3 days results in a faint stimulation of the uterine wet weight. In intact animals and in OVX animals co-treated with E2 no effects of GEN on uterine wet weight were detectable. GEN treatment did not affect the uterine epithelial height in intact animals but resulted in a decrease of the protein and mRNA expression of the proliferation marker PCNA. In OVX animals co-treated with E2, GEN antagonized the E2 stimulated increase of the uterine epithelial height and epithelial PCNA expression. Besides PCNA, GEN effects on the uterine mRNA expression of IGF-1, IGF-1R, Complement C3, estrogen receptor- (ER) and -β (ERβ), as well as progesterone receptor were investigated in intact and OVX co-treated animals. Overall there was a tendency in all combinatorial groups that GEN counteracts E2 function in uterine tissue. Surprisingly, while investigating estrogenic response markers in liver, we observed very strong effects of GEN on hepatic marker gene expression. GEN significantly down-regulated CaBP9K and IGFBP1 mRNA levels in intact animals. In OVX animals hepatic CABP9K and IGFBP1 mRNA levels were not affected by E2 treatment. GEN treatment, even in combination with E2, decreased the hepatic CaBP9K expression below the levels observed in untreated animals. Interestingly co-treatment of OVX rats with low dose E2 and GEN resulted in a significant increase of IGFBP1 mRNA expression. Summarising our results we conclude that (1) GEN treatment in the presence of E2 is safe regarding proliferative responses in the endometrium of adult animals; (2) the observation of differences of the GEN activity in intact and OVX/E2 substituted animals can be taken as a hint that GEN may interact mechanistically with progestins which has to be proven in detail in future investigations and (3) the detection of strong effects of the phytoestrogen GEN on hepatic gene expression may point to the need of future investigations to rule out the possibility of adverse responses in this organ.     

Diurnal pattern of proopiomelanocortin gene expression in the arcuate nucleus of proestrous, ovariectomized, and steroid-treated rats: a possible role in cyclic luteinizing hormone secretion

Opiate peptides are thought to modulate the pattern of LH release in female rats. We tested the hypothesis that changes in proopiomelanocortin (POMC) gene expression occur in proestrous (PRO) and ovariectomized (OVX) steroid-treated rats which may explain their unique patterns of LH secretion. Using in situ hybridization, we examined whether diurnal changes in POMC gene expression occur in the arcuate nucleus. Four groups of rats were used in this study. 1) PRO rats were used after exhibiting at least two consecutive 4-day estrous cycles; 2) OVX rats were killed 9 days after ovariectomy; 3) estradiol (E2)-treated rats were OVX for 7 days and then treated for 2 days; and 4) E2-progesterone (P4)-treated rats were treated with E2 as described above, and on day 9 at 1030 h, P4 was administered. Rats were killed at 2300, 0300, 1000, 1300, 1500, 1800, or 2300 h, beginning on the evening of diestrous day 2 or day 8 after ovariectomy. POMC gene expression exhibited a diurnal rhythm on PRO. Levels of mRNA rose during the morning, peaked between 0300-1000 h, and decreased by 2300 h. In E2-treated rats, which exhibited a LH surge similar in timing to the PRO surge, POMC mRNA levels exhibited a diurnal rhythm strikingly similar to that observed in PRO animals. OVX abolished the rhythm; however, average POMC mRNA levels across the 24-h period were not significantly different from those in PRO or E2-treated rats. P4 treatment increased POMC mRNA levels by 2300 h compared to those in all other experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)