E2-treated OVX Rats Research Articles

Estrogenic suppression of binge-like eating elicited by cyclic food restriction and frustrative-nonreward stress in female rats.

Because binge eating and emotional eating vary through the menstrual cycle in human females, we investigated cyclic changes in binge-like eating in female rats and their control by estrogens. Binge-like eating was elicited by three cycles of 4 days of food restriction and 4 days of free feeding followed by a single frustrative nonreward-stress episode (15 min visual and olfactory exposure to a familiar palatable food) immediately before presentation of the palatable food. Intact rats showed binge-like eating during the diestrous and proestrous phases of the ovarian cycle, but not during the estrous (periovulatory) phase. Ovariectomized (OVX) rats not treated with estradiol (E2) displayed binge-like eating, whereas E2-treated OVX rats did not. The procedure did not increase signs of anxiety in an open-field test. OVX rats not treated with E2 that were subjected to food restriction and sacrificed immediately after frustrative nonreward had increased numbers of cells expressing phosphorylated extracellular signal-regulated kinases (ERK) in the central nucleus of the amygdala (CeA), paraventricular nucleus of hypothalamus (PVN), and dorsal and ventral bed nuclei of the stria terminalis (BNST) compared with nonrestricted or E2-treated rats. These data suggest that this female rat model is appropriate for mechanistic studies of some aspects of menstrual-cycle effects on emotional and binge eating in human females, that anxiety is not a sufficient cause of binge-like eating, and that the PVN, CeA, and BNST may contribute to information processing underlying binge-like eating.

The effects of long-term estradiol treatment on social behavior and gene expression in adult female rats

This study tested the effects of long-term estradiol (E2) replacement on social behavior and gene expression in brain nuclei involved in the regulation of these social behaviors in adult female rats. We developed an ultrasonic vocalization (USV) test and a sociability test to examine communications, social interactions, and social preference, using young adult female cagemates. All rats were ovariectomized (OVX) and implanted with a Silastic capsule containing E2 or vehicle, and housed in same-treatment pairs for a 3-month period. Then, rats were behaviorally tested, euthanized, and 5 nuclei in the brain's social decision-making circuit were selected for neuromolecular profiling by a multiplex qPCR method. Our novel USV test proved to be a robust tool to measure numbers and types of calls emitted by cagemates that had been reintroduced after a 1-week separation. Results also showed that E2-treated OVX rats had profoundly decreased numbers of USV calls compared to vehicle-treated OVX rats. In a test of sociability, in which a female was allowed to choose between her cagemate or a same-treatment novel rat, we found few effects of E2 compared to vehicle, although interestingly, rats chose the cagemate over an unfamiliar conspecific. Gene expression results revealed that the supraoptic nucleus had the greatest number of gene changes caused by E2: Oxt, Oxtr and Avp were increased, and Drd2, Htr1a, Grin2b, and Gabbr1 were decreased, by E2. No genes were affected in the prefrontal cortex, and 1–4 genes were changed in paraventricular nucleus (Pgr), bed nucleus of the stria terminalis (Oxtr, Esr2, Dnmt3a), and medial amygdala (Oxtr, Ar, Foxp1, Tac3). Thus, E2 changes communicative interactions between adult female rats, together with selected expression of genes in the brain, especially in the supraoptic nucleus.

Chronic black tea extract consumption improves endothelial function in ovariectomized rats.

Purpose Menopause escalates the risk of cardiovascular diseases in women. There is an unmet need for better treatment strategy for estrogen-deficiency-related cardiovascular complications. Here we investigated the impact of chronic black tea extract (BT) consumption on cardiovascular function and lipid metabolism using a rat model of estrogen deficiency.MethodsFemale Sprague–Dawley rats were ovariectomized (OVX) and treated with BT (15 mg/kg/day, 4 weeks; active ingredients: theaflavins) or estrogen (E2) treatment for 4 weeks. Serum was collected for measuring cholesterol, triacylglycerol and estradiol levels. Changes in vascular reactivity were examined. The protein levels of NADPH oxidases were assessed by Western blotting. Reactive oxygen species (ROS) level was measured using dihydroethidium fluorescence imaging. The concentrations of cGMP were measured using ELISA kit.ResultsAortic rings from control, BT-treated and E2-treated OVX rats exhibited a greater increase in Phe-induced contraction after inhibition of NO synthase compared with those from OVX rats. ACh-induced endothelium-dependent relaxations were augmented in aortae and renal arteries in BT/E2-treated OVX rats than in OVX rats. BT/E2 treatment improved flow-mediated dilatation in small mesenteric resistance arteries of OVX rats. BT/E2 treatment restored the eNOS phosphorylation level and reversed the up-regulation of NADPH oxidases and ROS overproduction in OVX rat aortae. ACh-stimulated cGMP production was significantly elevated in the aortae from BT- and E2-treated rats compared with those from OVX rats. BT/E2 treatment reduced circulating levels of total cholesterol.ConclusionsThe present study reveals the novel benefits of chronic BT consumption to reverse endothelial dysfunction and favorably modifying cholesterol profile in a rat model of estrogen deficiency and provides insights into developing BT as beneficial dietary supplements for postmenopausal women.

17β Estradiol increases resilience and improves hippocampal synaptic function in helpless ovariectomized rats

Memory impairment is the most commonly reported cognitive symptom associated with major depressive disorder. Decreased hippocampal volume and neurogenesis in depression link hippocampal dysfunction with deficits in memory. Stress decreases hippocampal dendritic spine density and long-term potentiation (LTP) at glutamate synapses, a cellular correlate of learning and memory. However, elevated plasma levels of 17β estradiol (E2) during proestrus increase hippocampal structure and function, directly opposing the negative consequences of stress. In women, significant fluctuations in ovarian hormones likely increase vulnerability of hippocampal circuits to stress, potentially contributing to the greater incidence of depression compared to men. Using the learned helplessness model of depression and ovariectomized female rats, we investigated whether acquisition of helplessness and hippocampal synaptic dysfunction is differentially impacted by the presence or absence of plasma E2. We find that inescapable shock induces a greater incidence of helplessness in vehicle- versus E2-treated OVX rats. In the vehicle-treated group, LTP was absent at CA3-CA1 synapses in slices only from helpless rats, and CA1 spine density was decreased compared to resilient rats. In contrast, significant LTP was observed in slices from E2-treated helpless rats; importantly, spine density was not different between E2-treated helpless and resilient rats, dissociating spine density from the LTP magnitude. We also find that E2 replacement can reverse previously established helpless behavior. Thus, our results show that E2 replacement in OVX rats increases resilience and improves hippocampal plasticity, suggesting that E2 therapy may increase resilience to stress and preserve hippocampal function in women experiencing large fluctuations in plasma estrogen levels.

The Preventive Effect of Biochanin A on Bone Loss in Ovariectomized Rats: Involvement in Regulation of Growth and Activity of Osteoblasts and Osteoclasts

Biochanin A (BCA) is a major isoflavone abundant in red clover (Trifolium pretense). The protective effect of BCA on bone loss in an ovariectomized (OVX) animal model has never been clarified. The objective of this study was to investigate the biological effects of BCA on bone loss in OVX rats in vivo and on the development of osteoblasts and osteoclasts in vitro. Ovariectomy resulted in a marked increase in body weight and a decrease in femoral bone mineral density and trabecular bone volume that was prevented by BCA or 17β-estradiol (E2) treatment. However, an increase in uterine weight was observed in E2-treated OVX rats, but not in response to BCA treatment. Treatment with BCA increased the mRNA expression of osterix, collagen type I, alkaline phosphatase (ALP), and osteocalcin and decreased the mRNA expression of tartrate-resistant acid phosphatase (TRAP) and the receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG) ratio in the femur of OVX rats. Treatment with BCA or E2 prevented the OVX-induced increase in urinary deoxypyridinoline (DPD) and serum tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β). In vitro, BCA induced preosteoblasts to differentiate into osteoblasts and increased osteoblast mineralization. BCA inhibited preosteoclasts and osteoclast proliferation and decreased osteoclast bone resorption. These findings suggest that BCA treatment can effectively prevent the OVX-induced increase in bone loss and bone turnover possibly by increasing osteoblastic activities and decreasing osteoclastic activities.

Effect of the interaction between food state and the action of estrogen on oxytocinergic system activity

Increased plasma osmolality by food intake evokes augmentation of plasma oxytocin (OT). Ovarian steroids may also influence the balance of body fluids by acting on OT neurones. Our aim was to determine if estrogen influences the activity of OT neurones in paraventricular nucleus (PVN) and supraoptic nucleus (SON) under different osmotic situations. Ovariectomized rats (OVX) were treated with either estradiol (E(2)) or vehicle and were divided into three groups: group I was fed ad libitum, group II underwent 48 h of fasting, and group III was refed after 48 h of fasting. On the day of the experiment, blood samples were collected to determine the plasma osmolality and OT. The animals were subsequently perfused, and OT/FOS immunofluorescence analysis was conducted on neurones in the PVN and the SON. When compared to animals which were fasted or fed ad libitum, the plasma osmolality of refed animals was higher, regardless of whether they were treated with vehicle or E(2). We observed neural activation of OT cells in vehicle- or E(2)-treated OVX rats refed after 48 h of fasting, but not in animals fed ad libitum or in animals that only underwent 48 h of fasting. Finally, the percentage of neurones that co-expressed OT and FOS was lower in both the PVN and the SON of animals treated with E(2) and refed, when compared to vehicle-treated animals. These results suggest that E(2) may have an inhibitory effect on OT neurones and may modulate the secretion of OT in response to the increase of osmolality induced by refeeding.

Estrogen receptor hormone agonists limit trauma hemorrhage shock-induced gut and lung injury in rats.

BackgroundAcute lung injury (ALI) and the development of the multiple organ dysfunction syndrome (MODS) is a major cause of death in trauma patients. Earlier studies in trauma hemorrhagic shock (T/HS) have documented that splanchnic ischemia leading to gut inflammation and loss of barrier function is an initial triggering event that leads to gut-induced ARDS and MODS. Since sex hormones have been shown to modulate the response to T/HS and proestrous (PE) females are more resistant to T/HS-induced gut and distant organ injury, the goal of our study was to determine the contribution of estrogen receptor (ER)α and ERβ in modulating the protective response of female rats to T/HS-induced gut and lung injury.Methods/Principal FindingsThe incidence of gut and lung injury was assessed in PE and ovariectomized (OVX) female rats subjected to T/HS or trauma sham shock (T/SS) as well as OVX rats that were administered estradiol (E2) or agonists for ERα or ERβ immediately prior to resuscitation. Marked gut and lung injury was observed in OVX rats subjected to T/HS as compared to PE rats or E2-treated OVX rats subjected to T/HS. Both ERα and ERβ agonists were equally effective in limiting T/HS-induced morphologic villous injury and bacterial translocation, whereas the ERβ agonist was more effective than the ERα agonist in limiting T/HS-induced lung injury as determined by histology, Evan's blue lung permeability, bronchoalevolar fluid/plasma protein ratio and myeloperoxidase levels. Similarly, treatment with either E2 or the ERβ agonist attenuated the induction of the intestinal iNOS response in OVX rats subjected to T/HS whereas the ERα agonist was only partially protective.Conclusions/SignificanceOur study demonstrates that estrogen attenuates T/HS-induced gut and lung injury and that its protective effects are mediated by the activation of ERα, ERβ or both receptors.

Insulin signaling in the liver and uterus of ovariectomized rats treated with estradiol

We used rat hepatic and uterine tissues to examine the impact of estradiol (E2) on insulin (INS) signaling. Ovariectomized (OVX) female Wistar rats were treated with E2 (20 μg/kg b.wt., i.p.) and used for the experiment 6 h after E2 administration. To highlight E2 effects on tyrosine phosphorylation of INS receptor (IR) and INS receptor substrates (IRSs) and IRSs association with p85 subunit of phosphatidylinositol 3-kinase (PI3-K) in the context of INS signaling, E2-treated OVX rats were also injected with INS (20 IU, i.p.), 30 min before the experiment. Treatment with E2 did not change the levels of plasma INS and glucose (Glu). However, it significantly decreased the free fatty acid (FFA) level and increased uterine weight. Furthermore, the results show that E2 had no effect on the content of hepatic IR protein, but significantly increased IR protein content in the uterus and decreased IR tyrosine phosphorylation in both the liver and uterus. Compared to the control, hepatic IRS-1 and IRS-2 were significantly decreased and increased, respectively, after E2 treatment. Protein content of both molecules, IRS-1 and IRS-2, was increased in uterine tissue after E2 administration. Protein content of the p85 subunit of PI3-K and that of protein kinase B (Akt) were increased in the uterus, with no changes in the liver. The results suggest that E2 treatment induces tissue-specific changes in INS signaling. The consequences of E2 treatment on INS signaling molecules are more apparent in the uterus, but their physiological relevance for INS action is probably greater in the liver.

Female Sex Steroids Increase Adrenomedullin-Induced Vasodilation by Increasing the Expression of Adrenomedullin2 Receptor Components in Rat Mesenteric Artery

Based on the favorable effects of female sex steroids in vascular functions and the potent hypotensive effects of adrenomedullin (AM), we hypothesized that AM-induced vasodilation is gender dependent, and female sex steroids enhance this effect. In endothelium-intact rat mesenteric artery, AM (1 nm-0.3 microM)-induced concentration-dependent relaxation was significantly (P < 0.05) higher in females [pD2(-log EC50 of the molar concentration), 7.05 +/- 0.10; maximal relaxation response (Emax), 69.2 +/- 3.46%] than males (pD2, 6.53 +/- 0.08; Emax, 53.28 +/- 4.86%). The increased relaxation was lost when the females were ovariectomized (OVX) (pD2, 6.14 +/- 0.24; Emax, 39.68 +/- 5.68%). The reduced relaxation response in OVX rats was reversed by administration of either progesterone (P4; pD2, 7.18 +/- 0.07; Emax, 72.4 +/- 2.76%) or 17beta-estradiol (E2; pD2, 7.00 +/- 0.14; Emax, 70.4 +/- 4.79%). AM mediates its effects through either AM(22-52)-sensitive AM1 receptors [composed of calcitonin receptor-like receptors (CLs) and receptor activity-modifying protein (RAMP)2] or AM2 receptors (CL/RAMP3), which can be antagonized more potently by calcitonin gene-related peptide(8-37) than AM(22-52). Pharmacological characterization suggested the involvement of AM2 receptors in the increased vasodilatory effect of AM in both P4- and E2-treated animals as calcitonin gene-related peptide(8-37) (10 microM) was more potent in antagonizing the AM effects (Emax, P(4): 25.92 +/- 5.32%; E2: 29.11 +/- 7.41%) than AM(22-52) (100 microM). RT-PCR studies also supported the involvement of AM2 receptors because expression of mRNA levels encoding CL (previously reported) and RAMP3 were increased in P4- or E2-treated OVX rats. In conclusion, AM-induced vasodilation is gender-dependent and increased by female sex steroids by increased expression of AM2 receptor components.

Butyl benzyl phthalate, an endocrine disrupter, inhibits pulsatile luteinizing hormone secretion under an insulin-induced hypoglycaemic state in ovariectomized rats.

Butyl benzyl phthalate (BBP) and bisphenol A (BPA), termed endocrine disrupters, are known to mimic oestrogen in their actions, and therefore there is concern about their effect on reproductive functions. Since it is reported that the inhibitory action of oestrogen on the pulsatile secretion of luteinizing hormone (LH) is enhanced under insulin-induced hypoglycaemia, whether this also applies to BBP and BPA was examined in the present study. In adult ovariectomized (OVX) rats, the pulsatile LH secretion 24 h after subcutaneous injection of 10 mg BBP (BBP-treated), 10 mg BPA (BPA-treated) or 100 ng 17beta-oestradiol (E2-treated), all of which were dissolved in sesame oil, was not changed significantly compared to that after the injection of sesame oil only. Furthermore, in oil-treated OVX rats, the pulsatile LH secretion immediately after intravenous injection of insulin (1.0 U) was not changed compared to that after saline injection. In BBP-treated OVX rats, the injection of insulin (1.0 U) significantly decreased the number of LH pulses as in E2-treated OVX rats. The injection of insulin did not significantly affect the amplitude of LH pulses in BBP-, BPA- and E2-treated OVX rats. The results indicate that the oestrogenic action of BBP is significantly enhanced by insulin-induced hypoglycaemia and thus the pulsatile LH secretion is inhibited. We suggest that weak oestrogenic endocrine disrupters may become harmful to reproductive functions even in adult female rats, if acting under a low energy state.

Regulation of glutamic acid decarboxylase 65 and 67 gene expression by ovarian steroids: identification of two functionally distinct populations of GABA neurones in the preoptic area.

GABA neurones in the preoptic area (POA) are critical for oestradiol (E2)-dependent surge release of luteinizing hormone (LH); however, it is not clear which population(s) of POA GABA neurones is involved. The goals of the present studies were: (i) to determine whether E2 regulates GABA neurones similarly in two subdivisions of the POA that play a role in LH surge release, the rostral POA region that contains the organum vasculosum of the lamina terminalis (rPOA/OVLT), and the region containing the anteroventral periventricular nucleus (AVPV) and medial preoptic nucleus (MPN) and (ii) to determine whether GABA neurones in either or both regions exhibit temporal changes consistent with a role in the regulation of LH surge release. To accomplish these goals, we measured glutamic acid decarboxylase (GAD) 65 and 67 mRNA levels at several time points in ovariectomized (OVX), E2-treated OVX rats exhibiting LH surge release, and in E2-treated OVX rats in which LH surge release was blocked by prior administration of progesterone (P4). Our findings demonstrate that, despite their close proximity, GABA neurones in the AVPV/MPN region are regulated differently from those in the rPOA/OVLT. Only neurones in the AVPV/MPN region show temporal changes in GAD 67 mRNA expression that appear to be linked to positive-feedback effects of E2 on luteinizing hormone-releasing hormone (LHRH) and LH release. Our findings also indicate that a morning rise and an afternoon fall in GAD 67 mRNA levels marks two E2-dependent signals required for LHRH and LH surge release. Finally, our results suggest that there are distinct E2-induced signals to the rPOA/OVLT and AVPV/MPN regions and that these signals differentially regulate GAD 65 and 67 gene expression.

Intracerebroventricular Administration of Ghrelin Rapidly Suppresses Pulsatile Luteinizing Hormone Secretion in Ovariectomized Rats

Ghrelin, an endogenous growth hormone (GH) secretagogue, is shown to increase food intake, which action is similar to that of orexin, also a hypothalamic peptide. Since orexin suppresses pulsatile LH secretion in ovariectomized (OVX) rats, the present study was undertaken to investigate whether ghrelin also suppresses LH secretion. Effects of intracerebroventricularly injected ghrelin (0.1 nmol/0.3 μl) were examined in OVX rats treated with a small dose of 17β-estradiol (E2). After ghrelin injection, pulsatile LH secretions which were ongoing in these E2-treated OVX rats were significantly suppressed for about 1 h, whereas GH secretion increased, peaking at 30 min. The main parameter suppressed by ghrelin was the pulse frequency, not the pulse amplitude, suggesting the hypothalamus as the site of ghrelin action. This study provides evidence that ghrelin acts not only in the control of food intake but also in the control of LH secretion.

Effects of estrogen on leukocyte adhesion after transient forebrain ischemia.

Recent findings indicate that estrogen (ie, 17beta-estradiol [E(2)]) provides neuroprotection in models of transient global and focal ischemia. Enhanced postischemic leukocyte adhesion and infiltration have been linked to neuropathology in the brain as well as other tissues. We recently showed that estrogen reduces leukocyte adhesion in the cerebral circulation of female rats during resting conditions. We compared leukocyte adhesion in pial venules in vivo in intact, ovariectomized (OVX), and E(2)-treated OVX female rats subjected to transient forebrain ischemia (30-minute right common carotid artery occlusion and hemorrhagic hypotension) and reperfusion. Adherent rhodamine-6G-labeled leukocytes were viewed through a closed cranial window with the use of intravital microscopy. Leukocyte adhesion was measured before ischemia and at different times after reperfusion. Before ischemia, leukocyte adhesion (measured as a percentage of venular area occupied by adherent leukocytes) was 2 to 3 times greater in OVX versus intact or E(2)-treated OVX rats (7.0%, 3.4%, and 2.2%, respectively). This difference disappeared at 120 minutes of reperfusion, when comparable levels of enhanced leukocyte adhesion were observed in all groups. In OVX rats, leukocyte adhesion remained elevated after 4 and 6 hours of reperfusion (11.6% and 12.9%, respectively), while the other 2 groups showed significantly lower levels (5.0% and 5.8% for intact rats and 7.0% and 7.2% for E(2)-treated OVX rats). Present results demonstrate that estrogen modulates leukocyte adhesion in the cerebral circulation after transient forebrain ischemia. This effect suggests that decreased leukocyte adhesion may be an important mechanism in estrogen-mediated neuroprotection.

Nitric-Oxide-Dependent Pial Arteriolar Dilation in the Female Rat: Effects of Chronic Estrogen Depletion and Repletion

In this study, we compared endothelial nitric oxide synthase (eNOS)-mediated cerebral vasodilating responses in intact female rats, chronically ovariectomized (OVX) rats, and OVX rats treated for 2 weeks with 17β-estradiol (E2). Under anesthesia, using intravital microscopy and a closed cranial window system, pial arteriolar diameter changes were monitored during sequential cortical suffusions of an eNOS-dependent dilator [acetylcholine (ACh)] and a direct NO donor [S-nitrosoacetylpenicillamine (SNAP)]. In separate rats from the same groups, we compared eNOS and caveolin-1 (CAV-1) protein abundance in pial arterioles (via immunofluorescence analyses). In untreated and low-dose E2-treated (1.0 μg · kg−1 · day−1) OVX rats, ACh-induced vasodilations were virtually absent. High-dose E2 treatment (100 μg · kg−1 · day−1) restored ACh-induced pial arteriolar dilations to levels seen in intact females. The vasodilations elicited by SNAP and ADO were unaffected by chronic estrogen changes, indicating no direct estrogen influence on vascular smooth muscle (VSM) reactivity. Pial arteriolar eNOS protein abundance was diminished by ovariectomy and restored by high-dose E2 treatment. Pial arteriolar CAV-1 expression was higher in OVX versus intact and E2-treated OVX females. These results suggest that long-term changes in estrogen directly influence brain eNOS functional activity. The estrogen-related changes in eNOS-dependent vasodilating function appear to be related, in part, to a capacity for E2 to increase eNOS protein expression and, in part, to an E2-associated diminution in endothelial CAV-1 expression.

Cerebral vasodilating capacity during forebrain ischemia: effects of chronic estrogen depletion and repletion and the role of neuronal nitric oxide synthase.

The effects of chronic 17beta-estradiol (E2) depletion, via ovariectomy (OVX), and its repletion, on cortical cerebral blood flow (CBF) and EEG activities during forebrain ischemia, as well as post-ischemic recovery and neuropathology, were assessed and compared with results obtained in normal female rats. We also examined whether neuronal nitric oxide synthase (nNOS) activity is affected by OVX and E2 replacement and whether NOS-derived NO supports vasodilation during ischemia. OVX females displayed a significantly lower CBF during ischemia (10% of baseline) than did normal females (23% of baseline). In OVX rats, given chronic low-dose E2 treatment (0.1 mg kg(-1) day(-1)), intra-ischemic CBF was similar to normal females (25% of baseline). However, at supraphysiologic E2 doses (> or = 0.5 mg kg(-1) day(-1)), that benefit was diminished or lost. Intra-ischemic EEG power reductions and post-ischemic survival rates, neurological dysfunction, and histopathology displayed similar relative differences among groups as the CBF findings. Intra-ischemic CBF was reduced by nNOS inhibition, with ARL 17477, in normal and low-dose E2-treated OVX rats (4-8% baseline). The repressed intra-ischemic vasodilating function in OVX rats may be due to reductions in nNOS activity, because untreated OVX rats showed a 50% lower cortical nNOS activity than that in normal rats and in rats treated with low or high dose (5 mg kg(-1) day(-1)) E2. However, the inability to restore vasodilating function despite normalization of nNOS activity indicates that another mechanism is responsible for the repression of vasodilatory function in the high-dose group. These findings suggest that E2, at levels within the physiological range, promotes ischemic neuroprotection via improving vasodilating capacity. One possible mechanism may relate to E2 enhancing brain nNOS expression and activity.

Regional analysis of bone mineral density in the distal femur and proximal tibia using peripheral quantitative computed tomography in the ratin vivo

The use of peripheral quantitative computed tomography (pQCT) was investigated for the measurement of volumetric bone mineral density (BMD) in mg x cm-3. Two studies were undertaken. In the first study, the precision of pQCT in vivo and ex vivo was tested at 14 weeks postovariectomy (OVX). In the second study, the efficacy of a standard antiresorptive treatment, 17beta-estradiol (E2), was tested 6 weeks post-OVX. The precision for total (compact plus trabecular) BMD was 1.3-1.9%, and that for trabecular BMD was 2.4-2. 7%. There was excellent agreement between trabecular BMD measurements in vivo and ex vivo (r = 0.91). Significant reductions in trabecular BMD were observed in vivo at 14 and 6 weeks following ovariectomy in the femur, in each study. The loss of trabecular BMD depended on slice location, and varied from 0 to 22% at 6 weeks, and from 0 to 26% at 14 weeks (P < 0.001, at the affected locations). The antiresorptive effect of treatment was demonstrated in the 6-week study: there was no significant difference in BMD between sham-operated and E2-treated OVX rats.

L-arginine/nitric oxide amplifies the magnitude and duration of the luteinizing hormone surge induced by estrogen: involvement of neuropeptide Y

Although estrogen can induce LH hypersecretion in ovariectomized (ovx) rats, the magnitude and duration of the LH surge are invariably far less than those of the preovulatory LH surge in cycling rats. Recently, the crucial roles of hypothalamic nitric oxide (NO) and neuropeptide Y (NPY) in the induction of LHRH and the LH surge have been recognized. In this study, we have tested the hypothesis that low grade stimulatory feedback effects of 17 beta-estradiol (E2) on LH secretion in ovx rats is due to a deficit in NO signaling. As the NO substrate, L-arginine (L-Arg), can enhance NO-dependent physiological responses, the effects of L-Arg on the LH surge in E2-primed ovx rats was studied. Ovx rats bearing permanent cannulas in the lateral cerebroventricle received 15-mm long SILASTIC capsules filled with E2 (300 micrograms/ml oil, s.c.) at 1000 h. Two days later, saline (SAL) or L-Arg (0.1 or 1 nmol in 5 microliters SAL) or D-Arg (1.0 nmol) was injected intracerebroventricularly every hour from 1100-1400. Blood samples were collected at 1100 h and at hourly intervals from 1400-1800 h via intraatrial cannulae implanted the day before. Whereas an expected moderate LH surge occurred in SAL-injected control rats, L-Arg, but not D-Arg, greatly augmented the magnitude and duration of the LH surge, with peak values attaining the range normally seen on proestrus. Furthermore, L-Arg (1 or 10.0 mM) and the NO donor, sodium nitroprusside (1 or 10.0 mM), stimulated the basal and K(+)-induced in vitro release of LHRH and NPY from the hypothalami of ovarian steroid-primed ovx rats. As NPY is stimulatory to LHRH release, we next tested the possibility that L-Arg/NO may initially stimulate NPY release in the hypothalamus, which, in turn, elicits LHRH discharge. E2-treated ovx rats receiving intracerebroventricular L-Arg were injected with antisense or missense oligodeoxynucleotides (oligos) to NPY-messenger RNA. Antisense, not missense, NPY oligos blocked the L-Arg-induced potentiation of the LH surge. These results revealed for the first time the neurochemical cause underlying the E2 feedback action and show that a deficiency in either L-Arg itself or L-Arg-based NO signaling is responsible for the low grade LH surge in ovx rats treated with estrogen alone. Additionally, the L-Arg-dependent potentiation of the LH surge may involve increased signaling in the NO and NPYergic systems, resulting in optimal LHRH hypersecretion from the hypothalamus.

L-arginine/nitric oxide amplifies the magnitude and duration of the luteinizing hormone surge induced by estrogen: involvement of neuropeptide Y.

Although estrogen can induce LH hypersecretion in ovariectomized (ovx) rats, the magnitude and duration of the LH surge are invariably far less than those of the preovulatory LH surge in cycling rats. Recently, the crucial roles of hypothalamic nitric oxide (NO) and neuropeptide Y (NPY) in the induction of LHRH and the LH surge have been recognized. In this study, we have tested the hypothesis that low grade stimulatory feedback effects of 17 beta-estradiol (E2) on LH secretion in ovx rats is due to a deficit in NO signaling. As the NO substrate, L-arginine (L-Arg), can enhance NO-dependent physiological responses, the effects of L-Arg on the LH surge in E2-primed ovx rats was studied. Ovx rats bearing permanent cannulas in the lateral cerebroventricle received 15-mm long SILASTIC capsules filled with E2 (300 micrograms/ml oil, s.c.) at 1000 h. Two days later, saline (SAL) or L-Arg (0.1 or 1 nmol in 5 microliters SAL) or D-Arg (1.0 nmol) was injected intracerebroventricularly every hour from 1100-1400. Blood samples were collected at 1100 h and at hourly intervals from 1400-1800 h via intraatrial cannulae implanted the day before. Whereas an expected moderate LH surge occurred in SAL-injected control rats, L-Arg, but not D-Arg, greatly augmented the magnitude and duration of the LH surge, with peak values attaining the range normally seen on proestrus. Furthermore, L-Arg (1 or 10.0 mM) and the NO donor, sodium nitroprusside (1 or 10.0 mM), stimulated the basal and K(+)-induced in vitro release of LHRH and NPY from the hypothalami of ovarian steroid-primed ovx rats. As NPY is stimulatory to LHRH release, we next tested the possibility that L-Arg/NO may initially stimulate NPY release in the hypothalamus, which, in turn, elicits LHRH discharge. E2-treated ovx rats receiving intracerebroventricular L-Arg were injected with antisense or missense oligodeoxynucleotides (oligos) to NPY-messenger RNA. Antisense, not missense, NPY oligos blocked the L-Arg-induced potentiation of the LH surge. These results revealed for the first time the neurochemical cause underlying the E2 feedback action and show that a deficiency in either L-Arg itself or L-Arg-based NO signaling is responsible for the low grade LH surge in ovx rats treated with estrogen alone. Additionally, the L-Arg-dependent potentiation of the LH surge may involve increased signaling in the NO and NPYergic systems, resulting in optimal LHRH hypersecretion from the hypothalamus.

Droloxifene, a new estrogen antagonist/agonist, prevents bone loss in ovariectomized rats.

The purpose of this study was to determine the effects of droloxifene (DRO), a new estrogen antagonist/agonist, on bone turnover, bone mass, total serum cholesterol, and uterine weight in rats made estrogen deficient by ovariectomy. Sprague-Dawley female rats were ovariectomized (OVX) or sham operated (sham) at 5 months of age and treated with 17 beta-estradiol (E2) at 30 micrograms/kg, sc, daily or with DRO at 5, 10, or 20 mg/kg.day, orally, for 4 weeks. At the time of death, body weight gain, uterine weight, and total serum cholesterol were measured. Bone area, bone mineral content (BMC), and bone mineral density (BMD) of whole femora, distal femoral metaphyses, femoral shaft, and proximal femora were determined ex vivo using dual energy x-ray absorptiometry. Static and dynamic cancellous bone histomorphometric analysis of proximal tibial metaphyses was performed in double fluorescent labeled, undecalcified, 4- and 10-microns longitudinal sections. Body weight gain in E2-treated OVX rats was significantly reduced compared to that in OVX controls, but was not different from that in sham controls. Body weight gain in DRO-treated OVX rats was decreased significantly compared to that in both sham and OVX controls. In OVX rats, uterine weight was completely preserved by treatment with E2. Uterine weight in DRO-treated OVX rats was slightly, but significantly, increased from the vehicle-treated control value, and was significantly lower than that in sham controls and E2-treated OVX rats. Treatment with sc injection of E2 in OVX rats had no effect on total serum cholesterol, whereas OVX rats orally treated with DRO at 5-20 mg/kg.day decreased total serum cholesterol by 33-46% compared to levels in sham and OVX controls. Compared to sham controls, OVX decreased BMC and BMD of distal femoral metaphyses, increased BMD of the femoral shaft, and had no effect on BMC and BMD of whole femora and proximal femora. Treatment with either E2 or DRO prevented these changes induced by OVX. Proximal tibial metaphyseal trabecular bone volume and trabecular number were increased, and trabecular separation, percent osteoclast perimeter, osteoclast number, percent mineralizing perimeter, mineral apposition rate, bone formation rate, and bone turnover rate were decreased in 5, 10, or 20 mg/kg.day DRO-treated OVX rats compared to OVX controls. These cancellous bone histomorphometric indexes in DRO treated OVX rats did not differ from those in E2-treated OVX rats or sham controls, suggesting that DRO completely prevented the increases in bone turnover and the decrease in bone mass induced by OVX in rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Correlation of luteinizing hormone surges with estrogen nuclear and progestin cytosol receptors in the hypothalamus and pituitary gland. I. Estradiol dose response effects.

The present studies were designed to answer three questions: (1) how will a progressive increase in serum estradiol (E2) in ovariectomized (OVX) rats affect progesterone (P4)-induced luteinizing hormone (LH) surge concentrations? (2) Can steroid-induced LH surges be correlated with estrogen nuclear receptor (E2Rn) and progestin cytosol receptor (PRc) levels in brain regions known to regulate LH secretion, and (3) do differences in pituitary responsiveness to LHRH in E2- or E2P4-treated OVX rats parallel changes in E2Rn and PRc concentrations in this gland? 1 week after ovariectomy of adult cyclic rats (day 0), Silastic E2 capsules were placed subcutaneously at 09.00 h and produced serum E2 levels of 6-8 (low), 12-19 (medium) and 27-37 (high) pg/ml, respectively. 2 days later (day 2), some rats also received Silastic P4 capsules subcutaneously which elevated serum P4 concentrations to 10-12 ng/ml. In rats with low serum E2, P4 treatment induced peak serum LH levels of 913 ng/ml. When serum E2 was increased to the medium or relatively high physiologic range, P4 treatment resulted in LH surge levels of 4,686 and 5,030 ng/ml. OVX controls and E2-treated OVX rats were sacrificed at 10.00 h on day 2 and E2Rn and PRc were measured concurrently in the preoptic area (POA), mediobasal hypothalamus (MBH), corticomedial amygdala (CMA) and pituitary gland (PIT). Raising serum E2 from OVX levels to the low range significantly increased both E2Rn and PRc in MBH and PIT, but not in the POA or CMA.(ABSTRACT TRUNCATED AT 250 WORDS)