Increased vulnerability of brain to estrogen withdrawal-induced mitochondrial dysfunction with aging

In the present study, to determine whether aging could increase the vulnerability of the brain to estrogen withdrawal-induced mitochondrial dysfunction, we measured the cytochrome c oxidase (COX) activity and mitochondrial adenosine triphosphate (ATP) content in hippocampi of 2 groups of ovariectomized (OVX) Wistar rats aged 2 months (young) and 9 months (middle-aged), respectively. In addition, effects of genistein and estradiol benzoate (EB) were tested also. We observed only a transient alteration of COX activity and mitochondrial ATP content in hippocampi of young OVX rats but a prolonged lowering of COX activity and mitochondrial ATP content in hippocampi of middle-aged OVX rats. This suggested that with aging compensatory mechanisms of mitochondrial function were attenuated, thus exacerbated estrogen withdrawal-induced mitochondrial dysfunction in hippocampi. Significantly, EB/genistein treatment reversed this estrogen withdrawal-induced mitochondrial dysfunction in both young and middle-aged rats suggesting that genistein may be used as a substitute for estradiol to prevent age-related disease such as Alzheimer’s disease in post-menopausal females.     

Effects of Phytoestrogen on Mitochondrial Structure and Function of Hippocampal CA1 Region of Ovariectomized Rats

The present study was undertaken to evaluate whether estrogen deprivation might lead to mitochondrial alteration of hippocampal neurons of ovariectomized (OVX) rats, and to evaluate the protective effect of estrogen and phytoestrogen on the mitochondrial alteration. First, OVX rats were used to mimic the pathologic changes of neurodegeneration of postmenopausal female, and we looked into the alteration of the mitochondrial ultrastructure and ATP content of hippocampal CA1 region after ovariectomy on different phase by transmission electron microscope (TEM) and reversed-phase high-performance liquid chromatography (HPLC), and found the best phase points of the alteration of the mitochondrial ultrastructure and ATP content. Next, estrogen and phytoestrogen were administered to the OVX rats for the protective effects on the mitochondrial ultrastructure and ATP content. Meanwhile, the density, size, shape, and distribution parameters of mitochondrial ultrastructure were analyzed according to the morphometry principle. The experimental results presented that (1) The alteration of mitochondrial ultrastructure elicited by ovariectomy worsened with the days going on, and the changes were the most noteworthy in volume density (Vv), average surface area (S), specific surface area (δ), and particle dispersity (Cλz) on 12th day (P < 0.05 or P < 0.01). Moreover, there was no statistical significance of the numerical density (Nv) among the five groups in the first step experiment. (2) The treatment with estrogen, genistein (Gs), and ipriflavone (Ip) significantly reversed the effect elicited by ovariectomy on Vv, S, δ, Cλz, Nv, and particle average diameter (D) of mitochondria of hippocampal CA1 region (P < 0.05). (3) Furthermore, ATP content of hippocampal CA1 region after ovariectomy declined significantly on 7th day (P < 0.05), and estrogen and phytoestrogen could reverse the alteration (P < 0.05). Taken together, these results revealed that phytoestrogen may have a protective role against the neurodegeneration after menopause via protecting mitochondrial structure and functions. Phytoestrogen may be a good alternative as a novel therapeutic strategy for menopausal syndrome.     

Improved model for the induction of proestrus-like gonadotropin surges in long-term ovariectomized rats

In long-term (greater than 4 wk) ovariectomized rats the positive response of the gonadotropin release apparatus to a priming dose of estradiol is moderate as compared with that of proestrous rats exposed to endogenous estradiol. In the present study, high sensitivity to estrogen was restored in long-term ovariectomized rats by pretreatment with estradiol benzoate (EB, 20 micrograms, day 0) and progesterone (P, 2.5 mg, day 3). Estradiol benzoate (20 micrograms) given on day 5 induced proestrus-like surges of LH and FSH in the afternoon on day 6. Additional administration of P (2.5 mg at noon on day 6) had a facilitatory effect. Stimulation of LH release could be evoked in rats by the described regimen 1, 6 or 50 wk after ovariectomy. The long-term ovariectomized rat injected with EB and P as described might provide a useful model for neuroendocrinological investigations on the gonadotropin surge mechanism.     

Subchronic treatment with cyclosporin A decreases the binding properties of the GABAA receptor in ovariectomized rats

In this study, we investigated the effect of cyclosporin A on the binding properties of the GABAA receptor in the hippocampus, known to be responsible for the induction of seizures, to clarify the mechanism of cyclosporin A-inhibited GABA neurotransmission in ovariectomized rats, as a climacterium model. The effects of single and subchronic treatments with cyclosporin A were examined on [3H]muscimol binding to hippocampal synaptosomal membranes in sham, ovariectomized, and estradiol/ovariectomized rats. A single treatment with cyclosporin A (40 mg/kg, i.p.) failed to change [3H]muscimol binding in the 3 groups, when compared with each corresponding vehicle-treated group. Subchronic treatment with cyclosporin A (40 mg/kg, i.p., once a day for 5 days) significantly decreased the amount of [3H]muscimol binding in ovariectomized rats. However, this inhibitory effect was not observed in sham or estradiol/ovariectomized rats. These results demonstrated that the binding activity of the GABAA receptor was decreased in ovariectomized rats after subchronic cyclosporin A treatment. This study supports the hypothesis that ovariectomy elevates the susceptibility to cyclosporin A-induced convulsions by accelerating the inhibitory actions of cyclosporin A on GABA neurotransmission in the hippocampus.     

Changes in uterine estrogen receptor concentrations in persistent estrous and persistent diestrous rats

Changes in uterine weight and the estrogen receptor concentrations were examined in persistent estrous (PE) and persistent diestrous (PD) rats at 80 days of age. To prepare PE rats, 100 micrograms estradiol benzoate (EB) was injected sc into 3-day-old females. PD rats were obtained by daily injections of 10 micrograms EB into females for 10 consecutive days from the day of birth. The uterine weight in PE rats at 80 days was comparable to that in metestrous controls. The uteri of PD rats were smaller than those in PE rats. The concentrations of estrogen receptor in nuclear fractions in PE and PD rats were much lower than those in proestrous controls. Receptor concentrations in cytosol fractions were significantly lower in PE and PD rats than in control diestrous, proestrous and estrous rats. The dissociation constants and sedimentation coefficients of estrogen receptors in PE and PD rats were found to be in the same range as those in control rats. Thus, the reduction in the activity of cytosol receptors in these rats is attributable to a quantitative change in the amount of estrogen receptor protein. To study the response of the uterus to estrogen, ovariectomized rats were injected daily with 10 micrograms estradiol for 7 consecutive days. The uterine growth of PE and PD rats after administration of estradiol was less marked than in controls, indicating a reduction of estrogen sensitivity of the uterus. Seven daily administrations of estradiol continued to increase the concentration of uterine cytosol estrogen receptor in controls. In contrast, in PE and PD rats, the receptor concentrations continued to increase during the first 3 days, and then remained constant. These data suggest that EB in neonatal treatment may directly affect the mechanism of receptor synthesis in uterine tissues. This effect may contribute to the reduction of the uterine response to estrogen.     

The role of estradiol 17β in the activation of the uterus during premature labor and the effect of naproxen,an inhibitor of prostaglandin synthesis

Fifty-two pregnant rats were ovariectomized on day 16 of gestation to induce estrogen and progesterone deficiencies and the animals were divided into four Groups. Ovariectomy alone (Group A) resulted in the premature delivery of 21% of the fetuses. When ovariectomy was followed by estrogen treatment restoring normal estrogen levels (Group B), premature delivery of the fetuses increased to 96%. Daily injections of 25mg/kg b.w. Naproxen (Group C), given from the day of ovariectomy to reduce prostaglandin synthesis, completely prevented premature delivery if the animals received no estradiol treatment and reduced prematurity to 50% if estradiol had been administered (Group D).It is concluded that the estrogen and progesterone deficiency, induced by ovariectomy, provokes a regulatory imbalance which promotes premature delivery. This imbalance is enhanced when the estradiol levels are restored to normal values, probably because estradiol increases the synthesis of prostaglandin, the intrinsic myometrial stimulant. Naproxen, an inhibitor of prostaglandin synthesis, restores the regulatory balance, partially or completely, depending on the estrogen levels.     

A dominant negative ERβ splice variant determines the effectiveness of early or late estrogen therapy after ovariectomy in rats

The molecular mechanisms for the discrepancy in outcome of initiating estrogen therapy (ET) around peri-menopause or several years after menopause in women are unknown. We hypothesize that the level of expression of a dominant negative estrogen receptor (ER) β variant, ERβ2, may be a key factor determining the effectiveness of ET in post-menopausal women. We tested this hypothesis in ovariectomized nine month-old (an age when irregular estrous cycles occur) female Sprague Dawley rats. Estradiol treatment was initiated either 6 days (Early ET, analogous to 4 months post-menopause in humans), or 180 days (Late ET, analogous to 11 years post-menopause in humans) after ovariectomy. Although ERβ2 expression increased in all OVX rats, neurogenic and neuroprotective responses to estradiol differed in Early and Late ET. Early ET reduced ERβ2 expression in both hippocampus and white blood cells, increased the hippocampal cell proliferation as assessed by Ki-67 expression, and improved mobility in the forced swim test. Late ET resulted in either no or modest effects on these parameters. There was a close correlation between the degree of ERβ2 expression and the preservation of neural effects by ET after OVX in rats, supporting the hypothesis that persistent elevated levels of ERβ2 are a molecular basis for the diminished effectiveness of ET in late post-menopausal women. The correlation between the expression of ERβ2 in circulating white blood cells and brain cells suggests that ERβ2 expression in peripheral blood cells may be an easily accessible marker to predict the effective window for ET in the brain.     

The effects of ovarian steroids in controlling rat uterine prostaglandin F2-alpha during the peri-implantation period

Rats with delayed implantation, induced by ovariectomy or hypophysectomy, as well as those with normal pregnancy were used to examine the changes in uterine prostaglandin F2 alpha (PGF2 alpha) associated with implantation. In normal pregnant rats, while maximal uterine production of PGF2 alpha was found at 09:00, maximal catabolic enzyme activity (CEA) was seen at 17:00 of day 4. Uterine content of PGF2 alpha was high at 17:00 of day 4, but decreased by 80% within the next 24 h. There was no change in PGF2 alpha production during the first 6 h after injection of estradiol to hypophysectomized animals. There was, however, a dramatic decrease in production within the next 6 h. In contrast, CEA was not different in animals treated with estrogen than in those receiving only progesterone. In ovariectomized animals, uterine PGF2 alpha production also was lowered by estrogen but in these animals CEA was significantly elevated 18 h after injection of estradiol. Estrogen caused a greater increase in PGF2 alpha content in the hypophysectomized, compared to the ovariectomized, rats. The results are consistent with the view that ovarian steroids play an important role in controlling the changes in uterine PGF2 alpha around the time of implantation in rat.     

Effect of ovariectomy and estrogen supplementation on brain acetylcholinesterase activity and passive-avoidance learning in rats

The effect of ovariectomy and estrogen treatment on the brain acetylcholinesterase activity and cognition in rats was investigated in this study. Ovariectomized and nonovariectomized rats were treated subcutaneously with estradiol dipropionate for 8 d. In the single-trial, passive-avoidance test all the groups showed significant learning and retention of memory as evident by the increase in transfer latency time in trial 2 as compared with trial 1. No-transfer response was significantly increased in the estradiol-dipropionate-treated ovariectomized (80%) and nonovariectomized (60%) group as compared with the ovariectomized (30%) group. Specific activity of acetylcholinesterase was assayed spectrophotometrically in salt-soluble and detergent-soluble fractions of various brain areas: frontal cortex, cerebral cortex, striatum, hippocampus and hypothalamus, thalamus, pons, medulla, and cerebellum. The effect of ovariectomy and estradiol dipropionate was varied in both fractions of these brain areas. Estradiol dipropionate treatment could restore the acetylcholinesterase activity to the control level only in the detergent-soluble fraction of hypothalamus and salt-soluble fraction of hypothalamus, thalamus, and medulla in ovariectomized rats. The results indicate that ovariectomy alters acetylcholinesterase activity in the brain areas but not in a uniform manner and affects only qualitative aspects of cognitive function, which could be improved by estrogen supplementation.     

Influence of arginine vasotocin on the estrogen-induced surge of LH and FSH in adult ovariectomized rats

The effects of arginine vasotocin (AVT) on the estrogen-induced surge of LH and FSH were examined in ovariectomized adult rats. Two and one-half weeks after ovariectomy, animals that were treated with a single subcutaneous (s.c.) dose of 5 μg of estradiol benzoate (EB) exhibited a surge of LH and FSH at 1700 and 1900 hours, respectively, two days after the administration of the EB. AVT, antidiuretic hormone (ADH) and oxytocin (OT) were administered s.c. in 1 μg dose every 4 hours beginning at 1500 hours on day 1 after EB treatment and then every 2 hours beginning at 1200 hours on day 2 after EB treatment. AVT completely prevented the LH surge at 1700 hours but was without effect on the FSH surge at 1900 hours on the day 2 after steroid treatment. Neither ADH nor OT had any significant effect on the afternoon surge of these hormones. It is postulated that AVT may interfere with the mechanisms mediating the estrogen-induced afternoon surge of LH in ovariectomized rats.     

Effects of estradiol benzoate on learning-memory behavior and synaptic structure in ovariectomized mice

There is increasing evidence that estrogen is involved in CNS activity, particularly memory. Several studies have suggested that estrogen improves memory by altering neuronal plasticity, including increased hippocampus CA1 dendritic spine density and enhanced long-term potentiation (LTP). In the present study, we investigated the effects of estrogen on the ultrastructural modifications in cerebral frontal cortex and hippocampus of female ovariectomized mice. One week after ovariectomy (Ovx), ICR female mice received daily injection of estradiol benzoate (EB, 20, 100, 200 microg/kg, s.c.) for 4-5 weeks. Spatial memory was then tested in the water maze, and the overall locomotor activity was monitored in open field. Synaptic morphologic parameters were examined using a graph analyzer. The results from open field did not show any alterations in locomotor activity following Ovx and EB replacement. Both the latency to find the platform and the distance to reach the platform were significantly reduced in Ovx mice by EB at 20 or 100 microg/kg when compared to vehicle treated Ovx mice. The results from synaptic ultrastructural measurement and analysis did not show any differences in hemispheric or hippocampal volumes, the numeric synaptic density, the length of active zones, or the curvature of synaptic interface among Sham, Ovx, and Ovx plus EB replacement mice. However, EB replacement effectively normalized the changes induced by Ovx, reducing the width of the synaptic cleft, enlarging the thickness of postsynaptic density (PSD), and increasing the number of synaptic vesicles in the presynapse in both cerebral cortex Fr1 and hippocampus CA1 areas. These results suggest that the beneficial effects of EB on improving memory behavior of Ovx female mice are associated with the changes of some subtle structural parameters of synapses, including the width of PSD and synaptic cleft rather than some basic and permanent structure in frontal cortex and hippocampus regions.     

The influence of long-term estrogen treatments on daily plasma prolactin levels in ovariectomized rats

Prolactin levels were determined in the plasma of female rats from 4 to 54 days after ovariectomy or ovariectomy and treatment with a long acting polymer of estradiol, polyestradiol phosphate (PEP), or Silastic implants of crystalline estradiol-17β. Blood samples were withdrawn from indwelling aortic catheters in the morning (0900–1100) and afternoon (1500–1700). Both methods of estrogen delivery elevated plasma prolactin in the morning and afternoon compared to ovariectomized controls. However, the increases in the afternoon were significantly greater than those in the morning. The difference from ovariectomized controls and the morning-afternoon differences were maintained for 25–26 days in the polyestradiol phosphate-treated group whereas those differences in the group receiving the implants of estradiol were significant for the entire length of the experiment (54 days). In addition, there were periodic fluctuations in the morning and afternoon levels of prolactin in the estradiol implanted animals. It is suggested that the plasma prolactin response to estrogen varies with time of day, time after administration of estrogen and with the method of estrogen delivery.     

Role of estrogen in controlling the genital microflora of female rats

Plate counts of viable bacteria recovered by lavage from rat vaginae demonstrated that the number of bacteria associated with the vaginal epithelium varied cyclically and that this pattern was abolished by ovariectomy. After ovariectomy, vaginal bacterial counts remained relatively stable at low levels. The estrogen 17beta-estradiol (1,3,5(10)-estratriene-3,17beta-diol cypionate) administered to ovariectomized rats caused a significant increase in vaginal bacterial counts on day 3 post-treatment. A similar effect was seen in non-ovariectomized rats, but a larger dose of estrogen antagonist may have been present in non-ovariectomized animals. Progesterone (4-pregnene-3,20-dione) given with estradiol diminished the effect of the estrogen on vaginal bacterial counts, but did not abolish it. Progesterone administered without estradiol had no detectable effect on vaginal bacterial counts. These findings suggested that the cyclic variation in bacterial content of rat vaginae could be explained primarily as the effect of the secretory pattern of ovarian estrogen.     

Role of estrogen in controlling the genital microflora of female rats.

Plate counts of viable bacteria recovered by lavage from rat vaginae demonstrated that the number of bacteria associated with the vaginal epithelium varied cyclically and that this pattern was abolished by ovariectomy. After ovariectomy, vaginal bacterial counts remained relatively stable at low levels. The estrogen 17beta-estradiol (1,3,5(10)-estratriene-3,17beta-diol cypionate) administered to ovariectomized rats caused a significant increase in vaginal bacterial counts on day 3 post-treatment. A similar effect was seen in non-ovariectomized rats, but a larger dose of estrogen antagonist may have been present in non-ovariectomized animals. Progesterone (4-pregnene-3,20-dione) given with estradiol diminished the effect of the estrogen on vaginal bacterial counts, but did not abolish it. Progesterone administered without estradiol had no detectable effect on vaginal bacterial counts. These findings suggested that the cyclic variation in bacterial content of rat vaginae could be explained primarily as the effect of the secretory pattern of ovarian estrogen.     

Estrogenic regulation of body weight in the female rat.

For a period of weeks subsequent to bilateral ovariectomy, female rats given unlimited access to food increased their food intakes and the rates at which they gained body weight; this resulted in elevated levels of body weight. Restricting ovariectomized (ovx) rats to their preoperative level of food intake (restricted diet), prevented this excessive gain in body weight. Estradiol benzoate (EB) treatments of 0.5 μg per day for 15 consecutive days partially reversed pre-occurring weight gain in obese ovx rats; this was accompanied by a reduction in food intake. In contrast, identical EB treatment for nonobese ovx rats (restricted diet) did not result in any loss of body weight or change in food intake. Oil-treated nonobese ovx rats gained a small amount of weight relative to their EB-treated counterparts, despite the similarity in their food intakes. Thus, part of the increased weight gain observed after ovariectomy may be independent of changes in food consumption, and related to removal of estrogenic influences from metabolic and behavioral processes involved in energy balance. The weight limiting actions of estradiol were far more pronounced in animals already obese or facing impending obesity than in animals in which excessive weight gain was prevented. The data also suggest that estradiol is more effective in preventing than in reversing the weight gain associated with ovariectomy, and that estrogenic influences on the body weight set point are manifested with very short latencies. These findings support earlier conclusions that estradiol does not regulate food intake directly, but secondarily controls consumption as a means of weight regulation.     

雌激素在大鼠杏仁核与纹状体多巴胺代谢中作用的差异

为探讨雌激素对大鼠杏仁核（amygdala,Amy）与纹状体（striatum,Str）多巴胺（dopamine,DA）代谢的作用,本实验采用离体电化学检测技术--高效液相色谱法（high performance liquid chromatography,HPLC）测定正常雌鼠及经雌激素处理的去卵巢（ovariectomy,OVX）雌鼠Amy和Sr的DA及其代谢产物的组织含量。实验结果显示,OVX雌鼠经雌激素处理后,可引起Amy的DA及其代谢产物含量减少,而Str的DA及其代谢产物含量不受其影响。OVX雌鼠Amy的DA更新率低于正常及雌激素处理的OVX鼠,Amy组织的DA含量约是Str组织的1/6,而更新率是Str的2倍左右。以上结果提示,雌性大鼠血清雄激素浓度可影响其Amy组织的DA代谢及组织含量,而Str的DA组织含量不因雌激素浓度的改变而变化。     

Increasing Hippocampal Estrogen Receptor Alpha Levels via Viral Vectors Increases MAP Kinase Activation and Enhances Memory in Aging Rats in the Absence of Ovarian Estrogens

We previously demonstrated that aged ovariectomized rats that had received prior estradiol treatment in middle-age exhibited increased levels of estrogen receptor alpha (ERα) in the hippocampus as well as enhanced hippocampal dependent memory as compared to aged rats that had not received mid-life estradiol treatment. These effects persisted long after the estradiol treatment had been terminated. The goal of the current experiment was to determine if increased expression of ERα in the hippocampus, in the absence of exogenously administered estrogens, can impact the hippocampus and cognitive function in aging ovariectomized rats. Middle-aged rats were trained for 24 days on an eight-arm radial maze spatial memory task. All rats were then ovariectomized. Forty days later, rats received either lentiviral delivery to the hippocampus of the gene encoding ERα (lenti-ERα) or a control virus. Rats were tested on delay trials in the radial-maze in which delays of varying lengths were imposed between the fourth and fifth arm choices. Following behavior testing, hippocampi were immunostained using western blotting for ERα, the ERα-regulated protein choline acetyltransferase, and phosphorylation of the ERα-regulated kinases, ERK/MAPK and Akt. Results revealed that aging ovariectomized rats that received delivery of lenti-ERα to the hippocampus exhibited enhanced spatial memory as indicated by increased arm-choice accuracy across delays as compared to ovariectomized rats that received control virus. Western blot data revealed that lenti-ERα delivery significantly increased levels of ERα and phosphorylated ERK/MAPK and had no impact on levels of ChAT or phosphorylation of Akt. Results indicate that increasing hippocampal levels of ERα in aging females in the absence of ovarian or exogenously administered estrogens leads to increases in phosphorylation of ERK/MAPK as well as in enhanced memory.     

The modes of the anterior hypothalamic area as the regulatory center for the gonadotropin release

The effects of the anterior hypothalamic area (AHA) implants of gonadal steroid estrogen and progesterone as well as the effects of electrical stimulation and electrolytic lesion confined in this area on the gonadotropin secretion were investigated in ovariectomized estradiol (20 microgram sc)-primed adult Wistar rats housed in light and temperature controlled room. Progesterone implants evoked the rise of serum LH by 6 hr whereas estradiol implants suppressed serum FSH by 24 hr after implantation. Electrical stimulation effectively depleted both gonadotropins with a latency not shorter than 6 hr. The lesion significantly prevented FSH elevation investigated at 72 hr post ovariectomy and potentiated FSH secretion in response to estradiol treatment at 3 week post ovariectomy. The result revealed the involvment of the AHA in LH release mechanism which required progesterone activation while its involvement in FSH regulatory mechanism depended upon estrogen. The area was elucidated as the inhibitory as well as the stimulatory loci for the feedback action of estrogen on FSH release.     

Estrogen receptors and the activity of nitric oxide synthase in the artery of female rats receiving hormone replacement therapy

OBJECTIVE: To observe the expression of estrogen receptor and the activity of NOS in the arteries of female rats receiving estrogen replacement therapy. METHODS: Seventy-two female rats were randomly divided into four groups: group A: sham-ovariectomy; group B: ovariectomy; group C: ovariectomy with estrogen replacement therapy (benzoate estradiol, 5 microg i.m. once in 2 days); group D: ovariectomy with estrogen and progesterone replacement therapy (benzoate estradiol, 5 microg i.m. once in 2 days and progesterone, 1 mg i.m. once in 2 days). The rats were killed after 2 months. The receptor-binding assay was adopted to measure the estrogen receptors in the arteries of the rats, and the activity of NOS in the arteries was assessed by the hemoglobin reductase method. RESULTS: The ER number and NOS activity in the arteries of the ovariectomized group are less than those in sham-ovariectomy group (p<0.05). The ER number and NOS activity in the arteries of groups C and D are larger and higher than those in the ovariectomized group (p<0.05). No significant differences in the ER number and NOS activity were observed between groups C and D. CONCLUSION: The ER number and NOS activity in the rat artery significantly decrease after ovariectomy, while hormone replacement therapy can significantly increase the artery NOS activity and retain the ER number in the artery of the ovariectomized rats to normal level. The result may contribute to explaining the beneficial effect of estrogen in the prevention of coronary artery diseases in postmenopausal women.     

Effects of nomegestrol acetate administration on central and peripheral beta-endorphin and allopregnanolone in ovx rats

The aim of this study was to investigate the effects of nomegestrol acetate (NOMAc) on the central nervous system by analyzing the neurosteroid allopregnanolone and the opioid beta-endorphin (beta-endorphin). 104 Wistar female rats were used in this study; one group of fertile and one group of ovariectomized rats were used as control. The others were ovariectomized and they underwent a 2-week oral treatment of NOMAc (0.05, 0.1, 0.2, 0.5, 1mg/kg/day), alone or with 0.05 mg/kg/day of estradiol valerate (E2V). Allopregnanolone and beta-endorphin were assessed in different brain areas and in circulation. Ovariectomy decreased allopregnanolone anywhere except in the adrenal gland and E2V reversed the effects of ovariectomy. 0.5 and 1mg/kg/day of NOMAc increased allopregnanolone levels in hippocampus. Combined administration of 1mg/kg/day of NOMAc plus E2V induced a further increase of allopregnanolone levels in hippocampus, hypothalamus, and anterior pituitary. NOMAc (1mg/kg/day) decreased the adrenal content of allopregnanolone, both by itself and associated with E2V. NOMAc increased hippocampal and hypothalamic content of beta-endorphin at the highest doses, and it increased positively E2V action, at 1mg/kg/day, also in anterior pituitary and plasma. These findings reinforce the clinical data regarding the capability of NOMAc to modulate the pathways involved in mood and behaviour. In fact, due to the NOMAc action on hippocampus, hypothalamus, and anterior pituitary, our results highlight the selectivity of NOMAc on part of the limbic system and the anterior pituitary, regarding both allopregnanolone and beta-endorphin.