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Objective: Heart fat deposition has been linked to atherosclerosis, and both accelerate after menopause. Hormone therapy (HT) may differentially slow heart fat deposition and progression of atherosclerosis, depending on the specific HT agent or its route of administration. Our objective was to evaluate the effects of different HT agents, oral and transdermal, on associations between heart fat accumulation and atherosclerosis progression, measured by carotid intima-media thickness (CIMT), in recently menopausal women from the Kronos Early Estrogen Prevention Study (KEEPS) trial., Methods: KEEPS was a randomized, placebo-controlled trial of the effects of 0.45 mg/d oral conjugated equine estrogens (o-CEE) or 50 mcg/d transdermal 17β-estradiol (t-E2), compared with placebo, on 48 months progression of CIMT. Epicardial adipose tissue (EAT) and paracardial adipose tissue (PAT) volumes were quantified by computed tomography., Results: In all, 467 women (mean age [SD] 52.7 [2.5]; 78.2% White; 30% on o-CEE, 30.8% t-E2, 39.2% placebo) with heart fat volumes and CIMT at baseline and 48 months were included. EAT and PAT changes were not associated with CIMT progression; however, the assigned treatment significantly modified the association between PAT (but not EAT) change and CIMT progression. In the o-CEE group, adjusted CIMT progression was 12.66 μm (95% confidence interval [CI] 1.80, 23.52) lower than in t-E2 group (P = 0.02), and 10.09 μm (95% CI 0.79, 19.39) lower than in placebo group (P = 0.03), as per 1-SD increase in PAT., Conclusion: Compared with t-E2, o-CEE appears to slow down the adverse effect of increasing PAT on progression of atherosclerosis. Whether this beneficial association is specific to CEE or to the oral route of CEE administration is unclear and should be assessed further.

Changes in pituitary-ovarian hormones across the menopausal transition have multiple physiological consequences. However, little is known about how the major types of postmenopausal hormone therapy (HT) affect pituitary-ovarian hormonal relationships. This study evaluated these relationships in recently menopausal women (52.45 ± 2.49 yr of age) in the Kronos Early Estrogen Prevention Study (KEEPS) who were compliant to randomized, double-blinded treatment with oral conjugated equine estrogen (o-CEE; n = 109), transdermal 17β-estradiol (t-E2; n = 107), or placebo ( n = 146). Androstenedione, testosterone, 17β-estradiol, estrone, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) were measured in serum before (baseline) and 48 mo after randomization to treatment. Descriptive summaries of hormone levels were performed, and multiple regression analyses were used to examine the effects of o-CEE, t-E2, and placebo on these hormone levels at 48 mo, adjusting for baseline levels. A network analysis examined the covariance of changes in hormone levels over the 48 mo within treatment groups. As expected, at 48 mo of treatment, hormone levels differed between women in the two active treatment groups compared with placebo, and network analysis indicated stronger relationships among hormone levels in the t-E2 and o-CEE groups compared with placebo. Associations among testosterone, 17β-estradiol, FSH, and LH differed between the o-CEE group compared with t-E2 and placebo groups. Thus, two common HT regimens differentially alter pituitary-ovarian hormone levels, altering feedback cycles and interhormonal associations in recently menopausal women. These interactions provide the basis for future studies investigating the impact of hormonal modulation of aging, including cognitive decline in women.

Objective: This study determined whether two different formulations of hormone therapy (HT): oral conjugated equine estrogens (o-CEE; 0.45 mg/d, n = 209), transdermal 17β-estradiol (t-E2; 50 μg/d, n = 201) plus cyclic progesterone (Prometrium, 200 mg) or placebo (PBO, n = 243) affected sleep domains in participants of the Kronos Early Estrogen Prevention Study., Methods: Participants completed the Pittsburgh Sleep Quality Index at baseline and during the intervention at 6, 18, 36, and 48 months. Global sleep quality and individual sleep domain scores were compared between treatments using analysis of covariance, and correlated with vasomotor symptom (VMS) scores using Spearman correlation coefficients., Results: Global Pittsburgh Sleep Quality Index scores (mean 6.3; 24% with score >8) were similar across groups at baseline and were reduced (improved sleep quality) by both HT (average change -1.27 [o-CEE] and -1.32 [t-E2]) when compared with PBO (-0.60; P = 0.001 [o-CEE vs PBO] and P = 0.002 [t-E2 vs PBO]). Domain scores for sleep satisfaction and latency improved with both HT. The domain score for sleep disturbances improved more with t-E2 than o-CEE or PBO. Global sleep scores significantly correlated with VMS severity (rs = 0.170, P < 0.001 for hot flashes; rs = 0.177, P < 0.001 for night sweats). Change in scores for all domains except sleep latency and sleep efficiency correlated with change in severity of VMS., Conclusions: Poor sleep quality is common in recently menopausal women. Sleep quality improved with both HT formulations. The relationship of VMS with domains of sleep suggests that assessing severity of symptoms and domains of sleep may help direct therapy to improve sleep for postmenopausal women.

Midlife vascular risk factors influence later cognitive decline and Alzheimer's disease (AD). The decrease in serum estradiol levels during menopause has been associated with cognitive impairment and increased vascular risk, such as high blood pressure (BP), which independently contributes to cognitive dysfunction and AD. We describe the extent to which vascular risk factors relate to cognition in healthy, middle-aged, recently postmenopausal women enrolled in the Kronos Early Estrogen Prevention Cognitive and Affective Study (KEEPS-Cog) at baseline. KEEPS-Cog is a double-blind, randomized, placebo-controlled, parallel group, clinical trial, investigating the efficacy of low-dose, transdermal 17β-estradiol and oral conjugated equine estrogen on cognition. All results are cross-sectional and represent baseline data only. Analyses confirm that the KEEPS-Cog cohort (n = 571) was middle aged (mean 52.7 years, range 42-59 years), healthy, and free of cognitive dysfunction. Higher systolic BP was weakly related to poorer performance in auditory working memory and attention (p = 0.004; adjusted for multiple comparisons p = 0.10). This relationship was not associated with endogenous hormone levels, and systolic BP was not related to any other cognitive domain. BP levels may be more sensitive than other vascular risk factors in detecting subtle differences in cognitive task performance in healthy, recently menopausal women. Lower BP early in menopause may affect cognitive domains known to be associated with AD.

Rationale: The mechanism of atherogenesis includes leukocyte adhesion to endothelial cells followed by migration into the subendothelial space. The polysialylated neural cell adhesion molecules (PSA-nCAMs) are a group of hydrophilic neural cell adhesion molecule (NCAM) isoforms that inhibit NCAM: NCAM association, thereby blocking cell: cell adhesion. During previous studies, we demonstrated that sialylation of specific NCAMs are upregulated at proestrus in the rat and that PSA-nCAM is expressed by the rat vascular endothelium., Methods and Results: In this study, we sought the presence of PSA-nCAM in human vessels and regulation of its expression in estradiol-treated human umbilical vascular endothelial cells (HUVEC). Immunoreactive PSA-nCAM (ir-PSA-nCAM) was shown in blood and lymph vessels of adult rats and human brain, skin, liver, lung, cervix, endometrium, and ovary. Staining for ir-PSA-nCAM was present on the glycocalyceal surface of estradiol-treated HUVEC, but not in the presence of the estrogen receptor (ER)-blocker fulvestrant. Western blotting confirmed these findings., Conclusions: PSA-nCAM is widely present in the glycocalyx of human and rat vascular endothelium. It also is expressed by HUVEC, in which it is induced by estradiol. The estrogen-regulated presence of vascular PSA-nCAM could diminish NCAM-dependent interactions between vessels and circulating leukocytes, thereby impeding vascular inflammation and atherogenesis, and, contributing to estrogen-induced cardioprotection. This hypothesized action is presently under study.

Observational and epidemiological studies suggest that menopausal hormone therapy (MHT) reduces cardiovascular disease (CVD) risk. However, results from prospective trials showed neutral or adverse effects most likely due to differences in participant demographics, such as age, timing of initiation of treatment, and preexisting cardiovascular disease, which reflected in part the lack of basic science information on mechanisms of action of hormones on the vasculature at the time clinical trials were designed. The Kronos Early Estrogen Replacement Study (KEEPS) is a prospective, randomized, controlled trial designed, using findings from basic science studies, to test the hypothesis that MHT when initiated early in menopause reduces progression of atherosclerosis. KEEPS participants are younger, healthier, and within 3 years of menopause thus matching more closely demographics of women in prior observational and epidemiological studies than women in the Women's Health Initiative hormone trials. KEEPS will provide information relevant to the critical timing hypothesis for MHT use in reducing risk for CVD.

Gonadal steroids induce synaptic plasticity in several areas of the adult nervous system. In the arcuate nucleus of adult female rats, 17beta-estradiol triggers synaptic remodeling, resulting in a decrease in the number of inhibitory synaptic inputs, an increase in the number of excitatory synapses, and an enhancement of the frequency of neuronal firing. In the present paper, we studied the specificity of hormonal effects by determining the changes in synaptic connectivity of tyrosine hydroxylase (TH) immunoreactive (IR) neurons in the arcuate nucleus. We combined pre-embedding TH and post-embedding gamma-aminobutyric acid (GABA) immunostaining, and performed unbiased stereological measurements in gonadectomized and 17beta-estradiol-treated rats. We conclude that the synaptic connectivity of the TH-IR neurons is different from the other, nonlabeled population, and the response to estradiol is not uniform. TH-IR (dopaminergic) arcuate neurons of both male and female rats have more GABAergic (inhibitory) axosomatic inputs than the nondopaminergic population. Our study shows that the effect of 17beta-estradiol is sex and cell specific in the sense that not all arcuate neurons are affected by the structural synaptic remodeling. In ovariectomized females hormone treatment decreased the numerical density of GABAergic axosomatic synapses on TH-IR, but not on nondopaminergic, neurons, whereas in orchidectomized males, 17beta-estradiol treatment increased inhibitory synapses onto nondopaminergic neurons but did not affect the number of inhibitory terminals onto TH-IR neurons. The hormone-induced plastic changes in synaptic connectivity of TH-IR neurons may serve as the morphological basis for the cyclical regulation of the anterior pituitary.

Despite an abundance of data in animals, there is little research in humans regarding how estrogen and serotonin (5-HT) may interact to influence cognition. Through the use of estrogen treatment (ET) and tryptophan depletion (TRP-D) in a within-subject design involving healthy menopausal women, we have manipulated both estrogen and 5-HT in order to evaluate their individual and joint effects. Although neither manipulation influenced visuospatial learning, a significant interaction suggested that estrogen exerted a protective effect on verbal memory, such that TRP-D impaired performance to a greater extent before the administration of ET. In consonance with this finding, ET was associated with a small, but positive mood effect on the day following active TRP-D. In addition, ET significantly improved letter-cued verbal fluency with and without TRP-D. Finally, time since last menstrual period was significantly associated with verbal memory scores, such that longer length of hypogonadism resulted in decreased verbal memory performance. These data support the interaction of estrogen and 5-HT in nonreproductive behavior in humans as well as highlight the role of ovarian steroids in cognition.

Alzheimer's disease (AD) is three times more prevalent in women than men, and epidemiological studies have shown that estrogen replacement in aging women forestalls the onset of AD. Hyperphosphorylation of the tau protein that forms the neurofibrillary tangles found in AD brains might be responsible for the breakdown of microtubules in affected neurons. The mechanisms by which tau protein is phosphorylated in the AD brain are not fully understood. Using a human neuroblastoma cell line (SH-SY5Y) and primary cultures of newborn male or female rat cerebral cortical neurons, we investigated the effect of 17beta-estradiol on tau protein expression and phosphorylation. We found that estradiol increased total tau and induced dephosphorylation at the proline-directed site of the molecule. Further, estradiol prevented okadaic acid-induced hyperphosphorylation of tau in both proline- and non-proline-directed sites, and antiestrogens blocked this effect. To our knowledge, this is the first report of an effect of estradiol on naturally occurring and induced tau phosphorylation. This assumes special significance because the estrogen action was found to be sexually dimorphic in rat cortical neurons and differentiation-sensitive in human neuroblastoma cells.

We have for the first time exposed estrogen's role in the epithelial ovarian cancer (OVCA) metastatic cascade and discovered that it is related to the induction of ezrin over-expression. 17beta Estradiol (E2) was administered to SKOV3 (ERalpha>beta) and DOV13 (ERalpha

It has been shown that gonadal steroids have the capacity to induce synaptic plasticity in certain areas of the nervous system. Previously we have demonstrated that due to the effect of estradiol there is a transient decrease in the number of GABAergic axo-somatic synapses in the arcuate nucleus. By using systemic application of the tracer Fluorogold we retrogradely labeled a subpopulation of arcuate neurons that project to the median eminence. We than applied the disector method for synapse quantification and found that these "hypophysiotropic neurons" receive less axo-somatic inputs. We found that 17beta-estradiol induced a decrease in the numerical density of axo-somatic contacts of these retrogradely-labeled neoroendocrine cells. Our data support the hypothesis that the hormonally driven morphological synaptic plasticity is neuron specific within the arcuate nucleus and plays a decisive role in the regulation of anterior pituitary.

Background: High doses of estrogen can promote tumor regression in postmenopausal women with hormone-dependent breast cancer, but the mechanism is unknown. We investigated the molecular basis of this process by using LTED cells, which were derived by growing MCF-7 breast cancer cells under long-term (6-24 months) estrogen-deprived conditions., Methods: We treated LTED and MCF-7 cells with various concentrations of 17beta-estradiol (estradiol) and assayed their growth by counting the cells and measured apoptosis by annexin V staining and DNA fragmentation. Using western blot analysis, we also examined the expression of the apoptosis-inducing system of the Fas death receptor protein and its ligand, FasL, in these cells. To assess the involvement of Fas and FasL in the induction of apoptosis in LTED cells, we used activating anti-Fas antibodies and the universal caspase inhibitor Z-VAD. Finally, we examined the expression of Fas protein in E8CASS and BSK3 cells, two other cell lines derived by depriving MCF-7 cells of estrogen long term, and the responses of these cells to high-dose estradiol. All statistical tests were two-sided., Results: High concentrations of estradiol (>or=0.1 nM) resulted in a statistically significant, 60% reduction in the growth of LTED cells (P< .001) and in a sevenfold increase in apoptosis (P< .001) as compared with levels in vehicle-treated cells. Both LTED and MCF-7 cells expressed FasL, but only LTED cells expressed Fas. Treatment of LTED cells with 0.1 nM estradiol increased the expression of FasL. Activating anti-Fas antibodies increased apoptosis of LTED cells, which was further stimulated by estradiol. Z-VAD blocked estradiol-induced apoptosis. E8CASS cells, which express Fas protein, but not BSK3 cells, which do not, also responded to 0.1 nM estradiol by increasing apoptosis., Conclusion: Tumor regression induced by high-dose estrogen therapy in postmenopausal woman may result from estrogen activation of Fas-mediated apoptosis.

To assess their role in the regulation of gonadotropin secretion in primates, we determined the number of synaptic connections on gondotropin releasing hormone (GnRH)- and non-GnRH neurones of the arcuate nucleus of ovariectomized (OVX) and OVX plus oestradiol benzoate-treated African green monkeys. After 24 h (day 1), 48 h (day 2) and 8 days (day 8), we performed immunostaining for GnRH. Using electron microscopy, synapses on GnRH- and randomly selected non-GnRH neurones were counted and characterized according to the classification of Gray (symmetric/inhibitory or asymmetric/excitatory). Serum concentrations of oestradiol (OVX) needed to 232 pg/ml on day 1, 63 pg/ml on day 2 and 45 pg/ml on day 8. Concentrations of luteinizing hormone (LH) fell after ovariectomy to 9 microg/ml on day 1, surged to 93 microg/ml on day 2 and declined again by day 8. (a) Ten days after ovariectomy, there were no synapses on GnRH neurones, whereas non-GnRH cells received substantial inhibitory innervation and moderate excitatory input. (b) On day 1, GnRH neurones had highest numbers of inhibitory synapses, while inhibitory synapses on non-GnRH neurones decreased, whereas numbers of excitatory synapses remained relatively unchanged compared to OVX monkeys. (c) By day 2, synapses on GnRH neurones decreased, while synapses increased on non-GnRH cells compared to day 1. (d) On day 8, the most pronounced alteration on GnRH cells was an elevated inhibitory input while non-GnRH neurones received the fewest synapses compared to day 2. We conclude that during an oestrogen-induced LH surge, synapses on GnRH- and mixed non-GnRH neurones are differentially regulated. These findings suggest that oestrogen modulation of arcuate nucleus synapses may be important in the regulation of gonadotropin secretion in monkeys.

During neoplastic growth and metastasis, the immune system responds to the tumor by developing both cellular and humoral immune responses. In spite of this active response, tumor cells escape immune surveillance. We previously showed that FasL expression by breast tumor plays a central role in the induction of apoptosis of infiltrating Fas-immune cells providing the mechanism for tumor immune privilege. In the present study, we showed that FasL in breast tissue is functionally active, and estrogen and tamoxifen regulate its expression. We identified an estrogen recognizing element like-motif in the promoter region of the FasL gene, suggesting direct estrogen effects on FasL expression. This was confirmed by an increase in FasL expression in both RNA and protein levels in hormone sensitive breast cancer cells treated with estradiol. This effect is receptor mediated since tamoxifen blocked the estrogenic effect. Interestingly, tamoxifen also inhibited FasL expression in estrogen-depleted conditions. Moreover, an increase in FasL in breast cancer cells induces apoptosis in Fas bearing T cells and, tamoxifen blocks the induction of apoptosis. These studies provide evidence that tamoxifen inhibits FasL expression, allowing the killing of cancer cells by activated lymphocytes. This partially explains the protective effect of tamoxifen against breast cancer.

Adult sexual dimorphism in neuronal cell number is controlled by estrogen exposure during a tightly defined period of rat brain development. The mechanisms of estrogen's effect are unknown; one possibility is regulation of programmed cell death (apoptosis). In this study we have shown that estradiol can function as a neuroprotective agent or an inducer of apoptosis, depending on the estrogen receptor-subtype present in the cell. Thus, ERalpha has a neuroprotective effect, while ERbeta mediates the induction of apoptosis in neuronal cells. Moreover, we show that estrogen-induced apoptosis through ER-beta requires the expression of Fas- and Fas ligand (FasL) proteins, since the absence of FasL in neurons prevents this effect. Furthermore, we demonstrate that microglia-secreted products induce the expression of FasL necessary to mediate estradiol-ERbeta apoptotic effect. These findings may explain the dichotomous effect of fetal estradiol on the adult neuronal number., (Copyright 2000 John Wiley & Sons, Inc.)

Objective: The incidence of sleep apnea syndrome (SAS) in women increases after menopause. Progestins alone do not alleviate SAS in menopausal women. However, progestins may require concomitant estrogen administration and estrogen alone may stimulate breathing during sleep. To test these hypotheses, we studied the effects of estrogen alone and estrogen combined with progestin on SAS in menopausal women, using a prospective, cross-over, inception cohort study., Design: In this pilot study, five women who developed SAS after menopause underwent 2 nights of polysomnography to obtain a baseline, then returned for polysomnography after 3-4 weeks of taking micronized 17 beta-estradiol (E2) and after 10-12 days of taking E2 combined with medroxyprogesterone acetate (E2 + P). Sleep stages were scored according to Rechtshaffen and Kales, frequency and length of apneas were recorded for each subject each night, and the data were analyzed by Student's t test., Results: E2 and E2 + P both reduced the Respiratory Distress Index. E2 also raised the lowest oxygen desaturation associated with apneic episodes. Total minutes of rapid eye movement sleep increased, and the number of waking episodes decreased when the women were taking E2 and E2 + P, as previously reported., Conclusions: Within 1 month after initiating E2 or E2 + P, SAS was reduced in all patients. The Respiratory Distress Index decreased by 25%, and the addition of progestin brought the SAS reduction to 50% in this pilot study. A randomized study in a large group of patients is justified by the findings of this study. Because SAS increases the risk of cardiovascular disease and fatal accidents, the amelioration of SAS by sex steroid hormones could have significant implications for the health of menopausal women.

Estrogen is a major mitogenic stimulus to established breast cancer. Estrogen sources include ovarian, extraglandular sites and breast tissue. Which source primarily maintains benign and breast cancer tissue estrogen concentrations remains unclear. While macrophages may comprise up to 50% of the mass of breast carcinomas, previous studies neglected to study them as possible sources of estrogen. We present evidence that breast macrophages constitute an in situ source of estradiol and that the amount produced is sufficient to mediate cellular proliferation. We utilized immunohistochemistry and RT-PCR to study cell-specific aromatase expression in (i) 29 breast biopsies, (ii) human monocytes/macrophages and (iii) a myeloid cell line (THP-1) capable of differentiating into macrophages. Use of a breast cancer cell line (MCF-7) provided biologic confirmation of the role of aromatization in cell proliferation. We demonstrated considerable amounts of immunoreactive-aromatase (irARO) in breast tissue macrophages and a positive correlation between the proportion of irARO present in macrophages and lesion severity. Using in vitro techniques, we demonstrated that monocytes and THP-1 cells require differentiation into macrophages to produce aromatase in amounts approaching placental levels. The amount of estrogen produced by THP-1 cells stimulated MCF-7 cells to proliferate, an effect blocked by aromatase inhibitors. Estrogen production by macrophages in breast tissue appears sufficient to stimulate the proliferation of adjacent epithelial cells and to autoregulate cytokine production. These findings represent a new dimension of cellular regulation in breast tissue with major biologic implications, amenable to pharmacological manipulation.

Objective: It is well established that gonadal steroids have direct in vivo and in vitro effects on neurons. To further study these effects, we used rat PC 12 cells to examine the effects of estrogen receptor (ER) ligands on neuronal morphology., Design: PC 12 cells constitutively express ER beta, but only strongly express ER alpha after long-term priming with nerve growth factor (NGF). We therefore primed PC12 cells with NGF for 14 days before testing them for estradiol (10(-9)M)- and/or raloxifene (10(-7) M)-induced neurite growth. Neurite growth was assessed by quantitative light microscopy. As control, ER status of the PC12 cells was assessed by reverse transcription-polymerase chain reaction (RT-PCR)., Results: In this study, both estradiol and raloxifene induced the outgrowth of neurites in NGF-treated PC 12 cells (p < 0.05). The combination of estradiol- and raloxifene-induced neurite growth was statistically greater than the effects of either agent alone. RT-PCR confirms that NGF-treated PC 12 cells express both ERalpha and ERbeta., Conclusions: This report is the first on the neurotrophic effect of raloxifene. At 10(-7) M, raloxifene's effect equaled that of estradiol; moreover, raloxifene did not block the neurite growth of simultaneously estradiol-treated PC 12 cells, despite its functional antiestrogenic effects in vivo. We conclude that raloxifene is estrogen agonistic in this animal model and therefore studies are warranted to delineate the relationship between steroidal estrogen and raloxifene.

Bcl-2, a protein which negatively modulates apoptosis, is up-regulated by estrogen in several tissues. To determine the effect of estradiol on Bcl-2 in the adult brain, its immunoreactive distribution was examined in the hypothalamic arcuate nucleus of female rats under different endocrine conditions. The number of Bcl-2-immunoreactive neurons was significantly increased (p < 0.001) on the day of estrus compared with proestrus, diestrus and metestrus, was decreased by ovariectomy and showed a dose-response increase after estradiol administration to ovariectomized rats. Progesterone, when injected simultaneously with estradiol, reduced the effect of estradiol. These findings indicate that ovarian hormones regulate Bcl-2 in hypothalamic neurons and suggest that this protein may be involved in the neuroprotective effects of estrogen.

Interactions between glutamate and gonadal steroids are involved in the regulation of limbic and hypothalamic functions. We hypothesized that hormonal signals affect excitatory neurotransmission by regulating the expression of glutamate receptors (GluR) in limbic and hypothalamic regions. To test this hypothesis, first, the coexpression of dl-alpha-amino-3-hydroxy-5-methyl-4-isoxazone-propionate (AMPA) GluR1, GluR2/3, and androgen receptors or estrogen receptors was revealed in the same cells of septal, amygdaloid, and hypothalamic areas by double immunocytochemistry. The highest incidence of co-localization was detected in hypothalamic regions. To demonstrate a regulatory role of testosterone or estradiol on AMPA receptor expression, the hormonal milieu of male and female rats was manipulated by gonadectomy and hormonal treatment. GluR1 and GluR2/3 expression was assessed by Western blots. Statistical analysis demonstrated that testosterone and estradiol have a stimulatory influence on the expression of AMPA receptors in the hypothalamus. The regulatory effect of estradiol on AMPA receptors was found to be site and gender specific: after estradiol treatment, samples taken from the hypothalamus contained increased levels of GluR1 and GluR2/3, whereas in the septum, bed nucleus and amygdala, no changes could be detected. Furthermore, the increase in hypothalamic GluR 2/3 levels was two times higher in females, compared with males, whereas the changes in hypothalamic GluR 1 levels showed no sex differences. Our results support the hypothesis that the interaction between gonadal steroids and glutamate involves hormone regulation of GluR. This mechanism seems to be gender and site specific, suggesting that excitatory neurotransmission and related physiological mechanisms also may be distinctly different in males and females.

We have demonstrated that estrogen induces synaptic reorganization in the hypothalamic arcuate nucleus of female rats during the ovarian cycle and proposed that estrogen-induced synaptic retraction plays a role in the disinhibition of gonadotropin secretion that occurs during the afternoon of proestrus. This so-called positive feedback of gonadotropins is developmentally determined. It is present in female rats and absent in males. To confirm the role of the estrogen-induced synaptic retraction in positive feedback, we tested whether administration of estrogen to male rats also fails to induce synaptic remodeling of the arcuate nucleus. Male and female rats were gonadectomized and studied as follows. One month following gonadectomy, animals received either a single injection of estradiol (100 micrograms/animal in sesame oil; 12 males and 12 females) or vehicle (6 males and 6 females). Twenty-four hours following injections, all animals in the vehicle-injected group and 6 animals of each sex in the treatment groups were sacrificed, while the rest of the animals were killed 48 h following the hormone injections (6 per group). As expected, quantitative electron microscopic analysis of the female arcuate nuclei revealed that compared to oil-injected controls, estradiol induced drastic decreases in the overall synapse counts by 24 h (121 +/- 10 vs. 74 +/- 5 synapses/1,000 microns membrane; p < 0.05). Synaptic counts had recovered to control levels by 48 h. On the contrary, in males, estradiol treatment did not cause changes in the total synapse counts at either time. As a further control, the lack of an estrogen-induced gonadotropin surge in long-term castrate males was also confirmed. Our study confirmed that in males estradiol does not alter the net synaptology of the arcuate nucleus or cause gonadotropin positive feedback. This is in clear contradistinction to females which show both synaptic plasticity and gonadotropin-positive feedback upon receiving exogenous or endogenous estrogen. The lack of estrogen-induced synaptic plasticity may be an underlying mechanism in the abolishment of positive gonadotropin, feedback in developing males and the development of constant estrus in aging female rats.

We have shown that the ovarian cycle is accompanied by a fall in the axosomatic synapses on randomly selected neurons of the arcuate nucleus by the morning of estrus, with a return to the preovulatory levels by the morning of metestrus, indicating a possible role in positive feedback. However, it remains to be proven that the circulating estradiol is the actual regulator of this physiological synaptic plasticity, or that estrogen-induced synaptic retraction precedes in the surge of gonadotropins at midcycle. To resolve these questions, we used an estradiol-immunoneutralization protocol and studied arcuate nucleus axosomatic synapses during the critical points of the estrous cycle. In addition to blocking positive feedback, estrogen immunoneutralization abolished synaptic retraction in the arcuate nucleus. As a positive control, the nonbinding estrogen diethylstilbestrol maintained the gonadotropin surge and synaptic retraction in the antiestradiol-treated animals. Furthermore, in the diluent-treated cycling control females, the synaptic retraction was found to precede the preovulatory LH surge. We demonstrated that the midcycle synaptic retraction of arcuate nucleus synapses is induced by the preovulatory estradiol surge, and that these morphological events precede the preovulatory gonadotropin surge. Taken together, these observations strongly suggest that the hypothalamic mechanism underlying the physiological disinhibition of gonadotropins at midcycle (positive feedback) requires estrogen-induced synaptic retraction in the arcuate nucleus.

Neurotrophic effects of estradiol and insulin-like growth factor-I were assessed in primary cultures from fetal rat hypothalamus. Cultured neurons were immunostained with an antibody for the microtubule-associated protein-2. While both estradiol and insulin-like growth factor-I increased the number of microtubule-associated protein-2-immunoreactive neurons and the extension of immunoreactive processes, the effect of these two factors was not additive. The estradiol-induced increases in neuronal numbers and extension of neuronal processes were blocked by either the estrogen receptor antagonist ICI 182,780 or by an anti-sense oligonucleotide to the estrogen receptor. Furthermore, incubation of the cultures with an anti-sense oligonucleotide directed against the insulin-like growth factor-I messenger RNA also blocked the effect of estradiol. In turn, the effects of insulin-like growth factor-I were blocked by the estrogen receptor antagonist ICI 182,780 and by the anti-sense oligonucleotide to the estrogen receptor. These findings suggest that estradiol-induced activation of the estrogen receptor in developing hypothalamic cells requires the presence of insulin-like growth factor-I, and that both estradiol and insulin-like growth factor-I use the estrogen receptor as a mediator of their trophic effects on hypothalamic neurons.

During the prenatal period, gonadal steroid environment induces dramatic sexually dimorphic changes in the nervous system. We have used in vitro methods to study the mechanism and timing of hormonal influences on neuronal sprouting and myelination during the prenatal period. Organotypic cultures of hypothalamus and lumbar spinal cord (SC) slices from rat fetuses were grown on plasma clot or in hyaluronic acid and exposed to estrogen (17 beta estradiol) and testosterone (T) during cultivation. Both steroid hormones were active: 17 beta estradiol enhanced sprouting of hypothalamic neuronal fibers and increased the amount of synapses. In SC cultures T induced regeneration of thick nerve processes and an early onset of myelination, mainly of peripheral myelin.

1. In the rat hypothalamic arcuate nucleus, estradiol induces coordinated changes in the number of axosomatic synapses, the amount of glial ensheathing, and the ultrastructure of the membrane of neuronal somas. In the present study we used conventional electron microscopy and freeze-fracture to examine cellular mechanisms responsible for the estradiol-induced changes at the membrane level. 2. In freeze-fracture replicas taken 10-60 min and 24 hr after injection of 17 beta-estradiol to adult ovariectomized females, it was found that there was a rapid increase in the number of exoendocytotic images that reached a plateau by 30 min. 3. In thin sections from animals injected 24 hr earlier we demonstrated a significant increase in coated vesicles in the periphery of the neurons and coated pits in the perikaryal membranes and decreased axosomatic synapses. 4. We conclude that these morphological alterations are signaling estrogen-induced transport and/or turnover of perikaryal membrane constituents and extracellular components which may affect interneuronal and neuroglial interactions.

Recent evidence indicates that, in addition to their well known effects on neurons, gonadal steroids may exert part of their neural effects through astroglia. In adult female rats astroglia participate in the phasic remodelling of synapses that takes place during the estrous cycle in the arcuate nucleus of the hypothalamus under the influence of estradiol. Astroglia also appear to be involved in the genesis of sex differences in synaptic connectivity. Gonadal steroids influence hypothalamic astroglia differentiation in vitro and in vivo. In monolayer mixed neuronal-glial cultures from fetal rat hypothalami, estradiol induces a progressive differentiation of astrocytes from a flattened epithelioid morphology to bipolar, radial and stellate shapes. This effect of estradiol on astroglia is dependent on the expression of specific molecules on the neuronal surface, such as the polysialic acid-rich form of the neural cell adhesion molecule. In the rat arcuate nucleus in situ, perinatal androgen influences astroglia gene expression and differentiation, resulting in a sex difference in astroglia organization by postnatal day 20. By this day, the amount of neuronal surface covered by astroglial processes is higher in males than in females. This difference in the coverage of neuronal surface by astroglia may be directly related to the reduced number of synaptic contacts that is established on the soma of male neurons compared to females.

Gonadal steroids are known to affect astroglial morphology in developing and adult animals. Earlier studies of mixed neuronal-glial cultures from fetal rat hypothalamus showed that glial fibrillary acidic protein (GFAP)-immunoreactive cells with a polygonal shape were transformed into process-bearing cells upon exposure to the ovarian hormone estradiol. This effect was dependent on a direct contact of astroglia with living hypothalamic neurons. The present study shows that somata and processes of neurons in such cultures were immunoreactive for polysialic acid (PSA); astroglia were immunonegative. PSA appears to participate in the estradiol-induced shape changes since treatment with endoneuraminidase, an enzyme that specifically removes PSA from the cell surface, abolished PSA immunostaining and prevented the 17 beta-estradiol-induced morphological changes of astroglia. In contrast, treatment with endoneuraminidase did not affect astroglial shape changes induced by basic fibroblast growth factor (bFGF), nor those induced by the addition of neurons to glial cultures. These results suggest that PSA on neuronal membranes, probably linked to the highly sialylated isoform of the neural cell adhesion molecule, is necessary for the expression of certain hormonally-regulated neuro-glial interactions.

Light and electron microscopic triple immunostaining was performed on coronal vibratome sections prepared from the hypothalamus of ovariectomized (OVX) and OVX plus estrogen-treated African green monkeys (Cercopithecus aethiops). Immunoreactivity for progesterone receptors (PRs) and neuropeptide-Y (NPY) was visualized by a dark blue to black nickel diaminobenzidine reaction, while the tyrosine hydroxylase-containing perikarya were labeled with a light brown diaminobenzidine reaction. In the OVX plus estrogen-treated material, 30% of the tyrosine hydroxylase-immunoreactive neurons contained PR-immunopositive nuclei. The majority of these cells were found in the central portion of the periventricular area, and a few could be observed in the anterior hypothalamus and the arcuate and dorsomedial hypothalamic nuclei. These tyrosine hydroxylase-immunoreactive PR-containing cells were surrounded with NPY-immunoreactive axon terminals. A correlated electron microscopic analysis of the same sections revealed synaptic contacts between these NPY-immunoreactive boutons and the PR-containing tyrosine hydroxylase-immunoreactive neurons. In contrast, in the OVX animals, no PR-containing tyrosine hydroxylase-immunoreactive neurons could be detected. In these monkeys, the frequency of synaptic contacts between the NPY-immunoreactive axon terminals and tyrosine hydroxylase-immunopositive cells was similar to that in the OVX plus estrogen-treated monkeys. These observations indicate that in a population of hypothalamic dopamine cells, the presence of nuclear PRs is estrogen dependent, show that these cells are innervated by NPY axons, and suggest that these estrogen-induced PR-containing dopamine neurons are involved in mediation of the effect of NPY on hypophyseal hormone secretion, including ovarian steroid hormone-dependent LH and PRL release.

The number of axosomatic synapses, the length of the synaptic plates and the perimeter of the post synaptic neuronal perikarya were assessed on thin sections from the infundibular hypothalamic nucleus and from the ventrobasal thalamus of 3 adult ovariectomized African green monkeys that were treated with estradiol valerate and of 3 control animals that were injected with vehicle. Estradiol valerate treatment resulted in a 61% [corrected] decrease in the number of axosomatic synapses in the infundibular hypothalamic nucleus. The length of the synaptic plates and the perimeter of the postsynaptic cells were not affected by the hormonal treatment. The decrease in the number of axosomatic synaptic inputs in the infundibular hypothalamic nucleus was accompanied by a significant increase in the glial ensheathing of neuronal somas. No effect of the hormonal treatment was detected in the ventrobasal thalamus. The results indicate that estrogen may induce glial and synaptic plasticity in the hypothalamus of adult primates.

During the estrous cycle there is a phasic synaptic remodelling in the hypothalamic arcuate nucleus, consisting in a loss and regain of axo-somatic synapses during the 48 h period between the morning of proestrus and the morning of metestrus. Synaptic changes are accompanied by cyclic modifications in the number of intramembrane particles in the plasma membrane of arcuate neuronal somas. To test the effect of the ovarian steroids on arcuate axo-somatic synapses we treated castrated females either with oil vehicle, 17 beta-estradiol, progesterone, or a combination of estradiol and progesterone, and observed them for 48 h. The number of axo-somatic synaptic profiles showed a 33% fall by 24 h after estradiol treatment and returned to control levels by 48 h. The effect of estradiol on axo-somatic synapses was accompanied by a marked and reversible modification of the number of intramembrane particles in the plasma membrane of arcuate neuronal somas. Progesterone alone did not affect the number of axo-somatic synaptic profiles nor the number of intramembrane particles, but when administered together with estradiol, blocked the effects of estradiol on neuronal membrane and synapses.

The synaptic connectivity of the rat arcuate nucleus, a hypothalamic area rich in oestradiol receptors, is rapidly affected by physiological modifications of hormonal levels. A rise of oestradiol in plasma elicits a coordinated neuronal-glial response that begins with a rapid fall in the number of small (< 10 nm) intramembrane particles and a rapid increase in the number of large (> 10 nm) intramembrane particles in neuronal membranes, followed by a modification in the branching of astrocytic processes and finally results in decreased number of axo-somatic synapses and increased glial wrapping of the neuronal somas. In the course of a series of studies aimed to test possible non-genomic effects of oestradiol on neuronal membranes we analyzed the effect of the systemic administration of the protein synthesis inhibitor cycloheximide on the ultrastructure of arcuate neurons and granule cells of the cerebellar cortex, an area of the brain with low levels of estrogen receptors. Cycloheximide resulted in a significant inhibition of protein synthesis in hypothalmus and cerebellum of ovariectomized rats. Under these circumstances, the number of small intramembrane particles was reduced in hypothalamic and cerebellar neuronal membranes while the number of large intramembrane particles showed a decrease in cerebellar membranes and a transient increase in arcuate neuronal somas. Furthermore, cycloheximide resulted in an increased glial wrapping of arcuate neuronal somas but not of cerebellar granule cells. The ensheathing of arcuate neurons by glial was associated with a 41% decrease in the number of axo-somatic synapses. These results indicate that the protein synthesis inhibitor cycloheximide may elicit the integrated neuronal-glial response that is associated with the hormonally induced remodelling of synaptic contacts on arcuate neurons.

The preovulatory ovary is composed of two primary tissue components, stroma and follicles. To assess the role of these tissue compartments in ovarian steroidogenesis, stromal tissue, follicular tissue, and a mixture of both tissues from pregnant mare serum gonadotropin (PMSG)-treated, prepubertal rats were perifused simultaneously for 8 h. The basal level of estradiol secretion by stromal tissue was lower (24 +/- 3 pg/mg/30 min), than that secreted by follicles (64 +/- 5.6 pg/mg/30 min, n = 6; p < 0.05). On the other hand, the mean basal levels of progesterone and testosterone secreted by stromal tissue (252 +/- 14 pg/mg/30 min and 162 +/- 18 pg/mg/30 min, respectively) were greater than those secreted by follicular tissue (84 +/- 3 pg/mg/30 min and 81 +/- 4 pg/mg/30 min, respectively). When stromal and follicular tissue were combined the secretion of progesterone, testosterone and estradiol was intermediate to that of the separate tissues. Under gonadotropin stimulation (human menopausal gonadotropins plus follicle stimulating hormone), the follicular tissue secreted greater amounts of steroids than did the stromal tissue, or stromal plus follicular tissue. When stromal tissue and follicular tissue were combined, the levels of basal progesterone and testosterone secreted by both tissues were significantly lower than those of stromal tissue alone. However, the reduction in follicular estradiol secretion induced by stromal tissue under basal conditions, was in large part overcome during gonadotropin perifusion. These observations suggest that locally produced factors may play an inhibitory, paracrine role in the regulation of ovarian steroidogenesis.

In these studies, an in vitro perifusion model was used to compare stimulation of ovarian tissue with either human menopausal gonadotropin (hMG), which is an equal mixture of luteinizing hormone (LH) and follicle stimulating hormone (FSH), or with hMG plus added human FSH. Eight-hour perifusion studies were conducted on either whole, or dissected clusters of follicles from pregnant mare serum gonadotropin (PMSG)-treated rats. In the two groups, similar stimulatory protocols were used, consisting of a ramp stimulation over 60 min with either hMG (0-8 mIU/ml) or hMG plus FSH (0-8 mIU/ml hMG + 0-8 mIU/ml FSH), followed by hourly pulse stimulation with hMG (8-18 mIU/ml) or hMG plus FSH (8-18 mIU/ml hMG + 8-18 mIU/ml FSH), respectively. In the whole ovaries, no differences were detected in progesterone, testosterone, or estradiol secretion. However, in the cluster of follicles, an elevated hFSH/hMG ratio resulted in a significantly higher secretion of progesterone, testosterone and estradiol (n = 8; p < 0.05) than the steroids secreted by follicles perifused with hMG alone. In conclusion, an elevated FSH: LH ratio led to greater steroidogenic responses by the perifused cluster of follicles, but not by the whole ovary.

Insulin has been implicated as a regulatory factor in ovarian steroidogenesis. To assess this issue, we examined the role of insulin on steroid secretion by whole ovaries from pregnant mare serum gonadotropin (PMSG)-pretreated rats, using an in vitro perifusion ovarian model. Three concentrations of insulin were perifused in the absence and presence of gonadotropin pulse-stimulation. Bioactive insulin concentrations after perifusion of ovarian tissue were 4, 40 and 400 mIU/ml. In the absence of gonadotropin-pulse stimulation, acute perifusion with insulin had no effect on ovarian steroid secretion. During stimulation with luteinizing hormone (LH) plus follicle stimulating hormone (FSH), insulin significantly decreased the secretion of testosterone and estradiol but increased the output of progesterone. This effect was only evident after 180 min perifusion and with the highest concentration of insulin. We conclude that acute elevation of insulin concentration has no effect on basal ovarian secretion of progesterone, testosterone, or estradiol in gonadotropin-pretreated rats. However, insulin has a moderate effect on steroid secretion in ovaries exposed to LH/FSH pulsatile stimulation, resulting in a decrease in estradiol and testosterone release, and stimulation of progesterone secretion.

A computer-controlled perifusion apparatus has been used to investigate the effects of different patterns of hormonal stimulation on secretion of steroids by ovaries from untreated, or pregnant mare serum gonadotropin (PMSG)-pretreated, immature rats. With ovaries from untreated rats, a low rate of increasing concentration of gonadotropins (luteinizing hormone (LH) plus follicle stimulating hormone (FSH)) induced a maximum secretion of estradiol within 60 min (22.6 +/- 1.4 pg/mg/30 min). An intermediate and a high rate of increasing gonadotropin concentration stimulated maximum secretion (26.0 +/- 1.2 pg/mg/30 min) and 28.1 +/- 2.8 pg/mg/30 min), respectively) within 30 min. Peak secretion, however, was not maintained and was reduced despite continued LH/FSH pulses. Progesterone secretion increased during, and subsequent to, the decreasing estradiol output. Increasing amplitudes of LH/FSH or constant perifusion with LH/FSH did not change the profile, or the concentration of estradiol, but these measures increased progesterone release. An occasional transient increase in estradiol secretion was observed when ovaries from unstimulated rats were perifused with low LH/FSH pulse frequency. Thus, these studies support the hypothesis that in the prepubertal rat ovary, elements of pulse characteristics, such as rate of increasing LH/FSH concentration, and amplitude are important in differentially regulating steroid output. The steroid secretory pattern of ovaries from PMSG-treated prepubertal rats was different from that of untreated rats. With ovaries from PMSG-treated rats, an acute increase in secretion of progesterone, testosterone or estradiol was not observed, whether low or high rates of increasing gonadotropin concentration were used. Rather, the concentration of these steroids continued to rise following LH/FSH pulses. Thus, in contrast to the untreated ovary, the PMSG-treated ovary did not show differential regulation of steroid secretion in response to LH/FSH. In conclusion, we have shown the in vitro perifusion model to be a useful tool for studying the effects of different patterns of hormonal stimulation on ovarian steroidogenesis. In addition, differential effects of gonadotropins on steroid output were shown, depending upon prior maturation of the ovary.

In vitro perifusion of whole ovaries raises questions about tissue viability and its effects on the observed ovarian steroid secretion. To assess effects of tissue degeneration, whole and quartered ovaries from pregnant mare serum gonadotropin (PMSG-) treated immature rats were perifused for 8 h, employing various levels of pO2. Histological examination of both the whole and quartered ovary showed signs of degeneration in centrally located follicles but not in follicles located near the surface. However, basal and gonadotropin-stimulated (luteinizing hormone plus follicle stimulating hormone) secretion of estradiol, testosterone and progesterone were not significantly different in the whole ovary whether in the presence of high or low pO2 (n = 8; p > 0.05). Similarly, although the quartered ovary secreted greater amounts of steroids than did the whole ovaries (p < 0.05, n = 8), pO2 levels did not affect the steroid output. We conclude that during in vitro perifusion, minimal oxygen supply is sufficient for ovarian steroidogenesis to proceed. In addition, although quartered ovaries displayed some evidence of tissue degeneration, they were more responsive in terms of steroid output per mg ovary than were whole ovaries.

Progestin receptor-containing cells in the hypothalamus of the adult female green monkey (Cercopithecus aethiops) were examined by double-label immunocytochemical methods to determine their anatomical location, neurotransmitter content and afferent connections. Animals were ovariectomized and administered either estradiol valerate or the oil injection vehicle, and were sacrificed after 10 days of treatment. Using a monoclonal antibody raised against rabbit uterine progestin receptor (PR), the distribution of PR-immunoreactive cells in the mediobasal hypothalamus and the effect of estrogen treatment on this distribution was determined. PR-immunoreactive cells were found throughout the ventromedial nucleus (VMN), in the area between the VMN and fornix, and in the medial portion of the infundibular nucleus. Estrogen treatment dramatically increased both the number of labeled cells and the intensity of immunoreaction product in these regions. In double-immunostained sections, boutons immunoreactive for antigens indicative of serotonin, pro-opiomelanocortin derived peptides, GABA, catecholamine, neuropeptide Y, substance P, cholecystokinin, and somatostatin were demonstrated to establish synaptic contact with the soma of PR-immunoreactive hypothalamic neurons. In colchicine-pretreated animals, all PR-containing neurons in the mediobasal hypothalamus were found to contain immunoreactivity for glutamic acid decarboxylase, the enzyme required for synthesis of GABA. No evidence of colocalization with other antigens, including LHRH, was observed. Because LHRH neurons are known to receive a rich GABAergic innervation PR-containing GABAergic cells may represent steroid-sensitive sites of integration for inputs from other neural systems involved in the control of gonadotropin secretion.

The effects of subsaturating pulses of estradiol on cell nuclear retention of estrogen receptors in brain regions of male and female rats were determined. In the first experiment, age-matched adrenalectomized/gonadectomized (ADX/GDX) rats were killed 1 h after i.v. injection of a subsaturating dose of free estradiol (1.0 microgram/kg b. wt.) and cell nuclear estrogen receptor binding was measured in microdissected brain areas by an in vitro exchange assay. As we have previously reported with saturating doses of estradiol (3.6-36.0 micrograms/kg b. wt.), a greater concentration of receptors was measured in the ventromedial nucleus (VMN), periventricular preoptic area (PVPOA), and medial preoptic area (mPOA) of the female than in the same regions of the male brain. Binding levels did not differ between the sexes in the bed nucleus of the stria terminalis, arcuate nucleus, or the corticomedial amygdala. In the second experiment, GDX/ADX male and female rats received either a single or double pulse of 0.5 microgram estradiol/kg b. wt. administered 6 h apart. Rats were killed 1 h after the second pulse. In animals treated with a single pulse of estradiol 1 h before sacrifice, higher concentrations of estrogen binding were measured in the female VMN and PVPOA than in the male. Except in the mPOA, the concentration of estrogen receptor binding measured was not different from that measured in animals that had received an additional, earlier pulse of estradiol. In this area, receptor concentrations were lower in male rats treated with two pulses than in males treated with one pulse.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen treatment induces synaptic plasticity accompanied by damaged structures and aggregates of peroxidase in astrocytes in the hypothalamic arcuate nucleus of the rat. Synaptic plasticity also occurs within the arcuate nucleus after physiologic surges of estrogen. Although the function of estrogen-induced peroxidase is unclear at present, in other systems peroxidase can generate free radicals by catalyzing the oxidation of some molecules, including estrogen. Because free radicals underlie remodeling in a number of tissues, estrogen-induced free radicals could mediate synaptic remodeling within the arcuate nucleus. Although they contain estrogen-inducible peroxidase, astrocytes do not contain estrogen receptors as measured by conventional techniques, suggesting that estrogen-inducible peroxidase arises from some novel mechanism. Estrogen could induce peroxidase within receptor-deficient astrocytes by binding to receptors in neurons and stimulating the release of some factor that interacts with astrocytes. Alternatively, estrogen could act directly on astrocytes in the absence of estrogen receptors. Although astrocytes in the hypothalamus of the rat do not contain classical nuclear estrogen receptors, they do bind fluorescein-conjugated estradiol in extranuclear sites. The distribution of fluorescein-conjugated estradiol binding within the hypothalamus overlaps that of peroxidase-rich astrocytes, and double labeling reveals many cells with the stellate morphology of astrocytes, containing both peroxidase and fluorescein-conjugated estradiol binding. However, because peroxidase and fluorescein-conjugated estradiol always occupy different compartments of the cell, the fluorescein-conjugated estradiol is not binding to peroxidase.

Previous studies have shown sex differences in intramembrane particle content in the arcuate neurons of the rat hypothalamus. In this study, freeze-fracture replicas were prepared from the infundibular hypothalamus of adult African green monkeys (Cercopithecus aethiops) in order to determine whether primates also have sexual dimorphism in neuronal membranes. Intramembrane particles (IMP) were quantitatively assessed in the perikaryal plasma membranes of infundibular neurons. Four groups of monkeys were studied: intact males, intact females, ovariectomized females injected with 20 mg of estradiol valerate over 10 days and ovariectomized females injected with vehicle (castor oil). Membranes from females showed an increased numerical density of IMPs when compared to males. Ovariectomy of females did not affect IMP content, while estrogen administration resulted in a significant decrease in IMP numerical density to reach male values. These findings indicate a sex difference in neuronal membranes in the hypothalamus of monkeys and suggest that as in rodents, neuronal plasma membrane organization in higher primates may be modulated by gonadal steroids.

Sex differences in estrogen-induced progestin receptors have been described in specific regions of the rat brain. To determine if alpha 1-neurotransmission plays a role in the expression of the sex differences in progestin receptor induction, the effects of the alpha 1-antagonist, prazosin, on progestin binding in microdissected regions of the rat brain was determined. Adrenalectomized/gonadectomized male or female rats were administered various doses of estradiol benzoate (EB) in combination with prazosin. With all treatment paradigms, and in both sexes, no significant effect of prazosin treatment on progestin receptor levels was observed. These results are consistent with the idea that sex differences in the estrogen-induction of progestin receptors in the rat hypothalamus are not due to sex differences in the alpha 1-adrenergic regulation of progestin receptor synthesis.

Estrogen effects on tyrosine hydroxylase (TH), monoamine oxidase types A and B (MAO), and dopamine (DA) in microdissected regions of the hypothalamus, preoptic area and substantia nigra (SNR) of the female rat brain were investigated. Ovariectomized (OVX) young adult female rats were implanted with single silastic capsules containing 100% estradiol valerate (EV). Control rats received empty silastic capsules. Two weeks following capsule insertion, EV decreased TH activity and DA concentration in the arcuate nucleus (AN) while no significant changes in TH activity or DA concentration were observed in the SNR, ventromedial nucleus (VMN), suprachiasmatic nucleus, paraventricular nucleus, medial preoptic nucleus, or the periventricular preoptic nucleus. Although estrogen suppressed TH and DA in the AN, 2 weeks following removal of the estrogen containing capsules, TH activity and DA concentration were restored to control (OVX) levels. Suppression of MAO activity occurred in both the AN and the VMN of rats implanted with EV capsules and returned to OVX levels following the removal of the estradiol load. These results revealed that estrogen effects on TH and MAO activities and DA concentration in the midbrain are region specific and reversible; and that among the dopaminergic systems studied, estrogen effects on TH and DA are confined to the tuberoinfundibular dopaminergic system (TIDAS). Furthermore, these results support our hypothesis that estrogen is a key regulator of DA function in the TIDAS via effects on TH. The importance of these findings to the control of gonadotropin secretion and reproductive cyclicity is discussed.

Although it appears that the perinatal development of sexual phenotype in the rodent brain is determined by exposure to estradiol, generated locally via aromatization of androgen, the mechanisms underlying this process are not fully understood. We have, therefore, developed an in vitro model of hormone action based upon examining the effects of sex steroids on SV-40-transformed fetal rat hypothalamic cell lines. Using serum-free growth factor-deficient conditions the effects of 17 alpha- and 17 beta-estradiol, testosterone, dihydrotestosterone (DHT), and tamoxifen on survival of two estrogen-binding rat hypothalamic cell lines were examined. In one cell line, RCF-8, both 17 beta-estradiol and testosterone increased survival at picomolar concentrations. This effect was blocked by tamoxifen, but could not be reproduced by the nonaromatizable androgen DHT or the inactive isomer 17 alpha-estradiol. In the other cell line, RCA-6, addition of 17 beta-estradiol led to inhibition of cellular proliferation, which was reversed by the addition of tamoxifen. In an estrogen receptor-negative hypothalamic cell line, RCF-12, estradiol had no net effect on the growth pattern. In summary, the estrogen-binding capacity and the responsiveness to physiological concentrations of estradiol and testosterone, but not DHT, make the RCF-8 cell line a potential in vitro model of hypothalamic sexual differentiation. The use of estrogen-sensitive hypothalamic cell lines provides a unique opportunity for studying the cellular mechanisms underlying this process.

Freeze-fracture methodology was used to study the organization of the neuronal plasma membrane in the rat arcuate nucleus, an estrogen sensitive area of the hypothalamus. Freeze-fracture replicas were prepared from 6 adult ovariectomized rats injected with a single dose of 17 beta-estradiol and from 6 ovariectomized littermates injected with vehicle. Rats were sacrificed 2 days after the injection. Occasional gap junctions were observed in freeze-fractured neuronal membranes from both groups of animals and their incidence was increased (P less than 0.01) in estradiol treated rats. This study demonstrates gap junctions in arcuate neurons and suggests that these structures may be affected by gonadal hormones.

A macromoleclar component which binds 3H-estradiol with high affinity and limited capacity is present in the 150,000 x g supernatant fraction of pituitary, hypothalamus, cortex, and limbic system of both male and female human fetwses. 17 beta-estradiol and 17alpha-ethinylestradiol effectively compete with 3H-estradiol for binding sites while testosterone, dihydrotes-tosterone, and cortisol do not. The binding component is different from sexhormone-binding-globulin. It is present in these tissues in relatively large amounts and it is tentatively identified as an estrogen recptor.

Data accumulated from studies of several species indicate that sex steroids are metabolized by neuroendocrine tissues in a manner analogous to that of other target tissues. Evidence that androgens or their estrogenic metabolites affect the morphology and function of the nervous system in fetal, newborn, developing, and adult rats is presented. The destruction of neural processes subsequent to the administration of large doses of estrogen to intact rats can now be added to the previously known effects on synaptogenesis, cell morphology, and function. We believe this destruction to be a form of chemical deafferentation and that it may underlie age-related hypothalamic failure and the development of multifollicular ovaries in the rat. Implications for other species are not clear at present.

Homogenates of central neuroendocrine tissues from 2 male and 1 female midtrimester fetuses were incubated with (23H) estradiol-17beta. Metabolism at the C-2 position was monitored by measuring the tritium incorporated into water in the incubate. Liberation of tritium from the substrate by hypothalamus, limbic tissues, parietal cortex and pituitary occurred to the extent of 5.1-12.7%, 1.6-8.5%, 4.7-10.8%, and 0.9-4.1% of starting radioactivity, respectively. The nature of the product was confirmed by the isolation of 14C labelled 2-hydroxyestrone derivative from separate incubations with 14C-estrone as the substrate. With or without correction for weight of tissue incubated, catechol estrogen formation under these conditions occurs at levels similar to those seen in rat brain homogenates except that in contrast to the rat, the human cortex is also highly active.

2-Hydroxyest radiol-17 alpha and 4-hydroxyestradiol-17 alpha, the catechol derivatives of estradiol-17 alpha, have reduced affinity for hypothalamic, pituitary, and uterine estrogen receptors, but retain a potency for interaction with catechol-0-methyltransferase equal to that of the natural, 17 beta-hydroxy catechols. This dissociation of receptor binding and catecholamine interactions may allow the use of the 17 alpha catechols as a probe for the mechanism of action of the catechol estrogens.

The pharmacological effect of 2-hydroxyoestradiol (2-OHE2) and 4-OHE2 on concentrations of LH in the chronically castrated rat have been compared with that of oestradiol in order to determine whether the in-vivo activity is altered by insertion of a hydroxyl group at position 2 or 4 of the aromatic A ring; these derivatives are naturally occurring oestrogen metabolites. Four groups of six adult male rats were used 4 weeks after bilateral orchidectomy. The right jugular vein was exposed under ether anaesthesia and a basal blood sample taken (10.00 h) immediately before an intravenous injection of vehicle alone (0.1 ml ethanol with 0.01% ascorbic acid), oestradiol, 2-OHE2 or 4-OHE2 (10 micrograms of each in 0.1 ml vehicle). Blood was taken from each animal at 2, 4, 6, 8 and 24 h after treatment and serum assayed for LH. Baseline LH levels were similar in the four groups. At 2 h there was no change in 2-OHE2-treated rats but there was a significant decrease of serum levels of LH in rats treated with oestradiol and 4-OHE2 compared with vehicle-treated controls. The decrease in LH was quantitatively similar in oestradiol- and 4-OHE2-treated groups and was sustained at 4, 6 and 8 h, returning to control values at 24 h. In subsequent experiments the effects of lower doses of these two steroids were compared and the potency of 4-OHE2 was estimated to be about 25% that of oestradiol. In a further experiment, 2-OHE2 (100 micrograms) had no effect when given alone, but when injected i.v. immediately before treatment with 1 microgram oestradiol, it was able to inhibit the suppression of LH by oestradiol. In conclusion, 4-OHE2 had a potent effect in lowering plasma LH levels whereas 2-OHE2, even at a high dose (100 micrograms), did not suppress LH but it was able to inhibit the effect of oestradiol. These differences in biological activity may reflect more rapid metabolism of 2-OHE2 or differences in binding properties of these catechol oestrogens to the oestrogen receptor.

Young adult cyclic female rats were each injected with 2 mg estradiol valerate (EV) in sesame oil. Controls received an equivalent volume of sesame oil. Within 2 months after injection, most of the EV-treated animals showed persistent vaginal estrus and small polyfollicular ovaries as well as pathological changes in the hypothalamic arcuate nucleus. This pathological process was gradually progressive such that by 6 months after EV injection, the basal lateral region of the nucleus contained numerous reactive microglia, astrocytes, and degenerating elements of the neuropil. The experimental rats had elevated plasma PRL and GH concentrations which gradually diminished. Plasma estradiol concentration remained elevated 2 months after injection, while plasma LH and FSH concentrations stayed within the high and low normal range, respectively. The pituitary glands of injected animals weighed significantly more than those of controls 5.5 months after injection, but the enlarged glands did not cause hypothalamic compression. As mechanical anterior deafferentation of the medial basal hypothalamus has previously been shown to produce similar endocrine and reproductive alterations, it may be that estradiol treatment results in a functional-anatomical disconnection of the arcuate nucleus from the more anterior hypothalamic areas that regulate cyclicity. Whether this type of functional-anatomical phenomenon underlies other varieties of induced or secondary acyclicity in females remains to be determined.