Beneficial effects of the phytoestrogen genistein on hippocampal impairments of spontaneously hypertensive rats (SHR).

Hippocampal neuropathology is a recognized feature of the spontaneously hypertensive rat (SHR). The hippocampal alterations associate with cognitive impairment. We have shown that hippocampal abnormalities are reversed by 17ß-estradiol, a steroid binding to intracellular receptors (estrogen receptor α and ß subtypes) or the membrane-located G-protein coupled estradiol receptor. Genistein (GEN) is a neuroprotective phytoestrogen which binds to estrogen receptor ß and G-protein coupled estradiol receptor. Here, we investigated whether GEN neuroprotection extends to SHR. For this purpose, we treated 5-month-old SHR for 2 weeks with 10 mg kg-1 daily s.c injections of GEN. We analyzed the expression of doublecortin+ neuronal progenitors, glial fibrillary acidic protein+ astrocytes and ionized calcium-binding adapter molecule 1+ microglia in the CA1 region and dentate gyrus of the hippocampus using immunocytochemistry, whereas a quantitative real-time polymerase chain reaction was used to measure the expression of pro- and anti-inflammatory factors tumor necrosis factor α, cyclooxygenase-2 and transforming growth factor ß. We also evaluated hippocampal dependent memory using the novel object recognition test. The results showed a decreased number of doublecortin+ neural progenitors in the dentate gyrus of SHR that was reversed with GEN. The number of glial fibrillary acidic protein+ astrocytes in the dentate gyrus and CA1 was increased in SHR but significantly decreased by GEN treatment. Additionally, GEN shifted microglial morphology from the predominantly activated phenotype present in SHR, to the more surveillance phenotype found in normotensive rats. Furthermore, treatment with GEN decreased the mRNA of the pro-inflammatory factors tumor necrosis factor α and cyclooxygenase-2 and increased the mRNA of the anti-inflammatory factor transforming growth factor ß. Discrimination index in the novel object recognition test was decreased in SHR and treatment with GEN increased this parameter. Our results indicate important neuroprotective effects of GEN at the neurochemical and behavioral level in SHR. Our data open an interesting possibility for proposing this phytoestrogen as an alternative therapy in hypertensive encephalopathy.

1,25-Dihydroxyvitamin D3 increases testosterone-induced 17beta-estradiol secretion and reverses testosterone-reduced connexin 43 in rat granulosa cells.

BACKGROUND: Aromatase converts testosterone into 17beta-estradiol in granulosa cells, and the converted 17beta-estradiol contributes to follicular maturation. Additionally, excessive testosterone inhibits aromatase activity, which can lead to concerns regarding polycystic ovary syndrome (PCOS). Generally, 1,25-dihydroxyvitamin D3 (1,25D3) supplements help to improve the symptoms of PCOS patients who exhibit low blood levels of 1,25D3. Therefore, this study investigated the interaction effects of 1,25D3 and testosterone on estrogenesis and intercellular connections in rat granulosa cells. METHODS: Primary cultures of granulosa cells were treated with testosterone or testosterone plus 1,25D3, or pre-treated with a calcium channel blocker or calcium chelator. Cell lysates were subjected to western blot analysis to determine protein and phosphorylation levels, and 17beta-estradiol secretion was examined using a radioimmunoassay technique. Cell viability was evaluated by MTT reduction assay. Connexin 43 (Cx43) mRNA and protein expression levels were assessed by qRT-PCR, western blot, and immunocytochemistry. RESULTS: Testosterone treatment (0.1 and 1 microg/mL) increased aromatase expression and 17beta-estradiol secretion, and the addition of 1,25D3 attenuated testosterone (1 microg/mL)-induced aromatase expression but improved testosterone-induced 17beta-estradiol secretion. Furthermore, testosterone-induced aromatase phosphotyrosine levels increased at 10 min, 30 min and 1 h, whereas 1,25D3 increased the longevity of the testosterone effect to 6 h and 24 h. Within 18-24 h of treatment, 1,25D3 markedly enhanced testosterone-induced 17beta-estradiol secretion. Additionally, pre-treatment with a calcium channel blocker nifedipine or an intracellular calcium chelator BAPTA-AM reduced 1,25D3 and testosterone-induced 17beta-estradiol secretion. Groups that underwent testosterone treatment exhibited significantly increased estradiol receptor beta expression levels, which were not affected by 1,25D3. Neither testosterone nor 1,25D3 altered 1,25D3 receptor expression. Finally, at high doses of testosterone, Cx43 protein expression was decreased in granulosa cells, and this effect was reversed by co-treatment with 1,25D3. CONCLUSIONS: These data suggest that 1,25D3 potentially increases testosterone-induced 17beta-estradiol secretion by regulating aromatase phosphotyrosine levels, and calcium increase is involved in both 1,25D3 and testosterone-induced 17beta-estradiol secretion. 1,25D3 reverses the inhibitory effect of testosterone on Cx43 expression in granulosa cells.

[Effect and mechanism study of kidney tonifying and blood circulation activating Chinese drugs on development of mammary glands in female juvenile rats].

OBJECTIVE: To study the effect and mechanism of Kidney tonifying and blood circulation activating Chinese drugs (KB) on development of mammary gland. METHODS: Using the female juvenile rats as the experimental targets, the level of serum hormone, the amount and affinity of estradiol receptor (ER) and progesterone receptor (PR) as well as the DNA content and relative weight of the rats' mammary glands were measured to study the effect of KB at the pre-receptor, receptor and post-receptor level. RESULTS: KB markedly enhanced the serum level of estradiol, progesterone and the growth hormone (GH) at pre-receptor level, increased the number and affinity of estradiol and progesterone receptors both in the cytoplasm and nucleus at the receptor level. While at the level of post-receptor, it increased the DNA content and the weight index of mammary glands. CONCLUSION: The Kidney tonifying and blood circulation activating Chinese drugs are effective in promoting the growth and development of mammary glands.

[Effects of kidney reinforcing and blood circulation activating Chinese medicine on morphological structure of mammary glands in models of mammary atrophy and hyperplasia in female rats].

OBJECTIVE: To study the effects and mechanism of Kidney reinforcing and blood circulation activating Chinese medicine (K&B) on morphologic structure of mammary glands in female rat models of mammary atrophy and mammary hyperplasia. METHODS: The models of mammary gland atrophy and hyperplasia were established respectively by ovariectomy and diethylstilbestrol injection to observe the changes of gland's weight index, structure, estradiol receptor (ER) and progesterone receptor (PR) after treated by K&B. RESULTS: (1) K&B could ameliorate the atrophied structure of mammary gland with significant increase of weight index (P < 0.01), raise the number of ER and PR in mammary tissue and nuclei (P < 0.01, P < 0.05), normalize the ratios of ER and PR in plasma and those in nucleus (EnR/EcR and PnR/PcR). (2) K&B could also improve or restore the proliferated structure of mammary gland and its weight index as well as the EnR/EcR and PnR/PcR ratios. CONCLUSION: K&B has the effects in preventing and improving the mammary gland atrophy induced by hyposecretion of sexual hormone and promoting the development of mammary gland in adult rats. Although both K&B and estrogen can improve the development of mammary gland, their mechanisms are different. There is a bright future of K&B in treating underdevelopment of mammary gland.

Structures of 4-aryl-coumarin (neoflavone) dimers isolated from Pistacia chinensis BUNGE and their estrogen-like activity.

Activity-guided fractionation of twigs of Pistacia chinensis resulted in the isolation and characterization of two novel ingredients as potent estrogen agonists. On the basis of spectral analysis and comparison with a related compound their structures were elucidated as 3,3''-dimers of 4-aryldihydrocoumarins (3,4-dihydro-4-(4'-hydroxyphenyl)-7-hydroxycoumarin) differing only in the stereochemical disposition of the linkage between the two 4-arylcoumarin moieties. These compounds are the first examples of bis-flavonoids which have been proven to possess estrogen-like activity.

Prenylflavonoids: a new class of non-steroidal phytoestrogen (Part 1). Isolation of 8-isopentenylnaringenin and an initial study on its structure-activity relationship.

Bioassay-guided fractionation of a methanolic extract of a Thai crude drug, derived from heartwood of Anaxagorea luzonensis A. Gray (Annonaceae), resulted in the isolation of 8-isopentenylnaringenin (1) as an estrogen agonist with a activity of about an order of magnitude greater than genistein. Various flavonoids possessing isopentenyl side chains in the A-ring have been prepared and evaluated for their ability to bind estrogen receptor. In addition, enantiomers of 1 were separated and the respective enantiomers were assayed. These studies have demonstrated that the presence of an 8-isopentenyl group is an important factor for binding. Flavones, flavanones and flavonols having an isopentenyl substituent at C-8 exhibited an appreciable affinity for estrogen receptor. Conversely, isoflavones possessing an 8-isopentenyl substituent at C-8 did not show this activity. Movement of the isopentenyl group from position 8 to 6 resulted in loss of the activity. No significant difference was observed between 2(S)- and 2(R)-enantiomers of 1 in their binding affinity. Prenylflavonoids are reported to possess a wide range of biological activities; however, estrogenic activity has not been described.

[Study on relationship between ovulation inducing effect of drug-acupuncture and endometrial contents of estradiol receptor and progesterone receptor].

OBJECTIVE: To study the effect of Chinese herbal medicine for replenishing Kidney combined with acupuncture in treating anovulation and infertility, and the relationship between its ovulation inducing effect and endometrial contents of estradiol receptor (ER) and progesterone receptor (PR). METHODS: Twenty-nine cases were treated with replenishing Kidney drugs combined with acupuncture for 1-3 months. Patients' ER and PR were measured by immunohistochemical method. And patients were classified according to PR content into PR positive group and mild PR positive group. RESULTS: Fifteen cases of PR positive group, completed treatment for 45 cycles, among them, 40 cycles showed ovulation, the ovulation rate being 88.89%. Ten in 14 cases, who complicated with infertility, became pregnant, the pregnant rate being 71.43%. While in 11 cases of PR mild positive group, 9 complicated with infertility, completed treatment for 33 cycle, 10 cycles showed ovulation (30.30%), and pregnant rate 22.22% (2/9). The difference between the two groups was significant (P < 0.01). CONCLUSION: The replenishing Kidney drugs combined with acupuncture treatment could result a good effect in treating infertility due to anovulation, especially on those with high endometrial PR content.

Surface mapping of the ligand-filled C-terminal half of the porcine estradiol receptor by restricted proteolysis.

The ligand-filled 32-kDa fragment of the porcine estradiol receptor extending from His267 to the C-terminal Ile595 was purified to homogeneity by adsorption to mAb 13H2. The native protein was exposed at 4 degrees C to a panel of proteases: thermolysin, subtilisin, pronase, elastase, ficin, bromelain, endopeptidase Lys-C, both in the dimer and the monomer state, and chymotrypsin at pH 8.2 only. The digests were analysed by SDS/PAGE/Western blotting for Coomassie staining and immunostaining. Peptides were sequenced from blots. The majority of cleavage sites in upper domain E (8 out of 11) amassed in the Leu296-Leu310 stretch. Cleavage at Leu319 was seen with subtilisin and at Tyr328 with chymotrypsin. Susceptability to enzymic proteolysis was also pronounced in Thr465-Glu470 at the center of domain E. Three peptides, 13 kDa with thermolysin, beginning at Leu337, 6 kDa and, in low yield, 5 kDa with endopeptidase Lys-C beginning at Asp473 resp. Cys417 were only obtained from the monomer substrate. The various digests featured either 27-23-kDa peptides or mixtures of 17-13-kDa and 12-7-kDa peptides separable by SDS/PAGE. All peptides with N-termini between Leu297 and Ser329 reacted with mAb 13H2. The digests showed high peaks of bound estradiol in the dimer position of 32-kDa fragment controls on density gradient centrifugation at pH 7.4. However, the property of proton-driven dissociation was only preserved in the pronase, elastase and chymotrypsin digests with peptides extending beyond the His547-ArgLeuHis550 motif. The preservation of the estradiol-binding niche in the tightly complexed peptides of domain E was also demonstrated by refilling after steroid removal. The sites exposed to proteolytic enzymes and the epitope for 13H2 attachment are in good agreement with surface probability plots.

Neurochemical identity of neurones expressing oestrogen and androgen receptors in sheep hypothalamus.

Gonadal steroids exert important feedback influences on hypothalamic neurones involved in regulating reproductive behaviour and pituitary hormone secretion. The recent development of antibodies specific for individual gonadal steroid receptors has been of great use in determining precisely which cells in the hypothalamus express androgen, oestrogen and progesterone receptors. In the sheep brain, both oestrogen and androgen receptor antibodies have been used successfully and the distribution of cells expressing both receptors has now been determined in ewes and rams, respectively. In addition, the predominantly nuclear localization of the steroid receptors has enabled double-labelling immunocytochemical procedures to determine the neurochemical phenotype of neurones expressing the steroid receptor. Work in the sheep hypothalamus shows that gonadotrophin-releasing hormone neurones do not possess oestrogen or androgen receptors. However, substantial numbers of cells containing oestrogen receptors in the preoptic area of ewes contain the inhibitory neurotransmitter gamma aminobutyric acid, while most oestrogen receptor-immunoreactive neurones in the ventromedial nucleus synthesize the inhibitory neuropeptide somatostatin. Androgen receptors have been detected in many of the ventromedial somatostatin neurones in rams. In contrast, the neurochemical phenotype of the great majority of oestrogen and androgen receptor-immunoreactive cells in the arcuate nucleus remains unknown. The identification of the neurotransmitters and neuropeptides synthesized by neurones possessing androgen and oestrogen receptors in different regions of the ovine hypothalamus provides a neuroanatomical basis for understanding the mechanisms by which gonadal steroids regulate reproductive function.

Influence of chemotherapy on hormone receptor concentration in a xenotransplanted endometrial cancer.

There is growing evidence that chemotherapy may influence the hormone receptor capacity in human carcinomas. The consideration of this assumption may be of importance for the therapeutic management of tumors with metastatic spread which underwent previous adjuvant chemotherapy. Therefore we investigated the influence of six different kinds of chemotherapy on the hormone receptor concentration and the percentage of receptor positive cells in xenotransplanted endometrial cancer. Our results can be summarized as follows: (1) We find neither a significant decrease in hormone receptor capacity after chemotherapeutic treatment (biochemical determination), nor do we see a decrease in the percentage of ER/PR pos. cells (immunohistochemistry). (2) On the other hand, there is no increase in hormone receptor concentration inducible by chemotherapy and no increase in ER/PR pos. cells immunohistochemically.

Steroid receptors in breast cancer.

Recent advances in steroid receptor structure and function now indicate that oestrogen binds to the oestrogen receptor (ER) molecule at a specific site, denoted region E. This allows binding of the oestrogen-ER complex to DNA via cysteine residues in region C of the ER molecule, which tetrahedrally co-ordinate zinc. This modulates transcription and stimulates cell growth. A number of newly discovered growth factors are also regulated by ER, as is the progesterone receptor. Steroid receptor concentrations in tissues can now be measured on smaller tissue samples using enzyme immunoassay or on cells obtained by fine needle aspiration using monoclonal antibody technology. The prognostic value of steroid receptor is limited, but still constitutes the best marker for predicting response to endocrine therapy. The role of steroid receptors in selecting patients for adjuvant therapy is discussed.

Evidence for direct action of estradiol on growth hormone-releasing factor (GRF) in rat hypothalamus: localization of [3H]estradiol in GRF neurons.

Sex steroids have been shown to influence the secretion of GH. There appears to be no good evidence of the effect of estradiol on the anterior pituitary, while the central site of estradiol action on the regulation of GH secretion is not known. The present investigation was carried out to determine whether some of the GH-releasing factor (GRF) neurons and somatostatin (SRIF) neurons in the hypothalamus and GH cells in the pituitary contain estradiol receptors. Colocalization of [3H]estradiol and antibodies to GRF or SRIF in brain and antibodies to GH in pituitary was studied to show interrelationships between estrogen target cells and peptidergic cells. Eight female Sprague-Dawley rats were ovariectomized, each rat was treated with colchicine, and 24-48 h later the animals were given an iv injection of [2,4,6,7,16,17-3H]estradiol (SA, 166 Ci/mM) at a dose of 0.5 micrograms/100 g BW. One hour after the injection, the rats were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The hypothalami from the perfused rats and the pituitaries from unperfused rats were frozen in isopentane precooled in liquid nitrogen (-190 C) and processed for autoradiography. The brain autoradiograms were immunostained for GRF, SRIF, and tyrosine hydroxylase [TH; an enzyme for the synthesis of dopamine (DA)], and the pituitary autoradiograms were immunostained for GH by the avidin-biotin peroxidase method. The majority of GRF-containing neurons were found in the arcuate nucleus, with some scattered cells in the lateral region of the ventromedial nucleus and the basal lateral hypothalamus. In the central portion of the arcuate nucleus, 20-30% of GRF-containing neurons showed nuclear concentration of [3H]estradiol. In the anterior portion of the hypothalamus, 10-15% of immunoreactive GRF-containing neurons were labeled with [3H]estradiol. In the lateral basal hypothalamus and the lateral region to the ventromedial nucleus, a few GRF neurons showed nuclear concentration of radioactivity. In contrast, a few SRIF cells in hypothalamic periventricular nucleus showed nuclear labeling with [3H]estradiol. Dual immunostaining with GRF and TH antibodies revealed that the estradiol-labeled GRF neurons did not contain TH immunoreactivity. In addition, 80-90% of GH cells in the anterior pituitary showed nuclear concentration of [3H]estradiol. The present studies demonstrate for the first time that certain populations of GRF neurons are targets for estradiol and indicate that estradiol acts directly on certain hypothalamic GRF neurons. The results suggest that estradiol may have a role in the regulation of GH secretion by modulating GRF release and acting directly on the somatotrophs.

The effect of neonatal castration of male rats on the level of sex-hormone receptors in the hypothalamus and hypophysis of adult animals.

The concentration of oestradiol (E2) and testosterone (T) cytosolic and nuclear receptors was studied in the pituitary and hypothalamus of adult male rats gonadectomized either on the first day after birth (long-term castrates) or in adulthood (short-term castrates). Intact male rats and short-term castrates had the same levels as each other of cytosolic and nuclear oestrogen and androgen receptors in the pituitary, mediobasal hypothalamus (MBH), and preoptic anterior hypothalamus (POAH). In neonatally castrated males the number of nuclear T-binding sites in the pituitary and both areas of the hypothalamus decreased, whereas the number of nuclear E2-receptors was reduced only in the MBH. The number of E2-receptors in the POAH of such animals increased. The number of E2- and T-binding sites in the nuclear fraction of the MBH and POAH was the same in long-term castrates whether they did not receive testosterone propionate (TP) or received it from 7 days after birth until sexual maturity. Conversely, the T-receptor concentration in the hypophysis of neonatally castrated males who received TP was higher than in such animals which did not, but still lower than the level in intact adult rats; the number of hypophyseal nuclear E2-binding sites in long-term castrates which received TP was 1.5 times higher than in all the other groups of animals. The data demonstrate that in male rats sex-hormone receptors are involved in the sexual differentiation of the brain.

In vitro effects of cytosolic inhibitor and opiates on the binding of [3H]oestradiol to nuclear type II binding sites of rat uterus and hypothalamus.

The effect of cytosolic ultrafiltrates prepared from intact rat uteri, brain hemispheres and hypothalami and of some opiate analogues on oestradiol binding to nuclear type II sites in rat uterus and hypothalamus was studied. Opiate binding in nuclear fraction of rat uteri was also evaluated. Both uterine and hypothalamic low affinity nuclear oestradiol binding was inhibited by filtrate from uteri, while only hypothalamic nuclear binding was decreased in presence of hypothalamic filtrate. Filtrate from brain was ineffective on nuclear oestradiol binding of the studied tissues. Concentration dependent inhibition of uterine nuclear oestradiol binding could be demonstrated by some opiate analogues in vitro. Specific low affinity nuclear binding of opiate antagonist naloxone and agonist dihydromorphine was observed in rat uteri which could be inhibited by uterine filtrate and oestradiol but not by hypothalamic filtrate or other steroids. Present findings support the probable intracellular interplay of opiates and oestradiol action and suggest that cytosolic inhibitor factor might be involved.

[Effect of alcohol on the content of sex steroid receptors in the hypothalamus and hypophysis of male rats]. / Vliianie alkogolia na soderzhanie retseptorov polovykh steroidov v gipotalamuse i gipofize samtsov krys.

In experimental dipsomania model (formation of physical dependence by method of intensive alcoholization) we have studied receptor binding of testosterone (T) and estradiol (E2) in the hypothalamus and pituitary body of mature male rats. Administration (at 10 and 16 h) of 25% ethanol-saline solution at a dose of 7.5 g/kg of body weight in the course of 5 days significantly decreased serum T level but did not change serum LH and FSH levels. Essential reduction of the nuclear androgen receptors in the preoptic-anterior hypothalamic area (POA), mediobasal hypothalamus (MBH) and adenohypophysis was noted in alcohol-treated rats. Unlike androgen receptors the number of the nuclear E2-binding sites in PaO was significantly increased in these males. Thus the results of the present paper demonstrate that multiple administration of ethanol stipulates deficit of serum T, androgen receptors in MBH and pituitary body that possibly results in separation of negative feedback mechanism between the gonads and pituitary body. Increase of specific binding of E2 to nuclear receptors in PoA might appear to explain feminization of alcohol-treated rats.

Differential extraction of estradiol- and tamoxifen-receptor complexes from hypothalamic cell nuclei.

These experiments compared nuclear estradiol- and tamoxifen-receptor complexes from brain by measuring the ability of a series of ionic agents and DNA intercalators to release estrogen receptors from hypothalamic cell nuclei following in vivo administration of the agonist or antagonist. Over the concentration ranges tested, intercalators released 50-80% of the total estrogen receptors from hypothalamic cell nuclei in a concentration-independent fashion whereas extraction of nuclear receptors by the ionic agents was concentration dependent. In general, brain cell nuclear estrogen receptors were extracted more easily by both ionic agents and intercalators following injections of tamoxifen than of estradiol. It was also found that the polyanion heparin releases a small population of nuclear estrogen receptors that are not extracted by 0.5 M KCl, suggesting the existence of KCl-resistant chromatin acceptor sites in hypothalamic nuclei. These data support the hypothesis that estrogen agonists and antagonists promote different conformational changes when they bind neural estrogen receptors, resulting in the formation of ligand-receptor complexes with different biological activity.

The metabolism of 19-nor contraceptive progestins modulates their biological activity at the neuroendocrine level.

In this communication, a series of studies from our laboratory dealing with the mechanism of action of 17 alpha-ethinyl derivatives of 19-nor testosterone are reviewed. The administration of norethisterone (NET) to long-term castrated female rats induces the nuclear translocation of pituitary estradiol receptors and is followed by some estrogenic-like effects at the hypothalamic-pituitary unit. It is established that an A-ring reduced metabolite of NET, the 3 beta,5 alpha-tetrahydro NET derivative, is responsible for the observed in vivo estrogenic effects of the parent compound. 3 beta,5 alpha-NET binds to the estrogen receptor and is efficient in inducing the pituitary estrogen-dependent progesterone receptor and in increasing the uterine weight in long-term castrated rats. Furthermore, administration of 3 beta,5 alpha-NET and the 5 alpha-reduced metabolite of NET (5 alpha-NET) are able to inhibit the release of gonadotropins in the castrated animal to a greater extent than NET. Moreover, pretreatment with tamoxifen, an estrogen binding site competitor, results in a significant diminution of the antigonadotropic potency of 3 beta,5 alpha-NET but not of the 5 alpha-NET, which is only inhibited by the administration of cyproterone acetate. These findings underline the importance of the metabolic rate of NET for the expression of its biological effects at the hypothalamic-pituitary unit.

The role of the hypothalamus in aging of the female reproductive system.

We have investigated the role of neuroendocrine and neurochemical changes in the age-related deterioration of cyclic female reproductive function. During middle age the timing and amplitude of the proestrous and estradiol-induced LH surge is altered. We have found that the diurnal pattern of norepinephrine turnover is altered in critical hypothalamic areas known to regulate the release of LHRH. These changes may contribute to alterations in the timing and the amplitude of LH release, which may, in turn, affect the ability of rats to maintain regular estrous cycles.

Cellular mechanisms of acute estrogen negative feedback on LH secretion: pituitary responsiveness to LHRH and estradiol receptor kinetics in the pituitary, preoptic hypothalamic area, and the caudal hypothalamic area of the rat brain.

In order to examine the cellular mechanisms by which estradiol (E2) exerts its acute negative feedback upon luteinizing hormone (LH) secretion, a temporal correlation was made among serum LH concentrations, pituitary responsiveness to luteinizing hormone-releasing hormone (LHRH), and the translocation of E2 to nuclear (NER) receptors of the pituitary (PIT), preoptic hypothalamic area (POA), and the caudal hypothalamic area (HYP). Rats on diestrous day 1 were ovariectomized (OVX) and killed 10 days later. LH and LHRH were measured by RIA. NER was measured by an exchange assay. Serum LH increased 10-12-fold 10 days following OVX as compared to diestrous controls. The injection of estradiol benzoate (Eb, 20 microgram in corn oil/rat, sc) did not affect LH concentrations at 30 min but decreased serum LH at both 60 and 180 min following its administration. Pituitary responsiveness to LHRH (measured as the increase in LH 10 min after iv injections of 100 ng LHRH/rat) was not altered at 60 min but was significantly decreased 180 min following Eb injection. Thus, serum LH decreased prior to a detectable alteration in pituitary responsiveness to LHRH. Translocation of E2 receptors to NER of the HYP and PIT began 60 min following Eb injection and was maximal at 180 min. In contrast, translocation of E2 receptors in the POA was maximal at 60 min, and had recovered to control values 180 min following Eb administration.(ABSTRACT TRUNCATED AT 250 WORDS)