Sites of aromatization of [H]testosterone in forebrain of male, female and androgen receptor-deficient Tfm mice: an autoradiographic study

The distribution of radioactivity after injection of [3H]testosterone was studied in the forebrain of adult mice by thaw-mount autoradiography. Nuclear labeling was high in neurons in the dorsal part of the medial nucleus of the amygdala and in the dorsocaudal part of the bed nucleus of the stria terminalis. Low nuclear uptake occurred in the medial preoptic nucleus, in mediobasal hypothalamic nuclei and in the ventromedial amygdala. Nuclear concentration of radioactivity was not influenced by competition with dihydrotestosterone and was present in androgen receptor deficient Tfm mice. It was totally abolished by competition with estradiol. This indicates that in the brain [3H]testosterone is converted to estrogenic metabolites which bind to estrogen receptors. Nuclear labeling after [3H]testosterone was restricted to a few of the brain nuclei, known to contain estrogen receptors indicating that aromatization occurs only in select regions. The results suggest that testosterone acts on the brain via estrogen receptors.     

Metabolism and binding of androgens in the spinal cord of the rat

The binding of [3H]androgens and estrogens, and the metabolism of [3H]androgens, were studied in the spinal cord of the adult rat. High-affinity, specific binding sites for [3H]testosterone and [3H]estradiol were detected in cytosol fractions from the spinal cords of castrate animals. Equilibrium dissociation constants for reaction of these sites with their respective ligands were similar to those of androgen and estrogen receptors from other regions of the central nervous system. Nuclear binding of [3H]estradiol was observed in the spinal cord 1 h after intravenous administration of the isotope. Likewise, exchange assay demonstrated the presence of high-affinity androgen binding sites in spinal cord nuclei from orchidectomized, testosterone propionate treated animals. 5 alpha-Reductase activity in homogenates of the spinal cord was relatively high, approximately 3 times that in the pooled hypothalamus, preoptic area, septum and amygdala. However, in contrast to the latter brain regions, estrogen formation was not detectable in spinal cord tissue. No sex differences were observed in the metabolism of [3H]testosterone by spinal cord homogenates. These results confirm the presence of androgen and estrogen receptors in the rat spinal cord. The lack of detectable aromatase activity in the spinal cord is consistent with the hypothesis that the effects of circulating testosterone on spinal reflex function are mediated primarily through the androgen receptor system.     

Biochemical and radioautographic analysis of estrogen-inducible progestin receptors in female ferret brain and pituitary: Correlations with effects of progesterone on sexual behavior and gonadotropin-releasing hormone-stimulated secretion of luteinizing hormone

Cytosolic binding molecules for the synthetic progestin [3H]R5020, were isolated in vitro from several brain regions including preoptic area/anterior hypothalamus, mediobasal hypothalamus, medial amygdala and parietal cortex as well as the pituitary gland of ovohysterectomized female ferrets. Binding of [3H]R5020 to cortical cytosols was saturable, of high affinity (apparent dissociation constant of 2.0 nM), and was steroid specific. Pretreatment of ferrets with a Silastic capsule containing estradiol caused significant increments in the concentration of cytosolic R5020 binding sites in hypothalamus and pituitary gland, but not in the other brain regions studied. Brains of additional ovohysterectomized ferrets, which had been primed with estradiol prior to receiving an i.v. injection of [3H]R5020, were processed radioautographically. Intense labeling of cells was seen in the medial and lateral preoptic area, in the lateral hypothalamus, and in the ventromedial and arcuate nuclei of the hypothalamus. Radioautograms from the brains of additional ovohysterectomized ferrets given an i.v. injection of [3H]estradiol revealed labeled cells in all of the above regions, in addition to the basolateral portion of the septum, the bed nucleus of the stria terminalis, and in the anterior amygdaloid area as well as the medial and cortical nuclei of the amygdala. This distribution of neural progestin and estrogen binding sites resembles those previously reported for these steroids in the rat, guinea pig, hamster and macaque. Functional studies showed that acute s.c. implantation of a Silastic capsule containing progesterone to ovohysterectomized ferrets, which had been primed with a low dosage of estradiol, failed to augment their sexual receptivity in limited tests with stimulus males given 4 and 8 h after progesterone treatment. This result contrasts with the well-established facilitatory effect of progesterone on sexual receptivity in rat and guinea pig. Chronic exposure to a progesterone capsule caused significant reductions in sexual receptivity in ovohysterectomized ferrets which were implanted concurrently with a second Silastic capsule containing a high dosage of estradiol. Similar effects of progesterone have been reported in rat and guinea pig, but not in the rhesus monkey. Thus species differences in the ability of progesterone to facilitate or inhibit sexual receptivity are not readily explained by species differences in the neural distribution of estrogen-induced progestin receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hypothalamic Estrogen Receptor-lmmunoreactivity in Prepubertal vs Adult Female Guinea Pigs

Juvenile guinea pigs (18–20 days old) rarely display lordosis in response to estradiol and progesterone treatments that elicit sexual behavior in adult females. Nor do immature animals release a preovulatory-like surge of luteinizing hormone in response to estradiol. In vitro radioligand binding assays have revealed similar concentrations of estrogen receptors in thehypothalamus and preoptic area of prepubertal and adult guinea pigs. The aim of the present study was to compare estrogen receptor-immunoreactivity in a variety of forebrain regions of immature and adult guinea pigs, to determine whether age differences in estrogen receptor levels inmore discrete portions of the hypothalamus and preoptic area exist. Forebrain tissue from juvenile (17 days) and adult females (> 6 weeks), ovariectomized 6 days previously, was processed forestrogen receptor-immunoreactivity, using Abbott Laboratories' H222 anti-human estrogen receptor antibody. Juveniles had estrogen receptor-immunoreactive cells in all of the same regions as adults: medial preoptic area, medial preoptic nucleus, bed nucleus of the stria terminalis, periventricular, paraventricular, dorsomedial and arcuate nuclei, ventrolateral and anterior hypothalamic regions, and amygdala. Among the areas in which estrogen receptor-immunoreactivity was quantified (medial preoptic area, medial preoptic nucleus, anterior periventricular nucleus, arcuate nucleus and ventrolateral hypothalamus), the only region in which an age difference in estrogen receptor-immunostaining was observed was the rostral portion of the ventrolateral hypothalamus. Juvenile females had, on average, 30% fewer estrogen receptor-immunoreactive cells in asample of this region than adults (440 ± 25 vs 626 ± 25, P = 0.001). These data are consistent with the hypothesis that insufficient estrogen receptors in the rostral ventrolateral hypothalamus may underlie, in part, the markedly deficient responses of juvenile female guinea pigs to estradiol.     

Uptake and retention of androgen in neurons of the brain of the golden hamster

The distribution of cells capable of concentrating androgen was studied in the male hamster after injection of 5α-dihydro-[1,2,4,5,6,7,(n)-³H]testosterone([³H]DHT) using the technique of thaw-mount autoradiography. Castrated adult male hamsters were injected with 0.2 μg/100 g body weight of [³H]DHT (107 Ci/mmol) and killed 1.5 h later. Brains were rapidly removed and processed for autoradiography. Localization of radioactivity in high concentrations occurred chiefly in limbic forebrain structures and hypothalamic nuclei associated with the control of reproductive function including the following areas: the septal-preoptic region, the amygdala, and the anterior, ventromedial and arcuate nuclei of the hypothalamus. In addition, labeled cells in lesser concentrations were found in the lateral preoptic area, lateral hypothalamus, hippocampus, mesencephalon and various cortical regions. Treatment with 100-fold excess of testosterone, but not estradiol or diethylstilbestrol, inhibited nuclear localization. These studies provide information on the precise anatomical localization of androgen concentrating cells in the hamster brain and demonstrate the similarity of distribution of anrdrogen binding in the rat, mouse and hamster.     

Characteristics of [3H]prazosin binding to guinea pig brain alpha-adrenergic receptors: effects of estradiol and progesterone

[3H]Prazosin was found to bind to sites on guinea pig brain membranes with alpha 1-adrenergic receptor characteristics. Treatment of ovariectomized guinea pigs with estradiol benzoate (EB) or EB followed by progesterone (P) did not affect [3H]prazosin binding to membranes from hypothalamus, preoptic area, amygdala or cerebral cortex. When added to the incubation mixture of the assay, estradiol, P, and other steroids decreased [3H]prazosin binding but only at high concentrations. These results do not support the idea that estrogen and progestin influence reproductive physiology through effects on brain alpha 1-receptors, although limitations of the methodology employed do not completely rule out this possibility.     

Sites in the male primate brain at which testosterone acts as an androgen

Quantitative autoradiographic analysis was used to identify regions in the brain of the male primate where androgen binding sites may be involved in the actions of testosterone. Three days after castration, adult male rhesus monkeys received a subcutaneous injection of either dihydrotestosterone propionate (DHTP, 20 mg, n = 6), testosterone propionate (TP, 100 mg, n = 2), or oil vehicle (control males, n = 4). Three hours later, 5 mCi [3H]testosterone was administered as an i.v. bolus. At 60 min, brains were rapidly removed and the left halves were used for autoradiography. In control males, highest percentages of labeled neurons (20-84% using a rigorous Poisson criterion) were observed in the ventromedial, arcuate and premammillary nuclei (n.) of the hypothalamus, medial preoptic n., bed n. of stria terminalis, intercalated mammillary n., lateral septal n. and the medial, cortical and accessory basal n. of the amygdala. Pretreatment with DHTP eliminated labeling in androgen target tissues of the genital tract, and reduced the percentages of labeled neurons to 4-22% of control values in the arcuate, lateral septal, premammillary and intercalated mammillary n., indicating that in these regions testosterone acted predominantly at androgen binding sites. However, in the medial preoptic n., the ventromedial hypothalamic n. and the accessory basal amygdaloid n., DHTP pretreatment resulted in much less blocking which, together with other data, suggested that in these sites, testosterone's actions involved aromatization and interaction with estrogen-binding sites.     

Simultaneous presence of estrogen receptors and gonadotropin-releasing hormone in the hypothalamus and of estrogen receptors and follicle-stimulating hormone in the pituitary of the fetal guinea pig

Early studies in our laboratory have shown the presence of estrogen receptors (ER) in the fetal brain of guinea pig. Ontogenetic evaluation indicated maximal values of these specific binding sites between 50 and 62 days of gestation. We have now characterized ER in both the hypothalamus and the pituitary at the end of gestation. Autoradiographic studies after subcutaneous injection of [3H]estradiol to the fetus showed the nuclear localization of radioactivity in the hypothalamus to be in the neurons of the preoptic area and the nucleus arcuatus where gonadotropin-releasing hormone was also present. Nuclear localization of radioactivity also occurred in the anterior pituitary where the number of labeled cells increased from the border of the intermediate part to the periphery of the anterior lobe. These labeled cells were predominantly gonadotropes containing follicle-stimulating hormone. Since the fetal uterus responds very actively to estrogens, it is suggested that reciprocal influences of the hypothalamus-hypophyseal axis and gonadal steroid hormones are present in the fetal guinea pig.     

Immunocytochemical localization of progesterone receptor in the guinea pig central nervous system

The distribution of neurons containing progesterone receptors in the brain of guinea pigs ovariectomized and primed by estradiol was investigated immunohistochemically using monoclonal antibodies to the progesterone receptor. We found that the picric acid paraformaldehyde perfusion provided satisfactory conditions of fixation for visualizing the progesterone receptor in sections of frozen tissue. Among the different techniques of immunocytochemical detection used, the indirect antibody peroxidase-antiperoxidase method gave the best results. The immunostained neurons were mainly located in two specific regions of the hypothalamus: the preoptic area and the mediobasal hypothalamus. Within the preoptic region, the majority of immunoreactive cells were present in the nucleus preopticus periventricularis particularly at the level of the pars suprachiasmatica. Within the mediobasal hypothalamus, immunostained neurons were found in the nucleus periventricularis, arcuatus, ventromedialis and premamillaris. Differences in the intensity of immunoreactivity appeared from one region to another. A marked cellular heterogeneity was observed: in each neuroanatomical structure, labeled cells alternated with non-labeled cells. The receptor, even in absence of progesterone, was localized in the nucleus. The nucleolus was not stained and only neurons were labeled. There was no progesterone receptor immunoreactivity in other regions of the brain, especially in the amygdala, hippocampus and cortex where biochemical studies have shown the presence of a non-estrogen regulated protein binding the progestin [3H]R 5020. Control experiments with antibody pretreated with purified progesterone receptor or with mouse receptor-unrelated monoclonal antibody did not show fluorescent or immunoreactive cells.     

Characteristics of [H]prazosin binding to guinea pig brain α-adrenergic receptors: effects of estradiol and progesterone

[³H]Prazosin was found to bind to sites on guinea pig brain membranes with α₁-adrenergic receptor characteristics. Treatment of ovariectomized guinea pigs with estradiol benzoate (EB) or EB followed by progesterone (P) did not affect [³H]prazosin binding to membranes from hypothalamus, preoptic area, amygdala or cerebral cortex. When added to the incubation mixture of the assay, estradiol, P, and other steroids decreased [³H]prazosin binding but only at high concentrations. These results do not support the idea that estrogen and progestin influence reproductive physiology through effects on brain α₁-receptors, although limitations of the methodology employed do not completely rule out this possibility.     

Estrogen-receptor immunoreactivity in hamster brain: preoptic area, hypothalamus and amygdala

The distribution of estrogen-receptor containing cells in the preoptic area, hypothalamus and amygdala of female Syrian hamster brain was studied by immunocytochemical methods. Dense populations of estrogen-receptor immunoreactive (ER-IR) cells were found in the medial preoptic area, the bed nucleus of the stria terminalis, amygdala, ventral and lateral parts of the hypothalamus, and the arcuate nucleus. Injection of estradiol caused a decrease in estrogen-receptor immunoreactivity (ERIR) containing cells within one hour, a decrease that may reflect a change in the ability of the occupied estrogen receptor to bind the particular antibody (H222) used rather than down-regulation of the estrogen receptor. Our findings on the distribution of estrogen-receptor containing cells in these areas using an immunocytochemical technique are consistent with and extend the findings of others using autoradiographic and in vitro binding techniques to study estrogen receptor distribution in hamster brain.     

Small apomorphine-induced increase in the concentration of cytosol estrogen receptors in female rat hypothalamus and pituitary

A series of experiments was performed to investigate the previously-reported modulation of estradiol binding in female rat brain and pituitary gland by drugs that influence the dopaminergic system. Injection of the dopamine agonist, apomorphine, at minimum doses of 1-2 mg/kg body weight caused slight increases (in most cases, less than 10%) in the concentration of cytosol estrogen receptors in the mediobasal hypothalamus and anterior pituitary gland without influencing the concentration in the preoptic area. However, after subsequent injection of a saturating dose of estradiol, the level of nuclear estrogen receptors accumulating did not differ significantly between apomorphine-treated animals and vehicle-injected controls. These results extend, in part, previous reports that have shown an apomorphine-induced increase in the concentration of [3H]estradiol in brain and pituitary cell nuclei after an injection of [3H]estradiol. However, we failed to observe differences in the concentration of cytosol estrogen receptors as large as would be expected by previous work, and we failed to observe differences in the concentration of nuclear estrogen receptors after estradiol injection.     

Progestin receptor cells in mouse cerebral cortex during early postnatal development: a comparison with preoptic area and central hypothalamus using autoradiography with [125I]progestin

The distribution of progestin target cells in the cerebral cortex and the effect of estrogen treatment was assessed during the critical period of brain development and compared with the preoptic/central hypothalamic regions. [125I]progestin was injected into 0, 2, 8, and 12 day postnatal mice pretreated for 3 days with oil, 5 micrograms/100 g b, wt., or 100 micrograms/100 g b. wt. of estradiol dissolved in oil. Two hours after injection of radiolabeled ligand, brains were frozen and processed for thaw-mount autoradiography. At birth, labeled cells were detected in the deep (lamina VI) and intermediate (lamina V) layers of the lateral cortical regions, increased in laminae V-VI of the lateral cortex and laminae II-VI of the cingulate/paracingulate cortex at days 2 and 8, and decreased throughout the cortex by day 12. Pretreatment of animals with estradiol had no noticeable effect on the nuclear concentration of [125I]progestin in cortical cells, while estrogen weakly enhanced labeling in preoptic/central hypothalamic regions at day 2 and markedly augmented labeling in the 8 and 12 day brain. The results demonstrated that progestin receptor cells are present in the postnatal dorsal cortex, preoptic area, and hypothalamus and that the topography of cortical progestin target cells differs in part from that of estrogen target cells reported earlier.     

The ontogeny of estrogen receptors: brain and pituitary

This study examines the prenatal and neonatal development of estrogen receptors in the central nervous system of the mouse. [3H]Diethylstilbestrol (DES) was injected into pregnant mice on days 4, 7, 10, 13, 14, 15 and 17 of gestation or into neonates. DES is an estrogen agonist that circumvents the alpha-fetoprotein barrier, thereby gaining access to intracellular estrogen receptors. Sixty minutes after injection whole embryos, fetuses or neonates were rapidly frozen and processed for autoradiography. Although the transplacental movement of the isotope was confirmed in all age groups evidence for nuclear estrogen receptors was not seen in the brain until day E14. On this day a few labeled cells first appeared in the basal hypothalamus, preoptic area, amygdala, midbrain and spinal cord. The number and the labeling intensity of target cells increased in each of these areas on days E15, E17 and P0. The first appearance of estrogen receptors closely follows the reported birthdates of neurons in these regions.     

Brain stem projections to lobule VII of the posterior vermis in the squirrel monkey: as demonstrated by the retrograde axonal transport of tritiated horseradish peroxidase.

In streptozotocin-diabetic female rats acute (24 h) withdrawal of insulin significantly impairs both estradiol+progesterone-induced sexual receptivity and cell nuclear concentration of [³H]estradiol in hypothalamus, preoptic area, and pituitary gland. Omission of insulin treatment for the first 24 h of a 30-h or 54-h estradiol benzoate-conditioning period significantly reduced mean lordosis ratings of ovariectomized-diabetic rats. Insulin withdrawal at the time of progesterone treatment and behavioral testing did not diminish sexual receptivity. One-half or 2 h after an intravenous injection of [³H]estradiol-17β diabetic rats without insulin exhibited reduced cell nuclear [³H]estradiol concentrations (2 h) and/or diminished cell nuclear/whole homogenate concentration ratios (0.5 and 2 h). Twenty-four hour insulin withdrawal affected neither whole tissue [³H]estradiol uptake nor hypothalamus-preoptic area cytoplasmic estrogen-receptor content. These results: (1) suggest that diminished estradiol binding by target tissue cell nuclei may contribute to the well-known reproductive failures of female diabetics, and (2) support the concept that estradiol acts at the level of brain cell nuclei to induce female sexual behavior.     

Identification of medial preoptic neurons that concentrate estradiol and project to the midbrain in the rat

Fluorescent dye retrograde tracing was combined with steroid hormone autoradiography to study the midbrain projections of the estrogen-concentrating neurons in the preoptic region of the rat brain. Microinjections of the dyes DAPI, true blue, or a mixture of DAPI and primuline were made into the ventral tegmental area and into the midbrain central gray of ovariectomized, adrenalectomized 2-3-month-old female rats; 3 or 4 days later these animals were injected with [3H]estradiol; the brains were then processed for autoradiography. After exposures of from 3 to 12 months, the autoradiograms were developed and examined for reduced silver grains under cell nuclei (indicating binding of [3H]estradiol) and retrogradely transported fluorescent dye in the cytoplasm (indicating an efferent projection to the midbrain). Numerous [3H]estradiol-concentrating neurons in the medial preoptic region were found to send their axons to the medial midbrain. The largest numbers of estrogen target neurons that were afferent to the ventral tegmental area and to the midbrain central gray were found in the medial preoptic nucleus, in the surrounding medial preoptic area, and in the ventral bed nucleus of the stria terminalis. Double-labeled neurons were also identified in the preoptic suprachiasmatic area, in the lateral preoptic area, and in the rostral anterior hypothalamic area. Thus, a subset of the gonadal steroid target cells of the preoptic region have long projections to the medial midbrain, and a subset of the medial preoptic neurons that project to the ventral tegmental area and to the midbrain central gray concentrate estrogen. Behaviors (for example, maternal behavior, male copulatory behavior, and wheel-running) that are regulated by estrogen action in the medial preoptic region may be controlled by the direct estrogen-sensitive pathway to the medial midbrain revealed in this study.     

Perinatal development of estrogen receptors in mouse brain assessed by radioautography, nuclear isolation and receptor assay

The development of estrogen receptors was investigated in vivo in the brains of fetal and neonatal mice 2 h after administering [3H]moxestrol to the pregnant mothers or neonates. Moxestrol bypasses the alpha-fetoprotein 'protective barrier' and gains access to estrogen receptors. Analysis of [3H]moxestrol uptake by radioautography and by cell nuclear isolation and counting of radioactivity revealed a marked increase in the number of estrogen receptors and estrophilic cells in the brain during late fetal and early postnatal development. Assays of cytosol estrogen receptors were conducted in parallel and revealed a comparable pattern of development. The increase in estrogen receptors and labeling was especially great from embryonic day (E) 15 to E18. Cytosol assays revealed a low level of receptors in the whole brain on E13. Radioautography revealed that clearly labeled cells in the hypothalamus and preoptic area were virtually absent on E13 but were evident on E15, with marked increases occurring between E15 and E18, both in number of labeled cells and in intensity of labeling per cell. Within the cerebral cortex the dorsal cingulate cortex was the most extensively labeled area; however, clearcut labeling was not evident on E13 or E15. Thus, the development of cortical estrogen receptors occurs somewhat later than that in the hypothalamus and preoptic area. The perinatal increase in estrogen receptors usually begins several days after the birthdates of neurons in these estrophilic regions of the brain, and corresponds to the early responsiveness of these neurons to the organizational and activational influences of estrogen.     

Age‐related changes in hypothalamic androgen receptor and estrogen receptor α in male rats

The control of reproductive function involves actions of sex steroids upon their nuclear receptors in the hypothalamus and preoptic area (POA). Whether hypothalamic hormone receptors change their expression in aging male mammals has not been extensively pursued, although such changes may underlie functional losses in reproductive physiology occurring with aging. We performed a stereologic analysis of immunoreactive androgen receptor (AR) and estrogen receptor alpha (ERα) cells in three POA nuclei of male Sprague‐Dawley rats (anteroventral periventricular nucleus [AVPV], median preoptic area [MePO], and medial preoptic nucleus [MPN]), at young (3 months), middle‐aged (12 months), and old (20 months) ages. Serum testosterone and estradiol levels were assayed. Testosterone concentrations decreased significantly and progressively with aging. Estradiol concentrations were significantly higher in middle‐aged than either young or old rats. Stereologic analyses of the POA demonstrated that AR‐immunoreactive cell numbers and density in the AVPV, MePO, and MPN were significantly higher in old compared with young or middle‐aged rats. No change in the total number or density of ERα‐immunoreactive cells was detected with age, although when cells were subdivided by intensity of immunolabeling, the most heavily labeled ERα cells increased in number with aging in the AVPV and MePO, and in density in the AVPV. There are several interpretations to our finding of substantially increased AR cell numbers during aging, including a potential compensatory upregulation of the AR under diminished testosterone concentrations. These results provide further information about how the neural targets of steroid hormones change with advancing age. J. Comp. Neurol. 512:688–701, 2009. © 2008 Wiley‐Liss, Inc.     

Nuclear retention charactristics of [H]estrogen by cells in four estrogen target regions of the rat brain

Nuclear retention of radioactivity was studied in neural estrogen target cells 15 min–7 h after i.v. injection of [³H]estradiol. Maximal uptake occured by 15 min. Cells of the medial preoptic nucleus retained the label longer than did those of the medial amygdaloid nucleus. Cells of the ventromedial nucleus and arcuate nucleus exhibited similar retention profiles which were intermediate between the medial preoptic and medial amygdaloid cells. These data are discussed in relation to observations that the duration of nuclear occupancy by estrogens is proportional to the magnitude of the cellular response.     

Estrogen receptor-immunoreactive forebrain neurons project to the ventrolateral hypothalamus in female guinea pigs

In rodents, the facilitation of sexual receptivity by estradiol and progesterone is suspected to be mediated by a network of neurons containing estrogen and progestin receptors. In female guinea pigs, this network would include estrogen receptor-immunoreactive (ER-ir) neurons located within the rostro-ventral ventrolateral hypothalamus (r-vVLH). This hypothesis predicts that a proportion of the neurons projecting to the r-vVLH contains estrogen receptors. This prediction was tested through retrograde tracing combined with immunocytochemistry for estrogen receptors. Retrogradely labelled neurons were particularly abundant within the medial preoptic nucleus (MPN), bed nucleus of the stria terminalis (BST), anterior hypothalamus, amygdala, and lateral parabrachial nucleus. As predicted by the hypothesis, retrogradely labelled neurons were mostly observed in estrogen receptor-rich areas. Retrogradely labelled neurons also containing estrogen receptor-immunoreactivity (ER-IR) were primarily found within the MPN, BST, and amygdala. However, a majority of retrogradely labelled neurons did not contain ER-IR. As the preoptic area and the r-vVLH are both responsive to estradiol in the facilitation of sexual receptivity by progesterone, these data are consistent with the hypothesis tested. However, our data also suggest that the network of neurons controlling sexual receptivity may include elements not directly sensitive to estradiol. Finally, the location of retrogradely labelled neurons is discussed with respect to the stimuli provided to the r-vVLH in the context of sexual receptivity facilitated by estradiol and progesterone. © 1993 Wiley-Liss, Inc.