Sex differences in androgen receptors and aromatase activity in microdissected regions of the rat brain

Males are generally more responsive than females to the behavioral and neuroendocrine actions of androgens. The present experiments were performed to determine whether these differences may result from sex differences in the number of androgen receptors (AR) in specific brain areas. For this reason, AR binding was compared in both cytosol (ARc) and cell nuclear KCl extracts (ARn) from microdissected brain regions of gonadectomized male and female rats treated with doses of testosterone (T) that produced equivalent physiological circulating androgen levels. In addition, microsomal aromatase activity was measured as a biochemical index of tissue responsiveness to T, since estrogen formation in certain brain areas is regulated by androgen. One week after exogenous T administration, males exhibited significantly higher levels of ARn than females in the bed nucleus of the stria terminalis, periventricular preoptic area, and ventromedial nucleus. Males also had significantly higher aromatase levels in these same areas plus the medial preoptic nucleus and anterior hypothalamus. There were no significant differences in ARn concentrations in eight other nuclei that were examined for significant sex differences in ARc levels observed under these experimental conditions. When ARc levels were compared in untreated gonadectomized male and female rats, males had greater levels of ARc in the bed nucleus of the stria terminalis only, indicating that new receptor synthesis may be responsible for the sex differences observed in T-treated rats. These results suggest that sex differences in neural responsiveness to androgens may be due in part to sex differences in ARn occupation in specific brain regions.     

Distribution and regulation of aromatase activity in the rat hypothalamus and limbic system

Conversion of androgen to estrogen in the rat brain is catalyzed by aromatase enzymes. The maximum concentrations of these enzymes are found within the hypothalamus and amygdala, where they appear to play an important role in the process by which androgens affect both behavior and neuroendocrine function. In the present study, we measured the levels of aromatase activity (AA) in 20 nuclei and brain regions of the adult rat brain. Individual nuclei were microdissected from 600-micron frozen sections. Tissues from 3 animals were pooled, and AA was measured by an in vitro radiometric assay that quantifies the stereospecific production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. We report that AA is heterogeneously distributed within the rat brain. The greatest amounts of activity were found in the bed nucleus (n.) of the stria terminalis (700 protein fmol/h . mg) and in the medial (MA) and cortical amygdala (400-600 fmol/h . mg protein) of the male. There was an evident rostral-caudal and medial-lateral gradient in AA throughout the diencephalon. Activity was high in the periventricular preoptic n. and medial preoptic n.; intermediate in the suprachiasmatic preoptic n., anterior hypothalamus, periventricular anterior hypothalamus, and ventromedial n.; and low in the arcuate n.-median eminence, lateral preoptic n., supraoptic n., dorsomedial n., and lateral hypothalamus. Regions devoid of measurable AA included the medial and lateral septum, caudate-putamen, hippocampus, and parietal cortex. In the female, AA was greatest in the MA and cortical amygdala. We found that AA in the MA, stria terminalis n., suprachiasmatic preoptic n., periventricular preoptic in., medial preoptic n., anterior hypothalamus, and ventromedial n. was significantly greater (P less than 0.05) in males than in females. Orchidectomy reduced AA to levels seen in females, and administration of testosterone to castrated males restored AA in these areas. No significant sex differences were observed in any other hypothalamic or amygdaloid nuclei, although AA was increased by testosterone treatment in the periventricular anterior hypothalamus, arcuate n.-median eminence, and lateral hypothalamus. Our results provide a quantitative profile of AA in specific hypothalamic and limbic nuclei of the rat brain as well as information on the control of AA within these discrete regions.     

Estrogen induction of progestin receptors in microdissected hypothalamic and limbic nuclei of female guinea pigs

Estrogen induction of cytosolic progestin receptors (CPRs) in the hypothalamus-preoptic area of the female guinea pig is correlated with facilitation of female sexual behavior and gonadotropin secretion by progesterone. The present study validated a CPR microassay and determined where, within microdissected areas of the hypothalamus-preoptic area of the female guinea pig, induction of CPRs by estradiol occurs. Ovariectomized adult guinea pigs were given 20 micrograms estradiol benzoate (EB) or oil vehicle for 3 successive days. CPRs were measured using the synthetic progestin [3H]-R5020. The highest basal (no estrogen treatment) level of CPRs was seen in the arcuate-median eminence (34.1 +/- 3.7 fmol/mg). With EB treatment, the highest level of CPRs was again in the arcuate-median eminence (178.0 +/- 12.0 fmol/mg). EB-treated females also had high CPR levels in the periventricular area (88.5 +/- 10.8 fmol/mg) and the medial preoptic area (86.3 +/- 9.3 fmol/mg). Moderate levels were seen in the ventromedial nucleus of the hypothalamus (32.7 +/- 3.0 fmol/mg) and in the anterior hypothalamic nucleus (13.0 +/- 2.1 fmol/mg), but these were not significantly different from the low levels in the medial amygdala (4.5 +/- 1.2 fmol/mg) and in the dorsomedial nucleus of the hypothalamus (5.4 +/- 1.1 fmol/mg) of EB-treated females. However, EB caused a significant induction over baseline levels not only in the arcuate-median eminence, periventricular area, and medial preoptic area, but also in the ventromedial nucleus of the hypothalamus and the anterior hypothalamic nucleus. EB did not increase CPRs in the medial amygdala or the dorsomedial nucleus of the hypothalamus.     

Progesterone modulation of androgen receptors in the brain and pituitary of male guinea pigs

Androgen receptors (AR) were determined in cytosol and nuclear extracts of pituitary and neural tissue from intact male guinea pigs by a binding assay using [3H]dihydrotestosterone as ligand. Saturation analyses of cytosol from hypothalamus-preoptic area (POA)-amygdala regions and anterior pituitary revealed receptors (ARc) with apparent Kd values of 2.52 and 3.83 X 10(-10) M, respectively. Nuclear salt extracts from the same tissues contained receptors (ARn) with Kd values of 4.38 and 5.12 X 10(-10) M. Reproductive behavior of 10 males was observed with receptive females for 10 min once a week. After 4 weeks, half of the animals received 10 mg progesterone (P)/day for an additional 4 weeks. P treatment significantly (P less than 0.05) increased latency to first mount and decreased mounts per test period. After behavioral testing, analysis of the AR content of specific brain regions revealed that the highest concentrations of ARc and ARn were in the POA and medial basal hypothalamus, and the lowest were in the cerebral cortex. The ARn content was significantly suppressed in POA and medial basal hypothalamus (P less than 0.05) from P-treated males compared to the control value. These data show that AR content is highest in areas thought to control behavior and gonadotropin release within the brain of the male guinea pig. In addition, the antiandrogenic actions of P on the central nervous system, which in this experiment were expressed as a significant decline in reproductive behavior, may be explained by its interference with the retention of the AR in the nucleus.     

The quantitative distribution of cytosolic androgen receptors in microdissected areas of the male rat brain: effects of estrogen treatment

Estrogen and androgen synergize in the regulation of various neuroendocrine functions. To determine a potential cellular basis of this synergism, we measured androgen receptor (AR) in the cytosol of 16 hypothalamic and limbic nuclei and subregions in castrated male rats and castrated rats treated with estradiol. Androgen receptor was measured by a previously validated in vitro binding assay using the synthetic androgen methyltrienolone [( 3H]R1881). Male Sprague-Dawley rats (250-350 g) were castrated 2 weeks before the implantation of a 2.5-cm Silastic capsule filled with crystalline 17 beta-estradiol. Control rats were sham implanted. Estrogen treatment lasted for 1 week, after which time the animals were killed, their brains were frozen and sectioned, and individual nuclei and subregions were removed by a tissue punch technique. Tissue from six rats were combined for each determination. The highest levels of AR were found in the ventromedial nucleus (16.5 +/- 1.4 fmol/mg protein), medial preoptic area (12.1 +/- 1.4 fmol/mg protein), bed nucleus of the stria terminalis (11.6 +/- 1.4 fmol/mg protein), lateral septum (11.4 +/- 1.4 fmol/mg protein), arcuate nucleus-median eminence (10.9 +/- 2.1 fmol/mg protein), and medial amygdala (10.3 +/- 0.9 fmol/mg protein). Estrogen treatment resulted in significant increases in AR in medial preoptic area (14.8 +/- 0.6 fmol/mg protein; P less than 0.05) and medial amygdala (14.6 +/- 1.2 fmol/mg protein; P less than 0.02). Subsequent studies using block-dissected hypothalamus-preoptic area, anterior pituitary, and prostate revealed significant estrogen-mediated elevations in AR in anterior pituitary cytosol [42.2 +/- 3.0 vs. 26.4 +/- 1.6 fmol/mg protein (control); P less than 0.01], but not in hypothalamus-preoptic area or prostate cytosols. Estrogen treatment had no effect on AR affinity. The binding of [3H]R1881 was specific for AR and was not affected by the addition of radioinert progesterone to the incubation tube. Estimates of AR concentration were similar regardless of whether [3H]R1881 or [3H]dihydrotestosterone was used as the ligand. In this study, we describe the distribution of AR throughout the hypothalamus and limbic areas using biochemical techniques. In addition, we have identified some cellular events that may mediate the synergistic actions of estrogen and androgen on the neuroendocrine system.     

Regional sex differences in cell nuclear estrogen-binding capacity in the rat hypothalamus and preoptic area

Estrogen binding was compared in cell nuclear KCl extracts from microdissected brain regions of gonadectomized-adrenalectomized male and female rats treated with a near-saturating dose of 17 beta-estradiol. Injection of 3.6 or 36.0 micrograms 17 beta-estradiol/kg BW, iv, 1 h before death resulted in a higher level of estrogen binding in the periventricular preoptic area (PVPOA), medial preoptic area, and ventromedial nucleus of the hypothalamus (VMN) of the female than in comparable tissue samples from the male. No significant sex differences in nuclear estrogen binding were observed in the arcuate-median eminence region, bed nucleus of the stria terminalis, or corticomedial amygdala. Scatchard analysis of saturation binding data revealed that the sex differences in cell nuclear estrogen binding in the PVPOA, medial preoptic area, and VMN reflect a difference in binding capacity rather than binding affinity. These in vitro biochemical findings were confirmed by autoradiographic studies. Gonadectomized-adrenalectomized animals were injected with 125I-labeled 11 beta-methoxy-16 alpha-iodoestradiol (2.0 micrograms/kg BW). Thin frozen sections (10 microns) through the preoptic area and hypothalamus were thaw-mounted onto microscope slides, then exposed against LKB Ultrofilm for 21 days. The autoradiographic images exhibited similar silver distributions and densities in males and females in the arcuate-median eminence region bed nucleus of the stria terminalis, and amygdala. However, 11 beta-[125I]methoxy-16 alpha-iodoestradiol uptake was lower in males than in females in the PVPOA and VMN. These results suggest that sex differences in responsiveness to estrogen stimulation in the rat may be due in part to sex differences in estrogen-binding capacity in specific regions of the hypothalamus that play important roles in the control of pituitary function and reproductive behaviors.     

Regulation of androgen metabolism and luteinizing hormone-releasing hormone content in discrete hypothalamic and limbic areas of male rhesus macaques

The conversion of androgens to active metabolites by neural tissue is believed to be an essential component in the cellular mechanism of androgen-induced neuroendocrine responses. In this study, we measured the in vitro aromatization and 5 alpha-reduction of androgens in incubations of microdissected brain regions from four intact and five castrated (6 weeks) adult male rhesus monkeys. Individual nuclei were microdissected from 600-microns frozen brain sections and homogenized in a potassium phosphate buffer. Aromatase activity was measured by a radiometric assay that uses the incorporation of tritium from [1 beta-3H]androstenedione into 3H2O as an index of estrogen formation. We estimated 5 alpha-reductase activity by isolating 5 alpha-dihydrotestosterone on two different chromatography systems and measuring the amount of this product formed from [1 alpha,2 alpha-3H]testosterone. We acidified a portion of each homogenate and determined LHRH content by RIA. Between brain nuclei, aromatase activity varied 1500-fold, whereas 5 alpha-reductase activity varied only 3-fold. Both enzyme activities were highest in amygdaloid, medial preoptic, and medial diencephalic nuclei and lowest in the caudate nucleus. Aromatase activities in the supraoptic nucleus, periventricular area, medial preoptic area-anterior hypothalamus, and lateral hypothalamus were significantly (P less than 0.05) lower in castrated males. Castration did not significantly affect 5 alpha-reductase activity, except for an increase in the basolateral amygdala. The highest concentrations of LHRH were in the infundibular nucleus-median eminence and were 30 times greater than amounts measured in preoptic and medial hypothalamic nuclei. The LHRH contents of the infundibular nucleus-median eminence, ventral medial nucleus, and lateral hypothalamus were significantly lower in castrated males (P less than 0.05). In addition, we observed a significant correlation between aromatase activity and LHRH content in the basal hypothalamus of intact males (r = 0.947; P less than 0.05; n = 8), but not in the preoptic-anterior hypothalamus (r = 0.068; P greater than 0.05; n = 10). No correlation was observed between 5 alpha-reductase activity and LHRH content in either area. These data indicate that castration selectively affects androgen metabolism and LHRH content in discrete regions of the brain of male monkeys and suggest that aromatase and 5 alpha-reductase are regulated differentially in the primate brain.     

Changes in androgen receptor mRNA expression in the forebrain and oviduct during the reproductive cycle of female leopard geckos,Eublepharis macularius

Successful reproduction requires the coordination of reproductive physiology with behavior. The neural correlates of reproductive behavior have been elucidated in a variety of amphibians, mammals, and birds but relatively few studies have examined reptiles. Here we investigate differences in androgen receptor (AR) mRNA expression in the forebrain and oviduct between previtellogenic and late vitellogenic female leopard geckos, Eublepharis macularius. Plasma concentrations of testosterone (T) are low when females are previtellogenic and sexually unreceptive but increase dramatically during late vitellogenesis when females are receptive. In addition, receptivity can be induced by treatment with exogenous T. The relative abundance of AR-mRNA across various nuclei was greater in late vitellogenic than in previtellogenic females. This general pattern was observed in the medial preoptic area, anterior hypothalamus, external nucleus of the amygdala, dorsolateral aspect of the ventromedial hypothalamus, lateral septum, and periventricular hypothalamus. There were also clear differences in AR-mRNA expression among these nuclei. The pattern of gene expression observed in the brain was reversed within stromal cells of the oviduct where expression of AR-mRNA decreased from the previtellogenic stage to the late vitellogenic stage. Overall, these data demonstrate that T concentration in the plasma, abundance of AR-mRNA in the brain and oviduct, and sexual behavior change coordinately during the reproductive cycle of female leopard geckos. Although the function of AR in the female leopard gecko is not yet clear, our results are in accord with growing evidence that androgens regulate numerous aspects of female physiology and behavior in vertebrates.     

Differential regulation of brain aromatase by androgen in adult and fetal ferrets

The contribution of androgens to the regulation of aromatase activity (AA), measured by quantifying the in vitro formation of [3H]estrone from 19-[3H]hydroxyandrostenedione precursor, was studied in equivalent microdissected brain regions of adult and fetal ferrets. In adulthood, AA was similar in the bed nucleus of the stria terminalis, medial (M) and lateral (L) preoptic area (POA), medial (MA) and lateral amygdala (LA), ventromedial hypothalamus (VMH), and parietal cortex of gonadectomized males and females given no concurrent steroid treatment. Daily sc injections of the androgen dihydrotestosterone propionate significantly stimulated AA in MPOA, MA, and VMH of adult males and in MA of females; a similar trend was seen in MPOA and VMH of females. By contrast, no evidence of androgenic regulation of AA was obtained in these three brain regions microdissected from fetuses killed on embryonic day 35 (E35; 41-day gestation). Transplacental administration of the antiandrogen flutamide beginning on day E24 failed to affect AA in MPOA, LPOA, MA, LA, or parietal cortex, although this treatment significantly reduced AA in bed nucleus of the stria terminalis of fetal males. The results suggest that the responsiveness of aromatizing enzymes to androgenic induction is similar in several subcortical brain regions of adult ferrets of both sexes. In breeding males such an action of androgen may augment the neural production of estrogen, which has previously been implicated in the control of sexual behavior and the feedback regulation of LH secretion. By contrast, androgen apparently contributes minimally to the regulation of AA in brain regions of fetal ferrets, particularly in the MPOA, in which a sexually dimorphic nucleus differentiates in males around E37 in response to estrogen produced locally.     

Sex difference in the cytosolic and nuclear distribution of androgen receptor in mouse submandibular gland

Cytosol and nuclear androgen receptors in submandibular glands of male and female mice were measured by an exchange assay at 0 degree C. The binding of [3H]methyltrienolone to cytosol receptors in females was mostly saturated within a short period of incubation (3 h), whereas the saturation was much slower in males; suggesting that almost all of the cytosol receptors were unoccupied in females and the receptors were partially occupied in males. Nuclear receptors were extracted with pyridoxal 5'-phosphate (5 mmol/l) from nuclear fractions with 93-95% efficiency. The exchange of the bound steroids occurred by 24-48 h at 0 degree C, suggesting that most of the nuclear androgen receptor was occupied. The binding was low at higher temperatures, probably due to inactivation of the receptor. Scatchard analysis showed that the apparent dissociation constants of cytosol and nuclear receptors were similar (0.8 and 0.9 nmol/l respectively) in both sexes. On the other hand, the number of androgen-binding sites in the nucleus was much higher in males than in females (1052 fmol/mg DNA and 32 fmol/mg DNA respectively), while the number in the cytosol was higher in females than in males (512 fmol/mg DNA and 368 fmol/mg DNA respectively). These observations show that androgen receptors exist mainly (74%) in the nuclei of males, while they exist mostly (94%) in the cytosol of females.     

Autoradiographic localization of angiotensin II receptors in rat brain.

The 125I-labeled agonist analog [1-sarcosine]-angiotensin II ( [Sar1]AII) bound with high specificity and affinity (Ka = 2 X 10(9) M-1) to a single class of receptor sites in rat brain. This ligand was used to analyze the distribution of AII receptors in rat brain by in vitro autoradiography followed by computerized densitometry and color coding. A very high density of AII receptors was found in the subfornical organ, paraventricular and periventricular nuclei of the hypothalamus, nucleus of the tractus solitarius, and area postrema. A high concentration of receptors was found in the suprachiasmatic nucleus of the hypothalamus, lateral olfactory tracts, nuclei of the accessory and lateral olfactory tracts, triangular septal nucleus, subthalamic nucleus, locus coeruleus, and inferior olivary nuclei. Moderate receptor concentrations were found in the organum vasculosum of the lamina terminalis, median preoptic nucleus, medial habenular nucleus, lateral septum, ventroposterior thalamic nucleus, median eminence, medial geniculate nucleus, superior colliculus, subiculum, pre- and parasubiculum, and spinal trigeminal tract. Low concentrations of sites were seen in caudate-putamen, nucleus accumbens, amygdala, and gray matter of the spinal cord. These studies have demonstrated that AII receptors are distributed in a highly characteristic anatomical pattern in the brain. The high concentrations of AII receptors at numerous physiologically relevant sites are consistent with the emerging evidence for multiple roles of AII as a neuropeptide in the central nervous system.     

Regional specificity in estradiol effects on [3H]uridine incorporation in rat brain

In this study, the effects of 15 days of estradiol (E2) on tritiated-uridine incorporation were autoradiographically examined, on a cell-by-cell basis, in 4 E2-concentrating regions of the female rat brain. These areas included the ventromedial hypothalamus and medial amygdala nucleus, 2 regions involved in the behavioral components of reproduction, and the medial preoptic area and arcuate nucleus of the hypothalamus, 2 regions involved in the endocrine components of reproduction. Chronic E2 caused a 50% and 52% increase in tritiated-uridine incorporation in the arcuate nucleus and medial preoptic area, but was without effect in the ventromedial hypothalamus and medial amygdala nucleus. Somal area was also increased with E2 in the arcuate nucleus and medial preoptic area (16% and 43%, respectively) but not in the ventromedial hypothalamus and medial amygdala nucleus. The results indicate that the effects of estradiol on levels of newly synthesized RNA vary in a functionally and regionally specific manner within the brain.     

Estradiol-induced progestin receptor immunoreactivity is found only in estrogen receptor-immunoreactive cells in guinea pig brain

A fluorescent immunocytochemical technique was developed to determine if cells in the guinea pig hypothalamus and preoptic area that contain estradiol-induced progestin receptors also contain estrogen receptors. With this technique little or no progestin receptor-immunoreactivity (PR-IR) was observed in the absence of estrogen treatment in ovariectomized guinea pigs. As has been reported previously, priming with estradiol caused a large increase in the concentration of PR-IR cells in discrete regions of the hypothalamus and preoptic area, primarily in the arcuate nucleus, ventrolateral area of the hypothalamus, periventricular preoptic area, medial preoptic nucleus, medial preoptic area, anterior hypothalamic nucleus and anterior hypothalamus. A range of lightly to intensely labeled estrogen receptor-immunoreactive (ER-IR) cells were observed in high concentration in each of these areas, as well as in some areas in which no PR-IR cells have been identified, such as the amygdala. PR-IR was only observed in cells that also had ER-IR. In some areas such as the ventrolateral hypothalamic area and arcuate nucleus, nearly all medium to highly-fluorescent ER-IR cells also contained estradiol-induced PR-IR, while in the amygdala no PR-IR was observed despite a high concentration of ER-IR cells. These results confirm the hypothesis that progestin receptors are produced in estrogen receptor-containing cells in the brain, and they suggest that these cells are the sites where estradiol and progesterone act to influence behavior and physiology.     

Sex differences in progesterone receptor expression: a potential mechanism for estradiol-mediated sexual differentiation

The differential exposure of males and females to testosterone (T) and its metabolite estradiol (E) contributes to the development of sex differences in the brain. However, the mechanisms by which T and E permanently alter neural development remain virtually unknown. Two regions of the rat preoptic area, the anteroventral periventricular nucleus (AVPv) and the medial preoptic nucleus (MPN), are sexually dimorphic and serve as models for studying the hormonal mechanisms of sexual differentiation. Around birth, these regions express dramatically higher levels of progesterone receptor immunoreactivity (PRir) in males than they do in females. The present study examined the possibility that sexually dimorphic induction of PR expression in these two regions constitutes a potential mechanism of E-mediated sexual differentiation. Prenatal exposure to either T propionate or the synthetic estrogen, diethylstilbestrol, but not dihydrotestosterone propionate, significantly increased PRir levels in the MPN and AVPv of fetal females compared with controls. Prenatal exposure to the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione, significantly reduced PRir in the MPN and AVPv of fetal males, whereas the androgen receptor antagonist flutamide had no effect. This suggests that aromatization of T into E is crucial for the sex difference in PR expression in the MPN and AVPv during development.     

Opiate receptor ontogeny in the rat medial preoptic area is androgen-dependent

The relationship of opiate receptors in the medial preoptic area of the hypothalamus (MPOA) to the gonadal steroid hormone environment during development was assessed using regional densitometric analysis of [3H]naloxone binding in autoradiographs prepared using brain sections from 5-day-old male and female rats treated postnatally either with tamoxifen (0.5 mg/kg), flutamide (20 mg/kg), dihydrotestosterone (DHT; 12.5 mg/kg), or sesame oil vehicle. Tamoxifen, a specific estrogen receptor antagonist, did not alter MPOA binding density in either males or females. Flutamide, a specific androgen receptor antagonist, and DHT, a nonaromatizable androgenic compound, altered the MPOA binding density in males and females, respectively. No treatment altered the binding density in several other brain regions. The results suggest that androgen, not estrogen, regulates the differentiation of MPOA opiate receptors. Since the neuronal development in the region is thought to be mediated by estrogen, both hormones probably act concurrently to affect the ontogeny of different parameters in the same brain region.     

Comparison of the effects of castration and steroid replacement on incertohypothalamic dopaminergic neurons in male and female rats

The activities of incertohypothalamic (IH) and tuberoinfundibular (TI) dopamine (DA) neurons were compared in selected brain regions of male and female rats by measuring the rate of DA turnover (alpha-methyltyrosine-induced decline in brain DA concentrations). The rates of DA turnover in regions containing TIDA (median eminence) and rostral IHDA (rostral periventricular and medial preoptic nuclei) neurons were greater in diestrous females than in intact males. In contrast, the rate of DA turnover in the caudal IHDA neurons (medial zona incerta), was greater in intact males than diestrous females. These results indicate that the activities of IHDA neurons, like those of TIDA neurons, differ between the sexes but that the sexual differentiation of IHDA neurons is not homogeneous. Two weeks following orchidectomy, the rates of DA turnover were increased in the median eminence and decreased in the medial preoptic nucleus. Testosterone replacement in orchidectomized males produced opposite effects, causing a decrease in DA turnover in the median eminence and an increase in the medial preoptic nucleus. In female rats, the rates of DA turnover were decreased in the median eminence and medial zona incerta and increased in the medial preoptic nucleus 2 weeks following ovariectomy. Only in the median eminence did 2 days of estrogen replacement in ovariectomized rats produce effects opposite those seen after ovariectomy alone. These data show that the activities of IHDA neurons, as estimated from measurements of DA turnover, can be altered by the removal and replacement of the gonadal steroids.     

Regulation of Kiss1 expression by sex steroids in the amygdala of the rat and mouse

Kisspeptin (encoded by the Kiss1 gene) is an important regulator of reproduction. In rodents, Kiss1 is expressed in two hypothalamic regions, the arcuate nucleus and anteroventral periventricular/ periventricular continuum, where it is regulated by sex steroids. However, the distribution, regulation, and functional significance of neural kisspeptin outside of the hypothalamus have not been studied and are poorly understood. Here, we report the expression of Kiss1 in the amygdala, predominantly in the medial nucleus of the amygdala (MeA), a region implicated in social and emotional behaviors as well as various aspects of reproduction. In gonadally intact rats and mice, Kiss1-expressing neurons were identified in the MeA of both sexes, with higher Kiss1 expression levels in adult males than females in diestrus. In rats, Kiss1 expression in the MeA changed as a function of the estrous cycle, with highest levels at proestrus. Next, we tested whether Kiss1 in the MeA is regulated by the circulating sex steroid milieu. Kiss1 levels in the MeA were low in gonadectomized mice and rats of both sexes, and treatment with either testosterone or estradiol amplified Kiss1 expression in this region. Testosterone's inductive effect on Kiss1 expression in the MeA likely occurs via estrogen receptor-dependent pathways, not through the androgen receptor, because dihydrotestosterone (a nonaromatizable androgen) did not affect MeA Kiss1 levels. Thus, in rodents, Kiss1 is expressed and regulated by sex steroids in the MeA of both sexes and may play a role in modulating reproduction or brain functions that extend beyond reproduction.     

Estrogen receptor beta messenger ribonucleic acid expression in the forebrain of proestrous,pregnant, and lactating female rats

Estrogen receptor (ER)beta is present in hypothalamic and limbic neurons of female rat brains, but little is known about its regulation under physiological conditions. To determine whether ER beta expression varies during physiological conditions in which sex steroid hormone profiles are significantly different, we used in situ hybridization to assess ER beta mRNA expression in the periventricular preoptic area, bed nucleus of stria terminalis, paraventricular nucleus, supraoptic nucleus, and the posterodorsal medial amygdala of female rats on proestrus, on d 22 of pregnancy, or on d 10 of lactation (L10). In the periventricular preoptic area, d-22 pregnant females had fewer ER beta-mRNA-expressing cells than did females at proestrus, but the level of ER beta mRNA expression per cell in pregnant females was higher than in the two other groups. In the paraventricular nucleus, no changes in ER beta mRNA expression were observed; whereas in the supraoptic nucleus, proestrous females had fewer ER beta-mRNA-expressing cells than L10 females. In the posterodorsal medial amygdala, proestrous females had a greater number of ER beta-mRNA-expressing cells than did L10 females. These results demonstrate that ER beta mRNA expression is differentially regulated in a brain-region-specific and temporal manner under physiological conditions and suggest that ER beta may participate in the regulation of estrogen-sensitive reproductive functions in female rats.     

Sexual dimorphism characterizes steroid hormone modulation of rat aortic steroid hormone receptors

Quantification of aortic androgen and estrogen receptor content and distribution in AXC/SSh rats established that the total androgen receptor content in intact young mature males (mean +/- SD, 55 +/- 13 fmol/mg DNA) was indistinguishable (P greater than 0.05) from that in proestrous females (50 +/- 3 fmol/mg DNA). However, 60% of male aortic androgen receptors were in the nuclear fraction, whereas all proestrous female aortic androgen receptors were in the cytoplasmic fraction. The total aortic estrogen receptor content of intact young mature males (70 +/- 16 fmol/mg DNA) was indistinguishable (P greater than 0.05) from that of proestrous (92 +/- 12) or diestrous (77 +/- 4) females. However, 50% of proestrous female aortic estrogen receptors were in the nuclear fraction, whereas male or diestrous female aortic estrogen receptors were restricted to the cytoplasmic fraction. To assess estrogen receptor function, we characterized aortic cytoplasmic progesterone receptors and established that the receptor content of intact male aortae (101 +/- 3 fmol/mg DNA) was not significantly different (P greater than 0.05) from that of diestrous female aortae (100 +/- 11). 17 beta-Estradiol injection of intact males failed to affect aortic progesterone receptor content (93 +/- 17 fmol/mg DNA). However, injection of orchiectomized males with 17 beta-estradiol significantly (P less than 0.05) increased progesterone receptor content to 208 +/- 24 fmol/mg DNA. This value is twice that of intact males and is not significantly different (P greater than 0.05) from the aortic cytoplasmic progesterone receptor content (190 +/- 32 fmol/mg DNA) of 17 beta-estradiol-injected oophorectomized females. These studies establish that intracellular distribution of aortic androgen and estrogen receptors of male or female AXC/SSh rats is regulated by endogenous hormones. The observation that 17 beta-estradiol modulates aortic progesterone receptor content indicates that rat aortic estrogen receptors are physiologically functional. Our data imply that steroid hormones directly regulate aspects of rat cardiovascular cell function and that sexually dimorphic differential regulation may characterize male and female aortic metabolism.