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Endocrinology, doi:10.1210/en.2003-1363
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Endocrinology Vol. 145, No. 6 2906-2917
Copyright © 2004 by The Endocrine Society

Brain Estradiol Content in Newborn Rats: Sex Differences, Regional Heterogeneity, and Possible de Novo Synthesis by the Female Telencephalon

Stuart K. Amateau, Jesse J. Alt, Carolyn L. Stamps and Margaret M. McCarthy

Program in Neuroscience and Department of Physiology, University of Maryland at Baltimore, School of Medicine, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Stuart K. Amateau, Department of Physiology, University of Maryland at Baltimore, School of Medicine, 655 West Baltimore Street, Bressler RB 5020, Baltimore, Maryland 21201. E-mail: samat001{at}umaryland.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Accurate assessment of gonadal steroid levels in the developing brain is critical for understanding naturally occurring steroid-mediated sexual differentiation as well as determining the physiological relevance of exogenous steroid treatments commonly used in the study of this phenomenon. Using RIA, we measured the estradiol (E2) content of six regions of the developing brain immediately post partum, 1 d post partum, and after injection of exogenous estradiol benzoate, testosterone propionate, or the aromatase inhibitor formestane. We found sexually dimorphic E2 content in several regions of the newborn brain. At 2 h of life, there was significantly higher E2 content in males vs. females in the frontal cortex, hypothalamus and preoptic area but not in the hippocampus, brainstem, or cerebellum. Surprisingly, the female hippocampus had significantly higher E2 content than all other female regions examined. By d 1 post partum, E2 levels had decreased precipitously in most brain regions, and only the hypothalamus maintained a sex difference. Injection of female pups with estradiol benzoate raised tissue levels to that of the male in the hypothalamus but 2- to 3-fold higher in the other five regions. Testosterone administration increased E2 content exclusively in the preoptic area, suggesting local variation in aromatase activity and/or substrate availability. Central administration of formestane decreased estrogen content in the male cortex, hypothalamus, and preoptic area. Formestane treatment also decreased endogenous E2 in female cortex and hippocampus, suggesting de novo synthesis selectively in these brain regions. These data corroborate and extend previous findings of sex differences in brain E2 levels perinatally and reveal an unexpected regional heterogeneity in E2 synthesis and/or metabolism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXPERIMENTS INVESTIGATING THE effects of perinatal hormone exposure on adult behavior date as far back as the early 1930s (1). These and later findings were codified as the organizational hypothesis of hormone action on the developing brain in the seminal paper of Phoenix, Goy, Gerall and Young in 1959 (1A ). This decisive precept states that during specific periods of development, hormone secreted by the male testes exerts permanent effects on the organization of the male brain, ultimately determining adult male sex behavior and reproductive physiology (cf. Refs.2 , 3). By contrast, the female gonad remains quiescent and as a result, the brain is exposed to lower levels of hormone. In the absence of testosterone (T) exposure, the default pathway organizes as feminine neural patterns, resulting in female-typic adult behaviors. After these early organizational events, subsequent hormonal exposure in adulthood exerts activational effects, producing reversible changes in morphology, physiology, or behavior that is dependent on the continued presence of the hormone and the functioning of its receptors (4, 5).

Sexual differentiation of the male brain is, at minimum, a two-step process requiring masculinization and defeminization of neural circuits regulating sex-typic behavior and gonadotropin secretion. Achieving this end requires that during critical periods of development, adequate amounts of T gain access to what will ultimately become sexually dimorphic structures (6, 7, 8). In the rat, the fetal male gonad secretes T, peaking on embryonic d 18 and continuing through the first few days of life, resulting in significantly higher levels in males transiently during gestation and again at birth (9). Whereas some components of brain sexual differentiation are mediated by T acting at the androgen receptor (10, 11, 12), many are the result of estradiol (E2) action subsequent to metabolism of T and therefore involve the estrogen receptor. Most sexually dimorphic areas of the brain contain substantial levels of both aromatase cytochrome P450 (CYP19), the enzyme responsible for the conversion of T to E2, and high densities of estrogen receptors (8, 13, 14, 15, 16). Maternal estrogens are sequestered in the peripheral circulation of the fetus by {alpha}-fetoprotein, a steroid-binding glycoprotein that has very little affinity for androgens, allowing the testicular T to reach and influence fetal target tissues, including the brain. Treatment with T, but not the nonaromatizable androgen dihydrotestosterone, mimics many of the trophic effects of estrogen (17, 18, 19), and normal masculinization of the brain is prevented or at least disturbed subsequent to disruption of aromatase during the sensitive period (20, 21, 22, 23). The basic principle that the male brain is masculinized by local conversion of E2 is elucidated by the aromatization hypothesis first proposed by Naftolin et al. in 1975 (24). Advancing knowledge reveals that the regulation of aromatase is highly complex, being influenced by both substrate and product as well as nonsteroidal regulators, including catecholamines (15, 25, 26). It is also apparent that so-called gonadal steroids can originate from sources other than the gonads, and evidence suggests the brain may act as a major endocrine organ (27, 28).

Whereas our concept of steroids and their myriad mechanisms of action continue to expand, there remains a relative dearth of data on brain region-specific levels, uptake, and metabolism of gonadal steroids in the developing brain. Most data on the hormonal milieu of the perinatal rodent are measures of circulating plasma levels or whole-body content as opposed to content within brain (9, 29, 30, 31, 32). Despite its central importance, there exists only sparse information describing E2 levels within defined brain regions during the sensitive developmental window surrounding birth. Neonatal gonadal hormone concentrations in serum and hypothalamus have been quantified (33), but to date, other steroid receptor-rich regions have not been carefully examined. Recent advances elucidating the molecular mechanisms by which steroids masculinize the developing brain necessitate a reexamination of the hormonal milieu during this critical period. Using RIA, we measured the E2 content of six regions of the developing brain immediately post partum. We also measured E2 content in the neural tissue of animals 1 d post partum, and after 28 h of exposure to E2 benzoate, testosterone propionate, or the aromatase inhibitor formestane. These two developmental time points were chosen because they are during the sensitive period and at a time when circulating T is higher in males, which is believed to be the fundamental basis for establishment of sex differences in the brain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All animal experimentation was conducted in accord with accepted standards of humane animal care and approved by the University of Maryland, Baltimore Institutional Animal Care and Use Committee. Female Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) were mated in our animal facility, and pregnant females were isolated and allowed to deliver normally. Animals were maintained on a reverse 12-h light, 12-h dark cycle and provided food and water. Cages were checked hourly beginning at 0700 h for evidence of dams in labor, and only the litters of those observed in the process of delivery were used for experiments. Onset of delivery was therefore defined as the time of this first observation of labor and time points for killing are relevant to this observation. Pups from the litters used for experiment II received a sc ink injection in a paw for group identification. Table 1Go summarizes the litter of origin and the sex of the 93 animals used for each collection and treatment paradigm.


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  		TABLE 1. Group assignment for collection and treatment paradigms

 
Experiment I: endogenous hormone levels immediately post partum
Under aseptic conditions, animals from three litters were killed by decapitation at 2 h of life and brains removed from both males (n = 13) and females (n = 11). The frontal cortex, hippocampus, hypothalamus, preoptic area, cerebellum, and brainstem were microdissected from each brain as illustrated in Fig. 1Go and immediately flash frozen in isopentane cooled on dry ice. Briefly, portions of cortex, cerebellum, and brainstem were sliced from the appropriate location on the surface of the brain. To collect the three remaining areas, brains were placed inverted on a brain matrix (Ted Pella, Redding, CA) and 1-mm sections were collected both rostral and caudal to the anterior-most portion of the diencephalon, which was delineated by the optic chiasm. The preoptic area was collected from the rostral section and the hypothalamus from the caudal. The anterior commissure and the fornix provided the lateral and dorsal boundaries for the preoptic area and the hypothalamus, respectively. Depending on the angle of brain placement within the matrix, the hippocampus was collected from either of the two slices. For this brain region, the lateral ventricles defined the lateral limits, the corpus callosum the dorsal limit, and the dorsal portion of the third ventricle the ventral limit. A separate group of six animals (n = 3 for each sex) from two litters were exsanguinated by perfusion with ice-cold 0.9% NaCl saline, and the frontal cortex, hippocampus, preoptic area, and hypothalamus were then immediately collected as described above. One of these litters also provided pups that were not exsanguinated for a total of four litters used in experiment I (Table 1Go). For some experiments, the brainstem and cerebellum were not collected. All samples were stored at –80 C until homogenized.



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  		FIG. 1. Microdissection of the neonatal rat brain. Sagittal drawing of the developing rat brain with shaded regions demonstrating the areas collected: (1) frontal cortex, (2) hippocampus (hi), (3) hypothalamus, (4) preoptic area (POA), (5) cerebellum, and (6) brainstem. Also depicted are the landmarks used to guide the microdissections, including the anterior commissure (ac), optic chiasm (ox), fornix, corpus callosum, and dorsal portion of the third ventricle (d3v).

 
Experiment II: endogenous E2 content at 32 h and effects of exogenous manipulation
Animals from six litters received hormonal treatments within 2 h of birth and again 24 h later. Females received two sc doses of 100 µg 17ß-estradiol 3-benzoate (EB, E8515, Sigma, St. Louis, MO) in 0.1 cc sesame oil (n = 5), 100 µg testosterone propionate (TP, T1875, Sigma) in 0.1 cc sesame oil (n = 6), 100 µg 4-hydroxyandrostenedione [formestane (4-OHA), F2552, Sigma] in 0.1 cc sesame oil (n = 4), or 0.1 cc sesame oil vehicle (n = 6). The steroid doses were based on those routinely used in this laboratory for mechanistic studies of sexual differentiation of the brain. For the 4-OHA experiment, males received two sc injections of either 100 µg 4-OHA in 0.1 cc sesame oil (n = 4) or 0.1 cc sesame oil (n = 10). A separate group of animals received two bilateral intracerebroventricular (icv) infusions of either 5 µg 4-OHA in 1 µl sesame oil (n = 7 for each sex) or 1 µl sesame oil (n = 7 for each sex). Animals were killed by decapitation 4 h after the second injection, approximately 32 h after birth, and tissue immediately collected as described for experiment I.

Homogenization and protein quantification
Each of the 450 brain tissue chunks was placed in 500 µl lysis buffer consisting of 150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 1% Na-deoxycholate, 0.25% Nonidet P-40, and protease inhibitors (1 µg/ml aprotinin, leupeptin, and pepstatin; 1 mM phenylmethylsulfonyl fluoride) and homogenized on ice by mechanical trituration. Aliquots of homogenate were examined by Bradford assay to determine protein concentration and allow for standardization of RIA results.

Extraction
Four milliliters of diethyl ether were added to each 500-µl sample of homogenized tissue in Teflon-capped glass extraction tubes and rotated horizontally for 30 min. Tubes were then placed vertically and allowed to stand for 15 min to ensure complete separation. The aqueous phase was frozen within a dry ice and isopentane slurry and the organic phase decanted. The former was then placed under a nitrogen stream until the evaporation of the ether. The remaining solid was reconstituted in 500 µl of E2 calibrator solution, a medium certified to contain no detectable E2 (Diagnostic Systems Laboratories, Webster, TX) and rotated horizontally for 1 h at room temperature.

RIA
From each reconstituted sample, 25 µl were diluted in 475 µl of calibrator solution to obtain a concentration appropriate for the range of the RIA (20–6000 pg E2/ml). Samples were assayed in triplicate for E2 concentration by The Center for Research in Reproduction (University of Virginia, Charlottesville, VA) using commercially available double-label immunoassay kits (DSL-4400, Diagnostic Systems Laboratories). Briefly, 50 µl of standard, control, or sample, 500 µl of hormone 125I reagent, and 100 µl of hormone antiserum were added to a tube, vortexed, and incubated at 37 C for 60 min. For nonspecific counts, 150 µl of hormone calibrator substituted for the sample and antiserum. To these tubes, 1 ml of the precipitating reagent was added and the tube vortexed and incubated at room temperature for 15 min. The mixtures were then centrifuged at 1500 x g for 20 min, decanted, and the tubes counted in a {gamma}-counter for 1 min. Similar methods were used to obtain total counts. Standard curves were generated allowing for steroid level determination. The sensitivity of the assay was at least 4.7 pg/mg protein, and the intra- and interassay coefficients of variation were less than 6.2 and 15.0%, respectively, for all experiments. All samples had some detectable level of E2. The potential that a portion of this constituted background, due to the high lipid content of brain tissue, cannot be excluded.

Extraction efficiency for E2
To assess the efficiency of ether extraction across brain regions and sex, the six targeted brain regions were dissected from each of two males and two females and homogenized in calibrator solution before addition of approximately 2,000,000 cpm of tritiated E2 [American Radiolabeled Chemicals, St. Louis, MO; catalog no. ART-820 Estradiol (6,7-3H[N]) SA = 50 Ci/mmol], the equivalent of 40 µM. Samples were incubated for 2 h at 37 C and then extracted according to the above protocol. Determination of E2 remaining vs. that extracted from the brain homogenate was done indirectly by scintillation counting of both the extracted sample and the aqueous portion remaining. Approximately 29% of the total counts were accounted for and a extraction efficiency of 79.7% with a range of 71.3–90.0% found for the six brain regions. There were no differences between males or females or between brain regions.

Statistical analyses
The average of the triplicates (picograms E2 per milliliter) obtained through the RIA was corrected for the 20-fold dilution factor and standardized for protein concentration (milligrams protein per milliliter giving values in units of picograms E2 per milligram protein). For experiment I, because exsanguination had no significant effect on E2 content, immediately postpartum data were collapsed to include data from exsanguinated and nonexsanguinated animals from groups of same sex and region for all comparisons. Sex differences were assessed by comparing the mean values for each region of the male and female brains using Student’s t tests with Bonferroni correction for multiple comparisons. Differences between the mean values of the various areas within each sex were analyzed by one-way ANOVAs. For experiment II, data from vehicle-treated animals were analyzed separately for both regional differences within each sex, assessed by one-way ANOVA, and sex differences, assessed by comparing the mean values for each region of the male and female brains using Student’s t test. These data were also compared with the remaining 1 d postpartum data and by three separate ANOVAs run within each region. Groups for these comparisons included: male+vehicle, female+vehicle, female+EB, and female+TP; male+vehicle vs. both male+ 4-OHA groups; and female+vehicle vs. both male+4-OHA groups. Route of vehicle delivery had no significant effect; therefore, the sc and icv vehicle 1 d postpartum groups were collapsed for all comparisons. Comparisons of E2 levels within each region across the two time points used the same data collected for experiments I and II. The E2 content of the males and females from experiment I were compared with the sesame oil-treated male and female data from experiment II using two-way ANOVAs to access the effect of sex and exogenous hormone by brain region and one-way ANOVAs to access the effect of 4-OHA within sex by brain regions and two-way ANOVAs. For both experiments, each ANOVA was followed by the Fisher least significant differences (LSD) post hoc multiple comparison test to determine significant differences between groups. All statistical tests used {alpha} < 0.05 as the predetermined criterion for significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A summary of all group means, SEM, and statistical comparisons can be found in Table 2Go. The mean E2 content per brain region for all groups ranged from approximately 5 pg/mg protein to 18 pg/mg protein.


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  		TABLE 2. Estradiol content measured within six regions of the developing brain immediately postpartum and at 32 h of life with and without exogenous treatment

 
Brain E2 at birth
Within 2 h of birth, tissue was collected and E2 extracted from six regions of the developing brain. There were significant differences in E2 content between regions within the male brain [F (5, 71) = 12.554; P < 0.001; Fig. 2AGo]. For the most part in the males, the more caudal the region, the less the overall E2 content. Specifically, E2 content of the male cortex was significantly greater than all areas except the hippocampus. The male hippocampus, hypothalamus, and preoptic area also displayed higher levels than either the cerebellum or brainstem [Fisher’s LSD post hoc comparison; cortex vs. hypothalamus (P < 0.05), preoptic area (P < 0.005), cerebellum (P < 0.001), or brainstem (P < 0.001); hippocampus vs. cerebellum (P < 0.001) or brainstem (P < 0.001); hypothalamus vs. cerebellum (P < 0.01) or brainstem (P < 0.001); preoptic area vs. cerebellum (P < 0.05) or brainstem (P < 0.001)]. Interestingly, in the female there was also a main effect of brain region [F (5, 60) = 12.975; P < 0.001], but post hoc analysis indicated this involved only the hippocampus having significantly higher E2 than all of the other five areas sampled from the female brain (Fisher’s LSD; P < 0.001 for all significant comparisons). No other region within the female brain differed statistically from the other five. Three of the six regions examined exhibited a sex difference, with males having significantly higher levels of E2, compared with females in the frontal cortex (P < 0.001; Student’s t test with Bonferroni correction), hypothalamus (P < 0.001), and preoptic area (P < 0.001). Exsanguination before tissue collection had no statistical effect on E2 content (Table 2Go). There were no significant differences in total protein levels between sexes regardless of region.



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  		FIG. 2. Endogenous E2 content at birth and at 32 h of life. A, Microdissection of six brain areas within 2 h of birth reveals sexually dimorphic levels of E2 in the frontal cortex (t; *, P < 0.001), hypothalamus (*, P < 0.001), and preoptic area (*, P < 0.001) with higher E2 content in males (n = 13), compared with females (n = 11). E2 content of the male cortex was significantly greater than all areas besides the male hippocampus (ANOVA; {dagger}, P < 0.05). The female hippocampus had significantly higher E2 than all of the other five areas sampled from the female brain [ANOVA; f (P < 0.001)]. Exsanguination had no statistical effect on E2 content in any of the regions examined. Data graphed are means of nonexsanguinated samples. Dashed lines represent mean of E2 content after exsanguination. Analyses collapse data from exsanguinated and nonexsanguinated animals. Note that the y-axis intercept is 4.0, representing the detection limit of the assay. B, Microdissection of six brain areas at 32 h of life reveals sexually dimorphic levels of E2 in the hypothalamus (t; *, P < 0.001) and preoptic area (*, P = 0.006) with higher E2 content in males (n = 17), compared with females (n = 13). The female hippocampus displayed significantly higher levels than the male hippocampus at this later time point (*, P = 0.008). There were significant differences in E2 content between regions within the male brain (ANOVA; P < 0.001). E2 content of the male hypothalamus was significantly greater than all areas investigated (ANOVA; #, P < 0.01), except the male cortex, which itself displayed levels greater than the four other regions ({dagger}, P < 0.05). The female hippocampus had significantly higher E2 than all of the other five areas sampled from the female brain [ANOVA; f (P < 0.001)], and the female cortex had greater levels than the female preoptic area, cerebellum, and brainstem (IP < 0.05). Route of vehicle delivery had no significant effect; therefore, the sc and icv vehicle 1 d postpartum groups were collapsed for all comparisons. C, Comparison of endogenous E2 levels immediately post partum vs. 1 d of life revealed that regardless of sex, E2 levels displayed a negative slope within all regions, with the exception of the female cortex. Moreover, the E2 content of the male frontal cortex (ANOVA; *, P < 0.001), male hippocampus (ANOVA; *, P < 0.001), male preoptic area (ANOVA; *, P < 0.001), and male cerebellum (ANOVA; *, P < 0.02) significantly declined. Endogenous E2 levels of the preoptic area and hippocampus of the females significantly decreased by 1 d of life (ANOVAs; *, P = 0.001).

 
Brain E2 at 32 h post utero
Tissue was also collected from pups approximately 32 h after birth and E2 extracted and measured as above. There were significant differences in E2 content between regions within the male brain [F (5, 80) = 6.330; P < 0.001; Fig. 2BGo]. At this later time point, E2 content was significantly greater in the male hypothalamus than all areas investigated, save the male cortex, which itself displayed levels greater than the four other regions [Fisher’s LSD; cortex vs. hippocampus (P < 0.05), preoptic area (P < 0.001), cerebellum (P < 0.01), or brainstem (P < 0.001); hypothalamus vs. hippocampus (P < 0.01), preoptic area (P < 0.001), cerebellum (P < 0.001), or brainstem (P < 0.001)]. The female brain also exhibited regional heterogeneity in E2 content at 32 h post utero [F (5, 56) = 13.133; P < 0.001; Fig. 2BGo]. Estradiol levels within the female hippocampus remained significantly greater than all other regions examined, and the female cortex had greater levels than the female preoptic area, cerebellum, and brainstem [Fisher’s LSD; cortex vs. preoptic area (P < 0.001), cerebellum (P < 0.03), or brainstem (P < 0.05); hippocampus vs. all regions (P < 0.001)]. Three of the six regions examined on postnatal d 1 exhibited a sex difference, with males maintaining significantly higher levels of E2, compared with females in the hypothalamus (P < 0.001; Student’s t test) and preoptic area (P = 0.006). Intriguingly, the female hippocampus had significantly higher levels than that of the male (P = 0.008). As with data obtained immediately after birth, there were no significant differences in total protein levels between sexes regardless of region.

Changes in endogenous brain E2 from birth to 1 d post partum
By 1 d post partum, the endogenous content of E2 decreased throughout all brain regions examined and ranged from the lower limits of detectability (approximately 4 pg/mg protein) to 15 pg/mg protein (Table 2Go). Regardless of sex, E2 levels decreased within all regions over the course of the first 32 h of life, with the exception of the female cortex, in which E2 content remained unchanged (Fig. 2CGo). The E2 content of the male frontal cortex [F (3, 55) = 12.428; P < 0.001; Fisher’s LSD, P < 0.001], male hippocampus [F (3, 53) = 12.016; P < 0.001; Fisher’s LSD, P < 0.001], male preoptic area [F (3, 55) = 38.557; P < 0.001; Fisher’s LSD, P < 0.001], and the male cerebellum [F (3, 24) = 3.353; P < 0.05; Fisher’s LSD, P < 0.02] at 2 h was significantly greater than at 32 h (Fig. 2CGo). Within the female preoptic area, the decrease was particularly precipitous, with E2 levels falling by 49% (Fisher’s LSD, P < 0.001). E2 levels also decreased significantly in the female hippocampus (Fisher’s LSD, P < 0.001). The decline of E2 content in the other four regions examined within the female brain were not statistically significant across the two time points. The percent decrease of E2 content was larger within the male cortex, hippocampus, and to lesser extents in the brainstem and cerebellum, compared with that of the same regions in the female. On the contrary, the magnitude of the percent decrease in the hypothalamus was larger in the female than the male, which in part contributed to maintaining the sex difference in E2 levels at 32 h post partum. The percent decrease from 2 h to 32 h post partum was greatest, yet comparable between the sexes in the preoptic area.

Effect of exogenous hormone treatment
Females treated with two daily sc injections of EB (100 µg) had E2 content in the hypothalamus comparable with male hypothalami at 32 h of age but nearly 3 times greater than female controls [F (3, 23) = 5.507, P = 0.005; Fisher’s LSD, P < 0.01; Fig. 3Go]. Elsewhere, these treatments resulted in E2 levels 2- to 3-fold greater than those of the control animals regardless of sex [Fig. 3AGo, cortex-F (3, 22) = 9.124, P < 0.001; Fisher’s LSD, P < 0.001; Fig. 3BGo, hippocampus-F (3, 21) = 7.529, P = 0.001; Fisher’s LSD, P < 0.01; Fig. 3DGo, preoptic area-F (3, 22) = 4.452, P = 0.014; Fisher’s LSD, P < 0.01; Fig. 3EGo, cerebellumF (3, 22) = 6.301, P = 0.003; Fisher’s LSD, P < 0.001; Fig. 3FGo, brainstem-F (3, 23) = 3.494, P < 0.032; Fisher’s LSD, P < 0.02].



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  		FIG. 3. Effects of hormonal manipulation on E2 content 1 d post partum. Control male and female pups were injected with sesame oil vehicle by one of two routes, sc or icv. The latter was to control for icv injections of 4-OHA used in subsequent experiments. Because there was no effect of route of administration of sesame oil on endogenous E2 levels in males or females, controls were combined for each sex and used as the comparison group for this and the experiments involving 4-OHA treatment (see Table 2Go for sc data). Control males (sc: n = 10, icv: n = 7) and females (sc: n = 6, icv: n = 7) were treated with sesame oil within 2 h of birth and again approximately 32 h after birth. For comparative purposes, the data on control males and females at 1 day post partum presented in Fig. 2BGo is repeated here. Additional female pups received two daily sc injections of either EB (100 µg; n = 5) or TP (100 µg; females, n = 6), within the first 2 h of birth, and tissue microdissected from frontal cortex (A), hippocampus (B), hypothalamus (C), preoptic area (D), cerebellum (E), and brainstem (F) 4 h after the second treatment approximately 32 h after birth. Females treated with EB had comparable E2 content to that found in the male hypothalamus 1 d post partum (C) and 2- to 3-fold greater levels than those of the sesame oil controls in other regions irrespective of sex (ANOVAs: A, *, P < 0.001; B, *, P < 0.01; D, *, P < 0.01; E, *, P < 0.001; F, *, P < 0.02). TP treatment of females increased E2 content only within the preoptic area to levels comparable with that observed after exogenous E2 treatment, resulting in levels significantly greater than that observed for either of the sexes 32 h post utero (ANOVA, **, P < 0.01). Note the nonstandardization of the axes and that the y-axes intercept is 4.0, representing the detection limit of the assay as well as the nonstandardization of the axes.

 
Peripheral application of TP should allow for local aromatization to E2 within brain regions previously found to contain significant levels of the aromatase enzyme, and the use of the proprionated form does not interfere with this process (34). Treatment with TP (100 µg) significantly increased E2 within the preoptic area, compared with both control females (Fisher’s LSD, P < 0.01; Fig. 3Go) or males (Fisher’s LSD, P < 0.01). Exogenous TP had no effect on the other regions examined, which was contrary to expectation. As with E2 content at birth, there were no significant differences in total protein levels between sexes regardless of region.

Regional response to aromatase inhibition
To begin to discern the source of the high levels of E2 in the female hippocampus and frontal cortex that remained through 32 h of life, administration of 4-hydroxyandrostenedione (4-OHA) during the first 2 d of life was used. 4-OHA is a potent and selective irreversible-competitive inhibitor of the aromatase enzyme (35). As with other aromatase inhibitors, peripheral administration of 4-OHA gains access to all compartments of the body, including the privileged central nervous system (CNS) (36, 37), therefore reducing estrogen synthesis throughout the neonate. In the interest of definitively differentiating the source of E2, we administered 4-OHA both peripherally and directly to the lateral ventricles. In no instance was peripheral 4-OHA effective at reducing brain E2 in the absence of an effect of central administration. However, in some brain regions, only icv administered 4-OHA effectively reduced E2, possibly due to incomplete penetrance into those areas of the CNS by peripherally administered 4-OHA. Estradiol content decreased significantly in the male cortex [F (2, 25) = 8.848, P = 0.001; Fisher’s LSD, P < 0.001] and male preoptic area [F (2, 24) = 4.061, P = 0.030; Fisher’s LSD, P < 0.03] only after central 4-OHA administration, whereas in the male hypothalamus, both peripheral [F (2, 25) = 8.103, P = 0.002; Fisher’s LSD, P < 0.01] and central administration (Fisher’s LSD, P < 0.01) decreased E2 content (Fig. 4Go, see Table 2Go for means of sc treatment groups). 4-OHA had no effect on E2 levels in the male hippocampus. Estradiol content decreased in the female cortex after central 4-OHA [F (2, 19) = 5.321, P = 0.015; Fisher’s LSD, P < 0.01] and in the female hippocampus after either route of treatment [F (2, 20) = 7.280, P = 0.004; Fisher’s LSD: sc (P < 0.01), icv (P < 0.01); Fig. 5Go]. The female hypothalamus and preoptic area were unaffected by both peripheral and icv 4-OHA, in large part due to the very low levels of endogenous E2 in these brain regions at this time.



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  		FIG. 4. Regional heterogeneity of response to 4-OHA in male brain. Male pups received two daily treatments of sc sesame oil (n = 10), icv sesame oil (n = 7), or icv 4-OHA (5 µg; n = 7), within the first 2 h of birth, and tissue microdissected from frontal cortex (A), hippocampus (B), hypothalamus (C), and preoptic area (D) 4 h after the second treatment approximately 32 h after birth. E2 content decreased significantly in the cortex (ANOVA, *, P < 0.001), hypothalamus (ANOVA, *, P < 0.01), and preoptic area (ANOVA, *, P < 0.03) after exposure to icv 4-OHA. 4-OHA had no effect on E2 in the male hippocampus. Male control animals are the same as those described in Fig. 3Go. Statistical comparisons included sc 4-OHA treatment data but were omitted from the graph for simplicity. Note that the y-axis intercept is 4.0, representing the detection limit of the assay.

 


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  		FIG. 5. Regional heterogeneity of response to 4-OHA in female brain. Female pups received two daily treatments of sc sesame oil (n = 6), icv sesame oil (n = 7), or icv 4-OHA (5 µg; n = 7), within the first 2 h of birth, and tissue microdissected from frontal cortex (A), hippocampus (B), hypothalamus (C), and preoptic area (D) 4 h after the second treatment approximately 32 h after birth. E2 levels decreased in the female frontal cortex (ANOVA, *, P < 0.01) and hippocampus (ANOVA, *, P < 0.01) after icv 4-OHA treatment. 4-OHA had no effect in the hypothalamus or preoptic area in which E2 levels were already exceedingly low. Statistical comparisons included sc 4-OHA treatment data but were omitted from the graph for simplicity. Note that the y-axis intercept is 4.0, representing the detection limit of the assay.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found sexually dimorphic E2 content in several regions of the newborn rat brain immediately post partum. The male frontal cortex, hypothalamus, and preoptic area had significantly higher levels of E2 at 2 h after birth, compared with that of females. By d 1 post partum, E2 content had decreased substantially in the majority of brain regions, although not in female cortex. As a result, most sex differences in E2 content were eliminated, excluding the hypothalamus in which males continued to have twice the level of females. While exploring the impact of exogenous hormone treatment on brain E2 levels, we determined that masculinizing doses of EB raised brain levels in most regions only 2- to 3-fold and in the hypothalamus equalized the level in females to that of males. Administration of exogenous TP to females failed to raise E2 levels in most brain regions with the notable exception of the preoptic area. Central administration of 4-OHA, an aromatase inhibitor, decreased E2 content in the male cortex, hypothalamus, and preoptic area as well as the female cortex and hippocampus. The latter finding was surprising and indicated the potential for de novo synthesis of E2 by telencephalic regions of the female brain.

These findings corroborate and expand existing information on endogenous brain E2 levels. Our observation of a sexually dimorphic E2 content immediately post partum in the hypothalamus is consistent with previous data from Rhoda et al. (33) in which RIA of hypothalamic tissue collected from Sherman rats indicated a 2.5-fold greater picogram per gram content in the male than female, similar to the nearly 2-fold increase found by our method. To our knowledge, few if any other data exist directly describing brain levels of endogenous E2 in the developing brain. For the most part, investigators have relied on the assumption that either serum titer levels of steroid or regional aromatase activity allow for a reliable extrapolation to brain hormone levels, a supposition that does not necessarily account for regional specificity of hormone delivery, sequestration, and metabolism. We have found regional heterogeneity in E2 content at the time of birth and 1 d post partum with and without exogenous steroid treatment.

Among the surprising findings in the current study was the impact of peripheral hormone administration on brain E2 levels. We administered to female pups what would be considered a pharmacological dose of EB even if given to a fully mature adult (100 µg in oil sc twice) and an equally large dose of TP. This choice of hormone dose was based in part on the desire to determine the impact of what is a frequently used paradigm in our hands and others to study the phenomenon of masculinization of the developing brain. Use of the esterified forms of the hormones, benzoate and propionate, engenders lower water solubility to the steroids and increases lipid solubility. This causes the steroid to deposit in muscle tissue, from which it will slowly enter into circulation. Esters have no effect on the ability of the parent steroid to convert to E2 (34). Early studies on brain masculinization used a wide range of E2 and T doses (38, 39, 40) ranging from 5 to 100 µg and 10 µg to 1.25 mg, respectively. More recent studies, including those from our laboratory, employed 100 µg EB and 100–500 µg testosterone benzoate (or propionate) (41, 42, 43, 44, 45). This narrowed range has in part elucidated thresholds of hormone exposure necessary and sufficient to androgenize the female CNS. In fact, a series of studies by Mong et al. (46) demonstrated that a single injection of 50 µg TP only partially masculinized an end point that was fully masculinized by two doses of 100 µg TP (42). Nonetheless, the criticism that the doses of steroid employed were pharmacological was difficult to deflect in light of no data regarding the actual brain E2 levels achieved. Interestingly, whereas E2 content was increased 2- to 3-fold in several brain regions after two doses of 100 µg EB, it was certainly not outside the physiological range. Moreover, in the female hypothalamus, the increase in E2 content by exogenous administration made levels comparable with that naturally occurring in the male hypothalamus at the same developmental time point, which may explain why the lower dose of 50 µg TP was previously found to be submasculinizing (46). Thus, two things are clear: the doses currently employed are necessary and appropriate, and the system for buffering or retaining circulating E2 in the periphery is extremely effective in the neonatal rat.

The glycoprotein {alpha}-fetoprotein binds and sequesters extracellular maternal and placental rodent estrogens. The fetal yolk sac, liver, and gastrointestinal tract manufacture {alpha}-fetoprotein throughout gestation and its synthesis halts shortly after parturition (47). The protein is cleared by hepatocytes and, in rodents, has a half-life of approximately 24 h (48). At the time of birth, levels peak at 1–6 mg/ml and quickly decrease to 0.01% of fetal concentrations for the remainder of life (49). This brief, high concentration of {alpha}-fetoprotein is necessary to protect the developing brain from the deluge of maternal E2, especially considering the hormone has a lower affinity for the globulin (affinity constant 10–8) than for its primary receptor (ER{alpha}, affinity constant 10–11) (50, 51). Indeed, levels of {alpha}-fetoprotein reach a maximum during the same perinatal period that T surges and remain at significant levels in the bloodstream of pups for several days after birth (52).

Aromatase protein and activity have been detected in brain tissue of all mammalian species studied thus far, and in rats the activity is expressed within neurons of discrete hypothalamic, preoptic, limbic, hippocampal, neocortical, and midbrain regions (cf.53). In general, these sites of aromatase expression are areas involved in regulating neuroendocrine function and reproductive behavior. Neither the protein nor the activity is distributed uniformly across these regions, and both vary greatly across the stages of life. The highest levels of brain aromatase are detected during perinatal development within both the preoptic and hypothalamic areas (53, 54, 55). Several studies using various assays detected that levels in the majority of these regions peak at 18 d of gestation and slowly decline to baseline by postnatal d 8–10 (54, 56). Notable exceptions include the hippocampus and cortex, which do not exhibit aromatase activity until shortly after parturition (57). Aromatase has yet to be localized to the cerebellum and brainstem in rodent, although this is not the case for avian species (58). Importantly, within the diencephalon, males display slightly to moderately higher activity levels than females, independent of age (15, 55, 56, 59, 60, 61). No sex differences have been observed in areas outside this region. In the current experiments, exogenous TP treatment resulted in raised E2 levels only in the female preoptic area and not the hypothalamus as would be expected. This could reflect a sex difference such that there is insufficient aromatase in the female hypothalamus to effectively convert T to detectable E2.

Interpretation of the 4-OHA data requires one to address the following: where in the female did the substrate for the aromatase enzyme, i.e. T, originate? Whereas {alpha}-fetoprotein sequesters extracellular estrogens in utero, testicular T synthesized by the males of the litter seeps across the fetal compartments to potentially influence neighboring female fetuses (62). Local, high levels of aromatization within restricted regions of the brain, in this case the hippocampus and cortex, may then ultimately result in elevated E2 in selective brain areas. Alternatively, there is some evidence that {alpha}-fetoprotein can gain access to the interior of neurons and may then actually serve as a selective vehicle for maternal estrogen (63, 64). A third potential source of androgen for the brain is the fetal adrenal glands, which may be capable of androgen synthesis at this early stage of development. In fact, we found detectable levels of T in female circulation as late as postnatal d 3 (Amateau, S. K., and M. M. McCarthy, unpublished observation). However, if the adrenals were providing sufficient levels of androgens to the brain to account for the high levels of E2 we observed in the telencephalon, delivery would not be expected to be restricted to the hippocampus and frontal cortex. In fact, given the evidence for a high level of aromatase activity in the developing female preoptic area, one would anticipate that E2 levels would be equally high, if not higher here, and that there would be a reduction in the levels in the preoptic area after 4-OHA treatment. Thus, a peripheral source of androgen that selectively increases E2 in the telencephalon of females seems unlikely.

Instead, local synthesis of E2 de novo from cholesterol in the female telencephalon provides a parsimonious explanation for the selective effects of 4-OHA. There is precedent for de novo synthesis of E2 in the avian brain, which has significance to the sexual differentiation of the song system (27, 28). To our knowledge, there have been no previous reports of de novo E2 synthesis in the in vivo mammalian brain, but a recent report indicates that cultured hippocampal neurons release E2 into the medium (65). There is also substantial converging evidence indicative of de novo E2 synthesis by the mammalian brain. The aromatase enzyme is certainly the most rigorously and broadly studied steroidogenic protein in the brain as a result of its crucial role in sexual differentiation, and there is little doubt that the brain is capable of additional steroidogenesis. Enzymes displaying regional heterogeneity in expression within the brain have been reported to regulate the synthesis of the so-called neurosteroids, which interact with the {gamma}-aminobutyric acidA receptor. These include, but are not limited to, 3ß-hydroxysteroid dehydrogenase, 5{alpha}-reductase, and 21-hydroxylase (66, 67, 68). The detection of both allopregnanolone, a 5{alpha}-reduced metabolite of dihydroprogesterone, and dehydroepiandrosterone (DHEA), albeit at subpicomolar concentrations, in the brains of adult adrenalectomized and gonadectomized male rats further substantiates this theory (69, 70). Moreover, the mRNA for the biosynthesis of DHEA has been detected in the cortex (67). Cultured astrocytes and neurons from neonatal rodent cerebral cortex are also capable of synthesizing DHEA and subsequently converting the steroid to T and ultimately E2 (71). Finally, hippocampal neurons in particular have been found to produce progesterone at high levels, particularly during the neonatal period (72). Thus, all of the requisite machinery appears to be in place for the de novo synthesis of E2 by the brain. The current findings suggest this is occurring selectively in the female telencephalon and perhaps only during perinatal development, which may in part explain why this synthesis has not been previously detected.

The functional significance of elevated E2 in the female hippocampus and cortex during development is unknown. Nonetheless, the hippocampus shows modest sex differences in volume, which are apparent by the first week of life (73), and E2 appears to have trophic effects on hippocampal development (74). A role for E2 in feminization has been previously postulated based on the effects of either targeted disruption of the aromatase gene (22) or interference with the functioning of estrogen receptors (75, 76). These studies placed emphasis on reproductive behavior and physiology. Cognitive functions, such as learning and memory, in which the hippocampus and cortex play prominent roles, are much less sexually dimorphic than reproductive-related responses. The sex differences that are observed are largely limited to acquisition of a task with no sex differences in memory capacity or steady-state performance (77). Perhaps increased E2 synthesis by the developing female telencephalon contributes to maintaining parity in cognitive function between males and females. Recent evidence in aromatase null mice indicates both males and females show deficits on hippocampal-dependent tasks (78). Alternatively, E2 has pronounced neuroprotective effects in the adult brain (cf.73). Traumatic events such as birth are often accompanied by asphyxia. Moreover, premature parturition is frequently associated with cerebrovascular bleeding, hypoxia, and other teratogenic incidents. Elevated E2 in the female telencephalon may serve a neuroprotective function equal to that of males during this high-risk period of life, a view supported by the ability of E2 to reduce kainic acid-induced damage to the dentate gyrus of newborn female rats (73). A full understanding of the functional significance of elevated E2 in the telencephalon of developing females awaits further study.


   Footnotes
 
This work was supported by a predoctoral National Research Service Award (MH12862) and grant from the Women’s Health Research Group, University of Maryland at Baltimore (to S.K.A.), a grant from the National Institute of Mental Health (MH52716) (to M.M.M.), and National Institute of Child Health and Human Development/National Institutes of Health through cooperative agreement (U54 HD28934) as part of the Specialized Cooperative Centers Program in Reproductive Research.

Abbreviations: CNS, Central nervous system; DHEA, dehydroepiandrosterone; E2, estradiol; EB, 17ß-estradiol 3-benzoate; icv, intracerebroventricular; LSD, least significant differences; 4-OHA, formestane; T, testosterone; TP, testosterone propionate.

Received October 10, 2003.

Accepted for publication February 19, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C. D. Toran-Allerand, A. A. Tinnikov, R. J. Singh, and I. S. Nethrapalli
17{alpha}-Estradiol: A Brain-Active Estrogen?
Endocrinology, September 1, 2005; 146(9): 3843 - 3850.
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C. A. Cornil, C. Dalla, Z. Papadopoulou-Daifoti, M. Baillien, C. Dejace, G. F. Ball, and J. Balthazart
Rapid Decreases in Preoptic Aromatase Activity and Brain Monoamine Concentrations after Engaging in Male Sexual Behavior
Endocrinology, September 1, 2005; 146(9): 3809 - 3820.
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N. R. Miller, T. Jover, H. W. Cohen, R. S. Zukin, and A. M. Etgen
Estrogen Can Act via Estrogen Receptor {alpha} and {beta} to Protect Hippocampal Neurons against Global Ischemia-Induced Cell Death
Endocrinology, July 1, 2005; 146(7): 3070 - 3079.
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