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Endocrinology Vol. 139, No. 4 1738-1745
Copyright © 1998 by The Endocrine Society

ARTICLES

Ontogeny of Region-Specific Sex Differences in Androgen Receptor Messenger Ribonucleic Acid Expression in the Rat Forebrain1

Michael D. McAbee and Lydia L. DonCarlos

Program in Neuroscience (M.D.M.) and the Department of Cell Biology, Neurobiology, and Anatomy (L.L.D.C.), Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois 60153

Address all correspondence and requests for reprints to: Dr. Lydia L. DonCarlos, Department of Cell Biology, Neurobiology, and Anatomy, Stritch School of Medicine, Loyola University of Chicago, 2160 South First Avenue, Maywood, Illinois 60153. E-mail: ldoncar{at}luc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Testosterone and its metabolites are the principal gonadal hormones responsible for sexual differentiation of the brain. However, the relative roles of the androgen receptor (AR) vs. the estrogen receptor in specific aspects of this process remain unclear due to the intracellular metabolism of testosterone to active androgenic and estrogenic compounds. In this study, we used an 35S-labeled riboprobe and in situ hybridization to analyze steady state, relative levels of AR messenger RNA (mRNA) expression in the developing bed nucleus of the stria terminalis, medial preoptic area, and lateral septum, as well as the ventromedial and arcuate nuclei of the hypothalamus. Each area was examined on embryonic day 20 and postnatal days 0, 4, 10, and 20 to produce a developmental profile of AR mRNA expression. AR mRNA hybridization was present on embryonic day 20 in all areas analyzed. In addition, AR mRNA expression increased throughout the perinatal period in all areas examined in both males and females. However, between postnatal days 4 and 10, sharp increases in AR mRNA expression in the principal portion of the bed nucleus of the stria terminalis and the medial preoptic area occurred in the male that were not paralleled in the female. Subsequently, males exhibited higher levels of AR mRNA than females in these areas by postnatal day 10. There was no sex difference in AR mRNA content in the lateral septum, ventromedial nucleus, or arcuate nucleus at any age. These results suggest that sex differences in AR mRNA expression during development may lead to an early sex difference in sensitivity to the potential masculinizing effects of androgen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEXUAL differentiation of the brain is dependent upon exposure to the gonadal hormone testosterone during the perinatal period (1). In the absence of circulating testosterone, the mammalian brain develops an essentially female phenotype. In contrast, early exposure to testosterone permanently masculinizes numerous behavioral and physiological functions, including, but not limited to, reproduction, play, feeding, learning and memory, aggression, sleep/wake cycles, and GH secretion (1, 2, 3).

Testosterone may be metabolized to dihydrotestosterone via the 5{alpha}-reductase enzyme or to estrogen via aromatase, or it may remain unmetabolized (4, 5). Each of these metabolites acts through intracellular receptors to dramatically alter the transcription of hormone-responsive genes (6, 7). Both testosterone and dihydrotestosterone act through a single androgen receptor (AR), whereas estrogen acts via estrogen receptors to produce unique effects on gene expression (6, 8). Therefore, cells that express steroid hormone receptors are integral to the action of gonadal hormones and represent essential components of the mechanism underlying masculinization of the brain.

The relative contributions of androgens vs. estrogens in the sexual differentiation of the brain have not been fully determined. The impact of estrogen on the sexual differentiation of neuroendocrine and morphological parameters has been well characterized and is dramatic (9, 10). However, additional evidence challenges the assumption that estrogen acts alone in this process. For example, AR antagonists given during the perinatal period prevent full expression of male sexual behavior in adult male rats and inhibit testosterone-induced masculinization in female rats (11, 12). The development of certain male-typical behaviors, such as male-typical social play and open field behavior, is dependent primarily on androgens, not estrogen (13, 14, 15). Further, estrogen receptor knockout male mice retain some aspects of normal male-typical sexual behaviors (16). Therefore, we and others (17, 18, 19) have hypothesized that androgens, AR, and the cells that express AR participate in sculpting a male-typical brain. For this hypothesis to be true, AR must be present during the masculinization process.

Early work performed in the rodent hypothalamus demonstrated low levels of androgen binding during the last week of prenatal life, with increasing levels through the first 3 weeks of postnatal development (17, 20, 21). However, these studies were performed in gross blocks of hypothalamic tissue and may have provided insufficient spatial resolution to allow determination of subtle, region-specific sex differences in AR content. To determine whether AR expression is present during the perinatal period and provide improved spatial resolution, AR messenger RNA (mRNA) expression was assessed using a specific 35S-labeled riboprobe and in situ hybridization. We examined AR mRNA expression in five forebrain regions that are known to participate in sexually differentiated behaviors and physiological functions in the adult rat (22). These areas were the lateral septum, the bed nucleus of the stria terminalis, the medial preoptic area, and the ventromedial and arcuate nuclei of the hypothalamus. In addition to involvement in sex-specific functions, each of these areas demonstrates expression of both AR (18, 23, 24, 25, 26, 27, 28, 29, 30) and estrogen receptors (29, 31, 32) in the adult rat and represents a potential site for the interaction of androgens and estrogens during development. Five distinct perinatal ages were selected to encompass the perinatal period. These ages were embryonic day 20 (ED-20) and postnatal days (PND) 0, 4, 10, and 20 (PND-0 = day of birth).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Timed pregnant Sprague-Dawley rats were obtained from Zivic-Miller (Pittsburgh, PA). Animals were housed separately in a controlled environment on a 12-h light, 12-h dark cycle (lights on from 0700–1900 h), with food and water available ad libitum. ED-20 (ED-0 = day of conception) animals were obtained via cesarean section of pregnant females anesthetized with ether. On the day of birth, PND-0, each litter containing animals scheduled for death on PND-4, PND-10, or PND-20 was adjusted to five females and five males.

Before death, body weight and ano-genital distances were recorded. ED-20, PND-0, and PND-4 animals were anesthetized by hypothermia and decapitated. PND-10 and PND-20 animals were anesthetized with ether and decapitated. Brains were quickly removed, rapidly frozen in powdered dry ice, and stored at -70 C. Frozen sections were cut at a thickness of 16 µm (Kryostat, Leitz, Rockleight, NJ). Every third section was designated for AR mRNA detection. Sections were collected from the rostral forebrain to the midbrain, mounted onto Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA), and stored at -70 C until processing for in situ hybridization.

All animal procedures were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee at Loyola University; the animal facility is American Association for Accreditation of Laboratory Animal Care accredited.

In situ hybridization
Probe synthesis. AR mRNA was detected using a complementary RNA probe transcribed from subcloned rat AR complementary DNA (cDNA; gift from Dr. R. Handa, original cDNA from Dr. E. M. Wilson). The cDNA corresponded to nucleotides 3350–3840 in the region encoding the ligand-binding domain of the mature AR protein. The linearized cDNA template was incubated with 5 x transcription buffer (Promega, Madison, WI), dithiothreitol, RNAsin, ATP-CTP-GTP-UTP, [35S]UTP (Amersham, Arlington Heights, IL), and SP6 RNA polymerase (Promega) for 2 h at 30 C. The cDNA template was then digested with RQ1 deoxyribonuclease (Promega) for 20 min at 37 C. The riboprobe was PCI extracted, ethanol precipitated overnight, and examined by autoradiography after acrylamide gel electrophoresis (7.5 M urea and 5% acrylamide). Only probes demonstrating a clear full-length transcript were used for in situ hybridization.

Hybridization reaction. Standard hybridization procedures were employed (33). On the morning of hybridization, tissue was thawed briefly, fixed in 4% paraformaldehyde, acetylated, dehydrated, delipidated, and air-dried. Each slide was apposed to a coverslip with 100 µl hybridization solution (0.6 M NaCl, 10 mM Tris, 1 x Denhart’s solution, 1 mM EDTA, 10% dextran sulfate, 0.1 mg/ml salmon sperm DNA, 0.5 mg/ml total yeast RNA, 0.05 mg/ml yeast transfer RNA, 0.1% sodium thiosulfate, 50 µM dithiothreitol, 0.01% SDS, and 50% formamide) containing the 35S-labeled AR riboprobe diluted to a final activity of 1.5 x 107 cpm/ml. Slides were incubated for 20 h at 60 C in a humidified oven. Coverslips were removed in 2 x SSC (standard saline citrate). Slides were then washed in 2 x SSC, treated with ribonuclease A at 60 C, and washed in decreasing concentrations of SSC to a final stringency of 0.1 x SSC at 65 C for 30 min. Slides were dehydrated, air-dried, and apposed to Hyperfilm Betamax film (Amersham) for 10 days to obtain autoradiograms for image analysis. The specificity of AR mRNA hybridization was examined using sense-directed RNA probes. No hybridization was detected in this control. Furthermore, no specific hybridization was detected in tissue pretreated with ribonuclease A.

Analysis. The nomenclature used for analysis was according to the atlas of Swanson (34). AR mRNA hybridization signal on the film autoradiograms was localized using a Macintosh IIci computer (Apple Computer, Lakewood, NJ) with Scion video card (Scion Corp., Walkersville, MD) attached to a Sony video camera (Imaging Research, St. Catherines, Canada). The relative abundance of AR mRNA was quantified using the public domain NIH IMAGE analysis software (developed at the NIH, Bethesda, MD; available at http: rsb.info.nih.gov/nih-image) calibrated to 14C-labeled standards (Amersham) included in each cassette. The entire area of detectable label within each region was outlined in each brain section regardless of the intensity of the signal (Fig. 1Go). All sections exhibiting label in the region of interest were analyzed for the area and intensity of label. The average pixel value within the outlined region was measured and expressed as a mean gray level. The number of sections analyzed for each animal in each area ranged from three to six, depending on age. To eliminate possible differences between experimental runs and multiple films from the same run, mean background measures taken from the caudate-putamen were subtracted from each individual mean gray level for that animal. Each raw minus background measure constitutes a corrected gray value. One-way ANOVA with Fisher’s protected least significant difference post-hoc test (P <= 0.05) demonstrated that mean corrected gray levels from left and right hemispheres were not different when separated into a given age, region, and sex. Therefore, left and right values were combined to yield one mean corrected gray value for each animal. These mean corrected gray values were analyzed by two-way ANOVA for each area, with planned post-hoc tests (Fisher’s protected least significant difference and Scheffe’s multiple comparison tests). Differences were considered significant at P <= 0.05. In addition, the data were separated by sex, and one-way ANOVA was used to examine age effects. Differences were considered significant at P <= 0.05. Individual t tests, with P <= 0.01, were performed to examine sex differences at each age in each region. The mean area of label, ano-genital distances, and body weights were each analyzed by two-way ANOVA (age and sex) with planned post-hoc tests (P <= 0.05).



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  		Figure 1. Digitized images of sections from a PND-10 male rat, illustrating how mean gray level measures were taken in the medial preoptic area (MPO), lateral septum (LS), principal portion of the bed nucleus of the stria terminalis (BSTpr), and the ventromedial (VMH) and arcuate (ARH) nuclei of the hypothalamus.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
General results
AR mRNA expression was detected in the developing forebrain of male and female rats at all ages examined and was present in the same regions throughout the perinatal period (Fig. 2Go). In the preoptic area, AR mRNA hybridization density was strongest in the medial preoptic area. In the hypothalamus, the strongest AR mRNA expression was detected in the ventral premammillary nucleus. Other hypothalamic regions that demonstrated moderate levels of AR mRNA hybridization density included the ventromedial hypothalamic nucleus, the arcuate nucleus, the paraventricular hypothalamic nucleus, and the supraoptic nucleus. AR mRNA expression was also detected in specific septal regions, with intense AR mRNA expression in the principal bed nucleus of the stria terminalis and moderate hybridization density in the lateral septum. AR mRNA expression in the hippocampal formation was strongest in the CA1, moderate in the ventral CA3, and low in the CA2 and subiculum. In the amygdala, AR mRNA hybridization density was strongest in the medial nucleus and moderate in the cortical and central nuclei. Moderate cortical AR mRNA expression was also detected at each age in the entorhinal cortex and layers II and III of the neocortex. Additional areas expressing low levels of AR mRNA corresponded to those previously reported in the adult rat (29).



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  		Figure 2. Digitized images of sections from a PND-10 male rat, demonstrating expression of AR mRNA in the cerebral cortex (CTX), lateral septum (LS), anterodorsal bed nucleus of the stria terminalis (BSTad), medial preoptic area (MPO), principal bed nucleus of the stria terminalis (BSTpr), supraoptic nucleus (SO), paraventricular hypothalamus (PVH), ventral posterior thalamic complex (VP), medial amygdala (MeA), ventromedial hypothalamus (VMH), arcuate (ARH), central amygdala (CeA), cortical amygdala (CoA), posterior amygdala (PA), ventral premammillary (PMv), entorhinal cortex (ENT), CA1 field of Ammon’s horn (CA1), and CA3 field of Ammon’s horn (CA3).

 
There was no significant effect of sex on the area of label in any cell group examined: principal bed nucleus of the stria terminalis: F(1,50) = 2.251; P = 0.1185; anterodorsal bed nucleus of the stria terminalis: F(1,70) = 0.397; P = 0.5307; medial preoptic area: F(1,50) = 0.261; P = 0.6117; lateral septum: F(1,70) = 0.291; P = 0.5911; ventromedial hypothalamus: F(1,46) = 0.782; P = 0.3801; and arcuate nucleus of the hypothalamus: F(1,46) = 0.002; P = 0.9577. However, the area of label increased significantly with age (principal bed nucleus of the stria terminalis: F(4,50) = 4.458; P = 0.0035; anterodorsal bed nucleus of the stria terminalis: F(4,70) = 3.092; P = 0.0064; medial preoptic area: F(4,50) = 2.518; P = 0.0053; lateral septum: F(4,70) = 5.323; P = 0.0008; ventromedial hypothalamus: F(4,46) = 15.066; P < 0.0001; and arcuate nucleus of the hypothalamus: F(4,46) = 2.891; P = 0.0324).

General physical parameters were also assessed. The body weights of rats increased with age, but at any given age, male and female weights were not different (group mean ± SEM: PND-0 males, 6.8 ± 0.7 g; PND-0 females, 6.7 ± 0.2 g; PND-4 males, 11.9 ± 0.6 g; PND-4 females, 11.4 ± 0.6 g; PND-10 males, 22.7 ± 0.7 g; PND-10 females, 20.8 ± 0.7 g; PND-20 males, 55.5 ± 1.4 g; PND-20 females, 55.8 ± 2.1 g). Ano-genital distances increased with age and were longer in males than in females at all ages (by individual t tests, P <= 0.01; PND-0 males, 3.7 ± 0.6 mm; PND-0 females, 1.6 ± 0.1 mm; PND-4 males, 5.8 ± 0.2 mm; PND-4 females, 3.1 ± 0.2 mm; PND-10 males, 6.6 ± 0.2 mm; PND-10 females, 3.9 ± 0.3 mm; PND-20 males, 12.6 ± 0.1 mm; PND-20 females, 8.5 ± 0.6 mm).

Developmental profile of AR mRNA expression
Bed nucleus of the stria terminalis. AR mRNA expression was intense in the principal portion of the bed nucleus of the stria terminalis (Figs. 2Go, 3Go, and 5Go), but was low to moderate in the anterodorsal region (Fig. 2Go). These subnuclei were analyzed separately.



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  		Figure 3. Digitized images of AR mRNA expression in the developing principal bed nucleus of the stria terminalis (BSTpr) of male rats.

 


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  		Figure 5. Digitized images demonstrating AR mRNA in the principal portion of the bed nucleus of the stria terminalis (BSTpr) of PND-10 rats.

 
Bed nucleus, principal: In the principal portion of the bed nucleus of the stria terminalis, there were significant differences in AR mRNA hybridization density in males and females with age (Figs. 3Go and 4Go), due to sex (Figs. 4Go and 5Go), and there was an interaction of age and sex (age: F(4,50) = 45.104; P < 0.0001; sex: F(1,50) = 31.154; P < 0.0001; interaction: F(4,50) = 5.739; P < 0.0001). Significant increases in AR mRNA expression occurred in males and females with age. AR mRNA hybridization density was equivalent in males and females through PND-4, but was significantly higher in males than in females on PND-10 (P <= 0.01) and PND-20 (P <= 0.01).



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  		Figure 4. AR mRNA hybridization signal in the principal portion of the bed nucleus of the stria terminalis of perinatal rats. Each bar represents the mean corrected gray level. Error bars represent the SEM (n = 6 animals in each group). a, Different from ED-20 animal of the same sex, P <= 0.01; b, different from PND-0 and earlier ages of the same sex, P <= 0.01; c, different from PND-4 and earlier ages of the same sex, P <= 0.01; *, different from male of the same age, P <= 0.01.

 
Bed nucleus, anterodorsal: AR mRNA hybridization density increased gradually with age (F(4,70) = 4.850; P = 0.0016) in the anterodorsal bed nucleus (Fig. 6Go). In contrast with the principal portion, the anterodorsal region demonstrated no sex difference in AR mRNA levels at any age (F(1,70) = 0.664; P = 0.4181).



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  		Figure 6. AR mRNA hybridization signal in the anterodorsal bed nucleus of the stria terminalis of perinatal rats. Each barrepresents the mean corrected gray level. Error bars represent the SEM (n = 8 animals in each group). a, Different from ED-20 animal of the same sex, P <= 0.01.

 
Medial preoptic area. AR mRNA expression in the medial preoptic area was strongest in the central portion and was moderate in the area immediately surrounding the central portion (Figs. 2Go and 8Go). Although AR mRNA hybridization density increased significantly with age in both males and females (F(4,50) = 40.562; P < 0.0001), after PND-4 the increase was steeper in males than in females (Fig. 7Go). Thus, by PND-10 (P <= 0.01) and continuing to PND-20 (P <= 0.01), males exhibited higher levels of AR mRNA expression than females (Fig. 8Go).



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  		Figure 8. Digitized images demonstrating AR mRNA within the medial preoptic area (MPO) of PND-10 rats.

 


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  		Figure 7. AR mRNA hybridization signal in the medial preoptic area of perinatal rats. Each bar represents the mean corrected gray level. Error bars represent SEM (n = 6 animals in each group). a, Different from ED-20 animal of the same sex, P <= 0.01; c, different from PND-4 and earlier ages of the same sex, P <= 0.01; *, different from male of the same age, P <= 0.01.

 
Lateral septum. AR mRNA hybridization density was detected in the lateral septum throughout the dorsal, intermediate, and ventral subdivisions and was analyzed as a whole (Figs. 2Go and 9Go). AR mRNA expression in the lateral septum demonstrated parallel increases in males and females (Fig. 10Go), and no sex differences in AR mRNA expression were detected at any age examined (F(1,70) = 0.002; P = 0.9729).



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  		Figure 9. Digitized images demonstrating expression of AR mRNA in the lateral septum (LS) of PND-10 male and female rats.

 


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  		Figure 10. AR mRNA hybridization signal in the lateral septum of perinatal rats. Each bar represents the mean corrected gray level. Error bars represent the SEM (n = 8 animals in each group). a, Different from ED-20 animal of the same sex, P <= 0.01.

 
Ventromedial hypothalamus. AR mRNA hybridization density was detected in the dorsomedial and ventrolateral portions of the ventromedial hypothalamus at moderate to low levels (Figs. 2Go and 12Go). AR mRNA expression in the ventromedial hypothalamus as a whole increased gradually with age in both males and females (F(4,46) = 9.373; P < 0.0001; Fig. 11Go). There was no significant sex difference in AR mRNA expression at any age examined in the ventromedial hypothalamus (F(1,46) = 0.012; P = 0.7945).



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  		Figure 12. Digitized images demonstrating AR mRNA in the ventromedial hypothalamic nucleus (VMH) and arcuate nucleus (ARH) of PND-10 male and female rats.

 


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  		Figure 11. AR mRNA hybridization signal in the ventromedial hypothalamus of perinatal rats. Each bar represents the mean corrected gray level. Error bars represent the SEM (n = 4 for ED-20 animals; n = 6 for all remaining groups). a, Different from ED-20 animal of the same sex, P <= 0.01; b, different from PND-0 and earlier ages of the same sex, P <= 0.01; c, different from PND-4 and earlier ages of the same sex, P <= 0.01.

 
Arcuate nucleus. AR mRNA hybridization density in the arcuate nucleus of the hypothalamus was present in moderate to low amounts (Figs. 2Go and 12Go). Levels of AR mRNA hybridization did not change significantly with age (F(4,46) = 1.655; P = 0.1776; Fig. 13Go). However, possible regional variations may have contributed to high variability in AR mRNA hybridization signal and may have masked increases in AR mRNA expression. No sex difference was present at any age examined (F(1,46) = 0.001; P = 0.9663).



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  		Figure 13. AR mRNA hybridization signal in the arcuate nucleus of perinatal rats. Each bar represents the mean corrected gray level. Error bars represent the SEM (n = 4 for ED-20 animals; n = 6 for all remaining groups).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To our knowledge, this investigation represents the first report on the developmental profile of AR mRNA expression in the brain during the perinatal period. In these experiments, we have demonstrated that AR mRNA is present in the developing rat forebrain as early as ED-20 in both males and females. In addition, throughout the perinatal period, AR mRNA levels increase in each of the areas examined. The developmental profile of AR mRNA in the forebrain is consistent with a hypothetical role for androgens acting via the AR in the organization of male typical neural circuitry.

Although males and females both exhibit increases in relative AR mRNA expression between ED-20 and PND-10, the progression of steady state AR mRNA levels is sex and region specific. On ED-20, PND-0, and PND-4, the relative expression of AR mRNA is not different in males and females in any of the areas analyzed. After PND-4, sex differences in AR mRNA expression arise in the principal portion of the bed nucleus of the stria terminalis and the medial preoptic area, but not in the lateral septum, ventromedial hypothalamus, anterodorsal bed nucleus of the stria terminalis, or arcuate nucleus. These region-specific patterns in the profile of AR mRNA expression suggest a functional difference in the sensitivity of individual areas to the potential developmental actions of androgens.

Other evidence supports the view that AR mRNA is actively translated perinatally in all of these brain regions. First, the overall temporal pattern of AR mRNA expression parallels that of androgen binding (21). Second, the spatial pattern of AR mRNA expression is consistent with autoradiographic detection of nonaromatizable androgen uptake in the neonatal rat brain (30). Furthermore, even at the earliest age examined, the regional distribution of AR mRNA expression in the brain approaches that reported for AR mRNA (29), AR immunoreactivity (18, 23), and androgen-concentrating cells (27, 35) in the adult rat.

Early work on the sexual differentiation of preoptic area morphology described a narrow window of perinatal development, from ED-18 through PND-5, designated the critical period, as being essential to the masculinization of the nervous system (1). Originally, it was thought that androgen exposure after this critical period would have no impact on the organization of neural circuitry (9, 36). A surge in circulating testosterone occurs in males on ED-18, consistent with the onset of the critical period, and a second surge occurs in males within 2 h after birth (37, 38). However, even after testosterone levels decline after the surges, males continue to exhibit higher levels of circulating and hypothalamic testosterone than females through PND-10 (37, 39). This persistent sex difference in androgen levels coupled with the developmental increase in AR expression reported here and a revised view of the critical period, discussed below, are consistent with a later role for androgens in masculinization of the brain.

Various recent studies have indicated that the critical period extends well beyond the first postnatal week (40, 41, 42, 43). For example, Bloch et al. (40, 41) have shown that when males and females are gonadectomized on the day of birth and then treated with testosterone from PND-15 to PND-30, the behavior and endocrine responses of males are more masculinized and more defeminized than those of females. Additionally, castration of males as late as 4 weeks postnatally reduces the size of the sexually dimorphic nucleus of the preoptic area in adulthood (42). Together, these data suggest that the organizing effects of testosterone persist beyond the first postnatal week and that the development of a sex difference in AR mRNA between PND-4 and PND-10 is not too late to play a role in the organizational phase of brain masculinization.

As mentioned above, the contrast in the development of sex differences in AR mRNA in different brain regions suggests that AR activation plays a role in developmental organization of some neural substrates, but not others. In this regard, it may be important that levels of aromatase mRNA expression in the bed nucleus of the stria terminalis and the medial preoptic area are among the highest of any brain region (44, 45). Colocalization studies have not been reported for neonatal rats, but in adult hamsters, of the areas we examined, the bed nucleus of the stria terminalis and medial preoptic nucleus display the highest percentage of cells that colocalize AR and estrogen receptor (46). Moreover, estrogen induces AR expression in adult rats (47). Therefore, we propose that the region-specific sex differences in AR mRNA levels develop as a result of region-specific induction of AR mRNA by estrogenic metabolites of testosterone. Interestingly, previous reports have demonstrated a difference in estrogen receptor mRNA in the medial preoptic area by PND-0 (48), yet sex differences in AR mRNA do not arise until sometime between PND-4 and PND-10. This lag between the onset of estrogen receptor mRNA sex differences and AR mRNA sex differences is consistent with a cascade hypothesis whereby estrogens may prime the male brain for subsequent actions of androgens (17, 19, 49).


   Acknowledgments
 
The authors thank Dr. R. J. Handa for providing the AR cDNA template, Drs. R. J. Handa and K. J. Jones for critical review of the manuscript, the reviewers for substantive contributions to this report, and Diane Stancik, George Hejna, and Linda Fox for technical assistance.


   Footnotes
 
1 This work supported by NIMH Grant MH-48794 and NSF Grant IBN-9604487. Back

Received August 4, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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