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Endocrinology Vol. 140, No. 4 1807-1814
Copyright © 1999 by The Endocrine Society

ARTICLES

Regulation of Androgen Receptor Messenger Ribonucleic Acid Expression in the Developing Rat Forebrain1

Michael D. McAbee2 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
 
By postnatal day 10 (PND-10), males express more androgen receptor (AR) messenger RNA (mRNA) than females in the principal portion of the bed nucleus of the stria terminalis (BSTpr) and medial preoptic area (MPO), but not in the ventromedial hypothalamus. The development of these region-specific sex differences in AR mRNA expression may be critical for the organization of male-typical neural circuitry and may represent the onset of sex differences in the sensitivity of the rat brain to the actions of androgens. In this study, we used a 35S-labeled riboprobe and in situ hybridization to address whether postnatal testosterone exposure is important for the up-regulation of AR mRNA content in the developing rat forebrain.

In the BSTpr and the MPO of PND-10 rats, males gonadectomized on PND-0 or PND-5 had lower levels of AR mRNA compared with intact or sham-operated control males. Daily replacement of testosterone to animals gonadectomized on PND-0 maintained AR mRNA content in the BSTpr and the MPO at levels equal to those in intact males. In contrast, there was no effect of gonadectomy or testosterone replacement on AR mRNA expression in the ventromedial hypothalamus. Thus, the postnatal hormonal environment may permit the development of region-specific sex differences in AR mRNA.

Significant alterations in AR mRNA expression in the BSTpr and MPO in PND-10 male rats were induced by gonadectomy as late as PND-8. Males gonadectomized on PND-8 had levels of AR mRNA significantly lower than those in intact males, but significantly higher than those in intact females. Further, when animals were gonadectomized on PND-0 and given testosterone on PND-8 and PND-9, levels of AR mRNA were also intermediate between those found in intact males and intact females. The exact time course for transcriptional regulation of AR mRNA in the developing rat brain is unknown. However, others have shown significant regulation of AR mRNA within hours of hormone treatment, so that 2 days of hormone withdrawal or replacement are probably sufficient to achieve new steady state levels of message. Moreover, sexually dimorphic neuronal loss has been documented to peak in hypothalamic cell groups during the first postnatal week. Thus, it is likely that changes in the number of AR mRNA-expressing cells as well as the amount of AR mRNA expression per cell are responsible for the development of male-typical AR mRNA content.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXPOSURE of the rodent nervous system to the male gonadal hormone testosterone during the perinatal period permanently masculinizes numerous behavioral, physiological, and neuroanatomical functions (1). During this period, testosterone may be converted to either androgenic or estrogenic metabolites (2, 3). Although there is abundant evidence that estrogen (4) and its receptor (5) are critical for masculinization, other evidence suggests that estrogen alone is not sufficient for complete sexual differentiation and implicates androgen action in this process. For example, testicular feminized rats, which are sensitive to estrogen but insensitive to androgens, are not completely masculinized (6). Further, androgen exposure during development is integral to the maturation of certain social behaviors (7, 8, 9) and the complete expression of male sexual behavior in adulthood (10, 11).

The impact of testosterone and its metabolites on the organization of forebrain morphology and function is most dramatic during the classically defined critical period from embryonic day 18 (ED-18) to postnatal day 5 (PND-5) (12, 13), but sensitivity to the developmental effects of testosterone extends beyond the first week of postnatal development (14). We have recently demonstrated that androgen receptor (AR) messenger RNA (mRNA) is present in the rat forebrain during this critical period and that expression of AR mRNA increases with age (15). This confirms that the receptor necessary for androgen-specific action is actively transcribed during this period. Additionally, between PND-4 and PND-10, a region-specific sex difference in AR mRNA expression develops in the principal bed nucleus of the stria terminalis (BSTpr) and medial preoptic area (MPO), with males having higher AR mRNA levels than females (15). This sex difference is not present in the lateral septum, ventromedial hypothalamus (VMH), or arcuate nucleus (15). The BSTpr and MPO are part of an integrated neural circuitry that controls male sexual behavior in the adult rat (16). Numerous studies have shown sex differences in the capacity of testosterone to induce male sexual behaviors in the adult rat; for example, males exhibit a more complete pattern of male sexual behavior than females in response to treatment with exogenous testosterone (1). Therefore, sex differences in AR mRNA in these areas may be indicative of sex differences in the sensitivity of the developing rat brain to the actions of androgens.

During the perinatal period, circulating levels of testosterone are higher in the male than in the female rat (17, 18). Further, testosterone regulates AR expression in the BST and MPO of the adult rat (19, 20). Thus, we hypothesized that the development of sex differences in AR mRNA occurs as a result of exposure to testosterone during the perinatal period. The potential regulation of AR expression may represent a fundamental step in the cascade of events leading to brain masculinization. In this study, we used an 35S-labeled riboprobe and in situ hybridization to directly address whether postnatal testosterone exposure is important for the regulation of AR mRNA content and to indirectly determine whether testosterone regulates the number of AR mRNA-expressing cells or the amount of AR mRNA per cell during this process.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Timed pregnant Sprague-Dawley rats were obtained from Zivic-Miller Laboratories, Inc. (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. On PND-0 (day of birth), each litter was adjusted to five females and five males.

Before sacrifice, body weight and ano-genital distances were recorded. All animals were anesthetized with ether and decapitated on PND-10. Brains were quickly removed, rapidly frozen in powdered dry ice, and stored at -70 C. Frozen sections were cut from the rostral forebrain to the midbrain at a thickness of 16 µm and mounted on SuperFrost Plus slides (Fisher Scientific International, Inc., Pittsburgh, PA). Slides were stored at -70 C until processing for in situ hybridization.

Surgeries
Bilateral gonadectomies were performed on male rats on PND-0 or PND-5 for Exp 1 and 2 and on PND-0 or PND-8 for Exp 3. Gonadectomies on PND-0 were started more than 2 h after birth, which is after the peak surge of neonatal testosterone. All animals were chilled on ice to induce anesthesia. A small incision, approximately 1 mm long, was made through the skin and abdominal wall just rostral and lateral to the phallus. The testis was removed, and a single suture of 6.0 chromic gut was used to close the incision. The surgery was repeated on the opposite side. The incisions were covered with collodion to aid healing. Control male rats underwent sham operations in which the gonads were visualized but were not removed. Before being returned to the dam, all pups were warmed under a heat lamp until a normal body temperature and level of activity were achieved.

Testosterone treatments
Testosterone propionate (TP; 10 µg/0.1 cc in sesame oil) was administered sc in the dorsum through a 20-gauge needle to decrease resistance and ensure rapid administration of hormone. Control animals received injections of sesame oil vehicle. A drop of collodion was placed over the site of injection to prevent any leakage. All treatments were made daily beginning on the day of gonadectomy and continued through PND-9, the day before death. The site of administration was rotated each day to avoid damage to the skin as well as to maximize the uptake of testosterone. In those litters receiving hormone treatment, all animals in a litter were given the same substance (TP or oil) to ensure that excreted metabolites or leakage from the injection site did not affect littermates.

Treatment groups
Exp 1 had six different treatment groups: intact males, intact females, males gonadectomized on PND-0 (GDX-0), males gonadectomized on PND-5 (GDX-5), males sham operated on PND-0 (sham-0), and males sham operated on PND-5 (sham-5). Exp 2 had four different treatment groups: intact males, intact females, GDX-0, and males gonadectomized on PND-0 and given testosterone replacement on PND-0 through PND-9 (GDX-0 + TP-0–9). Exp 3 had five different treatment groups: intact males, intact females, GDX-0, males gonadectomized on PND-8 (GDX-8), and males gonadectomized on PND-0 and given testosterone on PND-8 and PND-9 (GDX-0 + TP-8,9). Each treatment group was comprised of animals from at least three different litters.

In situ hybridization
In situ hybridization procedures were conducted as previously described (15). AR mRNA was detected using a 35S-labeled complementary RNA probe transcribed from a rat AR complementary DNA corresponding to nucleotides 3350–3840. The complementary RNA probe was diluted with hybridization buffer to a final activity of 1.5 x 107 cpm/ml. The tissue was prepared for hybridization by acetylation, delipidation, and dehydration. Each slide was hybridized with 100 µl hybridization solution for 20 h at 60 C. After hybridization, slides were rinsed in sodium chloride-sodium citrate (SSC), treated with ribonuclease, and rinsed again to a final stringency of 0.1 x SSC at 60 C. After the rinses, slides were dehydrated, allowed to air-dry, and apposed to Hyperfilm Betamax (Amersham, Arlington Heights, IL) to produce film autoradiograms for analysis.

Analysis
Analysis of the film autoradiograms was carried out using NIH IMAGE analysis software (developed at the NIH available at http://rsb.info.nih.gov/nih-image/) and a Macintosh IIci computer with Scion videocard (Scion Corp., Walkersville, MD) attached to a Sony video camera (Imaging Research, Inc., St. Catherines, Canada). Three sections per animal were analyzed bilaterally per region of interest. The analysis focused on two regions that were previously shown to have a sex difference in AR mRNA on PND-10 (15): the BSTpr and the MPO. The VMH, a region with no sex difference in AR mRNA expression at PND-10, was also examined (15). The atlas of Swanson was used to aid in the identification of cell group boundaries (21). Each cell group was analyzed based on detectable signal as previously described (15). The entire area of label was outlined regardless of signal intensity. The average pixel value of the outlined region was measured and expressed as a mean gray level. These mean gray levels represent a semiquantitative index of steady state levels of AR mRNA.

To minimize the potential for variability due to interrun differences, brain sections from an equivalent number of animals from each treatment were processed in each hybridization run. In addition, to eliminate differences due to nonspecific hybridization, five background measures were taken from the caudate-putamen of each animal. The mean background measure for each animal was subtracted from each individual mean gray level to obtain a corrected gray value. As one-way ANOVA (P <= 0.05) demonstrated that average corrected gray levels from left and right hemispheres were not different, values from both hemispheres were combined to yield one mean corrected gray value for each animal. The effects of treatment on mean corrected gray values were analyzed by one-way ANOVA for each area, with planned post-hoc tests (Fisher’s protected least significant difference and Scheffe’s multiple comparison test). Differences were considered significant at P <= 0.05. Individual t tests, with P <= 0.01, were performed to examine specific differences due to treatment. Body weight and ano-genital distances were also analyzed for each experiment by one-way ANOVA with planned post-hoc tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight and ano-genital distance
In each experiment, there was no effect of treatment on body weight (Table 1Go). Ano-genital distances were significantly lower in intact females than in all other treatment groups (P <= 0.01; Table 1Go). Gonadectomy had no effect on ano-genital distance in any experiment.


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  		Table 1. Mean body weights and ano-genital distances for each treatment group in each experiment

 
Exp 1: does gonadectomy on PND-0 or PND-5 decrease AR mRNA content in the developing male rat forebrain?
In this experiment, we asked whether postnatal exposure to testicular hormones up-regulated AR mRNA content. AR mRNA expression was examined in PND-10 male rats that had undergone gonadectomy on either PND-0, after prenatal development, or PND-5, after the end of the classically defined critical period (12). Specifically, the BSTpr and MPO were examined because they were previously shown to express sexually dimorphic AR mRNA content (15). The VMH was examined as a control area that demonstrates no sex difference in AR mRNA expression (15).

In the BSTpr, in agreement with our previous findings (15), intact males expressed more AR mRNA than intact females on PND-10. Further, gonadectomy on either PND-0 or PND-5 resulted in significantly lower levels of AR mRNA compared with those in intact males (P <= 0.01; Fig. 1Go). In the MPO, as observed previously (15), males also expressed more AR mRNA than intact females. In addition, AR mRNA expression was significantly lower after gonadectomy (P <= 0.01; Fig. 2Go). Gonadectomy on PND-0 or PND-5 lowered AR mRNA content in the BSTpr and MPO to levels not significantly different from those in intact females. In contrast to the MPO and BST, AR mRNA in the VMH, was not altered by gonadectomy (P <= 0.992; Fig. 3Go). AR mRNA expression in sham-operated animals was not significantly different from that in intact males in any area examined (data not shown). Thus, hormone exposure from PND-5 through PND-10 is necessary to achieve male-typical levels of AR mRNA in PND-10 males.



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  		Figure 1. The effect of postnatal gonadectomy on AR mRNA expression in the BSTpr of PND-10 rats. The digitized images show comparisons among intact males, intact females, and males gonadectomized on PND-0 (GDX-0) or PND-5 (GDX-5). Semiquantitative analysis of AR mRNA levels is represented in the graph. Each column represents the mean gray level, and error bars represent the SEM (n = 6 animals in each group). *, Different from intact male animals, P <= 0.01.

 


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  		Figure 2. The effect of postnatal gonadectomy on AR mRNA expression in the MPO of PND-10 rats. The digitized images show comparisons among intact males, intact females, and males gonadectomized on PND-0 (GDX-0) or PND-5 (GDX-5). Semiquantitative analysis of AR mRNA levels is represented in the graph. Each column represents the mean gray level, and error bars represent the SEM (n = 6 animals in each group). *, Different from intact male animals, P <= 0.01.

 


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  		Figure 3. The effect of postnatal gonadectomy on AR mRNA expression in the VMH of PND-10 rats. The digitized images show comparisons among intact males, intact females, and males gonadectomized on PND-0 (GDX-0) or PND-5 (GDX-5). Semiquantitative analysis of AR mRNA levels is represented in the graph. Each column represents the mean gray level, and error bars represent the SEM (n = 6 animals in each group).

 
Exp 2: does treatment with exogenous testosterone after gonadectomy sustain AR mRNA expression at intact male levels?
This experiment specifically addressed whether postnatal testosterone exposure is sufficient for up-regulation of AR mRNA to male-typical levels. In this experiment, male rats were gonadectomized on PND-0 and received daily injections of TP (10 µg/day) on PND-0 through PND-9, with death on PND-10.

AR mRNA expression in males gonadectomized on PND-0 and treated with TP was not significantly different from that in intact males in the BSTpr or MPO (Figs. 4Go and 5Go). In both areas, levels of AR mRNA in gonadectomized animals given TP replacement were significantly higher than those in intact females (BSTpr: P <= 0.01; Fig. 4Go; MPO: P <= 0.01; Fig. 5Go). Further, levels of AR mRNA in males gonadectomized on PND-0 given TP were significantly higher than those in males gonadectomized on PND-0 that did not receive TP (BSTpr: P <= 0.01; Fig. 4Go; MPO: P <= 0.01; Fig. 5Go). As in Exp 1, levels of AR mRNA in the VMH were not different between intact males and intact females and were not affected by gonadectomy or TP replacement after gonadectomy (Fig. 6Go). Thus, postnatal exposure to TP was sufficient to produce male-like AR mRNA content in the developing BSTpr and MPO and had no little or no effect on the expression of AR mRNA in the VMH.



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  		Figure 4. The effect of TP treatment after neonatal gonadectomy on AR mRNA expression in the BSTpr of PND-10 rats. The digitized images show comparisons among intact males, intact females, males gonadectomized on PND-0 (GDX-0), and males gonadectomized on PND-0 that received TP treatment on PND-0 through PND-9 (GDX-0 + TP 0 to 9). Semiquantitative analysis of AR mRNA levels is represented in the graph. Each column represents the mean gray level, and error bars represent the SEM (n = 6 animals in each group). *, Different from intact male animals, P <= 0.01.

 


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  		Figure 5. The effect of TP treatment after neonatal gonadectomy on AR mRNA expression in the MPO of PND-10 rats. The digitized images show comparisons among intact males, intact females, males gonadectomized on PND-0 (GDX-0), and males gonadectomized on PND-0 that received TP treatment on PND-0 through PND-9 (GDX-0 + TP 0 to 9). Semiquantitative analysis of AR mRNA levels is represented in the graph. Each column represents the mean gray level, and error bars represent the SEM (n = 6 animals in each group). *, Different from intact male animals, P <= 0.01.

 


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  		Figure 6. The effect of TP treatment after neonatal gonadectomy on AR mRNA expression in the VMH of PND-10 rats. The digitized images show comparisons among intact males, intact females, males gonadectomized on PND-0 (GDX-0), and males gonadectomized on PND-0 that received TP on PND-0 through PND-9 (GDX-0 + TP 0 to 9). Semiquantitative analysis of AR mRNA levels is represented in the graph. Each column represents the mean gray level, and error bars represent the SEM (n = 6 animals in each group).

 
Exp 3: does short-term gonadectomy or short-term replacement of testosterone after gonadectomy alter AR mRNA expression?
This experiment addressed indirectly whether regional sex differences in AR mRNA content and the potential regulation of AR mRNA by testosterone are due to alterations in the number of AR mRNA-expressing cells, the amount of AR mRNA per cell, or both. The packing density of neurons in the regions of interest is too high during development to permit direct cellular analysis. However, making use of previous reports that the peak period of developmental cell death in these areas is before PND-8 (22), and that a 2-day time course provides ample time for hormone-dependent AR mRNA gene regulation (23), two treatments were designed to address this question. First, male rats underwent gonadectomy on PND-8, with death on PND-10. These rats would be expected to have a male-typical number of neurons. Second, some animals were gonadectomized on PND-0, followed by short-term treatment with TP on PND-8 and PND-9 and death on PND-10. These animals would be expected to have a female-typical number of neurons.

After gonadectomy on PND-8, AR mRNA expression was significantly lower in the BSTpr (P <= 0.01; Fig. 7Go) and MPO (P <= 0.01; Fig. 8Go) than that in intact males when AR mRNA content was examined on PND-10. AR mRNA expression was significantly higher in males gonadectomized on PND-8 than in intact females or in males gonadectomized on PND-0 in BSTpr (P <= 0.01; Fig. 7Go) and MPO (P <= 0.01; Fig. 8Go). As levels of AR mRNA were significantly reduced after gonadectomy on PND-8 but remained significantly higher than those in males gonadectomized on PND-0, it is likely that the production of male-like AR mRNA content in the BSTpr and MPO was due to alterations in both the number of AR-expressing cells and the amount of AR mRNA per cell.



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  		Figure 7. The effect of short term hormone manipulation on AR mRNA expression in the BSTpr of PND-10 rats. The digitized images show comparisons among intact males, intact females, males gonadectomized on PND-8 (GDX-8), and males gonadectomized on PND-0 that received TP on PND-8 and PND-9 (GDX-0 + TP-8,9). Semiquantitative analysis of AR mRNA levels is represented in the graph. Each column represents the mean gray level, and error bars represent the SEM (n = 6 animals in each group). *, Different from intact male animals, P <= 0.01. #, Different from intact female and gonadectomized male animals on PND-0, P <= 0.01.

 


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  		Figure 8. The effect of short term hormone manipulation on AR mRNA expression in the MPO of PND-10 rats. The digitized images show comparisons among intact males, intact females, males gonadectomized on PND-8 (GDX-8), and males gonadectomized on PND-0 that received TP on PND-8 and PND-9 (GDX-0 + TP-8,9). Semiquantitative analysis of AR mRNA levels is represented in the graph. Each column represents the mean gray level, and error bars represent the SEM (n = 6 animals in each group). *, Different from intact male animals, P <= 0.01. #, Different from intact female and gonadectomized male animals on PND-0, P <= 0.01.

 
In males gonadectomized on PND-0 and treated with TP on PND-8 and PND-9, levels of AR mRNA expression in the BSTpr and MPO were intermediate between levels observed in the intact males and intact females (Figs. 7Go and 8Go). AR mRNA expression in the BSTpr and MPO of TP-treated animals was significantly higher than that in males that did not receive TP treatment (Figs. 7Go and 8Go). These results, similar to those reported above, are indicative of changes in both the number of AR mRNA-expressing cells and the amount of AR mRNA per cell in response to short-term hormone replacement. As expected, AR mRNA expression in the VMH was not altered 2 days after gonadectomy or after gonadectomy with 2-day hormone replacement (Fig. 9Go).



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  		Figure 9. The effect of short term hormone manipulation on AR mRNA expression in the VMH of PND-10 rats. The digitized images show comparisons among intact males, intact females, males gonadectomized on PND-8 (GDX-8), and males gonadectomized on PND-0 that received TP on PND-8 and PND-9 (GDX-0 + TP-8,9). Semiquantitative analysis of AR mRNA levels is represented in the graph. Each column represents the mean gray level, and error bars represent the SEM (n = 6 animals in each group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The first experiment of this study demonstrated that gonadectomy on PND-0 or PND-5 decreased AR mRNA expression by PND-10 in a region-specific manner. AR mRNA expression in the BSTpr and MPO was significantly reduced in males after gonadectomy at either age. In contrast, AR mRNA expression in the VMH was not altered in gonadectomized males. These experiments confirm our previous reports of region-specific sex differences and demonstrate that the pattern of regulation of AR mRNA parallels the pattern of region-specific sex differences (15).

In addition, the first experiment demonstrated that sex differences in AR mRNA content in the developing rat forebrain are primarily dependent on the postnatal hormonal environment. Prenatal hormone exposure is not sufficient to produce male-typical levels of AR mRNA in these areas, as gonadectomy on either PND-0 or PND-5 eliminated the sex difference in AR mRNA expression exhibited on PND-10 in the MPO and BST.

In addition to confirming the effect of gonadectomy on PND-0, the second experiment addressed the capacity of exogenous testosterone to maintain male-like levels of AR mRNA expression after gonadectomy. Replacement of testosterone to male rats gonadectomized on PND-0 sustained AR mRNA hybridization intensity at levels not significantly different from those in intact males. However, testosterone may be converted to estrogenic and androgenic metabolites, and both estrogen and androgens regulate AR mRNA expression in the adult rat (20). Thus, production of male-typical levels of AR mRNA may be due to androgenic regulation, estrogenic regulation, or both. Moreover, regional specificity of AR mRNA regulation in the neonatal brain may depend upon the concerted actions of androgens and estrogens. This idea is supported by studies demonstrating that aromatase levels in the BST and MPO are among the highest of any forebrain region (19). Further, colocalization studies in the rat and hamster BST and MPO also show a high degree of AR and ER colocalization in the adult (24, 25). Therefore, up-regulation of AR mRNA may require estrogenic metabolites of testosterone and expression of the estrogen receptor, consistent with our earlier hypothesis that one role of estrogen in the neonatal brain is to enhance sensitivity to subsequent androgen exposure (15). Experiments are underway to determine whether the development of sex differences in AR mRNA content in the neonate are dependent upon androgenic or estrogenic mechanisms.

As mentioned earlier, because of the high packing density of neurons in the BSTpr and MPO during development, we are unable to perform a direct cellular analysis to determine whether the regulation of AR mRNA content by testosterone during the neonatal period is due to alterations in the number of AR mRNA-expressing cells, the amount of AR mRNA per cell, or both. Therefore, we have addressed this question indirectly basing our experimental design and conclusions on the results of previous studies showing that 1) the peak period of developmental cell death in these areas is before PND-8 (22); 2) 2 days provide ample time for hormone-dependent AR mRNA gene regulation (23); and 3) in vitro, maximal regulation of AR mRNA by androgens occurs 48–49 h after hormone addition (26, 27). In the third experiment, males with presumably more cells and without testosterone for 2 days (GDX-8) had more AR mRNA than females, but less AR mRNA than intact males; perhaps the GDX-8 males did not approach the lower female levels because they had more AR mRNA-expressing cells. Similarly, males with presumably the same number of cells as females and receiving testosterone replacement for 2 days (GDX-0 + TP-8,9) had more AR mRNA than females but less than intact males. The GDX-0 + TP-8,9 males may not have achieved intact male levels of AR mRNA because they had fewer AR mRNA-expressing cells. Both sets of results from the third experiment suggest that the development of sex differences in AR mRNA content are dependent upon regulation by testosterone of both the amount of AR mRNA per cell and the number of AR mRNA-expressing cells. There are other possible explanations for these findings; the most obvious is that 48 h of hormone depletion or replacement are insufficient to maximally regulate AR mRNA content in vivo. Additional experiments are in progress to determine the time course of testosterone-dependent regulation of AR mRNA content.

Interestingly, in the adult rat, short term gonadectomy produces a transient up-regulation of AR mRNA expression in the MPO and BST (20). As, in contrast, our study demonstrates a rapid down-regulation of AR mRNA after gonadectomy, the mechanism regulating AR gene transcription may be different in the neonatal vs. the adult rat.

One explanation for a possible difference in the capacity of androgens to regulate the AR during the perinatal period vs. adulthood is that an integral factor is missing or has yet to develop in the perinatal animal. Candidates for such a factor include AR-associated proteins, transcription cofactors, and growth factors. Examples of each of these have been shown to modify AR function. Specifically, the ARA-70 protein increases the transcriptional efficiency of the AR (30), whereas the presence of TFIIF alters the trans-activation function and ultimately the transcriptional capacity of the AR (31). In addition, recent studies have shown that other proteins, including receptor-associated coactivator-3 (32) and steroid receptor coactivator-1 (33), mediate the activation of nuclear hormone receptors. Further, both platelet-derived growth factor and transforming growth factor-ß have been shown to affect AR mRNA gene expression in cultures of smooth muscle cells (34, 35). The absence of each of these or other similar factors during the perinatal period may result in inefficient AR-mediated transcription and may account for putative differences in the regulation of AR mRNA expression between the neonatal period and adulthood.


   Acknowledgments
 
The authors thank Dr. R. J. Handa for providing the AR complementary DNA template, Drs. R. J. Handa and K. J. Jones for critical review of the manuscript, and Karen Schwenk for technical assistance.


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

2 Present address: Department of Biology, University of Massachusetts, Morrill Science Center, Amherst, Massachusetts 01003. Back

Received May 15, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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M. D. McAbee and L. L. DonCarlos
Estrogen, But Not Androgens, Regulates Androgen Receptor Messenger Ribonucleic Acid Expression in the Developing Male Rat Forebrain
Endocrinology, August 1, 1999; 140(8): 3674 - 3681.
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