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Biology of Reproduction 60, 1251-1256 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Androgen Receptor Messenger Ribonucleic Acid in Brains and Pituitaries of Male Rhesus Monkeys: Studies on Distribution, Hormonal Control, and Relationship to Luteinizing Hormone Secretion1

Salah E. Abdelgadira, Charles E. Rosellia, Jerome V.A. Choatea, and John A. Resko2,a

a Department of Physiology and Pharmacology, School of Medicine, Oregon Health Sciences University, Portland, Oregon 97201-3098


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Because the distribution and hormonal regulation of the androgen receptor (AR) mRNA in brains and pituitaries of adult rhesus monkeys have not been studied, we cloned and sequenced a 329-base pair segment of the 5' coding region of the rhesus AR cDNA. Monkey AR cDNA was 99% identical with the human sequence and 96% homologous with the rat sequence. Using a ribonuclease protection assay, we studied the distribution and regulation of AR mRNA in brains and anterior pituitary glands of three groups of male rhesus monkeys: intact (n = 3), castrated (Cx, n = 4), and Cx treated with testosterone (n = 6). Serum testosterone levels of Cx males treated with testosterone differed significantly (p < 0.05) in the morning but not in the evening hours from those in intact controls. Serum LH concentrations were significantly suppressed (p < 0.05) in both morning and evening serum samples of testosterone-treated males compared to intact controls. We found the highest concentrations of AR mRNA in the medial basal hypothalamus, the bed nucleus of the stria terminalis, the medial preoptic area-anterior hypothalamus, and the lateral dorsomedial hypothalamus. Intermediate amounts were found in the septum and amygdala. Low amounts were found in the hippocampus, cingulate cortex, parietal cortex, and cerebellum. The anterior pituitary gland also contained a large amount of AR mRNA. Surprisingly, neither Cx for 3 wk nor Cx plus testosterone replacement for 3 wk significantly affected AR mRNA in any brain area or in the pituitary gland.

The present study demonstrates that the effectiveness of testosterone as a regulator of LH secretion in male monkeys is not related to changes of AR mRNA in the brain or pituitary gland. It appears that AR mRNA in the monkey brain and pituitary gland is not regulated at the transcriptional level by androgen.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In nonhuman primates, circulating androgens can be correlated with an array of biological effects throughout the life cycle of the animal [1, 2]. In prenatal life, testosterone of fetal origin [3] organizes brain areas that control reproductive behaviors in infancy and adulthood [4, 5]. In adult males, androgens regulate reproductive [6] and aggressive [7] behaviors, growth and secretion of accessory organs of reproduction [8], and gonadotropin secretion [9].

The distribution of androgen receptor (AR) within the central nervous system of rats, hamsters, guinea pigs, and two species of nonhuman primate, Macaca fasciculars and Macaca mulatta [10–15], has been determined by the immunocytochemical visualization of these receptors. In the guinea pig, staining is localized in the nucleus [13], whereas in the rat, hamster, and nonhuman primate, cytoplasmic as well as nuclear staining was found [12, 14, 16].

The distribution and control of AR have been studied in the central nervous system and pituitary gland in rats [17, 18], guinea pigs [19, 20], and nonhuman primates [21–23] using binding techniques that employ assays of AR in nuclear (ARn) and cytosolic (ARc) cell fractions. In these preparations the ratio of ARn to ARc depends upon the hormonal state of the animal. This ratio increases in the presence of androgens such as testosterone or dihydrotestosterone [20] and decreases after castration (Cx) [20]. In male rats, androgens affect AR mRNA differently in target tissues such as the prostate and seminal vesicles as compared to brain tissues. In the former, androgens decrease and Cx increases AR mRNA [24–27], whereas in the latter, no treatment effect was observed [27]. Changes in AR mRNA concentrations in the prostate correspond to changes in ARc, suggesting that ARc is newly synthesized protein [27]. In nonhuman primates, the distribution of AR mRNA within the central nervous system and the effects of androgen on AR mRNA have not been reported, to our knowledge.

In this study, we synthesized a monkey AR cDNA by reverse transcription-polymerase chain reaction (RT-PCR) and used it to map the distribution and study the control of AR mRNA in the rhesus monkey brain.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Thirteen adult male rhesus monkeys (Macaca mulatta, 5–10 yr of age) were used in this study. The housing and care of the animals have been described previously [28]. Experiments and animal care were conducted in accordance with the principles and procedures outlined by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Blood samples (3 ml) were obtained from each animal through a cardiac catheter at 0900 and 2100 h, 4 days before they were either Cx, or Cx and treated with testosterone. Testosterone concentrations in serum obtained from these blood samples were used for comparison with testosterone concentrations after Cx. The placement of the catheter for remote drawing of blood has been described previously [29]. Testosterone was administered at the time of Cx by placing four (4 cm) silicone elastomer capsules (0.330 cm, i.d.; 0.465 cm, o.d.) filled with steroid s.c. between the scapulae. Empty capsules placed in the same location in Cx males were used as a control for implant placement. Animals were bled for 3 wk after Cx, as described above, before collection of tissues.

The tissues used for AR mRNA measurements in ribonuclease protection assays were from 3 intact, 4 Cx, and 6 Cx males treated with testosterone. On the day of autopsy, each animal was anesthetized with ketamine hydrochloride (15 mg/kg, i.m.) for transport from its cage to the autopsy room and then killed by injection of a lethal dose of pentobarbital (11 mg/kg BW, i.m.). The brain was rapidly removed from the cranium and rinsed in ice-cold 0.9% saline. The basal forebrain and the limbic lobes were dissected according to previously published methods [28] and immediately frozen on dry ice and stored at -80°C until they were assayed.

RNA Isolation

Total RNA was isolated by homogenizing tissues in 4 M guanidinium isothiocyanate, 10 mM EDTA, 2% sodium N-lauryl sarcosine, 1% (v:v) ß-mercaptoethanol, 50 mM Tris, pH 7.6, in the presence of 10 mM vanadyl ribonucleoside complexes. The guanidinium isothiocyanate extract was then centrifuged through 5.7 M cesium chloride [30], and the RNA was extracted twice with phenol and concentrated by ethanol precipitation.

Isolation of cDNA Probe for Rhesus Monkey AR

PCR, using primers selected from the 5' coding region of the published sequences of human [31] and rat [32] AR cDNAs, was employed to isolate a rhesus monkey-specific AR cDNA. The two primers selected for PCR encompass a 330-base pair (bp) sequence in human and rat AR cDNAs. The sense primer (5'-TCTCAAGAGTTTGGATGGCTCC-3') is identical to the sequences starting at nucleotides 2733 and 3313 of the human and rat AR cDNA, respectively. The antisense primer (5'-GAGATGATCTCTGCCATCATTTC-3') starts at nucleotide 3061 and 3641 in the human and rat AR cDNA, respectively. An internal sequence (5'-TCTACCAGCTCACCAAGC-3'), starting at nucleotides 2929 and 3509 of the human and rat AR cDNA, respectively, was labeled by means of [{gamma}-32P]ATP and T4 polynucleotide kinase and was used in a Southern blot analysis to determine the specificity of the RT-PCR products and in the selection of specific colonies for sequencing.

RT-PCR

RT of 5 µg total RNA extracted from a rhesus monkey prostate was carried out for 2 h at 37°C in a 20-µl reaction mixture containing RT buffer (50 mM Tris-HCl, pH 8.3; 40 mM KCl; 3 mM MgCl2), 10 mM dithiothreitol, 0.5 mM of each dNTP (dATP, dCTP, dGTP, dTTP), 20 U RNasin, 25 pmol of oligo(dT) primer, and 200 U of SuperScript RNase H- Reverse Transcriptase (Gibco BRL, Grand Island, NY). The RT reaction was then added to 80 µl PCR-cocktail containing Taq DNA polymerase buffer (100 mM Tris-HCl; 500 mM KCl; 1.0% Triton X-100), 1.5 mM MgCl2, 0.2 mM dNTP, 2.5 units Taq DNA polymerase (Promega, Madison, WI), 100 pmol of 5'-AR gene-specific primer, and 100 pmol 3'-AR gene-specific primer. The PCR consisted of 35 cycles of denaturing (92°C, 2 min), annealing (55°C, 2 min), and extension (72°C, 3 min), 40 sec at 92°C, 2 min at 55°C, and 3 min at 72°C. A blank tube (control) was carried out in parallel during the RT-PCR procedures and received all ingredients except RNA. Aliquots (10 µl) of the RT-PCR and control reactions were electrophoresed in a 1% agarose gel for visualization of PCR products. The PCR amplification produced a single DNA product of approximately 330 bp that was not present in the control reaction. After denaturation (1.5 M NaCl, 0.5 M NaOH) and neutralization (1.5 M NaCl, 0.5 M Tris, pH 7.5), the contents of the agarose gel were transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH), cross-linked, and hybridized to the internal sequence, which was labeled by 5' end-labeling using [{gamma}-32P]ATP and T4 polynucleotide kinase. The Southern analysis detected a single band of approximately 330 bp DNA that corresponded to the RT-PCR product (data not shown). We ligated this product in a pCR II vector (TA Cloning System; Invitrogen, San Diego, CA) and transformed it into competent Escherichia coli cells (INV{alpha}F') as supplied by the TA Cloning System (Invitrogen). The transformed cells were plated in the presence of 40 mg X-Gal on LB agar plates containing 50 µg/ml ampicillin and were incubated overnight at 37°C. Four white colonies were lifted, and each one was placed in 5 ml Luria broth medium containing ampicillin and grown overnight at 37°C in a metabolic shaking incubator. A 1.5-ml aliquot of each culture was subjected to the mini-prep method for isolation of plasmid DNA [33]. Each harvested DNA pellet was digested with DNase-free RNase/10 U of EcoRI to remove the RT-PCR insert and electrophoresed in 1% agarose to visualize the cloning products. The insert from a positive colony was sequenced using the chain termination reaction method (Sequenase, version 2.0; United States Biochemicals, Cleveland, OH). Briefly, the pCR II vector containing the insert was denatured and annealed to T7 and Sp6 primers for 1 h at 37°C, after which an initial extension and elongation (labeling reaction) was achieved using [35S]dATP, dCTP, dGTP, and dTTP in the presence of sequenase and pyrophosphatase. In the termination step, aliquots of the extension, elongation reaction were added to each of four separate reactions, each of which contained one of the 2',3'-dideoxynucleoside triphosphates as substrate. The products of these four reactions were then separated in adjacent lanes in an 8% acrylamide/urea gel and used to expose a Kodak (Eastman Kodak, Rochester, NY) x-ray film.

Ribonuclease Protection Assay

To develop a ribonuclease protection assay for measuring AR mRNA, we subcloned the first 292 nucleotides (nt) of the AR insert onto the EcoRI site of the pGEM 7Zf(-) vector. The ribonuclease protection assay was performed as described previously [34–36]. Briefly, the rhesus monkey AR cDNA-pGEM7Zf(-) complex was linearized with XhoI and used to synthesize a 32P-labeled antisense AR probe by SP6 RNA polymerase that was purified by electrophoresis in a 7.1 M urea, 5% polyacrylamide gel. Total RNA (10 µg) was incubated for 16 h at 45°C with 5 x 105 dpm of the gel-purified 32P-labeled antisense probe in 30 µl of hybridization buffer (80% formamide in 40 mM Pipes, pH 6.4, 0.4 M NaCl, 1 mM EDTA). After hybridization, the samples were digested with ribonuclease T1 (900 units/380 µl; Gibco BRL) for 1 h at 37°C. The ribonuclease digestion was terminated by addition of 10 µl 20% SDS and 50 µg proteinase K; then the reaction mixture was incubated for 20 min at 37°C, extracted with phenol:chloroform, and precipitated with ethanol. The pellets were dissolved in 80% formamide containing 0.4% xylene cyanole/0.4% bromophenol blue and resolved in a 7.1 M urea, 5% polyacrylamide gel at 250 V for 1.5 h. AR sense RNA was transcribed in vitro from the AR cDNA template by means of T7 RNA polymerase and used to construct standard curves (31.25, 62.5, 125, 250, 500, 1000, 2000 fg) for each ribonuclease protection assay. The ribonuclease protection assay protected a single RNA fragment of approximately 307 nt in length that included 15 nt of vector origin.

Cyclophilin mRNA [37, 38] was measured using a 185-nt [32P]cRNA probe that was transcribed from a rhesus monkey p1B15 cyclophilin cDNA cloned into pGEM-3Z vector. The protected cyclophilin mRNA fragment in the ribonuclease protection assay was 158 nt long.

Protected areas on the polyacrylamide gels were quantified using a Molecular Imaging System (Bio-Rad GS-525, Hercules, CA). Exposure time was 24 h. For each standard curve, a linear regression was used to relate the autoradiographic signal (i.e., relative optical density) to known amounts of AR sense RNA (31.25–2000 fg). The amount of AR mRNA in each sample was then determined from the ribonuclease protection assay standard curve. Levels of cyclophilin mRNA were used to normalize results to equal amounts of sample RNA.

Hormone Assays

Testosterone and biologically active LH were quantified in systemic serum by a specific RIA [39] and a dispersed mouse Leydig cell bioassay [40], respectively, which had been validated previously in our laboratory.

Statistical Analysis

Data were examined by one-way ANOVA, and differences between means were assessed by the Newman-Keuls multiple range test [41]. Probability values of p < 0.05 were considered statistically significant. Correlation coefficients were computed using a computer program (GraphPad Inplot, San Diego, CA). Differences in hormone concentrations in serum collected in the morning and in the evening, among treatment groups, were determined by paired t-tests.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The nucleotide sequence of AR cDNA insert is shown in Figure 1. Comparison of the rhesus, human, and rat AR cDNA sequences revealed 99% homology between rhesus and human and 96% homology between rhesus and rat sequences.



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  		FIG. 1. Nucleotide sequence of the open reading frame of the rhesus monkey AR cDNA insert compared with the human and rat sequence. The RT-PCR-amplified rhesus monkey cDNA was sequenced using the chain termination reaction method. See Materials and Methods for more detail. Star indicates homologies between the 3 species. The GenBank accession number is BankIt223976 AF092930. The corresponding amino acids coded by the nucleotide sequence in the monkey are identical to those found in the rat and human (BLAST protein database search, National Center for Biotechnology Information).

We used this cDNA sequence as a probe to quantify AR mRNA in brain tissue by a ribonuclease protection assay. A representative autoradiogram of a gel showing increasing concentrations of AR mRNA measured by the ribonuclease protection assay is shown in Figure 2A. In Figure 2B, we show a linear regression analysis of the optical densities of the AR sense RNA standard curve. The ribonuclease protection assay was able to detect as little as 62.5 fg of AR sense RNA, and the standard curve was constructed with the following amounts of AR sense RNA: 31.2, 62.5, 250, 500, 1000, and 2000 fg.



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  		FIG. 2. A) A representative ribonuclease protection assay autoradiogram showing the distribution of AR mRNA in brain tissues of an adult rhesus monkey (10 µg total RNA/brain area). Exposure time was 24 h. Lanes are 1) incubated undigested probe, 2) incubated ribonuclease-digested probe, 3) MPAH, 4) lateral dorsomedial hypothalamus, 5) MBH (infundibular nucleus/median eminence/ventromedial nucleus), 6) bed nucleus stria terminalis, 7) septum, 8) amygdala, 9) hippocampus, 10) cingulate cortex, 11) P.CTX, 12) cerebellum, 13) anterior pituitary gland, 14–20) AR sense RNA standard curve (31.2–2000 fg, respectively). See text for individual values of the standard curve. The standard curve generated was used to quantify AR mRNA levels in discrete regions of adult intact rhesus monkey brains and pituitary glands. B) A linear regression analysis of the optical densities of AR sense RNA standard curve as depicted in A.

A representative autoradiogram showing the distribution of AR mRNA in brain tissues and anterior pituitary gland of an intact adult male rhesus monkey is presented in Figure 2A (lanes 3–13).

We studied the effects of Cx and testosterone treatment on AR mRNA in various brain regions and anterior pituitaries of adult male monkeys. Before Cx, testosterone concentrations in serum from blood samples obtained on 4 consecutive days were determined by RIA. Significant elevations in testosterone concentrations were found in the evening hours compared to the morning hours, 5.3 ± 0.7 (SE) ng/ml serum vs. 8.0 ± 0.6 (SE) ng/ml serum, respectively, p < 0.05. Testosterone was not detected in 100 µl serum of Cx males. Serum from Cx males treated with silicone elastomer capsules filled with testosterone, however, contained morning concentrations of 10.3 ± 1.1 (SE) ng/ml serum and evening concentrations of 9.4 ± 1.5 (SE) ng/ml serum. These values did not differ significantly (p > 0.05). Testosterone concentrations in the latter serum samples, also, did not differ significantly from the concentrations found in serum obtained from intact males at 2100 h. These results are shown in Table 1.


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  		TABLE 1. Testosterone and LH concentrations in serum from male rhesus monkeys before and after castration and treatment with testosterone.

In the same serum, we quantified LH by a mouse Leydig cell bioassay. The results are shown in Table 1. In intact males, significantly higher concentrations of LH were observed in serum obtained in the evening hours as compared to samples collected in the morning, p < 0.05. LH was significantly elevated in serum from Cx males compared to intact or Cx males treated with testosterone (p < 0.05), but no morning/evening differences were found. LH concentrations in serum from Cx males treated with testosterone were significantly lower in comparison to those of both intact and Cx males (p < 0.05).

A representative phosphoimage of a ribonuclease protection assay is shown in Figure 3. The medial preoptic anterior hypothalamus (MPAH) and the parietal cortex (P.CTX) were chosen for this demonstration because these tissues represent the extremes of high and low quantities of AR mRNA. As one can determine from this figure, no apparent change in AR mRNA was found in lanes 3 and 4 (MPAH) or 9 and 10 (P.CTX) from Cx males as compared to lanes 5 and 6 (MPAH) or 11 and 12 (P.CTX) from Cx males treated immediately with testosterone, in quantities that did not differ significantly from quantities found in the evening hours in intact males. AR mRNA quantities from intact males are contained in lanes 7 and 8 (MPAH) and 13 and 14 (P.CTX).



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  		FIG. 3. A representative ribonuclease protection assay phosphoimage showing the distribution of AR mRNA in the MPAH (lanes 3–8), which contains high levels of AR mRNA, and in the P.CTX (lanes 9–14), which contains low amounts of AR mRNA. Lanes 3, 4, 9, and 10 are from Cx males; lanes 5, 6, 11, and 12 are from testosterone-treated males; and lanes 7, 8, 13, and 14 are from intact males. See legend for Figure 2 for other details. Exposure time was 24 h.

Since no significant treatment effects were found, the within-brain area data were collapsed to determine among-brain area differences. In Figure 4, the distribution of AR mRNA in all the brain areas and anterior pituitaries is shown. All the brain areas contained AR mRNA, but significant differences were found among these areas. The medial basal hypothalamus (MBH), which contained the greatest amounts of AR mRNA, differed from all other brain areas and the pituitary gland, p < 0.05. The statistical comparisons among the other brain areas are summarized in Figure 4.



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  		FIG. 4. AR mRNA distribution in various brain regions of adult (intact [n = 3], Cx [n = 4], and Cx treated immediately with testosterone [n = 6]) rhesus monkeys. Protected areas were quantified using a Bio-Rad molecular imager with an exposure time of 24 h. Data are presented as means (bars) ± SEM (vertical lines). Data were log transformed because of heterogeneity of variance and examined by ANOVA followed by the Neuman-Keuls multiple range test. No significant treatment effects were found. Therefore, the data from the various treatment groups were combined to determine significant effects among tissues. Thus the bracketed bars represent the pooled data from the three treatment groups (n = 13 for each tissue) and if marked with letters that differ represent significant differences between tissues, p < 0.05. BNST, bed nucleus stria terminalis; Sept, septum; Amyg, amygdala; Hippoc, hippocampus; C.Ctx, cingulate cortex; P.Ctx, parietal cortex; Cereb, cerebellum; Pit, anterior pituitary gland.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this experiment we cloned a part of the rhesus monkey AR gene (approximately 329-bp segment) by RT of the rhesus monkey RNA extracted from monkey prostate. We amplified the DNA by PCR using two primers from the published human and rat AR sequences. Sequence analysis of the monkey clone showed that it possessed a 99% homology with the human sequence and a 96% homology with that of the rat.

The sensitivity of the ribonuclease protection assay enabled us to reliably measure small amounts of AR mRNA in discrete regions of the rhesus monkey brain. Generally speaking, the distribution of AR mRNA corresponds to the distribution of AR in the nonhuman primate brain as determined by binding assays [21, 23]. More experiments must be completed on the nonhuman primate, however, before we will understand the relationship between factors that regulate AR message and the synthesis of the intracellular protein that binds specifically to androgen and probably represents the first step in androgen action. We did not find an effect of Cx or testosterone replacement on AR mRNA in any of the tissues studied. This observation on male monkey brains is similar to results previously reported from our laboratory on the male rat, with use of similar methodologies [27], but differs from data reported by others in male rats [42].

Using a different endpoint, however, namely in situ hybridization, AR mRNA is found in both the MBH and the bed nucleus stria terminalis of the male rat [43]. Hybridization densities of AR mRNA are elevated in both brain areas after short-term (4 days) Cx, but after long-term Cx (2 mo), the opposite was observed [43]. Treatment of Cx males with dihydrotestosterone reversed the effects of Cx in both long- and short-term Cx groups [43].

Likewise, an understanding of the relationship of AR protein to AR mRNA in brain tissues is hampered by experimental inconsistencies. Many of these inconsistencies may be attributable to the fact that AR has been localized in target tissues by different methods, e.g., by immunocytochemistry [10–14], binding of nuclei and cytosolic fractions [20, 22], and autoradiography [44], making it difficult to make comparisons with AR mRNA levels that have been determined in different laboratories, on different groups of animals, and even different species of animals.

AR gene expression appears to be regulated differently in different tissues. In granulosa cells, androgens and FSH synergize in regulating AR mRNA expression [45]. In cells from many target tissues such as prostate, hepatoma cells, and mammary tumor cells, androgens down-regulate AR mRNA [25, 26, 46, 47]. In other target tissues, androgens up-regulate AR mRNA [48–51].

Previous studies demonstrated that exogenous testosterone (which elevated serum concentrations to approximately 6 ng/ml), administered at the time of Cx, is unable to exert negative feedback effects on gonadotropin secretion in male rhesus monkeys [52]. If, however, Cx males are treated with larger doses of testosterone, at the time of Cx, for example serum levels of approximately 10 ng/ml in the present experiment and approximately 15 ng/ml in studies published by others [53], testosterone is able to exert negative feedback effects on LH secretion. Treatment with large doses of dihydrotestosterone also produced the same effect [54]. Refractoriness to the actions of testosterone appears to develop within a week after Cx in this species, which can be prevented either by treatment at the time of Cx with estradiol-17ß (E2, serum levels of ~100 pg/ml) or by a combination of a lower dose of E2 (serum levels of ~40 pg/ml) with a low lose of testosterone (serum levels of ~6 ng/ml), neither of which alone is effective [52]. In long-term Cx animals, however, the combined treatment with E2 and testosterone, mentioned above, as well as large doses of testosterone, was ineffective in suppressing gonadotropin secretion [52, 55].

The present study demonstrates that the effectiveness of testosterone as a regulator of LH secretion in male rhesus monkeys is not associated with changes of AR mRNA in the brain or anterior pituitary gland. Our data also suggest that the evening elevations of testosterone in serum from intact males are driven by elevations of LH that were also found in the same serum. It appears that the refractoriness that develops after Cx in this species is not due to the lack of AR mRNA in the hypothalamus, because the quantities of AR mRNA in the MBH and the MPAH 3 wk after Cx did not differ significantly among the three treatment groups. Additional studies will be needed to determine whether Cx for longer periods of time might affect AR mRNA.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. Sergio Ojeda for his advice and help in the beginning stages of this project and Henry Stadelman for his excellent technical assistance.


   FOOTNOTES
 
1 Supported by NIH grants HD-18196 and HD-18185. Back

2 Correspondence: FAX: 503 494 4352; reskoj{at}ohsu.edu Back

Accepted: December 22, 1998.

Received: March 12, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Goy RW, Resko JA. Gonadal hormones and behavior of normal and pseudohermaphroditic nonhuman female primates. Recent Prog Horm Res 1972; 28:707–733.
  2. Goy RW. Differentiation of male social traits in female rhesus macaques by prenatal treatment with androgens: variations in type of androgen, duration, and time of treatment. In: Novy MJ, Resko JA (eds.), Fetal Endocrinology. New York: Academic Press; 1981: 319–339.
  3. Resko JA, Ellinwood WE, Pasztor LM, Buhl AE. Sex steroids in the umbilical circulation of fetal rhesus monkeys from the time of gonadal differentiation. J Clin Endocrinol Metab 1980; 50:900–905.[Abstract]
  4. Phoenix CH, Goy RW, Resko JA. Psychosexual differentiation as a function of androgenic stimulation. In: Diamond M (ed.), Prospectives in Reproduction and Sexual Behavior. Bloomington and London: Indiana University Press; 1968: 33–49.
  5. Pomerantz SM, Goy RW, Roy MM. Expression of male typical behavior in adult female pseudohermaphroditic rhesus: comparisons with normal males and neonatally gonadectomized males and females. Horm Behav 1986; 20:483–500.[CrossRef][Medline]
  6. Phoenix CH, Chambers KC. Sexual behavior in adult gonadectomized female pseudohermaphrodite, females and male rhesus macaques (Macaca mulatta) treated with estradiol benzoate and testosterone propionate. J Comp Psychol 1982; 96:823–833.[CrossRef]
  7. Rose RM, Holaday JW, Bernstein IS. Plasma testosterone, dominance rank and aggressive behavior in male rhesus monkey. Nature 1971; 231:366–368.[CrossRef][Medline]
  8. Dinakar N, Arora R, Prasad MRN. Effects of microquantities of testosterone on the epididymis and assessory glands of the castrate rhesus monkey, Macaca mulatta. J Endocrinol 1974; 60:399–408.[Medline]
  9. Plant TM. Effects of orchidectomy and testosterone replacement treatment in pulsatile luteinizing hormone secretion in the adult rhesus monkey (Macaca mulatta). Endocrinology 1982; 110:1905–1913.[Abstract]
  10. Sar M, Lubahn DB, French FS, Wilson EM. Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology 1990; 127:3180–3186.[Abstract]
  11. Takeda H, Chodak G, Mutchnik S, Nakamoto T, Chang C. Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor. J Endocrinol 1990; 126:17–25.[Abstract]
  12. Clancy AN, Bonsall RW, Michael RP. Immunohistochemical labeling of androgen receptors in the brain of rat and monkey. Life Sci 1992; 50:409–417.[CrossRef][Medline]
  13. Choate JVA, Resko JA. Androgen receptor immunoreactivity in intact and castrate guinea pigs using antipeptide antibodies. Brain Res 1992; 597:51–59.[CrossRef][Medline]
  14. Wood RI, Newman SW. Intracellular partitioning of androgen receptor immunoreactivity in the brain of the male syrian hamster: effects of castration and steroid replacement. J Neurobiol 1993; 24:925–938.[CrossRef][Medline]
  15. Choate JVA, Slayden OD, Resko JA. Immunocytochemical localization of androgen receptors in brains of developing and adult male rhesus monkeys. Endocrine 1998; 8:1–10.[CrossRef][Medline]
  16. Prins GS, Birch L. Immunocytochemical analysis of androgen receptor along the ducts of the separate rat prostate lobes after androgen withdrawal and replacement. Endocrinology 1993; 132:169–178.[Abstract]
  17. Handa RJ, Roselli CE, Horton L, Resko JA. The quantitative distribution of cytosolic androgen receptors in microdissected areas of the male rat brain: effects of estrogen treatment. Endocrinology 1987; 121:233–240.[Abstract]
  18. Roselli CE, Handa RJ, Resko JA. Quantitative distribution of nuclear androgen receptors in microdissected areas of the rat brain. Neuroendocrinology 1989; 49:449–453.[Medline]
  19. Connolly PB, Resko JA. Progestins affect reproductive behavior and androgen receptor dynamics in male guinea pig brain. Brain Res 1989; 503:312–316.[CrossRef][Medline]
  20. Choate JVA, Resko JA. Effects of androgen on brain and pituitary androgen receptors and LH secretion of male guinea pigs. J Steroid Biochem Mol Biol 1996; 59:315–322.[CrossRef][Medline]
  21. Handa RJ, Roselli CE, Resko JA. Distribution of androgen receptor in microdissected brain areas of the female baboon (Papio cynocephalus). Brain Res 1988; 445:111–116.[CrossRef][Medline]
  22. Connolly PB, Choate JVA, Resko JA. Effects of exogenous androgen on brain androgen receptors of the fetal rhesus monkey. Neuroendocrinology 1994; 59:271–276.[Medline]
  23. Resko JA, Connolly PB, Roselli CE, Abdelgadir SE, Choate JVA. Selective activation of androgen receptors in subcortical brain of male cynomolgus macaques and its relationship to androgen-dependent aromatase activity. J Clin Endocrinol Metab 1993; 76:1588–1593.[Abstract]
  24. Tan JA, Joseph DR, Quarmby VE, Lubahn DB, Sar M, French FS, Wilson EM. The rat androgen receptor: primary structure, autoregulation of its messenger ribonucleic acid, and immunocytochemical localization of the receptor protein. Mol Endocrinol 1988; 2:1276–1285.[Abstract]
  25. Quarmby VE, Yarbrough WG, Lubahn DB, French FS, Wilson EM. Autologous down-regulation of androgen receptor messenger ribonucleic acid. Mol Endocrinol 1990; 4:22–28.[Abstract]
  26. Shan L-X, Rodriguez MC, Jänne OA. Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells. Mol Endocrinol 1990; 4:1636–1646.[Abstract]
  27. Abdelgadir SE, Connolly PB, Resko JA. Androgen regulation of androgen receptor messenger ribonucleic acid differs in rat prostate and selected brain areas. Mol Cell Neurosci 1993; 4:532–537.[CrossRef]
  28. Roselli CE, Stadelman H, Horton LE, Resko JA. Regulation of androgen metabolism and luteinizing hormone-releasing hormone content in discrete hypothalamic and limbic areas of male rhesus macaques. Endocrinology 1987; 120:97–106.[Abstract]
  29. Cameron JL, McNeill TH, Fraser HM, Bremner WJ, Clifton DK, Steiner RA. The role of endogenous gonadotropin-releasing hormone in the control of luteinizing hormone and testosterone secretion in the juvenile male monkey, Macaca fascicularis. Biol Reprod 1985; 33:147–156.[Abstract]
  30. Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 1979; 18:5294–5298.[CrossRef][Medline]
  31. Lubahn DB, Joseph DR, Sar M, Tan JA, Higgs HN, Larson RE, French FS, Wilson EM. The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. Mol Endocrinol 1988; 2:1265–1275.[Abstract]
  32. Tan J, Joseph DR, Quarmby VE, Lubahn DB, Sar M, French FS, Wilson EM. The rat androgen receptor: primary structure, autoregulation of its messenger ribonucleic acid, and immunocytochemical localization of the receptor protein. Mol Endocrinol 1988; 2:1276–1285.
  33. Agarwal VR, Bulun SE, Leitch M, Rohrich R, Simpson ER. Use of alternative promoters to express the aromatase cytochrome P450 (CYP19) gene in breast adipose tissues of cancer-free and breast cancer patients. J Clin Endocrinol Metab 1996; 81:3843–3849.[Abstract/Free Full Text]
  34. Gilman M. Ribonuclease protection assay. In: Ausubel FM, Brent R, Kingston RE (eds.), Current Protocols in Molecular Biology. New York: Green Publishing Associates and Wiley-Interscience; 1990: 4.7.1–4.7.6.
  35. Hill DF, Dissen GA, Ma YJ, Ojeda SR. Detection of nerve growth factor and one of its receptors. In: Conn PM (ed.), Gene Expression in Neural Tissue. Methods in Neuroscience. New York: Academic Press; 1992: 179–196.
  36. Abdelgadir SE, Roselli CE, Choate JVA, Resko JA. Distribution of aromatase cytochrome P450 messenger ribonucleic acid in adult rhesus monkey brains. Biol Reprod 1997; 57:772–777.[Abstract]
  37. Danielson PE, Forss-Petter S, Brow MA, Calavetta L, Douglass J, Milner RJ, Sutcliffe JG. p1B15: a cDNA clone of the rat mRNA encoding cyclophilin. DNA 1988; 7:261–267.[Medline]
  38. Kelly MJ, Garrett J, Bosch MA, Roselli CE, Douglass J, Adelman JP, Ronnekleiv OK. Effects of ovariectomy on GnRH mRNA, proGnRH and GnRH levels in the preoptic hypothalamus of the female rat. Neuroendocrinology 1989; 49:88–97.[Medline]
  39. Hess DL, Resko JA. The effects of progesterone on the patterns of testosterone and estradiol concentrations in the systemic plasma of the female rhesus monkey during the intermenstrual period. Endocrinology 1973; 92:446–453.[Medline]
  40. Ellinwood WE, Resko JA. Sex differences in biologically active and immunoreactive gonadotropins in the fetal circulation of rhesus monkeys. Endocrinology 1980; 107:902–907.[Abstract]
  41. Winer BJ. Statistical Principles of Experimental Design. New York: McGraw Hill; 1962: 46–104.
  42. Burgess LH, Handa RJ. Hormonal regulation of androgen receptor mRNA in the brain and anterior pituitary gland of the male rat. Mol Brain Res 1993; 19:31–38.[Medline]
  43. Handa RJ, Kerr JE, DonCarlos LL, McGivern RF, Hejna G. Hormonal regulation of androgen receptor messenger RNA in the medial preoptic area of the male rat. Mol Brain Res 1996; 39:57–67.[Medline]
  44. Bonsall RW, Rees HD. The distribution, nuclear uptake and metabolism of [3H]dihydrotestosterone in the brain, pituitary gland and genital tract of the male rhesus monkey. J Steroid Biochem 1985; 23/4:389–398.
  45. Tetsuka M, Hillier SG. Androgen receptor gene expression in rat granulosa cells: the role of follicle-stimulating hormone and steroid hormones. Endocrinology 1996; 137:4392–4397.[Abstract]
  46. Krongrad A, Wilson CM, Wilson JD, Allman DR, McPhaul MJ. Androgen increases androgen receptor protein while decreasing receptor mRNA in LNCaP cells. Mol Cell Endocrinol 1991; 76:79–88.[CrossRef][Medline]
  47. Wolf DA, Herzinger T, Hermeking H, Blaschke D, Hörz W. Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen. Mol Endocrinol 1993; 7:924–936.[Abstract]
  48. Dai JL, Maiorino CA, Gkonos PJ, Burnstein KL. Androgenic up-regulation of androgen receptor cDNA expression in androgen-independent prostate cancer cells. Steroids 1996; 61:531–539.[CrossRef][Medline]
  49. Gonzalez-Cadavid NF, Vernet V, Navarro AF, Rodriguez JA, Swerdloff RS, Rajfer J. Up-regulation of the levels of androgen receptor and its mRNA by androgens in smooth-muscle cells from rat penis. Mol Cell Endocrinol 1993; 90:219–229.[CrossRef][Medline]
  50. Kerr JE, Allore RJ, Beck SG, Handa RJ. Distribution and hormonal regulation of androgen receptor (AR) and AR messenger ribonucleic acid in the rat hippocampus. Endocrinology 1995; 136:3213–3221.[Abstract]
  51. Nastiuk KL, Clayton DF. Seasonal and tissue-specific regulation of canary androgen receptor messenger ribonucleic acid. Endocrinology 1994; 134:640–649.[Abstract]
  52. Resko JA, Quadri SK, Spies HG. Negative feedback control of gonadotropins in male rhesus monkeys: effects of time after castration and interactions of testosterone and estradiol-17ß. Endocrinology 1977; 101:215–224.[Medline]
  53. Dubey AK, Herbert J, Abbott DH, Martensz ND. Serum and CSF concentrations of testosterone and LH related to negative feedback in male rhesus monkeys. Neuroendocrinology 1984; 39:176–185.[Medline]
  54. Abbott DH, Batty KA, Dubey AK, Herbert J, Shiers HM. The passage of 5{alpha}-dihydrotestosterone from serum into cerebrospinal fluid and LH negative feedback in castrated rhesus monkeys. J Endocrinol 1985; 104:325–330.[Abstract]
  55. Plant TM, Hess DL, Hotchkiss J, Knobil E. Testosterone and the control of gonadotropin secretion in the male rhesus monkey (Macaca mulatta). Endocrinology 1978; 103:535–541.[Medline]



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