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Androgenic Regulation of Hypothalamic Aromatase Activity in Prepubertal and Postpubertal Male Golden Hamsters¹

Department of Psychology, Neuroscience Program, Michigan State University, East Lansing, Michigan 48824

Address all correspondence and requests for reprints to: Cheryl L. Sisk, Department Psychology, Neuroscience Program, Michigan State University, East Lansing, Michigan 48824. E-mail: sisk{at}pilot.msu.edu

	Abstract

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Doses of testosterone that fully activate male reproductive behavior in castrated adult male hamsters fail to elicit mounting and intromissions in prepubertal castrates, even when circulating levels of testosterone are equivalent in the two age groups. We hypothesize that this differential responsiveness to testosterone is mediated at least in part by the efficacy with which testosterone in the hypothalamus is aromatized to estradiol, an important hormone mediating male sexual behavior. Therefore, hypothalamic aromatase activity, as measured by the conversion of [³H]testosterone to [³H]estradiol in tissue homogenates, was assessed in four separate experiments: 1) intact prepubertal and adult male golden hamsters, 2 and 3) castrated adult or prepubertal males that received either a 0- or 2.5-mg dose of testosterone, and 4) castrated adult and prepubertal males treated with the 2.5-mg dose of testosterone. These studies demonstrate that hypothalamic aromatase activity is significantly higher in adult males compared with prepubertal males, and that hypothalamic aromatase activity is increased by testosterone to the same extent in both the adult and prepubertal male hamster. Therefore, the failure of testosterone-treated castrated prepubertal male hamsters to engage in the full suite of male reproductive behaviors is not due to the inability of testosterone to be converted into estradiol in the hypothalamus. Differences in the ability of testosterone to increase aromatase activity in other brain regions, or differences in the action of testosterone and/or estradiol on other cellular processes must account for the inability of testosterone to facilitate male reproductive behavior in juvenile males.

	Introduction

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TESTOSTERONE (T) is converted intracellularly to estradiol (E₂) in peripheral and central tissues by the aromatase enzyme. In the brain, estrogenic metabolites of T play a major role in the expression of male sexual behavior (1, 2). For example, systemic injections of E₂ benzoate to castrated male hamsters induce mounting behavior (3), whereas aromatase inhibitors decrease copulatory behaviors (4). The aromatase enzyme is present in brain regions that mediate male sexual behavior, such as the amygdala and hypothalamus (5, 6, 7), and appears to be regulated by androgens in some regions of the hypothalamus (5, 7–10; but see Ref. 6).

The capacity to engage in steroid-dependent reproductive behavior increases during pubertal maturation. Not only is there an increase in circulating levels of T during this time, but responsiveness of neural circuits to the behavioral actions of T increase as well. For example, in male hamsters, doses of exogenous T that fully activate male reproductive behavior in castrated adult male hamsters fail to elicit mounting and intromissions in castrated juveniles, even when circulating levels of T are equivalent in the two age groups (11). We hypothesize that this increased behavioral responsiveness to T in adults is mediated, at least in part, by the efficacy with which T is aromatized to E₂ in the hypothalamus. This hypothesis leads to two related predictions. First, in intact males, aromatase activity within the behavioral neural circuit should be greater in adults than in juveniles. Second, T treatment of castrated males should increase aromatase activity to a greater extent in adults than in juveniles, which would result in higher local concentrations of E₂ to activate male reproductive behavior in adults. As a test of this hypothesis, aromatase activity, as measured by the conversion of [³H]T to [³H]E₂, was assessed in hypothalamic homogenates obtained from intact and from castrated and T-treated adult and prepubertal male golden hamsters.

	Materials and Methods

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Animals and housing
Male golden hamsters (Mesocricetus auratus) were bred at Michigan State University (E. Lansing, MI) from stock obtained from Charles River Laboratories, Inc. (Kingston, NY). Animals were weaned at 21 days of age and at that time housed singly in clear polycarbonate cages (37.5 x 33 x 17 cm) with wood chips as substrate (Aspen Chip Laboratory, Warrensburg, NY) and with ad libitum access to both food (Teklad Rodent Diet No. 8640; Harlan Bioproducts for Science, Inc., Madison, WI) and water. The animal colony was maintained at 21 ± 2 C and the light-dark schedule was 14 h light/10 h dark (lights on at 0600 h EST). All animals were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and protocols were approved by the Michigan State University All-University Committee for Animal Use and Care.

Experimental design
Four experiments were conducted because the number of samples that can be run in a single assay is limited. Exp 1 characterized the amount of hypothalamic aromatase activity in untreated, intact prepubertal and adult male hamsters. In this experiment, 63-day-old (adult, n = 8) or 28-day-old (prepubertal, n = 8) male hamsters were weighed and rapidly decapitated. Hypothalami, blood samples, and testes were collected as described below. Exps 2–4 investigated the effects of T on hypothalamic aromatase activity before and after puberty in male hamsters. Exp 2 assessed the effects of T on aromatase activity in adult males. Adult males (60 days of age) were castrated under methoxyflurane anesthesia and implanted with a 3-week time-released pellet (Innovative Research of America, Sarasota, FL) containing either 0 mg (n = 7) or 2.5 mg of T (n = 7). One week after castration and implantation, hamsters were weighed, rapidly decapitated, and hypothalami and blood samples were collected as described below. Previous work has shown that aromatase activity is maximally increased within a week of T treatment (12). Furthermore, the difference in T-stimulated sexual behavior observed in prepubertal and adult male hamsters is observed 1 week after T treatment (11). Exp 3 assessed the effects of T on aromatase activity in juvenile males. In Exp 3, prepubertal males (21 days of age) were castrated and implanted with either a 0 mg (n = 7) or 2.5 mg (n = 6) pellet of T. One week after treatment, tissues were collected as in Exp 2. Exp 4 directly compared the effect of T on aromatase activity in juvenile and adult males. Prepubertal (21 days of age, n = 6) and adult (60 days of age, n = 8) males were castrated and implanted with a 2.5-mg pellet of T. One week after treatment, tissues were collected as in Exp 2.

Tissue collection
Animals were rapidly decapitated by a guillotine. Trunk blood samples were collected and centrifuged. Plasma was removed and stored at -20 C until RIAs were performed (see below). Brains were quickly removed, and the hypothalamus was dissected on a stainless steel surface on wet ice with a razor blade. Coronal cuts were made directly anterior to the optic chiasm and at the posterior end of the hypothalamus, just anterior to the mammillary bodies. Then a horizontal cut was made just ventral to the anterior commissure as it crossed the midline. Finally, the brain was placed on the dorsal surface and the optic chiasm, and tissue lateral to the hypothalamus was removed. The dissected hypothalamus was then snap frozen in dry ice and stored at -70 C until the aromatase assays were performed (see below).

Assay for steroid metabolizing enzymes
Individual hypothalami were homogenized in 600 µl of 250 mM sucrose/50 mM potassium phosphate buffer. Assays were conducted with minor modifications from those used in lizard brain tissue (13). Initially, validation assays that varied the incubation time and substrate concentration were run on adult male hamster hypothalamic homogenates to determine the appropriate assay conditions (details presented in Results). Once the assay was validated, experiments were conducted using duplicate 200-µl aliquots of hypothalamic homogenates incubated for 25 min with 250 nM substrate. The tissue homogenates were added to test tubes in which [³H]T (New England Biolabs, Inc., Boston, MA) had been dried. In all cases, substrate was repurified by TLC before use. Samples were incubated at 37 C with a NADH/NADPH-generating system, and the reaction was terminated by freezing the tubes in a methanol/dry ice bath.

Steroids were extracted from homogenates three times with diethyl ether. Androgens were then separated from estrogens twice by phenolic partition, and estrogens extracted three times with ethyl acetate. Androgenic and estrogenic products were applied to TLC plates following the addition of radioinert carrier steroids (Steraloids, Wilton, NH). TLC plates containing estrogens were run twice in ether/hexane (3:1), and the products visualized by exposure to iodine vapors. Plates containing androgens were run twice in chloroform/ethyl acetate (4:1), and the products were visualized under UV irradiation following a primulin spray. Regions containing the steroids of interest were scraped from the plates, and after the addition of 400 µl H₂O, steroids were eluted from the silica-gel in 2 ml methanol. A fraction of the eluate was mixed with Bio-safe cocktail II (Research Products International, Mt. Prospect, IL) and counted in a liquid scintillation counter (Beckman Instruments, Inc., LS6500, Fullerton, CA). Each sample was corrected for counter efficiency, volume, and background counts in tubes incubated with buffer and cofactors but no tissue. Results were also corrected for recovery efficiency, which was determined by the addition of a known quantity of [³H]E₂ or [³H]T (150,000 dpm) to tubes processed in parallel.

Protein content in each assay tube was determined with the method of Bradford (14) (Bio-Rad Laboratories, Inc., Hercules, CA) using BSA as the protein standard. To confirm their authenticity, samples of all steroid products were recrystallized with radioinert steroids (Steraloids) to constant specific activity using ethanol and water (details presented in Results).

T RIA
Plasma concentrations of T were measured in two different assays using the Coat-A-Count Total Testosterone Kit (Diagnostic Products, Los Angles, CA). This assay has been validated in our laboratory for the measurement of plasma T concentration in the Syrian hamster. The lower limit of detectability of both assays was 0.1 ng/ml. The intraassay coefficients of variation were 5.8% and 4.1%, and the interassay coefficient of variation was 10%.

Testis histology
Following storage in Bouin’s fixative, one testis from each animal in Exp 1 was cut in half, dehydrated in increasing concentrations of ethanol, cleared in xylene, and embedded in paraffin. The tissue was sectioned at 10 µm, stained with hematoxylin and eosin, and examined for the presence of mature sperm.

Data analysis
The data for each experiment were analyzed using two-tailed t tests. Differences were considered significant when P < 0.05. All data are reported as mean ± SEM. Values for the Michaelis-Menten constant (K_m) and maximum velocity (V_max) were generated from a Lineweaver-Burk plot using a regression line in Statview 4.1 (Abacus Concepts, Inc., Berkeley, CA).

	Results

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Validation of aromatase assay
Samples of E₂ were twice verified by recrystallization to constant specific activity in ethanol and water (93.6/85.8, 77.0/77.7; crystals_(dpm/mg)/mother liquor_(dpm/mg)). In both cases, recovery of the specific activity was 100%. This measure reflects the ratio of the specific activity (disintegrations per minute per milligram) in the crystals following the final recrystallization compared with that before the first recrystallization (disintegrations per minute of assay product used per milligram cold steroid used). Although androgens were detectable, the incubation time and substrate concentration for these assays were not optimized to measure the rate of androgen production. Estrone was not detected in any sample. Therefore, quantification of aromatase activity consisted solely of the rate of E₂ production.

Time course and saturation curve
An initial time-course study was performed using a 222 nM substrate concentration (specific activity = 45.1 Ci/mmol). Pooled hypothalamic tissue was incubated for 10, 30, 60, or 180 min. E₂ production increased linearly at incubation times up to 60 min, at which point it slowed. Based on these results, an assay used to generate a saturation curve was incubated for 35 min using substrate concentrations ranging from 4–2225 nM (specific activity = 45.1 Ci/mmol). The reaction rate increased to the 224 nM concentration at which point E₂ production began to level off. For this saturation curve, the K_m was 27.4 nM and V_max was 7.58 fmol•mg^-1•protein^-1•min^-1. A second time course experiment was performed using a 250 nM (specific activity = 92.4 Ci/mmol) substrate concentration at 15, 30, 60, and 120 min incubation times. E₂ production increased linearly through 30 min, then at a slightly lower rate between the 30- and 120-min time points (Fig. 1). Based on the results of these assays, an incubation time of 25 min and a substrate concentration of 250 nM (specific activity = 92.4 Ci/mmol) were used for the four experiments.

View larger version (17K): [in this window] [in a new window]   Figure 1. [3H]E2 production after incubation of hypothalamic homogenates with 250 nM [3H]T for 15, 30, 60, or 120 min. E2 production increased at a lower rate between the 30 and 120 min time points.

Exp 1: Hypothalamic aromatase activity in prepubertal and adult males
Paired testis weight and plasma T were significantly greater in adults compared with prepubertal males (t = 15.86 and t = 4.68, respectively, both P < 0.05, Table 1). Mature sperm were observed in the testes of adult but not prepubertal males (Fig. 2, A and B). Furthermore, adults had significantly higher hypothalamic aromatase activity compared with that of the prepubertal animals (t = 4.04, P < 0.05, Fig. 3). Specifically, adults had a 2-fold increase in aromatase activity compared with that of their prepubertal counterparts.

View larger version (166K): [in this window] [in a new window]   Figure 2. Testicular histology from an adult (A) and prepubertal (B) hamster in Exp 1. Note the presence of mature sperm in the adult testis only (A). Bar, 100 µm.

View larger version (44K): [in this window] [in a new window]   Figure 3. [3H]E2 production (femtomoles per milligrams protein per minute) in hypothalamic homogenates from intact prepubertal and adult male hamsters. Asterisk indicates significant difference. Values are means ± SEM.

View larger version (24K): [in this window] [in a new window]   Figure 4. [3H]E2 production (femtomoles per milligrams protein per minute) in hypothalamic homogenates from castrated adult male hamster treated with either 0 or 2.5 mg of T (A), castrated prepubertal male hamsters treated with either 0 or 2.5 mg of T (B), and castrated adult and prepubertal male hamsters treated with 2.5 mg of T (C). Asterisks indicate significant differences. All values are means ± SEM.

Exp 4: Comparison of androgenic regulation of hypothalamic aromatase activity in prepubertal and adult males
Adults had significantly heavier paired testis weight on the day of castration compared with the castrated prepubertal animals (t = 29.62, Table 1). Plasma T levels and hypothalamic aromatase activity were equivalent between the castrated adults and the castrated prepubertal animals treated with the 2.5-mg dose of T (t = -1.03 and t = -1.27, respectively, Table 1 and Fig. 4C, respectively).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This series of studies demonstrates that hypothalamic aromatase activity is significantly higher in adult male hamsters compared with prepubertal males, and that hypothalamic aromatase activity is positively regulated by T in both the adult and prepubertal male hamster. Furthermore, the ability of T to increase hypothalamic aromatase activity appears to be equivalent before and after puberty. The androgenic regulation of hypothalamic aromatase activity most likely accounts for the developmental difference in enzyme activity observed in intact males in Exp 1, because the prepubertal males in this experiment had significantly lower levels of circulating T compared with the adults.

The present data do not support the hypothesis that increased behavioral responsiveness to T in adults is mediated by the efficacy with which T is aromatized to E₂ in the hypothalamus. However, we cannot rule out the possibility that aromatase activity is differentially regulated in the hypothalamus of prepubertal and adult males in more discrete hypothalamic nuclei by adult-physiological levels of androgens. Indeed, Hutchison et al. (7) reported that regulation of hypothalamic aromatase activity by T in the adult male hamster is brain-region specific. The measurement of aromatase activity in distinct hypothalamic nuclei of prepubertal and adult male hamsters will help resolve this issue.

The present series of experiments has unequivocally demonstrated that aromatase activity in whole hypothalamic homogenates is positively regulated by T in both adult and prepubertal male hamsters, and this regulation is equivalent at these two developmental stages. Thus, the question remains as to why the same dose of T is unable to activate reproductive behavior in juvenile males. Our experiments suggest that the insensitivity of the juvenile male nervous system to steroid hormone lies downstream of T’s action on androgen receptors (11) and on aromatase activity (present data). One possibility is that the aromatized E₂ may have differential effects on the hypothalami of prepubertal vs. adult male hamsters. For instance, there is precedence in the literature suggesting that estrogen receptor expression can be differentially regulated by estrogens depending on the current physiological condition of the animal (18, 19, 20). Therefore, increased local concentration of estrogen in the hypothalamus may lead to an increase in steroid receptors in adults but not in prepubertal males, leading to behavioral activation in the adult males only.

Another possibility is that the increased estrogen levels resulting from T-induced increases in aromatase activity could alter the connectivity and/or morphology of the hypothalamic neurons differently in the prepubertal and adult males. Estrogenic metabolites of T are important in the prenatal and early postnatal differentiation and development of the mammalian central nervous system (21, 22, 23, 24, 25). Furthermore, the hormonal milieu has been implicated in the morphological plasticity exhibited by both neuronal and nonneuronal (e.g. glial cells) elements of the central nervous system in adult animals (26, 27, 28, 29, 30, 31). However, the nature of the plasticity of the juvenile brain in response to the hormonal milieu has not been established. Thus, age-related changes in E₂-induced alterations in the hypothalamic cytoarchitecture may underlie the pubertal increase in responsiveness to T and the maturation of male sexual behavior.

In rats, Lephart and Ojeda (32) observed a pubertal decrease in aromatase activity and hypothesized that the presumptive decrease in E₂ availability was responsible for the pubertal decline in steroid negative feedback regulation of gonadotropin secretion (32). However, Roselli and Klosterman (33) found a pubertal increase in aromatase activity in male and female rats. Variations in experimental methodologies and animal species used could account for the different results in the two experiments mentioned above and the present data. For example, tissue dissection was different in all of these experiments. In the present experiments, a hypothalamic tissue fragment containing both the preoptic area and the hypothalamus was used, whereas Lephart and Ojeda (32) and Roselli and Klosterman (33) assayed more discrete areas of the hypothalamus. It is also possible that the pubertal decrease in responsiveness to steroid negative feedback does involve a change in aromatase activity, whereas the pubertal increase in responsiveness to behavioral activation by steroids does not.

In conclusion, the present series of studies has established that adult male hamsters have approximately a 2-fold increase in hypothalamic aromatase activity compared with prepubertal males, and that, in both prepubertal and adult male hamsters, hypothalamic aromatase activity is under androgen regulation. Therefore, the failure of T-treated castrated prepubertal male hamsters to engage in the full suite of male reproductive behaviors is not due to the inability of T to be converted into E₂ in the hypothalamus. Differences in the ability of T to increase aromatase activity in other brain regions, or differences in the action of T and/or E₂ on other cellular processes must account for the inability of T to facilitate male reproductive behavior in juvenile males.

	Acknowledgments

 
We thank Heather Richardson, Chung Lee, and Stefani Diedrich for their technical assistance.

	Footnotes

 
¹ This work was supported by National Science Foundation (NSF) Grant IBN-9602169 (to C.L.S.) and NSF Grant IBN-9733074 (to J.W.).

² Supported by NIH Training Grant NS-07279.

Received June 5, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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