Effects of Age and Estradiol on Gene Expression in the Rhesus Macaque Hypothalamus (original) (raw)
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Research Articles| February 26 2015
aDivision of Neuroscience, Oregon National Primate Research Center, Beaverton, Oreg., and Departments of
bBehavioral Neuroscience and
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aDivision of Neuroscience, Oregon National Primate Research Center, Beaverton, Oreg., and Departments of
bBehavioral Neuroscience and
cPhysiology and Pharmacology, Oregon Health and Science University, Portland, Oreg., USA
Search for other works by this author on:
Neuroendocrinology (2015) 101 (3): 236–245.
Received:
September 22 2014
Accepted:
February 18 2015
Published Online:
February 26 2015
Abstract
Background: The hypothalamus plays a key role in mediating the effects of estrogen on many physiological functions, including reproduction, metabolism, and thermoregulation. We have previously observed marked estrogen-dependent gene expression changes within the hypothalamus of rhesus macaques during aging, especially in the KNDy neurons of the arcuate-median eminence (ARC-ME) that produce kisspeptin, neurokinin B, and dynorphin A. Little is known, however, about the mechanisms involved in mediating the feedback from estrogen onto these neurons. Methods: We used quantitative real-time PCR to profile age- and estrogen-dependent gene expression changes in the rhesus macaque hypothalamus. Our focus was on genes that encode steroid receptors (ESR1, ESR2, PGR, and AR) and on enzymes that contribute to the local synthesis of 17β-estradiol (E2; STS, HSD3B1/2, HSD17B5, and CYP19A). In addition, we used RT2 Profiler™ PCR Arrays to profile a larger set of genes that are integral to hypothalamic function. Results: KISS1, KISS1R, TAC3, and NPY2R mRNA levels increased in surgically menopausal (ovariectomized) old females relative to age-matched ovariectomized animals that received E2 hormone therapy. In contrast, PGR, HSD17B, GNRH2, SLC6A3, KISS1, TAC3, and NPY2R mRNA levels increased after E2 supplementation. Conclusion: The rhesus macaque ARC-ME expresses many genes that are responsive to changes in circulating estrogen levels, even during old age, and these may contribute to causing the normal and pathophysiological changes that occur during menopause.
Introduction
Menopause in women and female rhesus macaques is associated with a marked decline in the production and secretion of 17β-estradiol (E2) from the ovaries [1,2,3]. This attenuation of circulating estrogen levels adversely impacts many physiological processes and is thought to play a major role in the etiology of age-related pathologies such as reproductive quiescence, hot flashes, and disrupted sleep-wake cycles [4,5]. Little is known, however, about the mechanisms involved in mediating the feedback from E2 onto neurons in the hypothalamus.
Evidence from human and nonhuman primate studies suggests that loss of circulating estrogen can affect the cytoarchitecture of the arcuate region of the hypothalamus and alter the pattern of gene expression [6,7,8,9,10,11]. For example, using in situ hybridization, Rance et al. [6] demonstrated a significant increase in the size of estrogen receptor (ER)-expressing neurons of postmenopausal compared to premenopausal women. ERα-expressing neurons in the arcuate-median eminence (ARC-ME) colocalize with kisspeptin (KISS1), neurokinin B (NKB), and dynorphin A (DYN) (referred to as KNDy) and exert a major influence on the neuroendocrine reproductive axis by modulating the secretion of gonadotropin-releasing hormone (GnRH). Moreover, KNDy neurons show marked changes in their pattern of gene expression after menopause or ovariectomy, and these changes can be blocked by exposure to exogenous sex steroids [12,13,14,15,16,17]. Consequently, it is plausible that the influence of sex steroids on GnRH neuronal function is mediated by ERα, ERβ, and the progestin receptor, associated with the KNDy neural systems [18,19,20,21,22,23,24,25]. Furthermore, the expression of these receptors may change, as animals show the characteristic menopausal decrease in circulating sex steroid levels, and may represent a mechanism by which the hypothalamus undergoes a compensatory increase in its sensitivity to sex steroids. Another possible menopause-associated compensatory mechanism could involve an increased expression of enzymes involved in the local intracrine synthesis of sex steroids from precursor steroids such as dehydroepiandrosterone (DHEA) [26,27,28]. The circulating levels of DHEA and DHEA sulfate are especially pronounced in adult humans and nonhuman primates, and there is evidence that all of the key enzymes involved in DHEA-to-E2 conversion are expressed in the primate hypothalamus [29]. This suggests that local synthesis of sex steroids may also be contributing to the hormone milieu of the hypothalamus.
The aim of the present study was to help resolve these issues by examining the effect of age and E2 treatment on gene expression in the ARC-ME of female rhesus macaques. These nonhuman primates show age-related hormonal changes similar to those in women, though late in their lifespan [2,30,31], and thus they represent a pragmatic translational animal model in which to study the neuroendocrine mechanisms that contribute to healthy human aging. Our primary focus was on genes that encode the main sex steroid receptors [32,33,34,35,36,37]: ERα (encoded by ESR1), ERβ (encoded by ESR2), the progestin receptor (encoded by PGR), and the androgen receptor (encoded by AR). In addition, we examined the expression of genes that play a key role in the intracrine conversion of DHEA to testosterone and E2: steroid sulfatase (encoded by STS), 17β-hydroxysteroid dehydrogenase type 5 (encoded by HSD17B5), 3β-hydroxysteroid dehydrogenase types 1 and 2 (encoded by HSD3B1/2), and aromatase (encoded by CYP19A1). Our second goal was to profile the expression of a larger set of genes that contribute to the control of hypothalamus-mediated functions such as reproduction and metabolism. Our prediction was that the expression of hypothalamic genes would increase during aging, ensuring that the hypothalamus remains responsive to sex steroids even when the levels of these hormones in the circulation are attenuated.
Materials and Methods
Animals
Adult female rhesus macaques (Macaca mulatta) were cared for by the Division of Comparative Medicine at the Oregon National Primate Research Center (ONPRC) in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals, and used in a study approved by the Institutional Animal Care and Use Committee. The animals were housed indoors under controlled environmental conditions: 24°C temperature; 12-hour light/12-hour dark photoperiods (lights on at 07.00 h); regular meals at 08.00 and 15.00 h (Purina High Protein Monkey Chow; Purina Mills, Inc., St. Louis, Mo., USA) supplemented with fresh fruit and vegetables, and fresh drinking water available ad libitum.
Design of Experiments
Effect of Aging
Experiment 1 used quantitative real-time (qRT-)PCR to compare mRNA levels between young (∼13 years) and old (∼26 years) ovary-intact animals.
Effect of E2
Experiment 2 used qRT-PCR to compare mRNA levels between young (∼10 years) bilaterally ovariectomized (OVX) animals and young (∼10 years) OVX animals that had been treated with E2 for ∼1 month. This experiment also compared mRNA levels between old OVX controls and old OVX animals that had been treated with E2 for ∼4 years. The two older groups of animals were ∼21 and ∼22 years of age at the onset of the study, respectively.
Effect of Menopause and Hormone Therapy
Experiment 3 used custom-made RT2 Profiler™ PCR Arrays (SABiosciences) to compare gene expression in the ARC-ME of young animals (∼12 years) with that of old, surgically menopausal animals (old OVX) from experiment 2, and also with the long-term estradiol-treated animals (old OVX + E2).
Menstrual Cycle Status and Serum Hormone Concentrations
Experiment 1
The serum levels of E2 were 36.5 ± 6.3 pg/ml in the young gonad-intact animals and 62 ± 16.5 pg/ml in the old gonad-intact animals. All but 1 of these animals had progesterone (P4) levels below the assay detection limit. One young animal showed elevated P4 concentrations (2.4 ng/ml) and was assumed to still be in the luteal phase of the menstrual cycle. Based on individual animal menstruation records, all 4 young intact animals were normal cyclers, and the old intact animals consisted of 2 cycling animals, 1 irregular cycler, and 1 postmenopausal animal.
Experiment 2
One month following ovariectomy, half of the animals in the young and old OVX groups were implanted subcutaneously with empty Silastic capsules (Dow Corning, Midland, Mich., USA), while the other half received capsules containing crystalline E2 (Steraloids, Wilton, N.H., USA), as previously described [38]; these were designed to last up to 1 year and to maintain circulating E2 levels between 100 and 200 pg/ml, which is similar to what is observed during the late follicular phase of the menstrual cycle. In the old OVX + E2 group, the E2 capsules were replaced annually to ensure sustained long-term delivery of the steroid for the entire duration of the study (∼4 years); the untreated OVX animals maintained the same capsules throughout. Terminal serum E2 levels were undetectable in the young untreated OVX animals and had a mean level of 117 ± 7.0 pg/ml in the young OVX + E2 group. Serum E2 concentrations were measured at various time points across the 4-year experiment in the old OVX animals to confirm that the target hormone concentrations were being maintained. The serum E2 levels in the old untreated OVX group were <30 pg/ml at all time points and often fell below the limit of assay sensitivity. In the old OVX + E2 animals, the mean E2 concentration during the final 2 years of the study was 118.8 ± 15.9 pg/ml, and at the time of tissue collection it was 94.3 ± 20.5 pg/ml.
Experiment 3
At the time of postmortem tissue collection, the young intact animals had a mean serum E2 level of 58.9 ± 24.5 pg/ml and very low serum P4 levels (<0.3 ng/ml). These hormone levels are indicative of the follicular phase of the menstrual cycle. In 1 of these animals, however, the E2 level was considerably higher (128 pg/ml), suggesting that the animal was most likely in the late follicular phase. Note that the surgically menopausal animals used in experiment 3 were the same old OVX and old OVX + E2 animals from experiment 2.
The E2 and P4 assays were performed by the ONPRC Endocrine Technology and Support Core using a chemiluminescence-based automatic clinical platform (Immulite 2000; Siemens Healthcare Diagnostics, Deerfield, Ill., USA). The sensitivity limits of these assays were 20 pg/ml and 0.2 ng/ml for E2 and P4, respectively [39], and the intra-assay and inter-assay coefficients of variation were all <15%.
RNA Isolation
All the animals had previously been involved in various cross-sectional aging studies; their hypothalami and terminal blood serum became available for postmortem analysis through the ONPRC Tissue Distribution Program. At the time of necropsy, the mean (± SEM) age of the gonad-intact young animals from experiment 1 was 12.8 ± 2.3 years (n = 4), and the age of the old animals was 25.8 ± 0.4 years (n = 4). The animals from experiment 2 were 9.7 ± 0.3 years old (young OVX; n = 4), 9.6 ± 0.8 years old (young OVX ± E2; n = 4), 25.0 ± 1.7 years old (old OVX; n = 4), and 27.0 ± 0.9 years old (old OVX + E2; n = 4). The age of the young animals from experiment 3 was 12.3 ± 0.9 years (n = 4).
After sedation with ketamine (15-25 mg/kg, i.m.) and pentobarbital sodium (25-30 mg/kg, i.v.), a procedure consistent with the recommendations of the American Veterinary Medical Association's Panel on Euthanasia, each brain was flushed with 1 liter of 0.9% saline via a vascular catheter, and the hypothalamus was removed and preserved for ∼2 weeks in RNAlater (Ambion, Austin, Tex., USA). A coronal slice encompassing the ARC-ME was dissected and stored frozen at -80°C. The boundaries for this tissue block included the exterior ventral edge of the ME, lateral cuts midway between the third ventricle and the optic nerve, an anterior cut along the posterior edge of the optic chiasma, a posterior cut just anterior to the mammillary bodies, and a cut 1 mm dorsal to the base of the third ventricle (i.e., based on stereotaxic coordinates, this represents the border between the arcuate and the ventromedial hypothalamus). Subsequently, each ARC-ME block was individually homogenized using a PowerGen rotor-stator homogenizer (Fisher Scientific, Pittsburgh, Pa., USA), and RNA was extracted using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, Calif., USA). An Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif., USA) was used to determine the quality of the RNA, and a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, Mass., USA) was used to determine the concentration. For each sample, 1 μg of RNA was then converted to cDNA using random hexamers and the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen Life Technologies, Carlsbad, Calif., USA).
Gene Expression Profiling
The ARC-ME expression levels of genes encoding the sex steroid receptors ESR1, ESR2, PGR, and AR were determined using qRT-PCR. Similarly, the ARC-ME expression levels of genes encoding the following steroidogenic enzymes were examined: STS, HSD3B1/2, HSD17B5, and CYP19A1. Finally, RT2 Profiler™ PCR Arrays were used to profile a more extensive set of genes in the ARC-ME during aging and after treatment with E2.
Primers and Probes
Table 1 depicts nucleotide sequences of the primers and probes that were used for detection of the steroid receptor genes (ESR1, ESR2, PGR, and AR), steroidogenic enzyme genes (STS, HSD3B1/2, HSD17B5, and CYP19A1), and also two housekeeping genes (ALG9 and RPL13A) that we have previously shown to be stably expressed in the rhesus ARC-ME across various sex steroid environments [17,40]. The nucleotide sequences for these genes were designed using Primer Express 2.0 software [Applied Biosystems (ABI), Foster City [,]Calif., USA] and were based on NCBI rhesus macaque reference sequences. The primers and probes were purchased from Invitrogen Life Technologies and ABI, respectively. Reverse transcriptase-PCR (RT-PCR), using ARC-ME and ovarian RNA samples (as positive controls), was used to validate each of the primer sets by producing amplicons of a predicted size.
Table 1
TaqMan qRT-PCR primer and probe nucleotide sequences (5′-3′)
TaqMan qRT-PCR
A 7900HT Fast Real-Time PCR thermal cycler and sequence detection system software (version 2.2.1; ABI) was used to obtain qRT-PCR data. Initially, pooled cDNA was used to create standard curves for each gene, and the experimental samples were subsequently diluted accordingly so as to fall within the linear part of the curve. The PCR mixtures contained 5 μl TaqMan Universal PCR Master Mix, 0.3 μl of each specific forward and reverse primer (300 nM final concentration), 0.25 μl of specific probe (250 nM final concentration), and 2 μl of cDNA. The reaction sequence included 2 min at 50°C, 10 min at 95°C, and 50 cycles of 15 s at 95°C and 1 min at 60°C. Automatic baseline and threshold levels were determined by ABI sequence detection system software (version 2.2.1.), and the final expression values were normalized to the arithmetic mean of two reference housekeeping genes, ALG9 and RPL13A. Individual genes from experiments 1 and 2 were examined together on the same 384-well optical plate. A negative control included the omission of cDNA templates from the reaction mixture.
RT_2_ Profiler_TM_ PCR Array
Total RNA samples (0.5 μg) were reverse transcribed using the RT2 First Strand Kit (Qiagen). Each reaction was performed in 25 μl of solution containing cDNA, 2 × RT2 SYBR Green Mastermix, and RNase-free water, using custom-made RT2 Profiler™ PCR Arrays (Custom PCR Arrays; Qiagen) and a QuantStudio™ 12K Flex thermocycler (Life Technologies, Grand Island, N.Y., USA). The reaction sequence included 10 min of incubation at 95°C, followed by 40 cycles of 15 s at 95°C, 1 min at 60°C, 1 min at 60°C, and 15 s at 95°C. The relative gene expression was calculated using the ΔΔCt method, and the results are expressed with reference to the arithmetic mean of three reference housekeeping genes (ALG9, GAPDH, and RPL13A).
Statistical Analysis
Paired Student's t tests were used to compare differences in ESR1, ESR2, PGR, AR, STS, HSD3B1/2, HSD17B5, and CYP19A1 expression between young and old gonad-intact animals (experiment 1) and between OVX and OVX + E2 animals (experiment 2). ANOVA followed by the Dunnett multiple-range test was used to assess between-group differences in expression for all other analyses (experiment 3). Significance was considered at p < 0.05.
Results
The ovary-intact animals showed no detectable effect of age on the expression of genes encoding steroid receptors or steroidogenic enzymes in the ARC-ME (table 2). In both young and old OVX animals, however, PGR expression was significantly enhanced by E2 supplementation (fig. 1). Similarly, ESR1 and ESR2 expression was enhanced by E2 supplementation in the young (fig. 1a) but not in the old (fig. 1b) animals. In the OVX animals, there was no detectable effect of E2 supplementation on the expression of AR (fig. 1) or on genes encoding steroidogenic enzymes (fig. 2), except for a significant (p < 0.05) decrease in HSD17B5 expression in the old OVX + E2 animals relative to the untreated age-matched controls.
Table 2
Effect of age on gene expression in the ARC-ME of female rhesus macaques
Fig. 1
Effect of E2 treatment on steroid receptor gene expression in the ARC-ME of OVX rhesus macaques. The animals either served as controls or were treated with E2 for ∼1 month (young animals) or for ∼4 years (old animals). Each bar represents the mean (± SEM) mRNA levels of 4 animals, normalized to the arithmetic mean of two housekeeping genes, ALG9 and RPL13A. In the young animals (a), ESR1, ESR2, and PGR mRNA levels were significantly higher in the OVX + E2 group than in the OVX group, whereas AR mRNA levels were not significantly different. In the old animals (b), only PGR mRNA levels were significantly higher in the OVX + E2 group relative to the OVX group. * p < 0.05, ** p < 0.01 (paired Student's t test).
Fig. 1
Effect of E2 treatment on steroid receptor gene expression in the ARC-ME of OVX rhesus macaques. The animals either served as controls or were treated with E2 for ∼1 month (young animals) or for ∼4 years (old animals). Each bar represents the mean (± SEM) mRNA levels of 4 animals, normalized to the arithmetic mean of two housekeeping genes, ALG9 and RPL13A. In the young animals (a), ESR1, ESR2, and PGR mRNA levels were significantly higher in the OVX + E2 group than in the OVX group, whereas AR mRNA levels were not significantly different. In the old animals (b), only PGR mRNA levels were significantly higher in the OVX + E2 group relative to the OVX group. * p < 0.05, ** p < 0.01 (paired Student's t test).
Fig. 2
Effect of E2 treatment on steroidogenic enzyme gene expression in the ARC-ME of OVX rhesus macaques. Each bar represents the mean (± SEM) mRNA levels of 4 animals, normalized to the arithmetic mean of two housekeeping genes, ALG9 and RPL13A. In the young animals (a), there was no significant effect of E2 on any of the mRNA levels examined. In the old animals (b), HSD17B5 mRNA levels were significantly higher in the OVX + E2 group relative to the OVX group. * p < 0.05 (paired Student's t test).
Fig. 2
Effect of E2 treatment on steroidogenic enzyme gene expression in the ARC-ME of OVX rhesus macaques. Each bar represents the mean (± SEM) mRNA levels of 4 animals, normalized to the arithmetic mean of two housekeeping genes, ALG9 and RPL13A. In the young animals (a), there was no significant effect of E2 on any of the mRNA levels examined. In the old animals (b), HSD17B5 mRNA levels were significantly higher in the OVX + E2 group relative to the OVX group. * p < 0.05 (paired Student's t test).
To corroborate and extended the findings from the qRT-PCR experiments, we also performed gene expression analyses on RNA extracts obtained from young gonad-intact, old OVX, and old OVX + E2 animals, using custom-made RT2 Profiler™ PCR arrays. These three groups were selected to provide insights into ARC-ME gene expression during sexual maturity, after menopause, and after subsequent hormone therapy (HT). Our focus was on genes that encode steroid receptors, as well as genes associated with the control of the reproductive system, metabolism, and neurotransmission (table 3). As expected, the array analysis revealed a significant decrease (p < 0.01) in PGR expression in old OVX animals and showed that this decrease could be blocked by long-term HT. The expression of GNRH2 and SLC6A3 was also significantly enhanced by E2 (p < 0.05 and p < 0.01, respectively). In contrast, a significant increase in expression levels was observed for several genes associated with the KNDy neuronal system or metabolism (KISS1, p < 0.01; KISS1R, p < 0.05; TAC3, p < 0.01; NPY2R, p < 0.05), and in most cases the increase was reversed by E2.
Table 3
Gene expression in the ARC-ME of female rhesus macaques during reproductive aging
Discussion
Previous studies of the rhesus macaque hypothalamus relied on RT-PCR and in situ hybridization to examine the expression of ESR1, ESR2, and PGR [41,42]. Although these studies were essentially qualitative or semiquantitative, taken together their findings suggested that ESR1 and ESR2 expression is unaffected by circulating E2 levels, whereas PGR expression is highly stimulated by E2. In the present study, we used qRT-PCR, a more quantitative molecular approach, to corroborate these previous observations and to examine if these genes show age-associated changes in their expression. First of all, ESR1, ESR2, and PGR were all expressed in the ARC-ME region of the hypothalamus, thus confirming the results from previous studies. Despite their old age, however, we failed to detect a significant change in the expression of these genes in our old ovary-intact animals. This most likely stems from the fact that most of these animals had not completely entered menopause and their circulating E2 levels had not yet fallen to a sustained basal level. Consequently, to more closely mimic the human postmenopausal condition, we also examined age-related differences in the ARC-ME of young and old OVX animals and exposed some of them to supplementary E2 in order to gain insights into the influence of HT.
E2 HT had an age-related effect on ESR1 and ESR2 expression in OVX animals. While young OVX + E2-treated animals expressed more ESR1 and ESR2 mRNA than did untreated age-matched controls, the old animals did not respond to E2 supplementation. Although the reason for this difference is unclear, one possibility is that the hypothalamus of older animals is itself less responsive to exogenous E2. Another possibility is that the stimulatory effect of E2 on ESR1 and ESR2 mRNA is transitory and that it gradually decreases during long-term HT. In contrast to ESR1 and ESR2, PGR expression was highly stimulated by E2 regardless of age. It should be emphasized that the two major isoforms of the progestin receptor (PGR-A and PGR-B) show different expression patterns [43,44] and independent functions [45,46,47]. In the present study, however, we amplified both isoforms of PGR and thus cannot establish which form plays the dominant physiological role in the primate hypothalamus. Expression of the androgen receptor in the female rhesus macaque hypothalamus has not previously been described, but in the present study we found AR to be highly expressed in the female hypothalamus and to be unaffected by circulating E2 levels. This result is consistent with, and complements, the finding from a previous study in which a ribonuclease protection assay showed no effect of testosterone on the hypothalamic expression of AR in male rhesus macaques [48]. Taken together, the quantitative data from the present study clearly establish that gonadal nuclear steroid receptors are highly expressed in the female rhesus macaque hypothalamus, and that circulating E2 levels can significantly modulate progestin receptor gene expression even in old animals.
Recently, Naugle et al. [49] used immunohistochemistry to examine the effects of long-term E2 treatment on ERα and PGR expression in the rhesus macaque ARC-ME. Semiquantitative analysis of the immunoreactive cells revealed no effect of E2 on the number and density of ERα-positive cells in old OVX females, which is in agreement with the mRNA findings from the present study. On the other hand, E2 also failed to significantly affect the number and density of PGR-positive cells, which does not agree with our mRNA findings or those previously reported by Bethea et al. [33]. It is unclear whether this discrepancy is a reflection of the relative insensitivity of the semiquantitative immunohistochemistry methodology or whether it indicates that E2-induced PGR expression changes are not reflected at the translation level. Western blot analysis would help to resolve this issue in the future.
Given that the brain has an intrinsic capacity to synthesize sex steroids de novo [26,27,28,29], it is plausible that the hypothalamus can also compensate for the menopausal loss of sex steroids by increasing its local production of these steroids. There are several key enzymes involved in the synthesis of testosterone and E2 in the brain, using DHEA as a precursor. In the present study, we examined the hypothalamic expression of STS, HSD3B1/2, HSD17B5, and CYP19A1. These genes encode key enzymes in the conversion of DHEA to testosterone and E2 and are expressed in the rhesus macaque hypothalamus [29]. The findings from the current study corroborate these earlier observations. Moreover, they show that the expression of HSD17B5 becomes enhanced in old animals when circulating levels of E2 are low, which may result in a compensatory increase in hypothalamic E2 levels due to enhanced intracrine synthesis from DHEA. On the other hand, circulating levels of DHEA (particularly in the sulfated form as DHEA sulfate) show a significant age-related decline [29,50]. Consequently, it is unclear if the expression of STS, HSD3B1/2, HSD17B5, and CYP19A1 alone is sufficient to maintain high hypothalamic sex steroid levels during aging. Nevertheless, the results suggest that local steroid synthesis in the hypothalamus is likely to be preserved in older animals, provided that there is a sufficient amount of DHEA precursor available.
Other significant age-related gene expression changes have previously been observed in the human and nonhuman primate ARC-ME, especially in the KNDy neurons [12,13,14,15,16,17]. We observed the same neuronal hypertrophy-related increase in KISS1 and TAC3 gene expression observed in postmenopausal women, and this increase could be blocked by the administration of exogenous E2[17]. This suggests that the observed gene expression changes stemmed from an age-associated decline in circulating E2 levels, rather than from a sex steroid-independent aging mechanism. Although the physiological significance of the E2-dependent changes in KISS1, KISS1R, and TAC3 expression is unclear, these changes may serve to maintain homeostatic control of the reproductive neuroendocrine axis after secretion of sex steroids from the ovaries has declined [2]. In addition, we found that the expression of NPY2R, a central mediator of food intake, was also suppressed following chronic E2 supplementation in old OVX animals [51]. In postmenopausal women, therefore, NPY2R of the arcuate region may help to mediate the beneficial effects of estrogen HT in the treatment of disorders such as diabetes, obesity, and metabolic syndrome [52].
Analysis of the RT2 Profiler™ PCR arrays also established that most of the selected genes are stably expressed during reproductive aging and after HT. On the other hand, E2 supplementation was found to significantly stimulate the expression of GNRH2. We previously reported that E2 exerts a stimulatory action on GNRH2 gene expression in the hypothalamus of young OVX animals [53,54,55]. The finding that GNRH2 expression in old OVX animals also responds to E2 suggests that this second form of primate GnRH may play an important role in mediating E2-positive feedback to the reproductive neuroendocrine axis, as previously suggested [54,55]. E2 supplementation was also found to stimulate the expression of SLC6A3, the gene that encodes the dopamine transporter. Voytko et al. [56] reported that chronic E2 therapy did not have an effect on dopaminergic activity in the ventral tegmentum and dorsolateral prefrontal cortex. However, the present results show that chronic E2 supplementation was able to significantly increase the expression of the dopamine transporter in arcuate neurons of aged OVX animals. The influence of estrogen on the dopaminergic system is less well understood, but our results suggest that chronic E2 treatment may facilitate reuptake of dopamine by presynaptic neurons in the arcuate region.
Menopause in women and female rhesus macaques is associated with attenuated circulating E2 concentrations [1,2,3], which is thought to contribute to the development of many age-related pathologies. On the other hand, the results from the present study suggest that the hypothalamus retains its capacity to respond to sex steroids even in old age, and that the sensitivity of the hypothalamus to E2 shows some compensatory changes that may help to maintain local homeostasis.
Acknowledgements
This work was supported by NIH grants AG-019100, AG-023477, AG-029612, AG-036670, and OD-011092. We wish to thank Laurie Renner for managing the in vivo component of the study, and Dr. Steven Kohama for collecting and processing the postmortem tissues. We also wish to thank the ONPRC Endocrine Technology and Support Core for conducting the hormone assays, and the ONPRC Molecular and Cell Biology Core for assistance with the mRNA quantitation.
Disclosure Statement
The authors have no conflicts of interest to declare.
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2015
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