Regional specific regulation of steroid receptor coactivator-1 immunoreactivity by orchidectomy in the brain of adult male mice

Steroids 88 (2014) 7–14

Contents lists available at ScienceDirect

Steroids journal homepage: www.elsevier.com/locate/steroids

Regional specific regulation of steroid receptor coactivator-1 immunoreactivity by orchidectomy in the brain of adult male mice Chen Bian a,1, Kaiyuan Zhang a,b,1, Yangang Zhao a, Qiang Guo a, Wenqin Cai a,⇑, Jiqiang Zhang a,⇑ a b

Department of Neurobiology, Chongqing Key Laboratory of Neurobiology, Third Military Medical University, Chongqing 400038, China Cadet Brigade, Third Military Medical University, Chongqing 400038, China

a r t i c l e

i n f o

Article history: Received 26 February 2014 Received in revised form 13 May 2014 Accepted 4 June 2014 Available online 16 June 2014 Keywords: Androgen Orchidectomy Sex hormone Nuclear receptor

a b s t r a c t Androgens including testosterone and dihydrotestosterone play important roles on brain structure and function, either directly through androgen receptor or indirectly through estrogen receptors, which need coactivators for their transcription activation. Steroid receptor coactivator-1 (SRC-1) has been shown to be multifunctional potentials in the brain, but how it is regulated by androgens in the brain remains unclear. In this study, we explored the effect of orchidectomy (ORX) on the expression of SRC-1 in the adult male mice using nickel-intensified immunohistochemistry. The results showed that ORX induced dramatic decrease of SRC-1 immunoreactivity in the olfactory tubercle, piriform cortex, ventral pallidum, most parts of the septal area, hippocampus, substantia nigra (compact part), pontine nuclei and nucleus of the trapezoid body (p < 0.01). Significant decrease of SRC-1 was noticed in the dorsal and lateral septal nucleus, medial preoptical area, dorsomedial and ventromedial hypothalamic nucleus and superior paraolivary nucleus (p < 0.05). Whereas in other regions examined, levels of SRC-1 immunoreactivity were not obviously changed by ORX (p > 0.05). The above results demonstrated ORX downregulation of SRC-1 in specific regions that have been involved in sense of smell, learning and memory, cognition, neuroendocrine, reproduction and motor control, indicating that SRC-1 play pivotal role in the mediating circulating androgenic regulation on these important brain functions. It also indicates that SRC-1 may serve as a novel target for the central disorders caused by the age-related decrease of circulating androgens. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction It is well known that male sex steroids androgens, including testosterone and dihydrotestosterone, are necessary for the development and maintenance of normal brain structure and function such as neural circuits and plasticity, behavioral and sexual modulation, cognition, neuroendocrine, feeding and sleep-wakefulness [1–4]. In the humans, age-related decrease of androgens is associated with increased risk of Alzheimer’s disease (AD), and testosterone replacement has been shown to improve cognitive deficits in rodents [5]. In the brain, androgens can be aromatized into 17beta-estradiol, therefore function through androgen receptor (AR) directly or estrogen receptors (ERa and ERb) indirectly [6]. Studies have revealed different levels of AR or ERs expression in specific brain regions that are mostly related to neurogenesis, learning

⇑ Corresponding authors. Tel./Fax: +86 23 68752232 (J. Zhang). Tel./Fax: +86 23 68753460 (W. Cai). E-mail addresses: [email protected] (W. Cai), [email protected] (J. Zhang). 1 Equal contribution. http://dx.doi.org/10.1016/j.steroids.2014.06.006 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

and memory, cognition, motor control, reproduction, neuroendocrine, pain and social decision-making [7–9]. Furthermore, ERb knockout induces changes of hippocampal synaptic plasticity and impairs long-term potentiation [10]. It is well established that steroid receptors need coactivators for their efficient transcriptional activity. Among which steroid receptor coactivator-1 (SRC-1; or NCoA-1) has been shown to dramatically enhance the transcriptional activity of nuclear receptors including AR and ERs in a ligand-dependent manner [11–13]. Accumulated studies have shown that brain SRC-1 may play a role in the modulation of neural plasticity, development of olfactory epithelium and cerebellar Purkinje cells [14,15], the defeminizing actions of estrogen [16], HPA axis function and thyroid hormone function [17,18]. It might also function to regulate reproduction and acute stress [19–21], motor learning [15], the anti-obesity effects of estrogen-ERa signals [22], optical and auditory regulation [23,24]. In our previous studies, we have demonstrated agerelated significant decrease of SRC-1 in specific brain regions related to learning and memory, motor and sense [25]. Furthermore, in the hippocampus, males and females shared similar postnatal developmental profiles for SRC-1 [26] and levels of

8

C. Bian et al. / Steroids 88 (2014) 7–14

hippocampal SRC-1 were regulated by postnatal development but not ovariectomy [27]. In the mice brain, a significant malepredominant expression profile of SRC-1 when compared to the females was also reported [28]. Limited references have reported the testosterone regulation of brain SRC-1 expression in specific regions with discrepant results. For example, testosterone did not regulate expression of SRC-1 in the adult Siberian hamster [23] or zebra finch brain [29]; however, in the male preoptic area/hypothalamus of Japanese quail it was up-regulated by testosterone [30]. In a recent study, we found that expression of hippocampal SRC-1 was significantly regulated by gonadectomy (GDX) in a sex-dependent manner: while orchidectomy (ORX) caused persistent decreases of SRC-1, ovariectomy (OVX) only induced a transient decrease of SRC-1 at two weeks after surgery [31]. These results indicated that androgens are the main regulator of hippocampal SRC-1; but how about the androgenic regulation on SRC-1 in other brain regions is not clear. In order to explore the multiple roles that SRC-1 plays in steroids regulation on the central nervous system, in this study we investigated the effects of androgens deprivation on the expression of SRC-1 immunoreactivities in the brain of adult male mice. 2. Materials and methods 2.1. Animals and surgery Adult male SPF grade C57BL/6 mice (12 weeks old, 22 ± 2 g, n = 10) were obtained from the Experimental Animal Center of Third Military Medical University. All the animal-related procedures were conducted in strict compliance with Approved Institutional Animal Care and Use Protocols. The animals were randomly divided into two groups. ORX was carried out according to previous surgical procedures [31]. In brief, mice were anesthetized with 100 mg/kg sodium pentobarbital, the hair was clipped over the surgical area and scrubbed with Betadine and ethanol swipe, the skin of the scrotum was opened, the epididymis was cut and the testes were removed completely, then the wound was sutured. A sham-operated group animal was used as control. 2.2. Tissue preparation Four weeks after surgery, the mice were deeply anaesthetize with 100 mg/kg sodium pentobarbital, perfused transcardially with saline and followed by 4% paraformaldehyde in phosphate buffer (pH 7.4). The brains were carefully dissected, removed, post-fixed overnight with the same fixative, and then transferred to the fixative containing 30% sucrose until they sank to the bottom of the container. Tissue preparation was carried out according to our previous reports [7,25,28]. In brief, brains were serially cut frozen into 25 lm-thick coronal sections with a cryostat (CM1900, Leica Microsystems, Germany). By following the principles of unbiased and systematic random sampling, the serially cut sections were transferred into one of six wells, with every sixth section being placed in the same well. 2.3. Immunohistochemistry (IHC) Nickel-intensified SRC-1 IHC was carried out according to our previous description [7,25,28]. Free-floating sections were first washed with PBS (phosphate buffered saline, 10 mmol/L; pH 7.4), quenched for 15 min in 3% H2O2 in PBS, and blocked in 5% normal goat serum for 30 min at room temperature. The sections were then incubated overnight at 4 °C with the primary rabbit polyclonal antiserum (1:200; sc-8995, Santa Cruz, USA) diluted with Antibody Diluent (S3022, Dako Inc., Glostrup, Denmark). After

washes, the sections were incubated with the biotinylated secondary goat-antirabbit antibody (1:200; ZB2010, Zhongshan Biotech; Beijing, China) for 1 h at room temperature. The sections were washed in PBS again, incubated with the HRP-labeled streptavidin reagent (1:200; ZB2404, Zhongshan Biotech; Beijing, China) for 1 h at room temperature and then visualized using a DAB-nickel chromogen kit (SK-4100; Vector Laboratories Inc., USA) for 5 min at room temperature. Finally, the sections were dehydrated, cleared in xylene and mounted with DPX. Blank control was carried out using the same procedure, but PBS was used instead of the primary antiserum. 2.4. Data analysis and statistics Data analysis and statistics were conducted according to our previous studies [25,28]. All the images were recorded by using a digital camera (DP70, Leica Microsystems, Germany) equipped with an Olympus microscope (BX60, Japan) as previous reports [26,31]. SRC-1 expression pattern was determined from images of the brain regions guided by The Mouse Brain in Stereotaxic Coordinates (2nd edition) [32]. The average optical density from 2 to 5 sections from each brain region or sub-region was used to represent the regional expression level for each animal. The representative SRC-1 immunostaining in specific brain regions was measured by Image Pro Plus software 6.0, values in each group were averaged and reported as means ± SEM. Independent-sample T-test was carried out with software SPSS (version 13.0) and a level of P < 0.05 was considered to be statistical significant. 3. Results In the sham animals, expression of SRC-1 immunopositive materials was in well agreement with our previous observations [28]; and it was distinctly regulated by ORX in a region-specific manner as indicated in Figs. 1–3 and summarized in Figs. 4–6. 3.1. Telencephalon 3.1.1. Sham animals Concentrated SRC-1 immunopositive cell nuclei were detected in most part of the olfactory bulb, including anterior olfactory nucleus, mitral cell layer of the accessory olfactory bulb, granular cell layer of the accessory olfactory bulb and external plexiform layer (Fig. 1A). In the cerebral cortex, sense positive cells were detected in most part of the cortex including cingulate cortex (Cg1) and motor cortex (M1 and M2) as shown in Fig. 1C, piriform cortex, ventral pallidum and olfactory tubercle (Fig. 1E). In the septal area, moderate immunostaining nuclei were detected in the dorsal part of lateral septal nucleus; relative weak levels of SRC-1 were detected in the medial septal nucleus (Fig. 1G) and the horizontal limb of the diagonal band, while strong expression of SRC-1 was detected in the vertical limb of the diagonal band (Fig. 2A). In the hippocampal formation, the CAs and dentate gyrus (Fig. 2C) and the ventral taenia tecta showed the highest expression, lower levels of SRC-1 were detected in the bed nucleus of stria terminalis and subiculum. Different levels of SRC-1 immunopositivities were also detected in other parts of the forebrain such as accumbens nucleus (shell; Fig. 2A) and amygdaloid. 3.1.2. ORX animals In the olfactory bulb and cerebral cortex, dense SRC-1 immunopositive cells were detected as that of the sham animals as shown in Fig. 1B, D and F. Significant decrease of SRC-1 immunoreactivity was only detected in the piriform cortex and olfactory tubercle (p < 0.01; Fig. 4). In the septal area, ORX induced significant

C. Bian et al. / Steroids 88 (2014) 7–14

Fig. 1. SRC-1 immunoreactivity detected in specific forebrain nuclei of sham (CON) and orchidectomized (ORX) adult male mice. (A and B): olfactory bulb; (C and D): cerebral cortex; (E and F) olfactory cortex; (G and H): septal area. Cg1: cingulate cortex, area 1; M1: primary motor cortex; M2: secondary motor cortex; Tu: olfactory tubercle; VP: ventral pallidum; MS: medial septal nucleus; LSV: lateral septal nucleus, ventral part; LSI: lateral septal nucleus, intermediate part. Significant decreases were detected in Tu, VP, LSI, MS and LSV (p < 0.05). Bar = 500 lm.

decrease of SRC-1 was detected in the septal nucleus (dorsal and intermediate part) and the medial septal nucleus (Fig. 1H) as well as the vertical limb of the diagonal band (Figs. 2B and 4). In the hippocampal formation, levels of SRC-1 were dramatically decreased in CAs and dentate gyrus (Figs. 2D and 5); while in the bed nucleus of stria terminalis, the ventral taenia tecta and the subiculum, levels of SRC-1 were comparable to that detected in the sham animals. 3.2. Diencephalon 3.2.1. Sham animals SRC-1 immunopositive cells were extensively localized in the thalamus. Dense SRC-1 immunopositive cells were detected in the medial habenular nucleus and the thalamic paraventricular nucleus (Fig. 2E); relative less dense SRC-1 immunopositive cells were detected in the lateral habenular nucleus (Fig. 2E) and the medial geniculate nucleus and scattered SRC-1 immunopositive cells were found in other sub-regions. In the hypothalamus, relative higher levels of SRC-1 were detected in the dorsal medial nucleus and ventral medial nucleus (Fig. 2G), anterior hypothalamic area (posterior part; Fig. 3A) and arcuate nucleus; relative lower levels were detected in the medial optical nucleus and the hypothalamic paraventricular nucleus (Fig. 3A). SRC-1 immunopositive cells were also detected in the other sub-regions of

9

Fig. 2. SRC-1 immunoreactivity detected in specific forebrain nuclei of sham (CON) and orchidectomized (ORX) adult male mice. (A and B): septal area; (C and D): hippocampus; (E and F): thalamus; (G and H): hypothalamus. AcbSh: accumbens nucleus, shell; VDB: nucleus of the vertical limb of the diagonal band; DG: dentate gyrus. Mhb: medial habenular nucleus; Lhb: lateral habenular nucleus; D3 V; third ventricle, dorsal. PV: paraventricular thalamic nucleus; DMH: dorsomedial hypothalamic nucleus; VMH: ventromedial hypothalamic nucleus; 3 V: third ventricle; Significant decreases were detected in the VDB, hippocampus, DMH and VMH (p < 0.05). Bar = 500 lm.

hypothalamus such as the hypothalamic periventricular nucleus, the mammillary body and supraoptic nucleus. 3.2.2. ORX animals SRC-1 immunopositive cells were also extensively detected in these animals. Significant decrease of SRC-1 was detected in the dorsal medial nucleus and ventral medial nucleus of the hypothalamus as shown in Fig. 2H (p < 0.05). In other paired nucleus examined such as thalamic medial habenular nucleus and paraventricular nucleus (Fig. 2F), hypothalamic paraventricular nucleus (Fig. 3B) and arcuate nucleus, expression of SRC-1 maintained similar levels as that detected in the sham animals. 3.3. Brainstem and cerebellum 3.3.1. Sham animals In the brainstem, SRC-1 immunopositive cells were extensively detected. In the substantia nigra, the positive cells were predominantly localized in the compact part (Fig. 3C), dense SRC-1 immunopositive cells were also detected in the periaqueductal gray area including dorsomedial and lateral periaqueductal gray (Fig. 3E) as well as pontine nuclei (Fig. 3G). Scattered SRC-1 positive cells were

10

C. Bian et al. / Steroids 88 (2014) 7–14

Fig. 3. SRC-1 immunoreactivity detected in specific nuclei in the diencephalon and brainstem of sham (CON) and orchidectomized (ORX) adult male mice. (A and B): hypothalamus; (C and D): substantia nigra; (E and F): periaqueductal gray; (G and H): pontine nucleus. Pa: hypothalamic paraventricular nucleus; AHP: anterior hypothalamic area, posterior part; SNC: substantia nigra, compact part; DMPAG: dorsomedial periaqueductal gray; LPAG: lateral periaqueductal gray; Aq: cerebral aqueduct. cp: cerebral peduncle; Pn: pontine nucleus. 3 V: third ventricle. Significant decreases were detected in the SNC and Pn (p < 0.05). Bar = 500 lm.

localized in other sub-regions such as the superior colliculus and dorsal medial raphe. In the medulla oblongata, higher levels of SRC-1 were detected in the trapezoid body, pontine reticular nucleus, superior paraolivary nucleus and solitary nucleus as well as spinal trigeminal nucleus. In the cerebellar cortex, very strong SRC-1 immunoreactive materials were clearly localized in the cell nuclei of Purkinje cells, dense but light-stained immunopositive cell nuclei were detected in the granular cell layer. 3.3.2. ORX animals Dramatic decrease of SRC-1 immunostaining was detected in the substantia nigra (Fig. 3D), pontine nuclei (Fig. 3H) and the nucleus of the trapezoid body (p < 0.01); significant decrease was also detected in the superior paraolivary nucleus (p < 0.05). While in the periaqueductal gray (Fig. 3F) and other sub-regions examined, ORX did not cause any significant changes (p > 0.05). The above results were summarized in Figs. 4–6. 4. Discussion In this study, based on our previous findings that hippocampal SRC-1 was differently regulated by gonadectomy [31], we

investigated the effect of testicular androgen deprivation on the expression of SRC-1 in the adult male mice. The results showed that ORX induced decrease of SRC-1 in specific brain regions. The most dramatic decrease of SRC-1 was detected in the piriform cortex, olfactory tubercle, lateral septal nucleus(intermediate and lateral part), ventral part medial septal nucleus, nucleus of the vertical limb of the diagonal band, hippocampus, substantia nigra (compact part), nucleus of the trapezoid body and pontine nucleus (p < 0.01). Significant decrease was noticed in the dorsal lateral septal nucleus, dorsomedial and ventromedial hypothalamic nucleus and superior paraolivary nucleus (p < 0.05). In other brain regions examined, such as in the olfactory bulb, cerebellum and most part of the brainstem, expression of SRC-1 was not obviously changed by ORX (p > 0.05). It is clear that androgens have many important physiological roles on the central nervous system. Androgens exposure has been shown to modulate brain development, resulting in changes of behavioral, spatial orientation and cognitive functions [1]; decreases of androgens are associated with increased risk of AD while testosterone has been shown to improve cognitive deficits seen in animal models [5]. Testosterone is also thought to masculinize and defeminize the male brain and induce male-typical behaviors in the adult [33]. Additionally, circulating testosterone has been correlated to the magnitude of the cochlear response to auditory stimuli [34]. AR and/or ERs directly or indirectly mediate the androgenic action on the brain, studies have shown that AR immunoreactivity or mRNA were detected in many brain regions [9,35–37] and similar profiles of ERa and ERb were also detected in these brain regions [7,38]. The extensive expression of SRC-1 in the brain of control mice was in agreement with the above expression patterns of nuclear receptors (as summarized in Table 1) and our previous findings [28], indicating its multifunctional potentials in the regulation of brain structure and function. We noticed that ORX induced significant decrease of SRC-1 in specific brain regions with different functions. For example, the piriform cortex and olfactory tubercle belong to the olfactory cortex [39], the former has been shown to be involved in encoding the predictive value of olfactory stimuli [40] and the later serves as an interaction center between smells and sounds [41]. The lateral septum has been implicated in the modulation of social behavior, anxiety, fear conditioning, memory-related behaviors, and the mesolimbic rewarding pathways [42]. The other parts of the septal area (medial septa and the vertical limb of the diagonal band) and hippocampus have been demonstrated to play crucial roles in the regulation of learning and memory and cognition [43,44], indicating SRC-1 plays a role in the regulation of learning and memory. The dorsomedial nucleus of the hypothalamus (DMH) has long been involved in the regulation of reproduction, thermogenesis, stress response, pancreatic nerve activity, plasma glucose levels, food intake and circadian rhythms [45]; while the ventromedial hypothalamic nucleus has been related to regulate many physiological processes, such as reproduction, defensive responses, locomotor activity, glucose homeostasis and appetite control [46–51]. In the midbrain, it has been well established that the compact part of substantia nigra contains dopaminergic neurons, which have been related to locomotor symptoms such as slowness of movement, tremor, rigidity and postural instability especially PD [52]. The pontine nucleus belongs to the proprioceptive system, provides continuous positional information on the limbs and body [53] and previous studies suggested that auditory-conditioned stimulus information is routed to the cerebellum by this nucleus [54]. The nucleus of the trapezoid body and superior paraolivary nucleus are key nuclei of brainstem auditory system [55,56]. The significant decrease of SRC-1 in the above nuclei after ORX strongly suggested the potential roles of SRC-1 in the regulation on these specific regions. For example, Nishihara et al. demonstrated that

C. Bian et al. / Steroids 88 (2014) 7–14

11

Fig. 4. Statistical analysis of SRC-1 immunoreactivity in the cerebral cortex and septal area of sham (CON) and orchidectomized (ORX) adult male mice. AO: anterior olfactory; M1: primary motor cortex; M2: secondary motor cortex; Pir: piriform cortex; ventral pallidum; Tu: olfactory tubercle; LSD: lateral septal nucleus, dorsal part; LSI: lateral septal nucleus, intermediate part; LSV: lateral septal nucleus, ventral part; MS: medial septal nucleus; VDB: nucleus of the vertical limb of the diagonal band. Results were expressed as mean ± S.E.M. ⁄p < 0.05, ⁄⁄p < 0.01.

Fig. 5. Statistical analysis of SRC-1 immunoreactivity in the hippocampal formation and hypothalamus of sham (CON) and orchidectomized (ORX) adult male mice. VTT: ventral taenia tecta; Hippo: hippocampus; BST: bed nucleus of stria terminalis; S: subiculum; Pa: paraventricular hypothalamic nucleus; AHP: anterior hypothalamic area, posterior part; MPA: medial preoptic area; Arc: arcuate hypothalamic nucleus; DMH: dorsomedial hypothalamic nucleus; VMH: ventromedial hypothalamic nucleus. Results were expressed as mean ± S.E.M. ⁄p < 0.05, ⁄⁄p < 0.01.

12

C. Bian et al. / Steroids 88 (2014) 7–14

Fig. 6. Statistical analysis of SRC-1 in the thalamus, brainstem and cerebellum of sham (CON) and orchidectomized (ORX) adult male mice. MHb: medial habenular nucleus; LHb: lateral habenular nucleus; PV: paraventricular thalamic nucleus; MG: medial geniculate nucleus; SNC: substantia nigra, compact part; DMPAG: dorsomedial periaqueductal gray; Pn: pontine nucleus; Tz: nucleus of the trapezoid body; SPO: superior paraolivary nucleus; Pc: cerebellar Purkinje cells. Results were expressed as mean ± S.E.M. ⁄p < 0.05, ⁄⁄p < 0.01.

Table 1 Selected references regarding the expression of SRC-1, AR and ERs in specific brain regions. SRC-1 protein

AR

ERa

Anterior olfactory Cerebral cortex Amygdaloid complex Lateral septal nucleus Basal forebrain Hippocampus Bed nucleus of stria terminalis Paraventricular hypothalamic nucleus Anterior hypothalamic area Medial preoptic area Arcuate nucleus Dorsomedial hypothalamic nucleus Ventromedial hypothalamic nucleus Habenular nucleus Paraventricular thalamic nucleus Substantia nigra, compact part Central gray Pontine nucleus Nucleus of the trapezoid body Cerebellar Purkinje cells

mRNA [58] Protein [9,59] Protein [9,61,62] mRNA [58] protein [9,35,36,59,63–65] mRNA [35,58] protein [63,64,68] Protein [9,61] Protein [9,62,69] mRNA [35] protein [9,36,59,63,64,68] mRNA [58] protein [9] mRNA [58] protein [9,63,65] mRNA [58] protein [9,36,59,62,68] Protein [9,62] Protein [62] mRNA [58] protein [9,35,62] mRNA [35] mRNA [35] protein [9] Protein [71] Protein [36] Protein [9,36,65,71] Protein [9] Protein [65]

Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein Protein

ERb [59,60] [60] [64,66,67] [64,66,67] [67] [60,67,69] [59,64,67] [67] [60,66,67] [59,67] [66,67] [66,67] [66,67] [67] [67] [71] [66]

Protein & mRNA [36]

Protein [7] Protein [7] Protein [7,63,64] Protein [63] Protein [7] Protein [7,69] Protein [7,63,64] Protein [7] Protein [63,64,70] Protein [7,64] Protein [7] Protein [7] Protein [7] Protein [7] Protein [7,70] Protein [7,71] Protein [7] mRNA [36] protein [7,36] Protein [7] mRNA [36] protein [7,36]

Notice: The first column shows SRC-1 immunoreactivities reported in this study.

SRC-1 deficiency caused delayed escape latencies in Morris maze test (hippocampus-dependant) [15], Charlier et al. found that infusion of antisense SRC-1 oligonucleotides into hypothalamus could significantly reduce male-typical sexual behaviors and alter neural plasticity [19]. However, in other brain regions which ORX did not affect SRC-1 levels significantly such as in the cerebellar Purkinje cells, SRC-1 might also play important roles, the strong supporting evidences came from Nishihara’s report that knockout SRC-1 gene delayed the development and maturation of Purkinje cells and caused motor deficiency [15]. All these indicate the complicated function of SRC-1 in the brain.

In summary, in this study we reported ORX regulation of brain SRC-1 expression in a region-specific manner, interestingly these results were highly in agreement with our recently finding that aromatase inhibitor letrozole injection down-regulates brain SRC1 [57] in a similar manner and similar regions. These findings may have strong implications for the elucidation of the mechanisms underlying circulating androgenic regulation on many pivotal brain functions and structures. It also indicates that SRC-1 may serve as a novel target for the central disorders caused by the age-related decrease of circulating androgen such as AD, PD and hearing loss. Future works about the colocalization of AR

C. Bian et al. / Steroids 88 (2014) 7–14

and/or ERs with SRC-1 in specific brain regions and how SRC-1 coactivates these receptors under androgen treatment are urgently needed to clarify the functional roles of SRC-1 in the androgenic regulation on brain functions.

Conflict of interest The authors declare that they have no conflict of interest. Acknowledgement This work was supported by the National Science Foundation of China (No. 81171035). References [1] Wojniusz S, Ropstad E, Evans N, Robinson J, Solbakk AK, Endestad T, et al. Sexspecific development of spatial orientation is independent of peripubertal gonadal steroids. Psychoneuroendocrinology 2013;38:1709–16. [2] Lagunas N, Calmarza-Font I, Grassi D, Garcia-Segura LM. Estrogen receptor ligands counteract cognitive deficits caused by androgen deprivation in male rats. Horm Behav 2011;59:581–4. [3] Kawato S. Role of androgen in the elderly. Modulation of synaptic plasticity by brain-synthesized androgens. Clin Calcium 2013;23:1141–50. [4] Silveyra P, Cataldi NI, Lux-Lantos V, Libertun C. Gonadal steroids modulated hypocretin/orexin type-1 receptor expression in a brain region, sex and daytime specific manner. Regul Pept 2009;158:121–6. [5] George S, Petit GH, Gouras GK, Brundin P, Olsson R. Nonsteroidal selective androgen receptor modulators and selective estrogen receptor beta agonists moderate cognitive deficits and amyloid-beta levels in a mouse model of Alzheimer’s disease. ACS Chem Neurosci 2013. [6] Lephart ED. A review of brain aromatase cytochrome P450. Brain Res Brain Res Rev 1996;22:1–26. [7] Zhang JQ, Cai WQ, Zhou DS, Su BY. Distribution and differences of estrogen receptor beta immunoreactivity in the brain of adult male and female rats. Brain Res 2002;935:73–80. [8] Sheng Z, Kawano J, Yanai A, Fujinaga R, Tanaka M, Watanabe Y, et al. Expression of estrogen receptors (alpha, beta) and androgen receptor in serotonin neurons of the rat and mouse dorsal raphe nuclei; sex and species differences. Neurosci Res 2004;49:185–96. [9] Feng Y, Weijdegard B, Wang T, Egecioglu E, Fernandez-Rodriguez J, Huhtaniemi I, et al. Spatiotemporal expression of androgen receptors in the female rat brain during the oestrous cycle and the impact of exogenous androgen administration: a comparison with gonadally intact males. Mol Cell Endocrinol 2010;321:161–74. [10] Liu F, Day M, Muniz LC, Bitran D, Arias R, Revilla-Sanchez R, et al. Activation of estrogen receptor-beta regulates hippocampal synaptic plasticity and improves memory. Nat Neurosci 2008;11:334–43. [11] Onate SA, Tsai SY, Tsai MJ, O’Malley BW. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 1995;270:1354–7. [12] Ikonen T, Palvimo JJ, Janne OA. Interaction between the amino- and carboxylterminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators. J Biol Chem 1997;272:29821–8. [13] McInerney EM, Tsai MJ, O’Malley BW, Katzenellenbogen BS. Analysis of estrogen receptor transcriptional enhancement by a nuclear hormone receptor coactivator. Proc Natl Acad Sci USA 1996;93:10069–73. [14] Misiti S, Koibuchi N, Bei M, Farsetti A, Chin WW. Expression of steroid receptor coactivator-1 mRNA in the developing mouse embryo: a possible role in olfactory epithelium development. Endocrinology 1999;140:1957–60. [15] Nishihara E, Yoshida-Komiya H, Chan CS, Liao L, Davis RL, O’Malley BW, et al. SRC-1 null mice exhibit moderate motor dysfunction and delayed development of cerebellar Purkinje cells. J Neurosci 2003;23:213–22. [16] Auger AP, Tetel MJ, McCarthy MM. Steroid receptor coactivator-1 (SRC-1) mediates the development of sex-specific brain morphology and behavior. Proc Natl Acad Sci USA 2000;97:7551–5. [17] Schmidt MV, Oitzl M, Steenbergen P, Lachize S, Wurst W, Muller MB, et al. Ontogeny of steroid receptor coactivators in the hippocampus and their role in regulating postnatal HPA axis function. Brain Res 2007;1174:1–6. [18] Iannacone EA, Yan AW, Gauger KJ, Dowling AL, Zoeller RT. Thyroid hormone exerts site-specific effects on SRC-1 and NCoR expression selectively in the neonatal rat brain. Mol Cell Endocrinol 2002;186:49–59. [19] Charlier TD, Ball GF, Balthazart J. Inhibition of steroid receptor coactivator-1 blocks estrogen and androgen action on male sex behavior and associated brain plasticity. J Neurosci 2005;25:906–13. [20] Molenda HA, Griffin AL, Auger AP, McCarthy MM, Tetel MJ. Nuclear receptor coactivators modulate hormone-dependent gene expression in brain and female reproductive behavior in rats. Endocrinology 2002;143:436–44.

13

[21] Bousios S, Karandrea D, Kittas C, Kitraki E. Effects of gender and stress on the regulation of steroid receptor coactivator-1 expression in the rat brain and pituitary. J Steroid Biochem Mol Biol 2001;78:401–7. [22] Zhu L, Yang Y, Xu P, Zou F, Yan X, Liao L, et al. Steroid receptor coactivator-1 mediates estrogenic actions to prevent body weight gain in female mice. Endocrinology 2013;154:150–8. [23] Tetel MJ, Ungar TC, Hassan B, Bittman EL. Photoperiodic regulation of androgen receptor and steroid receptor coactivator-1 in Siberian hamster brain. Brain Res Mol Brain Res 2004;131:79–87. [24] Tahera Y, Meltser I, Johansson P, Salman H, Canlon B. Sound conditioning protects hearing by activating the hypothalamic–pituitary–adrenal axis. Neurobiol Dis 2007;25:189–97. [25] Zhang D, Guo Q, Bian C, Zhang J, Lin S, Su B. Alterations of steroid receptor coactivator-1 (SRC-1) immunoreactivities in specific brain regions of young and middle-aged female Sprague–Dawley rats. Brain Res 2011; 1382:88–97. [26] Bian C, Zhu K, Guo Q, Xiong Y, Cai W, Zhang J. Sex differences and synchronous development of steroid receptor coactivator-1 and synaptic proteins in the hippocampus of postnatal female and male C57BL/6 mice. Steroids 2012;77:149–56. [27] Zhang D, Guo Q, Bian C, Zhang J, Cai W, Su B. Expression of steroid receptor coactivator-1 was regulated by postnatal development but not ovariectomy in the hippocampus of rats. Dev Neurosci 2011;33:57–63. [28] Bian C, Zhang D, Guo Q, Cai W, Zhang J. Localization and sex-difference of steroid receptor coactivator-1 immunoreactivities in the brain of adult female and male mice. Steroids 2011;76:269–79. [29] Duncan KA, Jimenez P, Carruth LL. Distribution and sexually dimorphic expression of steroid receptor coactivator-1 (SRC-1) in the zebra finch brain. Gen Comp Endocrinol 2011;170:408–14. [30] Charlier TD, Ball GF, Balthazart J. Plasticity in the expression of the steroid receptor coactivator 1 in the Japanese quail brain: effect of sex, testosterone, stress and time of the day. Neuroscience 2006;140:1381–94. [31] Bian C, Zhu K, Yang L, Lin S, Li S, Su B, et al. Gonadectomy differentially regulates steroid receptor coactivator-1 and synaptic proteins in the hippocampus of adult female and male C57BL/6 mice. Synapse 2012;66:849–57. [32] Paxinos G, Franklin KB. The mouse brain in stereotaxic coordinates. 2nd ed. San Diego: Academic Press; 2001. [33] Sato T, Matsumoto T, Kawano H, Watanabe T, Uematsu Y, Sekine K, et al. Brain masculinization requires androgen receptor function. Proc Natl Acad Sci USA 2004;101:1673–8. [34] Snihur AW, Hampson E. Click-evoked otoacoustic emissions: response amplitude is associated with circulating testosterone levels in men. Behav Neurosci 2012;126:325–31. [35] Simerly RB, Chang C, Muramatsu M, Swanson LW. Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. J Comp Neurol 1990;294:76–95. [36] Hamson DK, Jones BA, Watson NV. Distribution of androgen receptor immunoreactivity in the brainstem of male rats. Neuroscience 2004;127:797–803. [37] Scott CJ, Clarke IJ, Rao A, Tilbrook AJ. Sex differences in the distribution and abundance of androgen receptor mRNA-containing cells in the preoptic area and hypothalamus of the ram and ewe. J Neuroendocrinol 2004;16:956–63. [38] Shughrue PJ, Lane MV, Merchenthaler I. Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. J Comp Neurol 1997;388:507–25. [39] Ceci ML, Pedraza M, de Carlos JA. The embryonic septum and ventral pallium, new sources of olfactory cortex cells. PLoS One 2012;7:e44716. [40] Garske AK, Lawyer CR, Peterson BM, Illig KR. Adolescent changes in dopamine D1 receptor expression in orbitofrontal cortex and piriform cortex accompany an associative learning deficit. PLoS One 2013;8:e56191. [41] Wesson DW, Wilson DA. Smelling sounds: olfactory–auditory sensory convergence in the olfactory tubercle. J Neurosci 2010;30:3013–21. [42] Talishinsky A, Rosen GD. Systems genetics of the lateral septal nucleus in mouse: heritability, genetic control, and covariation with behavioral and morphological traits. PLoS One 2012;7:e44236. [43] Tsurusaki M, Gallagher JP. A distinct group of non-cholinergic neurons along the mid-line of the septum and within the rat medial septal nucleus. Neurosci Lett 2006;410:20–4. [44] Widmer H, Ferrigan L, Davies CH, Cobb SR. Evoked slow muscarinic acetylcholinergic synaptic potentials in rat hippocampal interneurons. Hippocampus 2006;16:617–28. [45] Lee S, Bookout AL, Lee CE, Gautron L, Harper MJ, Elias CF, et al. Laser-capture microdissection and transcriptional profiling of the dorsomedial nucleus of the hypothalamus. J Comp Neurol 2012;520:3617–32. [46] Cheung CC, Kurrasch DM, Liang JK, Ingraham HA. Genetic labeling of steroidogenic factor-1 (SF-1) neurons in mice reveals ventromedial nucleus of the hypothalamus (VMH) circuitry beginning at neurogenesis and development of a separate non-SF-1 neuronal cluster in the ventrolateral VMH. J Comp Neurol 2013;521:1268–88. [47] Segi-Nishida E, Sukeno M, Imoto Y, Kira T, Sakaida M, Tsuchiya S, et al. Electroconvulsive seizures activate anorexigenic signals in the ventromedial nuclei of the hypothalamus. Neuropharmacology 2013;71:164–73. [48] Kim KW, Li S, Zhao H, Peng B, Tobet SA, Elmquist JK, et al. CNS-specific ablation of steroidogenic factor 1 results in impaired female reproductive function. Mol Endocrinol 2010;24:1240–50.

14

C. Bian et al. / Steroids 88 (2014) 7–14

[49] Krebs CJ, Jarvis ED, Chan J, Lydon JP, Ogawa S, Pfaff DW. A membraneassociated progesterone-binding protein, 25-Dx, is regulated by progesterone in brain regions involved in female reproductive behaviors. Proc Natl Acad Sci USA 2000;97:12816–21. [50] Xu Y, Hill JW, Fukuda M, Gautron L, Sohn JW, Kim KW, et al. PI3K signaling in the ventromedial hypothalamic nucleus is required for normal energy homeostasis. Cell Metab 2010;12:88–95. [51] Unger TJ, Calderon GA, Bradley LC, Sena-Esteves M, Rios M. Selective deletion of Bdnf in the ventromedial and dorsomedial hypothalamus of adult mice results in hyperphagic behavior and obesity. J Neurosci 2007;27:14265–74. [52] Hennis MR, Marvin MA, Taylor 2nd CM, Goldberg MS. Surprising behavioral and neurochemical enhancements in mice with combined mutations linked to Parkinson’s disease. Neurobiol Dis 2013. [53] Bermingham NA, Hassan BA, Wang VY, Fernandez M, Banfi S, Bellen HJ, et al. Proprioceptor pathway development is dependent on Math1. Neuron 2001;30:411–22. [54] Clark RE, Gohl EB, Lavond DG. The learning-related activity that develops in the pontine nuclei during classical eye-blink conditioning is dependent on the interpositus nucleus. Learn Mem 1997;3:532–44. [55] Alamilla J, Gillespie DC. Maturation of calcium-dependent GABA, glycine, and glutamate release in the glycinergic MNTB–LSO pathway. PLoS One 2013;8:e75688. [56] Felix 2nd RA, Vonderschen K, Berrebi AS, Magnusson AK. Development of on– off spiking in superior paraolivary nucleus neurons of the mouse. J Neurophysiol 2013;109:2691–704. [57] Bian C, Zhao Y, Guo Q, Xiong Y, Cai W, Zhang J. Aromatase inhibitor letrozole downregulates steroid receptor coactivator-1 in specific brain regions that primarily related to memory, neuroendocrine and integration. J Steroid Biochem Mol Biol 2014;141C:37–43. [58] Rhen T, Crews D. Distribution of androgen and estrogen receptor mRNA in the brain and reproductive tissues of the leopard gecko, Eublepharis macularius. J Comp Neurol 2001;437:385–97. [59] Portillo W, Diaz NF, Cabrera EA, Fernandez-Guasti A, Paredes RG. Comparative analysis of immunoreactive cells for androgen receptors and oestrogen receptor alpha in copulating and non-copulating male rats. J Neuroendocrinol 2006;18:168–76. [60] Navarro A, Del Valle E, Ordonez C, Martinez E, Perez C, Alonso A, et al. Aging and substitutive hormonal therapy influence in regional and subcellular distribution of ERalpha in female rat brain. Age (Dordr) 2013;35:821–37.

[61] DonCarlos LL, Garcia-Ovejero D, Sarkey S, Garcia-Segura LM, Azcoitia I. Androgen receptor immunoreactivity in forebrain axons and dendrites in the rat. Endocrinology 2003;144:3632–8. [62] Choate JV, Slayden OD, Resko JA. Immunocytochemical localization of androgen receptors in brains of developing and adult male rhesus monkeys. Endocrine 1998;8:51–60. [63] He F, Tai F, Zhang Y, Zhang X. Effects of castration on aggression and levels of serum sex hormones and their central receptors in mandarin voles (Microtus mandarinus). J Comp Physiol A Neuroethol Sens Neural Behav Physiol 2012;198:347–62. [64] Stanic D, Dubois S, Chua HK, Tonge B, Rinehart N, Horne MK, et al. Characterization of aromatase expression in the adult male and female mouse brain. I. Coexistence with oestrogen receptors alpha and beta, and androgen receptors. PLoS One 2014;9:e90451. [65] Waragai M, Yamada T. Moroo I [The distribution of androgen receptor like protein in the central nervous system of rat and human medulla and spinal cord–immunohistochemical study]. No To Shinkei 1993;45:661–7. [66] Blurton-Jones MM, Roberts JA, Tuszynski MH. Estrogen receptor immunoreactivity in the adult primate brain: neuronal distribution and association with p75, trkA, and choline acetyltransferase. J Comp Neurol 1999;405:529–42. [67] Mufson EJ, Cai WJ, Jaffar S, Chen E, Stebbins G, Sendera T, et al. Estrogen receptor immunoreactivity within subregions of the rat forebrain: neuronal distribution and association with perikarya containing choline acetyltransferase. Brain Res 1999;849:253–74. [68] Lu SF, McKenna SE, Cologer-Clifford A, Nau EA, Simon NG. Androgen receptor in mouse brain: sex differences and similarities in autoregulation. Endocrinology 1998;139:1594–601. [69] Bian C, Zhao Y, Guo Q, Xiong Y, Cai W, Zhang J. Aromatase inhibitor letrozole downregulates steroid receptor coactivator-1 in specific brain regions that primarily related to memory, neuroendocrine and integration. J Steroid Biochem Mol Biol 2014;141:37–43. [70] Fan X, Warner M, Gustafsson JA. Estrogen receptor beta expression in the embryonic brain regulates development of calretinin-immunoreactive GABAergic interneurons. Proc Natl Acad Sci USA 2006;103:19338–43. [71] Ravizza T, Galanopoulou AS, Veliskova J, Moshe SL. Sex differences in androgen and estrogen receptor expression in rat substantia nigra during development: an immunohistochemical study. Neuroscience 2002;115:685–96.