Testosterone regulation of sex steroid-related mRNAs and dopamine-related mRNAs in adolescent male rat substantia nigra

Animal experiments

All animal experiments were approved by the Animal Care and Ethics Committee of the University of New South Wales in accordance with the National Health and Medical Research Council of Australia’s Code of Practice for the Care and Use of Animals for Experimental Purposes, which also conforms to standard international guidelines. Male Sprague–Dawley rats were used for all experiments (Animal Resource Centre, Perth, WA, Australia). Rats were group housed (3-4/cage) in 12/12 hr light/dark phases with constant humidity and temperature and free access to water and standard rat chow.

Gonadectomy and sex steroid replacement

Adolescent male rats were gonadectomised at 45 days of age, prior to the adolescent testosterone increase, and given continuous replacement testosterone (T), dihydrotestosterone (DHT) or 17β-estradiol (estradiol, E) by subdermal silastic implant [22-24] for two weeks. By this age, intact animals have experienced the increase in testosterone associated with adolescence. There were five groups (~15 rats per group): intact (Intact), gonadectomy alone (Gdx); gonadectomy plus testosterone (Gdx+T); gonadectomy plus DHT (Gdx+DHT), gonadectomy plus estradiol (Gdx+E). Male rats (45 days old) were anaesthetized with an intraperitoneal (i.p.) injection of ketamine hydrochloride (60 mg/kg) and xylazine hydrochloride (10 mg/kg) (Provet, Castle Hill, Australia). The intact animals underwent sham abdominal surgery but gonads were left in place. Silastic implants were placed under the skin between the shoulder blades at time of gonadectomy. Gdx and Intact groups were given empty implants. Implants were 1 cm long, internal diameter 1.47 mm, outer diameter 1.95 mm (ends sealed with silastic adhesive). These implants have been characterized in previous studies and achieve supraphysiological, steady-state hormone levels and maintain seminal vesicle weights equal to that in untreated animals [23,25]. Seminal vesicles depend on androgen action for normal development and maintenance of structural and functional integrity [26]. Weight of seminal vesicles at the end of experiment served as an index of the restoration of androgen action by the implants as previously reported [25].

At 60 days of age rats were anaesthetized with 60 mg/kg sodium pentobarbital (Euthal, Delvet, Seven Hills, Australia). Brain was removed from the skull and a tissue block containing the midbrain was dissected following the Rat Brain Atlas as a guide [27]. The block was trimmed transversely at the cerebral aqueduct and two lateral segments of midbrain on either side of the ventral tegmental area (VTA) containing the SN were collected. Trunk blood was collected on day of euthanasia in 0.8 ml serum gel tubes (Z serum sep MiniCollect tube, Greiner Bio One, Wemmel, Belgium) and serum collected by centrifugation. Left and right hemisphere midbrain segments including SN were randomly assigned for either protein or RNA extraction.

Immunohistochemistry

For immunohistochemical studies five normal intact male rats (50–53 days old) were deeply anaesthetized with pentobarbital (Euthal) followed by transcardial perfusion with 4% paraformaldehyde (PFA, pH 7.2). Midbrain was dissected in the coronal plane and post-fixed (4% PFA) overnight, washed with phosphate buffered saline (PBS) and cryoprotected in 30% sucrose/PBS.

Midbrain sections (40 μm) were collected in PBS and sections from each brain between Bregma −5.52 and −5.64 mm was incubated for 1 h with 10% horse serum (Sigma) and 0.1% Triton X-100 (Sigma) in PBS. This was followed by incubation with primary antibodies overnight at 4°C, washes and incubation with fluorescence-conjugated secondary antibodies for 2 h at room temperature. The antisera used were: TH (host species mouse, 1:500; MAB318, Millipore, Temecula, CA, USA) or TH (host species rabbit, 1:500; AB152, Millipore), ERα (host species mouse, 1:50; 6 F-11, Nova Castra, Leica-Microsystems, Wetzlar, Germany) and AR (host species rabbit, 1:200, PA1-110, Thermo Scientific, Australia, Scoresby, Victoria). ERβ immunohistochemistry was not performed as there are currently no well-validated commercial ERβ antibodies available. Primary antibody binding was visualized with fluorophore-conjugated species-specific secondary antibodies (donkey-anti-mouse-Alexa Fluor488 and 594, donkey-anti-rabbit-Alexa Fluor488 and 594, all 1:500, Life Technologies, Carlsbad, CA, USA). Sections incubated with only secondary antibody or with each primary antibody with the alternate (incorrect) secondary antibody confirmed that no fluorescent signal was due to non-specific or cross-over binding. Nuclei were visualized by incubation with 1 μg/ml DAPI (Sigma). Sections were mounted with glycerol/PBS (Citifluor AF1, London, UK). Sections were viewed under a Nikon Eclipse 80i fluorescence microscope (Nikon) with a CX9000 digital camera attached (MBF Biosciences, Williston, VT, USA). SN and VTA were demarcated under low power (4x) using StereoInvestigator (v8.27, MBF Biosciences) and referring to a Rat Brain Atlas [27]. Under higher power (20x) using StereoInvestigator, TH positive cells (100–200) were identified within the SN pars compacta, they were then assessed for receptor immunoreactivity and percentage of TH positive neurons expressing either receptor was determined. Images were also collected on a Nikon C1 laser scanning confocal unit (Nikon D-Eclipse C1, Nikon Australia) attached to a fluorescence microscope (Nikon Eclipse 90i). Images were acquired digitally and processed using the software EZ-C1 (v 3.5) for Nikon C1 confocal microscopes (Nikon Australia).

Immunoblotting

The substantia nigra blocks were homogenized [0.1 M Tris, pH 7.5, 50% glycerol, proteinase inhibitor cocktail (Sigma Cat# P8340) and aprotinin (0.015 mM, Sigma, St Louis, MO, USA)] using a handheld electric homogenizer (Polytron, Kinematica, Lucerne, Switzerland). Protein concentration was determined using the Bradford protein assay (Sigma). An aliquot of each sample was combined and used as a standard and run in duplicate on each gel to allow standardization between blots (internal control). Standard curves with between 0.5 and 20 μg SN protein were run and TH expression was determined to be within a linear range using 3 μg protein. SDS-PAGE (10% acrylamide) was performed on 3 μg of protein per sample. Proteins were transferred to nitrocellulose (45 μm, Biorad, CA, USA). Primary antibodies were anti-TH (host species mouse, 1:5000; Chemicon) and anti-β-actin (host species rabbit, 1:5000; MAB1501, Millipore). Secondary antibodies were goat anti-mouse or anti-rabbit horseradish peroxidase (HRP) conjugated (1:4000, Millipore). Immunoreactive bands were detected using the LumiGlo detection kit (LumiGlo Reagent; Cell Signaling, Danvers, MA, USA) on hyperfilm (Amersham, GE Healthcare, Uppsala, Sweden). The immunoblots were scanned and band densities converted to numerical values using ImageJ software (ImageJ 1.43u, National Institutes of Health, USA). Relative intensity of TH bands were normalized to relative intensity of β-actin bands.

RNA Extraction and cDNA Synthesis

Total RNA was extracted from substantia nigra samples in 800–1000 μl TRIzol reagent (Life Technologies Inc., Grand Island, NY, USA) with a handheld electric homogenizor (Polytron) as recommended by the manufacturer. RNA was quantified using a ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, USA). RNA integrity (RIN) was assessed with high resolution capillary electrophoresis (Agilent Bioanalyzer 2100, Agilent Technologies, Palo Alto, CA, USA). Two separate aliquots of 3 μg RNA from each sample were reverse transcribed to cDNA with SuperScript III First-Strand Synthesis Supermix and random hexamers, according to the manufacturer’s protocol (Life Sciences). A parallel reaction was performed without reverse transcriptase as a negative control. The 2 aliquots of cDNA from each sample were pooled and diluted for RT-PCR.

Quantitative Real-Time PCR (qPCR)

Messenger RNA (mRNA) levels of genes of interest were measured by TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) using an ABI Prism 7900HT Fast Real-Time PCR System and a 384-well format. Expression of three housekeeping genes, beta-glucuronidase (GusB), 18 S ribosomal RNA (18 S rRNA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), was used to calculate the normalizing control for gene expression (termed geometric mean), and were selected on the basis that they were unchanged by the treatment. The Taqman probes used were: GusB (Rn00566655); 18 s rRNA (Hs99999901); GAPDH (Rn01775763). The geometric mean of the three housekeeping genes was calculated as described previously [28]. There were no group differences when the geomean of the three housekeepers was compared (F=1.67, df=4, p=0.168). Genes of interest were targeted by the following Taqman probes (Applied Biosystems): TH (Rn00562500), COMT (Rn00561037), MAOA (Rn01430950), MAOB (Rn00566203), ERα (Rn01640372), ERβ (Rn00562610), AR (Rn00560747) and Cyp19A1 (Rn00567222), 5αR-1 (Rn00567064) and 5αR-2 (Rn00575595, 5αR-2 mRNA levels in midbrain were too low to be accurately quantitated). Samples were run alongside a seven point standard curve using serial dilutions of cDNA derived from midbrain RNA pooled from a subset of 25 rats (taken from all treatment groups). No template controls were included which produced no signal for any mRNA examined. Measurements were performed in triplicate. PCR cycling conditions were: 50°C for 2 minutes, 95°C for 10 minutes, 50 cycles of 95°C for 15 seconds and 60°C for 1 minute. PCR data were captured with Sequence Detector Software (SDS version 2.4, Applied Biosystems). SDS software plotted real-time fluorescence intensity and the threshold was set within the linear phase of the amplification profiles.

Sex steroid measurements

Testosterone (T), dihydrotestosterone (DHT) and 17β-estradiol (estradiol, E) were quantified in extracts of serum using a stable isotope dilution liquid chromatography-tandem mass spectroscopy (LC-MS/MS) method as described [29] and adapted for rodents [30]. The limit of quantitation for this method is 0.025 ng/ml T, 0.1 ng/ml DHT and 5 pg/ml E. T and DHT were also measured in serum extracts using a gas chromatography MS (GC-MS) method adapted from Horning et al., [31] (data presented in additional material).

Statistics

Unless otherwise stated statistical analyses were conducted using SPSS software (IBM SPSS Statistics, version 19) and p < 0.05 was considered statistically significant. Seminal vesicle and body weight (g) data are shown as mean ± standard error of the mean (SEM). Immunoblotting data are presented as percent change of relative intensity compared to the Gdx group ± SEM. Immunoblotting data were normalized to β-actin expression and population outliers removed by performing Grubbs test for outliers (GraphPad Software). Data was then analyzed by one-way ANOVA followed by Fisher’s least significant differences (LSD), post hoc.

QPCR data are presented as percent change of mRNA levels relative to the Gdx group ± SEM. Outlier detection of the triplicates obtained from the qPCR raw data was used to exclude measurement errors [32]. qPCR raw data was normalized by the geomean of the housekeepers. Population outliers were removed by performing Grubb’s test on the normalized data and data were analyzed by one-way ANOVA followed by Fisher’s LSD.

Confirmation of sex steroid replacement by organ weight

There were no significant differences in overall body growth between any of the five experimental groups (the mean - of all 5 groups - increase in body weight over the course of study was 44.0 ± 2.0%) (Figure 1A). There were no significant group differences in brain weight (results not shown). There was a significant effect of treatment group when examining seminal vesicle weights (F=149.2, df=4, p < 0.0001). Gdx reduced seminal vesicles to 9.0% weight of Intact group (Figure 1B). Seminal vesicles were maintained at 94.2% of intact weights by replacement testosterone and at 44% of intact weights by replacement DHT. Estradiol replacement had no effect on seminal vesicle weights such that the Gdx+E group was not significantly different to the Gdx group.

Figure 1

Effect of gonadectomy and sex steroid replacement on body weights and seminal vesicle weights in adolescent male rats.(A) There were no significant differences in overall growth between any of the five experimental groups (F=2.09, df=4, 221, p > 0.05). (B) Seminal vesicles weighed less following gonadectomy (n=16) compared with Intact rats (n=15) and this effect was completely prevented by T replacement (n=17), partly prevented by DHT (n=14) but unchanged by E (n=15). * denote comparisons with Intact, # denote comparisons with Gdx, ### and ***=p < 0.0001.

Confirmation of sex steroid replacement by blood levels

DHT and T were measured by LC-MS/MS and GC-MS methods. Estradiol was detectable only by LC-MS/MS. Comparisons of GC-MS and LC-MS/MS androgen measurements in sera samples are included in additional material (see Additional Material File 1). As LC-MS/MS proved to require less serum (200 μl) and was able to detect circulating estradiol we report these measurements. Average circulating serum testosterone was 0.03 ± 0.001 ng/ml in the Gdx group (n=9), 2.8 ± 0.6 ng/ml (n=12) in the Intact group and 23.1±12.0 ng/ml in the Gdx+T group (n=14). Average circulating serum DHT was not detectable in the Gdx group (n=8), 0.2 ± 0.03 ng/ml in the Intact group (n=7), and 21.42 ± 10.6 ng/ml in the Gdx+DHT group (n=12). Circulating serum estradiol was 7.3 ± 0.15 pg/ml (duplicate measure of 8 pooled samples) in the Intact group and 17.0 ± 6.7 pg/ml in the Gdx+E group (n=8).

Anatomical co-localization of AR and ER and TH in substantia nigra dopamine neurons

Co-localization of TH and sex steroid receptors was indicated by AR immunoreactivity in ~65.5 ± 3.5% of TH positive (TH+) neurons and ERα-immunoreactivity in ~39.9 ± 4.4% of TH+ neurons in the normal intact male adolescent substantia nigra (50–53 days old, n=5) (Figure 2), indicating that many dopamine neurons in the substantia nigra could respond directly to sex steroids. Sex steroid receptor immunoreactivity was also present in TH negative cells.

Figure 2

AR and ERα immunoreactivity in dopamine neurons of the adolescent male rat substantia nigra. Midbrain tissue sections (approximately bregma −5.52) were probed with antibodies raised against tyrosine hydroxylase (TH) and either androgen receptor (left column) or estrogen receptor α (right column). Blue staining indicates DAPI labeled nuclei (top row). Arrowheads indicate TH positive neurons (green) that are also immuno-positive for sex steroid receptors (red). Neurons with co-localized sex steroid receptors and TH immunostaining appear yellow in the merged images (bottom row, arrowheads). Stars indicate TH negative, sex steroid receptor positive (red) neurons, solid arrow indicates a TH positive, ERα negative neuron. Scale bar=20 μm.

Regulation of substantia nigra TH protein by sex steroids

TH protein was detected at the expected molecular weight of 60 kDa and β-actin at 43 kDa (Figure 3A). There was a significant effect of treatment group on TH/β-actin protein levels in the substantia nigra in adolescent male rats (F=4.43, df=4, p=0.0043). TH protein in the substantia nigra was increased 70% in the Gdx+T group relative to the Gdx group and 106% relative to the Intact group (Figure 3B). There were no significant differences in TH protein levels between Intact, Gdx, Gdx+DHT or Gdx+E groups.

Figure 3

Effect of gonadectomy and sex steroid replacement on TH protein and mRNA in adolescent male rats.(A) Representative immunoblots of a subset of substantia nigra samples from all five groups showing TH protein (60 kDa) and β-actin protein (43 kDa). (B) TH protein was increased by testosterone (n=10) compared to Intact (n=10) and Gdx (n=10) groups, whereas DHT (n=8) and E (n=10) did not increase TH protein. Gdx+T was significantly different to Gdx+DHT and Gdx+E, ***=p < 0.001, and significantly different to Gdx and Intact, p < 0.05 and p < 0.01, respectively. (C) TH mRNA was measured using real time qPCR. Midbrain TH mRNA relative expression was not significantly different according to treatment group.

Regulation of substantia nigra TH mRNA by sex steroids

There was no overall effect of treatment group on TH mRNA (F=1.97, df=4, p=0.109) in the SN of adolescent male rats. Fisher’s LSD post hocs were run as we hypothesized, a priori, that there would be an increase in TH mRNA with testosterone. There was a trend for TH mRNA to be increased in the Gdx+T group when compared to the Intact (13.6%) and Gdx (15.4%) groups (intact vs. Gdx+T; p=0.073; Gdx vs. Gdx+DHT, p=0.092) (Figure 3C).

Modulation of dopamine metabolite enzyme mRNAs by sex steroids

There was a significant effect of treatment group on COMT (F=6.65, df=4, p < 0.0001), MAOA (F=9.983, df=4, p < 0.0001) and MAOB (F=5.35, df=4, p < 0.001) mRNA expression in the adolescent male rat substantia nigra. All three breakdown enzyme mRNAs were significantly increased in the Gdx+T and Gdx+DHT groups when compared to the Intact or Gdx groups (all p’s <0.05). None of the breakdown enzyme mRNA expression levels were changed in the Gdx+E group compared to the Intact or Gdx groups (Figure 4A, B, C).

Figure 4

Effect of gonadectomy and sex steroid replacement on dopamine metabolic enzyme COMT, MAOA and MAOB mRNA in the substantia nigra of adolescent male rats.(A) COMT mRNA levels were significantly increased by T and DHT when compared to both Gdx and Intact groups. (B) MAOA mRNA and (C) MAOB mRNA levels were significantly increased by T and DHT when compared to both Gdx and Intact groups. * denotes comparisons with the Intact group, # denotes comparisons with the Gdx group. * and #=p < 0.05, ** and ##=p < 0.01, *** and ###=p < 0.0001.

Modulation of androgen and estrogen receptor mRNAs by sex steroids

We detected a significant effect of treatment group on AR mRNA expression (F=11.3, df=4, p < 0.0001) in the male SN. AR mRNA was increased by 45%, 32.5% and 26% in the Gdx+T, Gdx+DHT and Gdx+E groups, respectively, compared to the Gdx group and 39.8%, 27.7% and 21.4% in the Gdx+T, Gdx+DHT and Gdx+E groups, respectively, compared to the Intact group (Figure 5A). There was no significant difference in AR mRNA relative expression between the Intact and Gdx groups. We also found a significant effect of treatment on ERα mRNA expression (F=2.8, df=4, p=0.032) in the male SN. ERα mRNA was significantly decreased 24.3% in the Gdx+DHT group compared to the Intact group. There were no significant differences in ERα mRNA relative expression between GDX+T and GDX+E relative to the Gdx and Intact groups. The decrease in ERα mRNA relative expression (14.2%) in the Gdx group compared to the Intact group did not reach statistical significance (Intact vs. Gdx, p=0.089) (Figure 5B). We detected an effect of treatment group on ERβ mRNA expression in the male adolescent SN (F=2.6, df=4, p=0.045). ERβ mRNA expression was significantly increased in the Gdx+T (27%) and Gdx+DHT (20%) groups (Figure 5C).

Figure 5

The effect of gonadectomy and sex steroid replacement on sex steroid receptor mRNA expression in adolescent male rat substantia nigra.(A) AR mRNA levels were increased by T, DHT and E compared to Intact (*) and Gdx (#) groups (n=12-14/group). (B) ERα mRNA was decreased by DHT replacement compared to the Intact group. T and E did not change ERα mRNA levels and were significantly different to the DHT treated group. (C) ERβ mRNA was increased by T (compared to Intact and Gdx groups) and by DHT (compared to Gdx only), but not by E. *=p < 0.05, **=p < 0.01, *** and ###=p < 0.0001.

Modulation of sex steroid conversion enzyme mRNAs by sex steroids

We found a significant effect of treatment group on Cyp19A1 mRNA (F=2.8, df=4, p=0.034) with decreased expression (40.4%) in the Gdx group compared to the Intact group and this was not reversed by replacement of any of the three sex steroids compared to Gdx and all replacement groups were significantly decreased compared to Intact group (Figure 6A). We also found a significant effect of treatment group on 5αR-1 mRNA expression (F=3.76, df=4, p=0.009). Relative expression of 5αR-1 mRNA was increased by 17.4% in the Gdx+DHT group when compared to the Intact group. There was a trend for 5αR-1 mRNA expression to be increased (9.7%) in the Gdx+T group compared to the Intact group (intact vs. Gdx+T, p=0.07) and 5αR-1 was unchanged by estradiol (Figure 6B).

Figure 6

The effect of gonadectomy and sex steroid replacement on steroid conversion enzyme mRNA expression in adolescent male rat substantia nigra.(A). Cyp19A1 mRNA was decreased by gonadectomy and this was not prevented by T, DHT or E replacement. * denotes comparisons with Intact, * = p < 0.05, ** = p < 0.01 (B). 5αR-1 mRNA was increased by DHT compared to the Intact group but not by T or E replacement. ** p = < 0.01.