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

Tertia D Purves-Tyson, David J Handelsman, Kay L Double, Samantha J Owens, Sonia Bustamante, Cynthia Shannon Weickert, Tertia D Purves-Tyson, David J Handelsman, Kay L Double, Samantha J Owens, Sonia Bustamante, Cynthia Shannon Weickert

Abstract

Background: Increased risk of schizophrenia in adolescent males indicates that a link between the development of dopamine-related psychopathology and testosterone-driven brain changes may exist. However, contradictions as to whether testosterone increases or decreases dopamine neurotransmission are found and most studies address this in adult animals. Testosterone-dependent actions in neurons are direct via activation of androgen receptors (AR) or indirect by conversion to 17β-estradiol and activation of estrogen receptors (ER). How midbrain dopamine neurons respond to sex steroids depends on the presence of sex steroid receptor(s) and the level of steroid conversion enzymes (aromatase and 5α-reductase). We investigated whether gonadectomy and sex steroid replacement could influence dopamine levels by changing tyrosine hydroxylase (TH) protein and mRNA and/or dopamine breakdown enzyme mRNA levels [catechol-O-methyl transferase (COMT) and monoamine oxygenase (MAO) A and B] in the adolescent male rat substantia nigra. We hypothesized that adolescent testosterone would regulate sex steroid signaling through regulation of ER and AR mRNAs and through modulation of aromatase and 5α-reductase mRNA levels.

Results: We find ERα and AR in midbrain dopamine neurons in adolescent male rats, indicating that dopamine neurons are poised to respond to circulating sex steroids. We report that androgens (T and DHT) increase TH protein and increase COMT, MAOA and MAOB mRNAs in the adolescent male rat substantia nigra. We report that all three sex steroids increase AR mRNA. Differential action on ER pathways, with ERα mRNA down-regulation and ERβ mRNA up-regulation by testosterone was found. 5α reductase-1 mRNA was increased by AR activation, and aromatase mRNA was decreased by gonadectomy.

Conclusions: We conclude that increased testosterone at adolescence can shift the balance of sex steroid signaling to favor androgenic responses through promoting conversion of T to DHT and increasing AR mRNA. Further, testosterone may increase local dopamine synthesis and metabolism, thereby changing dopamine regulation within the substantia nigra. We show that testosterone action through both AR and ERs modulates synthesis of sex steroid receptor by altering AR and ER mRNA levels in normal adolescent male substantia nigra. Increased sex steroids in the brain at adolescence may alter substantia nigra dopamine pathways, increasing vulnerability for the development of psychopathology.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
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.
Figure 5
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.
Figure 6
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.

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