Androgen and estrogen receptor-mediated mechanisms of testosterone action in male rat pelvic autonomic ganglia

Abstract

Although male reproductive function is primarily androgen dependent, many studies suggest that estrogens have direct actions on the male reproductive organs. Pelvic autonomic neurons provide the motor control of the internal reproductive organs and the penis and various properties of these neurons are affected by endogenous androgens. However, the possible role of estrogens at this site has not been examined. Here we have investigated the significance of estrogens produced by aromatization of testosterone (T) in the physiological actions of androgens on adult male rat pelvic ganglion neurons. Reverse transcriptase polymerase chain reaction (RT-PCR) studies showed that aromatase and both estrogen receptors (ERalpha and ERbeta) are expressed in these ganglia. Western blotting also showed that aromatase is expressed in male pelvic ganglia. Using immunohistochemical visualization, ERalpha was predominantly expressed by nitric oxide synthase (NOS)-positive parasympathetic pelvic ganglion neurons. In vivo studies showed that the decrease in pelvic ganglion soma size caused by gonadectomy could be prevented by administration of T or dihydrotestosterone (DHT), but not 17beta-estradiol (E2), showing that this maintenance action of testosterone is mediated entirely by androgenic mechanisms. However, in vitro studies of cultured pelvic ganglion neurons revealed that T, DHT and E each stimulated the growth of longer and more complex neurites in both noradrenergic and cholinergic NOS-expressing neurons. The effects of T were attenuated by either androgen or estrogen receptor antagonists, or by inhibition of aromatase. Together these studies demonstrate that estrogens are likely to be synthesized in the male pelvic ganglia, produced from T by local aromatase. The effects of androgens on axonal growth are likely to be at least partly mediated by estrogenic mechanisms, which may be important for understanding disease-, aging- and injury-induced plasticity in this part of the nervous system.

Figure 1. Aromatase and estrogen receptors in male rat pelvic ganglion

1. Aromatase RT-PCR yielded a 102 bp band in prostate and pelvic ganglion but not skeletal muscle; lane 1, 100 bp ladder; 2, blank; 3, prostate; 4, pelvic ganglion; 5, skeletal muscle.

2. ER RT-PCR yielded a 345 bp band for ERα and a 262 bp for ERβ in pelvic ganglion but only the ERβ transcript was present in prostate; lane 1, 100 bp ladder; 2, pelvic ganglion GAPDH (207 bp); 3, pelvic ganglion ERβ (262̣ bp); 4, prostate ERβ; 5, pelvic ganglion ERα(345 bp); 6, prostate ERα; 7 blank, ERα primers; 8, prostate GAPDH. For all primer sets, experiments were repeated twice.

3. Aromatase protein was identified in extracts of pelvic ganglia from three rats, each showing a single band at ∼55 kD.

4. Triple staining immunofluorescence showing examples of ERα expression in noradrenergic (TH-positive) and nitrergic (NOS-positive) pelvic ganglion neurons. Arrows show examples of ERα expression in NOS-positive, TH-negative neurons, and arrowheads show examples of weaker ERα expression in TH-positive, NOS-negative neurons. In merged colour images, ERα is red, NOS is green and TH is blue. Calibration in D represents 20 μm.

Figure 2. Effects of gonadectomy and steroid replacement on seminal vesicle weight and size of pelvic ganglion somata

1. Seminal vesicles (mean of paired seminal vesicles in each animal) were much smaller after gonadectomy (n=5, “control”) compared to naïve intact animals (n=4) and this effect was completely prevented by testosterone (T, n=6), partly prevented by dihydrotestosterone (DHT, n=5) but unchanged by estradiol (E2, n=6). *, significantly different from control and from E2, P<0.001; not indicated, but DHT vs. naïve or T, P<0.001.

2. Size of NOS-immunoreactive somata decreased after gonadectomy (“control”) and this effect was completely prevented by T or DHT but not by E2. Data is from the same animals as shown in A. *, significantly different from control and from E2, P<0.05.

3. Size of TH-immunoreactive somata decreased after gonadectomy (“control”) and this effect was completely prevented by T or DHT but not by E2. Data is from the same animals as shown in A. *, significantly different from control and from E2, P<0.001.

Figure 3. Effects of estradiol (E2), testosterone (T) and dihydrotestosterone (DHT) on neurite outgrowth in vitro

Typical examples of NOS- or TH-positive pelvic ganglion neurons cultured overnight in the absence of steroid (control) or steroid (E2, 10 nM; T and DHT, 100 nM). Each steroid caused an increase in neurite complexity. Images have been inverted to show neurite structure more clearly.

Figure 4. Effects of testosterone (T), dihydrotestosterone (DHT) and estradiol (E2) on neurite complexity in vitro

1. In NOS neurons, all three steroids increased neurite branching, both androgens increased neurite length and only DHT increased primary neurite number.

2. In TH neurons, all three steroids increased neurite length and branching but only DHT increased primary neurite number.

Groups significantly different from control are indicated: *P<0.05, **P<0.01

Figure 5. Testosterone (T)-stimulated neurite growth involves both androgen and estrogen receptors

1. In NOS neurons, T (10 nM) caused an increase in neurite length (P<0.01), branching (P<0.001) and number (P<0.05) compared with untreated controls. The effects on length and branching were blocked by the AR blocker, hydroxyflutamide (HF, 10μM; T vs. T/HF, P<0.01 and P<0.001 for length and branching respectively) and by the ER blocker, ICI182780 (ICI, 10μM; T vs. T/ICI, P<0.01 for both parameters). The modest effects of T on neurite number appeared to be reduced by each blocker but neither effect reached statistical significance. Groups significantly different from T are indicated, *P<0.05, **P<0.01, ***P<0.001.

2. In TH neurons, T (10 nM) caused an increase in neurite length (P<0.05), and number (P<0.05) compared with untreated controls. A small effect of T on neurite branching appeared to occur but this was not statistically significant compared with controls; however, an effect of T on this parameter was indicated by the difference between T and T/HF groups (P<0.001). The effects on length and number were blocked by the AR inhibitor, hydroxyflutamide (HF, 10μM; T vs. T/HF, P<0.001 for length and P<0.05 for number). The ER blocker, ICI182780 (ICI, 10μM) had no significant effect on the T-induced increase in neurite length and branching. Groups significantly different from T are indicated, *P<0.05, ***P<0.001.

C, D. Estradiol (E2, 10 nM) actions on neurite growth were blocked by ICI182780 (ICI, 10μM). E2 caused a significant increase in neurite length and neurite branching in both NOS (C) and TH (D) neurons. Both effects in both neuron types were abolished with ICI182780. E2 did not affect the number of primary neurites. Groups significantly different from E2 are indicated, *, P<0.05, **, P<0.01.

Figure 6. Effects of aromatase inhibition on testosterone responses

1. In NOS neurons, both T and DHT (each 10nM) stimulated neurite length and branching. The aromatase inhibitor letrozole (L, 100 nM) appeared to partially reduce the effects of testosterone (10 nM) on neurite length and branching but T/Lwas not significantly different to either control or T alone. Letrozole had no effect on the response to DHT (10 nM). In this group of experiments, androgens had weak or no effects on the number of primary neurites, but it should be noted that the controls showed a relatively high number of primary neurites compared with experiments shown in Figure 5. Groups significantly different from control are indicated, * P< 0.05, ** P < 0.01.

2. In TH neurons, the effects of T on neurite length and number of primary neurites were abolished by letrozole (L, 100 nM) (T vs T/L, P < 0.05), whereas the effect on neurite branching appeared to be only slightly reduced, i.e. T/L not significantly different to either control or T alone. The effects of DHT were unchanged by letrozole. Groups significantly different from control are indicated, * P< 0.05, ** P < 0.01. Groups significantly different from T are indicated, # P < 0.05.