Localization and regulation of reproductive steroid receptors in the raphe serotonin system of male macaques

Abstract

We previously showed that tryptophan hydroxylase 2 (TPH2) and serotonin reuptake transporter (SERT) mRNAs are increased by the androgens, testosterone (T) and dihydrotestosterone (DHT) in serotonin neurons of male macaques. In addition, we observed that serotonin in axons of a terminal region were markedly decreased by aromatase inhibition and lack of estradiol (E) from metabolism of T. These observations implicated androgen receptors (AR) and estrogen receptors (ER) in the transduction of steroid hormone actions in serotonin neurons. Due to the longer treatment period employed, the expression of the cognate nuclear receptors was sought. We used single and double immunohistochemistry to quantitate and phenotypically localize AR, ERα and ERβ in the dorsal raphe of male macaques. Male Japanese macaques (Macaca fuscata) were castrated for 5-7 months and then treated for 3 months with [1] placebo, [2] T, [3] DHT (non-aromatizable androgen) plus ATD (steroidal aromatase inhibitor), or [4] Flutamide (FLUT; androgen antagonist) plus ATD (n = 5/group). After single labeling of each receptor, quantitative image analysis was applied and receptor positive neurons were counted. Double-label of raphe neurons for each receptor plus TPH2 determined whether the receptors were localized in serotonin neurons. There were significantly more AR-positive neurons in T- and DHT+ATD-treated groups (p = 0.0014) compared to placebo or FLUT+ATD-treated groups. There was no difference in the number of positive-neurons stained for ERα or ERβ⋅ Double-immunohistochemistry revealed that serotonin neurons did not contain AR. Rather, AR-positive nuclei were found in neighboring cells that are likely neurons. However, approximately 40% of dorsal raphe serotonin neurons contained ERα or ERβ⋅ In conclusion, the stimulatory effect of androgens on TPH2 and SERT mRNA expression is mediated indirectly by neighboring neurons contain AR. The stimulatory effect of E, derived from T metabolism, on serotonin transport is partially mediated directly via nuclear ERs.

Keywords: Androgen receptors; Estrogen receptors; Macaque; Male; Serotonin.

Figure 1. Photomicrographs of ERα in the endometrial glands of a female macaque (top, arrows) and ERβ in the prostate gland of a male macaque (bottom, arrows)

Top panels. Nuclear ERα was observed in an isolated endometrial gland (left), and in a gland that is surrounded by endometrial stromal tissue (right) with the commercial antibody used in this study (C1355, Millipore). Bottom panels. The prostate gland exhibits robust expression of nuclear ERβ with the commercial antibody used in this study of the brain (GTX70174, GeneTex). Black arrows point to positively stained nuclei.

Figure 2. Photomicrographs of AR, ERα and ERβ in the dorsal raphe of male monkeys

AR immunostaining at different magnifications is shown in panels A, B, and C. ERα immunostaining at different magnifications is shown in panels D, E, and F. ERβ immunostaining at different magnifications is shown in panels G, H and I. The insert (to panel I) shows the different types of cellular staining patterns observed for ERβ. Panels A, D, and G illustrate the widespread and dense receptor staining observed in the dorsal raphe region. Black arrowheads point to positively stained nuclei. White arrowhead points to a neuron with largely cytoplasmic staining for ERβ.

Figure 3. Quantitation of AR immunostaining in the dorsal raphe of male macaques

AR-positive pixel area and the number of AR-positive neurons are illustrated in the top and bottom panels, respectively. AR–positive pixel area and the number of AR-positive neurons were significantly increased in the T and DHT+ATD groups compared to placebo or FLUT+ATD. * significantly increased with Newman-Keuls posthoc comparison

Figure 4. Quantitation of ERα immunostaining in the dorsal raphe of male macaques

ERα-positive pixel area and the number of ERα-positive neurons are illustrated in the top and bottom panels, respectively. There was no difference in ERα-positive pixel area or -positive neurons between the groups.

Figure 5. Quantitation of ERβ immunostaining in the dorsal raphe of male macaques

ERβ-positive pixel area and the number of ERβ-positive neurons are illustrated in the top and bottom panels, respectively. There was no difference in ERβ-positive pixel area or -positive neurons between the groups.

Figure 6. Double immunolabelling for each steroid receptor and serotonin neurons using TPH as a specific marker

Panels A – B. AR + TPH staining is illustrated at different magnifications. Asterisks indicate TPH-positive neurons with an overlapping neuron containing an AR-positive nucleus. Panels C – D. ERα + TPH staining is illustrated at different magnifications Panels E – F. ERβ + TPH staining is illustrated at different magnifications. Black arrowheads point to steroid receptor positive nuclei that are not in serotonin neurons. Black arrows point to neurons that are double labeled for TPH and a steroid receptor. White arrows point to neurons that are single labeled for TPH.