Distribution of androgen receptor mRNA in the prepubertal male and female mouse brain

Abstract

Androgens are steroid hormones that play a critical role in brain development and sexual maturation by acting upon both androgen receptors (AR) and estrogen receptors (ERα/β) after aromatization. The contribution of estrogens from aromatized androgens in brain development and the central regulation of metabolism, reproduction, and behavior is well defined, but the role of androgens acting on AR has been unappreciated. Here, we map the sex specific expression of Ar in the adult and developing mouse brain. Postnatal days (PND) 12 and 21 were used to target a critical window of prepubertal development. Consistent with previous literature in adults, sex-specific differences in Ar expression were most profound in the bed nucleus of the stria terminalis (BST), medial amygdala (MEA) and medial preoptic area (MPO). Ar expression was also high in these areas at PND 12 and 21 in both sexes. In addition, we describe extra-hypothalamic and extra-limbic areas that show moderate, consistent and similar Ar expression in both sexes at both prepubertal time points. Briefly, Ar expression was observed in olfactory areas of the cerebral cortex, the hippocampus, several thalamic nuclei, and cranial nerve nuclei involved in autonomic sensory and motor function. To further characterize forebrain populations of Ar expressing neurons and determine whether they also coexpress estrogen receptors, we examined expression of Ar, Esr1 and Esr2 in prepubertal mice in selected nuclei. We found populations of neurons in the BST, MEA and MPO that coexpress Ar, but not Esr1 or Esr2, whereas others express a combination of the three receptors. Our findings indicate that various brain areas express Ar during prepubertal development and may play an important role in female neuronal development and physiology.

Keywords: gonadal steroids; postnatal development; puberty; sex differences.

FIGURE 1

Validation of androgen receptor (AR) immunohistochemistry and Ar in situ hybridization probe. (A, B) Fluorescent images showing AR‐immunoreactivity (AR‐IR) in the adult female mouse brain (postnatal day [PND] 56–70). AR‐IR was observed in sections incubated in primary antibody (A), but not in sections without primary antibody (B). (C, D) Darkfield images showing silver grain deposition corresponding to Ar hybridization signal in adjacent sections from the same brain (PND 12 male mouse). A signal was observed in sections hybridized with an antisense probe (C), but not with a sense probe (D). PMv, ventral premammillary nucleus; 3V, third ventricle. Scale bar = 100 µm (A, B), 200 µm (C, D)

FIGURE 2

Ar mRNA hybridization signal expression in male and female prepubertal and adult brain. Images from scanned autoradiographic film of adult, postnatal day [PND] 56–70 (A), and prepubertal, PND 12 (B) and PND 21 (C), male and female mouse brain. Select coronal sections are shown in rostral to caudal order. Darker signal indicates higher expression of Ar mRNA. Approximate distance from bregma (left column) derived from adult mouse brain (Paxinos and Franklin atlas). Scale bar = 4000 µm

FIGURE 3

Androgen receptor (AR) immunoreactivity (AR‐IR) in adult mouse brain. (A–H) Fluorescen images showing AR‐IR in the adult female mouse brain (postnatal day [PND] 56–70). AR‐IR was observed in almost all areas where we observed Ar mRNA. Selected areas from (A) cerebral cortex (dorsal and ventral anterior cingulate area, ACAd, ACAv), (B) hippocampal formation (pyramidal layer of fields CA1 and CA2), (C) cerebral nuclei (lateral septal nucleus, caudodorsal, LSc), (D) thalamus (paraventricular nucleus of the thalamus, PVT), (E) hypothalamus (subparaventricular zone, SBPV), (F) midbrain (periaqueductal gray, PAG), (G) pons/medulla (medial vestibular nucleus, MV) and (H) circumventricular organs (subfornical organ, SFO) are shown. AHN, anterior hypothalamic nucleus; alv, alveus; AQ, cerebral aqueduct; IG, induseum griseum; MS, medial septal nucleus; PRP, nucleus prepositus; PT, parataenial nucleus; 3V, third ventricle; 4V, fourth ventricle. Scale bar = 100 µm

FIGURE 4

Ar mRNA expression in cerebral cortex in prepubertal male and female mice. Images showing thionin staining for neuroanatomical reference (left), silver grains corresponding to Ar mRNA (right). Low Ar expression was observed in the piriform area (PIR) (A, B), dorsal and ventral anterior cingulate area (ACAd and ACAv) (C, D), induseum griseum and septohippocampal nucleus (IG and SH) (E, F), and CA3, with high expression in field CA1 and CA2 (G, H). ACB, nucleus accumbens; ccg, genu of corpus callosum; DG, dentate gyrus; lot, lateral olfactory tract; LS, lateral septal nucleus; MOs, secondary motor area; OT, olfactory tubercle. PND, postnatal day. Scale bar = 200 µm

FIGURE 5

Ar mRNA expression in cerebral nuclei of male and female prepubertal mice. Silver grain deposition corresponding to Ar mRNA hybridization signal in prepubertal postnatal day (PND) 12 (A, B, G, H) and PND 21 (D, E, J, K) male (M) (A, D, G, J) and female (F) (B, E, H, K) mice. (A–F) Lateral septal nucleus, caudodorsal (LSc) and (G–L) bed nucleus of the stria terminalis, principal nucleus (BSTpr). Bar graphs showing the mean ± SEM integrated optical density (IOD) of silver grains (C, F, I, L). IOD was analyzed by a t test with Welch's correction for LSc male vs. female PND 12 (p = .16, n = 7–8 per sex), PND 21 (p = .96, n = 8 per sex), BST male vs. female PND 12 (p = .39, n = 5–7 per sex), and BST male vs. female PND 21 (p = .75, n = 8 per sex). Abbreviations: cc, corpus callosum; LSr, lateral septal nucleus, rostral (rostroventral); MS, medial septal nucleus; PVT, paraventricular nucleus of the thalamus; RE, nucleus of reuniens; VL, lateral ventricle. NS, not significant. Scale bar = 200 µm

FIGURE 6

Ar mRNA expression in thalamic nuclei of male and female prepubertal mice. Images showing thionin staining for neuroanatomical reference (left), silver grains corresponding to Ar mRNA (right). (A, B) Low silver grain deposition in the paraventricular nucleus of the thalamus (PVT), with (C, D) low to moderate deposition in the nucleus of reuniens (RE), (E, F) ventral posterolateral and posteromedial nuclei of the thalamus (VPL and VPM), and (G, H) subthalamic and parasubthalamic nuclei (STN and PSTN). AD, anterodorsal nucleus of the thalamus; AV, anteroventral nucleus of the thalamus; cpd, cerebral peduncle; DG, dentate gyrus; em, external medullary lamina of the thalamus; fr, fasciculus retroflexus; ml, medial lemniscus; PF, parafascicular nucleus; RH, rhomboid nucleus; sm, stria medullaris; VM, ventral medial nucleus of the thalamus; ZI, zona incerta; 3V, third ventricle. PND, postnatal day. Scale bar = 200 µm

FIGURE 7

Ar mRNA expression in hypothalamic nuclei of male and female prepubertal mice. Silver grain deposition corresponding to Ar mRNA hybridization signal in prepubertal postnatal day (PND) 12 (A, B, I, J) and PND 21 (E, F, M, N) male (M) (A, E, I, M) and female (F) (B, F, J, N) mice. (A–H) Suprachiasmatic nucleus (SCH) and subparaventricular zone (SBPV), and (I–P) dorsal and ventral premammillary nuclei (PMd and PMv). Note the higher expression of Ar in the SCH of males at PND 21 (E). Bar graphs showing the mean ± SEM integrated optical density (IOD) of silver grains (C, D, G, H, K, L, O, P). IOD was analyzed by a t test with Welch's correction for SCH male vs. female PND 12 (p = .38, n = 5 per sex), SCH male vs. female PND 21 (p = .009, n = 6–7 per sex), SBPV male vs. female PND 21 (p = .45, n = 6–7 per sex), PMv male vs. female PND 21 (p = .21, n = 8–9 per sex), and PMd male vs. female PND 12 (p = .58, n = 7–8 per sex) and PND 21 (p = .19, n = 8–9 per sex), and a Mann–Whitney non‐parametric test for SBPV male vs. female PND 12 (p = .12, n = 6 per sex), and PMv male vs. female PND 12 (p = .57, n = 8 per sex). fx, fornix; 3V, third ventricle. NS, not significant. Scale bar = 200 µm

FIGURE 8

Ar mRNA expression in brainstem nuclei of prepubertal male and female mice. Images showing thionin staining for neuroanatomical reference (left), silver grains corresponding to Ar mRNA (right). (A, B) Very low to low silver grain deposition in the periaqueductal gray (PAG) and low deposition in the dorsal tegmental nucleus (DTN). (C, D) Low expression in the superior olivary complex (SOC) and (E, F) facial motor nucleus (VII). (G, H) Moderate expression in the nucleus ambiguus (AMB). (I, J) Low to moderate expression in the dorsal motor nucleus of the vagus nerve (DMX) and hypoglossal nucleus (XII). AP, area postrema; AQ, cerebral aqueduct; c, central canal of the spinal cord/medulla; DR, dorsal nucleus raphe; IRN, intermediate reticular nucleus; LRN, lateral reticular nucleus; MARN, magnocellular reticular nucleus; PRNc, pontine reticular nucleus, caudal part; PRNr, pontine reticular nucleus; py, pyramid; sctv, ventral spinocerebellar tract; VIIn, facial nerve. PND, postnatal day. Scale bar = 200 µm

FIGURE 9

Ar mRNA expression overlaps with Esr1 and Esr2 in specific forebrain nuclei of prepubertal mice. (A–P) Images showing fluorescent in situ hybridization signal for Ar (magenta, A, E, I, M), Esr1 (yellow, B, F, J, N) and Esr2 (green, C, G, K, O). Merge of all three channels shown in (D), (H), (L) and (P). Areas with Ar and Esr1 and/or Esr2 co‐expression include the bed nucleus of the stria terminalis, principal nucleus (BSTpr) (A–D), medial amygdalar nucleus, posterodorsal (MEApd) (E–H), medial preoptic area (MPO) (I–L) and suprachiasmatic nucleus (SCH) (M–P). Arrows show dual or triple‐labeled neurons. Images shown are from postnatal day (PND) 12 female (BSTpr, MEApd, MPO) and male (SCH) mice. Scale bar = 100 µm