Androgen receptors in a cichlid fish, Astatotilapia burtoni: structure, localization, and expression levels

Abstract

Androgens are an important output of the hypothalamic-pituitary-gonadal (HPG) axis that controls reproduction in all vertebrates. In male teleosts two androgens, testosterone and 11-ketotestosterone, control sexual differentiation and development in juveniles and reproductive behavior in adults. Androgenic signals provide feedback at many levels of the HPG axis, including the hypothalamic neurons that synthesize and release gonadotropin-releasing hormone 1 (GnRH1), but the precise cellular site of androgen action in the brain is not known. Here we describe two androgen receptor subtypes, ARalpha and ARbeta, in the cichlid Astatotilapia burtoni and show that these subtypes are differentially located throughout the adult brain in nuclei known to function in the control of reproduction. ARalpha was expressed in the ventral part of the ventral telencephalon, the preoptic area (POA) of the hypothalamus and the ventral hypothalamus, whereas ARbeta was more widely expressed in the dorsal and ventral telencephalon, the POA, and the ventral and dorsal hypothalamus. We provide the first evidence in any vertebrate that the GnRH1-releasing neurons, which serve as the central control point of the HPG axis, express both subtypes of AR. Using quantitative real-time PCR, we show that A. burtoni AR subtypes have different expression levels in adult tissue, with ARalpha showing significantly higher expression than ARbeta in the pituitary, and ARbeta expressed at a higher level than ARalpha in the anterior and middle brain. These data provide important insight into the role of androgens in regulating the vertebrate reproductive axis.

Fig. 1

a: Comparison of A. burtoni ARα and ARβ predicted protein sequences. The canonical functional steroid receptor domains (A—F) are represented schematically, and their roles are given accordingly. Numbers indicate amino acid position from the start of the coding sequence. ARα contains a 23-aa insert in the hinge region (domain C) absent in ARβ. b: Phylogenetic comparison of A. burtoni ARα and ARβ protein sequences with AR in other species. As shown, A. burtoni ARβ clusters with the other ARβ/AR2 family members identified to date in teleosts, and this AR subtype is more similar to mammalian ARs than is A. burtoni ARα. A. burtoni ARα clusters with other ARα/AR1-type receptors previously described in teleosts. ARs that are marked with an asterisk have nomenclature that does not correspond to their position in the ARβ/AR2 family, defined by their position in the phylogenetic tree. Some of these species (marked with a dagger) likely have two forms of the ARβ/AR2 family as a result of tetraploidy. Bootstrap values are shown.

Fig. 2

In situ hybridization showing the distribution of ARα expression in A. burtoni brain. Cartoon of a sagittal section through the A. burtoni brain (top) shows the positions of the coronal sections shown in a1—c3. OB, olfactory bulb; Tel, telencephalon; OT, optic tectum; C, cerebellum; Pit, pituitary. Cartoon of coronal sections through the A. burtoni brain (a1,b1,c1) show the region magnified in a2,3, b2,3, and c2,3, respectively. Brightfield images (a2,b2,c2) show cresyl violet counterstain (purple) of coronal brain sections, with the brain nuclei depicted schematically. Darkfield images (a3,b3,c3) show silver grains specific for the antisense ARα probe in the corresponding nuclei. Specific expression of A. burtoni ARα can be seen in the ventral portion of the ventral telencephalon (Vv; a2,a3), anterior portion of the parvocellular preoptic nucleus (aPPn) and extending dorsally along the boundary of the third ventricle (3V; b2,b3, arrow) and scattered diffusely in the ventral hypothalamus (VH; c2,c3). Scale bar = 250 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 3

In situ hybridization showing the distribution of ARβ in A. burtoni brain. Cartoon of a sagittal section through the A. burtoni brain (top) shows the positions of the coronal sections shown in panels a1—h3. OB, olfactory bulb; Tel, telencephalon; OT, optic tectum; C, cerebellum; Pit, pituitary. Cartoon of coronal sections through the A. burtoni brain (a1,b1,c1,d1,e1,f1,g1,h1) show the region magnified in a2,3, b2,3, c2,3, d2,3, e2,3, f2,3, g2,3, and h2,3, respectively. Brightfield images (a2,b2,c2,d2,e2,f2,g2,h2) show cresyl violet counterstain (purple) of coronal sections, with the corresponding brain nuclei depicted schematically. Darkfield images (a3,b3,c3,d3,e3,f3,g3,h3) show silver grains specific for the antisense ARβ probe in the corresponding nuclei. Specific A. burtoni ARβ expression can be seen in the central nucleus of the dorsal telencephalon (Dc; a2,a3), at the lateral edge of part 3 of the medial zone of the dorsal telencephalon (Dm3; b2,b3), dorsal (Vd) and ventral (Vv) portion of the ventral telencephalon (c2,c3), in the anterior portion of the parvocellular preoptic nucleus (aPPn), the magnocellular portion of the anterior preoptic nucleus (maPn) and the parvocellular portion of the magnocellular preoptic nucleus (pMPn; d2,d3). More posterior sections through the A. burtoni brain reveal ARβ expression in the ventral (VH; e2,e3) and dorsal (DH; f2,f3) hypothalamus. Scale bars = 250 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 3

In situ hybridization showing the distribution of ARβ in A. burtoni brain. Cartoon of a sagittal section through the A. burtoni brain (top) shows the positions of the coronal sections shown in panels a1—h3. OB, olfactory bulb; Tel, telencephalon; OT, optic tectum; C, cerebellum; Pit, pituitary. Cartoon of coronal sections through the A. burtoni brain (a1,b1,c1,d1,e1,f1,g1,h1) show the region magnified in a2,3, b2,3, c2,3, d2,3, e2,3, f2,3, g2,3, and h2,3, respectively. Brightfield images (a2,b2,c2,d2,e2,f2,g2,h2) show cresyl violet counterstain (purple) of coronal sections, with the corresponding brain nuclei depicted schematically. Darkfield images (a3,b3,c3,d3,e3,f3,g3,h3) show silver grains specific for the antisense ARβ probe in the corresponding nuclei. Specific A. burtoni ARβ expression can be seen in the central nucleus of the dorsal telencephalon (Dc; a2,a3), at the lateral edge of part 3 of the medial zone of the dorsal telencephalon (Dm3; b2,b3), dorsal (Vd) and ventral (Vv) portion of the ventral telencephalon (c2,c3), in the anterior portion of the parvocellular preoptic nucleus (aPPn), the magnocellular portion of the anterior preoptic nucleus (maPn) and the parvocellular portion of the magnocellular preoptic nucleus (pMPn; d2,d3). More posterior sections through the A. burtoni brain reveal ARβ expression in the ventral (VH; e2,e3) and dorsal (DH; f2,f3) hypothalamus. Scale bars = 250 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 4

In situ hybridization showing the distribution of ARα (a1,a2) and ARβ (b1,b2) in the A. burtoni pituitary. Brightfield images (a1,b1) show cresyl violet counterstain (purple) and silver grains (black spots) of coronal sections through the posterior pituitary, and darkfield images. b1 and b2 show silver grains specific for antisense ARα (a2) and ARβ (b2). Expression of both A. burtoni ARα (a1,a2) and ARβ (b1,b2) is seen restricted to the ventral pituitary. Scale bars = 250 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 5

Double in situ hybridization showing coexpression pattern of GnRH1 with ARα (a—c) and ARβ (g—i) in the POA of A. burtoni. Brightfield images (a,d,g,j) show DAB-labeled GnRH1 mRNA (brown), cresyl violet counterstain (purple) and silver grains (black spots). Darkfield (b,e,h,k) and merged (c,f,i,l) images show silver grains pseuocolored in green to facilitate demonstration of colocalization. Both A. burtoni ARα (c) and ARβ (i) are expressed in GnRH1-releasing neurons (c, i, arrows) and surrounding neurons (c, i, arrowheads). Double in situ hybridization with the equivalent sense probes for ARα (d—f) and ARβ (j—l) demonstrate the specificity of the antisense probe above background. Scale bars = 50 μm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Fig. 6

Relative expression levels of ARα and ARβ in brain and pituitary of A. burtoni. QRT-PCR reveals that ARα and ARβ are differentially expressed in regions of the brain (see Materials and Methods) and in the pituitary of adult A. burtoni males. ARβ mRNA is expressed at significantly higher levels than ARα in the anterior (a) and middle (b) brain regions (*P < 0.001), but ARα expression is significantly higher than ARβ expression in the pituitary (d, **P = 0.001). Although AR expression levels in the posterior brain are low compared with those in other brain regions (c), expression of ARα is significantly higher than ARβ in this area (†P = 0.003). Data are plotted as mean percentage of 18s mRNA expression as a control for variation in total mRNA levels between samples, with standard errors designated by error bars.