Biochemical Characterization of a Membrane Androgen Receptor in the Ovary of the Atlantic Croaker (Micropogonias undulatus)1

Abstract

Membrane androgen receptors have been biochemically characterized in only a few vertebrate species to date. Therefore, the purpose of the current study was to comprehensively investigate the binding characteristics of a putative membrane androgen receptor in the ovary of the teleost, Atlantic croaker (Micropogonias undulatus). Specific androgen binding to an ovarian plasma membrane fraction was demonstrated using a radioreceptor assay protocol consisting of a short-term incubation with [(3)H]testosterone (T) and subsequent filtration of bound steroid from free steroid. Saturation and Scatchard analyses of T binding to an ovarian plasma membrane fraction indicated the presence of a single, high-affinity (K(d) = 15.32 +/- 2.68 nM [mean +/- SEM]), low-capacity (B(max) = 2.81 +/- 0.31 pmol/mg protein), androgen-binding site. Specific androgen binding to the receptor was readily displaceable, and the association and dissociation kinetics were rapid (half-time = 3.7 +/- 1.7 and 4.7 +/- 0.2 min, respectively). Competitive binding assays showed that 5alpha-dihydrotestosterone, T, and 11-ketotestosterone had relative binding affinities (RBAs) of 193%, 100%, and 13%, respectively, whereas none of the C(18) or C(21) steroids tested bound with high affinity except for progesterone (RBA = 191%). This androgen-binding moiety with high affinity for progesterone is unlikely to mediate the physiological actions of progestins in croaker, because it has low binding affinity for fish progestin hormones. Androgen-binding sites were also detected in membrane fractions of the brain, liver, kidney, and drumming muscle, whereas little or no binding was detected in the trunk muscle, heart, gills, or intestine. Receptor levels increased 10-fold during ovarian recrudescence, reaching maximum levels in fully mature ovaries, which suggests a likely physiological role for this receptor during the reproductive cycle of female croaker. It is concluded that the androgen-binding moiety identified in the plasma membrane fraction of Atlantic croaker ovarian tissue fulfils all the criteria for its designation as a steroid receptor.

Differential ligand selectivity of androgen receptors α and β from Murray–Darling rainbowfish (Melanotaenia fluviatilis)

“…Because sex determination can be altered in response to environmental cues such as temperature or exposure to exogenous steroid hormones, even in fish with chromosomal sex determination, the susceptibility of androgen-mediated aspects of male sexual differentiation to environmental AR modulators is likely to be higher at critical developmental stages. Furthermore, while nuclear androgen receptors have been the focus of the majority of studies, putative membrane-associated ARs have also been described (Braun and Thomas, 2004;Papadopoulou et al, 2009), which may represent additional targets of (anti)androgenic aquatic environmental endocrine disruptors in fish. While the present study highlights intraspecies differences between ligand specificity of receptor subtypes, it is clear that interspecies differences in hormone receptor ligand specificity need to be better defined so that environmental risk assessments are conducted in a manner that is relevant to the ecosystem in a particular geographic region.…”

Cellular phenotype of androgen receptor‐immunoreactive nuclei in the developing and adult rat brain

“…Therefore, the observation of AR in the astrocytic cytoplasm in proximity to the nucleus is not unexpected; moreover, because the rim of cytoplasm surrounding the astrocytic nucleus is thin (Peters et al, 1991), it may be difficult to discriminate the cytoplasmic from nuclear label based solely on the shape of the immunoreactive profile. Alternatively, increasing evidence for membrane-associated actions of an- drogens, acting via an AR that is identical to or distinct from nuclear AR, has accumulated in recent years (Braun and Thomas, 2004;Lu et al, 2001;Lutz et al, 2003;Rosner et al, 1999), and, if cytoplasmic AR exists in astrocytes, it may represent a form of AR that acts via nongenomic mechanisms. Extranuclear AR has been observed not only in neurons, within cortical axons and dendrites (DonCarlos et al, 2003), but also in astrocytes of the rat hippocampal formation (Tabori et al, 2005).…”

GPRC6A Mediates the Non-genomic Effects of Steroids

“…Binding Assay-Binding assays were performed as previously described (32 (33). We performed the analysis by liquid scintillation counting using a liquid scintillation counter (Beckman Instruments).…”