Mechanisms of Hormone Action

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

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 present in the ovary of the teleost, Atlantic croaker. Specific androgen binding to an ovarian plasma membrane fraction was demonstrated using a radioreceptor assay protocol consisting of a short-term incubation with [³H ]-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, Kd = 15.32 ± 2.68 nM, low capacity, Bmax = 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, with t_1/2's of 3.7 ± 1.7 min and 4.7 ± 0.2 min, respectively. Competitive binding assays showed that 5-dihydrotestosterone, T, and 11-ketotestosterone had relative binding affinities (RBA) of 193 %, 100 %, and 13 %, respectively, whereas none of the C₁₈ or C₂₁ steroids tested bound with high affinity except 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 intestines. Receptor levels increased 10-fold during ovarian recrudescence reaching maximum levels in fully mature ovaries, suggesting 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.

Key words: Ovary • Androgen receptor • Steroid hormones • Steroid hormone receptors • Testosterone

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