Characterization of a progestogen receptor in the ovary of the spotted seatrout, Cynoscion nebulosus

Abstract

A nuclear progestogen receptor was identified in the ovary of the spotted scatrout, Cynoscion nebulosus. A single class of high-affinity, low-capacity cytoplasmic binding sites for 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β- P) was characterized by saturation and Scatchard analyses (K(D) = 1.89 ± 0.61 nM, B(max) = 1.80 ± 0.63 pmol/g ovary, n = 4), as well as by one-point assay (B(max) = 1.41 ± 0.26 pmol/g ovary, n = 12). Analysis of the binding kinetics indicated a fairly rapid association rate (T( 1/2 ) = 72 ± 10.2 min) and a slightly slower dissociation rate (T( 1/2 ) = 99 ± 9.4 min). Competition studies revealed that several steroids exhibited the same range of affinity (17α,20β-P > 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S) > 11- deoxycorticosterone > progesterone) while others displayed an order of magnitude less affinity (17α-hydroxy-4-pregnen-3-one > pregnenolone > 11- deoxycortisol > testosterone). No displacement was found with 1000-fold excess estradiol-17β or cortisol. Binding activity was also present within the testis, but not in the brain, gill, muscle, or plasma. Nuclear binding was detected by DNA-cellulose column chromatography and was inhibited by the addition of molybdate, a characteristic of nuclear steroid receptors. Isolation and analysis of the nuclear fraction revealed specific binding with an order of magnitude lower affinity (K(D) = 20.7 ± 5.0 nM, n = 6) and capacity (0.22 ± 0.03 pmol/g ovary, n = 6), and steroid specificity nearly identical to that of the cytoplasmic receptor, although estradiol-17β competed for the nuclear binding sites (relative binding affinity ≃ 1%).