CHARACTERIZATION OF MEMBRANE-RECEPTOR ACTIVITY FOR 17-ALPHA,20-BETA,21-TRIHYDROXY-4-PREGNEN-3-ONE IN OVARIES OF SPOTTED SEA-TROUT (CYNOSCION-NEBULOSUS)

The proposed maturation-inducing substance (MIS) of spotted seatrout (Cynoscion nebulosus) is 17α,20β,21-trihydroxy-4-pregnen-3-one (20β-S). In this study, we characterized the binding of radioactive 20β-S to plasma membranes from the ovaries of spotted seatrout. Bound 20β-S was isolated by filtration of membrane suspensions and quantified by measurement of the radioactivity content of the filters. The saturable component of 20β-S binding reached equilibrium within 5 min at 0°, showed linearity with membrane concentration, and was pH dependent (optimum, 7.5-7.8). Scatchard analyses suggested a single class of high-affinity (KD, 10-9 M), low-capacity (10-13-10-12 mol/g ovary) binding sites for 20β-S. High levels of saturable binding were found in membrane preparations from the ovary, testis, and liver, but not from the gills. 17α,20β-Dihydroxy-4-pregnen-3-one (17α,20β-P) showed relatively little affinity for the 20β-S binding site. However, this steroid was converted to a compound immunologically and chromatographically similar to 20β-S by intact ovarian follicles, a finding which may explain its previously reported high potency in an in vitro oocyte maturation bioassay. Conversely, although reportedly a weak inducer of oocyte maturation, progesterone readily displaced 20β-S from its binding site. Thus, progesterone appears to be a relatively inactive ligand with high affinity for the 20β-S receptor. The concentration of 20β-S binding sites in ovaries was significantly higher during final oocyte maturation (germinal vesicle migration) than at earlier stages of development. These results strongly suggest that the 20β-S binding activity characterized in our study represents authentic MIS receptors. A distinct, soluble binding site for 17α,20β-P was also identified in seatrout ovaries, but its biological function remains unclear. A hypothesis is presented for the significance of this 17α,20β-P binding site. © 1990.