Characterization of membrane receptor activity for 17 alpha, 20 beta, 21-trihydroxy-4-pregnen-3-one in ovaries of spotted seatrout (Cynoscion nebulosus).
The proposed maturation-inducing substance (MIS) of spotted seatrout (Cynoscion nebulosus) is 17 alpha, 20 beta, 21-trihydroxy-4-pregnen-3-one (20 beta-S). In this study, we characterized the binding of radioactive 20 beta-S to plasma membranes from the ovaries of spotted seatrout. Bound 20 beta-S was isolated by filtration of membrane suspensions and quantified by measurement of the radioactivity content of the filters. The saturable component of 20 beta-S binding reached equilibrium within 5 min at 0 degree, 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 20 beta-S. High levels of saturable binding were found in membrane preparations from the ovary, testis, and liver, but not from the gills. 17 alpha, 20 beta-Dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-P) showed relatively little affinity for the 20 beta-S binding site. However, this steroid was converted to a compound immunologically and chromatographically similar to 20 beta-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 20 beta-S from its binding site. Thus, progesterone appears to be a relatively inactive ligand with high affinity for the 20 beta-S receptor. The concentration of 20 beta-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 20 beta-S binding activity characterized in our study represents authentic MIS receptors. A distinct, soluble binding site for 17 alpha, 20 beta-P was also identified in seatrout ovaries, but its biological function remains unclear. A hypothesis is presented for the significance of this 17 alpha,20 beta-P binding site.
Patiño
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R
University of Texas, Marine Science Institute, Port Aransas, Austin 78373-1267.
Thomas
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P
D015065:17-Hydroxycorticosteroids; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D003350:Cortodoxone / Q000031:analogs & derivatives* / Q000378:metabolism; D005260:Female; D005880:Gills / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism; D008297:Male; D010053:Ovary / Q000378:metabolism; D011863:Radioimmunoassay; D011987:Receptors, Steroid / Q000378:metabolism / Q000493:pharmacokinetics*; D012487:Salmonidae / Q000378:metabolism*; D013737:Testis / Q000378:metabolism; D014337:Trout / Q000378:metabolism*
D015065:17-Hydroxycorticosteroids; D011987:Receptors, Steroid; C051954:17,20,21-trihydroxy-4-pregnen-3-one; D003350:Cortodoxone