Specific binding sites for progesterone in subcellular fractions of the porcine corpus luteum.
Subcellular fractionation of porcine corpus luteum (CL) homogenates on continuous sucrose gradients has previously demonstrated that most of the endogenous progesterone of the CL was associated with a unique particulate fraction. Exogenous radiolabelled steroids were also sequestered with some specificity by this fraction. We now report that this particulate fraction is capable of binding high levels of exogenous 3H-labelled progesterone (and pregnenolone) in vitro, but only in the presence of the saponin, digitonin. Binding was dependent on the pH, temperature and duration of incubation, and showed specificity and high affinity for progesterone (Kd, 79 nM). Androgens, oestrogens and pregnenolone competed for porcine luteal [3H]progesterone binding sites, but only at much higher concentrations, whereas cholesterol, a number of progesterone receptor agonist and antagonist analogues and inhibitors of 3 beta-hydroxysteroid dehydrogenase and C17-hydroxylase/C17,20-lyase did not compete. Analysis of profiles for a number of luteal cell-surface membrane and intracellular organelle markers confirmed previous studies showing the association of an NADH-cytochrome C reductase with this fraction. Moreover, the content of endogenous progesterone associated with particulate subcellular fractions isolated from porcine granulosa cell (GC) and CL homogenates at different stages of the luteal phase and early pregnancy waxed and waned with the stage of the luteal phase (and the secretory activity of the CL). Binding of [3H]progesterone in vitro equilibrated at the same buoyant density as endogenous progesterone: levels of both were highest during the mid-luteal phase and during early pregnancy, lower in early and late luteal CL, and undetectable in corpora albicantia. In contrast, relaxin secretory granules were readily resolved from progesterone binding sites. We propose that these particulate progesterone binding sites may be involved in the sequestration and/or packaging of newly-synthesized steroid for secretion by the luteal cell, or may mediate actions of progesterone within the luteal cell.
University of Edinburgh, Department of Obstetrics and Gynaecology, UK.
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Menzies
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Bramley
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive; D002499:Centrifugation, Density Gradient; D003338:Corpus Luteum / Q000378:metabolism*; D004072:Digitonin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004971:Estrus / Q000502:physiology; D005260:Female; D006863:Hydrogen-Ion Concentration; D011284:Pregnenolone / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine; D013696:Temperature; D013997:Time Factors
D011374:Progesterone; D011284:Pregnenolone; D004072:Digitonin