Characterization of progesterone-binding moieties in the little skate Raja erinacea

In this study we report evidence of a [3H]progesteronebinding moiety in the liver and oviduct of the little skate Raja erinacea. It is characterized by high affinity, low capacity, and DNA-cellulose-binding activity. Furthermore Western blot analysis revealed that monoclonal antibodies against the chicken progesterone receptor (PR) subunits A and B cross-reacted with a 110-kDa band in the liver and a 80-kDa band in the oviduct. When analyzed by DEAE-Sepharose ion-exchange column chromatography, [3H]progesterone-binding molecules resolved into two peaks, one nonadherent and one adherent to the column. The liver adherent peak eluted in a linear gradient at a NaCl concentration of about 0.07 M and resolved on Western blot as a single band of a 110 kDa. The oviduct adherent peak eluted at about 0.14 M NaCl and resolved onWestern blot as a single band of 80 kDa. Competition studies showed that the progesteronebinding moiety in the cytosol was specific for progesterone. On the contrary, the nuclear component is not specific for progesterone; it also binds testosterone and estradiol 17b in the oviduct, and progesterone, testosterone, dihydrotestosterone, estradiol 17b, mibolerone, and R5020 in the liver. The [3H]progesterone-binding activity was monitored in both liver and oviduct of females in different reproductive stages. Females were separated into three groups: laying, nonlaying, and immature. [3H]Progesterone-binding activity levels were higher in the liver of immature than of nonlaying skates, and it was undetectable in laying skates. [3H]Progesterone binding was higher in the oviduct of laying and nonlaying skates than of immature skates. This PRbinding moiety has many characteristics of a true receptor: high affinity, low capacity, binds to DNA, and cross-reacts with antibodies against chicken PR. However, while the cytosolic form of this progesteronebinding component was quite specific for P, nuclear extracted material was nonspecific. If these progesteronebinding components are homologous with the PR A and PR B forms of other vertebrates, as we believe, it is clear that there are species differences that probably relate to phylogenetic level and physiology of the organism.