Search for a Progesterone-Binding Protein in Secretory Granules of the Ovine Corpus Luteum*

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Abstract
The hypothesis that electron-dense granules present in the corpus luteum contain progesterone in a proteinbound form was examined using differential and sucrose gradient centrifugation. Ovine luteal tissue was fractionated by differential centrifugation at 1,000 × g (P, pellet), 10,000 × g (P2), and 82,000 × g (P3; supernatant S3). Samples of P2, P3, and S3 were further fractionated on 20–40% (P2 and P3) or 5–25% (S3) sucrose gradients and examined for progesterone-binding activity by measuring the progesterone content and/or the specific binding of [3H]progesterone of sucrose gradient samples. In addition, saturation binding assays were performed with steroid-free samples of P2 and S3. Saturable binding of progesterone was not found in P2, the fraction containing electron-dense granules. In S3, two progesterone-binding proteins with sediment tion rates of 3.2S and 8.6S and an af inity of 7.1 × 105 M-1 for progesterone were detected. The sedimentation behavior of these proteins was distinct from that of ovine plasma transcortin, a 4S protein. The view that a binding protein is released into the interstitial fluid during the exocytosis of granules was examined by measuring the progesterone-binding activity of protein released by slices of corpus luteum in vitro. No binding activity was found. The results of this investigation do not support the hypothesis that putative progesterone-secreting granules observed in luteal tissue contain a binding protein.