Binding characteristics of the ovine membrane progesterone receptor alpha and expression of the receptor during the estrous cycle

Abstract

Abstract Background Classically, progesterone has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors and subsequent modulation of gene expression. However, there is increasing evidence for rapid, non-genomic effects of progesterone in a variety of mammalian tissues and it is possible that a membrane PR (mPR) is causing these events. We recently isolated and characterized an ovine mPR referred to as mPR-alpha, distinct from the nuclear PR. Based on predicted structural analysis, the ovine mPR-alpha possesses seven transmembrane domains typical of G protein-coupled receptors. Despite the homology to other reported mPRs, information pertaining to the steroid binding characteristics of the ovine mPR-alpha was lacking. Additionally, the ovine mPR-alpha transcript has been identified in the hypothalamus, pituitary, uterus, ovary and corpus luteum, yet changes in expression of the ovine mPR-alpha in these tissues were not known. Consequently, the purpose of this work was to determine the steroid binding characteristics of the ovine mPR-alpha and to investigate possible changes in expression of the ovine mPR-alpha in reproductive tissues throughout the estrous cycle. Methods Binding studies were performed using crude membrane fractions from CHO cells expressing the mPR-alpha. Using quantitative Real-time PCR we determined the expression pattern of mRNA for the ovine mPR-alpha during the ovine estrous cycle in tissues known to express the mPR-alpha. Jugular blood samples were also collected and analyzed for serum concentrations of P4 to ensure ewes were at the appropriate stage of their cycle. Results Only progesterone, 20alpha-hydroxyprogesterone and 17alpha-hydroxyprogesterone were able to displace binding of 3H-P4 (P < 0.001) to membrane fractions from CHO cells expressing ovine mPR-alpha. The average B-max and Kd values for three separate experiments were 624 +/- 119 fmol/micro gram protein and 122 +/- 50 nM, respectively. Significant changes in expression of mRNA for the mPR-alpha during the estrous cycle were noted in the corpus luteum and uterus. Conclusion The mPR-alpha specifically binds progestins and its expression was correlated to progesterone secretion during the ovine estrous cycle. Results from the present studies suggest that mPR-alpha may have an important physiological role during the ovine estrous cycle.