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


CONCLUSIONS
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.
Department of Biomedical Sciences, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Colorado, USA. ryashley22@gmail.com
Ryan L
RL
Ashley
J Alejandro
JA
Arreguin-Arevalo
Terry M
TM
Nett
D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D030762:Estrous Cycle / Q000502:physiology*; D005260:Female; D007031:Hypothalamus / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism; D010053:Ovary / Q000502:physiology; D010902:Pituitary Gland / Q000502:physiology; D011374:Progesterone / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics* / Q000378:metabolism*; D012756:Sheep; D014316:Tritium; D014599:Uterus / Q000502:physiology
D008565:Membrane Proteins; D012333:RNA, Messenger; D011980:Receptors, Progesterone; D014316:Tritium; D011374:Progesterone