Graduation date: 2007
Experiments were conducted to characterize the nongenomic effects of
progesterone (P4) on binding of oxytocin (OT) to the ovine oxytocin
receptor (OTR) and signal transduction. The dose-response relationship of
P4 to OT binding to the OTR in endometrium was examined. Progesterone
interfered with the binding of OT to the OTR in a dose-dependent manner.
Endometrium was then recovered from cyclic ewes and divided into
explants that were analyzed for total inositol mono- (IP), bis- (IP2), and
trisphosphate (IP3) content after exposure to 2.5 ng/ ml P4 and (or) OT.
Pre-incubation with P4 for 10 min significantly interfered with OT
stimulation of IP3 synthesis. In the next experiment endometrial explants
were analyzed for PGF2α response after exposure to OT, arachidonic acid
(AA) +OT, P4+OT, and P4 + AA + OT. Treatment of explants with AA
increased PGF2α content compared to that of controls. Brief exposure to P4 significantly decreased OT-induced PGF2α secretion from explants
previously exposed to medium or AA. The goal of the next study was to
determine if P4 would inhibit OT-stimulated phosphoinositide hydrolysis in
transfected COS-7 cells with little or no nuclear P4 receptors (nPR). Lack
of nPR was confirmed by use of immunocytochemistry and RT-PCR. Pretreatment
of COS-7 cells transiently transfected with a plasmid containing
the ovine OTR for 10 min with 2.5 ng/ ml P4 significantly interfered with
OT-stimulated IP3 production (P4 x OT interaction; P= 0.03). Specificity of
steroid inhibition of OT-induced IP3 production was performed. Only P4
was able to significantly inhibit OT-induced IP3 production. A binding assay
for R5020, (a synthetic progestin), was performed. There was no
measurable specific binding of steroid to both transfected and
nontransfected cells. It is concluded that P4 can inhibit OTR-mediated
phosphoinositide hydrolysis in COS-7 cells that express little or no nPR
protein via some mechanism other than by binding to a membrane
progestin receptor.