title: Comparison of mRNA levels for the PGF(2α) receptor (FP) during luteolysis and early pregnancy in the ovine corpus luteum.
abstract: Prostaglandin F(2α) (PGF(2α)) is the physiological signal that triggers luteolysis in the non-pregnant ewe. This is associated with a decline in circulating levels of progesterone, beginning around day 14 of a 16-17 day estrous cycle. Recently, the receptor for PGF(2α) (FP) was cloned from an ovine day 10 large luteal cell cDNA library. The purpose of this study was to measure relative abundance of FP mRNA as it may change with luteolysis during the luteal phase. Corpora lutea (CL) were collected from ewes on day 10, 12, 14 or 16 (n≥4/day; day 0=synchronized estrus); 12 h after PGE(2α)-treatment on day 10, 12 or 14 (n≥3/day) of the estrous cycle; or on day 16 of pregnancy (n=6). Pregnancy was confirmed by visualization of the conceptus. Blood samples were collected 12 h prior to and at the time of tissue collection to determine levels of progesterone. Serum concentrations of progesterone declined with the onset of luteolysis in control animals (day 14, day 16;P<0.05) as well as 12 h following PGF(2α)-treatment (day 10, day 12;P<0.05). Genomic DNA from these tissues was prepared and visualized by agarose gel electrophoresis. Internucleosomal fragmentation (indicative of apoptosis) was seen in CL from animals in which luteolysis had been initiated (all PGE(2α)-treated and day 16 control ewes), but not in ewes with functional CL. Total RNA isolated from each CL was separated through a denaturing 1% agarose gel, transferred to nylon membranes and hybridized to a radioactive ovine FP cDNA probe. Hybridization to a radiolabeled 18S ribosomal cRNA probe was used to confirm equal loading of RNA in each lane. By northern analysis, a major transcript was seen at ∼6.1 kb. A relatively high level of FP mRNA was measured in CL collected from control non-pregnant ewes during the mid luteal phase (day 10, 2.73±0.17; day 12, 2.47±0.91; FP/18S ratio), but varied among animals (3.09±1.59) on day 14. Administration of PGF(2α) resulted in the lowest amounts of FP mRNA on days 10, 12, and 14 (P<0.05). Amounts of FP mRNA were higher (P<0.05) on day 16 of pregnancy as compared to day 10 (by 1.9-fold) or to day 16 (by 5.9-fold) of the estrous cycle. From these observations we conclude that PGF(2α) or some event associated with luteolysis appears to down regulate amounts of the FP mRNA. Furthermore, pregnancy, and/or the antiluteolytic signals associated with maternal recognition of pregnancy may prevent the decline in the amount of FP mRNA.
authors.affiliation: Department of Physiology, University of Arizona, 85724, Tucson, AZ.
authors.forename: B R
authors.initials: BR
authors.lastname: Rueda
authors.forename: I W
authors.initials: IW
authors.lastname: Botros
authors.forename: K L
authors.initials: KL
authors.lastname: Pierce
authors.forename: J W
authors.initials: JW
authors.lastname: Regan
authors.forename: P B
authors.initials: PB
authors.lastname: Hoyer