Cellular mechanisms responsible for development of sensitivity of the bovine corpus luteum to prostaglandin F2 alpha

Abstract

Cellular Mechanisms Responsible for Development of Sensitivity of the Bovine Corpus Luteum to Prostaglandin F2 alpha Madhusudan P. Goravanahally Prostaglandin F2 alpha (PGF 2α) brings about regression of the bovine corpus luteum (CL). This luteolytic property of PGF 2α is used in beef and dairy cattle to synchronize estrus. A limitation of this protocol is an insensitivity of the early CL to luteolytic actions of PGF 2α. The mechanisms underlying this differential luteal sensitivity are poorly understood. Therefore the main objective of the current study is to understand the cellular mechanism of luteal insensitivity. The developing CL has a maximum number of PGF 2α receptors; therefore differences in signaling events might be responsible for luteal insensitivity. Hence differential gene expression at two developmental stages of CL, days 4 (D-4) and 10 (D-10) post estrus, might account for differences in signal transduction pathways associated with luteal sensitivity. For example, differential expression of protein kinase C epsilon (PKCε /PRKCE) and its ability to regulate PGF 2α-stimulated rise in intracellular calcium concentration have been proposed to be part of luteal resistance mechanism. Therefore the current study investigates the: 1) physiological role of PRKCE in regulating the ability of PGF 2α to inhibit progesterone synthesis, 2) role of PGF 2α-stimulated rise in intracellular calcium in progesterone inhibitory actions of PGF 2α , 3) differential expression of a large portion of the luteal transcriptome during its developmental transition from early to mature stage, and 4) role of differentially expressed CAMKK2 in acquisition of luteolytic sensitivity to PGF 2α. Down-regulation of PRKCE significantly reduced the ability of PGF 2α to inhibit LH-stimulated progesterone accumulation. A pharmacological increase in intracellular calcium concentration [Ca 2+ ]i significantly inhibited LH-stimulated progesterone accumulation irrespective of luteal developmental stage. More importantly, buffering the rise in [Ca 2+ ]i reduced the ability of PGF 2α to inhibit progesterone accumulation. Microarray analysis identified 167 genes that were expressed differentially (p < 0.05). These were categorized into genes involved in cell signaling (12%), steroidogenesis and metabolism (10.2%), protein degradation (5.3%), transcription regulation and DNA biosynthesis (18.5%), protein biosynthesis and modification (18.5%), extracellular matrix and cytoskeletal proteins (9.5%), antioxidant property (3%), miscellaneous (17%), and unknown functions (6%). In addition, the in vivo administration of PGF 2α increased the expression of a guanine nucleotide binding protein (G protein), beta polypeptide 1 (GNB1) in D-4 CL and calcium/calmodulin dependent kinase kinase 2, beta (CAMKK2) in D-10 CL. Furthermore, large and small luteal steroidogenic cells, known to be targets for actions of PGF 2α were demonstrated to be a cellular source for CAMKK2. More importantly, in vitro, a CAMKK2 inhibitor significantly reduced the ability of PGF 2α...