Genetic Profiling of the Bovine Pituitary Gland Using cDNA Microarray Technology

Abstract

Goals of this dissertation were to 1) use custom-made cDNA microarrays to identify genes in the bovine pituitary gland that are differentially expressed during the estrous cycle and 2) characterize their patterns of gene expression. The estrous cycle is a dynamic process that requires coordination between the hypothalamus, pituitary gland, and ovaries. The anterior pituitary gland synthesizes and secretes the gonadotropins, luteinizing hormone (LH) and follicle stimulating hormone (FSH), which regulate steroidogenesis and follicular development. Currently, intrapituitary factors that modulate gonadotropin synthesis, storage, and release are not well described, thus, requiring investigation. To investigate the validity of the microarray results, we performed real-time PCR on 35 genes identified by cDNA microarray as being differentially regulated. Overall, microarray and real-time PCR results were consistent among our experiments suggesting that cDNA microarray is an efficacious tool for profiling gene expression in the bovine pituitary gland. Our first experiment was designed to identify genes that were regulated during the early luteal phase. This period is characterized by steadily increasing concentrations of progesterone (P4) from nadir to maximum. Samples from three different time points, d 2, d 6, and d 10 following initiation of the first follicular wave, were compared. One hundred and sixty nine genes were determined to be differentially expressed. Ten of these genes were validated using real-time PCR. The other two studies were designed to identify genes that were regulated during the preovulatory period as induced by the administration of prostaglandin F2α (PGF2α). This period is characterized by a decrease in circulating concentrations of P4 coincident with an increase in circulating concentrations of estradiol. Prior to the surge, FSH and LH are disconcordinately released but the underlying mechanisms regulating their release is unknown. The second study identified 1406 genes to be differentially regulated during the 72 h following administration of PGF2α. Twenty-seven of these transcripts were validated by real-time PCR. The third study identified 503 genes to be differentially regulated during the 48 h following administration of PGF2α. Twenty two of these transcripts were validated by real-time PCR. Together these experiments have identified several genes as potential intrapituitary factors that may function to regulate the reproductive axis.