Comprehensive analysis of ovarian gene expression during ovulation using differential display

Mammalian ovulation is a normal biological process that is initiated when a gonadotropic hormone stimulates G protein-coupled receptors in the plasma membrane of cells in ovarian follicles. This article outlines differential display (DD) protocols and associated methods that have been used to discover more than 30 genes that are expressed in the rat ovary during the ovulatory process. Details are provided regarding the methods for total RNA extraction, reverse transcription (RT), DD-polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE), Northern analysis of the differentially expressed cDNA fragments, cloning of the cDNA fragments, sequencing of the cDNA, and in situ hybridization of the cDNA fragments with sections of ovarian tissue. These methods provide clear evidence of the temporal and spatial patterns of expression of ovulation-specific genes in the ovary. Most of the genes that have been discovered to date have been associated previously with cascades of gene expression in acute inflammatory reactions. Therefore, the data support the working hypothesis that the ovary becomes inflamed at the time of ovulation, and this acute condition softens local connective tissues and causes ovarian follicles to rupture and release fertile eggs.