Detection of differential gene expression in ovarian granulosa cells from swine and cattle using microarray technology

Scope and Method of Study. Differential gene expression in ovarian granulosa cells from swine and cattle was studied. Two experiments were performed. In the first, porcine granulosa cells collected from peripubertal gilts were cultured in vitro and RNA was collected and hybridized to Affymetrix Porcine Geome Arrays. In the second study, granulosa cells and follicular fluid were isolated from dominant and cystic bovine follicles collected through ultrasound guided aspiration or from a slaughterhouse. Hormone analysis was performed by radioimmunoassay for levels of progesterone, estradiol and androstenedione in follicular fluid. RNA was extracted from granulosa cells and hybridized to Affymetrix GeneChip Bovine Genome Arrays. In both studies differentially regulated DNA sequences were annotated using bioinformatic tools. Validation of the level of expression of selected genes was performed through one-step real-time RT-PCR.;Findings and Conclusions. Results from our first study showed that treatment of granulosa cells with stimulatory doses of IGF-I promotes differential gene expression in granulosa cells, with 388 DNA sequences detected to be differentially regulated. Validation of microarray results led to identification of two genes that were previously unknown to be regulated by IGF-I: fibroblast growth factor receptor 2 splice variant III c (FGFR2IIIc) and Thrombospondin 1. Our second study showed that there are dramatic differences in gene expression in granulosa cells between cystic and noncystic follicles, with 163 DNA sequences differentially expressed. Further validation of microarray results allowed to confirm dramatic changes in the level of expression of two genes previously unrelated to the cystic condition: Angiogenin and G-protein coupled receptor 34 (GPCR34). Overall, results from our two studies contribute to the knowledge of genes involved in the process of follicular development in mammals, which is of vital importance for the design of strategies to improve the reproductive efficiency of food producing species as well for the development of treatments for ovarian diseases not only in animals but also in humans.