Effect of follicular aging on ATP content and mitochondria distribution in bovine oocytes.
Microarray analysis was used to compare the gene expression of granulosa cells from dominant follicles with that of those after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation and control, n=6/group). A new follicular wave was induced by ablation of follicles ≥5 mm in diameter, and a progesterone-releasing device controlled internal drug release (CIDR) was placed in the vagina. The superstimulation group was given eight doses of 25 mg FSH at 12-h intervals starting from the day of wave emergence (day 0), whereas the control group was not given FSH treatment. Both groups were given prostaglandin F2α twice, 12 h apart, on day 3 and the CIDR was removed at the second injection; 25 mg porcine luteinizing hormone (pLH) was given 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification, and microarray hybridization. A total of 190 genes were downregulated and 280 genes were upregulated. To validate the microarray results, five genes were selected for real-time PCR (NTS, FOS, THBS1, FN1, and IGF2). Expression of four genes increased significantly in the three different animals tested (NTS, FOS, THBS1, and FN1). The upregulated genes are related to matrix remodeling (i.e. tissue proliferation), disturbance of angiogenesis, apoptosis, and oxidative stress response. We conclude that superstimulation treatment i) results in granulosa cells that lag behind in maturation and differentiation (most of the upregulated genes are markers of the follicular growth stage), ii) activates genes involved with the NFE2L2 oxidative stress response and endoplasmic reticulum stress response, and iii) disturbs angiogenesis.