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, nZ6/group). A new follicular wave was induced by ablation of follicles R5 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 F 2a 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.

The objective was to determine how maternal age influences the transcriptome of the dominant follicle during the preovulatory period. We tested the hypotheses that delayed ovulation in aged cows is associated with 1) altered gene expression of granulosa cells of the preovulatory follicle and 2) decreased synthesis of progesterone by granulosa cells of the preovulatory follicle. Granulosa cells of preovulatory follicles were obtained 24 h after LH treatment from aged Hereford cows (17.0 ± 2.5 years; n = 6) and their daughters (9.0 ± 0.6 years; n = 6), and compared using bovine specific microarrays (EMBV3, EmbryoGENE, Québec, QC, Canada). Results were confirmed by RT-qPCR. A total of 1340 genes or gene isoforms were expressed differentially (≥2-fold change; P ≤ 0.05) in aged cows v. their younger daughters. Differentially expressed up- and down-regulated genes were related to 1) LH response (↑RGS2, ↑SERPINE2, ↑PTGS2), 2) cellular differentiation and luteinization (↑TNFAIP6, ↑GADD45B, ↓VNN1), and 3) progesterone synthesis (↑STAR, ↑HSD3B2, ↑NR5A2, ↑NR4A1). Intra-follicular concentration of progesterone was lower (P < 0.05) in aged v. young cows. Pathway analysis of the dataset revealed that mechanisms of delayed ovulation in aged cows may involve 1) post-receptor desensitization of G-coupled protein receptors, 2) inactivation of tissue plasminogen activator, and 3) delayed production of prostaglandin E2. In conclusion, transcriptome analysis of granulosa cells from aged cows revealed a delayed or suboptimal response to the preovulatory LH stimulus, represented by delayed cellular differentiation, luteinization, and progesterone synthesis. This study was supported by grants from the National Science and Engineering Research Council of Canada, and the EmbryoGene Network, Canada. M.I.R. Khan was supported by graduate assistant scholarship from the Higher Education Commission of Pakistan.

Wenyou Huang, Dan Yü and Song Huang contributed equally to the work. Article Information

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