Date of Graduation

2008

Document Type

Dissertation/Thesis

Abstract

Mammalian oogenesis usually occurs concomitantly with folliculogenesis, coordinated through paracrine effects and physical contacts. During this time, the oocyte acquires the ability to resume meiosis, undergo fertilization and sustain embryonic development. Synthesis of RNA occurs during oogenesis and is largely responsible for early embryonic development. Any perturbations that delay either activation or ovulation can result in early embryonic loss. Therefore, the interacting effects at the genomic level were investigated using a persistent follicle model. Experiment 1. The present study was conducted to analyze global gene expression profiles in granulosal cells from ovarian follicles in cows. Analysis of the microarray data revealed up-regulation of 272 genes (M-value ≥ 0.9) and down-regulation of 203 genes (M-value ≤ -0.9) in granulosal cells from persistent follicles in comparison to growing follicles. Grouping of these genes into themes revealed altered expression of many genes involved in energy and protein metabolism, amino acid transport and apoptosis in granulosal cells of persistent follicles. It is suggested that aged granulosal cells may have a reduced capacity to provide energy and amino acids to the oocyte. Experiment 2. An experiment was done to determine if persistence of a follicle altered mRNA expression of important genes in the oocyte. Quantitative real-time PCR was used to measure the mRNA abundance of 10 selected genes important for early embryogenesis in oocytes obtained from growing and persistent follicles. Relative abundances of MSY2, PARN and YY1 mRNA were lower (p < 0.05) in oocytes from persistent than from growing follicles. Oocytes from persistent follicles, however, had greater abundances of PAP and eIF-4E transcripts (p < 0.05). It is suggested that persistence of a follicle alters amounts of mRNA for genes important for regulation of transcription and protein translation in the oocyte, which could compromise development of early embryos in cows that ovulate a persistent follicle. Experiment 3. Protein expression of NPM2 was, however, barely detectable in blastocyst stage embryos. Recent reports indicate microRNAs might play an important role in degradation of maternal RNAs in developing early embryos. It was hypothesized that, as a maternal transcript, NPM2 is targeted by microRNAs for silencing and/or degradation. Therefore, the presence of potential microRNA binding sites in the 3’ UTR of bovine NPM2 mRNA sequence was investigated using bioinformatic analysis. Using MicroInspector, (http://mirna.imbb.forth.gr/microinspector/), an algorithm for detection of possible interactions between microRNAs and target mRNA sequences, a microRNA binding site (miR-181a) in the 3’-UTR of the bovine NPM2 mRNA was identified. Alignment of the 3’-UTR sequence of the bovine NPM2 mRNA with the human and mouse orthologous sequences revealed that the “seed” sequence for microRNA:mRNA interactions was conserved in the aligned sequences. Real time PCR analysis indicated that miR-181a was expressed in both oocytes and embryos and tended to increase at the morula and blastocyst stages (p = 0.07). To test if miR-181a regulates bovine NPM2 protein expression in the context of its native mRNA sequence, the full-length cDNA of bovine NPM2 was cloned into a pcDNA3.1 vector. A plasmid designed to deliver bovine miR-181a was constructed by inserting a 330 nt genomic fragment containing the pre-miR-181a sequence into pcDNA3.1. HeLa cells stably expressing the bovine miR-181a were first established. Constructs expressing bovine NPM2 were then used to transfect both stable cells, expressing the bovine miR-181a, and control HeLa cells without miR-181a. Cells were harvested 24 hrs after transfection and subjected to western blot analysis using antibodies against bovine NPM2. A control western blot against GAPDH also was performed in six independent transfection experiments. Expression of bovine NPM2 protein was reduced (p = 0.006) in cells expressing bovine miR-181a compared to control cells without miR-181a, indicating that translation of NPM2 is repressed by miR-181a. (Abstract shortened by UMI.).

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