Semester

Spring

Date of Graduation

2011

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Biochemistry

Committee Chair

Jianbo Yao.

Abstract

Newborn ovary homeobox (NOBOX) is an oocyte-specific transcription factor essential for folliculogenesis and expression of many germ-cell specific genes in mice. However, the temporal and cell specific expression of NOBOX in bovine oocytes and potential function and regulation of NOBOX in early embryogenesis have not been described previously. Messenger RNA for bovine NOBOX is preferentially expressed in ovaries and undetectable by RT-PCR in somatic tissues examined. NOBOX protein is present in oocytes throughout folliculogenesis and is stage-specifically expressed during oocyte maturation and early embryonic development. Depletion of NOBOX in early embryos demonstrated that NOBOX is an essential maternal derived transcription factor during bovine early embryogenesis, which functions in regulation of embryonic genome activation, pluripotency gene expression and blastocyst cell allocation. Collectively, our results establish a novel functional role for NOBOX during early embryonic development. Results also provide evidence that miR-196a is a bona fide negative regulator of NOBOX during bovine early embryogenesis.;Factor In the GermLine Alpha (FIGLA), a basic helix-loop-helix transcription factor, is essential for primordial follicle formation and expression of many oocyte specific genes that are required for fertilization and early embryonic survival. Here we report the characterization of bovine FIGLA gene and its regulation during early embryogenesis. Expression of bovine FIGLA mRNA is restricted to gonads and is abundant in germinal vesicle and metaphase II stage oocytes, as well as in embryos from pronuclear to eight-cell stage, but barely detectable in embryos collected at morula and blastocyst stages, suggesting that FIGLA might be a maternal effect gene. Collectively, our results demonstrate that FIGLA is spatio-temporally regulated during bovine early embryogenesis and miR-212 is an important negative regulator of FIGLA during the maternal to zygotic transition in bovine embryos.;Small endogenous RNA molecules, termed microRNAs (miRNAs), are evidenced for their involvement in the regulation of gene expression during folliculogenesis and early embryonic development. To identify miRNAs expressed in bovine oocytes/ovaries, we constructed a bovine fetal ovary miRNA library. Sequence analysis of random clones from the library identified 679 miRNA sequences, which represent 58 distinct bovine miRNAs. Of these distinct miRNAs, 42 are known bovine miRNAs and the remaining 16 miRNAs include 15 new bovine miRNAs that are homologous to miRNAs identified in other species, and one novel miRNA, which does not match any miRNAs in the database. This study reports the cloning of 58 miRNAs from bovine fetal ovaries, discovery of new bovine miRNAs including one that is completely novel, and identification of two miRNAs that are highly abundant in oocytes and early cleavage embryos.;Sumoylation is a post-translational modification in which a SUMO (small-ubiquitin-related modifier) moiety is conjugated to specific proteins involved in transcriptional regulation, protein transport, chromosome segregation and signal transduction via an enzymatic cascade. SUMOconjugating enzyme Ubc9, which is highly conserved across species, is essential for several developmental processes. As the first step towards understanding the physiological role of bovine Ubc9, we analyzed the expression of Ubc9 mRNA in bovine tissues including fetal ovaries of different developmental stages, and characterized the temporal expression patterns of Ubc9 mRNA and protein during oocyte maturation and early embryogenesis. Bovine Ubc9 is ubiquitously expressed in all tissues and is highly abundant in fetal ovaries of late gestation. Real time PCR analysis and immunocytochemical analysis demonstrated that expression of Ubc9 mRNA and protein is abundant in germinal vesicle (GV) and metaphase II (MII) stage oocytes, as well as in early embryos, but decreases between eight-cell and blastocyst stage, suggesting that Ubc9 might be a maternal effect gene. Collectively, our results suggest that Ubc9 is developmentally regulated in cattle and it interacts with ATF7IP2, a gonad-specific protein, which might play an important role during folliculogenesis and early embryonic development. (Abstract shortened by UMI.).

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