Following fertilization, the embryo undergoes a sequence of precisely timed cleavage cycles to produce a blastocyst. The timing of these cycles likely depends in part on appropriate levels of gene activity. I have investigated whether high mobility group (HMG) proteins 14 and 17, which are associated with chromatin containing transcribed genes, are expressed in mouse embryos and are required to maintain normal early developmental timing. As assayed using RT-PCR, mRNAs encoding both HMG-14 and HMG-17 were present throughout preimplantation development to the blastocyst stage. By immunofluorescence, both proteins were detected in the nuclei of prophase I-arrested oocytes and embryos beginning at the 2-cell stage. To investigate their function, antisense oligonucleotides targeting the 5' end of each mRNA species were injected into 1-cell stage embryos which were then cultured to develop to the blastocyst stage. At the 2- and 4-cell stages, only weak nuclear immunofluorescence was observed; however, by the 8-cell stage, the staining pattern of injected embryos was indistinguishable from controls. Thus, the injected antisense oligonucleotides transiently depleted the cellular supply of HMG-14 and HMG-17. Furthermore, the embryos in which both HMG-14 and MAG-17 had been depleted progressed significantly more slowly through successive stages of preimplantation development, as compared with embryos in which the proteins were individually depleted or injected with nonsense oligonucleotides. Therefore, it can be concluded that depletion of HMG-14 and HMG-17 from embryonic chromatin transiently delays preimplantation development, demonstrating a crucial role for these proteins in maintaining the normal temporal coordination of development.

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published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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Glutamine serves as a major energy source for all stages of preimplantation mouse embryo development, whether the embryos are raised in vivo or in vitro from the one-cell stage. Glutamate dehydrogenase (GDH) and glutamine synthetase (GS) are enzymes that are involved in the metabolism of glutamine. GDH catalyzes the conversion of glutamate into a-ketoglutarate, a primary component of the tricarboxylic acid cycle. GS catalyzes the conversion of glutamate to glutamine. The expression of GDH RNA and GS RNA were analyzed in preimplantation mouse embryos using reverse transcription (RT) with an oligo dT primer followed by Polymerase Chain Reaction (PCR) amplification of GDH and GS cDNAs using gene specific primers. Data show that GDH RNA is expressed in mouse embryos grown in vivo at the one-cell, two-cell, eight-cell, and blastocyst stages of development. GS RNA is not expressed at the one-cell stage, but first appears at the two-cell stage and is expressed at the eight-cell and blastocyst stages. Semiquantitative PCR analysis using a globin internal standard demonstrated that GS RNA is present at high levels at the two-cell stage and declines by 51 % by the blastocyst stage. These results suggest that, within the preimplantation mouse embryo, GDH RNA is expressed by both the maternal genome as well as the embryonic genome, while GS RNA is only expressed by the embryonic genome. This study provides an explanation for why glutamine is utilized as an energy source during preimplantation development, which allows for a better understanding of glutamine metabolism and its role during early mouse development. / Department of Biology

The cell cycle is a highly regulated process driven by endogenous factors that have regulatory functions. Certain proteins such as cyclins and cyclin-dependent kinases (cdks) are needed to progress through the four phases of the cell cycle. Cell cycle regulatory proteins have been characterized in somatic cells and exhibit phase specific expression patterns. However, the changes in expression of these proteins have not been characterized in early cleavage stage mouse embryos. This study utilized indirect immunofluorescence microscopy to determine the expression pattern of cell cycle regulators cyclin B 1, cyclin E, and CDK2 during the first three cell cycles of preimplantation mouse embryo development. Results suggest unique and specific patterns of expression for all three cell cycle regulators at different stages of the cell cycle. In G1 of the first cell cycle, cyclin E is expressed at high levels, whereas cyclin B 1 and CDK2 are expressed at moderate levels. During DNA synthesis (S phase), CDK2 levels slightly increase. However, cyclin B 1 and cyclin E levels begin to decline in S and continue to decrease to minimal levels in G2. CDK2 expression follows a similar trend during G2, decreasing considerably. During the second cell cycle, cyclin B 1 and CDK2 show staining patterns similar to the first cell cycle. The expression of cyclin E is maintained at a moderate level throughout the entire second cell cycle. Cyclin B 1, cyclin E, and CDK2 are all expressed at moderate levels during GI of the third cell cycle. During S phase, cyclin B 1 and CDK2 are maintained at moderate levels, but cyclin E is decreased to minimal levels. The expression of all three proteins was minimal during G2. This study provides baseline information on the unique expression patterns of cell cycle regulators in early mouse embryos. The determination of cell cycle protein expression will allow for a better understanding of the complex mechanisms in the division process during preimplantation mouse embryo development. / Department of Biology

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The preimplantation mouse embryo undergoes many metabolic changes as development proceeds. One major change is the switch from a pyruvate based metabolism, to a glucose based metabolism. The phosphofructokinase enzyme is the regulatory enzyme of glycolysis and is thought to be a major contributor in controlling the block to glycolysis in early preimplantation mouse embryos. This study was undertaken to construct a system that would allow detection of RNA for the highly glycolytically active subunit (muscletype) of the phosphofructokinase (PFK) enzyme. A muscle specific mutant PFK plasmid was generated to provide mutant internal control RNA. Using this internal control, initial reverse transcriptionpolymerase chain reaction data collected from early embryo stages suggest that the muscle type PFK subunit RNA is not expressed in the preimplantation mouse at the 1-cell or blastocyst stages. This result suggests that PFK activity detected at the later morula and blastocyst stages must be from either a different PFK subunit or a novel embryonic form of PFK. / Department of Biology