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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

At fertilization, the remodelling of the sperm nucleus into the male pronucleus is critical for normal development. Morphological and functional changes to the nucleus are underpinned by biochemical changes in the chromatin composition, most notably the removal of sperm specific protamines and assembly of histones onto the paternal DNA. This exchange is controlled by oocyte factors, as exemplified in Xenopus by nucleoplasmin. Though mammalian factors remain unidentified, a functional assay based on antibodies recognizing core histones has been developed to test the activity in oocytes that transfers histones onto sperm DNA, named histone transfer activity (HTA). The assay was applied to growing and maturing murine oocytes to determine when during oogenesis HTA develops, and to probe potential regulatory mechanisms. Fully-grown oocytes develop HTA upon maturation, in a protein-synthesis dependent manner. Large, growing oocytes also develop HTA upon entry into M-phase. Small growing meiotically incompetent oocytes, ones that do not spontaneously enter M-phase, do not develop HTA, though this can be overcome by culture of oocytes to meiotic competence, or by treatment with strontium to induce intracellular calcium oscillations. Taken together these findings form a model of how HTA develops throughout oogenesis. Finally, an attempt is made to identify a potential mammalian HTA factor. Transcripts for two remodelling factors, mNAP and Npm3, are identified in the murine oocyte, and injection of anti-sense oligonucleotides reveals that Npm3 plays a significant role in the deposition of histories and the remodelling of sperm chromatin at fertilization. Combined with the findings of the HTA assay, the data forms a testable model of how Npm3 may be regulated throughout oogenesis.

Thesis (M.S.)--University of Hawaii at Manoa, 2007. / Includes bibliographical references (leaves 70-81). / vii, 86 leaves, bound ill. (some col.) 29 cm

Thesis (Ph. D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1984. / Includes bibliographical references (leaves 113-128).

Thesis (Ph. D.)--University of North Texas, Aug., 2007. / Title from title page display. Includes bibliographical references.

Thesis (Ph.D.) -- McMaster University, 1999. / Includes bibliographical references (leaves 228-273). Also available via World Wide Web.

Thesis (M.S.)--University of North Carolina Wilmington, 2007. / Includes bibliographical references (Leaves: 37-44)

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / "December 2007" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.