No description available.

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

No description available.

No description available.

Three experiments were conducted to evaluate in vitro and in vivo development of zona pellucid a-intact (ZPI) and zona pellucida-free (ZPF) mouse embryos after encapsulation in either 2% sodium alginate or 0.1% poly-L-Iysine (PLL). In Experiment 1, rate of development of ZPI embryos (n = 150) from morulae to hatched blastocysts was measured after encapsulation in alginate or PLL and as unencapsulated controls. Following encapsulation, developmental stages were recorded every 24 h for 120 h. Percentage of encapsulated embryos completely hatched from the zona pellucid a were not different from each other but were lower than unencapsulated controls at 48, 72, 96 and 120 h. Development of ZPI and ZPF mouse embryos after encapsulation in either alginate or PLL was examined in Experiment 2. Developmental stages and diameters were recorded every 24 h for 72 h. At 72 h, embryos were stained and fixed on slides to examine nuclei. Percentage of ZPI embryos developing to expanded blastocysts, their diameters and nuclear counts were not different from each other or from ZPF embryos. Percentage of ZPI embryos initiating hatching or completely hatched from the zona pellucida, their diameters and nuclear cell numbers were also similar. In the final experiment, ZPI mouse morulae were unencapsulated or encapsulated in either alginate or PLL and transferred into recipients to examine in vivo development. Recipients were allowed to develop fetuses to term. Recipients receiving encapsulated embryos failed to deliver pups. However, five of six recipients of unencapsulated embryos (n = 71) delivered a total of 16 live pups. Additional transfers were performed to examine viable fetuses and resorption sites on day 10 of gestation. Pregnancy rates, diagnosed by the presence of via bIe fetuses or resorption sites, were similar for all treatments: unencapsulated (71.4%), a1ginate (87.5%) and PLL (87.5%). However, the total number of viable fetuses present was higher for unencapsulated embryos (42.1 %) when compared to embryos in alginate microgels (17%) and embryos in PLL microcapsules (14.60/0). Additionally, recipients of alginate and PLL encapsulated embryos had more resorption sites (4 0/0 and 13.4%) when compared to recipients of unencapsulated embryos (0%). These investigations demonstrated that development of encapsulated ZPI mouse morulae is impaired at the hatched blastocyst stage; however, encapsulated ZPI and ZPF mouse morulae develop similarly in size and nuclear counts. In vivo development of ZPI morulae was also impaired due to an asynchronous condition between the uterine environment and the developing embryos. / Master of Science

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Type II collagen is the major extracellular matrix (ECM) protein present in cartilage and is detected in non-chondrogenic tissues such as the heart and the neural tube during developmental stages involving rapid tissue morphogenesis indicating an active role played by the collagen in embryogenesis. Type II collagen is synthesized as a procollagen precursor which has amino- and carboxyl-terminal globular extensions (N- and C-propeptides) flanking a central triple helical domain. Two isoforms of type II procollagen are generated by alternative mRNA splicing of the exon 2: IIA and IIB. Sequence present in the N-propeptide of IIA, translated from the spliced-in exon 2, encodes a von Willebrand factor-like C cysteine rich (CR) domain. This domain is homologous to those present in regulators of the bone morphogenetic protein (BMP) signaling such as chordin (Chd), twisted gastrulation (Tsg) and crossveinless (Cv). Previous in vitro binding assays and overexpression studies in frog embryo suggest that the CR domain of IIA antagonized BMP signaling. In order to give a better understanding of the function of IIA in embryonic development and cellular signaling, several approaches including expression pattern analyses, phenotypic analyses of null mutant and gain of function studies are employed in this study. Expression studies of IIA mRNA in early postimplantation mouse embryos find that it is present in the axial mesendoderm (including the anterior definitive endoderm [ADE] and the prechordal plate) which is a critical head organizer at neural plate (E7.5) and head process (E8.0) stages. Characterization of the IIA deficient mice (IIA-/-), constructed by removing exon 2 from type II collagen (Col2a1) gene by homologous recombination, indeed reveals that the anterior-most neural tissue is deficient at early somitogenesis denoted by reduction/loss of the forebrain/optic cup markers. Marker studies indicate that the ADE may already be affected at the neural plate stage in IIA-/-. The neural phenotype of IIA-/- displays significant similarities with mutants deficient in BMP pathway components such as Chd-/-;Nog+/-, Tsg-/- and Tsg-/-;BMP4+/- suggesting that IIA plays a role in maintaining the specification and/or regulating the signaling properties of the anterior midline tissue which involves regulation of BMP signaling. Results of ectopic expression of IIA in Xenopus laevis embryos suggest that IIA regulate BMP and the related Nodal signaling pathways in a context dependent manner which has significant implications in normal anterior neural plate development. Based on the work described in this thesis and the body of existing evidence, a model is presented which suggests that IIA promote/maintain anterior neural plate development by regulating the range and extent of BMP signaling in the anterior neural plate. This study sheds light on the role of an ECM component in regulating tissue patterning and cellular signaling during early mouse development and also provides putative function for the CR domain of other fibrillar procollagens including type I, III and V which is poorly understood currently. This work will provide the framework for the design of subsequent studies in re-examining the role of these fibrillar procollagens in embryogenesis. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy