Japanese quail hens were used to study the effect of feeding palmitic, oleic or linoleic acids on the reproductive performance, tissue fatty acid composition and embryo lipid metabolism. Quail fed palmitic acid consumed more feed than those fed either oleic or linoleic acids. The highest level of reproductive performance was observed in quail fed palmitic acid followed by those fed oleic and linoleic acids. The highest level of embryo survival, observed in the palmitic acid fed group, was associated with more rapid mobilization and assimilation of yolk material by the embryo during incubation and it was not related to changes in eggshell quality. High levels of oleic and linoleic acids were found in egg yolk, plasma and liver lipids from quail fed oleic and linoleic acids, respectively. However, feeding palmitic acid resulted in elevated levels of palmitoleic acids in all three tissues. The fatty acid profiles of phospholipid, triglyceride and cholesterol esters of embryonic tissues were consistently influenced by the fatty acid composition of the yolk lipids and the stage of development. Feeding palmitic acid promoted more retention of labeled fatty acids in embryo lipids. Labeled oleic acid was preferentially esterified in the cholesterol ester fraction of yolk sac membrane lipids, and it appears that this fatty acid is utilized to a great extent by the quail embryo during its development.

No description available.

Although many of the genes and pathways involved in Drosophila embryogenesis have been thoroughly investigated, a complete understanding of the mechanisms behind these processes is still lacking. In order to gain a better perspective, the main objective of current research is to identify additional components of the signaling pathways that are crucial for normal Drosophila development. / One such developmental process is germ band retraction, which occurs in mid-embryogenesis and consists of the movement of the tail end of the germ band, or embryo proper, to its final posterior position. One of our primary objectives is to identify the signaling pathways behind this process. To this end, we investigated the 7T2 mutant, which fails to retract. This zygotic lethal mutant was originally uncovered in a screen for maternal-effect U-shaped embryonic phenotypes. Using a combination of meiotic recombination with molecularly mapped P-element insertions and complementation tests with deficiencies, we mapped the 7T2 mutant to the chromosomal region containing the gene pellino. Here, we show that both pellino mRNA and Pellino protein are missing in the 7T2 mutant tissue, indicating that 7T2 is a loss of function allele of pellino. / Further characterization of the 7T2 mutant revealed three distinct phenotypes: germ band retraction defects, twisted germ bands and head defects. Based on these observations, we propose that pellino is involved in several biological processes during early Drosophila development. Here we show that pellino is involved in the JNK pathway through genetic interaction with hemipterous, an upstream member of the JNK pathway. In addition, we provide preliminary evidence suggesting that the expression of Twist, a protein induced by the Toll pathway, is affected in the absence of pellino, suggesting a role for pellino in dorsal-ventral pattern formation.

No description available.

A mouse embryo culture medium which would allow for in vitro development from 1-cell stage to blastocyst stage could offer many benefits for human research. Previous researchfrom our lab has demonstrated a mouse embryo culture medium named CZB seems to allow for in vivo-like conditions for development. Compared to other commonly used mouse embryo culture media, CZB medium promotes a higher frequency of 1 cell mouse embryos developing to blastocyst stage (Chatot et 1989). A key difference between CZB and other mouse embryo culture media is that CZB contains the amino acid glutamine metabolism is glutamate dehydrogenase (GDH). In order to determine if CZB cultured embryos follow in vivo-like patterns of gene expression for GDH, a quantitative competitive RT-PCR system was designed. A mutant GDH mRNA template was created which lacked a specific restriction enzyme site and was used as a competitive template in quantitative RT-PCR. This system was used to determine the amount of GDH mRNA present in in vivo grown blastocyst stage mouse embryos. It was determined that the amount of GDH mRNA present in in vivo blastocyst stage embryos was 282 fg/embryo. It is believed this system will also allow for quantitation of GDH mRNA in the earlier preimplantation stages of in vivo grown embryos, as well as the preimplantation stages 2-cell to blastocyst of CZB cultured embryos. / Department of Biology

The proteins and molecular machinery mediating the release of cortical granule (CG) contents from fertilized embryos is not completely understood. The process of vesicle fusion involves linking chaperones prior to vesicle to membrane contact. Rab3A, a member of a low-molecular weight GTP-binding protein superfamily has been detected in mouse embryos from the unfertilized meiotic II stage to the 2-cell. It is believed to positively regulate the final step of CG exocytosis by binding to Rabphillin, calcium ions (Ca2+), and phospholipids. SNAP23 a member of soluble NSF [N-ethylmaleimidesensitive factor] attachment protein receptors (SNAREs) binds together with parts of the Rab3A-rabphilin3A complex and is believed to be involved in the Ca2+-dependent exocytosis of non-neuronal systems. In this study we observed the mRNA expression for SNAP23 and Rab3A in pre-Meiotic I, post-Meiotic I unfertilized eggs (pre-MI UFE and post-MI UFE), and fertilized eggs (FE) utilizing RT-PCR. The products were analyzed in 2% agarose gel stained with ethidium bromide. Density analysis using a globin external standard showed that the levels of mRNA transcripts declined from the UFE to the FE in both genes, SNAP23 and Rab3A. Immunofluorescence was used for the detection and localization of Rab3A protein within the pre-MI and post-MI UFE and FE mouse egg. Eggs were stained with anti-Rab3A primary antibody and lens culinaris agglutinin (LCA) conjugated to FITC. Rab3A showed punctate staining in pre- and post-MI UFEs on small vesicles assumed to be CGs and in FEs on vesicles of a larger size. Uniform cytoplasmic expression was also seen, throughout the cells cortical and subcortical regions in each stage (pre- and post-MI UFEs and FEs), but with decreasing intensity as the eggs matured. This cytoplasmic stain may represent inactive Rab3A in the cytosol. The LCA stain showed punctate expression of cortical granules with localization within the cortical region and the plasma membrane. The addition of information on SNAP23 and Rab3A will aid in the process of studying CG exocytosis as well as in understanding the temporal and spatial development pathways involved in stimulating the cortical reaction. / Department of Biology

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Biology

Dilantin (DPH) is a common anticonvulsant drug used to prevent seizures. It is known to be a human teratogen causing fetal hydantoin syndrome (FHS). FHS is characterized by multiple developmental and growth related abnormalities and mental retardation. Previous studies demonstrated that DPH slowed growth and division in preimplantation mouse embryos in vivo and in vitro. DHP exposure in utero decreased the crown to rump length and weight of 25-35% of embryos and reduced the rate of endochondral bone conversion from cartilage. In vitro preimplantation mouse embryos treated with DPH at 5, 10 and 20 μg/ml showed a reduction of 25-35% in their development, and block at 2-cell or 3-4-cell stages. These embryos also showed a prolonged DNA synthesis (S) phase during the second cell cycle. Nuclear localization and concentration levels of cyclin A , the S phase cyclin, were also altered in vivo in 2-cell DPH treated embryos compared with NaOH control embryos during G1, S phase and G2 of the first, second and third cell cycles. DPH altered patterns of expression of cyclin A were associated with cell cycle disregulation during preimplantation development. The purpose of the current study was to determine whether DPH also affects the concentration of DNA pol δ catalytic subunit in 2-cell preimplantation mouse embryos at G1 and S phases, thus delaying DNA synthesis and contributing to FHS. Immunofluorescence and confocal microscopy were used as tools to determine relative levels and distribution of DNA pol δ (for consistency with text) in the cytoplasm and the nuclei of DPH and NaOH treated 2-cell embryos at G1 and S phase of the second cell cycle. DPH decreased DNA pol δdelta total embryo and nuclear levels by 43% and 36%, respectively, in G1 compared with NaOH controls. Similarly, nuclear levels of DNA pol δ in DPH embryos in S phase near the G2 transition of the second cell cycle increased to 144% of NaOH control levels; there was not a statistically significant difference between total embryonic levels of late S phase DNA pol δ in DPH and NaOH treated control embryos. The results indicated that DPH affects the levels of DNA pol δduring G1 and S phase near the G2 transition of the second cell cycle in preimplantation mouse embryos. The significant alteration in the levels of DNA pol δ during S phase and its probable consequent altered polymerase activity could contribute to an explanation for the extension of S phase in preimplantation embryos observed by Blosser and Chatot. Even more, the alteration in the levels of DNA pol δ and potentially in its exonuclease activity could lead to an increase in the rate of mismatches and mutations suggesting a likely explanation for some features of FHS. / Department of Biology

Cyclin B1 is a cell cycle protein typically associated with the regulation of cellular division (mitosis). Previous studies in this laboratory involving preimplantation mouse embryos found that cyclin B1, or a cyclin B 1-related protein, were present at both G1 and G2/M phase of the cell cycle. Not only was cyclin Bi detected during G1 phase in this study, it was found to be present in higher concentrations at G1 phase through the first three cell cycles. These findings were unexpected, because most of the literature suggests that cyclin B1 is normally degraded during G1 phase. Using immunoprecipitation and immunoblot techniques, a more detailed study of cyclin B1 expression was inititated. Using two different primary antibodies direct against cyclin B1, a 48.97 kDa protein band, which is believed to be cyclin B1, was detected at both G1 and G2/M phases in 1-cell mouse embryos. Using another antibody directed against Cdk1, the kinase that forms a complex with cyclin B1 in order to direct the G2/M transition, a 37 kDa protein band was also detected at both G1 and G2/M phases in 1-cell mouse embryos. In order to determine whether cyclin B1 was present as a complex with Cdk1, immunoblotting with the anti-Cdk1 antibody. Again, a 37kDa protein band was detected at both G1 and G2/M phases. Finally, in order to determine whether the cyclin B1/Cdk1 complex exists in its active form, histone kinase assays were performed using anti-cyclin B1 immunoprecipitates. Kinase activity was detected in immunoprecipitates collected from G2/M phase 1-cell embryos, but no kinase activity was detected from immunoprecipitates collected from G1 phase 1-cell embryos. These data indicate that cyclin B1 and Cdk1 are present and exist as a complex in both G1 and G2/M phases of 1-cell mouse embryos, although the complex only appears to be active at the G2/M phase. / Department of Biology

The blastoderm embryo of Drosophila melanogaster consists of a unicellular syncytium with a large number of peripheral nuclei. The cytoplasm surrounding each peripheral nucleus is compartmentalised into apical periplasm above each nucleus and basal periplasm below it. The expression of different genes in the syncytial blastoderm is crucial for the genetic control of development. The pair-rule genes are involved in controlling the pattern of metamerisation of the embryo. Pair-rule mRNAs are expressed in alternate metameres, in a pattern of stripes. Within each stripe, mRNA is found in the apical periplasm of the syncytial blastoderm. By analysing the distribution of mRNA of a number of hybrid constructs, I show that the 3' untranslated part of three pair-rule genes are required for the apical localisation of their transcripts. A 1.2kb region in the 3' end of fushi tarazu (ftz), a 700bp region in the 3' end of hairy (h) and a 160bp fragment of the 3' untranslated part of the even-skipped (eve) pair-rule gene are shown to contain apical localisation signals. I show that the mechanism of apical localisation is unlikely to involve a cytoplasmic process and that the 3' untranslated part of the bicoid (bed) gene contains sequences necessary for apical localisation. I propose that apical localisation involves a nuclear mechanism which exports mRNA from the apical side of the nuclear membrane. I demonstrate that apical localisation is achieved by an RNA-mediated process and not by a DNA-mediated mechanism. Finally, I demonstrate that the intracellular localisation of transcripts encoding cytoplasmic proteins influences the distribution of the protein in the periplasm. I propose that the function of apical localisation is to limit the diffusion of pair-rule proteins so that the pattern of protein expression resembles precisely the transcriptional domain.