Thesis (M.S.)--California Polytechnic State University, 2009. / Title from PDF title page; viewed on February 2, 2010. Major professor: Matthew A. Burd, D.V.M. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Agriculture." "December 2009." Includes bibliographical references (p. 47-52).

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Implantation is a critical step in reproduction. It is complicated and well-coordinated consisting of apposition, attachment and invasion of embryo into the endometrium. The mechanism of implantation is unclear. Our previous proteomic study showed an increase of annexin A2 in the endometrium during the implantation window of mice, consistent with the increased annexin A2 expression in the receptive human endometrium. The hypothesis of this project was that annexin A2 mediatedthe embryo-endometrium attachment. The first objective was to study the spatio-temporal expression of endometrial annexin A2 immunoreactivities in humans and mice. The cyclical change in annexin A2 expression in the mouse and human reproductive cycle suggested the involvement of a steroid regulatory mechanism. Interestingly, annexinA2 was transiently expressed on the membrane between the mouse uterine luminal epithelium and the implanting embryos from Day 4 (pre-implantation) to Day 5 (post-implantation) of pregnancy. No such signal change was observed at the inter-implantation sites, showing that the implanting embryos partially regulated annexin A2 expression. These observations and the high expression of the molecule in the luminal epithelium of human endometrium in the mid-and late luteal phase were consistent with a role of annexin A2 in implantation. The second objective was to verify the action of steroids on annexin A2 expression. It was found that a combination of 6675 pmol/L of estrogen and 429.8nmol/L of progesterone increased the total and apical surface expression of annexin A2. In mice, estrogen but not progesterone, increased annexin A2 expression in the uterine luminal epithelium of ovariectomized mice. The third objective was to study the function of annexin A2 in embryo-endometrium attachment using an Ishikawa (endometrial epithelial cells)-JEG-3 trophoblast spheroids (embryo surrogate) coculture model. Knockdown of the expression of annexin A2 in either or both cell lines significantly decreased the attachment rate of the spheroids onto the endometrial cells. The suppressive action on the two cell lines was additive. The attachment was also suppressed in the presence of anti-annexin A2 antibody during coculture. Annexin A2 was also involved in mouse implantation as demonstrated by a significant decrease in implantation sites after injection of anti-annexin A2 antibody into the mouse uterine horn. The final objective was to study the action of annexin A2 as an adhesive molecule in embryo attachment. It was found that loss of P11, the binding partner of annexin A2, reduced the attachment rate of the JEG-3 spheroids probably by decreasing the translocation of annexin A2 to the surface of the endometrial cells. Recombinant P11 and annexin A2 protein failed to bind significantly to the Ishikawa cells and the JEG-3 cells. In summary, this study demonstrates the involvement of annexin A2 as an adherent molecule in the embryo-endometrium interaction. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Previous studies have shown that NACHT, leucine rich repeat and PYD domain containing 5 (Nlrp5) deficient embryos fail to develop beyond the two-cell stage. Despite this strong phenotype, little is known of the function of NALP5 and the pathways affected by its deficiency. We showed that Nlrp5 deficient oocytes and embryos exhibit a decrease in caspase activity. In addition, the kinetics of NF-κB-p65 nuclear translocation is altered, which leads to negative downstream effects. Autophagy is known to be regulated downstream of NF-κB and is a key event during the oocyte-to-embryo transition. We observed defective execution of autophagy in Nlrp5 deficient two-cells evidenced by absence of autophagosome formation and abnormal lysosomal maturation. We found that inactivating autophagy leads to an accumulation of lipid droplets and embryos lacking Nlrp5 exhibit this accumulation. Thus, NALP5 may be an integral component responsible for autophagy mediated lipid metabolism, which when compromised causes developmental arrest.

Previous studies have shown that NACHT, leucine rich repeat and PYD domain containing 5 (Nlrp5) deficient embryos fail to develop beyond the two-cell stage. Despite this strong phenotype, little is known of the function of NALP5 and the pathways affected by its deficiency. We showed that Nlrp5 deficient oocytes and embryos exhibit a decrease in caspase activity. In addition, the kinetics of NF-κB-p65 nuclear translocation is altered, which leads to negative downstream effects. Autophagy is known to be regulated downstream of NF-κB and is a key event during the oocyte-to-embryo transition. We observed defective execution of autophagy in Nlrp5 deficient two-cells evidenced by absence of autophagosome formation and abnormal lysosomal maturation. We found that inactivating autophagy leads to an accumulation of lipid droplets and embryos lacking Nlrp5 exhibit this accumulation. Thus, NALP5 may be an integral component responsible for autophagy mediated lipid metabolism, which when compromised causes developmental arrest.

Although cottonseed is an important source of oil and fiber, the development of cotton embryos has not been investigated as well as development of cotton fiber. The development of cotton embryos in late heart-stage and early cotyledonary stage is less well investigated than the first 10-14 days after anthesis, or the late stages of embryo development during seed-fill and desiccation. This analysis focused on cotton embryos in the late heart-stage and early cotyledonary stage of development (1.5-4.0 mm or about 13-18 DPA). In vitro analyses are important tools for studying embryos in isolation from the endosperm and fiber and when it is necessary to monitor the developing embryo continuously. The original goal of this work was to develop an in vitro culture method that would support continued development of excised zygotic embryos from the early cotyledonary stage into complete plants with true shoots, i.e. true leaves or visible buds and then to use this method to study aspects of developmental regulation during cotyledonary stage and the transition to later stages. Not all embryos were competent to develop true shoots (an apical bud or a leaf plus a bud) in culture. A number of cultural variables were tested and eliminated. Embryo maturity at the time embryos were excised and the presence or absence of light during the first 14 days of culture affected the competence of immature embryos to developed true shoots. The effect of light was verified in several large replicated experiments. Morphological changes occurring during in vivo development were examined microscopically. The transition from heart-stage to early cotyledonary stage and the development of the first leaf from initials to a large structure were identified. Embryonic shoot apices continued to grow in cultured 1-3 mm embryos. The size and shape of light-treated and dark-treated embryonic apices was compared. A germination test of mature seeds identified seedlings with a similar phenotype occurring at similar rates in seedlings and light-cultured embryos and possible causes were discussed.

No description available.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Initially, we aimed to identify novel genes regulating vertebrate neurogenesis and somitogenesis by screening cDNAs derived from gastrulation/neurulation stage zebrafish embryos for clones revealing corresponding genes with expression patterns suggestive of roles in these processes. The lack of suitable cDNA libraries prompted us to devise a simplified method for producing randomly-primed, directionally cloned cDNA libraries from small amounts of embryonic tissue. To achieve this, several techniques were combined, including cDNA synthesis on a solid carrier, random priming of 1st cDNA strand synthesis, non-specific priming of 2nd cDNA strand synthesis and amplification of initially small amounts of cDNAs by suppression-PCR. A pilot-scale in situ screen using a cDNA library produced by the above method identified a gene, spadetail, that is expressed in presomitic mesoderm and in unidentified, apparently irregularly distributed cells of the spinal cord. spt functions in mesodermal development, yet its role in neural tissue remains unknown. Analysis of the spadetail-expressing neural cells' gene co-expression profile and dorsoventral location implied that they are Dorsal Longitudinal Ascending interneurons. Quantitative analysis of these cells' rostrocaudal distribution showed that there is a tendency to higher cell numbers in rostral spinal segments. The observation that spadetail-expressing neurons are frequently juxtaposed to somitic cells expressing spadetail at low levels suggests that the distribution of spadetail-expressing neurons may be 'inefficiently' patterned by spadetail-expressing somitic cells or that the expression of spadetail in both tissues is induced by a common positional cue. The strategy for non-specific was then extended to develop a simple technique for cloning unknown DNA sequences flanking known DNA. An initial nonspecific PCR amplification was performed with a single primer that binds specifically within known sequence and non-specifically in the unknown DNA region. In a second reaction, the sequences of interest were amplified from the primary reaction mixture (that also contains undesired sequences) with nested PCR using a primer that had been extended further downstream from the primer used in the initial PCR. This enabled isolation of a 0.5 kb region of amphioxus Notch cDNA, that, in turn, contributed to the subsequent analysis of the evolution of vertebrate Notch genes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1108027 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2004

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Initially, we aimed to identify novel genes regulating vertebrate neurogenesis and somitogenesis by screening cDNAs derived from gastrulation/neurulation stage zebrafish embryos for clones revealing corresponding genes with expression patterns suggestive of roles in these processes. The lack of suitable cDNA libraries prompted us to devise a simplified method for producing randomly-primed, directionally cloned cDNA libraries from small amounts of embryonic tissue. To achieve this, several techniques were combined, including cDNA synthesis on a solid carrier, random priming of 1st cDNA strand synthesis, non-specific priming of 2nd cDNA strand synthesis and amplification of initially small amounts of cDNAs by suppression-PCR. A pilot-scale in situ screen using a cDNA library produced by the above method identified a gene, spadetail, that is expressed in presomitic mesoderm and in unidentified, apparently irregularly distributed cells of the spinal cord. spt functions in mesodermal development, yet its role in neural tissue remains unknown. Analysis of the spadetail-expressing neural cells' gene co-expression profile and dorsoventral location implied that they are Dorsal Longitudinal Ascending interneurons. Quantitative analysis of these cells' rostrocaudal distribution showed that there is a tendency to higher cell numbers in rostral spinal segments. The observation that spadetail-expressing neurons are frequently juxtaposed to somitic cells expressing spadetail at low levels suggests that the distribution of spadetail-expressing neurons may be 'inefficiently' patterned by spadetail-expressing somitic cells or that the expression of spadetail in both tissues is induced by a common positional cue. The strategy for non-specific was then extended to develop a simple technique for cloning unknown DNA sequences flanking known DNA. An initial nonspecific PCR amplification was performed with a single primer that binds specifically within known sequence and non-specifically in the unknown DNA region. In a second reaction, the sequences of interest were amplified from the primary reaction mixture (that also contains undesired sequences) with nested PCR using a primer that had been extended further downstream from the primer used in the initial PCR. This enabled isolation of a 0.5 kb region of amphioxus Notch cDNA, that, in turn, contributed to the subsequent analysis of the evolution of vertebrate Notch genes. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1108027 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2004

Thesis (M.S.)--Brigham Young University. Dept. of Plant and Animal Sciences, 2004. / Includes bibliographical references.