Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

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The high incidence of pregnancy loss is a major issue facing the cattle industry. Use of in vitro fertilized (IVF) bovine embryos has become increasingly popular to help alleviate several of these reproductive issues and provide a means to enhance genetic gain for production traits. An uterine paracrine factor cocktail containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1) (collectively termed EFI) was recently identified as a means for improving in vitro derived bovine embryo development and trophectoderm cell numbers. The objectives of this work were to determine if EFI treatment during in vitro bovine embryo culture improves transferable embryo quality and post-transfer placental and fetal development. For each replicate (3 total), slaughterhouse-derived bovine oocytes were matured and fertilized in vitro. At day 4 post-fertilization, ≥8 cell embryos were harvested, pooled, and exposed to either the EFI treatment (10ng/ml EGF, 10ng/ml FGF2, 50ng/ml IGF1) or carrier only (1% Bovine Serum Albumin). At day 7, individual embryos were transferred to estrous synchronized beef cattle. Artificial insemination (AI) was completed on a subset of cows. The EFI treatment increased (P<0.05) the percentage of transferable embryos. Pregnancy rate at day 28 post-estrus was similar among treatments. Circulating concentrations of pregnancy-associated glycoproteins (PAGs) were determined from plasma harvested at day 28, 42 and 56. Transrectal ultrasonography was used to measure fetal crown-rump length (CRL) at day 42 and 56 and to determine fetal sex at day 60. There were no main effect differences observed across days for PAG concentration. Fetus sex by ET/AI group interactions were absent at day 28 but existed at days 42 and 56 (P<0.05). At both days, this interaction reflected fetus sex-dependent changes within the ET control group, where PAG concentrations were greater (P<0.05) in male fetuses than female fetuses. No CRL differences or interactions existed among fetal sex and pregnancy group. In summary, addition of the EFI cocktail during bovine embryo culture improved the quality of transferable embryos, but did not affect placental function or embryonic/fetal development. Increasing the numbers of transferable embryos is of value given the cost of in vitro embryo production, but no apparent increases in embryo or placental competency were detected. The EFI treatment increased (P<0.05) the percentage of transferable embryos. / Master of Science / The high incidence of pregnancy loss is a major issue facing the cattle industry. Use of in vitro fertilized (IVF) bovine embryos has become increasingly popular to help alleviate several of these reproductive issues and provide a means to enhance genetic gain for production traits. An uterine paracrine factor cocktail containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF1) (collectively termed EFI) was recently identified as a means for improving in vitro derived bovine embryo development and trophectoderm cell numbers. The objectives of this work were to determine if EFI treatment during in vitro bovine embryo culture improves transferable embryo quality and post-transfer placental and fetal development. For each replicate, slaughterhouse-derived bovine oocytes were matured and fertilized in vitro and at day 4 post-fertilization, embryos were exposed to either the EFI treatment (10ng/ml EGF, 10ng/ml FGF2, 50ng/ml IGF1) or carrier only (1% Bovine Serum Albumin). Artificial insemination (AI) was completed on a subset of cows and the remaining cattle receive embryos at day 7. The EFI treatment increased (P<0.05) the percentage of transferable embryos. Pregnancy rate at day 28 post-estrus was similar among treatments. Circulating concentrations of pregnancy-associated glycoproteins (PAGs) were determined from plasma harvested at day 28, 42 and 56. Transrectal ultrasonography was used to measure fetal crown-rump length (CRL) at day 42 and 56 and to determine fetal sex at day 60. There were no main effect differences observed across days for PAG concentration. Fetus sex by ET/AI group interactions were absent at day 28 but existed at days 42 and 56 (P<0.05). At both days, this interaction reflected fetus sex-dependent changes within the ET control group, where PAG concentrations were greater (P<0.05) in male fetuses than female fetuses. No CRL differences or interactions existed among fetal sex and pregnancy group. In summary, addition of the EFI cocktail during bovine embryo culture improved the quality of transferable embryos, but did not affect placental function or embryonic/fetal development. Increasing the numbers of transferable embryos is of value given the cost of in vitro embryo production, but no apparent increases in embryo or placental competency were detected. The EFI treatment increased (P<0.05) the percentage of transferable embryos.

Despite the importance of zinc in cell proliferation and development, mechanisms of zinc redistribution during these processes remain largely elusive. Given the limited external supply of nutrients during embryogenesis, developing organs most likely redistribute zinc from neighboring cells to satisfy their increased demand, thus raising the intriguing and fundamental question of how the limited supply of zinc in a fertilized egg is redistributed in the course of embryonic development. To systematically explore this question, we employed both cell culture and zebrafish as model systems in combination with a Zn(II)-selective fluorescent probe and synchrotron X-ray fluorescence (SXRF) microtomography studies. Using the Zn(II)-selective emission ratiometric fluorescent probe designed in our lab, we followed the redistribution dynamics of labile Zn(II) pools in a zebrafish embryo during the first 24 hours post fertilization. Furthermore, SXRF microtomography studies were used to visualize the 3D distribution of total zinc in fixed zebrafish samples. From this method we successfully reconstructed a 3D elemental distribution map at 5 μm resolution. The volumetric map revealed a distinct zinc distribution that could be correlated with characteristic anatomical features at this stage of embryonic development. Together these powerful techniques allow us to study both labile zinc in live samples and total zinc content in fixed samples in order to achieve a more detailed understanding of the zinc redistribution dynamics during embryogenesis.

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Shoot epidermal identity is critical for plant survival, growth, and interaction with the environment. Epidermal identity is specified during very early embryogenesis, and maintained in the outermost cells of the plant throughout the entire life cycle. In this work I aimed to generate a model for the establishment of basal epidermal cell fate during embryogenesis based on the analysis of both known and novel regulators. Loss of function of two HD-ZIP IV transcription factors, ATML1 and PDF2 had previously been shown to lead to embryo lethality due to loss of epidermal specification. In this study I uncover dosage dependency of ATML1 and PDF2 function during embryogenesis. By expressing functional ATML1 and PDF2 fusion proteins specifically in the epidermis, I developed a novel tool allowing demonstration of homo- and heterodimerization of these two transcription factors in planta. Using genetic and proteomic analysis I provide evidence that other HD-ZIP IV proteins are involved in epidermal specification together with ATML1 and PDF2, suggesting the presence of multiple regulatory protein complexes. Based on previous published and unpublished work, I tested the hypothesis that ATML1 and PDF2 form part of a regulatory feedback loop necessary for maintenance of epidermal identity, and involving cell-cell signalling mediated by the receptor kinase ACR4. Using a genetic approach I confirm that ATML1 and PDF2 likely act together with ACR4 in the specification of embryonic epidermal identity. I show that ATML1 and PDF2 negatively regulate both ACR4, and their own expression, most likely by binding to L1 box motifs. In contrast, I provide evidence that ACR4-mediated signalling participates in maintaining expression levels of ATML1 and PDF2. Mathematical modelling of the properties of the feedback loop supported by my results, suggests that it is capable of maintaining a robust epidermal cell fate, and predicts possible changes in network interactions during the process of epidermal cell fate specification. Finally I used a combination of bioinformatics approaches to integrate in silico and experimental data with the aim of discovering potential novel epidermal regulators and targets of epidermal fate specifying pathways. This work highlighted potential roles for WOX-family transcription factors in epidermal fate specification, which were further analysed genetically. In addition, bioinformatics analysis pinpointed an intriguing overlap between the targets of epidermal specification pathways and targets of abiotic stresses signalling.