Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 79-99).

Ulm, Univ., Diss., 2001.

Ulm, Univ. Diss., 2006.

Thesis (Ph.D.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 127-168) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Many attempts have been made to improve the in vitro production of cattle embryos by refining in vitro maturation (IVM) and culture systems. Cysteine supplementation to IVM media of bovine oocytes increases cellular glutathione production, which reduces reactive oxygen species (ROS). Similarly, beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of the antioxidant cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on subsequent embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mM cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/mL); EGF (10 ng/mL); and IGF-I+EGF (100 ng/mL+10 ng/mL) for all IVM treatments. Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the IVM media (12 h C IGF-I+EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I+EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD; SOD1) and manganese (Mn) SOD (SOD2) in embryos was assessed. No significant treatment effect was observed on the expression of apoptotic and oxidative stress genes. Bax was expressed strongly (4-fold) in morulae with the addition of IGF-I, but was less prevalent in all other morula and blastocyst groups relative to FCS. There was slightly less expression of both SOD1 and SOD2 with treatments compared to FCS in morulae and blastocysts, indicative of low mitochondrial activity and/or a low level of oxidative stress in treatments. There was no significant treatment effect on total cell number, apoptotic nuclei, or apoptotic index. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS. / Master of Science

Scientists have developed techniques like ovum pick up (OPU) and follicular ablation as a large source of oocytes for creating IVP bovine embryos. These techniques have allowed for more efficient dissemination of valuable female genetics compared to traditional artificial insemination or embryo flushing. IVP embryos have lower embryo development rates and quality, leading to lower pregnancy rates. Nerve growth factor-beta (NGF), however, has been previously shown to improve 48-hour cleavage rates and the number of hatching/ hatched blastocysts out of total presumptive zygotes. We hypothesize that NGF will improve IVP embryo development by positively influencing cleavage and blastocyst rates. The first two experiments' objectives were to determine the effect of recombinant bovine (60 or 90% purity) and human NGF (97% purity) supplementation during in vitro fertilization on 24- and 48-hour cleavage and day 8 blastocyst development rates. The objective of the third experiment was to assess the effect of the supplementation of bovine NGF (90% purity) on heat shocked and non-heat shocked in vitro-matured cumulus-oocyte complexes, assessing cleavage rates at 48 and 72 hours post insemination and blastocyst development rates. The results of experiment 1 show there were no differences between any of the three treatment groups (bNGF60, hNGF95, and control) for 24 hour (P = 0.66) or 48 hour (P = 0.33) embryonic cleavage rates. Additionally, there were no differences between treatments in the total percentage of blastocysts per oocyte (P = 0.91) or the percentage of blastocysts per cleaved embryo (P = 0.32). The results of experiment 2 also showed no differences between any of the three treatment groups (bNGF90, hNGF95, and control) for 24 hour (P = 0.16) or 48 hour (P = 0.18) embryonic cleavage rates. Additionally, there were no differences between treatments in the total percentage of blastocysts per oocyte (P = 0.42) or the percentage of blastocysts per cleaved embryo (P = 0.57). In the 3rd experiment, there was not a significant effect of treatment (P ≤ 0.05) at all stages of embryonic development assessed. On the contrary, in the third experiment, non-heat stressed NGF treatment had an interestingly detrimental effect on early cleavage rates of embryos compared to the non-treated control embryos. These results showed that NGF could not improve in vitro embryonic development rates in standard conditions; however, this negative impact of NGF on early cleavage was not observed in heat-shocked embryos. Suggesting that there could be a protectant factor in NGF that warrants further investigation. / Master of Science / Nerve growth factor (NGF) was initially thought to only play a role in nerve cell development, but research has since shown an influence on female reproduction in cattle. NGF and its receptors have been identified in the follicular fluid and reproductive cell types of females, contributing to egg maturation. Previous data on NGF supplementation with IVP embryos, which took place during the summer, showed that NGF positively affected in vitro-produced embryo development when added to fertilization media, specifically on cleavage rates (division without growth, must be two cells or greater) and blastocyst development. The actual role of NGF on embryo development is still unclear. Therefore, replication of this study is essential. First, we added either recombinant human nerve growth factor (90% pure) or bovine nerve growth factor (60% or 90% pure) to the IVF medium. The goal was to determine if NGF would have the same effects on cleavage rates as bovine purified NGF when supplemented during the fertilization stage, as well as to decide if protein purity and species affected how NGF influenced embryo development rates. For the second part of the study, we heat-shocked oocytes during maturation in a "hot incubator" and supplemented them with bovine 90% NGF. This was done to mimic the summer month heat stress that may have occurred in the abstract data. Our objective was to determine if NGF could mitigate the detrimental heat shock during development and potentially improve embryo developmenNerve growth factor (NGF) was initially thought to only play a role in nerve cell development, but research has since shown an influence on female reproduction in cattle. NGF and its receptors have been identified in the follicular fluid and reproductive cell types of females, contributing to egg maturation. Previous data on NGF supplementation with IVP embryos, which took place during the summer, showed that NGF positively affected in vitro-produced embryo development when added to fertilization media, specifically on cleavage rates (division without growth, must be two cells or greater) and blastocyst development. The actual role of NGF on embryo development is still unclear. Therefore, replication of this study is essential. First, we added either recombinant human nerve growth factor (90% pure) or bovine nerve growth factor (60% or 90% pure) to the IVF medium. The goal was to determine if NGF would have the same effects on cleavage rates as bovine purified NGF when supplemented during the fertilization stage, as well as to decide if protein purity and species affected how NGF influenced embryo development rates. For the second part of the study, we heat-shocked oocytes during maturation in a "hot incubator" and supplemented them with bovine 90% NGF. This was done to mimic the summer month heat stress that may have occurred in the abstract data. Our objective was to determine if NGF could mitigate the detrimental heat shock during development and potentially improve embryo development rates under these stressful conditions. The results of all experiments indicated that NGF could not influence development rates. positively. On the contrary, in the third experiment, non-heat stressed NGF treatment had an interestingly detrimental effect on early cleavage rates of embryos when compared to non-treated control embryos. This negative impact of NGF on early cleavage was not observed in heat-shocked embryos pointing to a possible protectant factor in NGF that needs further investigation.t rates under these stressful conditions. The results of all experiments indicated that NGF could not influence development rates. positively. On the contrary, in the third experiment, non-heat stressed NGF treatment had an interestingly detrimental effect on early cleavage rates of embryos when compared to non-treated control embryos. This negative impact of NGF on early cleavage was not observed in heat-shocked embryos pointing to a possible protectant factor in NGF that needs further investigation.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen involved in angiogenesis, the formation of new blood vessels. sFlt-1, a secreted form of the signal-transducing VEGF receptor Flt-1, can inhibit cellular responses to VEGF both in vitro and in vivo. sFlt-1 is generated by alternative pre-mRNA processing; removal of Flt-1 intron 13 by splicing produces the mRNA for transmembrane Flt-1, whereas cleavage/polyadenylation within this intron, preserving the exon 13/intron 13 junction, yields sFlt-1 mRNA. Despite the likely importance of sFlt-1 in VEGF signaling, little is known about the regulation of its expression. Previous studies using an Flt-1 minigene (pFIN13) revealed that intronic cleavage/polyadenylation signals can affect Flt-1 expression, and, conversely, that 3' intronic splice signals can affect sFlt-1 expression. The goal of present work was to test the hypothesis that splicing and cleavage/polyadenylation factors compete functionally on Flt-1 transcripts, by 1) assessing the influence of exon 13/14 splicing determinants on expression of Flt-1 RNA processing variants in a transfected cell model system; 2) determining the effects of altering the relative abundance of proteins principally involved in splicing or cleavage/polyadenylation; and 3) characterizing a previously-unknown splice variant, predicted to encode a novel sFlt-1 protein isoform, in cells overexpressing the spliceosomal RNA binding protein U2AF65. When the upstream exon in pFIN13 was decreased from 2135 to 309 bp, the sFlt-1:Flt-1 mRNA ratio decreased 8.9-fold and an aberrant 5'UTR/exon 14 splice decreased 60-fold, indicating that "exon definition" is a key parameter of successful Flt-1 RNA processing. Mutation of 5' or 3' intronic splice signals had little effect on Long sFlt-1:Total sFlt-1 mRNA ratio, suggesting that splicing and cleavage/polyadenylation factors may not compete physically for Flt-1 transcripts. Although co-transfection with RNA processing factor cDNAs did not generally produce the predicted pattern of effects on sFlt-1:Flt-1 mRNA ratio, a cryptic exon within intron 13 was revealed in cells overexpressing U2AF65. sFlt-1 protein apparently can be encoded by mRNAs either cleaved/polyadenylated within intron 13 or, surprisingly, by splicing of the cryptic exon "13b." Thus, the cellular decision to produce sFlt-1 or Flt-1 from a nascent RNA can no longer be viewed as a simple choice between cleavage/polyadenylation and splicing. / Ph. D.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

<p>Collagen crosslinking with a polypropyleneimine octaamine dendrimers, via carbodiimide chemistry, was further exploited to demonstrate the ability of this technology for various tissue engineering strategies, including tissue engineered corneal equivalents (TECE) and blood-contacting biomaterials. In addition, modification with extracellular matrix components and other biomimetic molecules may enhance tissue-host interactions for greater <em>in vivo </em>compatibility.</p> <p>First, the efficacy of the dendrimer crosslinking technology was further validated with commercially available collagen-based materials, from bovine or human sources (Chapter 4: Paper 1), as determined via transmittance, water uptake, differential scanning calorimetry, collagenase stability and <em>in vitro </em>cell compatibility. Despite gel formation, the matrix integrity was compromised with collagen-based materials manufactured under acidic conditions and purified via freeze-drying.</p> <p>To continue the theme of dendrimer crosslinked collagen gels as TECE materials, growth factor incorporation was investigated with epidermal growth factor (EGF) and heparin-binding EGF (HB-EGF), as a method for improving device epithelialization and subsequent host integration. However, given the short half lives of these growth factors, an effective growth factor delivery system is necessary to protect growth factor bioactivity. As heparan sulphate proteoglycans sequester and release heparin-binding growth factors <em>in vivo</em>, the use of heparinized dendrimer crosslinked collagen (CHG) gels for HB-EGF delivery would provide prolonged, controlled delivery, while maintaining growth factor effectiveness (Chapter 5: Paper 2). HB-EGF release was prolonged and capable of inducing human cornea epithelial cell (HCEC) proliferation. Thus, HB-EGF delivery from CHG gels could aid in TECE device retention through enhanced device-host integration via epithelialization.</p> <p>Alternatively, tethering EGF or HB-EGF to dendrimer crosslinked collagen (CG) gels could also supply growth factor stimulation in a manner that maintains bioactivity, while stimulating growth factor receptors continually with minute concentrations (Chapter 6: Paper 3). Growth factor uptake and bioactivity was assessed with radiolabeled growth factor and through <em>in vitro </em>epithelial cell culture, respectively. Surface-modification of CG gels with growth factors demonstrated greater bioactivity, compared to growth factor bulk-modification of CG gels.</p> <p>Finally, dendrimer crosslinked collagen gels, with pre-activated heparin (PH gels) were investigated as a tissue engineered blood-contacting biomaterial (Chapter 7: Paper 4), as we hypothesized that biomaterial induced coagulation is not only influenced by an anticoagulant surface, but also by the underlying material and that improved blood-biomaterial interactions may be achieved by utilizing a natural polymer that emulates biomimetic properties. Pre-activation of heparin was utilized to increase heparin gel content, while antithrombotic properties were evaluated via antithrombin and fibrinogen adsorption and plasma recalcification times. PH gels had increased heparinization, but extensive crosslinking compromised antithrombin-heparin interactions, compared to CHG gels. CHG gels demonstrated improved antithrombotic properties and further evaluation of these gels for blood-contacting applications is warranted.</p> / Doctor of Philosophy (PhD)