Dissertations / Theses on the topic 'Whole embryo culture'

Using the rodent embryo culture techniques of New (1978), it can be demonstrated that, over the 48-hours culture period, the in-vitro growth and development, of explanted 9.5 days old post-implantation rat conceptuses, in immediately-centrifuged and heat-inactivated homologous serum is almost indistinguishable from that which occurs in-vivo up-to 11.5 days. Moreover, using these methods, it has become possible to grow rat conceptuses in a culture medium made up of 90 per cent human serum supplemented with glucose and 10 per cent rat serum. Embryonic growth and developmental indices achieved in culture, using supplemented human serum, were reported to be comparable to those observed in whole rat serum (Reti et al., 1982). The successful growth of rat conceptuses during the major part of the organogenetic stage, is an important technical departure, since it enables the effect of potential teratogens, in human serum to be investigated. In order to validate the current embryo culture model, it was demonstrated initially that reproducible embryonic growth and development can be achieved in appropriately prepared serum samples, obtained from healthy women during the various stages of their menstrual cycles, pregnancy and the post-natal period. Furthermore, using serum from patients with ovarian malignancies, on antimitotic chemotherapy, it was demonstrated that the modified whole rat embryo culture model is highly sensitive in distinguishing between normal and embryotoxic human sera. This method of maintaining embryos in culture, was adapted to investigate the differences in the species specific embryopathic properties of valproic acid. This anticonvulsant agent, commonly used during pregnancy, was found to be embryopathic at a lower serum concentration in rats than in humans. The implications of these findings are discussed with a view to investigate potential embryotoxicity of drugs used during human pregnancy. Finally, sera from women with histories of neural tube defect (NTDS) affected pregnancies and those with unexplained recurrent miscarriages were used as a culture medium to investigate serum related environmental embryopathic factors. Experimental work reported in this thesis indicates, that culture of rat conceptuses in appropriately prepared sera from 76 per cent of women with NTD affected pregnancies and 25 per cent of the habitual aborters resulted in an abnormal growth.

碩士
中興大學
生命科學系所
95
Primordial germ cells (PGCs) are the precursors of germline cells. Vertebrates’ PGCS originate in an extra-embryonic region, and subsequently migrate into the embryonic gonads after gastrulation. This aim of this thesis is to establish an effective method for in vitro yolk-free culture of chick embryos for time-lapse observation of the migratory route of chick PGCs. Since chick PGCs first appear in the germinal crescent at HH 8, travel through circulation from stage 11, and arrive in the prospective gonadal area at HH 18. The culture method used in this study must able to sustain normal embryo development through these stages. The in vitro yolk-free culture system provides an opportunity to combine fluorescence labeling with microsurgery, histological procedures. In this study, we created a culture chamber that supports a three-dimensional space to the growing embryo that greatly reduced the distortion seen in the plane surface culture method. We also modified FBS/F12 medium to enhance the nutritional conditions. Moreover, by monitoring embryos with different amount of culture medium or with different time of nutrient replenish; we found that these two factors affected the growth of embryos. According to our analysis, over 50% of the cultured embryos developed normally to HH22. Besides their external morphology, we investigated the spatial-temporal gene expression of SDF-1，CXCR4 and Vasa by in situ hybridization and confirmed that normal gene expression pattern of the cultured embryo in cell differentiation and organogenesis. Using this improved culture method, we can trace the migratory pathway of PGCs from germinal crescent towards the gonadal anlagen. Since this culturing method is suitable for embryos from HH8 to HH22, we think is can be used for studying the molecular mechanisms of other organogenesis that occur during this period.

by Chiu Pui Yu.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 86-118).
Abstracts in English and Chinese.
Title Page --- p.i
Abstract --- p.ii-iv
Acknowledgement --- p.v
Table of Contents --- p.vi-viii
List of Tables --- p.ix
List of Figures --- p.x-xii
List of Abbreviations --- p.xiii
Chapter Section I: --- Introduction
Chapter Chapter 1: --- Overview --- p.1-2
Chapter Chapter 2: --- Teratogenic Effects of Hyperglycemia
Chapter 2.1 --- What is Hyperglycemia --- p.3
Chapter 2.2 --- Teratogenic Effects of Hyperglycemia --- p.4-6
Chapter 2.2.1 --- Human Studies
Chapter 2.2.2 --- Animal Studies
Chapter 2.3 --- Timetables for Embryogenesis: Rats versus Humans --- p.7
Chapter 2.4 --- Mechanisms of Hyperglycemia Induced Teratogenesis --- p.8-12
Chapter 2.4.1 --- What are Free Radicals?
Chapter 2.4.2 --- Major Free Radical Species Involvedin Hyperglycemic Teratogenesis
Chapter 2.4.3 --- Molecular Damage Induced by Reactive Oxygen Species
Chapter 2.4.4 --- Supporting Evidence of Reactive Oxygen Species Causing Anomalies
Chapter 2.4.5. --- Hyperglycemia and Formation of Free Radicals
Chapter Chapter 3: --- Caffeine as Teratogen and Antioxidant
Chapter 3.1 --- Popularity of Caffeine --- p.13
Chapter 3.2 --- Basic Metabolism of Caffeine --- p.14
Chapter 3.3 --- Biological Actions of Caffeine --- p.15
Chapter 3.4 --- Teratogenicity of Caffeine --- p.16-20
Chapter 3.4.1 --- Animal Studies
Chapter 3.4.1.1 --- Teratogenic Effects of Caffeine in Animals
Chapter 3.4.1.2 --- Teratogenic Dose of Caffeine
Chapter 3.4.1.3 --- Interspecies Sensitivity
Chapter 3.4.2 --- Human Studies
Chapter 3.5 --- Possible Mechanisms for the Teratogenic Actions of Caffeine --- p.21
Chapter 3.6 --- Caffeine as an Antioxidant --- p.22
Chapter 3.7 --- Combined Effects of Caffeine with Other Substances --- p.23
Chapter Chapter 4: --- Combined Effects of Hyperglycemia and Caffeine on Early Embryogenesis- A Question to be Answered
Chapter 4.1 --- Possible Links between Hyperglycemia and Caffeine --- p.24
Chapter 4.2 --- Objectives of the Present Study --- p.25
Chapter 4.3 --- Hypothesis --- p.26
Chapter Section II: --- Research Designs and Methods
Chapter Chapter 5: --- Materials and Methods
Chapter 5.1 --- Licenses --- p.27
Chapter 5.2 --- Overall Study Design --- p.28-40
Chapter 5.2.1 --- Whole Embryo Culture Model
Chapter 5.2.1.1 --- Animals
Chapter 5.2.1.2 --- Explantation of Embryos and Serum Collection
Chapter 5.2.1.3 --- Preparation of Serum
Chapter 5.2.1.4 --- Culture Media
Chapter 5.2.1.5 --- Embryo Culture
Chapter 5.2.2 --- Experimental Groups
Chapter 5.2.3 --- Morphological Assessment
Chapter 5.2.4 --- Quantitation of Oxidative Stress
Chapter 5.2.5 --- Protein Assay
Chapter 5.3 --- Statistical Evaluation --- p.41
Chapter Chapter 6: --- Laboratory Considerations
Chapter 6.1 --- Whole Embryo Culture Model --- p.42-43
Chapter 6.1.1 --- Subjects
Chapter 6.1.2 --- Time Mating
Chapter 6.1.3 --- Culture Medium
Chapter 6.1.4 --- Gas Phase and Rotating Bottle Culture Method
Chapter 6.2 --- Quantification of Oxidative Stress --- p.47-49
Chapter 6.2.1 --- 8-Isoprostaglandins F2a as a Marker
Chapter 6.2.2 --- Assay for 8-Isoprostaglandins F2a
Chapter 6.2.2.1 --- Enzyme Immunoassay versus Gas Chromatography/ Mass Spectrometry
Chapter Section III: --- Results
Chapter Chapter 7: --- Results
Chapter 7.1 --- Justifications of Methods of Statistical Analysis --- p.50
Chapter 7.2 --- Effects of Hyperglycemia on Early Embryogenesis --- p.51-56
Chapter 7.2.1 --- Effects of Hyperglycemia on Morphological Development
Chapter 7.2.2 --- Effects of Hyperglycemia on Production of 8-isoprostaglandins F2a
Chapter 7.2.3 --- Effects of Hyperglycemia on Total Protein Content
Chapter 7.3 --- Effects of Caffeine on Early Embryogenesis --- p.57-61
Chapter 7.3.1 --- Effects of Caffeine on Morphological Development
Chapter 7.3.2 --- Effects of Caffeine on Total Protein Content
Chapter 7.4 --- Combined Effects of Hyperglycemia and Caffeine on Early Embryogenesis --- p.62-66
Chapter 7.4.1 --- Combined Effects of Hyperglycemia and Caffeine on Morphological Development
Chapter 7.4.2 --- Combined Effects of Hyperglycemia and Caffeine on Production of 8-isoprostaglandins F2a
Chapter 7.4.3 --- Combined Effects of Hyperglycemia and Caffeine on Total Protein Content
Chapter Section IV: --- Discussion and Conclusions
Chapter Chapter 8: --- Discussion --- p.67-83
Chapter Chapter 9: --- Conclusions and Future Directions --- p.84
Appendices --- p.85
References --- p.86-118

This thesis investigates the applicability of novel approaches designed to study the molecular mechanisms required for the initiation of organogenesis within the early endoderm. The endoderm is the germ layer that gives rise to the gut-tube and associated organs including the thyroid, lung, liver and pancreas. Our laboratory focuses on understanding the molecular mechanisms governing the developmental transition from endoderm to liver and pancreas. Several signaling pathways including Wnt, Retinoic Acid (RA), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor-β (TGFβ) have been implicated in the emergence of the liver bud from the endoderm in the mouse or other vertebrate species. However, neither the exact signals nor the precise roles during budding process have been identified, due to the complexity of specifically altering these essential pathways using traditional genetic approaches during the earliest stages of endoderm organogenesis. These traditional techniques include transgenic, knockout or conditional knockouts strategies. To overcome the difficulties of genetic accessibility, our laboratory has optimized two complementary approaches, electroporation and addition of activators or inhibitors directly to the culture media, to study the earliest stages of organ formation using an ex vivo culture system (whole embryo culture), that allow us for normal embryonic development for up to two days. This ex-vivo technique also provides the opportunity to access and manipulate the endoderm, specifically the liver and pancreas precursor cells, prior to organ specification. Because the endoderm undergoes normal liver and pancreas specification in our ex vivo system by 24 hours after culture begin, we reason that it is possible to manipulate gene expression at the onset of culture. We then determine the effects of this manipulation on liver or pancreas development by molecular and morphological analysis after culture. The first approach we developed is the use of directional electroporation of nucleic acids to manipulate a specific region of the endoderm, particularly on liver and pancreas developmental processes. The second method is global inhibition or activation using inhibitors or growth factors activators, focusing on the TGFβ signaling pathway. These techniques will be performed prior to, or concurrent with, liver and pancreas specification, followed by embryo culture until after the onset of organogenesis. The combination of these techniques constitutes a practical approach to stage-manage the endoderm in a temporally and spatially distinct manner. In addition, it will allow us to alter specific signaling pathways without the labor-intensive generation of genetically modified animals. Indeed, establishment of these methodologies may provide a robust tool for rapid screening of candidate genes and signaling molecules underlying organogenesis in any endodermally derived organ in mouse embryos.