Dissertationen zum Thema „Pre-implantation development“

The first cell differentiation in the mammalian embryo occurs during pre implantation development. At blastocyst stage two cell lineages can be distinguished: the inner cell mass (ICM), situated at the embryonic pole and the trophectoderm (TE) at the abembryonic pole. In murine embryos, it has been suggested that the first cleavage plane might be related with the embryonic-abembryonic (Em-Ab) axis at the blastocyst stage. So the daughter cells of the two-cell embryo might be already predisposed to a specific cell lineage further on development. The objective of the present thesis is to investigate the effects of assisted reproductive technologies (ART) factors affecting cell allocation patterns during pre implantation development. This was addressed by observing cell allocation patterns during mammalian pre implantation development on embryos produced using different ARTs. Using live-cell tracing, it has been concluded that cell allocation patterns during pre-implantation embryo development are potentially conserved among mammals, or at least among mouse and bovine embryos. Pre determined (orthogonal and deviant patterns), as well as stochastic development (random pattern), have been identified in mouse and bovine embryos. The incidence of these cell allocation patterns was not affected by maternal age, oocyte production, oocyte fertilisation/activation method, cleavage-stage biopsy or species. In addition, differences on epigenetic profiles, coping mechanisms after cell removal and further organ development were present among patterned embryos. Future work is advised to understand the basis of the mechanism(s) driving or driven by cell allocation patterns; particularly its relation with organ development.

All eukaryotic cells possess mitochondrial DNA (mtDNA), which is maternally inherited through the oocyte, its replication being regulated by nuclear-encoded replication factors. It was hypothesised that mtDNA replication is highly regulated in oocytes, pre-implantation embryos and embryonic stem cells (ESCs) and that this may be disrupted following nuclear transfer (NT). MtDNA copy number decreased between 2-cell and 8-cell staged porcine embryos and increased between the morula and expanded blastocyst stages, coinciding with increased expression of mtDNA replication factors. Competent porcine oocytes replicated their mtDNA prior to and during in vitro maturation to produce and maintain the 100000 mtDNA copies required for fertilisation. Those oocytes in which mtDNA replication was delayed had reduced developmental ability. Expression of pluripotency-associated genes decreased as murine ESCs differentiated into embryoid bodies, although expression of mtDNA replication factors did not increase until the stage equivalent to organogenesis. Cross-species NT embryos in which the donor cell-derived mtDNA was replicated produced decreased developmental outcomes compared to those in which no mtDNA replication took place. Disruption of the strict regulation of mtDNA replication that occurs during early embryogenesis, as is likely following NT, may therefore contribute to the reduced developmental ability of embryos produced using such techniques.

The goal of the work presented here was to investigate the hypothesis that cytoplasmic SLBP is required for translation of somatic H1 mRNA, and their translational repression is due to a lack of SLBP in the cytoplasm of oocytes, and early cleavage-stage embryos. To this end, the expression of SLBP in murine oocytes and pre-implantation embryos was characterized by RT-PCR, Western blotting, and immunocytochemical. techniques.
mRNA encoding SLBP was detected throughout oogenesis and pre-implantation development, from small growing oocytes to the late blastocyst stage. SLBP protein was found in the nucleus and cytoplasm of growing and fully-gown prophase I-arrested oocytes. SLBP accumulated to extremely high levels during meiotic maturation in a process requiring translation. The protein remained abundant both in the nucleus and cytoplasm throughout the 1- and 2-cell stages. SLBP was depleted in 4-cell embryos in a process independent of DNA replication, and was not detected again until the late 8-cell stage. From the late 8-cell stage to the early blastocyst stage, SLBP was detected exclusively in the cytoplasm. Interestingly, in late blastocysts, SLBP was translocated to the nucleus. (Abstract shortened by UMI.)

Präzise regulierte Genexpression, ist der Schlüssel zu erfolgreicher Embryonal-entwicklung. Die Expression von Zelltyp-spezifischen Transkriptionsfaktoren kann durch räumliche Interaktionen von Promotoren und Enhancern im Nukleus kontrolliert werden, aber auch durch 3D Faltung der DNA in größere organisatorische Einheiten wie “Topologically Associating Domains” (TADs) oder “A/B compartments”. Um die 3D Faltung in den Zelltypen des prä-implantations Embryos zu untersuchen, nutze ich ES und XEN Zellen, die stark dem Epiblast und dem primitiven Endoderm in der inneren Zellmasse des E4.5 Embryos ähneln. Um den Zusammenhang zwischen 3D DNA Faltung und zellulärer Identität zu erforschen, habe ich GAM, ATAC-seq und RNA-seq Daten von ES und XEN Zellen produziert. Um die Genom-Architektur im Embryo zu untersuchen, habe ich außerdem die GAM Methode an den Mausembryo angepasst und kann dadurch erstmals genomweit DNA-Faltung in den spezifischen Zelltypen der inneren Zellmasse des prä-implantations Embryos zeigen. ES und XEN Zellen zeigen viele differentiell exprimierte Gene, sowie starke Veränderungen in der Chromatin-Organisation, beispielweise in der Bildung von reprimierten Chromatinnetzwerken in ESCs, die wichtige XEN Gene wie Gata6 und Lama1 enthalten, während diese nicht aktiv sind. XEN-spezifische Genexpression ist oft mit der Präsenz von XEN-spezifischen “TAD boundaries” gekoppelt. Der Sox2 Locus zeigt eine ESC-spezifische Organisation mit aktiven Genen, und Regionen die von den Transkriptionsfaktoren SOX2, NANOG und OCT4 gebunden sind. Die starke Reorganisation der Genom-Architektur in wichtigen Loci wie Gata6 und Sox2 konnte ich mit in vivo GAM Daten bestätigen und finde ähnliche Unterschiede zwischen den beiden Zelltypen der inneren Zellmasse wie im in vitro Model. Diese Ergebnisse zeigen, wie wichtig es ist, Zelltypen getrennt zu untersuchen und, dass eine Verbindung zwischen zellulärer Identität und der Faltung des Genoms in der Embryonalentwicklung besteht.
Tightly controlled gene regulation is key to functional metazoan embryonic development. The expression of cell-fate determining transcription factors orchestrates the establishment of the various lineages of the embryo. Gene expression is often regulated via specific chromatin organisation. To investigate cell type-specific differences in chromatin folding in early embryonic development, I used in vitro models of the two distinct cell populations in the blastocyst ICM. In mouse ES and XEN cells, I mapped 3D genome conformation using Genome Architecture Mapping (GAM), chromatin accessibility using ATAC-seq, and gene expression using total RNA-seq. To enable the mapping of 3D genome folding directly in the blastocyst ICM, I adapted GAM for cell type-specific selection of nuclei, by integrating immunofluorescence detection of markers, and generated the first genome-wide chromatin contact maps that distinguish ICM cell types. I report that the ES and XEN cell lineages undergo abundant large scale rearrangements of genome architecture and exhibit high numbers of differentially expressed genes. For example, extra-embryonic endoderm genes, such as Lama1 and Gata6, form silent hubs in ESCs, potentially connecting maintenance of pluripotency to 3D structure of the genome. Further, I show that the expression of XEN cell-specific genes relates to the formation of XEN cell-specific TAD boundaries. Chromatin contacts at the Sox2 locus exhibit an ESC-specific organisation around binding of pluripotency transcription factors OCT4, NANOG and SOX2, into hubs of high gene activity. The observations detected in in vitro models, were investigated in smaller GAM datasets produced using the in vivo counterparts in the ICM. Overall, in vivo data confirmed the high degree of chromatin rearrangement among the two cell types, specifically in loci of lineage driving genes. The findings from in vivo data further underscore the connection of genome topology and cellular identity.

Since the number of pre- and postimplantation human polyploid embryos available for analysis is very small and relatively few are available at any one centre, I have used appropriate experimental mouse models to facilitate a large scale investigation. One aim was to analyse the influence of ploidy on the cleavage rate of preimplantation embryos. A deviation from the optimum embryonic (diploid fertilized) cell number during this time might explain the poor development and failure of genetically abnormal embryos. Therefore, experimentally produced haploid, diploid, triploid and tetraploid embryos were analysed together with appropriate control embryos. In addition, the analysis of parthenogenetic embryos and diandric and digynic triploid and homozygous tetraploid embryos during this time would also enable the influence of the parental genomes on development to be investigated. I also wished to examine whether a predictable relationship existed between ploidy and cell size and number in the tissues of postimplantation mammalian polyploid embryos. Confirmation of such a relationship may explain the abnormal morphological features encountered in some of these embryos and the premature death of all triploid and tetraploid mouse embryos, and the majority of human embryos with similar genetic abnormalities. My results show that the duplication of one or both parental genomes leading to the triploid or tetraploid status, respectively, still allows apparently normal preimplantation development to occur. However, the loss of a haploid genome from the diploid status is invariably detrimental to normal development. My results confirm that the presence of a maternal genome is important for normal early embryogenesis since parthenogenetic diploid embryos and diandric and digynic triploid embryos developed as well as fertilised diploid embryos during the pre- and very early postimplantation period.

The majority of human pre-implantation embryos created through in vitro fertilization (IVF) are mosaic as they are constituted of a mixture of diploid and aneuploid cells. Chromosome abnormalities are widely believed to contribute towards the relatively low success rates of IVF treatment. Consequently major efforts have been undertaken to develop effective tools to aid the selection of embryos with minimal abnormalities with the aim of improving clinical outcomes. However, the ultimate fate of mosaic embryos is not known. Human embryo research is limited by practical and ethical constraints, and directly relevant animal studies are sparse. To circumvent many of these limitations, a mouse model for pre-implantation chromosome mosaicism was developed. Acute chromosome segregation errors were induced in cleavage stage mouse blastomeres by bypassing the spindle assembly checkpoint (SAC). This model was used to investigate the fate of abnormal cells within the developing pre-implantation embryo, and the ultimate developmental outcome of mosaic embryos. Time-lapse imaging of pre-implantation development revealed that cells with chromosome abnormalities were progressively depleted during blastocyst maturation; inner cell mass (ICM) cells exhibited higher rates of apoptosis, while in the trophectoderm (TE) lineage effects on the cell-cycle predominated. Depletion continued throughout post-implantation development. Significantly, the presence of a critical number of control blastomeres within the embryo could rescue the early post-implantation lethality that occurred in embryos containing high rates of abnormalities. Thus it was demonstrated that mosaic embryos can achieve full developmental potential and that abnormal cells are progressively depleted as development proceeds. Finally, the mechanisms responsible for eliminating the abnormal cells from the embryo were investigated, revealing that embryos containing chromosome abnormalities may have increased metabolic requirements which could contribute to their clonal depletion; a feature previously characterised in aneuploid cells in the context of cancer research.

Administration of cyclophosphamide to males targets the germ cells and causes DNA damage including single strand breaks and DNA-DNA cross links. When males are treated with a chronic low dose of cyclophosphamide and then mated to normal females, progeny loss is manifested at the pre- and post-implantation stages of development. The earliest events that lead to embryonic loss were traced to day 2 of gestation when embryos had a decreased DNA synthesis profile and lower cell numbers than control litters. I investigated the hypothesis that chronic exposure of male rats to cyclophosphamide alters zygotic gene expression thus leading to embryonic loss. To assess DNA damage in the embryo, the Comet Assay was performed on 1-cell stage embryos. A significant number of embryos sired by cyclophosphamide-treated males showed the appearance of the Comet indicative of the effect of damaged sperm on the embryos starting from the 1-cell stage. Using a candidate gene approach, the antisense RNA (aRNA), I described the presence of several DNA repair gene families in normal embryos. Progeny sired by cyclophosphamide-treated males manifested a differential expression profile for several of these genes when compared to controls, suggestive of the ability of the embryo to respond to damaged sperm through the major DNA repair systems. To study the functional capacity of progeny sired by cyclophosphamide-treated males, I assessed total RNA synthesis in both groups; while control litters showed a peak of RNA synthesis at the 4-cell stage, the treatment group showed constant low expression throughout the stages examined. I mapped the profile of a number of gene families whose roles are essential for early development in both control and cyclophosphamide-treated groups. While control embryos showed a peak of expression for the majority of genes at the 8-cell stage, that of the cyclophosphamide-group showed an early induction at the 2-cell stage, indicative of loss of the tightly regulated

Doctor of Philosophy (PhD)
An in-depth, longitudinal study of the speech and oral language development of eight infants with a profound hearing loss who receive early interventions focused on developing their auditory, speech and oral language capacity is presented in this thesis. Infants were tracked for two years, during the period when they are changed from a hearing aid to a cochlear implant. All infants in this study had their hearing loss identified early and were fitted with hearing aids between 1 and 7 months of age and received their cochlear implant between 8 and 16 months of age. They attended a number of different auditory-verbal early intervention programs (depending on where they lived) all of which focused on developing speech and language skills through listening. Attendance at their particular early intervention program at least once a week was in addition to weekly attendance the Sydney Cochlear Implant Centre for therapy and audiological services. A broad range of measures has been used to track the infants’ acquisition of oral language skills, including measures of communicative intention, pre-speech and speech development, and oral language development. Despite a wide range of individual differences across the group of infants, the results suggest some general trends. In the area of communicative intent most infants followed typical development patterns in terms of both the types (e.g. requesting, answering etc) and forms (gestural, vocal, verbal) used, but they showed delays in their frequency of usage of these types and forms. For speech development the infants demonstrated typical speech skills by 18-months post-cochlear implantation in the areas of consonant inventories, severity of phonological involvement (speech intelligibility) and phonological process development, but they showed delays in vowel and consonant acquisition. Finally, for language development the infants were delayed relative to typical development at 18 months post-implantation. The findings support and extend previous studies which have demonstrated the benefits of early intervention for communication development in infants with hearing loss (Calderon & Naidu, 2000; Mayne, Yoshinaga-Itano & Sedey, 2000; Moeller, 2000; Yoshinaga-Itano & Apuzzo, 1998). However, the delays in the oral communication skills of the infants in the current study suggest that more intensive long-term intervention is required if the infants are to attain typical oral speech and language development. The findings capture the complexity of early oral language development, which has been lacking in previous studies of infants with significant hearing loss, receiving a cochlear implant (Dettman, Briggs, & Dowell, 2005; Houston, Ying, Pisoni, & Iler Kirk, 2003; Schauwers, Gillis, Daemers, De Beukelaer, & Govaerts, 2004). The present data also provide some limited support for earlier implantation, that is, before 12 months of age, as the infants made little progress in oral language development while using hearing aids. The reduced amount of auditory signal available to them prior to implantation may be the determining factor in their inability to follow typical rates and patterns of development. However, rates of development with the implant were not straightforward and further research on this population is needed. Universal neonatal screening programs for hearing loss will potentially provide a larger population of early identified infant for future research. This will create the opportunity for large scale, prospective, longitudinal, studies examining the acquisition of speech and oral language development. Limitations of this study, tracking the early stages of speech and language development over a two year period are identified. Future studies are needed to follow the infants for a longer time to determine if their rate of development is sufficient for them to catch up in areas of delay and maintain their performance in areas where they match their typically developing peers.

Thesis (Ph. D.)--University of Sydney, 2006.
Title from title screen (viewed 19 Dec. 2006). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Communication Sciences and Disorders, Faculty of Health Sciences. Includes bibliographical references. Also issued in print.

Unsuccessful fertilization, aberrant embryo development, implantation failure and recurrent miscarriages can occur despite any obvious reasons during assisted reproduction. DNA fragmentation and aneuploidy in gametes have been implicated as a possible cause for infertility, but the use of DNA fragmentation tests is controversial. The SCD-FISH test claims to analyse DNA fragmentation and aneuploidy in the same cell. In this thesis SCD-FISH was compared to single FISH and SCD, which showed that SCD-FISH was an unreliable method to study these parameters. Sperm preparation techniques in assisted reproduction technologies (ART) potentially generate exogenous stresses that cause additional DNA damage. This study subjected mature sperm to environmental insults that normally occur during ART, and highlighted the significant increase in sperm DNA fragmentation due to heat, freezing and oxidative stress. Since it is not possible to investigate the level of DNA damage in the egg or sperm, and still maintain its viability for use in fertilization, DNA damage in cumulus and granulosa cells were studied. There was no relationship between DNA fragmentation in these cells and fertilization or pregnancy outcome. DNA fragmentation was significantly higher in cumulus than granulosa cells. Allegedly minimising DNA damage, GM-CSF is a component added to IVF culture media. Its effect on murine embryo DNA fragmentation was studied and it was found to have no significant effect. The exposure of human embryonic stem cells to pre-tested toxins and its impact on DNA fragmentation and aneuploidy, and the correlation between these two parameters were also investigated. These studies emphasise the belief that the introduction of DNA fragmentation assays to the clinical arena is premature as its role is unsubstantiated, the lack of a transparent relationship between DNA fragmentation and pregnancy outcome and the importance of minimising damage to sperm and embryos due to external stresses during laboratory research and ART. Overall, the aim of this thesis was to examine the hypotheses that DNA fragmentation, aneuploidy and phosphatidylserine translocation are potential biomarkers of reproductive potential that exist in variable degrees of at different stages of gametogenesis and preimplantation embryo development, and are susceptible to environmental stresses.

The Hippo signalling pathway is a conserved kinase cascade involved in the regulation of tissue growth and organ size. Activation of the Hippo pathway through multiple upstream inputs results in phosphorylation and inactivation of the transcriptional co-activator, Yes associated protein (Yap). While the role of Yap as an oncogene and an effector of the Hippo pathway is well established, its role in cellular differentiation is less well known. In this present work I have utilised quantitative immunofluorescence analysis to examine the expression of Yap in the differentiation of mouse embryonic stem cells (mESCs). I show that differentiation of mESCs is accompanied by an initial increase in nuclear Yap expression. Furthermore, I show that this increase in nuclear Yap expression is associated with differentiation towards the primitive endoderm lineage (PrE). Moreover, small molecule inhibition of Yap was able to decrease the proportion of cells differentiating towards PrE. Following on from these in vitro studies, I examine the expression of Yap in vivo in the corresponding differentiation event in mouse preimplantation embryos. I show that increased nuclear Yap expression is associated with expression of the PrE-specific transcription factor Gata6 during specification and eventual sorting of the PrE. Culturing embryos in the presence of small molecule inhibitors of Yap resulted in decreased expression of Gata6 and a reduction in trophectoderm cell number. These studies demonstrate that Yap is involved in the process of cellular differentiation and is associated with specification of the PrE lineage. Finally I attempt to create an inducible knockout of Yap in mESCs using a serial targeting strategy, with the intention of creating a model system in which to examine the role of Yap in mESCs.

Cochlear implantation is a proven and accepted option for young children with profound hearing loss. Cochlear implantation requires a professional team which should inform, guide, support and collaborate with parents constantly throughout the process. Existing programs for children with hearing loss and their families are generally designed on the basis of what experts believe they should contain, rather than on what parents actually wish to receive, which may often lead to a mismatch between the professionals view and the parents’ views of parental needs. In order to ensure that parental needs are appropriately addressed it is imperative that professionals investigate and understand the individual needs and desires of the parents with whom they collaborate. This can be managed by carefully tailoring information to their individual needs and presenting information in an accessible format at the time it is most appropriate and digestible. The aim of this research study was to develop a pre-implantation tool to rate the individual support and information needs of parents of young cochlear implant candidates. Within the context of applied research, a qualitative descriptive intervention research design was used in the study. Ten parents of children with cochlear implants participated in a semi-structured interview to investigate their need for information and support during the pre-implantation phase of cochlear implantation. Their responses were analysed and compared to relevant literature in order to develop the pre-implantation rating tool for parents of cochlear implant candidates. The rating tool consists of ten areas for information and support. These areas are as follows: general, technical, surgery, social support, financial, communication options, education, outcomes, rehabilitation and parental role. Parents are able to rate which areas of information and support is important to them and what they would like to discuss with the professional involved. Parents are also encouraged to identify any area of information and support that is not included in the rating tool that they would want information on from the cochlear implant team. This rating tool was evaluated by eight speech-language pathologist/audiologist working in six cochlear implant programmes in South Africa to determine the value of the rating tool. Positive responses were given about the adaptability of the tool to identify individual needs for support and information and the tool would be useful to guide speech-language pathologist/audiologists to identify needs of parents that should initially be addressed. Respondents agreed that the rating tool provides an opportunity to express parent’s individual needs for information and support; that the tool correlates with a family centered approach and would be useful to include in cochlear implant programs. The majority of participants felt the rating tool possibly will be effective in identifying information and support needs of parents before cochlear implantation and respondents would be willing to implement the rating tool in their cochlear implant programme. The positive response from professionals working in the field of cochlear implantation validates the effectiveness of the rating tool. AFRIKAANS : Kogleêre inplantering is ‘n beproefde en aanvaarde opsie vir jong kinders met ‘n uitermatige gehoorverlies. Kogleêre inplanting vereis dat ‘n professionele span ouers deur die proses inlig, lei en ondersteun. Huidige programme vir kinders met gehoorverlies en hulle gesinne, is oor die algemeen gebaseer op grond van inligting wat volgens kundiges belangrik is om in te sluit. Hierdie programme is nie noodwendig gebaseer op inligting ouers graag wil ontvang nie. Dit kan lei tot ‘n verskil tussen die perspektief van professionele persone teenoor die van die ouer oor ouer- behoeftes aan inligting. Om te verseker dat ouers se behoeftes effektief aangespreek word, is dit noodsaaklik om dit te ondersoek en die individuele behoeftes van ouers te verstaan. Dit kan gedoen word deur inligting aan te pas volgens die individuele behoeftes van ouers en die inligting te verskaf in ‘n toeganklike wyse op ‘n gepaste tyd wanneer dit geskik is en die ouer die inligting kan prosesseer. Die doel van hierdie navorsing studie was om ‘n pre-inplantering instrument te ontwikkel om die individuele behoeftes aan inligting en ondersteuning van ouers van jong kogleêre inplantings kandidate te bepaal. Binne die konteks van toegepaste navorsing is ‘n kwalitatiewe beskrywings intervensie navorsingsontwerp gebruik. Tien ouers van kinders met kogleêre inplantings het deelgeneem aan ‘n semi-gestruktureerde onderhoud. Die onderhoud het ouers se behoefte aan inligting en ondersteuning tydens die pre-inplanterings fase van kogleêre inplantasie ondersoek. Die resultate is geanaliseer en vergelyk met relevante literatuur om sodoende die pre-inplantering bepaling instrument vir ouers van kogleêre kandidate te ontwerp. Die instrument bestaan uit tien areas van inligting en ondersteuning. Hierdie areas is as volg: algemeen, tegnies, chirurgie, sosiale ondersteuning, finansieel, kommunikasie opsies, onderrig, rehabilitasie en ouer rol. Ouers kan bepaal watter areas van inligting en ondersteuning vir hulle belangrik is en wat hulle graag wil bespreek met die professionele persone betrokke by die kogleêre inplanting proses. Ouers word ook aangemoedig om enige area van inligting en ondersteuning te identifiseer wat moontlik nie ingesluit is in die instrument nie, maar wat hulle graag met die kogleêre span wil bespreek. Agt spraak-taal patoloë/oudioloë van ses kogleêre inplantings programme in Suid-Afrika het die instrument geëvalueer om die waarde daarvan te bepaal. Positiewe insette is gegee oor die aanpasbaarheid van die instrument om die individuele behoeftes vir inligting en ondersteuning te bepaal; dat die instrument betekenisvol is om die spraak-taal patoloog/oudioloog te lei om die behoeftes van ouers te identifiseer en aan te spreek; dat die instrument ooreenstem met ‘n familie- gesentreede benadering en dat die instrument effektief ingesluit kan word in kogleêre inplantings programme. Die meeste deelnemers het aangedui dat die instrument effektief sal wees in die identifisering van inligting en ondersteunings behoeftes van ouers voor ‘n kogleêre inplanting. Deelnemers het aangedui dat hulle bereid sal wees om die instrument te implementeer in hulle kogleêre inplantingsprogram. Die positiewe respons van spraak-taal patoloë en oudioloë dui op die geldigheid en effektiwiteit van die instrument.
Dissertation (MCommunication Pathology)--University of Pretoria, 2010.
Speech-Language Pathology and Audiology
unrestricted

碩士
國立清華大學
奈米工程與微系統研究所
103
Assisted reproductive technologies (ART) are of increasing importance and impact worldwide. A crucial aspect of ART is to control and mimic the in vitro environment of mammalian to the in vivo one. Integration biotechnology with micro- and nanotechnology is one of the necessary ways to improve ART. There are many platforms capable of nurturing cells in 3D, including using gel, microwells or hanging drop techniques. Microwells were chosen for the culture and investigation of early mouse embryos in this study, since microwells made of polydimethylsiloxane (PDMS) are low-cost, easy to operate, and biocompatible. Each mouse early embryo could be maintained in an individual microwell, allowing for high-resolution time-lapse microscopy and collecting the data of developmental process from every single embryo without confounding factors. In addition, the fluidic environment of each microwell could be secluded from each other by layering oil on top, preventing the communication of soluble factors between embryos cultured in individual microwells. The initial step of ART involves in vitro fertilization (IVF), and we demonstrated a successful fertilization rate of 67.9% in this research. We successfully cultured mouse embryos from the two-cell stage to blastocyst stage inside different volume of microwells with a ~80% successful rate. Among the five different volumes of microwells evaluated, we chose 393 nL microwells as the cultured platform to tracing the development process of mouse embryos. After cultured in microwells for 72 hours, 22 embryos at blastocyst stage were transferred into a recipient female mouse, and 15 mice were successfully born after 19 days. The development timings of mouse embryos that developed into blastocysts were statistically different to those of embryos that failed to form blastocysts (p–value < 10-10), and could be robust indicators of the quality of the embryo with >93% sensitivity and 100% specificity. This microwell platform, which supports the development of pre-implant embryos and is low-cost, easy to fabricate and operate, we believe, opens opportunities for a wide range of applications in reproductive medicine and cell biology.

碩士
中原大學
奈米科技碩士學位學程
106
Matrine is a legume belonging to the dry roots of legumes extracted, in many studies found that matrine has many effects, such as: anti-arrhythmia, inhibition of cancer cell proliferation, anti-inflammatory effects and other effects. Many studies also found that matrine can regulate the function of leukopenia in the body, and also found that cound inhibit the proliferation and induce apoptosis in many cancer cell lines. Previous studies in our laboratory have found that co-culture of blastocysts with matrine inhibits the proliferation of embryonic cells, promotes apoptosis and affects DNA methylation. Study found that matrine increases H19 DNA methylation and induces high expression of IGF-2 gene, resulting in organ abnormalities and other effects. According the functional effects of matrine on inhibition of cell proliferation, induction of cell apoptosis, we further investigate the effects of matrin on early stage embryo development, including cell apoptosis, proliferation and in vitro embryonic development. The main purpose of this study was to investigate the effects of different concentrations of matrine (0,　12.5,　25,　50 μM) on the development of early stage embryos. Using the TUNEL assay, Hoechst staining analysis and in vitro development, studies found that the 12.5-50 μM matrin could inhibit cell proliferation and early embryonic development. Taken together, our study clearly point out that matrine has injury effects on cell proliferation and induce cell apoptosis, which can trigger developmental damage and cause embryo development injury. However, the detail regulatory mechanisms of matrin on early stage embryo development are unclear and need further investigation.

碩士
中興大學
動物科學系所
95
A group of homologous ribonucleases (RNases) belonged to the RNase A superfamily has been isolated and characterized from various species. During the past decades, a lot of efforts have been devoted to the study of RNases, including DNA cloning, analyses of their catalytic activities and structures. However, little is known about the biological functions of the ubiquitous RNases in vivo, especially RNase 5, the angiogenin. Recently, it was demonstrated that the gene expression could be blocked after injection of double-stranded RNA (dsRNA) into organisms and the phenomenon is called RNA interference (RNAi). The RNAi is mediated by 19-23 nucleotide dsRNAs homologous in sequence to the target genes to silence cognate genes post-transcriptionally, which involves mRNA degradation. Consequently, it shows the gene knockdown or knockdown-like effects. Hence, the aims of this study were to investigate the effects of angiogenin on the embryonic implantation by knocking down the expression of angiogenin (Ang) in mouse blastocysts, in addition to establishment of an in vitro culture system for early implantation study. In Experiment 1, the specific primers for Ang-1, Ang-2 and Ang-4 were designed to analyze the expression of pattern in mouse blastocysts by reverse transcription polymerase chain reaction (RT-PCR). The results showed that only Ang-1 was expressed at the blastocyst stage in mice. In Experiment 2, the different regions in the DNA sequences of Ang-1 and Ang-2 were selected and subcloned into RNAi expression vector, in which short hairpin RNAs were derived by U6 promoter. After transfected into B16-F10 mouse cell line, the expression of Ang-1 and Ang-2 were analyzed by RT-PCR. The RT-PCR results demonstrated that the expression of angiogenin was decreased to at least 40% by various RNAi expression vectors. In Experiment 3, the U6-sh-1 (RNAi expression cassette for knocking down Ang-1) or U6-shR-1 (RNAi expression cassette for knocking down RNase 1) fragments were injected into the pronuclei of mouse zygotes. The cleavage rates in the U6-sh-1-injected and U6-shR-1 injected groups were significantly lower than those in the uninjected control group after 72 h culture in vitro (77 % and 81% v.s. 95%, P < 0.05). No significant differences of morula/blastocyst formation were found between U6-sh-1-injected and U6-shR-1-injected groups (77% v.s. 81%, P > 0.05), but the development of morula/blastocyst in the U6-sh-1-injected or U6-shR-1 group was significantly decreased, compared to the control group (49% and 48% v.s. 90%, P < 0.05). The defective effects of U6-sh-1 and U6-shR-1 on the development of preimplantation embryos were demonstrated. Immunofluorescent staining showed that U6-sh-1 injection reduced Ang-1 protein levels in the blastocysts compared to those in the control group. In Experiment 4, the endometrial epithelial cells and stromal cells were isolated from mouse uteruses. For construction of the 3-dimensional culture system, epithelial cells were seeded on an artificial basal membrane (ECMatrix™) with underlying stromal cells embedded in the type I collagen matrix. The whole system was settled in a Millicell® (Millipore) hanging in a 24-well culture plate. The morphology of epithelial cells on the matrix became cuboidal after culture. Additionally, the columnar appearance with a basal nucleus was observed on the paraffin wax sections of epithelial cells. The mouse blastocysts were recovered and cultured in this model system. Normal hatching and attachment of the blastocyst were observed. The endometrial cells grown in the established in vitro culture system appeared similar morphology as those in vivo, suggesting this model system might facilitate further understanding of the cellular and molecular mechanisms involving in the implantation of mammalian embryos. In conclusion, the DNA-based shRNA constructs produced in this study could effectively decrease the expression of Ang-1. Further studies would be required to elicit the optimal culture conditions for the in vitro implantation model before applied to the study of the effect of angiogenin on the implantation.

It is recognised that the environment to which the fetus is exposed in utero, after implantation, can program longer term health outcomes and alter the possibility of disease onset later in life. It is becoming evident that the environment, to which the pre-implantation embryo is exposed, can also affect the ability of the embryo to form a viable pregnancy as well as altering fetal growth. Despite this understanding, little is known about the mechanism by which the environment can ‘program’ the pre-implantation embryo. Using model stress systems, either ammonium or DMO in the culture medium, this thesis addressed the hypothesis that suboptimal environmental conditions may alter mitochondrial homeostasis and function and/or epigenetic parameters and these are the possible mechanisms responsible for the altered fetal outcomes seen. While common measures of embryo quality such as on time blastocyst development were not affected by either stress, more in-depth investigations found several striking differences. Exposure to DMO significantly decreased blastocyst cell number and allocation to the inner cell mass and trophectoderm, as well as increased blastocyst apoptosis. After exposure to DMO, blastocysts were transferred to pseudopregnant recipients, and both the ability of the embryos to implant and develop into a fetus was impaired as well as fetal weights and crown rump length were significantly reduced indicative of altered growth. Similar results have also been demonstrated after pre-implantation embryos are exposed to ammonium in vitro. Exposure to ammonium during pre-implantation embryo development also altered placental gene expression and function, indicating a possible mechanism of the observed reduced fetal growth parameters. Interestingly, the pre-implantation embryo appears to be the most vulnerable to an environmental stress during the pre-compaction stage, in particular the zygote to 2-cell transition, as exposure to either stress during this stage alone shows similar perturbations to if the stress was present for the entire pre-implantation developmental period. At this early stage of embryo development, mitochondria are the sole energy generators and are therefore critical for embryo function. This study determined that either ammonium or DMO stress exposure, during the first cleavage division, significantly perturbed mitochondrial distribution, membrane potential and ATP/ADP levels. Removal of the stress did not allow these effects to be completely reversed, implicating mitochondrial perturbations as a possible mechanism behind altered embryo programming. During pre-implantation embryo development there are also significant epigenetic changes which are vital for re-programming the embryonic genome. Both in vitro stresses significantly altered DNA de-methylation at the 2-cell stage and reduced blastocyst gene expression levels of DNA methyltransferases (Dnmt3a and Dnmt3b), which are responsible for de novo methylation. Together these data highlight the importance of pre-implantation embryo development as a critical period of growth in which the presence of environmental stress can have an impact on metabolic homeostasis and critical epigenetic events that may be responsible for the downstream effects seen on fetal growth. These results are not only important for assisted reproductive therapy, where the presence of an in vitro laboratory stress can potentially alter embryo programming, but are also important for in vivo embryo development where the health and wellbeing of the mother can also potentially influence the in utero environment and thus the long-term health outcomes of her child.
http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1522143
Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2010

WNT ligands are secreted proteins that act as signals between cells. WNTs activate several interconnected signaling pathways that are required for embryonic development as well as tissue homeostasis in adults. The X-chromosomal Porcn gene encodes a membrane-bound O-acyl transferase that is required for the acylation of all 19 WNT ligands encoded in the mammalian genome. Non-acylated WNTs fail to be secreted from the producing cell and thus do not activate downstream signaling targets. In my thesis research, I have investigated the function of Porcn in mouse embryonic development. In vitro, I have shown that Porcn is required for canonical WNT signaling in ES cells and further, for their differentiation into endodermal and mesodermal derivatives. Taking advantage of a mouse line carrying a conditional (floxed) Porcn allele that I have generated, I have focused my studies on the early embryonic roles of Porcn using Cre recombinase-mediated and X chromosome inactivation-based ablation of Porcn function in vivo. I have found that the earliest requirement for Porcn in mouse development is the induction of gastrulation. In contrast to findings from in vitro studies, I have provided evidence that Porcn is not required for pre-implantation development in vivo. Dissecting embryonic and extra- embryonic roles of Porcn, I have been able to show that Porcn is required in the extra-embryonic chorion in order to mediate chorio-allantoic fusion, whereas ablation in the extra-embryonic visceral endoderm had no apparent effects. The extra-embryonic requirement for Porcn results in a parent-of-origin effect in Porcn heterozygous females due to X chromosome inactivation. In contrast to the placentation defect causing embryonic lethality of maternal allele mutants, deletion of the paternal allele caused variable fetal defects resulting in perinatal lethality with only rare survivors to adulthood. Both fetuses and adults represent a mouse model for Focal Dermal Hypoplasia (FDH), the syndrome caused by mutations in the human PORCN gene. My studies highlight the importance of PORCN-mediated WNT signaling for gastrulation, placentation, and fetal development, but suggest that endogenous WNT secretion does not play an essential role in either implantation or blastocyst lineage specification.

碩士
中原大學
生物科技研究所
99
Berberine is an alkaloid isolated from Coptis Chinensis, Phellodendron and other plant extracts. Berberine is a traditional Chinese medicine in China which has been applied for a long time. Berberine has been evaluated as anti-diarrhea, anti-bacterial, anti-arrhythmic, anti-vascular smooth muscle proliferation and cholesterol-lowering. Berberine also performs a wide range of applications in the cardiovascular system and nervous system diseases. Several studies have demonstrated that berberine is able to induce apoptosis process in cancer cells. However, a study regarding the embryonic toxicity of berberine has not yet been done. In this study, mouse embryos were used to study the effects of berberine on embryonic development and proliferation. The study results showed that blastocysts treated with 5 or 10 μM of berberine that resulted a significant dose-dependent increase in apoptosis and inhibited cell proliferation. To evaluate the alteration of inner cell mass (ICM) and trophoblast cell (TE) of mouse blastocysts, we used differential staining and TUNEL assay. Statistics showed, TE subject to more damage than ICM. Furthermore, the immunofluorescence staining, revealed that berberine induced apoptosis in mouse blastocysts by reducing expression of anti-apoptotic Bcl-2 protein and increasing expression of the pro-apoptotic Bax protein, which increases the ratio of Bax/Bcl-2. Conclusion, berberine induced embryo cell apoptosis and caused hazardous effects on embryonic development.

Preimplantation embryo development in the mouse is a time of rapid cellular morphological and molecular changes leading to embryo implantation for the generation of offspring. The Mager lab studies these events occuring between fertilization and implantation in order to better understand the initial events which set the stage for all future aspects of development. The result of this research impacts many scientific disciplines including in-vitro based means of embryo culture, establishment of epigenetic marks, differentiation and cellular reprogramming and can be used in translational research for the improvement of in-vitro culture techniques and develop novel therapies such as cell replacement in the case of macular degeneration (Bin, L., 2009). Through the use of in-vitro embryo culture, RNA interference (RNAi) approaches and daily observations, gene function required in preimplantation embryo development can be determined. In the initial published body of work evaluating gene knockdown using our RNAi approach (Maserati M 2011), WDR74 was characterized in preimplantation embryo development. We now understand that WDR74 is implicated in RNA production and/or stability as gene knockdown at the 1 cell stage significantly depletes mRNA within the embryo by the morula stage. Furthermore, double knockdown of Trp53 and Wdr74 results in a partial rescue of blastocyst formation suggesting p53 mediated apoptosis in the failure to make a blastocyst phenotype. The initial characterization of 4 RNA processing genes (SF3b14, SF3b1/SAP155, Rpl7l1 and Rrp7a) required for blastocyst formation was later evaluated. The results of this work has been submitted for publication and will be published soon in the journal Zygote. SF3b14 and SF3b1, identified as being part of the splicesome complex, disproportionally contributes to gene transcription of those genes containing more than 1 exon verifying a role in RNA splicing. Rpl7l1, identified by GO terms as a possible ribosomal gene, was found to be present in the cytoplasm and, surprisingly, in the nucleus. It is surmised this gene influences polymerase 2 activity as Rpl7l1 gene knockdown embryos demonstrate reduced active polymerase 2 activity at the morula stage. Rrp7a was identified as being critical in blastocyst formation and is present in the cytoplasm while excluded from the nucleus. Based on location and GO terms, this suggests a role in translation. Taken together, these 4 genes act in 3 different ways impacting RNA production, splicing or translation promoting blastocyst formation in the mouse. The final gene evaluated in this work was Bcl-6 corepressor (Bcor). As opposed to our previous work with RNA processing factors, this gene knockdown does not result in a failure to make a blastocyst. Bcor knockdown increases the rate of physiologically normal blastocysts in both murine and bovine models. Although further characterization must be done, temporary Bcor gene knockdown might be a useful improvement of in-vitro embryo culture systems including murine, bovine, equine and possibly even human. This manuscript is divided into 4 chapters, the first of which is a review of preimplantation embryo development. This covers selected and relevant events between fertilization and just before implantation of the embryo into the uterus. I mainly focus on events after fertilization and the necessary changes required for zygotic genome transcription and lineage specification. The second chapter characterizes WDR74, a gene we identified as critical in the formation of a blastocyst in a reverse genetic screen. As state before, we assess WDR74 function with the developing embryo and conclude the protein plays a role in RNA production and/or stability of RNA transcripts. We also test to rescue blastocyst formation in WDR74 knockdown embryos in an attempt to further evaluate WDR74 function. We continue the characterization of genes whose temporary reduction causes the failure of blastocyst formation in the third chapter. Here we report on four additional RNA processing genes in a body of work which has been published in the journal Zygote. Since these genes contained similar GO terms, we assumed they may all function in a similar way so they were assayed together as a group. As function of these genes were unknown, we determined protein localization within the cell, function in RNA splicing, alternative splicing and to determine if the failure to make a blastocyst is due to lineage specification. In the final chapter, BCOR gene expression is characterized in preimplantation embryo development as in the former 2 chapters. However, the result of this gene knockdown does not lead to the failure to make a blastocyst, rather this improves the number of blastocysts formed during the correct physiological time; the same time that blastocysts form invivo. Undoubtedly, this could lead to possible commercial applications which are reviewed along with the preliminary data we have been able to collect thus far. Specifically, the continuation of the BCOR gene knockdown research in preimplantation embryo development is pitched in the form of academic and international business collaboration with InvitroBrasil for the production of cloned bovine, equine and ICSI in equine.