Dissertations / Theses on the topic 'Pre-implantation embryo'

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.

There is considerable interest in the beneficial role of dietary polyunsaturated fatty acids (PUFA) on reproduction in ruminants. Detailed information regarding the mechanisms behind this beneficial effect is limited. The main objective of this thesis was to test the effects of dietary supplementation with omega-3 (n-3) or -6 (n-6) PUFA on gene expression, fatty acid (FA) composition and steroidogenesis in granulosa and theca cells and pre-implantation embryo development. A previous study in our laboratory reported increased follicular-fluid progesterone concentrations in ewes fed an n-3 compared to an n-6 PUFA-enriched diet, but detected no differential effect of n-3 and n-6 PUFA enriched high-density lipoproteins (HDL) on granulosa cell steroidogenesis in vitro. Also, n-6 PUFA enriched HDL reduced early embryo development, but in the absence of a net uptake of FA. In view of these observations it was hypothesised that (i) effects of n-3 PUFA on ovarian steroidogenesis are mediated by theca rather than granulosa cells and (ii) during embryo culture lipids are acquired solely from the albumin fraction of serum, so that albumin delivered n- 3 and n-6 PUFA would exert a greater differential effect on embryo development than either LDL or HDL delivered PUFA. Initial investigations into granulosa cell gene expression profiles using an ovine gonad-targeted cDNA macroarray were unsuccessful, highlighted by subsequent qRTPCR analysis. A thorough investigation confirmed that inconsistencies were due to poor array hybridisation. In vitro data confirmed that n-3 PUFA, via delivery by HDL, increase progesterone production solely in theca cells and that this is associated with an increase in STAR transcript expression. We also demonstrate that albumin is the only serum fraction that leads to a net uptake of FA during embryo culture. PUFA enriched serum and albumin accelerated the development of embryos and increased the yield of morphologically poorer quality blastocysts with increased transcript expression for the antioxidant enzyme SOD1. Important differential effects of n-3 and n-6 PUFA on ovarian steroidogenesis acting solely on theca cells are identified, but differentially effects of PUFA on embryo development are less apparent.

Lors du développement précoce de l’embryon de souris, divers évènements de spécification des destins cellulaires induisent la formation du blastocyste pré-implantatoire. Ces évènements sont majoritairement contrôlés par l’action de voies de signalisation activées via la fixation de molécules signal à la membrane de la cellule. L’activité de ces voies de signalisation permet la régulation de la transcription de gènes cible responsable de l’acquisition d’une identité cellulaire et de son arrangement sous forme de tissu. Ici je m’intéresse aux rôles des voies ACTIVINE/NODAL et βCATENIN dans la spécification de ces identités cellulaires lors de la formation du blastocyste de souris
During the early mouse embryogenesis, cell-fate specification events result in the formation of the pre-implantation blastocyst. Those events are mainly regulated by the action of signaling cascades activated upon fixation of the signaling molecules at the cell membrane. The activity of these signaling pathways allow the transcriptional regulation of a specific pool of genes responsible for cell-fate decisions and the formation of tissues. Here, I am interested in the roles of both ACTIVIN/NODAL and βCATENIN signaling pathways in the specification of cell identities during the maturation of the mouse blastocyst

Scientific discoveries, developments in medicine and health issues are the constant focus of media attention and the principles surrounding the creation of so called ‘saviour siblings’ are of no exception. The development in the field of reproductive techniques has provided the ability to genetically analyse embryos created in the laboratory to enable parents to implant selected embryos to create a tissue-matched child who may be able to cure an existing sick child. The research undertaken in this thesis examines the regulatory frameworks overseeing the delivery of assisted reproductive technologies (ART) in Australia and the United Kingdom and considers how those frameworks impact on the accessibility of in vitro fertilisation (IVF) procedures for the creation of ‘saviour siblings’. In some jurisdictions, the accessibility of such techniques is limited by statutory requirements. The limitations and restrictions imposed by the state in relation to the technology are analysed in order to establish whether such restrictions are justified. The analysis is conducted on the basis of a harm framework. The framework seeks to establish whether those affected by the use of the technology (including the child who will be created) are harmed. In order to undertake such evaluation, the concept of harm is considered under the scope of John Stuart Mill’s liberal theory and the Harm Principle is used as a normative tool to judge whether the level of harm that may result, justifies state intervention or restriction with the reproductive decision-making of parents in this context. The harm analysis conducted in this thesis seeks to determine an appropriate regulatory response in relation to the use of pre-implantation tissue-typing for the creation of ‘saviour siblings’. The proposals outlined in the last part of this thesis seek to address the concern that harm may result from the practice of pre-implantation tissue-typing. The current regulatory frameworks in place are also analysed on the basis of the harm framework established in this thesis. The material referred to in this thesis reflects the law and policy in place in Australia and the UK at the time the thesis was submitted for examination (December 2009).

A gestação inicial da égua é um período fascinante que abrange numerosas e intensas mudanças em seu desenvolvimento, muitas das quais são únicas para a espécie equina. Esse desenvolvimento depende da manutenção da função lútea, do estabelecimento de um ambiente uterino e de uma interação precisa e orquestrada entre o concepto e o ambiente uterino. O objetivo deste estudo foi verificar as alterações histológicas do endométrio e a produção histotrófica em éguas cíclicas e prenhes nos dias 7, 10 e 13 pós-ovulação. No primeiro ciclo, biópsias endometriais de 30 éguas foram coletadas no dia 7 (n = 10), 10 (n = 10) e 13 (n = 10) constituindo o grupo éguas cíclicas. No segundo ciclo, as mesmas éguas foram cobertas por um garanhão fértil, acompanhadas diariamente até detectar a ovulação, considerada o dia 0. Foram coletadas biópsias endometriais nos dias7 (n 10), 10 (n 10) e 13 (n 10). Imediatamente após a coleta, o útero foi lavado e as éguas em que foi obtido embrião, foram inseridas no grupo de éguas prenhes. Um maior calibre dos vasos sanguíneos foi observado em prenhez comparados às éguas cíclicas do dia 7 aos 13. No sétimo dia pós-ovulação, uma grande perda de células ciliadas foi evidente no grupo de éguas prenhes, comparadas ao grupo de éguas cíclicas, as células do epitélio endometrial estavam mais protusas e uma pequena quantidade de secreção histotrófica entre as dobras endometriais foi observada. No décimo dia de prenhez, secreção histotrófica glandular e do epitélio luminal estavam mais presentes comparadas às éguas do grupo cíclico. No dia 13 de prenhes, foi observado um grande conteúdo de histotrofo nas aberturas glandulares que estavam cercadas por células ciliares. Ocorreram alterações no ambiente uterino logo após a entrada do embrião no útero. No estroma e no lúmen, essas modificações parecem visar fornecer a nutrição necessária para o desenvolvimento inicial do embrião e estas mudanças nas estruturas celulares irão interagir na sinalização embrionária, futura fixação, implantação e placentação.
The early pregnancy of mare is a fascinating period that encompasses numerous and intense changes in its development, many of which are unique to the equine species. This development depends on the maintenance of the luteal function, the establishment of a favorable uterine environment and a precise and orchestrated interaction between the concept and the uterine environment. The aim of this study was to evaluate histological changes in the endometrium in days 7, 10 and 13 post-ovulation in pregnant and cyclic mares. In the first cycle, endometrial biopsies from 30 cyclic mares (Cyclic group) were collected on days 7, 10 and 13 post-ovulation. In the second cycle, the same mares were bred by a fertile stallion. At days 7, 10 and 13 post-ovulation intrauterine biopsies were collected. Immediately after sample collection, the mare‟s uteri were flushed, and those mares with embryo recovery were assigned to the Pregnant group. A larger blood vessel caliber was observed in pregnant mares than in cyclic from day 7 to 13. On the 7th day a large loss of ciliated cells was evident in the group of pregnant mares in comparison with the Cyclic group and the superficial cells of the endometrium were more protruded, and a small amount of histotrophic material between the folds was observed. On the 10th day of pregnancy, the glandular histotrophic secretion and the secretion of luminal epithelium became more intense than the secretion of cyclic mares. On the 13th day of pregnancy, a very large amount of histotroph was observed within large glandular openings surrounded by ciliated cells. Changes occurred in the uterine environment thereupon the entry of the embryo into the uterus. In the stroma and in the lumen, these modifications seem aim to provide the necessary nutrition for the initial development of the embryo and to promote changes at cellular structures that will interact in the embryonic signaling and future fixation, implantation and placentation.

Previous studies have shown that poor maternal nutrition during pregnancy may induce metabolic syndrome in adults. In this study, we explored the mechanisms of how maternal low protein diet during the first 3.5 days of pregnancy, programs embryo development and embryonic stem cell function. Our results showed that mouse maternal low protein diet (9% casein; Emb-LPD) enhanced both Clathrin dependent and independent endocytosis in both in vivo blastocysts and in vitro differentiated embryoid bodies (EB day 5) compared with maternal normal protein diet (18% casein; NPD) controls. This increase in endocytosis was accompanied by an increase in lysosome volume per cell. This was done by confocal microscopy and 3D image analysis. To determine whether this effect on the lysosome system was due to autophagy or simply due to increased endocytosis, we studied the expression of LC3 protein, Clathrin, Megalin (also named as low density lipoprotein receptor 2 (LRP2)) and Cubilin. Immunostaining and Western blot analysis revealed Megalin and Cubilin were significantly up-regulated in Emb-LPD embryos and EBs, whilst Clathrin protein level was marginally increased and LC3 protein unaltered. This enhanced nutrient uptake ability was maintained even after cells or embryos were re-introduced into a normal environment in vitro. Thus, stimulated nutrient uptake in day 5.5 EB showed compensatory growth, known to associate with long-term disease symptoms. To understand the mechanisms involved, we investigated elements of the mTOR pathway. In vitro culture of early embryos in the presence of reduced levels of the three branched-chain amino acids (Lecine, Valine and Isoleucine) as occurring in Emb-LPD uterine fluid resulted in stimulated endocytosis of Trophectoderm (TE). In addition, we found although mTORC1 was partially suppressed, mTORC2 downstream RhoA-Actin interaction was stimulated in blastocysts by observing more actin and RhoA protein in Emb-LPD blastocysts as well as that inhibiting RhoA function abolished the enhanced endocytosis by Emb-LPD. We also investigated epigenetic changes induced by Histone deacetylase 3 (Hdac3) in terms of regulation of genes involved in Extraembronic Endoderm (XEN) differentiation and cardiomyocyte differentiation. We found that Emb-LPD EBs expressed reduced Gata6 and exhibited increased histone deacetylation at promoter of Gata6, together with increased Hdac3 expression. Our results reveal for the first time at the cellular level how early embryos respond to poor nutrition environment and reprogram to protect fetal growth. This further helps us to understand the mechanism of how adult metabolic syndrome can be originated from environment which early embryos were exposed to.

The coordinated growth of the conceptus is sustained by reciprocal signalling between the epiblast and the extraembryonic ectoderm (ExE). In mice, FGF4 produced by the epiblast promotes Cdx2 expression in the trophoblast stem cell (TSC) niche located in the ExE. Cdx2, Eomes and EIf-5 expression in the ExE constitute an auto-regulatory circuit that maintains the mouse TSC gene network. Cells in the ExE, in turn secrete BMP4, which is a critical mesoderm determinant. The ExE however, is absent in non-rodent embryos, raising the question whether the cross-talk between embryonic and extraembryonic domains is conserved in mammals. Pig embryos represent "mammotypical" embryos in that the flat epiblast is surrounded by trophectoderm (TE). The pig TE undergoes a remarkable elongation, however it is not known whether the epiblast regulates this process and whether a TSC niche supports this remarkable growth.

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.

As the pre-implantation embryo traverses the female reproductive tract, it experiences fluctuations in the composition of the surrounding maternal environment, including the availability of nutrients, growth factors and cytokines. In particular, the cytokine milieu surrounding the early embryo is pivotal in programming optimal embryo development. The pre-implantation embryo is sensitive to a range of perturbations such as maternal diet or in vitro culture. These and other insults influencing the maternal environment including infection, stress and environmental toxins may in part act via impact on oviduct and uterine cytokine synthesis. However the effect of maternal perturbation to inflammation or infection, on the embryo and the role of cytokines in mediating this is not fully elucidated. The studies described in this thesis employed an in vivo mouse model of maternal systemic inflammation with the proinflammatory bacterial lipopolysaccharide (LPS), where a pro-inflammatory cytokine response was elicited on days 2.5 and 3.5 post coitum (pc), prior to implantation. This model was studied in wildtype C57Bl/6 (Il10 ⁺ʹ⁺) mice and mice with a null mutation in the Il10 gene (Il10 ⁻ʹ⁻) were studied to investigate the effects of maternal deficiency in the anti-inflammatory cytokine IL-10 during LPS treatment. We demonstrated that the altered cytokine signals resulting from a low level pro inflammatory LPS challenge (0.5 μg/mouse) in the pre-implantation period elicit changes in the embryo developmental trajectory that in turn alter fetal growth and delay postnatal growth in the male progeny from LPS-treated mothers. As LPS did not directly impact development of the embryo at low and moderate doses, this result appears to reflect indirect effects of LPS mediated via the maternal tract. This is consistent with data from day 3.5 pc oviduct and uterus tissues which revealed increased mRNA expression of proinflammatory cytokines including Il6, Tnfa and Il12b following maternal LPS treatment. Peri-conceptional low dose LPS treatment in Il10 ⁺ʹ⁺ and Il10 ⁻ʹ⁻ mice revealed that the number of viable fetuses and fetal weight were both significantly reduced after LPS treatment, particularly in the Il10 ⁻ʹ⁻ mice. Embryo transfer was then utilised to investigate the mechanism by which LPS acts on the embryo, where day 3.5 pc embryos from donors treated with 0.5 μg LPS or PBS on days 2.5 and 3.5pc were transferred into day 2.5 pc pseudopregnant Swiss female recipients. The effect of maternal LPS treatment on fetal and placental development was seen to be maintained even after embryo transfer, suggesting that any effects of altered cytokine expression in embryos are exerted during cleavage stages before embryo recovery from donors. In addition, postnatal investigation of male and female progeny derived from control PBS and LPStreated Il10 ⁺ʹ⁺ and Il10 ⁻ʹ⁻ females from birth until 19 weeks of age showed that maternal LPS treatment constrains postnatal growth in male progeny regardless of maternal Il10 genotype, compared to male progeny from PBS-treated mothers. While the adult male progeny from LPS-treated Il10 ⁺ʹ⁺ and Il10 ⁻ʹ⁻ mothers did not display changes in fat mass compared to their PBS-treated control counterparts, the combination of maternal LPS treatment and maternal IL-10 deficiency resulted in greater fat mass accumulation in the adult male progeny from LPS-treated Il10 ⁻ʹ⁻ mothers compared to adult male progeny from LPS-treated Il10 ⁺ʹ⁺ mothers. In addition, we investigated the effects of maternal systemic inflammation during the pre-implantation period on the response to LPS challenge during adulthood. Male progeny from LPS-treated Il10 ⁻ʹ⁻ mothers had a dampened response in LIF cytokine following a 100μg/kg LPS challenge at 18 weeks of age. This study implies a role for cytokines as mediators of programming the embryo during the preimplantation period, with altered responses in the event of maternal systemic inflammation impacting on later fetal and postnatal development. The anti-inflammatory cytokine IL-10 acts to protect the embryo from the adverse programming effects of exposure to LPS during the pre-implantation period, with absence of IL-10 resulting in altered postnatal phenotype and particularly fat mass accumulation in the male progeny during adulthood. It appears likely that the absence of IL-10 in the maternal environment delays the clearance of adverse pro-inflammatory cytokines induced during an inflammatory challenge, resulting in prolonged exposure of the embryo to circulating pro-inflammatory cytokines in the maternal tract, supporting a cytokine-mediated mechanism. These studies provide additional evidence for a role of cytokines in embryo sensing of environmental conditions, and indicate that IL-10 is a key regulator of this communication pathway.
Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2014

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
國立陽明大學
醫學生物技術研究所
95
RNF33, a mouse pre-implantation embryo-specific RING-finger protein, belongs to the TRIM/RBCC protein family. Each TRIM/RBCC protein is composed of a RING finger, one or two B-box fingers, a coiled-coil region and a carboxyl-terminal domain. Rnf33 transcripts are also detected in unfertilized eggs. To date, the biological functions of RNF33 in the mouse early embryo has not been determined. However, restrictive temporal expression and the highly conserved nature of RNF33 suggest that RNF33 should play an important role in pre-implantation embryonic development. In this study, we have identified three candidate RNF33-interacting proteins by screening of a yeast two-hybrid cDNA library. Expression of one of the candidate interacting protein genes, Kif3a, was detected in the egg and the pre-implantation stages by both bioinformatics analysis and direct PCR and Southern blot analysis. The KIF3A protein associates with KIF3B and KAP3 by the rod and the tail regions of the protein, respectively, to form a heterotrimeric complex which in turn acts as a motor protein for cellular transportation and cell division. Our experiments showed that the yeast two-hybrid system was not suitable for full-length KIF3A interaction tests. Hence, confirmation of interactions between full-length RNF33 and full-length KIF3A was demonstrated by an in vitro co-immunoprecipitation assay using baculovirus vector-expressed proteins. Interacting domains between RNF33 and KIF3A were further defined by domain mapping using the yeast two-hybrid system. The results showed that RNF33/KIF3A interaction occurs via the B30.2 domain of RNF33 and the coiled-coil rod region of KIF3A. Based on our data, we propose that the RNF33 may regulate KIF3A functions by blocking KIF3A/KIF3B heterodimeric complex formation, or, by interfering with the motility of the KIF3A/KIF3B/KAP3 heterotrimeric complex resulting in retarded cellular transportation or cell division, and critically affecting normal development.

碩士
國立陽明大學
解剖學及細胞生物學研究所
100
Implantation and placentation are critical precesses in pregnancy. During implantation, trophoblast increases the ability of migration and invasion to invade deeply enough into the endometrium in order to secure the embryo. Previous studies have shown that implantation failure and pre-eclampcia are caused by weak migration and invasion during implantation and placentation. Epithelial-mesenchymal transition (EMT) is a process of cell type changing from epithelial to mesenchymal type, during which the epithelial cells lose cell-cell junctions and cell-basal adhesions such that their ability of migration is increased and eventually these cells are transformed into mesenchymal cells at last. In this study, we hypothesize that the trophoblasts increase migratory ability during implantation and placentation through EMT. Experimentally, I first detected the expression of protein markers in human normal delivery placenta, and found that E-cadherin in cell column of choronic villi was decreased, and vimentin in extravillus cytotrophoblasts was increased. This suggests that EMT indeed occurs in trophoblast cells. Because previous studies have reported that myriad growth factors exist in the microenvironment of implantation to maintain the process of pregnancy. Therefore, I used mouse blastocysts to test 5 different growth factors for the ability to promote trophoblasts expansion as well as to trigger EMT in trophoblast stem (TS) cells. I found that EGF and FGF-II could enhance blastocyst expansion and trigger EMT on TS cells. On the other hand, I used an animal model to functionally inhibit uterus EGF and FGF-II receptors in mouse fertilized 3 ~ 3.5 days, and found that implantation rate was significantly reduced. When the uterus was treated with the EGFR inhibitor; PD153035; to expression of EMT protein markers, we found that E-cadherin expressed in giant cells but vimentin did not detected in giant cells. It imply that EMT could be inhibited by blocking EGF receptors. This study demonstrates that during embryo implantation and placentation, EGF-induced EMT promoted the increase in trophoblasts increasing migratory ability. Although FGF-II was also important to embryo implantation and placentation, but it had no effect on EMT. These results may be help to women suffering from low implantation rate.

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.

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.
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Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2010

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.

碩士
國立東華大學
物理學系
107
Currently, many types of research are about the toxicity of nanoparticle (NP) on embryonic development considering NP’s various applications such as NP-containing products. NPs can possibly be released into the environment, interact with the human body, and even has the possibility to penetrate the reproductive system. Thus, this presented study aims to investigate the effect of nanoparticle (NP) on the developing embryo. By employing Raman Spectroscopy, this study will propose a method to evaluate the embryo quality before and after interaction with amine-functionalized nanodiamond (NDA). Spectroscopic analysis using Raman spectroscopy is presented as an additional method of estimation of the embryo by determining the embryo quality based on molecular information obtained which is non-invasive and under safe parameters for the live embryo. The Raman spectra of the embryo blastomeres have been measured using 785 nm NIR and 532 nm excitation wavelengths.

Sexual dimorphisms in growth rate and metabolism have been observed in the early embryo suggesting a role for sex chromosome linked gene expression in the early embryo. It is hypothesized that Y chromosome gene expression is aiding male embryo development in vitro. Reverse transcription polymerase chain reaction revealed the expression of DDX3Y, EIF1AY, TSPY, USP9Y, ZFY, and ZRSR2 in blastocysts but not HSFY or SRY. Furthermore DDX3Y, USP9Y, and ZRSR2Y showed expression in 100% of individual male blastocyst analysed. USP9Y and ZFY showed expression as early as the 4-cell and 8-cell stage however not in embryos arrested at the 2- to 8-cell stage. RNA interference to knock down the transcripts of USP9Y resulted in a significant decreased the cleavage rate but no significant difference in the blastocyst rate. Given Y linked gene expression coincidence with embryonic genome activation, and the absence of USP9Y and ZFY transcripts in the arrested embryos it was concluded that these genes may be important for early male embryo development.