Dissertations / Theses on the topic 'Preimplantation embryo'

The experiments reported in this thesis were undertaken to study two aspects of the biology of mammalian preimplantation embryos. The first of these dealt with the utilization of the energy substrate, glucose, by the sheep embryo up to and including day 19 of pregnancy. The second dealt with the utilization of glutamine by mammalian embryos and the reciprocal effects of glutamine and glucose on each others metabolism. This latter study was prompted by the recent reports of the beneficial effects of medium containing glutamine, but excluding glucose, on the ability of mouse zygotes to undergo full preimplantation development in vivo . Prior to undertaking the metabolic studies with glutamine a series of experiments were undertaken to study the capacity of the mouse zygotes of different genetic background to develop in vitro and the constituents of the medium that may influence this development. Initial experiments were carried out to examine the effects of reducing oxygen concentration upon the catabolic and anabolic utilization of glucose by the preimplantation sheep conceptus as it develops and differentiates from the cleaving stage to the 19th day of pregnancy. The results of this study demonstrated that lowering O2 to 5% substantially altered both the catabolic and anabolic metabolism of glucose but the pattern of response depended on the stage at which the conceptus was explanted. Next the activity of the pentose phosphate pathway was estimated in both cleaving embryos and the advanced conceptus. There was a substantial utilization of glucose through this pathway and the activity of the pathway changed with development and differentiation. As it has been proposed that trophoblastic vesicles formed by in vitro culture of fresh trophoblast may be used as a model system for studying the biochemistry and physiology of the preimplantation embryos, a study was undertaken to examine glucose metabolism by sheep trophoblastic vesicles formed from day 13 trophoblast and cultured for 6 days. By assessing catabolic and anabolic metabolism of glucose, it was shown that these vesicles were active in the utilization of glucose but the pattern of metabolism did not mimic that seen with fresh tissue samples. The second major aspect dealt with the role of glutamine in the biology and metabolism of the embryo during preimplantation development. The first experiment investigated the development of mouse zygotes of different genotype through the "2-cell block" and the effect of medium components including glutamine, glucose and EDTA upon this development. The results showed that the glutamine containing medium, (CZB medium), significantly improved the development of embryos with more than 50% of zygotes from blocking strains (randombred) reaching the blastocyst stage. By using reciprocal crosses between blocking and non-blocking strains it was shown that the maternal genome is mainly responsible for the "2-cell block". As regards media constituents it was found that EDTA was essential for the 1-cell embryo to pass the 2-cell block. Glutamine, in general, did not exert a major effect upon development of mouse zygotes but appeared to benefit development at early stages. Glucose was necessary for successful development of embryos at least from the post-block stages and had no detrimental effects when added from the commencement of culture. Subsequent studies were undertaken to investigate the metabolism of glutamine and glucose by in vivo and in vitro derived mouse embryos. The data showed that glutamine can be utilized catabolically as an energy source by oxidation to CO2. Embryos from the blocking strain metabolised significantly less glutamine than did those from the non-blocking strain but glucose metabolism was similar between the embryos from the two strains, suggesting that strain differences in embryonic development in vitro may be related to the metabolism of glutamine rather than glucose. Finally, a study of glutamine utilization and its reciprocal relationship with glucose by the sheep conceptus was undertaken. These experiments showed that, as found in the mouse, glutamine can be substantially utilized by the conceptus via oxidation to CO2 through the TCA cycle. However, there was increasing preferential utilization of glucose for energy metabolism with development from the cleaving embryo through to the day 19 conceptus.

Au cours du développement embryonnaire, les animaux suivent une séquence complexe de changements de forme provoqués par des forces issues de la contractilé cellulaire, générées par le cytosquelette de l’actomyosine. Chez l’embryon de mammifère, la morphogenèse débute dès la phase préimplantatoire, qui commence par la fécondation et aboutit à la formation du blastocyste, la structure qui s’implante dans l’utérus maternel. Ainsi, l’embryon préimplantatoire suit une série de mouvements morphogénétiques contrôlés par la contractilité qui aboutissent avec la formation du premier lumen au sein du blastocyste.Nous avons mené la première étude examinant l’effet de la perte complète de contractilité sur le développement préimplantatoire. Nous avons généré les mutants maternels-zygotiques simples et double des gènes Myh9 et Myh10, codants pour les chaînes lourdes de la myosine non musculaire de type II, ce qui nous a permis d’étudier la contribution relative de ces deux paralogues dans la génération de la contractilité cellulaire. Nous avons constaté que Myh9 est le principal contributeur dans ce processus, car sa perte entraîne un retard du cycle cellulaire, une réduction du nombre de cellules, de la compaction et de la différenciation. La perte de Myh10 n’a pas d’impact fort par rapport aux embryons de type sauvage. Néanmoins, un phénotype plus sévère a pu être observé chez les embryons double mutants dont la cytocinèse échoue dans la plupart des cas, suggérant une compensation par Myh10 dans les mutants Myh9. Malgré le manque de cellules, la différenciation et la formation du lumen se poursuivent dans les double mutants. Dans les cas les plus extrêmes, des embryons composés d’une seule et unique cellule accumulent du fluide dans des compartiments intracellulaires, ce qui indique que le programme préimplantatoire est exécuté indépendamment du nombre de cellules.Dans les embryons sauvages, le blastocyste se forme suivant un processus de fracturation hydraulique produisant un réseau de microlumens qui murit en un lumen unique entourée de plusieurs cellules. Bien que le mécanisme global de ce processus soit compris, on ne sait toujours pas comment les cellules régulent localement la dynamique des microlumens. Ici, nous décrivons des blebs inversés aux contacts entre cellules. Les blebs inversés sont des invaginations membranaires de courte durée qui gonflent dans le cytoplasme avant d’être repoussées par la contractilité cellulaire. Les blebs inversés se forment à la suite de l’augmentation globale de la pression du fluide intercellulaire et du confinement local du fluide par l’adhésion cellulaire. Nous proposons que les blebs inversés agissent comme des pompes hydrauliques pour diriger le fluide au sein du réseau de microlumens, promouvant ainsi son murissement.Ces résultats approfondissent notre compréhension moléculaire, cellulaire et biophysique de la façon dont la contractilité cellulaire façonne l’embryon de mammifère
During development, embryos undergo a complex sequence of morphogenetic shape changes powered by cellular contractile forces which allow the embryo to correctly form tissues and organs. Contractile cellular forces in morphogenesis are chiefly generated by the actomyosin cytoskeleton. In the mammalian embryo, morphogenesis begins during preimplantation development, which commences with fertilization and leads up to the formation of the blastocyst, the structure that implants into the maternal uterus. During preimplantation development, the embryo undergoes a series of contractility-driven morphogenetic steps that culminate in the positioning of the first lumen of development in the blastocyst.Here, we conducted the first study investigating the effect of complete contractility loss on preimplantation development. We performed single and double maternal-zygotic knockout of the non-muscle myosin heavy chain genes Myh9 and Myh10, which allowed us to study the relative contribution of these two paralogs to contractility generation in preimplantation development. We found that Myh9 is the main contributor in this process, as its maternal-zygotic loss results in cell cycle delay, reduced cell number, compaction and differentiation. Loss of Myh10 did not have a strong detectable impact compared to wildtype embryos. Nevertheless, a more severe phenotype could be observed in Myh9 and Myh10 double knockout embryos as cytokinesis failed in most cases, suggesting some compensation by Myh10 in Myh9 single mutants. Despite severely reduced cell number, differentiation and lumen formation still somehow continued in double knockout embryos. In the most extreme cases, single-celled embryos accumulated fluid in intracellular compartments, indicating that the preimplantation developmental program is executed independently of cell number.In WT embryos, the blastocyst forms in a process of hydraulic fracturing producing hundreds of microlumen followed by their coarsening into a single lumen surrounded by multiple cells. While the global mechanism of this process is understood, it remains unclear how cells regulate microlumen dynamics locally. Here, we describe inverse blebs at cell-cell contacts during microlumen formation. Inverse blebs are short-lived membrane protrusions expanding into the cytoplasm before being retracted by actomyosin contraction. We show that inverse blebs form due to a global increase in intercellular fluid pressure and require local fluid confinement by cell-cell adhesion. We propose that inverse blebs serve as hydraulic pumps to push the fluid within the microlumen network, thereby supporting the coarsening of the first mammalian lumen.Together, these finding expand our molecular, cellular and physical understanding of how cell contractility shapes the early mammalian embryo

Comment le zygote acquiert la totipotence à partir de deux cellules complètement différenciées, et comment les décisions du destin cellulaire sont faites plus tard dans le développement sont des questions biologiques essentielles. Les études menées au cours de la première partie de mon doctorat ont contribué à l'annotation de la composition de la chromatine embryonnaire en ce qui concerne les variantes des histones et des modifications post-traductionnelles. L'expression ectopique de H2A.Z après la fécondation réduit la progression du développement, ce qui suggère que l'absence de H2A.Z au début du développement pourrait être importante pour l'organisation de la chromatine embryonnaire nouvellement formée. Deuxièmement, j'ai étudié la dynamique des histones dans l'embryon de souris en développement. La reprogrammation épigénétique après la fécondation est accompagnée par une étonnante forte mobilité des histones dans le noyau. Ma thèse a contribué à la compréhension des événements dynamiques affectant la chromatine embryonnaire pendant le remodelage épigénétique après la fécondation
How the zygote acquires totipotency from two differentiated cells, and how cell fate decisions are made later in development is a pivotal biological question. The studies conducted during the first part of my doctorate contributed to the annotation of embryonic chromatin composition with regards to histone variants and PTMs, and more specifically those correlated with active chromatin regions. The histone variant H2A.Z was shown to be present on embryonic chromatin in a stage-specific manner. Ectopic expression of H2A.Z after fertilization reduced developmental progression, suggesting that absence of H2A.Z at the onset of development might be important for the organization of the newly formed embryonic chromatin. Secondly, I investigated histone dynamics in the developing mouse embryo. Our work represents the first report on histone mobility during early mouse embryogenesis. My thesis contributed to the understanding of the dynamic events affecting embryonic chromatin during epigenetic remodeling after fertilization

It has previously been demonstrated that a large percentage of in vitro generated human embryos are chromosomally mosaic. The current thesis investigated this mosaicism in greater detail. It characterized the mosaicism present at each stage of preimplantation development in vitro. It examined the relevance of the different forms of the observed mosaicism to preimplantation embryo wastage, implantation failure, and fetal and placental mosaicism. Finally, it addressed the identification of the chromosomally mosaic embryos during preimplantation genetic diagnosis (PGD). For each of the studies presented within the thesis, blastomeres from "spare" in vitro generated embryos were assessed for chromosomal content using multi-color fluorescence in situ hybridization (FISH) DNA probes. Mosaicism was detected at all stages of preimplantation development, from the 2-cell stage to the blastocyst stage; it comprised of diploid, aneuploid, "chaotic", haploid, and polyploid chromosome patterns. Compared to blastocysts, arrested embryos or embryos at the earlier stages of development, showed a much higher incidence of mosaicism involving "chaotic" imbalances for multiple chromosomes and/or high percentages of abnormal cells. These results indicate that extensive post-zygotic abnormalities impair embryonic development to the blastocyst stage. The presence of mosaicism was not predicted by embryo morphology. Mosaicism may therefore contribute to the low rates of blastocyst formation in vitro and to the high rates of implantation failure following cleavage stage embryo transfer. Probe mixtures comprising of three autosomes, of one autosome and gonosomes, or of five autosomes could be applied for the identification of the mosaic embryos during cleavage stage PGD. Culture of isolated blastomeres from cleavage stage embryos for genetic diagnosis increases the number of cells available for analysis; however, the presence of nuclear defects and mosaicism among the cultured cells indica

During fertilisation the sperm causes a series of Ca2+ i oscillations in the egg that lead to events such as cortical granule exocytosis, pronuclei formation, exit from meiosis and entry into mitosis. These events are collectively termed "egg activation" and are essential for initiating development in the egg. Injection of PLC£ into mouse eggs has previously been shown to cause Ca2+ i oscillations, egg activation and development to the blastocyst stage. In this Thesis I show that injection of PLC£ cRNA also causes Ca2+ i oscillations in pig eggs and in human eggs which had failed to fertilise after assisted reproduction technologies (ART). The human eggs were shown to undergo further development up to the blastocyst stage. The Ca2+ i oscillations during egg activation are known to lead the immediate events of egg activation but the role of these Ca2+ i oscillations on later preimplantation development in the mouse has not been established. The work presented in this Thesis addresses this issue by comparing differences in mouse embryo development after parthenogenetic activation using stimuli that induce a Ca2+ i increase (e.g. Sr2"1" and ethanol) with those that do not cause any Ca2+ i change (e.g. cycloheximide). The data shows that parthenogenetic activation in the absence of a Ca2+ increase leads to embryo loss around the 8-cell stage, and that any blastocysts that form in absence of a Ca2+ i increase have a reduced inner cell mass and an increased incidence of apoptosis. Ca2+ i oscillations during activation do not appear to have an effect on the timing or amount of overall global gene expression during embryonic genome activation (EGA). However, when I used microarray analysis I found that a significant number of genes were differentially expressed in 8-cell stage embryos that were activated with a Ca2+ i increases compared to those that activated in the absence of a Ca2+ i change. These results provide evidence that Ca2+ i increases at egg activation have an important role in regulating events during the later stages of preimplantation development.

There have been phenomenal advances in the field of reproductive medicine and success rates following in vitro fertilisation have improved dramatically in recent years. The aim of this project was to improve our understanding of human preimplantation embryo development by identifying potential markers of viability that may aid us in selecting the best embryo for uterine transfer in the clinical embryology laboratory. Investigations into the distribution of cytoskeletal F-actin in human embryos demonstrated that a highly organised actin cortex is important for embryo cleavage and continued development to the blastocyst stage. Whilst they are polarised in the mouse from the oocyte to the blastocyst, the regulatory proteins leptin and STAT3 are co-localised only at the oocyte stage in humans, and are distributed within different cytoplasmic domains in human cleavage stage embryos and blastocysts. Whether polarity in humans is predetermined in the oocyte remains elusive, but none of the evidence generated in this thesis supports this idea. Leptin transiently activates STAT3 via the long form of the leptin receptor, and most significantly in the ICM of human day 6 blastocysts. Morphological features of blastocysts that can be visualised microscopically, such as a double ICM and cytoplasmic projections connecting the ICM to the TE, provide clues to their viability and may help us to choose the most suitable embryo from a cohort when deciding which to transfer. Nuclear volumes may in future contribute to this selection. Using time lapse technology to study cleavage patterns is now a routine occurrence in the clinical embryology laboratory. The results in this thesis show that distinctive patterns of divisions and the site at which blastocysts hatch can provide us with more information than a snap-shot morphological evaluation. Finally, contributing to the development of modelling software and predictive algorithms for the study of human embryos, particularly in time lapse imaging, means that our understanding of this fascinating area of medicine will continue to progress.

Genes that are associated with the response of cells to growth arrest and DNA damage include the growth arrest specific (gas) genes, the growth arrest and DNA damage (gadd) genes and the tumour suppressor gene p53. This study was undertaken to characterise the changes in gene expression as the embryo responds to growth arrest. The different types of stress that may be encountered by the embryo in culture, such as amino acid deficiency, DNA damaging agents (MMS and sodium arsenite) and metabolic inhibitors (tunicamycin and PALA), induced different patterns of gene expression. Two different pathways implicated with the negative regulation of growth were identified; one involving the transcriptional activator CHOP-10; the other mediated by arrest in the G₁ phase involving p53 and gadd45. Both gadd45 and p53 were expressed in the mouse embryo at the blastocyst-stage, suggesting a role for these genes in a system that arrests growth when embryos are exposed to DNA damage. CHOP-10 was expressed at a constant level from the 8-cell stage onwards and was induced when blastocysts were treated with MMS, the metabolic inhibitor sodium arsenite or an inhibitor or protein glycosylation tunicamycin, but not in blastocysts treated with the inhibitor of nucleotide synthesis PALA. The overexpression of CHOP-10 may be a marker of one of the pathways that lead to apoptosis in the blastocyst. Overall these findings suggest that there is more than one control system regulating growth arrest in the blastocyst and the fetal outcome may differ depending on the type of stress encountered in culture.

During preimplantation embryo development the zygote is transformed into the blastocyst. Metabolism during preimplantation development has been well studied but a major challenge is to understand the underlying control mechanisms. THe hypothesis tested in this thesis is that control mechanisms may be revealed by examining regulatory metabolic enzymes at the level of gene and protein expression alongside measurements of their biochemical activity. The gene expression, protein localisation and biochemical activity of three key enzymes of metabolism: Hexokinase (HK), Creatine Kinase (CK) and the Na+/K+ ATPase, were therefore studied during pig and mouse preimplantation embryo development. HK is the first enzyme of glycolysis, CK functions to buffer cellular ATP/ADP ratios and the Na+/K+ ATPase is a major consumer of ATP at cavitation. Pig zygotes were produced by in vitro fertilisation of in vitro-matured abattoir-derived immature oocytes; mouse zygotes were derived in vivo. Both pig and mouse zygotes were cultured to t~e blastocyst stage in vitro. Some in vivo-derived pig embryos at various stages of preimplantation development were also studied. The gene expression profiles of multiple isoforms ofHK and CK and the Na+/K+ ATPase al subunit were determined using quantitative RT-PCR. The biochemical activity of HK and CK were profiled in both species, using ultramicrofluorescence, with CK activity also measured during human preimplantation embryo development. There was little relationship between gene expression and biochemical activity of HK and CK throughout preimplantation -development. Protein expressio~ patterns of CKB and Na+/K+ ATPase al were determined at all stages of mouse preimplantation development and revealed ubiquitous expression of CK and a possible interaction with the mitotic spindle. The work suggests that the previously little studiedCK plays an important role in the metabolism of the developing preimplantation embryo ~d, consistent with the original hypothesis, that biochemical activity needs to be studied alongside gene and protein expression to understand fully the factors involved in the control of energy metabolism.

Periconceptional deficiencies in one-carbon (l-C) metabolites (e.g. folate, vitamin Bl2 and methionine) influence parental fertility and offspring health. Knowledge of l-C metabolism is limited to the liver where species-specific differences have been reported. Functionality in the ovary, germ cells and preimplantation embryo is poorly understood. The multitude of allosteric interactions involved in l-C metabolism makes it challenging to understand the principles by which this pathway is regulated. Mathematical models of this cycle, based on published enzyme kinetics and regulatory mechanisms within the liver, exist and can address this problem in part. We recently found that transcripts for specific l-C metabolism enzymes were either absent or poorly expressed in the bovine ovary and preimplantation embryo. Extending these initial observations, to include the sheep, pig, rat and human hepatocyte and granulosa cell lines, this thesis sought to increase the understanding of the regulation of l-C metabolism in the ovary and preimplantation embryo by combining experimental data with theoretical models. Experimental investigations at transcript, protein and enzyme activity levels revealed that l-C metabolism differs between the ovary and liver and also between the ovaries of different species. Variations considered primarily relate to the presence of MATII and the absence of BHMT within the ovary. Consequently, a mathematical model of hepatic methionine metabolism was modified to render it more representative for ovarian cells, particularly in the cow. Model predictions indicated that the ovary regulates methionine metabolism over a much narrower range of methionine input than the liver. Within this narrow range S-adenosylmethionine (SAM)-mediated transmethylation reactions are predicted to vary. Model predictions were tested in bovine granulosa cells cultured under various methionine concentrations (i.e. 0 to 500 J!M). Methionine adenosyltransferase 2A (MAT2A) transcript levels, de novo synthesis of SA M and the SAM:SAH (S-adenosylhomocysteine) ratio decreased with increasing methionine within the physiological range (i.e. 10 to 50 /!M). Homocysteine concentrations in spent culture media increased within the same range whereas progesterone synthesis and DNA methylation were unaffected by methionine dose. Results herein promote and support the importance of an optimal parental methionine status during the periconceptional period.

The purpose of this research was to define the effects of various growth-promoting factors on in vitro embryonic development of in vitro matured and in vitro fertilized bovine embryos. The control medium was a chemically defined medium which improves the possibility of closely determining the in vivo conditions the embryo is actually exposed to. The growth-promoting factors tested in this experiment included transferrin, IGF-I (insulin-like growth factor-one), IGF-II (insulin-like growth factor-two), TGF-a (transforming growth factor-alpha) , TGF-B1 (transforming growth factor-beta1) , PDGF (platelet derived growth factor), EGF (epidermal growth factor), NGF (nerve growth factor), and bFGF (basic fibroblast growth factor). Transferrin was included at 10 micrograms/milliliter , while all other factors were utilized at 10 nanograms/milliliter in the control medium. Bovine cumulus-oocytes were retrieved from slaughterhouse ovaries and were matured i n Medium-199 containing 10% feta l bovine serum for 24 hours at 39°C in a 5% C02 atmosphere. Frozen-thawed bull spe r m were s wim-up separated and capacitated in medium containing heparin for 3 hours prior to insemination. Gametes were co- incubated fo r 18 hours and then cumulus cells were stripped from the ova. Ova which did not cleave were removed from culture 36 hours after insemi nati on and were stained for evidence of fertilization. Embryos were cultured in one of the 10 conditions (including control) described above. A total of 150 total oocy.t.es were cultured per treatment for a tota l of 10 days. EGF improved embryo development, while TGF-Bl and TGF-a only slightly improved embryo development compared to the control. All other factors tested did not have a beneficial effect on embryo development in this culture medium. In summary, EGF improved in vitro development of bovine embryos obtained from in vitro maturated and in vitro fertilized bovine oocytes. Other factors which were t est ed did not significantly improve in vitro bovine embryo development. Further experiments are necessary fo r determining the requirements of bovine embryos in vitro.

Understanding the establishment of polarity and the cell fate specification of epithelial cells is important for developmental biology, regenerative medicine and the study of cancer. In this thesis, models of pre-implantation epithelial development are used to investigate the relationship between these two processes. The trophoblast is an extraembryonic epithelial tissue which contributes to the placenta. Addition of BMP4 to mouse and human embryonic stem (mES) cells grown in culture has been suggested to induce differentiation of cells to the trophoblast lineage. The use of this differentiation method was investigated as a possible model of trophoblast polarisation and cell fate specification. Unfortunately, with the protocol and reagents available this model did not appear to physiologically recapitulate trophoblast development and was not reliable. The primitive endoderm is an epithelium which arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. When mES cells are grown in suspension they form aggregates of differentiating cells known as embryoid bodies. The outermost cell layer of an embryoid body is an epithelial cell type comparable to the primitive endoderm. Embryoid bodies were used here to study the polarisation and cell fate specification of the primitive endoderm. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. The acquisition of epithelial polarity occurred prior to the maximal expression of cell fate markers. Fgfr/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier which normally blocks free diffusion across the epithelial cell layer occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erkmediated polarisation. This data shows that the formation of a polarised primitive endoderm layer in embryoid bodies requires the Fgfr/Erk signalling pathway.

Intracellular pH (pHi) impacts many cellular mechanisms including cellular metabolism, gene expression, cell volume regulation, cell survival and proliferation. Most cells use two general pHi regulatory mechanisms: HCO3-/Cl- antiporters (AE, Slc4a family) to reduce internal alkaline load, and Na+/H+ exchangers (NHE, Slc9a family) that protect cells from acidosis. Previous studies with preimplantation (PI) embryos have shown robust activity of HCO3-/Cl- exchanger in all stages of development. It was also determined that inhibition of this exchange with the stilbene AE inhibitor 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) was detrimental to embryo development from the 2‐cell stage to blastocyst when cultured at high external pH. In this study I investigated which of the five known plasma membrane NHE isoforms was present and active within mouse PI embryos and their role as pHi regulators throughout preimplantation embryo development. In mouse oocytes and preimplantation embryos, mRNAs were detected encoding NHE1 (SLC9A1), NHE3 (SLC9A3), and NHE4 (SLC9A4), with higher mRNA levels for each in fully-grown oocytes through one-cell stage embryos and then generally lower levels after the two-cell stage. No transcripts for NHE2 (SLC9A2) or NHE5 (SLC9A5) were detected. Measurements of intracellular pH during recovery from acidosis, induced by transient ammonium pulse, suggested that recovery occurred and was mediated by NHE activity at all preimplantation stages assessed (one-cell, two-cell, eight-cell and morula). This recovery was inhibited by 1 mM amiloride, a general NHE inhibitor. The observed residual recovery was attributed to passive passage of protons across the membrane, rather than the activity of NHE4 (an amiloride-resistant isoform), since no further decrease in recovery rates from acidosis was observed upon amiloride increase to 5 mM. Furthermore, recovery from acidosis at each stage was entirely inhibited by cariporide, which is very highly selective for NHE1. In contrast, the moderately NHE3-selective inhibitor S3226 did not preferentially block recovery, nor did adding S3226 increase inhibition over that achieved with cariporide alone, indicating that NHE3 did not play a functional role in pHi regulation at any stage assessed. Another regulator of intracellular pH against acidosis, previously reported to be active in oocytes and 1-cell embryos, the sodium-dependent bicarbonate/chloride exchanger (NDBCE; SLC4A8), had low or absent activity in two-cell embryos. This indicated that NHE1 is likely the only significant regulator of pHi in preimplantation mouse embryos, at least after the 1-cell stage. Culturing embryos from the one-cell or two-cell stages in acidotic medium inhibited their development, as assessed by development to the blastocyst stage and cell lineage allocation. However, inhibition of NHE1 with cariporide, NDBCE with DIDS, or both together did not further decrease embryo development to the blastocyst stage more extensively under conditions of chronic acidosis than at normal pH. This suggests that mouse PI embryos have a restricted ability to counteract chronic acidosis by means of pHi regulatory mechanisms, despite clearly being able to recover from acute acidosis via NHE1 activity.

DNA methylation is one of the principal epigenetic modifications that plays an essential role in transcriptional regulation. After fertilization, mammalian embryos undergo dynamic changes in genome-wide DNA methylation patterns and the changes are essential for normal embryo development. Ten-eleven translocation (TET) methylcytosine dioxygenases are implicated in DNA demethylation by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The three members of TET protein family, TET1, TET2, and TET3, are highly expressed in preimplantation embryos in a stage-specific manner. Previous studies demonstrated that TET proteins are involved in diverse biological processes such as gene regulation, pluripotency maintenance, and cell differentiation by mediating 5mC oxidation. My dissertation research was conducted to elucidate the mechanistic roles of TET proteins in epigenetic reprogramming of mammalian embryos using porcine embryos as a model. The first set of studies focused on the relationship between TET proteins and pluripotency. To understand the role of TET proteins in establishing pluripotency in preimplantation embryos, CRISPR/Cas9 technology and TET-specific inhibitors were applied. TET1 depletion unexpectedly resulted in an increased expression of NANOG and ESRRB genes in blastocysts, although the DNA methylation levels of NANOG promoter were not changed. Interestingly, transcript abundance of TET3 was increased in blastocysts carrying inactivated TET1, which might have had an effect on the increase of NANOG and ESRRB. When the activity of TET enzymes was inhibited to eliminate the compensatory increase of TET3 under the absence of functional TET1, the expression levels of NANOG and ESRRB were decreased and methylation level of NANOG promoter was increased. In addition, ICM specification was impaired by the inhibition of TET enzymes. These results suggest that the TET family is a critical component of the pluripotency network of porcine embryos by regulating expression of genes involved in pluripotency and early lineage specification. In the next set of studies, the presence of TET3 isoforms in porcine oocytes and cumulus cells was investigated to dissect the gene structure of TET3 that could assist in understanding mechanistic actions of TET3 in the DNA demethylation process. Among the three TET3 isoforms identified in cumulus cells, only the pTET3L isoform, which contains CXXC domain that carry DNA binding property, was verified in mature porcine oocytes. Expression level of the pTET3L isoform was much higher in mature oocytes compared to that in somatic cells and tissues. In addition, the transcript level of this isoform was significantly increased during oocyte maturation. These results suggest that pTET3L isoform is predominantly present in mature porcine oocytes and that CXXC domain may play an important role in DNA demethylation in zygotes. In a follow-up study, the role of the TET3 CXXC domain in controlling post-fertilization demethylation in porcine embryos was investigated by injecting TET3 GFP-CXXC into mature porcine oocytes. The injected CXXC was exclusively localized in the pronuclei, indicating that the CXXC domain may localize TET3 to the nucleus. The CXXC overexpression reduced the 5mC level in zygotes and enhanced the DNA demethylation of the NANOG promoter in 2-cell stage embryos. Furthermore, the transcript abundance of NANOG and ESRRB was increased in blastocysts derived from GFP-CXXC overexpressing zygotes. These results provide an evidence that the CXXC domain of TET3 is critical for post-fertilization demethylation of porcine embryos and proper expression of pluripotency related genes in blastocysts. In the last set of studies, the impact of MBD proteins on porcine embryo development was examined under the hypothesis that competitive binding of MBD and TET proteins to 5mC contributes to the proper maintenance of DNA methylation levels in embryos. Cloning of porcine MBD1, MBD3, and MBD4 from mature oocytes indicates that the genes are highly conserved among different species, implying the involvement of porcine MBD proteins in the maintenance of DNA methylation. MBD1 overexpression in oocytes impaired preimplantation development of porcine embryos, suggesting that the MBD1 overexpression may have negatively affected porcine embryo development because proper DNA methylation levels were not preserved under the high level of MBD1. Collectively, the studies in my dissertation demonstrate that TET family proteins are important epigenetic players involved in the regulation of pluripotency and reprogramming of DNA methylation, and are thus crucial for normal embryo development. The findings in the dissertation will improve our understanding of epigenetic events occurring in mammalian embryos, and have the potential to overcome epigenetic defects that are observed in pluripotent stem cells and in-vitro derived embryos.
Doctor of Philosophy
Epigenetic modifications are heritable changes affecting the level of gene expression without changing the sequence of the genome. DNA methylation, one of the biggest epigenetic marks in mammalian genome, is often correlated to gene repression. In mammals, DNA methylation patterns are dramatically changed during preimplantation development to acquire embryonic developmental potential. Understanding of the epigenetic changes occurring in preimplantation embryos is necessary for producing healthy domestic animals in agriculture and for developing strategies for the treatment of epigenetic defects in human. Ten-eleven translocation (TET) family enzymes, TET1, TET2, and TET3, are known to function as a DNA methylation modifier by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). My dissertation research was performed to elucidate the role of TET family during preimplantation development using porcine embryos as a model. Pluripotency refers to the ability of cells to differentiate into all cell types of a mature organism. Pluripotent cells emerge in embryos as embryonic cells acquire lineage-specific characteristics. The first set of studies focused on the role of TET enzymes in regulating the pluripotency of porcine embryos. The impacts of inhibited activities of TET enzymes on the expression of pluripotency related genes were examined. We found that the inhibition of all TET enzymes leads to a decreased expression of pluripotency related genes, an altered DNA methylation level on a gene segment controlling pluripotency, and the impaired formation of pluripotent cell lineage in porcine embryos. This study demonstrates that the TET family is critical for the acquisition of pluripotency in porcine embryos. In the following sets of studies, the function of TET3 protein in the demethylation process occurring in preimplantation embryos was investigated. Fertilized mammalian embryos undergo genome-wide demethylation process to reset germ cell specific epigenetic marks into the embryonic epigenome. Previous studies indicate that TET3 is responsible for the demethylation process in mammalian embryos, although detailed mechanistic action of TET3 is still elusive. Here, we identified a predominant expression of a specific TET3 gene in porcine oocytes. The TET3 gene contained a CXXC domain, a potential DNA binding module, suggesting that TET3 may mediate DNA demethylation through its DNA binding property. To examine the function of the CXXC domain in TET3-mediated DNA demethylation, isolated CXXC domain was injected into porcine oocytes. The injection of CXXC domain facilitated DNA demethylation in embryos, demonstrating that the DNA binding property of TET3 is important for its functionality. In the last study, we investigated the importance of genes known to interact with TET enzymes in porcine embryos. Methyl-CpG-binding domain proteins (MBDs) have the ability to bind methylated region on the genome and play a critical role in mediating DNA methylation and gene repression. Our hypothesis was that a competitive binding of MBD and TET proteins to methylated regions was critical for proper DNA methylation levels in embryos. We identified that porcine MBD sequences were very similar to other species in terms of gene structure, indicating that the genes could also possess gene repressing activity by competing with TET enzymes during porcine embryo development. Injection of MBD1 mRNA to oocytes impaired normal embryo development, suggesting that the injected MBD1 may have negatively affected early embryo development in pigs by disrupting the proper maintenance of DNA methylation levels. My dissertation researches demonstrate that maintaining proper DNA methylation levels through the TET family is critical for normal embryo development in pigs. This research assists in improving our understating of epigenetic dynamics occurring in mammalian embryos and offers a potential solution to the epigenetic defects frequently observed in mammalian embryos produced through artificial reproductive technologies and pluripotent stem cells reprogrammed from somatic cells.

The competence of oocytes to mature and undergo fertilization and embryonic development is known to be influenced by the conditions under which their maturation occurs. Suboptimal maturation in vitro currently limits the use of immature oocytes for embryo creation. This project examines the relationship between the conditions of in vitro maturation of human oocytes and aspects of their subsequent developmental competence through the in vitro creation and analysis of research embryos. This work is essential in defining effective and safe conditions for the use of human immature oocytes in programmes of clinical treatment to alleviate infertility. Human immature oocytes were exposed in vitro to various concentrations of meiosis activating sterol (FF-MAS), an endogenous mediator of follicle and oocyte function, or epidermal growth factor (EOP), in the absence of other hormonal support. Their survival and further development relative to controls were measured by assessing the proportions maturing, fertilizing by sperm injection (ICSI), and cleaving in vitro. Image analysis was used to measure various dimensions of oocytes and embryos daily. A pilot study of chromosome and spindle configurations at meiotic metaphase was also conducted. The major findings of this project are that FF-MAS supplementation of maturation medium has different positive effects upon immature oocytes arising from patient groups having different endocrine profiles and yielding differing oocyte populations. FF-MAS at 30J.lg/ml promotes survival of oocytes from unstimulated patients with polycystic ovaries (p