(English) Oocyte-to-embryo transition (OET) is one of the most complex developmental events, during which a differentiated oocyte gives rise to a totipotent zygote. During OET a transcriptionally silent oocyte undergoes massive reprogramming of gene expression, which transforms it into a transcriptionally active zygote. Although numerous studies have contributed to understanding the mechanism of OET, many genes involved in OET are yet to be identified. A whole new level of possible regulation of OET came with the discovery of long non-coding RNAs (lncRNA). LncRNAs are pol II transcripts longer than 200 nucleotides, that are typically spliced and polyadenylated but do not encode proteins. While lncRNAs have been studied in many model systems including embryonic stem cells, their expression in oocytes and early embryos and contribution to OET were largely unexplored at the beginning of this project. In my PhD project, I aimed to identify, annotate, and analyze lncRNAs expressed during OET. First, using RNA-Seq, 1600 highly reliable lncRNAs were identified and annotated in mouse oocytes and early embryos. Majority of lncRNAs were novel with expression exclusively at OET stages. A significant fraction of these lncRNAs was found associated with LTR retrotransposons, contributing to their novelty and...

La reproduction sexuée repose sur deux processus fondamentaux : la méiose qui permet la formation des gamètes dont le génome est haploïde et la syngamie qui permet, après fécondation, de restaurer la diploïdie par fusion des deux noyaux parentaux haploïdes. Alors que la méiose repose respectivement sur le génome maternel pour l'ovocyte et paternel pour le spermatozoïde, la restauration de la diploïdie dans le zygote repose exclusivement sur le génome maternel. Si un pronucleus maternel compétent pour la réplication est formé au terme de la méiose ovocytaire, le génome paternel quant à lui, n'acquiert cette compétence que sous l'influence de facteurs maternels. En effet, à la fin de la méiose, le génome paternel est « empaqueté » avec des protamines qui le rendent inactif pour toute fonction biologique, en particulier la réplication. L'éviction des protamines et leur remplacement par des histones maternelles sont des étapes indispensables à l'acquisition par le génome paternel de sa compétence à la réplication, préalable à la syngamie. Tous ces événements doivent être extrêmement coordonnés afin de permettre à un premier noyau zygotique comportant les deux lots de chromosomes parentaux de se former et d'entrer dans le premier cycle mitotique.Notre laboratoire a identifié yemanuclein-alpha, aussi appelé yemanuclein (yem) dans un crible moléculaire pour des gènes exprimés spécifiquement dans la lignée germinale femelle, et son premier allèle muté yem1. Cette mutation ponctuelle (V478E) a été identifiée dans un crible génétique de « stérilité femelle ». Une descendance exceptionnelle observée chez les femelles yem1, présente la propriété inattendue d'être parthénogénétique. Cette propriété révèle un double défaut chez le mutant : dans le processus de méiose ovocytaire qui conduit à la formation d'un pronucleus maternel haploïde mais aussi dans la formation d'un pronucleus paternel compétent pour la syngamie. Mes travaux de thèse ont porté sur les deux aspects de la fonction de la Yemanucléine. En conjuguant des méthodes de génétique, de biochimie, et de biologie cellulaire, nous avons pu mettre en évidence des fonctions essentielles de la Yemanucléine dans les étapes initiales de la prophase méiotique de l'ovocyte de drosophile. Nous avons pu montrer que la Yemanucléine joue un rôle clé dans la recombinaison méiotique et plus particulièrement dans la fréquence et la cinétique d'apparition des cassures double brin. Son association au complexe synaptonémal et au complexe cohésine, tous deux connus comme étant nécessaires à la ségrégation chromosomique, est un élément clé de cette fonction.Outre cette fonction méiotique, la Yemanucléine, facteur maternel, est aussi requise pour l'assemblage de la chromatine du pronucleus paternel. Nous montrons dans ce manuscrit qu'elle joue ce rôle à travers son action dans un troisième complexe, en partenariat avec la protéine HIRA. Le complexe multiprotéique contenant la protéine HIRA est connu pour sa fonction de chaperon du variant de l'histone H3.3 et son rôle dans l'assemblage de la chromatine du pronucleus paternel. La Yemanucléine est le premier membre de la famille HPC2/UBN1 caractérisé. Son rôle dans l'assemblage des nucléosomes découplé de la réplication est décrit pour la première fois dans ce manuscrit. C'est aussi la première fois qu'une protéine spécifique de la reproduction est décrite pour son implication à deux étapes clés de ce processus. / Sexual reproduction relies on two key events: formation of cells with a haploid genome through meiosis and restoration of diploidy through syngamy in the zygote. Meiosis completion is supported exclusively by the maternal genome for the oocyte and the paternal genome for the sperm cell. In contrast diploidy restoration in the zygote is entirely dependent on maternal factors. At the end of meiosis the maternal pronucleus is competent for replication, whereas the paternal genome is packed with protamines. These proteins need to be removed in the zygote and replaced by maternally provided histones before the paternal genome acquires competence for replication, a prerequisite for syngamy. All these events must be highly coordinated to allow the first zygotic nucleus to form with the two sets of parental chromosomes and enter the first mitotic cycle. Our laboratory has identified yemanuclein-alpha, also called yemanuclein (yem) in a molecular screen for genes specifically expressed in the female germ line and its first mutant allele yem1, in a female sterile screen. The role played by yem not only in the meiotic process through which a haploid maternal pronucleus is formed but also in the zygotic process that makes a paternal pronucleus competent for syngamy, is underscored by the obtention of exceptional parthenogenetic progeny from yem1 mothers.My thesis work is precisely dedicated to the analysis of both aspects of Yemanuclein function: in the oocyte and the zygote. Using genetic, biochemical and cell biology methods we were able to uncover essential functions of Yemanuclein in early meiotic prophase in the Drosophila oocyte. Using yem1 allele (V478E), we could show its requirement for meiotic recombination especially for the frequency and timing of the double strand breaks formation. Yemanuclein association with two protein complexes, the Synaptonemal Complex (SC) and the Cohesin complex known to be required for proper chromosome segregation, supports these findings. Beyond its meiotic function, Yemanuclein is also required in the zygote for assembly of paternal pronucleus chromatin. This is achieved through a third complex that acts as histone H3.3 chaperone. In the present manuscript we identify Yemanuclein as a partner of HIRA in its role in H3.3 nucleosome assembly and deposition on the paternal pronucleus. Interestingly Yemanuclein is the first member of the HPC2/UBN1 protein family ever characterized. The role of Yem/ HPC2/ UBN1 in replication independent chromatin remodeling remained elusive until very recently. Our work is original in that it is the first to report on a role of one member of this family in oocyte meiosis and paternal chromatin assembly in the zygote.

Reproductive efficiency in beef cattle can be improved with reproductive technologies at the herd, individual cow, and embryonic levels. Decreasing the bull:cow ratio for natural service after fixed time artificial insemination (FTAI) can alleviate economic burden associated with FTAI. In experiment 1, the total number of cows exposed per bull was negatively correlated with pregnancy rate to natural service on first return to estrus after FTAI in fall herds. The number of open cows per bull in fall herds using one natural service sire was negatively correlated with pregnancy rate on first return to estrus. There was no correlation between number of cows exposed per bull and pregnancy rates in fall herds with multiple sires or in spring herds. However, bull:cow ratio accounted for only 5–11% of the variation in pregnancy rates, thus we conclude that a reduced bull:cow ratio did not affect natural service return to estrus pregnancy rate. Experiment 2 examined how supplementing calcium salts of soybean oil (CSSO) improves beef cow fertility. Non-pregnant cows received supplement with either saturated fat or omega-6 rich CSSO. There were no changes in dominant follicle diameter, corpus luteum volume, plasma progesterone, or endometrial gene expression (PTGES and AK1B1, PPARA, PPARA, PPARD) between treatments. Plasma and follicular fluid fatty acid compositions were altered between treatments. Experiment 3 examined if size parameters of zygotes have potential as a non-invasive, objective embryo selection method. The outer diameter, area of ooplasm, and thickness of zona pellucida (ZP) was digitally measured on individual artificially activated oocytes and in-vitro fertilized (IVF) zygotes. Larger outer diameter increased probability of development to the blastocyst stage by days 7 and 8 for activated oocytes and tended to by day 8 for IVF zygotes. Thinner ZP increased probability of development to blastocyst stage on days 7 and 8 for oocytes, and to day 8 for IVF zygotes. Area did not affect development but was positively correlated with blastomere number on day 8. An interaction between diameter and ZP thickness was observed in zygotes, but not activated oocytes, suggesting oocyte activation is not always a suitable replacement for in-vitro fertilization. / Doctor of Philosophy / We need to improve reproductive efficiency in beef cows if we are to combat the challenges of producing more food while using less resources due to limited land availability and concerns with greenhouse gas emissions from agriculture. In cow-calf production systems, this means producing one healthy calf per cow per year. Cattlemen can implement a variety of assisted reproductive technologies to achieve this goal. Achieving maximal reproductive efficiency will require using technologies that are incorporated into herd management, individual animal care, and in vitro embryo production. Fixed time artificial insemination (FTAI) allows cattlemen to maximize the number of cows becoming pregnant and calving earlier in the season to increase efficiency. Unfortunately, use of FTAI is uncommon in cow-calf production systems because of labor and economic restraints. In order to improve economic feasibility of FTAI, bull-related costs need to be reduced, which can be done through increasing the number of cows serviced per bull (decreasing the bull:cow ratio). This study retrospectively examined correlations between the bull:cow ratio and pregnancy rate on first return to estrus after FTAI. There was little to no correlations between bull:cow ratio and pregnancy rates, and if they were significant, there was much variation in the data. With this we concluded that a reduced bull:cow ratio does not affect pregnancy rate on first return to estrus, allowing producers to increase the number of cows serviced by a single bull and reduce bull related costs. Supplementing calcium salts of soybean oil (CSSO) that are rich in omega-6 fatty acids can enhance beef cow fertility, but it is unclear why this happens. Using non-pregnant cows as a model, we studied the effects of either saturated fat or CSSO on reproductive parameters such as ovarian structures, hormone concentrations, and uterine gene expression. There were no changes in any of these parameters between treatments, but there were changes in the concentrations of certain plasma and follicular fluid fatty acids. There was also reduced activity of lipid metabolism enzymes. We were unable to pinpoint how CSSO supplementation improves reproduction, but the altered fatty acid content of tissues and altered enzyme activity likely plays a key role, thus ultimately impacting fatty acid utilization and growth of the embryo. In-vitro embryo production can increase the number of offspring produced from a single female and accelerate the incorporation of animals with high genetic merit into herds. To obtain optimal pregnancy rates with in vitro embryos, we should develop non-invasive, objective methods for identifying the most viable embryos. This study examined if size parameters of activated and fertilized oocytes are indicative of successful development. We discovered that oocytes with large diameters and those with thin zona pellucida were most likely to develop to the blastocyst stage, and that the area of the cell was positively correlated with blastocyst total cell number. An interaction between diameter and ZP thickness was observed in zygotes, but not activated oocytes, suggesting oocyte activation is not always a suitable replacement for in-vitro fertilization. This suggests that digital measurements of fertilized oocytes may have potential as objective selection criteria. Addressing issues of reproductive inefficiency in beef cows requires a comprehensive approach, as there is not one ideal solution. Management techniques can alleviate the cost of FTAI by reducing the number of bulls used without affecting pregnancy rates. Supplementing CSSO can alter tissue fatty acids to enhance fertility. Finally, the efficiency of in vitro embryo production can be improved by selecting better embryos for transfer without compromising the embryo. Combinations of all these techniques can create more reproductively efficient animals.

Pluripotence je schopnost buňky diferencovat do jakéhokoliv buněčného typu. Formuje se během časného embryonálního vývoje u savců a její vznik je spojen s reprogramací genové exprese na globální úrovni. Proces přirozeného vzniku pluripotence není stále zcela pochopen. Pro získání nového pohledu na události, které vedou ke vzniku pluripotence u savců, studovali jsme změny v genové expresi během oocyt-zygotického přechodu u myši. V tomto modelovém systému, oplodněné vajíčko podstoupí reprogramaci, která vede k vytvoření pluripotentních blastomer. Tyto blastomery zakládají samotné embryo. Cílem mé diplomové práce bylo analyzovat aktivaci transkripce během časného vývoje a vyvinout metodu pro monitorování exprese genů v oocytech, časných embryích a embryonálních kmenových buňkách. Metoda využívá kvantitativní PCR a umožnuje změřit expresi až 48 vybraných genů, které slouží jako markery pro maternální degradaci, aktivaci pluripotentního programu a diferenciaci do zárodečných linií. Dále ukazujeme, že náš systém monitoruje dynamiku transkriptomu během oocyt-zygotického přechodu, a získané výsledky jsou srovnatelné s daty naměřenými pomocí jiných metod. Díky našemu bioinformatickému přístupu jsme navíc identifikovali nové oocyt-specifické a zygotické nekódující RNA. Klíčová slova: pluripotence,...

Das Studium der Entwicklung von Tieren ist eine der ältesten Disziplinen in der Biologie. Die gesammelten Daten von unzähligen Untersuchungen an den verschiedensten Spezies wurden dazu benutzt, um ein generelles Verständnis des tierischen Lebenszykluses zu formulieren. Ein wichtiges Ergebnis der intensiven Untersuchungen war vor etwa einem Jahrhundert die Entdeckung spezifischer morphologischer Veränderungen, die sich während der Teilungsphase, der Zeitperiode die der Befruchtung und Aktivierung des Eies am Anfang der Embryogenese folgt, vollziehen. Diese Befunde führten schlussendlich zur Formulierung des Konzepts einer „Mid-Blastula Transition“ (MBT). Bisher gibt es nur eine Theorie die die Regulierung der MBT in befriedigender Weise erklärt. Dies ist das Model des Kern/Plasma-Verhältnis, welches sich aus dem Verhältnis DNA-Menge zu Zytoplasmavolumen ableitet. Es erklärt die MBT-Aktivierung durch bisher unbekannte, maternal deponierte Faktoren im Ei, welche die MBT Aktivierung kontrollieren, deren Konzentration allerdings mit jeder Zellteilung verdünnt wird, bis sie schließlich ihre blockierende Funktion verloren haben. Zwar wurde die Existenz dieses Mechanismuses schon in zahlreichen Spezies experimentell bewiesen, allerdings bleibt er nur eine ungenaue Beschreibung der ablaufenden Prozesse und lässt weiterhin viele Fragen unbeantwortet. Vor diesem Hintergrund hat diese Arbeit gezeigt, dass die Zellzyklen in Embryonen von Medaka (Oryzias latipes) ihre Synchronität schon nach dem vierten oder fünften Teilung verlieren, und diese durch ein Teilungsmuster ersetzt wird, das als „metasynchron“ bezeichnet wird. In diesem Teilungsmuster verlaufen die Zellteilungen in Wellen, die im Zentrum des Embryos beginnen und sich von dort nach außen hin radial ausbreiten. Noch ist der Sinn einer auf diese Art verlaufenden Zellteilung unbekannt, auch wenn es verschiedene Theorien gibt die versuchen den zugrunde liegenden Mechanismus zu erklären. Allen voran steht die Theorie eines unterschiedlichen Zugangs zu Faktoren innerhalb des Dotters. Allerdings wird diese Theorie durch die Beobachtungen in verformten Embryonen wiederlegt, in denen sich die Teilungswellen von einer Seite des Embryos zur gegenüberliegenden Seite ausgebreitet haben. Somit bleibt der Mechanismus für diese Art der Zellteilung weiterhin unklar. Nicht zu vergessen ist, dass diese deformierten Embryonen eine der möglichen Konsequenzen asymmetrischer Furchung während einer frühen Zellteilung sind. Asymmetrische Teilungen treten in Medaka in einer erheblichen Anzahl von Embryonen auf und haben einen direkten Einfluss auf die gleichmäßige Verteilung des Zytoplasma. Leider war es nicht möglich die Auswirkungen einer solchen ungleichmäßigen Verteilung aufzudecken, auch wenn man davon ausgehen kann, dass ein ausreichend großes Ungleichgewicht zu unterschiedlichen Zeitpunkten der MBT-Aktivierung in verschiedenen Zellgruppen führen müsste. Ähnliche Beobachtungen wurden bereits in anderen Spezies gemacht, und es wurde vermutet, dass diese in ungleichmäßigen Zellteilungen begründet lagen. Weiterhin wurde bewiesen, dass die zygotische Transkription schon wesentlich vor dem bisher angenommenen frühesten Zeitpunkt aktiv ist. Darüber hinaus wurden Hinweise gefunden, die darauf hindeuten, dass die Transkription in Embryonen von Medaka in zwei Schritten einsetzt. Der erste Zeitpunkt ist das 16-Zellen-Stadium, in dem die ersten Zellen identifiziert wurden, die Phosphorylierung für RNAPII zeigten, und der zweite das64-Zellen Stadium, in dem der Anteil an p-RNAPII positiven Zellen signifikant anstieg. Ein schrittweiser Anstieg der Transkription wurde bereits in anderen Spezies beobachtet, auch wenn in diesen Fällen nur eine Erhöhung der mRNA-Menge festgestellt wurde, und nicht die unterschiedliche Anzahl an transkriptionell aktiven Zellen untersucht wurde.Zusammenfassend bestätigen und erweitern die hier gezeigten Daten die grundliegenden Kenntnisse über die Prozesse vor und währen der MBT, liefern darüber hinaus aber auch Anzeichen für viele Prozesse vor und während der MBT, die nur wenig oder gar nicht verstanden sind. / The study of animal development is one of the oldest disciplines in the field of biology and the collected data from countless investigations on numerous species have formed a general understanding of the animal life-cycle. Almost one century ago, one consequence of these intense investigations was the discovery of specific morphological changes that occur during the cleavage phase, a period that follows fertilization and egg activation at the very beginning of animal embryogenesis. These observations resulted into the formulation of the concept of a midblastula transition (MBT). So far, the mechanism of the nucleo-cytoplasmic ratio model is the only one that explains MBT regulation in a satisfying way. It suggests that the MBT is controlled by several maternal repressive factors in the egg, which are titrated out by every cell division until they lose their repressing potential. Although this regulatory mechanism was proven for several species and in different approaches, it is still only a rudimentary model for MBT control and leaves numerous questions unanswered. On this conceptual background, this thesis has shown that embryos from the medaka fish (Oryzias latipes) lose their cell cycle synchrony already after the fourth or fifth round of cell divisions, and replace it by a metasynchronous divisions pattern, in which cell division occurs in clear waves beginning in the embryo's center. The reason for this change in division mode is still unknown, although several hypotheses were put forward, most notable a difference in yolk-access between cells. However, this theory was weakened by division waves that progressed from one embryonic pole to the opposing one, which were occasionally observed in deformed embryos, leaving the mechanism for this phenomenon furthermore unclear. Those deformed embryos were most likely the result of asymmetric cell divisions at very early stages, a phenomenon which occurred in a significant percentage of medaka embryos and which directly influenced the equal distribution of cytoplasmic material. It could not beuncovered what kind of effects this unequal distribution of cytoplasm exerted on the progression of embryonic development, but it can be argued that relevant differences in cell volumes could result in cell clusters that will enter MBT at different time points. Comparable observations were already made in other species and it was hypothesized that they were the direct results of early unequal cell cleavages. Finally, it was demonstrated that zygotic transcription in medaka embryos is activated prior to the hitherto assumed time of the first transcriptional initiation. Moreover, indications were found that strongly speak for a transcriptional activation that occurs in two steps; a first step at the 16-cell stage when first cells were identified positive for RNAPII phosphorylation, and a second step at the 64-cell stage, when the number of p-RNAPII positive cells significantly increased. A stepwise activation of zygotic transcription was already observed in other species, but only for the overall increasing amount of mRNAs and irrespective of the actual number of transcriptionally active cells within the embryos. .. Overall, these data confirm and expand the basic knowledge of pre-MBT embryos and about the MBT itself. Furthermore, they also suggest that many early processes in pre-MBT embryos are only rudimentarily understood or still totally unknown.

Ste2p and Ste3p are well-characterized yeast pheromone G-protein Coupled Receptors (GPCR) those are involved in the signaling of mating responses that lead to cell fusion. Their signaling–associated interactions with G-protein/MAPK signal transduction machinery are well established, homologous to those in mammalian systems, and serve as a simplified model system in GPCR research. While the arrestin- mediated biased signaling mechanism of mammalian GPCR has not been discovered for the pheromone receptors, a recent demonstration of α-arrestins being involved in the internalization of the pheromone GPCR, Ste2p was reported. The present study was designed to reevaluate and extend the alternate functionality for pheromone receptors and to determine the role of yeast arrestins in the yeast mating. Specific residues in the TM6 of Ste2p exhibiting strong mating and constitutive MAPK signaling were combined and investigated in terms of their effect on MAPK signal transduction leading to cell cycle arrest as well as their impact on downstream mating projection formation and zygote formation events. Our findings indicate that Ste2p possess as specific residues that govern its relative bias for mediating MAPK signaling or mating events. Relative dose response experiments accounting for systemic and observation bias for these mutations yielded evidence of mutational-derived functional biases for Ste2p and further validated the alternate pheromone dependent functionalities for Ste2p. Further, arrestin knockout and knock-in studies showed that Art1 (Ldb19) is selectively involved in the regulation of zygote formation but not MAPK signal transduction following the binding of ligand to Ste2p receptors. In addition, ligand stimulated selective localization of Art1 (Ldb19) to the mating projection, implicating it in the regulation of downstream mating functionalities. Overall, while leaving the full mechanism of alternate/biased Ste2p signaling to be elucidated, these results highlight the possibility of continued relevance of the yeast pheromone-mating pathway as a simplified model for GPCR research in the context of arrestin-mediated biased GPCR signaling.

ABSTRACT The purpose of this project was to investigate monocarboxylate (i.e. pyruvate and lactate) transport in the preimplantation stage of embryo development. Much progress has been made over the last 15 years towards understanding preimplantation and peri-implantation embryo physiology, including metabolic preferences during this period. It is known that as the cells (blastomeres) of an embryo compact via tight junctions and the embryo differentiates into a blastocyst, a metabolic “switch” occurs to allow the blastocyst to take up glucose at a rapid rate, obtaining energy derived from glycolysis. Glucose transporter molecules have been identified and characterized during this period of development and a paradigm for glucose transport has been described. However, during the early cleavage stages (days 1-3 post-fertilization), the embryo preferentially derives its metabolic energy from the monocarboxylate pyruvate. Evidence for the expression of pyruvate transporter molecules (a family of proton-coupled monocarboxylate co-transporters, MCT) has only been indicated via some kinetic studies on pH homeostasis and PCR analysis for MCT expression, and results have been conflicting (Gibb et al., 1997, Harding et al., 1999, Herubel et al., 2002). This project aimed to clarify discrepancies in reports for mRNA expression of MCT and to enhance the understanding of monocarboxylate transport processes during preimplantation development by pioneering investigations into protein expression for various MCT isoforms. Transport kinetics for monocarboxylate, DL-lactate, were examined by measuring the uptake of radioactive [3H]-DL-lactate from the medium by two-cell embryos and blastocysts. It was discovered that blastocysts demonstrate significantly higher affinity for DL-lactate compared to zygotes (Km 20 + 10 v 87 + 35 mM lactate; p=0.03), which suggested that alterations in the expression of various MCT isoforms might be expected as the embryo developed to a blastocyst. The rate of transport showed a trend towards a decrease from the zygote to blastocyst stages, although this could not be confirmed as significant within the limitations of this experiment. Mouse embryos, both in vivo and in vitro-derived, were collected and pooled at the zygote, two-cell, morula and blastocyst stages of development. RNA purification, reverse-transcription and PCR were used to analyze the expression of the four best-characterized MCT isoforms. MCT1, MCT2 and MCT4 were all found to be expressed in oocytes and mouse embryos from the zygote through to the preimplantation blastocyst. MCT3, an isoform uniquely expressed in the retina, was not detected at any stage in embryos. Since glucose has been implicated in regulatory processes involving glucose transporter expression in mouse embryos (Pantaleon et al., 2005, Pantaleon et al., 2001), mRNA expression was examined in the presence or absence of glucose in the culture media to determine whether the same phenomena applied to MCT. It was discovered that MCT1 and MCT4 isoforms were responsive to glucose-deprivation as evidenced by a reduction in mRNA expression in compacted morula cultured from the zygote stage without glucose. When glucose-deprived embryos were exposed to a brief high concentration of glucose during the 4-cell stage of development and continued in culture without glucose, the expression of mRNA for MCT1 and MCT4 persisted post-compaction, demonstrating that glucose exposure is necessary for the continued expression of these two isoforms in the mouse blastocyst. MCT2 mRNA did not respond to the absence of glucose in this way, and mRNA expression persisted in either the presence or absence of glucose. To follow these analyses of MCT gene transcription during early embryo development, confocal laser scanning immunofluorescence and western blotting were used to identify the expression of MCT proteins at various stages of development. Culture in the presence or absence of glucose was again employed to determine whether the changes seen in mRNA expression were conveyed at the protein level. All three proteins were identified throughout preimplantation development, though their locations were uniquely different. MCT1 was notably absent from plasma membranes at all stages, and was detected diffusely within the cytoplasm. In expanding blastocysts MCT1 tended to concentrate in the cortical cytoplasm of blastomeres and staining was more intense in the polar trophectoderm. In this cytoplasmic location its function is unclear. MCT1 does not appear to be a key transporter of monocarboxylates into and out of the embryo, but it may have a role in shuttling pyruvate and lactate within the cytoplasm to maintain metabolic and redox homeostasis. In embryos cultured without glucose, the immunostaining intensity for MCT1 gradually decreased as morulae degenerated and died. Protein loss occurred from the morula stage onwards, whilst mRNA was already undetectable at this stage. This would indicate that glucose signals which maintain mRNA expression most likely operate at the level of gene activation/transcription with latent effects on protein expression. MCT4 appeared to be located on the plasma membranes of oocytes and 2-cell embryos and nuclear staining was evident throughout preimplantation development, however plasma membrane expression was not apparent in morulae and blastocysts. This is consistent with earlier kinetic evidence of a low affinity lactate transporter (Km 87 + 35 mM lactate) operating at the early preimplantation stages. MCT4 has the lowest affinity for lactate of all the characterized MCT to date. Kinetic data also suggests that a change might occur in MCT protein expression as the embryo progresses to a blastocyst with a higher affinity lactate transporter taking precedence, and the loss of MCT4 from the plasma membrane at these later stages supports this view. Similarly to MCT1, MCT4 mRNA expression was also found to be dependent on glucose exposure during the early preimplantation period, and embryos cultured entirely without glucose demonstrated a loss of MCT4 mRNA expression at the morula stage. MCT4 typically exists as a lactate exporter in glycolytic tissues and it most likely exports lactate from the embryo for pH and redox homeostasis during this period of development. Protein localization studies found MCT2 to be located on the plasma membranes of oocytes, zygotes, 2-cell embryos, and polarized to the surface of the outer blastomeres of morulae and blastocyst trophectodermal cells. Throughout preimplantation development, MCT2 protein co-localized with peroxisomal catalase in peroxisome-sized granules throughout the cells. Known to be a high affinity pyruvate transporter, given its location in embryos it was proposed here that MCT2 most likely imports pyruvate to fuel early embryos, and later works as a bifunctional pyruvate/lactate importer/exporter on the transporting epithelium (trophectoderm) of blastocysts to maintain the pH, redox and metabolic status of the embryo. MCT2 was an enigma to the other MCT. Its expression in the absence of glucose behaved in an opposite way to that of MCT1 and MCT4, with mRNA expression persisting in the absence of glucose. In fact, MCT2 and catalase proteins demonstrated a quantitative increase in embryos lacking glucose, and the increase in staining was noticed as an increase in the density of peroxisome-like structures (or peroxisome proliferation) within the embryo. As such, it was decided to investigate the possibility that peroxisome proliferators (Peroxisome Proliferator Activated Receptors, PPARs) were involved in the control of MCT expression in the same way that they are known to control the expression of catalase and other peroxisomal proteins. At this stage, no MCT isoforms had been identified as being under the control of PPARs, although it was known that their expression was most likely controlled at the level of transcription, with no translational or post-translational controlling elements. PPARα, one of three isoforms (α, γ and β/δ) was selected as a likely candidate given that it controls peroxisomal proliferation and fatty acid β-oxidation processes at the level of transcription in other tissues, and it was known to be upregulated in conditions of starvation and oxidative stress. PPARα mRNA was shown to be expressed in early cleavage preimplantation mouse embryos, but its expression was reduced in morulae and blastocysts. Further, lack of glucose led to persistence of PPARα mRNA expression at the morula stage. PPARα protein was also demonstrated to stain more brightly in early preimplantation embryos compared to later stages. Further experimentation demonstrated that the phenomenon of increased catalase and MCT2 expression in embryos cultured without glucose could be mimicked in the presence of glucose by treating these embryos with the PPARα-selective agonist, WY14,643. The timing and quantitative nature of this upregulation were very similar, suggesting that PPARα was in some way involved in the glucose-deprived upregulation pathway for catalase and MCT2. To further investigate this pathway, oxidative stress was investigated in embryos cultured in the presence and absence of glucose to test whether the generation of reactive oxygen species contributed to the PPARα/MCT2 phenomenon. It was demonstrated that within 2 h of culture in the absence of glucose, hydrogen peroxide levels were significantly elevated in zygotes. Amelioration of increased peroxide generation in glucose-deprived embryos using a non-selective flavoenzyme inhibitor diphenyleneiodonium (DPI) eliminated any increases in PPARα and MCT2 protein expression that were earlier noted in the absence of glucose. To summarize, MCT1, MCT2 and MCT4 mRNA and protein expression were successfully demonstrated in mouse preimplantation embryos and all were confirmed to be in some way regulated by glucose in the culture medium. In the absence of glucose, mRNA expression for MCT1 and MCT4 were reduced to undetectable levels in morulae indicating that their expression was glucose-dependent. Paradoxically, glucose deprivation caused an increase in PPARα, catalase and MCT2 protein expression. PPARα-selective agonism in the presence of glucose induced similar timing and effects on catalase and MCT2 upregulation, implicating PPARα in this pathway. Hydrogen peroxide levels were significantly elevated within 2 h of culture in the absence of glucose. This peroxide elevation could be quenched to control levels by treating these embryos with DPI, and reducing hydrogen peroxide to control levels also eliminated the upregulation of PPARα and MCT2, implicating oxidative stress as an important component in the glucose-deprivation induced upregulation of MCT2. The experimental data presented in this thesis demonstrate that from its very conception, the embryo interacts with, adapts to, and is indeed affected by the external environment in which it develops. Even components like glucose, once considered simply as metabolic substrates, have profound effects on gene transcription and protein expression within the embryo which may impact on later its developmental competence, a reality we need to consider more deeply in light of the implementation of artificial reproductive technologies widely used today in zoology, agriculture and clinically, in humans.

This thesis examined aspects of the reproductive and molecular biology of Eucalyptus marginata (jarrah). The aims were to develop protocols for controlled pollination, that could be used in clonal orchard trees to breed jarrah seedlings that have a known genetic resistance to Phytophthora cinnamomi (dieback), for use in rehabilitation after mining and logging. An intimate knowledge of the breeding biology of jarrah was necessary to achieve this aim. The project also aimed to increase knowledge of the genetic diversity and structure of jarrah, in order to make informed decisions regarding the collection of material to be used for clonal propagation. Previous research has had little success in producing viable seed from any controlled pollinations, but clonal material resistant to P. cinnamomi has been produced using tissue culture. The question posed in this thesis was ‘Can we improve breeding and propagation techniques of jarrah?’ Techniques were developed for testing of in vitro pollen viability and pollen storage, pollination and fertilisation success after controlled pollinations, including determination of stigma receptivity and development of bud isolation techniques using alfoil. The variation in female fertility between genotypes was examined. The use of paclobutrazol was explored as a method of increasing the level of viable seed production in clonal orchard trees. The use of fertiliser as well as the growth retardant was also explored to see if it increased the level of seed production even more. Genetic diversity, genetic differentiation and phylogeny within Eucalyptus marginata were examined using nuclear and chloroplast DNA analysis with Restricted Fragment Length Polymorphisms. While it was first thought that the fertilisation rate was quite low, it was confirmed that the fertilisation rate is similar to other eucalypt species. The zygote abortion rate was quite high in one clone, but one wild tree had a similar seed production rate to other eucalypt species. The zygote and endosperm appeared to be different in the clone and the wild tree observed. The level of seed production was examined in clones and wild trees and it was found that the level was often quite low, particularly in the clones (0 – 13% in clones, 0 – 18% in wild trees) in comparison with other Eucalyptus species, and varied between genotypes. The use of a growth retardant such as paclobutrazol may increase the production of viable seed, if it is applied during autumn. The results were inconclusive for the fertiliser/paclobutrazol experiment, since the paclobutrazol was applied during spring which was the worst time of year for increasing seed production. There were differences between genotypes in reaction to both the paclobutrazol and the fertiliser/paclobutrazol. Genetic diversity was moderate in comparison with other Eucalyptus species, and there was a low level of genetic differentiation between populations in the nuclear genome. No differentiation was observed between the morphologically recognised subspecies in the nuclear genome, but differentiation between the populations on the Swan Coastal Plain and populations on the Darling Plateau was seen in the chloroplast genome, indicating that there was historical separation of these two areas. The conclusions arising from this work are that while controlled pollinations are possible in Eucalyptus marginata the clones that were used in these experiments have often behaved differently to the wild trees in the time of anthesis and levels of viable seed production, and in one clone (5J119) the zygote and endosperm nuclei appeared to be very different to the zygote and endosperm nuclei of a wild tree. Further investigation is necessary to see if these differences are related to the low level of seed production observed in the clonal populations. Paclobutrazol may be worth exploring further as a means of increasing seed production. Material to be used for rehabilitation and seed orchards can be collected from a wide area in the main distribution of the species, although trees on the Swan Coastal Plain are distinct from the trees in the main forest area in the chloroplast genome.

Orientador: Carlos Alberto Petta / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T05:22:09Z (GMT). No. of bitstreams: 1 Cossiello_RaquelDiFalco.pdf: 3774222 bytes, checksum: 96b4cbfa6f25f58d8240f552b5c83617 (MD5) Previous issue date: 2009 / Resumo: Objetivo: comparar os efeitos de quatro diferentes meios de cultura na morfologia dos zigotos e embriões. Materiais e métodos: estudo retrospectivo conduzido no Centro de Reprodução Humana de Campinas, em que 2.289 embriões de 319 ciclos de ICSI foram avaliados de setembro de 2006 a setembro de 2008. O protocolo longo foi usado para estimulação ovariana em todos os casos. Todos os oócitos foram cultivados em dois meios diferentes. O meio HTF (Irvine Scientific) foi usado como meio-padrão, enquanto que os meios Universal IVF Médium (Medicult), Global (LifeGlobal) e IVF-30 (Vitrolife) foram usados como secundários. A separação dos oócitos em meios diferentes foi realizada alternadamente após ICSI. A presença e a posição de pronúcleos e Nuclear Precusror Bodies (NPBs) foram checadas 18 a 20 horas após ICSI. Baseado na classificação descrita por Gianaroli et al., os zigotos foram identificados como: (A1) pronúcleos justapostos e centralizados com NPBs grandes e alinhados; (A2) pronúcleos justapostos e centralizados com NPBs grandes e dispersos. Os embriões foram avaliados 44 a 46 horas após ICSI, de acordo com o número de blastômeros, porcentagem de fragmentação e multinucleação. Os embriões considerados top apresentaram quatro blastômeros regulares, fragmentação menor que 20% do volume embrionário e blastômeros não multinucleados. Para a análise dos dados foram utilizados Z-test, odds ratio simples e múltiplo através de regressão logística com seu respectivo intervalo de confiança a 95%. Resultados: quando a classificação dos zigotos foi analisada, o meio IVF-30 mostrou maior porcentagem (55,2%) de zigotos A1+A2, em relação ao HTF, Global e Universal IVF Medium (49,1%, 44,7% e 44,2%, respectivamente). A porcentagem de embriões top foi significativamente maior no meio Global (40,4%) comparado com HTF (21,1%), IVF-30 (25,0%) e Universal IVF Medium (11,1%). No segundo dia de desenvolvimento, Medicult produziu mais embriões com três células em relação aos outros meios que produziram mais embriões com quatro células. Conclusão: Houve diferenças significativas entre os quatro meios de cultura sobre a morfologia dos zigotos e a morfologia embrionária. IVF-30 (Vitrolife) resultou em maior número de zigotos com pronúcleos centralizados e nucléolos justapostos e dispersos. Global (LifeGlobal) sustentou maior formação de embriões top no dia 2 e maiores taxas de clivagem em relação aos demais meios / Abstract: Objective: compare the effects of four different culture media on the quality of zygotes and embryos. Methods: This retrospective study, performed at the Center for Human Reproduction of Campinas-Brazil analyzed 2289 embryos were assessed from September 2006 to September 2008. Long protocol was used for ovarian stimulation in all cases. The oocytes of each patient were cultivated in two different culture media. The medium HTF - Irvine was set as the default for all cycles and IVF Medium - Medicult, GGG 20 - Global and IVF 30 - Vitrolife defined as secondary media. The sibling oocytes were divided in the two culture media after ICSI. The confirmation of fertilization and classification as described by Gianaroli were evaluated 18-20 hours after ICSI. On the second day (day 2) of development, the embryos were evaluated according the number of cells, percentage of fragmentation and number of nuclei. On day 2, the embryos that had four cells with less than 20% of fragmentation and were mononucleated embryos were classified as Top. Z-test and Odds ratios were used for statistical analysis. Results: IVF-30 showed a higher percentage (55.2) of zygotes A1 + A2 when compared to HTF, Global and Universal IVF Medium media (49.1%; 44.7%; 44.2% respectively) The percentage of Top embryos was significantly higher in Global medium (40.4%) compared to HTF (21.1%), IVF-30 (25.0%) and Universal IVF medium (11.1%). On day 2 Universal IVF Medium produced more embryos with three blastomeres when compared to other media that produced more embryos with four blastomeres. Conclusions: The use of IVF- 30 medium resulted in a higher number of zygotes with centralized pronuclei with juxtaposed or scattered nucleoli. Meanwhile, Global medium produced a greater number of morphologically good embryos (TOP) and higher cleavage rate on the second day of development / Universidade Estadual de Campi / Tocoginecologia / Mestre em Tocoginecologia

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