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Mitochondria are major powerhouses in all eukaryotic cells, producing ATP through oxidative phosphorylation and the citric acid cycle. An increase in ATP production is required during oocyte maturation, fertilization, and early embryo development in mammals [1, 2]. Previous studies have also reported an association between mitochondrial DNA (mtDNA) copy number and oocyte quality during maturation [1, 3]. For example, mtDNA copy number is an indicator of fertilization potential and oocyte maturation, and oocytes with a low mtDNA copy number have a significantly lower developmental potency [4, 5]. mtDNA copy number also increases during the in vitro maturation of porcine oocytes and after the treatment of oocytes with follicular fluids or EGF (epidermal growth factor), which likely affects the developmental potential of oocytes [6]. Mitochondrial membrane potential (Δφm) is also critical for the production of ATP. During oocyte maturation, there is a significant increase in mitochondrial Δφm [7], and in the absence of an increase, the developmental potential of oocytes decreases [8, 9]. In addition, a high mitochondrial Δφm in mouse and human oocytes and early preimplantation stage embryos is associated with ionic and metabolic regulation [10]. To date, few maternal genes in mammalian oocytes have been characterized. Among these maternal transcripts, C-mos, Cyclin B1, Cdc2 (cell division cycle 2), Gdf9 (growth differentiation factor 9), and Bmp15 (bone morphogenetic protein 15) are well-studied genes considered to be markers of female germ cells. One of the essential regulators of meiosis resumption is formed by Cyclin B1 and Cdc2 kinase [11]. It has been reported that the dynamic change in levels of cyclin B1 is mainly controlled by cytoplasmic polyadenylation during mouse [12] and bovine [13] oocyte maturation. GDF9 and BMP15 belong to the transforming growth factor-β (TGF-β) superfamily, which contains many members with important roles in regulating fertility [14]. GDF9 and BMP15 were recently identified as oocyte-secreted factors involved in folliculogenesis and oocyte maturation, as well as in cooperative regulation of granulosa cells [15]. Recently Ge et al. [16] reported a connection between mouse oocyte quality and both mitochondrial metabolic activity and DNA copy number, specifically with spindle formation, chromosomal alignment, and embryo development. However, the underlying molecular mechanism has not been addressed. In vitro maturation of pig oocytes is useful in the study of the molecular mechanisms that underlie meiosis and fertilization as well as in the production of cloned and transgenic embryos and pigs [17,18,19]. While in vitro culture conditions and/or micromanipulations such as enucleation and injection of DNA or sperm can affect mitochondrial activity in oocytes from several species, this information is not available for porcine oocytes. To determine the effects of mitochondrial metabolic activity and mtDNA copy number on oocyte maturation and developmental competence, we treated immature porcine oocytes with FCCP, which inhibited mitochondrial oxidative phosphorylation, and ddC (2’3-dideoxycytidine), which depleted mtDNA. The effects of these two inhibitors on oocyte dynamics were assessed by determining the mitochondrial Δφm, mtDNA copy number, ATP concentration, target mRNA expression and poly(A) tail length of maternal genes. P34cdc2 kinase activity and mitogen-activated protein kinase (MAPK) phosphorylation were also investigated following FCCP and ddC treatment. To the best of our knowledge, this is the first report to address the relationship among mitochondrial Δφm and copy number, the in vitro maturation of porcine oocytes, and the developmental competence of embryos.

Die isotherme Kalorimetrie ist eine Methode zur Untersuchung von chemischen Reaktionen in homogener Phase, die ohne den Einsatz von Markierungen auskommt. Die Reaktionswärme sorgt dabei für geringe Temperaturänderungen in einer Messzelle, welche über eine Thermosäule mit einer Wärmesenke in Kontakt steht. Durch die Temperaturdifferenz zwischen Zelle und Senke wird an der Thermosäule eine Spannung generiert, die in einem gewissen Temperaturbereich proportional zur Temperaturdifferenz ist. Die Signale stellen sich auf Werte ein, die proportional zur Wärmeleistung sind. Im Fall der isothermen Titrationskalorimetrie (ITC) ist es beispielsweise möglich, zwischenmolekulare Wechselwirkungen zu charakterisieren. Um detektierbare Signale zu erhalten, ist es allerdings nötig, ausreichend große Wärmemengen zu generieren. Um dies zu erreichen, muss meist eine relativ große Menge der Stoffe eingesetzt werden, die in einem großen Volumen gelöst werden müssen, um ausreichend geringe Konzentrationen zu erzielen. Im Gegensatz dazu wurde in dieser Arbeit ein von Herrn Dr. Lerchner an der TU Freiberg entwickeltes, miniaturisiertes Gerät genutzt. Dieses hat ein Zellvolumen von nur 20 µl, sodass es auf Reaktionen ausgelegt ist, bei denen eine hohe Wärmeleistung pro Volumen vorliegt. Dies ist beispielsweise bei Untersuchungen des Stoffwechsels von Mikroorganismen gegeben. Da aufgrund des geringen Probenvolumens die Wärmekapazität gering ist, liegen die Zeitkonstanten im Bereich von wenigen zehn Sekunden. Gegenüber gängigen ITC Aufbauten, bei denen die Zeitkonstanten typischerweise im Bereich von Minuten liegen, kann die Kinetik der Reaktionen in dem miniaturisierten Aufbau besser erfasst werden. Die vorliegende Arbeit widmet sich der Etablierung von Testverfahren zur Quantifizierung des Stoffwechsels von Hefemitochondrien, sowie Methoden zur Datenprozessierung zur weiteren Verbesserung der zeitlichen Auflösung. Bisher wurden diese Reaktionen vor allem elektrochemisch über den Verbrauch von Sauerstoff verfolgt. Kalorimetrische Verfahren haben den Vorteil, dass auch sauerstoffunabhängige Reaktionen beobachtet werden können. Dies ist insbesondere wichtig für Untersuchungen anderer Mikroorganismen, da beispielsweise viele interessante Bakterien ausschließlich anaeroben Stoffwechsel betreiben. Mitochondrien aus Saccharomyces cerevisiae (S. cerevisiae) stellen ein interessantes Modellsystem für das Verständnis dieser Zellorganellen dar. Da der Organismus nur auf wenige der Funktionen seiner Mitochondrien tatsächlich angewiesen ist, können Hefen in der Regel selbst mit schwerwiegenden Mutationen in mitochondrialen Genen überleben, bei deren Anwesenheit die Zellen anderer Organismen zugrundegehen würden. Viele mitochondriale Gene sind in S. cerevisiae und im Menschen homolog zueinander, einigen davon wird eine Schlüsselrolle in diversen Krankheiten zugeschrieben. Da das Genom von S. cerevisiae gut charakterisiert ist und viele Techniken zu Genmanipulation gut etabliert sind, handelt es sich um einen besonders interessanten Modellorganismus für ein grundlegendes Verständnis diverser Genfunktionen. Im Rahmen dieser Arbeit wurde in Kooperation mit Herrn Prof. Dr. Rapaport, Herrn Tan und Herrn Dr. Papic ein Messverfahren etabliert, mit dessen Hilfe der Energiestoffwechsel von Mitochondrien zeitaufgelöst beobachtet werden kann. Dieses Verfahren wurde genutzt, um zwei Mutationen in Genen für mitochondriale Proteine hinsichtlich ihres Effekts auf den Energiestoffwechsel zu untersuchen. Die Ergebnisse sind auf Stoffwechselreaktionen von Mikroorganismen übertragbar und können als Grundlage für diverse Untersuchungen dienen. Denkbar sind beispielsweise Studien zur Wirkung antimikrobieller Substanzen oder zur Ausbildung, Wachstum und Resistenzbildung von Biofilmen. Isothermal calorimetry is a label-free method for investigations of chemical reactions in homogenous phase. The heat of reaction results in small temperature changes in the measuring cell, that is in contact to a heat sink through a thermopile. The temperature difference between the cell and the sink results in a voltage generated in the thermopile that is proportional to the temperature difference within a certain temperature limit. The signals approach equilibrium values proportional to the heat power. For example, characterisation of molecular interactions is possible using isothermal titration calorimetry (ITC). However, in order to receive detectable signals, it is necessary to generate sufficient amounts of heat. Therefore, a relatively large amount of the interacting molecules needs to be used, which need to be dissolved in a large volume in order to keep the concentrations low enough. In contrast do this, a miniaturized device developed by Dr. Lerchener from TU Freiberg was used in this thesis. It has a cell volume of only 20 µl, so that it is suitable for reactions with a high heat power per volume. This is the case in microbial metabolism. The heat capacities are low due to the low sample volume, resulting in time constants of several ten seconds. Compared to ITC devices with time constants in the order of minutes, the kinetics of the observed processes can be measured more accurately. This thesis deals with method development for quantification of metabolic rates of yeast mitochondria, as well as methods for data processing for further improvement of time resolution. So far, these reactions were mainly observed electrochemically through oxygen consumption. Calorimetric methods provide the possibility of investigating oxygen independent reactions as well. This is particularly interesting for investigating other microorganisms, since e.g. many bacteria use anaerobic metabolism only. Mitochondria of Saccharomyces cerevisiae (S. cerevisiae) are an interesting model system for understanding of these organells in general. The organism only depends on few functions of its mitochondria, so yeasts often survive even severe mutations in mitochondrial genes that would be lethal in other organisms. Many mitochondrial genes in S. cerevisiae and humans are homologous, and many of them play a role in several diseases. With the well characterized genome of S. cerevisiae and many techniques for gene manipulation readily available, it is a particularly interesting model organism for basic understanding of deverse gene functions. Within this thesis, a test system was established in collaboration with Prof. Dr. Rapaport, Mr. Tan and Dr. Papic from the university of Tübingen, that can be used for time resolved observation of mitochondrial metablic rates. This method was used to study two mutations in genes coding for mitochondrial proteins with respect to their effect on energy metabolism. The results can be transferred to metabolic reactions of other microorganisms and serve as a basis for other investigations, such as studies on the effect of antimicrobial substances or the formation, growth and development of resistances in biofilms.

p32 [also known as HABP1 (hyaluronan-binding protein 1), gC1qR (receptor for globular head domains complement 1q) or C1qbp (complement 1q-binding protein)] has been shown previously to have both mitochondrial and non-mitochondrial localization and functions. In the present study, we show for the first time that endogenous p32 protein is a mitochondrial protein in HeLa cells under control and stress conditions. In defining the impact of altering p32 levels in these cells, we demonstrate that the overexpression of p32 increased mitochondrial fibrils. Conversely, siRNA-mediated p32 knockdown enhanced mitochondrial fragmentation accompanied by a loss of detectable levels of the mitochondrial fusion mediator proteins Mfn (mitofusin) 1 and Mfn2. More detailed ultrastructure analysis by transmission electron microscopy revealed aberrant mitochondrial structures with less and/or fragmented cristae and reduced mitochondrial matrix density as well as more punctate ER (endoplasmic reticulum) with noticeable dissociation of their ribosomes. The analysis of mitochondrial bioenergetics showed significantly reduced capacities in basal respiration and oxidative ATP turnover following p32 depletion. Furthermore, siRNA-mediated p32 knockdown resulted in differential stress-dependent effects on cell death, with enhanced cell death observed in the presence of hyperosmotic stress or cisplatin treatment, but decreased cell death in the presence of arsenite. Taken together, our studies highlight the critical contributions of the p32 protein to the morphology of mitochondria and ER under normal cellular conditions, as well as important roles of the p32 protein in cellular metabolism and various stress responses.