Vergleich des Einflusses der mTor-Inhibitoren Rapamycin und Torin2 auf die Reifung von Rindereizellen und Charakterisierung der Entwicklungskompetenz Torin2-behandelter Rindereizellen

Für einen ungestörten Ablauf der Oozytenreifung (Entwicklung vom GV- zum M II-Stadium) ist eine genaue räumliche und zeitliche Regulierung der in der Säugetier-Eizelle stattfindenden Genexpression auf Ebene der Translation von entscheidender Bedeutung. Einer der Initiationsfaktoren, die dabei eine wichtige Rolle spielen, ist eIF4E. Dieser Faktor bindet an die Cap-Struktur der mRNAs. Ebenfalls an der Kon-trolle der Translation beteiligt ist mTor, eine Ser/Thr Protein-Kinase, die 4E-BP1, ein Protein, das an eIF4E bindet und die Translation unterdrückt, reguliert. mTor agiert als katalytische Untereinheit zweier Multiprotein-Komplexe. Dies sind mTorc1, das Raptor enthält, und mTorc2, das Rictor als Komponente besitzt. Im Rahmen dieser Arbeit wurden zwei verschiedene mTor-Inhibitoren, Rapamycin und Torin2 wäh-rend der In-vitro-Maturation von Rindereizellen angewendet. Diese Substanzen können eine Abgrenzung der jeweiligen mTorc1- und mTorc2-Funktionen ermögli-chen. Die infolge der Inhibitor-Behandlungen erzielten Effekte wurden mittels mor-phologischer Beurteilung anhand der Chromatinkonfiguration, durch biochemische Untersuchungen mittels Western-Blot und durch immunhistochemische Analysen mittels konfokaler Laserscanning-Mikroskopie überprüft. Während Torin2 eine Arretierung von 60% der Eizellen in der M I-Phase verursacht, inhibiert Rapamycin bei einem Teil der behandelten Eizellen die asymmetrische Zellteilung einschließlich der Ausschleusung des ersten Polkörpers. Die biochemischen und immunhistochemischen Analysen ergaben ein weitgehend konstantes Vorkommen der untersuchten Faktoren und Zielmoleküle (eIF4E, BP1, mTor, Raptor, Rictor, rpS6) im Verlauf der In-vitro-Maturation. Für alle Faktoren, außer für Raptor, das sich gegensätzlich verhält, zeigt sich im GV-Stadium eine niedrige, im M II-Stadium dagegen eine hohe Phosphorylierung. Rapamycin hat keinerlei Auswirkungen auf den Phosphorylierungszustand der untersuchten Faktoren, Torin2 hingegen hemmt spezifisch die BP1-Phosphorylierung an Thr37/46. Überraschenderweise wird weder durch Rapamycin noch durch Torin2 die rpS6-Phosphorylierung blockiert. Im Rahmen der immunhistochemischen Analysen wurde außerdem für manche Faktoren ein spezifisches Verteilungsmuster innerhalb der Eizelle gesehen, was für eine nicht nur zeitlich, sondern auch räumlich regulierte Translationskontrolle bei Rindereizellen spricht. Schlussendlich konnte kein Rapamycin-sensitives Zielmolekül bei Rindereizellen identifiziert werden, das eine Erklärung für die beobachteten morphologischen Rapamycin-Effekte liefern könnte. Zusätzlich zu den bisher erwähnten Untersuchungen wurden im Rahmen dieser Arbeit Torin2 behandelte Rindereizellen außerdem fertilisiert und kultiviert und die Blastozystenrate bestimmt. Es zeigte sich, dass diese Eizellen fähig sind, sich zu Blastozysten weiterzuentwickeln. Die hier vorgestellten Untersuchungen ermöglichen Einblicke in regulatorische Me-chanismen, die bei der Oozytenreifung eine Rolle spielen (insbesondere in die räumliche und zeitliche Steuerung der Translation) und können als Grundlage für weitergehende Forschung dienen. Als ein langfristiges Ziel solcher Forschung wäre die Steigerung der Erfolgsrate beim Transfer durch IVP erzeugter Embryonen anzusehen.An exact spatiotemporal regulation of gene expression in mammalian oocytes at translation level is absolutely necessary to guarantee an unimpaired course of meiot-ic progression (transition from GV-stage to M II). One of the specific initiation factors playing an important role in this process is eIF4E. This factor binds the mRNA cap-structure. Also involved in translational control is mTor, a Ser/Thr kinase regulating 4E-BP1, a protein binding eIF4E and repressing translation. mTor acts as catalytic subunit of two multiprotein-complexes. These are mTorc1 containing Raptor and mTorc2 harboring Rictor. Within the framework of this study two different inhibitors of mTor, Rapamycin and Torin2, were used during bovine oocyte in vitro matura-tion. Those substances may allow discrimination between mTorc1 and mTorc2 com-plex functions. The effects of the inhibitors were monitored by morphological exami-nation via reference to chromatin configuration, biochemical analysis by means of Western blotting and immunohistochemical analysis by means of confocal laser scanning microscopy. Whereas Torin2 arrests approx. 60% of the oocytes at the M I stage, Rapamycin inhib-its asymmetric division including polar body extrusion within a part of the treated oocytes. Biochemical and immunohistochemical analysis revealed broadly equal abundance of the factors and targets investigated (eIF4E, BP1, mTor, Raptor, Rictor, rpS6) in the course of IVM. Phosphorylation analysis showed low phosphorylation in GV-stage and high signals in M II for all factors except Raptor, which shows an opposite behavior. Rapamycin has no impact on the phosphorylation status of the factors investigated, Torin2 however inhibits specifically BP1 phosphorylation at Thr37/46. Surprisingly, rpS6 phosphorylation is blocked neither by Rapamycin nor by Torin2. Moreover, within the scope of immunohistochemical analysis a specific pattern of distribution within the oocyte was seen for some factors, suggesting not only a temporal but also a spatial regulation of translational control within bovine oocytes. In conclusion, no Rapamycin sensitive target explaining the morphological effects of Rapamycin observed could be identified in bovine oocytes. In addition to the investigations mentioned, within the framework of this study, Torin2 treated bo-vine oocytes were also fertilized and cultured. The blastocyst rate was determined. It became apparent that these oocytes are capable to develop to the blastocyst stage. The investigations presented here provide insights into the regulatory events in-volved in meiotic maturation (especially into spatiotemporal control of translation) and can serve as basis for further research. The improvement of the yield of offspring after transfer of IVP-derived embryos could be a long-term target of such research

Strukturelle und funktionelle Charakterisierung essenzieller Faktoren für die Initiation der Translation im Endometrium des Schweins

Mit Beginn der Trächtigkeit tritt im Endometrium des Schweins eine neue 23 kDa-Variante des eIF4E auf. Gleichzeitig wird das eIF4E-Repressorprotein 4E-BP1 hypophosphoryliert. Der 23 kDa-eIF4E-Variante fehlen die Aminosäuren 1-21 am N-Terminus. Sie entsteht proteolytisch durch eine Ca-abhängige Protease. Die neue Variante kann mit der Cap-Struktur der mRNA interagieren, aber nur sehr schwach mit 4E-BP1. Möglicherweise kann die 23 kDa-Variante die Repression durch 4E-BP1 überwinden und die Translation spezifischer Proteine aufrechterhalten werden.At the onset of pregnancy a new variant of the eIF4E occurs in the endometrium. With a molecular mass about 23 kDa. At the same time the eIF4E repressor protein is hypophosphorylated. The 23 kDa variant lacks the amino acids 1-21 at the N-terminus. It is formed by cleaving of a Ca-dependent protease. This new variant is able to interact well with die Cap-structure of the mRNA, but poorly with the 4E-BP1. It is likely that the small sized variant might overcome translational repression caused by eIF4E-BP1 at the time of implantation to maintain translation of specific proteins

Role and modulation of maternal transcripts during the first cleavage divisions in bovine embryos

Ce travail porte sur l’identification, la fonction et la régulation des molécules maternelles d’ARNm qui dirigent la compétence développementale juste après la fécondation chez les bovins. Tout d’abord, en utilisant le modèle du temps écoulé jusqu’au premier clivage zygotique et à travers l’évaluation du transcriptome des embryons à 2-cellules, il fut possible de déterminer la signature moléculaire des niveaux extrêmes de compétence au développement et sélectionner des molécules candidates pour des études postérieures. Les résultats ont montré que les embryons de capacité développementale variable diffèrent dans certaines fonctions comme la réparation de l’ADN, le traitement de l’ARN, la synthèse de protéines et l'expression génique définies par des ARNm synthétisés par l’ovocyte. Pour obtenir une confirmation fonctionnelle, une paire de transcrits maternels (l’un détecté dans notre sondage précédent et l’autre étant une molécule reliée) ont été inhibés par « knock-down » dans des ovocytes. Les effets du knock-down de ces facteurs de transcription sont apparus avant la formation des blastocystes dû à une diminution de la capacité au clivage et celle à progresser après le stage de 8-cellules. L’analyse moléculaire des embryons knock-down survivants suggère qu’un de ces facteurs de transcription est un contrôleur crucial de l’activation du génome embryonnaire, qui représente une fenêtre développementale dans l’embryogenèse précoce. Dans la dernièr étude, nous avons testé si les facteurs de transcription d'intérêt sont modulés au niveau traductionnel. Des ARNm rapporteurs couplés à la GFP (Protéine fluorescente) contenant soit la version courte ou la version longue de la séquence 3’-UTR des deux molécules furent injectées dans des zygotes pour évaluer leur dynamique traductionnelle. Les résultats ont montré que les éléments cis-régulateurs localisés dans les 3’-UTRs contrôlent leur synchronisation traductionnelle et suggèrent une association entre la compétence développementale et la capacité de synthèse de ces protéines. Ceci conduit à l’idée que ces facteurs de transcription cruciaux sont aussi contrôlés au niveau traductionnel chez les embryons précoces. Les connaissances acquises ont joué un rôle essentiel pour définir le contrôle potentiel des molécules maternelles sur les embryons au début de leur développement. Cette étude nous montre aussi une utilisation potentielle de cette information ainsi que les nouveaux défis présents dans le secteur des technologies reproductives.This work explores the identity, the function, and the regulation of maternal mRNA molecules that drive developmental competence shortly after fertilization in cattle. First of all, by using the model of the time of first zygotic cleavage and assessing the transcriptome of 2-cell embryos, it was possible to determine the molecular fingerprint of extreme levels of developmental competence and select candidate molecules for further monitoring. Data implied that early embryos of variable developmental capacity differ in functions including DNA repair, RNA processing, protein synthesis, and gene expression that are dictated by oocyte-synthesized mRNA. To obtain a functional confirmation, a pair of maternal transcripts (one detected in our previous survey and other related molecule) were knocked-down in oocytes that were further cultured. The effects of ablating these transcription factors were evident before blastocyst formation due to a decrease in cleavage capacity, as well as progression past the 8-cell stage. The molecular analysis of surviving knocked-down embryos suggested that one of these transcription factors is a pivotal orchestrator of the activation of the embryonic genome, a critical developmental window in early embryogenesis. In the last survey, we asked whether the transcription factors of interest are modulated at the translational level. Reporter mRNAs containing either short or long versions of the 3’-UTR sequences of both molecules were injected in zygotes to look at their translational dynamics. Results showed that cis-acting elements located in the 3’-UTRs govern their timely translation and suggested an association between developmental competence and protein synthesis capacity. This led to the notion that these crucial transcription factors are also controlled at the translational level in early embryos. The acquired knowledge was instrumental to define the possible control operated by maternal molecules on embryos at the onset of their development, as well as some of the challenges and potential use of this information in the field of reproductive technologies

The translational oscillation in oocyte and early embryo development

Translation is critical for development as transcription in the oocyte and early embryo is silenced. To illustrate the translational changes during meiosis and consecutive two mitoses of the oocyte and early embryo, we performed a genome-wide translatome analysis. Acquired data showed significant and uniform activation of key translational initiation and elongation axes specific to M-phases. Although global protein synthesis decreases in M-phases, translation initiation and elongation activity increases in a uniformly fluctuating manner, leading to qualitative changes in translation regulation via the mTOR1/4F/eEF2 axis. Overall, we have uncovered a highly dynamic and oscillatory pattern of translational reprogramming that contributes to the translational regulation of specific mRNAs with different modes of polysomal occupancy/translation that are important for oocyte and embryo developmental competence. Our results provide new insights into the regulation of gene expression during oocyte meiosis as well as the first two embryonic mitoses and show how temporal translation can be optimized. This study is the first step towards a comprehensive analysis of the molecular mechanisms that not only control translation during early development, but also regulate translation-related networks employed in the oocyte-to-embryo transition and embryonic genome activation

Efficient isolation, biophysical characterisation and molecular composition of extracellular vesicles secreted by primary and immortalised cells of reproductive origin

Effective communication between the maternal reproductive tract, gametes and the pre-implantation embryo is essential for the successful establishment of pregnancy. Recent studies have recognised extracellular vesicles (EVs) as potent vehicles for intercellular communication, potentially via their transport of microRNAs (miRNAs). The aim of the current investigation was to determine the size, concentration and electrical surface properties (zeta potential) of EVs secreted by; (1) primary cultures of porcine oviductal epithelial cells (POECs) from the isthmus and ampullary regions of the female reproductive tract; (2) Ishikawa and RL95-2 human endometrial epithelial cell line cultures; and (3) the non-reproductive epithelial cell line HEK293T. In addition, this study investigated whether EVs secreted by POECs contained miRNAs. All cell types were cultured in EV-depleted medium for 24 or 48 h. EVs were successfully isolated from conditioned culture media using size exclusion chromatography. Nanoparticle tracking analysis (NTA) was performed to evaluate EV size, concentration and zeta potential. QRT-PCR was performed to quantify the expression of candidate miRNAs (miR-103, let-7a, miR-19a, miR-203, miR-126, miR-19b, RNU44, miR-92, miR-196a, miR-326 and miR-23a). NTA confirmed the presence of EVs with diameters of 50–150 nm in all cell types. EV size distribution was significantly different between cell types after 24 and 48 h of cell culture and the concentration of EVs secreted by POECs and Ishikawa cells was also time dependent. The distribution of EVs with specific electrokinetic potential measurements varied between cell types, indicating that EVs of differing cellular origin have varied membrane components. In addition, EVs secreted by POECs exhibited significantly different time dependant changes in zeta potential. QRT-PCR confirmed the presence of miR-103, let-7a, miR-19a, miR-203, miR-126, and miR-19b in EVs secreted by POECs (CT ≥ 29). Bioinformatics analysis suggests that these miRNAs are involved in cell proliferation, innate immune responses, apoptosis and cellular migration. In conclusion, reproductive epithelial cells secrete distinct populations of EVs containing miRNAs, which potentially act in intercellular communication in order to modulate the periconception events leading to successful establishment of pregnancy

Genomic Differences Between Highly Fertile and Sub-Fertile Holstein Dairy Heifers

Infertility in dairy cattle remains a major economic loss to dairy producers. Identifying dairy cattle with superior genetic potential for improved fertility would increase dairy farm profitability. Dairy heifers were classified into two groups based upon services per conception (SPC); those animals with a single SPC were determined to be highly fertile and animals with greater than or equal to 4 SPC were classified as sub-fertile. Whole genome association analysis was performed on 20 individual heifers from each group utilizing a 777K highly density (HD) single nucleotide polymorphism (SNP) chip. Genomic data were evaluated utilizing PLINK, a whole genome association analysis toolset, and 570,620 SNP were available for analysis with a total of 39 samples being analyzed. Forty-four SNP were determined to be associated with fertility classification (P <= 0.00001) and were located on Bos taurus chromosome (BTA) 2, 4, 9, 19, and 26. The SNP and ranges between SNP were analyzed using BLAST-Like Alignment Tool (BLAT); SNP were associated with 5 candidate genes for reproduction. The SNP on BTA 2 were located within the region coding for the non-imprinted Prader-Willi/Angelman syndrome 2 (NIPA2) gene, which is involved in gestational magnesium transport. Also on BTA 2, SNP were identified within the region encoding for cytoplasmic fragile X mental retardation 1 (FMR1) interaction protein 1 (CYFIP1). The CYFIP1 gene is involved with the functionality of FMR1 and has been linked to premature ovarian failure in humans. Additionally, 3 SNP on BTA 9 were located near monofunctional C1-tetrahydrofolate synthase (MTHFD1L), which has been linked to neural tube defects during gestation in humans A difference in allele frequency was observed between the two groups for SNP located on BTA19 in proximity to two genes, zinc finger 18 (ZNF18) and mitogen activated protein kinase 4 (MAP2K4). The ZNF18 motif and MAP2K4 were found to be involved in heart development of the early embryo and associated with toll-like receptors (TLR) involved in gonadotropin releasing hormone (GnRH) signaling, respectively. The involvement of one or all of these genes may further explain reduced fertility in dairy cattle

Estudios In Vitro de los mecanismos de toxicidad de las micotoxinas

Se ha llevado a cabo la evaluación in vitro de los efectos producidos por micotoxinas de Alternaria y Fusarium en células de mamífero. Se ha evaluado la citotoxicidad individual del alternariol (AOH), alternariol monometil éter (AME), beauvericina (BEA), deoxinivalenol (DON), eniatina B (ENN B), fumonisina B1 (FB1), zearalenona (ZEA) y α-zearalenol (α-ZOL) en células de adenocarcinoma de colon humano (Caco-2) donde únicamente se obtuvieron valores de IC50 para la ENN B, DON, BEA y α- ZOL. La evaluación de la citotoxicidad combinada entre mezclas de micotoxinas en células Caco-2 mostró efecto sinérgico en las combinaciones de AOH+AME, DON+AOH y ENN B+AOH; efecto aditivo en la combinación DON+ENN B y efecto antagonista en la combinación terciaria DON+AOH+ENN B. Se estudió la bioaccesibilidad y biodisponibilidad del AOH, ZEA y α-ZOL. Se evaluó la bioaccesibilidad mediante el método de digestión estático in vitro, siendo el α-ZOL más bioaccesible a nivel gástrico y duodenal que la ZEA. Se observó una baja biodisponibilidad en las tres micotoxinas ensayadas con las células Caco-2/TC7, siendo el AOH el más biodisponible. Teniendo en cuenta la biodisponibilidad de las micotoxinas y los escasos estudios de mecanismos de toxicidad conocidos, se estudió la interacción de las micotoxinas con los componentes y la alteración de actividades celulares. Los resultados obtenidos demostraron que el AOH bloquea el ciclo celular en la fase G2/M, causa pérdida del potencial de la membrana mitocondrial y produce apoptosis a través de la vía mitocondrial. Además, se evaluó el daño causado por el AOH a nivel del ADN mediante el ensayo del cometa y se observó un incremento del daño dependiente de la concentración. Debido a la biodisponibilidad de las micotoxinas, se determinó la actividad estrogénica de algunas muy prevalentes como la FB1 y la BEA, observándose que la BEA produce mayor actividad estrogénica sobre células que la FB1. Dado que un mecanismo de citotoxicidad es el estrés oxidativo, se determina la capacidad del AOH para generar especies reactivas de oxígeno (ROS), evidenciándose que el AOH en las células Caco-2 produce ROS inmediatamente tras la exposición en todas lasconcentraciones ensayadas. Una de las consecuencias de las ROS es la oxidación de los lípidos de las membranas celulares, es decir, la generación de peroxidación lipídica (LPO). Los resultados obtenidos demostraron que el AOH aumenta significativamente la producción de LPO. Tras los resultados obtenidos se procedió a determinar la eficacia del sistema de defensa intracelular (enzimático y no enzimático) frente al estrés oxidativo. Estos indicaron un incremento de la actividad de la superóxido dismutasa (SOD) a todas las concentraciones de AOH expuestas en las células Caco-2 y que la actividad enzimática de la catalasa (CAT) fue más eficaz que la glutatión peroxidasa (GPx), eliminando peróxido de hidrógeno. Además, se demostró que el glutatión (GSH) y las enzimas implicadas en el ciclo del glutatión participan de manera activa en la defensa celular frente al AOH. Por otra parte, se estudió el efecto protector de vitaminas y antioxidantes de la dieta mediterránea frente a la exposición al AOH. Los resultados demostraron que los antioxidantes del aceite de oliva virgen extra previenen el daño celular producido por el AOH cuando se exponen simultáneamente. Mientras que la quercetina, considerada el polifenol en mayor cantidad ingerido diariamente en la dieta, no presentó efecto citoprotector frente al AOH, la soyasaponina I, saponina en mayor proporción en las legumbres y con efecto antioxidante, sí mostró efecto citoprotector tras la exposición de AOH. Para concluir, se estudió el isotiocianato de alilo como estrategia de mitigación para prevenir el crecimiento de los hongos en los alimentos y evitar la presencia de micotoxinas en la dieta. El isotiocianato de alilo reaccionó mejor con la ZEA que con el α- ZOL, reduciendo más de la mitad de la concentración inicial de ZEA. De esta forma, el isotiocinato de alilo podría considerarse una buena estrategia de reducción de las micotoxinas de los alimentosIn vitro evaluation of the effects produced by Alternaria and Fusarium mycotoxins in mammalian cells has been carried out. Individual cytotoxicity of alternariol (AOH), alternariol monomethyl ether (AME), beauvericin (BEA), deoxynivalenol (DON), eniatin B (ENN B) fumonisin B1 (FB1), zearalenone (ZEA) and α-zearalenol (α-ZOL) in human colon adenocarcinoma (Caco-2) cells have been evaluated, where only IC50 values for ENN B, DON, BEA and α-ZOL were obtained. The evaluation of the combined cytotoxicity between mixtures of mycotoxins in Caco-2 cells showed synergistic effects in AOH + AME, DON + AOH and ENN B + AOH combinations; Additive effect on DON + ENN B combination and antagonist effect on the tertiary combination DON + AOH + ENN B. Bioaccessibility and bioavailability of AOH, ZEA and α-ZOL were studied. In order to evaluate the bioaccessibility, the static digestion method was applied in vitro, being α- ZOL more bioaccessible at the gastric and duodenal levels than ZEA. Low bioavailability was observed in the three mycotoxins tested with Caco-2 / TC7 cells, being AOH the most bioavailable. Taking into account the bioavailability of mycotoxins and the few studies on toxicity mechanisms, the interaction of mycotoxins with the components and the alteration of cellular activities were examined. The results obtained demonstrated that AOH blocks the cell cycle in the G2 / M phase and produces apoptosis and necrosis through the mitochondrial pathway. The loss of potential of mitochondrial membrane after AOH exposure suggested that mitochondria plays an important role in the induction of apoptosis / necrosis. In addition, the damage caused by AOH at the DNA level was assessed through the Comet assay and an increase in concentration-dependent damage was observed. Generally, mycotoxins are bioavailable, although some are absorbed faster than others. For this reason, the estrogenic activity of some high prevalent mycotoxins, such as FBI and BEA, was analised. It was noticed that BEA had greater estrogenic activity in cells than FB1.Because a mechanism of cytotoxicity is the oxidative stress, the ability of AOH to generate reactive oxygen species (ROS) is determined. The results obtained demonstrated that AOH produces ROS immediately after the exposure to every concentration tested. One of the most studied consequences produced by ROS is the lipid oxidation of cell membranes, namely, the generation of lipid peroxidation (LPO). The results obtained demonstrated that AOH significantly increases LPO production. Following the results obtained, we proceeded to determine the effectiveness of the intracellular defense system (enzymatic and non-enzymatic) against oxidative stress. The results indicated an increase in superoxide dismutase (SOD) activity at all concentrations of AOH exposed in Caco-2 cells and that catalase (CAT) enzyme activity was more effective than glutathione peroxidase (GPx) on the elimination of hydrogen peroxide. In addition, it was demonstrated that glutathione (GSH) and the enzymes involved in the glutathione cycle are actively involved in cell defense against AOH. On the other hand, the protective effect of vitamins and antioxidants of the Mediterranean diet in front of exposure to AOH was studied. The results showed that the antioxidants of the extra virgin olive oil prevent cell damage produced by the AOH when exposed simultaneously. Quercetin, considered the greatest amount of polyphenol ingested daily, did not present a cytoprotective effect against AOH; while the soyasaponin I, the saponin that is present in legumes in a higher proportion and that has antioxidant effect, showed a cytoprotective effect after exposure of AOH. In conclusion, allyl isothiocyanate was studied as a mitigation strategy to prevent the growth of fungi in food and to avoid the presence of mycotoxins in the diet. The allyl isothiocyanate reacted better with the ZEA than with the α-ZOL, reducing more than half the initial concentration of ZEA. Therefore, allyl isothiocyanate could be considered a good strategy to mitigate mycotoxins in food

NRF2 mediated oxidative stress response activity during early in vitro bovine embryo development

Overcoming oxidative stress is one of the various embryo challenges to survive under suboptimal conditions during in vitro production of bovine embryos. Thus, the present study aimed to examine the ability of preimplantation bovine embryos to activate nuclear factor erythroid-derived 2-like 2 (NFE2L2 or NRF2)-mediated oxidative stress response and trigger their survival under oxidative stress conditions. An in vitro model was used to culture embryos under low (5%) oxygen tension as in bovine oviduct or high oxygen tension (20%), which is widely used in vitro culture of embryos. Early stage embryos including 2-, 4-, 8-, 16-cell and blastocyst stage embryos were generated under low (5%) or high (20%) oxygen level culture conditions. NRF2, NRF2 cytoplasmic inhibitor (KEAP1) and selected NRF2 target antioxidant genes expression were measured in each stage using quantitative real time PCR (qPCR). Reactive oxygen species (ROS) were evaluated in the blastocysts using green fluorescent probe. Our results revealed that the ROS level was high under 20 % compared to 5 % oxygen level in blastocysts. The transcription level of NRF2 and its downstream antioxidant genes was dramatically increased in 8-, 16-cell and blastocyst stage embryos under high compared to low oxygen level, while NRF2 inhibitor showed opposite expression pattern. In order to know whether NRF2 activity is associated with the embryo developmental competence, consequently NRF2 activity was compared in developmentally competent versus incompetent embryos. For this, the mRNA and protein expressions of NRF2 and the transcription level of its downstream antioxidant genes were compared in early (competent) vs. late (incompetent) cleaving 2-cell and blastocyst stage embryos. In the early developing blastocysts accompanied by low ROS level, NRF2 and its antioxidant target genes expression were increased. Likewise, protein expression pattern was observed in similar manner with more active nuclear NRF2. In conclusion, this study demonstrated that under oxidative stress conditions, pre-implantation bovine embryos are able to activate the NRF2-mediated oxidative stress response pathway, which is found to be correlated with their survival under in vitro condition.NRF2 vermittelte oxidative Stressreaktion während der frühen bovinen in vitro Embryoentwicklung Für bovine Embryonen ist das Überwinden von oxidativem Stress eine wichtige Herausforderung um in suboptimalen in vitro Entwicklungsbedingungen überleben zu können. Das Ziel dieser Studie war es, die Reaktionsfähigkeit und Überlebensfähigkeit von pre-implantierten bovinen Embryonen auf den durch den nuclear factor erythroidderived 2-like 2 (NFE2L2 oder NRF2)-vermittelten oxidativen Stress zu untersuchen. Für diese Studie wurde ein in vitro Kulturmodell mit unterschiedlicher Sauerstoffkonzentration (5%, 20%) etabliert. Dabei ähnelte die niedrige Sauerstofftension (5%) der im bovinen Eileiter und die höhere (20%) der die normalerweise zur in vitro Kultur von Embryonen benutzt wird. Frühe embryonal Stadien, 2-, 4-, 8-, 16-Zell- und Blastozystenstadien wurden unter geringen (5%) oder hohen (20%) Sauerstoffkonzentrationen kultiviert. Anschließend wurden die Genexpressionen von NRF2, NRF2 cytoplasmic inhibitor (KEAP1) und ausgewählten NRF2 Antioxidans-Zielgenen in den verschieden Stadien mittels quantitative real time PCR (qPCR) analysiert. Reactive oxygen species (ROS) wurden in Blastozyten mittels green fluorescent probe untersucht. Das Ergebnis zeigte, dass der ROS Spiegel in Blastozysten bei einer Sauerstofftension von 20% höher war im Vergleich zu der 5% Gruppe. Die Expression von NRF2 und seinen nachgeschalteten Antioxidans-Genen war unter einem hohen Sauerstoffspiegel im Vergleich zu einem niedrigeren in 8-, 16- Zell- und Blastozytenstadien dramatisch erhöht. Demgegenüber zeigte NRF2 Inhibition ein gegenteiliges Expressionsmuster. Um festzustellen, ob die NRF2 Aktivität mit der Embryoentwicklungsfähigkeit zusammen hängt, wurde die NRF2 Aktivität im Vergleich von entwicklungsfähigen zu nicht entwicklungsfähigen Embryonen untersucht. Dafür wurden die NRF2 mRNA- und Proteinexpressionen und die der Antioxidans-Gene im Vergleich von früh (kompetent) zu spät (inkompetent) entwickelten 2-Zell- und Blastozytenstadien analysiert. Die Genexpression von NRF2 und deren Antioxidans-Zielgenen war in früh entwickelten Blastozyten bei einem geringen ROS Spiegel erhöht. Ein ähnliches Bild zeigte sich für die Proteinexpression mit einem größeren aktiven Anteil an nuklearem NRF2. Schlussendlich zeigte diese Studie, dass unter oxidativen Stress pre-implantiere bovine Embryonen in der Lage sind den NRF2-vermittelten oxidativen Stressreaktionssignalweg zu aktivieren. Dieser steht in Korrelation zu der Überlebensfähigkeit unter in vitro Bedingungen

The Relevance of Ribonuclease III in Pathogenic Bacteria

Dissertation presented to obtain the Ph.D degree in Biology.Ribonucleases (RNases) are key factors in the control of all biological processes, since they modulate the stability of RNA transcripts, allowing rapid changes in gene expression. Some RNases are up-regulated under stress situations and are involved in virulence processes in pathogenic microorganisms. RNases also control the levels of regulatory RNAs, which play very important roles in cell physiology.(...

Diaplazentare Deoxynivalenolintoxikation bei Schweinefeten. Lassen sich am 70. Trächtigkeitstag histomorphologisch und immunhistologisch diagnostisch verwertbare Befunde erheben?

Diaplacentar deoxynivalenol intoxication in porcine fetuses. Are histomorphological and immunohistochemical investigations at the 70th day of gestation a helpful diagnostic tool