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Additional file 1. Electronic Supplementary Material.

PDF file containing all supplementary material- Text S1 "Polyandry and Sperm Competition with an Arbitrary Number of Mating Partners", Text S2 "Analytical Results | Single Release", Text S3 "Analytical Results | Continued Release under Monandry"

Supplementary Methods, Figures and Table S1

Supplementary results and material and methods. (DOC 90Â kb)

Titel und Inhalt 1 Einleitung 1 1.1 Speiseröhrenkrebs (Ösophaguskarzinom) 1 1.2 Apoptose: der programmierte Zelltod 3 1.3 Zellzyklusregulation 5 1.4 Der periphere Benzodiazepinrezeptor und seine Liganden 5 1.5 Fragestellung 12 2 Material 13 2.1 Antikörper 13 2.2 PCR-Primer 13 2.3 Zellkulturmedien, Zusätze, Chemikalien 14 2.4 Kits 15 3 Methoden 16 3.1 Zellbiologische Methoden 16 3.2 Molekularbiologische Methoden 23 3.3 Proteinchemische Methoden 28 3.4 Statistik 31 4 Ergebnisse 32 4.1 Charakterisierung von Primärzellkulturen humaner Ösophaguskarzinome 32 4.2 PBR-Expression in humanen Ösophaguskarzinomen 32 4.3 Aufnahmekinetiken von NBD-FGIN-1-27 36 4.4 Wachstumsstudien an Ösophaguskarzinom-Zellkulturmodellen 37 4.5 Bestimmung der Zytotoxizität von PBR-Liganden 42 4.6 Apoptoseinduktion durch PBR-Liganden 42 4.7 Zellzyklusuntersuchungen 50 4.8 Modulation extrazellulär regulierter Kinasen durch PBR-Liganden 52 4.9 Transkriptionelle Effekte von PBR-Liganden 55 5 Diskussion 61 5.1 PBR-Liganden als innovative Therapeutika für fortgeschrittene Ösophaguskarzinome 61 5.2 Der PBR, ein antiapoptotisches und proliferationsförderndes Protein 72 5.3 Abschließende Betrachtungen 76 6 Zusammenfassung 77 7 Summary 79 8 Literaturverzeichnis 81 9 Publikationsverzeichnis 95 9.1 Originalarbeiten 95 9.2 Übersichtsarbeiten 95 9.3 Eingereicht zur Veröffentlichung 95 9.4 Vorträge 96 9.5 Kurzveröffentlichungen und Posterabstracts 96 9.6 Auszeichnungen und Preise 99 10 Danksagung 100 11 Lebenslauf 101, Der periphere Benzodiazepinrezeptor (PBR) ist ein mitochondriales, transmembranäres Protein. Es ist mit dem permeability transition pore complex assoziiert, der eine Bedeutung für die Induktion mitochondrialer Apoptosesignalwege besitzt. In verschiedenen Tumorentitäten ist der PBR überexprimiert und besitzt vermutlich eine funktionelle Bedeutung für die Entstehung und Progression der Tumore. Die Modulation des PBR mittels spezifischer, exogener Liganden stellt daher eine Möglichkeit zur Entwicklung neuartiger pharmakologischer Therapiestrategien und zur Aufklärung der Funktion des PBR dar. Ziel dieser Arbeit war es, diesen Ansatz am Beispiel des Ösophaguskarzinoms zu untersuchen, das für seine sehr schlechte Prognose bekannt ist und dessen chemotherapeutische Behandlungsmöglichkeiten bisher unbefriedigend sind. Hierzu wurden Experimente an zwei Zelllinien der beiden histologischen Typen des Ösophaguskarzinoms, des Plattenepithel- und des Adenokarzinoms, sowie an Primärzellkulturen von humanen Ösophaguskarzinomen durchgeführt. Bei den Zelllinien KYSE-140 (Plattenepithel-) und OE-33 (Adenokarzinom) sowie allen Primärzellkulturen und auch Gewebeschnitten von Ösophaguskarzinomen wurde die Expression des mitochondrialen PBR nachgewiesen. In etwa einem Drittel der untersuchten Schnitte wurde zudem eine Überexpression des PBR im Tumorgewebe gezeigt. Der knockdown des PBR mittels antisense-Technologie führte in Ösophaguskarzinomzellen zu einer Wachstumsinhibition. Daher könnte der PBR im Ösophaguskarzinom eine Bedeutung für die Proliferation besitzen. PBR-Liganden hemmten dosis- und zeitabhängig die Proliferation der Ösophaguskarzinomzelllinien. Die Wirkung der Liganden beruhte sowohl auf der Induktion von Apoptose als auch auf einem Zellzyklusarrest am G1/S-Kontrollpunkt. Am Beispiel des PBR-Liganden FGIN-1-27 konnte der Mechanismus der PBR-Ligand-induzierten Apoptose und des Zellzykusarrests gezeigt werden: Zunächst wird der Zusammenbruch des mitochondrialen Membranpotenzials induziert, anschließend die Caspase-3-Aktivierung, die wiederum zur Aktivierung der p38MAPK führt. Dies bewirkt in der Folge zum einen die Fragmentation der DNA, zum anderen vermittelt die p38MAPK-Aktivierung den Zellzyklusarrest am G1/S-Kontrollpunkt. Die antiproliferativen Wirkungen der PBR-Liganden waren mit transkriptionellen Veränderungen Apoptose- und Zellzyklus-relevanter Gene assoziiert: Mittels cDNA-Array-Technologie und semiquantitativer RT-PCR wurde in beiden Ösophaguskarzinomzelllinien nach Behandlung mit PBR-Liganden eine starke Überexpression der growth arrest and DNA-damage-inducible-Gene gadd153 und gadd45 nachgewiesen, die von der Phosphorylierung der p38MAPK abhängig war. Die gadds sind als Apoptoseinduktoren und Regulatoren des Zellzyklus bekannt. Weiterhin wurde eine Aktivierung der ERK1/2 durch PBR-Liganden nachgewiesen. Die Hemmung dieses antiapoptotischen und mitogenen Stimulus führte zu einer überadditiven Steigerung der Apoptoseinduktion und der Zellzyklusarretierung durch PBR-Liganden. Die Aktivierung von ERK1/2 durch PBR-Liganden könnte die Basis für künftige Kombinationstherapien bilden. Der PBR stellt damit eine interessante Zielstruktur für die innovative pharmakologische Therapie von Ösophaguskarzinomen dar. Die mögliche klinische Anwendbarkeit der hier vorgestellten innovativen Therapiekonzepte sollte künftig durch in-vivo- Modelle und klinische Studien evaluiert werden., The periphal benzodiazepine receptor (PBR) is a mitochondrial transmembrane protein. It is associated with the permeability transition pore complex which participates in mitochondrial apoptosis pathways. PBR is overexpressed in various tumor entities and may be of functional relevance for tumorigenesis and tumor progression. Thus, the modulation of PBR by specific exogenous ligands represents a promising strategy for the development of innovative drug therapies and for the elucidation of PBRýs function. The aim of this study was to investigate this approach using esophageal carcinoma as a model, which is known for its very poor prognosis and the lack of curative treatment modalities. Experiments were performed using two established esophageal cancer cell lines representing the two different histologies, squamous cell and adenocarcinoma, as well as primary cell cultures of human esophageal carcinoma. The expression of the mitochondrial PBR was shown both in KYSE-140 (squamous cell) and OE-33 (adenocarcinoma) cell lines, all primary cell cultures, and tissues of esophageal carcinoma. One third of the histological sections displayed an overexpression of PBR in the tumor tissue versus normal mucosa. In KYSE-140 cells, the specific knockdown of PBR by antisense technology led to growth inhibition. Thus, PBR may regulate the proliferation of esophageal cancer cells. PBR ligands were shown to inhibit the proliferation of esophageal cancer cells in a time- and dose-dependent manner. The drug effects were due to an induction of apoptosis and an arrest of the cell cycle at the G1/S checkpoint. The mechanism of PBR-ligand-induced apoptosis and cell cycle arrest was then elucidated: A disruption of the mitochondrial membrane potential precedes and is required for caspase-3 activation elicited by PBR ligands. Caspase-3-activation then leads to p38MAPK activation which in turn induces DNA fragmentation and G1/S cell cycle arrest. The antiproliferative effects of PBR ligands were found to be associated with transcriptional alterations of genes involved in the regulation of apoptosis and cell cycle: Using cDNA microarrays and semi-quantitative RT-PCR, a strong up-regulation of the growth arrest and DNA-damage-inducible genes gadd45 and gadd153 was observed in response to PBR ligands. The expression of gadd genes was shown to be regulated by p38MAPK. Gadd genes are known regulators of apoptosis and cell cycle. Moreover, a PBR-ligand-mediated activation of the mitogenic and anti-apoptotic ERK1/2 was demonstrated. The inhibition of ERK1/2 activation led to an over-additive increase of apoptosis and cell cycle arrest caused by PBR ligands. The activation of ERK1/2 by PBR ligands may help to design combination therapies in the future. In conclusion, PBR represents an interesting target for the innovative pharmacological treatment of esophageal carcinoma. The transfer of these innovative therapeutic concepts to the clinical situation should be evaluated in vivo and in clinical studies in the future.

Meiotic drivers distort transmission to the next generation in their favor, with detrimental effects on the fitness of their homologues and the rest of the genome. Male carriers of meiotic drivers commonly inflict costs on their mates through genetic incompatibility, reduced fecundity, or biased brood sex ratios. Given these costs, evidence for female discrimination against male carriers is surprisingly rare. One of few examples is the t haplotype in house mice, a meiotic driver that shows strong transmission distortion in males and is typically homozygote lethal. As a consequence, mating between 2 t heterozygous (+/t) mice leads to high embryo mortality. Previous experiments showing that +/t females avoid this incompatibility cost by preferring +/+ versus +/t males have inferred preference based on olfactory cues or brief social interactions. Evidence from mating contexts in laboratory settings and semi-natural populations has been inconclusive. Here, we investigated female choice from a large number of no-choice mating trials. We found no evidence for discrimination against +/t males based on mating, remating, and copulatory behavior. Further, we found no evidence for avoidance of incompatibility through selective interactions between gametes. The likelihood of mating showed significant effects of female weight and genotype, suggesting that our test paradigm enabled females to exhibit mate choice. We discuss the strengths and limitations of our approach. By explicitly considering selection at both the individual and gene level, we argue why precopulatory female discrimination by +/t females may be less evolutionarily stable than discrimination by all females based on postcopulatory mechanisms.

Background With female multiple mating (polyandry), male-male competition extends to after copulation (sperm competition). Males respond to this selective pressure through physiological, morphological and behavioural adaptations. Sperm competitiveness is commonly decreased in heterozygote carriers of male meiotic drivers, selfish genetic elements that manipulate the production of gametes in males. This might give carriers an evolutionary incentive to reduce the risk of sperm competition. Here, we explore this possibility in house mice. Natural populations frequently harbour a well-characterised male driver (t haplotype), which is transmitted to 90 % of heterozygous (+/t) males’ offspring. Previous research demonstrated strong detrimental effects on sperm competitiveness, and suggested that +/t males are particularly disadvantaged against wild type males when first-to-mate. Low paternity success in the first-to-mate role is expected to favour male adaptations that decrease the risk of sperm competition by preventing female remating. Genotype-specific paternity patterns (sperm precedence) could lead to genetically determined alternative reproductive tactics that can spread through gene level selection. Here, we seek confirmation that +/t males are generally disadvantaged when first-to-mate and address whether males of different genotypes differ in reproductive tactics (copulatory and morphological) to maximise individual or driver fitness. Finally, we attempt to explain the mechanistic basis for alternative sperm precedence patterns in this species. Results We confirmed that +/t males are weak sperm competitors when first to mate. When two +/t males competed, the second-to-mate was more successful, which contrasts with first male sperm precedence when wild type males competed. However, we found no differences between male genotypes in reproductive behaviour or morphology that were consistent with alternative reproductive tactics. Sperm of +/+ and +/t males differed with respect to in vitro sperm features. Premature hypermotility in +/t males’ sperm can potentially explain why +/t males are very weak sperm competitors when first-to-mate. Conclusions Our results demonstrate that meiotic drivers can have strong effects on sperm precedence patterns, and may provide a heritable basis for alternative reproductive tactics motivated by reduced sperm competitiveness. We discuss how experimental and evolutionary constraints may help explain why male genotypes did not show the predicted differences. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0710-4) contains supplementary material, which is available to authorized users.

Polyandry is widespread across animal taxa, and subjects males to intense post-copulatory sexual selection which favors adaptations that enhance a male’s paternity success, either by decreasing the risk of sperm competition and/or by increasing the competitiveness of the ejaculate. Copulatory plugs deposited by males are thought to have evolved in the context of sperm competition. However, experimental studies that assess the function of copulatory plugs remain scarce. Moreover, most studies have used unnatural manipulations, such as ablating plug-producing male glands or interrupting copulations. Here, we investigated whether repeated ejaculation affects plug size in a mammalian model species, the house mouse. When males experience short periods of sexual rest we found that plug size decreased over repeated ejaculations so that time since last ejaculation can be applied as an approximation for plug size. We induced natural variation in plug size arising from variation in male sexual restedness, and investigated the behavior and paternity success of rival males. Male behavior in the offensive mating role (second) was influenced, albeit not significantly, by the sexual restedness of the first male-to-mate, and therefore the size of his plug. However, second males sired a significantly greater proportion of embryos when competing against a male that had recently mated compared to a male that had not. This supports a potential role of the plug in promoting a male's competitive fertilization success when remating occurs, which could be mediated both by delaying female remating and by ensuring efficient sperm transport through the female reproductive tract.

Female multiple mating (polyandry) is widespread across many animal taxa and indirect genetic benefits are a major evolutionary force favouring polyandry. An incentive for polyandry arises when multiple mating leads to sperm competition that disadvantages sperm from genetically inferior mates. A reduction in genetic quality is associated with costly selfish genetic elements (SGEs), and studies in invertebrates have shown that males bearing sex ratio distorting SGEs are worse sperm competitors than wild-type males. We used a vertebrate model species to test whether females can avoid an autosomal SGE, thethaplotype, through polyandry. Thethaplotype in house mice exhibits strong drive intheterozygous males by affecting spermatogenesis and is associated with homozygousin uterolethality. We used controlled matings to test the effect of thethaplotype on sperm competitiveness. Regardless of mating order,theterozygous males sired only 11% of zygotes when competing against wild-type males, suggesting a very strong effect of thethaplotype on sperm quality. We provide, to our knowledge, the first substantial evidence that polyandry ameliorates the harmful effects of an autosomal SGE arising through genetic incompatibility. We discuss potential mechanisms in our study species and the broader implications for the benefits of polyandry.