Fettleibigkeit und Adipositas haben in den letzten Jahren weltweit drastisch zugenommen. Die Fettleibigkeit geht nicht nur mit einer verringerten Lebensqualität einher, sondern ist auch mit verschiedenen Folgeerkrankungen, wie kardiovaskulären Erkrankungen (z.B. Arteriosklerose) und metabolische Erkrankungen (z.B. Diabetes) assoziiert. Vorliegende Studien belegen einen Zusammenhang zwischen Diabetes und männlicher Infertilität. In der vorliegenden Arbeit wurden daher die Spermienmembran wie auch das Seminalplasma (SP) mittels matrix-assisted laser desorption and ionisation time-of-flight Massenspektrometrie (MALDI-TOF MS) analysiert, um mögliche Lipid-Biomarker für die Fertilität bzw. Infertilität zu finden. Dafür wurde zunächst die MALDI-TOF MS Methode mit relevanten Standardlipiden optimiert. Anschließend konnte sowohl das Phospholipidmuster des SP mit dem Spermien verglichen werden als auch die Spermienlipide von normalgewichtigen und fettleibigen Probanden. Durch diese Analyse konnte das Phosphatidylcholin/Lysophosphatidylcholin (PC/LPC)-Verhältnis, aber auch ein stark erhöhter Sphingomyelin (SM)-Anteil bei den fettleibigen Probanden als Qualitätsmarker gefunden werden. Des Weiteren wurden im Rahmen dieser Arbeit murine Spermien aus dem Caput und dem Cauda der Epididymis mittels MALDI-TOF MS analysiert und das Phospholipidmuster miteinander verglichen. Es konnte damit gezeigt werden, dass die murinen Spermien einen wesentlich höheren Anteil an Stearinsäureresten aufweisen, die humanen Spermien hingegen vor allem durch Palmitinsäurereste charakterisiert sind. In den Spermienmembranen aus dem Cauda und dem Caput gab es wesentliche Unterschiede im Phospholipidmuster. Spermienmembranen aus dem Caput besitzen einen hohen Anteil an PC und Phosphatidylethanolamin (PE). Die Spermienmembranen aus dem Cauda hingegen enthalten mehr SM, und auch einen höheren Anteil an LPCs und Formyl-LPC. Diese Arbeit konnte somit zeigen, dass die Reifungsprozesse in der Epididymis auch die Phospholipid-Zusammensetzung betreffen.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Huntington's disease (HD) is one of the incurable and fatal diseases. HD belongs to the monogenic neurodegenerative diseases. According to the number of the CAG repetitions in the gene coding huntingtin, the onset of the disease is in childhood (5%), in the middle age, which is the most common (90%) and in the older age (5%). Beginning of the disease is manifested by changes in behavior; including problems with coordination and movement. Later, there is a psychological change. The disease leads to death. Until now, there is no effective curative treatment. In 2009, we created a model of the transgenic minipigs (TgHD) carrying the N - terminal part of the human mutant huntingtin (mtHtt) at our Institute in Liběchov. The number of offsprings and the resemblance in physiology and morphology between the pig (Sus scrofa) and humans (Homo sapiens) give us opportunities not only to study changes not only in central nerve organs, but also in peripheral tissues. The reproductive problems of TgHD boars were observed as the first phenotypic changes. Therefore, my work focuses at first on a study of the reproduction parameters of TgHD boars as well as ultrastructural, immunocytochemical and biochemical changes in testes and spermatozoa. In PhD thesis, I described in details the reproductive defects in TgHD...

Stress can induce prolonged deleterious effects on many characteristics in chickens (Gallus gallus). Particular interest has been paid to early life stress. Social isolation as an early life stressor results in increased plasma corticosterone levels. Moreover, it induces behavioural and physiological changes as well as gene expression modifications in the hypothalamus. In the first part of my study, I aim to inquire into social isolation impacts on the short and long-term. Short and long-term effects were assessed by immune system, behaviour and weight. 82 male chickens were assigned to three groups (stress, control and enrichment). The stress group was exposed to social isolation, the enrichment group was provided with enrichment substrates while the control group was left untreated. According to my knowledge, this is the first study that investigates the effects of social isolation on the interuleikn-6 levels as an indicator of immune system response. My findings suggest that social isolation induces short and long-term effects on immune response as well as on body weight. In the second part of my study, I aim to develop a method investigating effects of early stress on DNA methylation in blood and sperm. For this purpose, two methods GBS (Genotyping by sequencing) and MeDIP (Methylated DNA immuneprecipitation) were f using pooled DNA from all individuals for the first time. Moreover, I developed a protocol for extracting sperm DNA from frozen testis. Combining both methods has many advantages, such as cost effectiveness and the ability to evaluate epigenetic signatures in large number of individuals

Fettleibigkeit und Adipositas haben in den letzten Jahren weltweit drastisch zugenommen. Die Fettleibigkeit geht nicht nur mit einer verringerten Lebensqualität einher, sondern ist auch mit verschiedenen Folgeerkrankungen, wie kardiovaskulären Erkrankungen (z.B. Arteriosklerose) und metabolische Erkrankungen (z.B. Diabetes) assoziiert. Vorliegende Studien belegen einen Zusammenhang zwischen Diabetes und männlicher Infertilität. In der vorliegenden Arbeit wurden daher die Spermienmembran wie auch das Seminalplasma (SP) mittels matrix-assisted laser desorption and ionisation time-of-flight Massenspektrometrie (MALDI-TOF MS) analysiert, um mögliche Lipid-Biomarker für die Fertilität bzw. Infertilität zu finden. Dafür wurde zunächst die MALDI-TOF MS Methode mit relevanten Standardlipiden optimiert. Anschließend konnte sowohl das Phospholipidmuster des SP mit dem Spermien verglichen werden als auch die Spermienlipide von normalgewichtigen und fettleibigen Probanden. Durch diese Analyse konnte das Phosphatidylcholin/Lysophosphatidylcholin (PC/LPC)-Verhältnis, aber auch ein stark erhöhter Sphingomyelin (SM)-Anteil bei den fettleibigen Probanden als Qualitätsmarker gefunden werden. Des Weiteren wurden im Rahmen dieser Arbeit murine Spermien aus dem Caput und dem Cauda der Epididymis mittels MALDI-TOF MS analysiert und das Phospholipidmuster miteinander verglichen. Es konnte damit gezeigt werden, dass die murinen Spermien einen wesentlich höheren Anteil an Stearinsäureresten aufweisen, die humanen Spermien hingegen vor allem durch Palmitinsäurereste charakterisiert sind. In den Spermienmembranen aus dem Cauda und dem Caput gab es wesentliche Unterschiede im Phospholipidmuster. Spermienmembranen aus dem Caput besitzen einen hohen Anteil an PC und Phosphatidylethanolamin (PE). Die Spermienmembranen aus dem Cauda hingegen enthalten mehr SM, und auch einen höheren Anteil an LPCs und Formyl-LPC. Diese Arbeit konnte somit zeigen, dass die Reifungsprozesse in der Epididymis auch die Phospholipid-Zusammensetzung betreffen.

Fertilization is a process that generates the first cell of a new organism. In mammals, fertilization occurs in the female reproductive tract. The male gametes (spermatozoa) are rendered fertilization-competent only after they undergo capacitation and acrosome reaction (AR). The set of physiological changes, characterised by the acquisition of hyperactivated motility, that render the spermatozoa fertilization competent is known as capacitation. Using in vitro models, the complex intracellular signaling events mediating this process are still being understood. This thesis explores the role of protein tyrosine phosphorylation during capacitation using the golden hamster (Mesocricetus auratus) spermatozoa. The knowledge about the molecular components involved in capacitation, apart from enriching our understanding about a basic cellular process could also provide leads in the management of male (in)fertility. A comprehensive review on the perspectives of male reproduction, spermatogenesis, the structural features of a spermatozoon and sperm maturation, relevant to the content of the thesis is provided in Chapter-1 (General Introduction). Molecular mediators that initiate capacitation include cAMP, Ca2+and HCO3- ions. These signalling molecules regulate activities of protein kinases and phosphatases, which control the level of protein phosphorylation in spermatozoa. Capacitation-associated increase in protein phosphorylation, specifically protein tyrosine phosphorylation (PYP) has been demonstrated in a few species such as mouse, rat and human. The unique nature of PYP signaling during sperm capacitation has been exemplified by discoveries of several male germ cell-specific signalling molecules like soluble adenylate cyclase. However,molecular identities of tyrosine-phosphorylated proteins and their functional role during sperm capacitation are yet to be investigated in detail. In this context, the effect of modulating intracellular levels of signaling molecules upstream of protein phosphorylation was sought using pentoxifylline (PF), a cAMP phosphodiesterase inhibitor. Interestingly, PF-induced capacitation was associated with an early induction of tyrosine phosphorylation of proteins (45-80 kDa) localized to the mid piece of the sperm tail. Interestingly, the ultrastructural localization of tyrosine-phosphorylated proteins in the sperm tail by immunoelectron microscopy (IEM) revealed most intense immunolabelling in the fibrous sheath, followed by outer dense fibers (ODFs)and the axoneme. Data pertaining to the effect of PF on sperm capacitation and the associated protein-phosphorylation is presented in Chapter-2. Since PYP was determined to be extremely critical for hyperactivation in spermatozoa, the involvement of protein tyrosine kinases (PTKs) in this process was assessed using a specific PTK inhibitor, tyrphostin A47 (TP-47: EGFR-TK specific). The third chapter deals with the effect of tyrphostins on sperm capacitation and PYP. A dose-dependent inhibition by TP-47 of capacitation and principal piece associated-PYP of ~45-60 kDa proteins was observed. Interestingly, TP-47 treated-spermatozoa exhibited a circular motility pattern; when assessed for kinematic parameters, by computer aided sperm analysis, sperm showed lower values for key kinematic parameters as compared to the controls. While sperm viability in TP-47- treated samples was not affected, the ATP content reduced towards latter (4-5 h) part of culture as compared to the controls. When spermatozoa were treated with two other PTK inhibitors, tyrphostin AG1478 (EGFR-TK specific) and tyrphostin AG1296 (PDGFR-TK specific), they did not show any changes in kinematic parameters or PYP, indicating that the TP-47-effect was compound-specific. The fourth chapter of this thesis involves the molecular analysis of proteins hypo-tyrosine phosphorylated in the presence of TP-47, which started with the enrichment of sperm flagellar proteins that are tyrosine phosphorylated during capacitation, using various detergents. Detergent extractions established that most tyrosine-phosphorylated proteins were non-membranous in nature, which complemented the IEM data. Therefore, phosphoproteome analysis of the untreated and TP-47-treated sperm samples was performed. For this, protein extracts were subjected to 2D-PAGE-phosphotyrosine immunoblots. A 51 kDa spot and two 45 kDa spots, corresponding to the hypo-tyrosine phosphorylated spots, were analyzed by MS/MS. While peptides from the 51 kDa protein matched with tektin-2 (a microtubular protein), those of the 45 kDa spots matched with ODF-2 protein of the sperm flagellum. Validation of the presence of tektin-2 and ODF-2 protein and their tyrosine-phosphorylated forms on sperm capacitation in the hamster spermatozoa has also been performed. In addition to detailing the role of PYP in hamster sperm capacitation, this study revealed the identities of a few of these proteins, whose tyrosine phosphorylated status could be critical for optimal sperm flagellar bending, required for sperm hyperactivation. By understanding causes that lead to altered sperm function, for example, as observed with hamster spermatozoa, new insights could be achieved into molecular regulatory mechanisms that govern sperm function in clinical cases of non-obstructive male infertility in the human.