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Univ., Diss., 2009--Marburg.

In sperm chromatin, DNA exists in a highly condensed state reaching a final volume roughly twenty times that of a somatic nucleus. For the vast majority (>90%) of sperm DNA in mammals, somatic-like histones are first replaced by transition proteins which in turn are replaced by arginine-rich protamines. This near crystalline organization of the DNA in mature sperm is thought crucial for both the transport and protection of genetic information since all DNA repair mechanisms are shut down. Recent studies show that increased DNA damage is linked to dysfunctions in replacing histones with protamines resulting in mispackaged DNA. This increased DNA damage correlates not only to infertility but also impacts normal embryonic development. This damage is currently poorly characterized, but is known to involve oxidative base damage by reactive oxygen species (ROS). Using a variety of biophysical methods, the effect of DNA condensation by polycations on the on free radical access and DNA damage in the packaged state was investigated. In Chapter 2, gel electrophoresis was used to quantify the ability of free radicals to damage both unpackaged and packaged DNA. DNA condensed by polycations shows significantly reduced levels of indirect damage from exposure to free radicals. Combining previous work on packaging density, it is also shown that differences in the packaged state, even by a few Angstroms, can result in significantly different degrees of damage to the DNA. In Chapter 3, we investigate the effects of protamine concentration on the ability to condense and protect DNA. Insufficient protamination is known to be a potential source of protamine dysfunction in mammalian sperm chromatin. Using gel retardation assays and UV-Vis studies, we examined the ability for DNA to condense with protamine at varying nitrogen to phosphate (N:P) charge ratios. Initial results on damage as a function of N:P are also discussed. Future work will more quantitatively determine the interrelationship between DNA packaging densities and the resulting accessibility of DNA to reactive oxygen species (ROS).

Heparin is an acid mixture of glycosaminoglycans with high negative charge density which naturally occurs in human body. Due to its ability to bind antithrombin III and thus accelerate inhibition of thrombin it has anticoagulant effect. This is abundantly used in clinical practice for operations, in case of embolia or heart-attacks. Protamine is a mixture of small basic peptides, which is used in clinical practice as a heparin antidote. The interaction between heparin and protamine is electrostatic and is also used for determination of heparin in human plasma or blood using affinity methods. In my study it was found that if protamine and heparin are mixed in one vial, a complex is formed. Its resulting charge depends on concentration ratio of protamine and heparin. On the other hand, in case the protamine is injected as a sample and heparin is added to background electrolyte as a protein-binding ligand, it is possible to determine heparin from decreasing protamine peak area. Because of the complexity of protamine-heparin interaction, tetraarginine was used as structurally close model of protamine to increase repeatability of measurements. The method for determination of heparin was optimalised. It uses 20 mM or 60 mM ortho-phosphoric acid as background electrolyte, 1 mg/mL solution of tetraarginine...

Le spermatozoïde des animaux à reproduction sexuée est une cellule extrêmement spécialisée, dont la chromatine très particulière est le siège de nombreux remodelages tant lors de la gamétogenèse que lors de la formation du zygote. Chez D. melanogaster comme chez les mammifères, lors de la spermiogenèse, les histones qui condensent l'ADN sont remplacées par des petites protéines basiques spécifiques du noyau spermatique : les protamines. Cette architecture est stabilisée par des liaisons disulfures. Lors de la fécondation, ces protéines sont éliminées du noyau paternel, qui réincorporent des histones pour former une chromatine fonctionnelle. Toutefois, les mécanismes régissant la mise en place et l'enlèvement des ponts disulfures et des protamines sont inconnus chez la Drosophile.Au cours de ma thèse, j'ai démontré l'importance de deux thiorédoxines sexuelles pour la reproduction.D'une part, j'ai pu montrer que DHD, qui est une thiorédoxine strictement maternelle, est essentielle à l'éviction des protamines de la chromatine paternelle lors de la fécondation. Sans cette protéine essentielle, la décondensation du noyau mâle n'a pas lieu, les protamines ne sont pas enlevées et le développement zygotique ne peut pas avoir lieu. Cette thiorédoxine est directement responsable de la réduction des liaisons disulfures qui stabilisent la chromatine spermatique.D'autre part, j'ai démontré que TrxT, une thiorédoxine exclusivement testiculaire, est nécessaire au bon déroulement de la spermiogenèse. Sans cette protéine, les spermatides subissent des dommages à l'ADN et sont éliminées.Ce travail met en évidence les rôles essentiels des thiorédoxines sexuelles pour la reproduction / In animal sexual reproduction, spermatozoon is a very specialized cell. Its very peculiar chromatin is remodeled both during spermiogenesis and fertilization. During mammalian and drosophilian spermiogenesis, histones involved in DNA condensation are replaced with sperm specific small nuclear basic proteins : the protamines. This sperm specific architecture is stabilized by disulfide bonds. At fertilization,protamines are removed from the male nucleus and maternally-provided histones are incorporated to form a functional paternal chromatin. However, the mecanisms involved in the incorporation and the removal of protamines of their disulfide bonds are unknown in Drosophila.During my PhD, I demonstrated that two sexual thioredoxins are important for spermiogenesis and fertilization in D. melanogaster. In one hand, I showed that DHD, a female specific thioredoxin, is essential for protamine eviction at fertilization. Without this major protein, male nucleus does not decondense, protamines are not removed from sperm chromatin and zygotic development does not occur. Besides, I demonstrated that DHD is directly responsible for the reduction of the disufide bonds which stabilize sperm chromatin.On the other hand, I showed that TrxT, a testis-specific thioredoxin, is needed for spermiogenesis. Without this protein, DNA damages appear on spermatid nuclei, and those spermatozoon are then eliminated during spermatogenesis.This work highlights that drosophilian sex-specific thioredoxins are essential for sexual reproduction success

Les gènes du chromosomeX sont majoritairement inactivés au cours de la méiose mâle. Chez la souris, seulement 6% d’entre eux sont réactivés au cours des stades post-méiotiques. Parmi eux le gène Uba1X codant pour l’enzyme activatrice de l'ubiquitine, UBA1 qui produit trois transcrits dont deux sont ubiquitaires mais le troisième prédomine dans les cellules post¬-méiotiques : les spermatides. Nos travaux montrent que les 5’UTR, seul différence entre ces trois transcrits, déterminent la localisation et la dose relative des isoformes nucléaire et cytoplasmique de la protéine UBA1. Nous avons mis en évidence chez la souris que le transcrit spermatide-spécifique code pour l'isoforme nucléaire, exprimée fortement dans les spermatides suggérant un rôle de la protéine UBA1 dans la dégradation des histones lors du remodelage chromatinien. Nous avons détecté deux mutations dans la région spermatide-spécifique du gène : une délétion de 13pb et une transition G>A, chacune portée par un patient infertile, et non retrouvée dans notre population témoin. Les analyses ont montré que la délétion de 13pb induit un épissage anormal du transcrit spermatide-spécifique et que la transition G>A pouvait réduire le taux d’expression du transcrit spermatide-spécifique. Ces mutations pourraient induire l'infertilité des deux patients. En parallèle nous avons pu démontrer que les mutations dans le gène codant pour la protamine PRM1 décrites dans la littérature c.102G>T et c.-107G>C ne sont pas liées à l'infertilité masculine et que le variant est un polymorphisme fréquemment retrouvé dans la population congolaise. / The majority of genes on the X chromosome are repressed during meiosis and only 6% of them are expressed in post meiotic germ cells. One of these genes is Ubal, encoding the ubiquitin-activating enzyme UBA1. Ubal produces three different transcripts, two of which are ubiquitously expressed while the third is predominant in the post meiotic germ cells: the spermatids. Our study shows that the 5’UTR, which is the only difference between these transcripts, determines the localization and the relative dose of the nuclear and cytoplasmic isoform of the UBA1 protein. The spermatid-specific transcript encodes for the nuclear isoform in the spermatids in the mouse suggesting that the UBA1 protein is implicated in chromatin remodeling during spermiogenesis. We have detected two mutations in the spermatid-specific region of the UBA1 gene in two infertile men: a deletion of 13bp and a G>A transition, neither of which was found in our cohort of fertile men. The deletion of 13bp diminishes the correct splicing of the spermatid-specific transcript and that the G>A transition may reduce expression of the spermatid-specific transcript. These results show that the UBA1 gene is involved in spermiogenesis, and reactivated in spermatids by its spermatid-specific transcript and that the mutations identified may induce infertility by reducing UBA1 levels in spermatids. We have also demonstrated that two variants described in the protamine codant gene PRM1C.102G>T and c.-107G>C are clearly not associated with male infertility and that the c.-107G>C is polymorphism frequently found in the congolese population.

The main objective of the study was to understand the mode of interaction of protamine (Ptm), a cationic antibacterial peptide from fish milt on the Gram negative bacterial envelope. The present study was designed to resolve the question of Ptm translocation across the seemingly impermeable Gram negative cell envelope. The Gram negative pathogen Pseudomonas aeruginosa was studied as an example of a microorganism that is Ptm-sensitive but doesn’t lyse even at bactericidal concentrations. Acquired resistance to Ptm was induced in P. aeruginosa by continuous sub-culturing in nutrient rich media containing increasing concentrations of Ptm. Alterations in bacterial surface charge, LPS composition, cell morphology and Ptm localisation on acquiring resistance were also examined. Expression of outer membrane proteins significantly decreased as P. aeruginosa acquired resistance to Ptm. OprF, the major porin in P. aeruginosa was found to be stably expressed in control, revertant (Ptm-Rev) and resistant (Ptm-Res) groups. No change in expression of efflux proteins was observed as a result of induced Ptm resistance, indicating that efflux is not among the Ptm resistance mechanisms at least in P. aeruginosa. OprM, which is part of the major efflux system (MexAB-OprM) in P. aeruginosa, was found to be down-regulated in Ptm-resistant P. aeruginosa. Another outer membrane protein down-regulated in Ptm-resistant P. aeruginosa was found to be petidyl-prolyl cis trans isomerase (PPIase) which plays a major role in proper folding and maturation of channel proteins in the outer membrane. Among the sarcosinate soluble proteins, DNA dependent RNA polymerase ? and ?’ subunits were found to be down-regulated in Ptm-resistant group indicating lower transcription levels in them. Lipopolysaccharide (LPS) from the three groups of P. aeruginosa under study was isolated and separated by SDS-PAGE. LPS composition of Ptm-Res P. aeruginosa was found to be significantly different from that of the control and Ptm-Rev but was found to be similar with that of LPS from O-antigenic mutant (A+B-, which possessed only A band structures). Comparison of the zetapotential of control, Ptm-Rev and Ptm-Res P. aeruginosa, proved that electrostatic shielding was coincidental in acquired resistance to Ptm in P. aeruginosa. The MIC of the parent strain of P. aeruginosa (A+B+) and the O-antigenic mutants (A+B-, A-B+ and A-B-) were found to be the same which may be indicating that alterations in O-antigenic components alone cannot contribute to Ptm resistance. Effects of Ptm treatment on morphologies of E. coli, S. typhimurium and P. aeruginosa whole cells and spheroplasts were also studied using transmission immuno-electron microscopy. Condensation of cytoplasmic contents was observed when whole cells and spheroplasts were treated with Ptm. Also, Ptm-treated cells and spheroplasts were stained with colloidal gold-labelled antibodies against Ptm to determine distribution within the target cells. It was quite evident that Ptm internalised in whole cells and spheroplasts without lysis and was found to be concentrated in the cytoplasm. Morphological changes observed in Ptm-Rev P. aeruginosa when exposed to Ptm were comparable with that of the control. Condensation of cytoplasmic contents was not observed in Ptm-Res P. aeruginosa when challenged with Ptm. Most of the Ptm was localized at or near the outer membrane of Ptm-treated Ptm-Res P. aeruginosa, indicating decreased outer membrane permeability. Results obtained from these experiments confirm that the resistance to Ptm observed in P. aeruginosa is at the very least, coincidental with the pleiotropic mutations involving change in outer surface including change in LPS composition, loss of porins and or alterations of porin size in OprF.

Protamine, a set of small basic proteins (P1 and P2), play a key role in compacting and protecting the DNA in sperm. As such, the structure of how P1 and P2 bind to DNA and potentially themselves and each other is of interest to several fields including forensics. In forensic DNA analysis, protamine binding of DNA is taken advantage of in the “differential extraction” procedure in which a sample that contains sperm and non-sperm cells can have DNA from the two different cell types separated and extracted at different points thus preventing a mixture of DNA. A key component of this greater structure and what makes the differential extraction functional are the disulfide bonds formed by protamine. So as a first step to elucidating the protamine-DNA complex, methods to analyze human protamine via 2D-Liquid Chromatography-Mass Spectrometry (2D-LC-MS) were developed in the hopes they could be used for disulfide bond mapping. Methods and multiple strategies for digestion, 2D-LC-MS were investigated and developed using chum salmon protamine. Digestion strategies were developed for Chymotrypsin and Lys-C, Trypsin or Arg-C with incubation times and substrate:enzyme mass ratios optimized. Various “trap and elute” 2D-chromatography configuration were tested for analysis intact and digested protein. Using H2O with 2% NH4OH as the loading mobile phase and H2O and Acetonitrile both with 0.5% formic acid as the eluting mobile phases with the first dimension column being an HLB (Hydrophilic Lipophilic Balance) 2.1 x 30 mm column and the second dimension being an C18 2.1 x 100 mm was found to produce the highest signal.

The impacts of the paternal genome and proteins transferred to the oocyte through spermatozoa cannot be neglected during mammalian embryonic development. Studies over the past 40 years suggest that sperm chromatin alterations (such as DNA fragmentation induced by either chromatin condensation errors, apoptosis and/or oxidative stress) might be negatively associated with fertilization and early embryonic development [1, 2], [3] [4].However, precise molecular mechanisms by which sperm chromatin integrity and sperm proteins impact early embryonic development still remain unclear. Therefore, the objectives of this study were 1) determine DNA fragmentation induced by apoptosis its relationship with male fertility in spermatozoa from bulls with varying fertility, and 2) identify expression dynamics of Protamine 1 and examine chromatin structure in spermatozoa from bulls with varying fertility. To accomplish our goals we determined 1) the DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) as well as 2) the expression and localization of Protamine 1 (PRM1) with chromatin condensation and protamination in sperm from bulls with varying fertility. Our results demonstrated that the most relevant fertility markers might be the percentage of necrotic spermatozoa detected by flow cytometry and live spermatozoa determined via eosin-nigrosin staining and that there was no relationship between apoptosis and male fertility. While BCL-2 was not expressed, BAX was identified in bovine spermatozoa. However, the expression of BAX did not differ among groups. In addition, defective chromatin condensation and protamination errors were significantly increased in sperm from low fertility bulls, while the expression of PRM1 was significantly abundant in high fertility bulls. Bull fertility was negatively correlated with protamination errors and defective chromatin condensation, and it was positively correlated with the expression of PRM1. We concluded that defective sperm DNA condensation, not abortive apoptosis, might be the major reason of male infertility in bulls and that sperm chromatin stability differs among bulls with varying fertility. Improper chromatin packaging during spermatogenesis might be caused by the limited expression and/or mislocalization of PRM1. Thus, inadequate chromatin dynamics were associated with bull infertility, which might lead improper fertilization.

To clarify the role of charge in protein movement across the pulmonary endothelial barrier, we simultaneously measured the permeability-surface area product (PS) for native [isoelectric point (pI) 4.4-5.1] and cationic (pI 7.2-8.0) albumin in isolated rabbit lungs perfused with and without protamine sulfate. We focused our measurement on the initial (endothelial) barrier by using a technique that is based on the very rapid (3 min) uptake of tracer. This allowed us to distinguish the charge properties of the endothelium separate from other barriers. In control studies, PS was greater for cationic than for native albumin (8.67 ± 0.93 vs. 2.55 ± 0.20 x 10-2 ml · min-1 · g dry lung-1). In the presence of 1 mg/ml protamine sulfate, cationic albumin permeability was not different from control (7.34 ± 0.49 x 10-2 ml · min-1 · g dry lung-1), whereas PS for anionic albumin increased to 8.82 ± 1.32 x 10-2 ml · min-1 · g dry lung-1. Thus the protamine sulfate eliminated the difference between native and cationic albumin PS. This selective increase in anionic albumin permeability is presumably due to the cation, protamine sulfate, binding to the anionic charges on the endothelium and reducing the anionic charge-charge repulsion. If protamine sulfate had produced a general endothelial injury, the PS for both albumins would have increased. Our results suggest that the normal pulmonary endothelium is an anionic charge barrier restricting the transcapillary movement of negatively charged molecules.