Taken together, the present studies showed that incubation of stallion sperm with different concentrations of X/XO can be used to induce defined levels of ROS resulting in sub-lethal oxidative stress. Oxidative stress was referred to as being sub-lethal, since plasma membrane and DNA integrity were not affected, whereas sperm motility and the mitochondrial membrane potential were reduced in a dose dependent manner. ROS accumulation in sperm can be detected using specific dyes like H2DCFDA, but only newly formed ROS that react with such dyes are visualized. ROS accumulation was predominantly detected in the sperm midpiece where the mitochondria are located. Sperm with increased levels of intracellular ROS exhibited a more rapid decrease in sperm motility and membrane intactness during refrigerated storage and after cryopreservation. Furthermore it was determined that increased intracellular ROS levels resulted in decreased survival (i.e.
membrane intact sperm) upon dilution in hypotonic saline solution. Moreover, reduced tolerance of sperm towards hypotonic stress pre-freeze correlated with decreased sperm cryosurvival. In the experimental setup used in the present studies addition of antioxidants did not significantly improve sperm motility and hypo-osmotic membrane resistance after exposure to induced oxidative stress and cryopreservation. Centrifugation for removal of X/XO seemed to result in reduced ROS levels and negative effects. Anyways, use of the X/XO-system for inducing defined ROS levels in sperm might be a useful tool for further studies on oxidative damage in sperm and effects of extenders supplements on sperm quality.
6 SUMMARY
Franziska Ertmer (2016):
Induced oxidative stress in stallion sperm: effects on osmotic resistance and cryosurvival
Reactive oxygen species (ROS), like the superoxide anion radical (∙O2
-), hydrogen peroxide (H2O2) and the hydroxyl radical (∙OH), are products of the physiological oxidative metabolism of sperm. In addition, when sperm samples are subjected to processing and storage, degenerative reactions may occur that give rise to formation of ROS. When imbalances between production and removal of ROS exist, ROS can accumulate and react with biomolecules, including membrane phospholipids, altering their structure and function.
This is referred to as oxidative stress, and is described to affect sperm motility as well as membrane and chromatin integrity.
The first aim of the studies described in this thesis was to use xanthine (X) and xanthine oxidase (XO) to induce defined levels of ROS and oxidative stress in stallion sperm, to validate presence of intracellular ROS accumulation, and examine effects of sub-lethal oxidative stress on sperm motility parameters, membrane and chromatin integrity, and tolerance towards hypo-osmotic stress. Sperm in extender supplemented with X/XO were incubated for 30 min at 37°C, after which samples were analyzed or used for refrigerated storage or cryopreservation. Sperm motility was analyzed using computer assisted sperm
analysis, whereas sperm mitochondrial membrane potential and plasma membrane intactness were evaluated by flow cytometric analysis of JC1- and SYBR/PI-stained samples, respectively. Chromatin intactness was evaluated using the sperm chromatin structure assay.
ROS accumulation was measured using flow cytometry as H2DCFDA-derived fluorescence in membrane intact sperm. It was confirmed that treatment with increasing X/XO-concentrations resulted in defined increasing levels of ROS accumulation. Microscopic observations revealed that accumulation of ROS (i.e. dyes reacting with them) occurred predominantly in the sperm midpiece. It was found that X/XO-treatment resulted in a dose-dependent decrease in sperm motility and sperm with high mitochondrial membrane potential. For the conditions used in the present studies, membrane and chromatin integrity were not affected by using up to 150 mU mL-1 XO, indicating that the induced oxidative stress was sub-lethal. Tolerance of sperm towards osmotic stress was determined by comparing membrane intactness after dilution in hypotonic saline solution. It was found that sperm membrane hypo-osmotic resistance decreased with increasing levels of sub-lethal ROS induced.
In the second part of this thesis, effects of different levels of induced ROS on sperm quality during refrigerated storage and cryopreservation were examined. Furthermore, it was determined whether hypo-osmotic tolerance pre-freeze correlated with sperm cryosurvival. It was found that during cooled storage sperm motility and osmotic resistance decreased, which occurred more rapidly after X/XO-treatment and higher intracellular ROS levels. Removal of X/XO from solution via centrifugation and replacement with fresh extender did result in decreased H2DCFDA-derived fluorescence intensities in membrane intact sperm to similar levels as detected in untreated samples. For the conditions used in the present studies, sperm membrane and chromatin integrity did not show drastic decreases during refrigerated storage,
indicating that the induced ROS levels were sub-lethal. Also in case of cryopreservation, the decrease in membrane intact and motile sperm was more pronounced after X/XO-treatment.
Using X/XO to induce defined levels of oxidative stress prior to cryopreservation, a correlation was found between sperm hypo-osmotic tolerance pre-freeze and cryosurvival.
The aim of the last part described in this thesis was to test whether antioxidant supplementation (α-tocopherol/vitamin E, catalase) or centrifugation clean-up are beneficial
for counteracting induced oxidative damage and increasing cryosurvival. Using the present setup, addition of catalase or vitamin E did not significantly improve percentages of membrane intact and motile sperm after induced oxidative stress pre-freeze and after cryopreservation. Removal of X/XO from solution via centrifugation and replacement with fresh extender did result in similar H2DCFDA-derived fluorescence intensities in membrane intact sperm as control levels.
Taken together, incubation of stallion sperm with X/XO can be used to induce defined levels of reactive oxygen species. Stallion sperm motility and hypo-osmotic resistance are negatively affected by exposure to sub-lethal levels of oxidative stress. Using X/XO to induce oxidative stress might be a useful tool for further studies on oxidative damage in sperm during processing and storage and testing effects of extenders supplements on sperm quality.
7 ZUSAMMENFASSUNG
Franziska Ertmer (2016)
Untersuchungen zur Induktion von oxidativem Stress und dessen Auswirkungen auf die osmotische Resistenz und Tiefgefrierfähigkeit von Hengstspermien
Reaktive Sauerstoffspezies (reactive oxygen species = ROS), wie das Superoxidanion (∙O2-), Wasserstoffperoxid (H2O2) und das Hydroxylradikal (∙OH), sind Produkte des physiologischen, oxidativen Metabolismus von Spermien. Zusätzlich können bei der Aufbereitung und Lagerung von Spermienproben schädigende Reaktionen auftreten, die zur Entstehung von ROS führen können. ROS können bei einer Imbalanz zwischen Produktion und Abbau akkumulieren und mit Biomolekülen, insbesondere Phospholipiden der Plasmamembran, reagieren und diese in ihrer Struktur und Funktion beeinträchtigen. Dieses Phänomen wird auch als oxidativer Stress bezeichnet, der die Spermienmotilität, sowie die Plasmamembran- und Chromatinintegrität beeinträchtigen kann.
Das Ziel des ersten Teils dieser Dissertation war es mittels Xanthin (X) und Xanthinoxidase (XO) definierte Mengen von ROS und oxidativem Stress in Hengstspermien zu induzieren, das Vorkommen von intrazellulärer ROS- Akkumulation zu überprüfen und Effekte von sublethalem, oxidativem Stress auf die Motilitätsparameter, die Plasmamembran-und Chromatinintegrität sowie auf die Toleranz von Hengstspermien gegenüber
hypoosmotischem Stress zu untersuchen. Die Spermienproben wurden in mit X/XO versetzten Verdünner für 30 Minuten bei 37°C inkubiert und anschließend entweder analysiert oder der gekühlten Lagerung oder Kryokonservierung zugeführt. Die Spemienmotilität wurde durch computer- assistierte Spermienanalyse ermittelt, wohingegen das mitochondriale Membranpotential und die Plasmamembranintegrität mittels flowzytometrischer Analyse von jeweils JC1- und SYBR/PI- gefärbten Spermienproben untersucht wurden. Die Chromatinintegrität wurde mit dem Spermien-Chromatin-Struktur-Assay (SCSA) ermittelt. Die Akkumulation von ROS wurde flowzytometrisch als H2DCFDA-abgeleitete Fluoreszenz in membranintakten Spermien bestimmt. Es wurde bestätigt, dass die Behandlung mit steigenden X/XO- Konzentrationen in definiert ansteigenden Mengen von ROS- Akkumulation resultiert. Die mikroskopischen Untersuchungen zeigten eine Akkumulation von ROS (bzw. den mit ROS reagierten Farbstoffen) überwiegend im Mittelstück der Spermien. Es wurde zudem festgestellt, dass eine Behandlung mit X/XO zu einer dosisabhängigen Verminderung der Anzahl motiler Spermien und Spermien mit einem hohen mitochondrialen Membranpotential führt. Unter den in dieser Studie verwendeten Bedingungen blieben die Plasmamembran- und Chromatinintegrität unbeeinflusst bei Verwendung von bis zu 150 mU mL-1XO. Dies deutet darauf hin, dass der induzierte oxidative Stress subletal ist. Die Toleranz von Spermien gegenüber hypotonem Stress wurde durch den Vergleich der Membranintaktheit nach Verdünnung mit hypotoner Kochsalzlösung bestimmt. Es konnte eine Verminderung der hypoosmotischen Resistenz der Spermien mit steigenden, subletalen Konzentrationen induzierter ROS festgestellt werden.
Im zweiten Teil dieser Dissertation wurden Effekte von verschiedenen induzierten ROS- Konzentrationen auf die Spermienqualität während gekühlter Lagerung und Kryokonservierung untersucht. Des Weiteren wurde die Korrelation zwischen der hypoosmotischen Toleranz der Spermien vor dem Einfrieren und deren Überlebensrate nach dem Auftauen bestimmt. Während gekühlter Lagerung zeigten die Spermienmotilität und die hypoosmotische Resistenz eine deutlichere Verminderung nach X/XO- Behandlung und mit steigenden intrazellulären ROS- Konzentrationen. Die Entfernung von X/XO aus der Lösung durch Zentrifugation und Ersatz mit frischem Verdünner, führte zu einem Abfall der H2DCFDA-abgeleiteten Fluoreszenzintensitäten in membranintakten Spermien auf ähnliche Niveaus wie in den unbehandelten Proben. Die Membran- und Chromatinintegrität zeigten unter den verwendeten Bedingungen keinen deutlichen Abfall während gekühlter Lagerung und mit steigenden X/XO- Konzentrationen. Daraus folgt, dass die induzierten ROS-Mengen als subletal bewertet werden können. Auch nach Kryokonservierung war die Reduktion von membranintakten und motilen Spermien deutlicher nach X/XO- Behandlung. Wenn definierte Mengen von oxidativem Stress mittels X/XO vor der Kryokonservierung induziert wurden, konnte eine Korrelation zwischen der hypoosmotischen Toleranz vor dem Einfrieren und der Überlebensrate der Spermien nach dem Auftauen festgestellt werden.
Das Ziel des letzten Teiles dieser Dissertation war es zu testen, ob die Supplementation von Antioxidantien (α-Tocopherol/Vitamin E, Katalase) oder eine Aufbereitung mittels Zentrifugation geeignet sind, um der induzierten, oxidativen Schädigung entgegen zu wirken und die Überlebensrate der Spermien nach dem Auftauen zu erhöhen. Mit dem hier verwendeten Versuchsaufbau konnte durch Zugabe von Katalase oder Vitamin E nach induziertem oxidativem Stress, der Anteil membranintakter und motiler Spermien vor
dem Einfrieren und nach Kryokonservierung nicht signifikant erhöht werden. Die Entfernung von X/XO aus der Lösung mittels Zentrifugation und Ersatz mit frischem Verdünner resultierte in ähnlichen Niveaus von H2DCFDA-abgeleiteten Fluoreszenzintensitäten in membranintakten Spermien, wie in unbehandelten Proben.
Zusammenfassend kann die Inkubation von Hengstspermien mit X/XO benutzt werden, um definierte Mengen reaktiver Sauerstoffspezies zu induzieren. Die Motilität und hypoosmotische Resistenz von Hengstspermien werden durch subletale Mengen von oxidativem Stress negativ beeinflusst. Die Verwendung von X/XO zur Induktion von oxidativem Stress kann eine geeignete Methode für weiterführende Studien von oxidativer Spermienschädigung während Aufbereitung und Lagerung, sowie für die Prüfung von Effekten von Verdünnerzusätzen auf die Spermienqualität, darstellen.
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