The effect of superoxide dismutase mimic on mitochondrial and functional state of human spermatozoa

Neplodnost je globalni problem u svetu. Uzroci i frekvenca različitih oblika neplodnosti variraju, a novije studije pokazuju da reaktivne vrste kiseonika (ROS) i azota (RNS), superoksid (O2 •─) i azot oksid (NO) pre svega, igraju važnu ulogu, posebno kod neplodnosti muškaraca. Pokazano je da su mitohondrije ne samo mesto visoke produkcije reaktivnih vrsta, već organele od čijeg funkcionog statusa zavisi, odnosno sa njim pozitivno korelira, fertilizacioni potencijal i kvalitet humanih spermatozoida. Međutim, molekulska osnova delovanja ROS/RNS na funkcionost spermatozoida, posebno na funkcioni status mitohondrija, još uvek je nepoznata. Cilj doktorske disertacije je da se ispitaju efekti modulacije redoks statusa spermatozoida na ključne parametre koji determinišu funkcioni status, odnosno aktivnost mitohondrija, kao i kvalitet spermatozoida, podjednako na molekulskom i strukturnom nivou. Iz tih razloga, redoks status spermatozoida je specifično moduliran pentaazamakrocikličnim Mn(II) mimetikom SOD (engl. superoxide dismutase), M40403, koji selektivno uklanja O2 •─ i indirektno modulira bioraspoloživost NO. U cilju rasvetljavanja signalnih puteva regulacije fertilizacionih procesa, naročito eventualnog uticaja NO na poboljšanje funkcionog statusa mitohondrija i fertilizacionih svojstava spermatozoida, ispitivali su se sledeći parametri: pokretljivost spermatozoida, koncentracija NO elektrohemijski, korišćenjem NO specifične elektrode, kao i korišćenjem fluorescentne probe specifične za NO (Daf2-DA, diamino-fluorescein-2- acetat); lokalizacija produkcije NO u spermatozoidima; endogena produkcija NO praćenjem ekspresije izoformi NOS (engl. NO synthase) – endotelske (eNOS), neuronalne i inducibilne; enzimi ključni za produkciju O2 •─ (NADPH oksidaza) i njegovo uklanjanje (CuZnSOD, engl. cooper zink SOD i MnSOD, engl. manganese SOD); ekspresija enzima odgovornih za uklanjanje H2O2, nastalog aktivnošću SOD i SOD mimetika, katalaze i GSH-Px (engl. glutathione peroxidase); mitohondrijalna dinamika i metabolički status, praćenjem mitohondrijalnog membranskog potencijala (katjonska fluorescentna proba Mitotracker Green FM) i ekspresija kompleksa oksidativne fosforilacije (kompleks I, kompleks III, kompleks IV, citohrom c, citohroma b i ATP sintaza)...Infertility is a global problem worldwide. The frequency and origin of different forms of infertility vary, but recent studies showed that reactive oxygen species (ROS) and reactive nitrogen species (RNS), first of all, superoxide (O2 •─) and nitric oxide (NO), play an important role, especially in male infertility. It has been shown that mitochondria play essential role in regulation of reactive species production. Moreover, mitochondrial functional status in spermatozoa positively correlates with fertilizing potential and quality of human sperm. However, the molecular basis of ROS/RNS action on sperm functionality, especially on the functional status of sperm mitochondria, is still unknown. The aim of the dissertation was to examine the effects of redox status modulation in human spermatozoa on the key parameters determining functional status (activity) of mitochondria and sperm quality, at the molecular and structural levels. The redox status of the sperm is specifically modulated using pentaazamacrocyclic Mn(II) mimic SOD (eng. superoxide dismutase), M40403, that selectively removes O2 •─ and indirectly modulates the bioavailability of NO. In order to shed more light on the signalling pathways underlying fertilizing processes, particularly possible beneficial effects of NO on mitochondria functional status and sperm fertilizing properties, the following parameters were examined: sperm motility; concentration of NO, electrochemically, using NO specific electrodes, as well as using fluorescent probes specific for NO (Daf2-DA, fluorescein-diamino-2-acetate); localization of NO production in the spermatozoa; endogenous NO production by determining the expression of NOS isoforms (NO synthase) – endothelial (eNOS), neuronal and inducible; enzymes essential for production of O2 •─ (NADPH oxidase) and its removing (CuZnSOD, cooper zinc SOD and MnSOD, manganese SOD); expression of enzymes removing H2O2, generated by activity of SOD and SOD mimic, catalase and glutathione peroxidase (GSH-Px); mitochondrial dynamic and metabolic status, using cationic fluorescent probe Mitotracker Green FM for monitoring mitochondrial membrane potential and finally the expression of complexes of the oxidative phosphorylation (Complex I, Complex III, Complex IV, cytochrome c, cytochrome b and ATP synthase)..

The effect of superoxide dismutase mimic on mitochondrial and functional state of human spermatozoa

Neplodnost je globalni problem u svetu. Uzroci i frekvenca različitih oblika neplodnosti variraju, a novije studije pokazuju da reaktivne vrste kiseonika (ROS) i azota (RNS), superoksid (O2 •─) i azot oksid (NO) pre svega, igraju važnu ulogu, posebno kod neplodnosti muškaraca. Pokazano je da su mitohondrije ne samo mesto visoke produkcije reaktivnih vrsta, već organele od čijeg funkcionog statusa zavisi, odnosno sa njim pozitivno korelira, fertilizacioni potencijal i kvalitet humanih spermatozoida. Međutim, molekulska osnova delovanja ROS/RNS na funkcionost spermatozoida, posebno na funkcioni status mitohondrija, još uvek je nepoznata. Cilj doktorske disertacije je da se ispitaju efekti modulacije redoks statusa spermatozoida na ključne parametre koji determinišu funkcioni status, odnosno aktivnost mitohondrija, kao i kvalitet spermatozoida, podjednako na molekulskom i strukturnom nivou. Iz tih razloga, redoks status spermatozoida je specifično moduliran pentaazamakrocikličnim Mn(II) mimetikom SOD (engl. superoxide dismutase), M40403, koji selektivno uklanja O2 •─ i indirektno modulira bioraspoloživost NO. U cilju rasvetljavanja signalnih puteva regulacije fertilizacionih procesa, naročito eventualnog uticaja NO na poboljšanje funkcionog statusa mitohondrija i fertilizacionih svojstava spermatozoida, ispitivali su se sledeći parametri: pokretljivost spermatozoida, koncentracija NO elektrohemijski, korišćenjem NO specifične elektrode, kao i korišćenjem fluorescentne probe specifične za NO (Daf2-DA, diamino-fluorescein-2- acetat); lokalizacija produkcije NO u spermatozoidima; endogena produkcija NO praćenjem ekspresije izoformi NOS (engl. NO synthase) – endotelske (eNOS), neuronalne i inducibilne; enzimi ključni za produkciju O2 •─ (NADPH oksidaza) i njegovo uklanjanje (CuZnSOD, engl. cooper zink SOD i MnSOD, engl. manganese SOD); ekspresija enzima odgovornih za uklanjanje H2O2, nastalog aktivnošću SOD i SOD mimetika, katalaze i GSH-Px (engl. glutathione peroxidase); mitohondrijalna dinamika i metabolički status, praćenjem mitohondrijalnog membranskog potencijala (katjonska fluorescentna proba Mitotracker Green FM) i ekspresija kompleksa oksidativne fosforilacije (kompleks I, kompleks III, kompleks IV, citohrom c, citohroma b i ATP sintaza)...Infertility is a global problem worldwide. The frequency and origin of different forms of infertility vary, but recent studies showed that reactive oxygen species (ROS) and reactive nitrogen species (RNS), first of all, superoxide (O2 •─) and nitric oxide (NO), play an important role, especially in male infertility. It has been shown that mitochondria play essential role in regulation of reactive species production. Moreover, mitochondrial functional status in spermatozoa positively correlates with fertilizing potential and quality of human sperm. However, the molecular basis of ROS/RNS action on sperm functionality, especially on the functional status of sperm mitochondria, is still unknown. The aim of the dissertation was to examine the effects of redox status modulation in human spermatozoa on the key parameters determining functional status (activity) of mitochondria and sperm quality, at the molecular and structural levels. The redox status of the sperm is specifically modulated using pentaazamacrocyclic Mn(II) mimic SOD (eng. superoxide dismutase), M40403, that selectively removes O2 •─ and indirectly modulates the bioavailability of NO. In order to shed more light on the signalling pathways underlying fertilizing processes, particularly possible beneficial effects of NO on mitochondria functional status and sperm fertilizing properties, the following parameters were examined: sperm motility; concentration of NO, electrochemically, using NO specific electrodes, as well as using fluorescent probes specific for NO (Daf2-DA, fluorescein-diamino-2-acetate); localization of NO production in the spermatozoa; endogenous NO production by determining the expression of NOS isoforms (NO synthase) – endothelial (eNOS), neuronal and inducible; enzymes essential for production of O2 •─ (NADPH oxidase) and its removing (CuZnSOD, cooper zinc SOD and MnSOD, manganese SOD); expression of enzymes removing H2O2, generated by activity of SOD and SOD mimic, catalase and glutathione peroxidase (GSH-Px); mitochondrial dynamic and metabolic status, using cationic fluorescent probe Mitotracker Green FM for monitoring mitochondrial membrane potential and finally the expression of complexes of the oxidative phosphorylation (Complex I, Complex III, Complex IV, cytochrome c, cytochrome b and ATP synthase)..

Level of NO/nitrite and 3-nitrotyrosine in seminal plasma of infertile men: Correlation with sperm number, motility and morphology.

Nitric oxide (NO) is a signaling molecule responsible for initiation of molecular events that influence sperm functionality in a concentration-dependent manner. It is still not fully understood how seminal plasma NO contributes to sperm pathologies. The aim of this study was to elucidate whether and how NO is implicated in etiology of different sperm abnormalities. To this end we determine NO, nitrite and 3-nitrotyrosine (3-NT) content in seminal plasma of infertile men with specific pathologies (terato-, oligoterato- and oligoasthenoteratospermia) and relate it to infertile normospermic samples. To gain further understanding of NO metabolism in seminal plasma we determine protein expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). Here we show that NO, nitrite and 3-NT levels in seminal plasma of men with suboptimal semen parameters are significantly lower compared to normospermic men. An increase in protein expression of eNOS and no change in protein expression of iNOS is observed in men with sperm pathologies. Association of seminal plasma 3-NT level with functional sperm parameters is observed - positive correlation with sperm count, motility and morphology in normospermia, teratospermia and oligoteratospermia, as well as negative correlation with sperm morphology and motility in oligoasthenoteratospermia. Present study revealed that suboptimal seminal plasma NO content is found in all examined sperm pathologies. This result unequivocally shows the importance of NO for sperm function and involvement of suboptimal NO level in etiology of sperm abnormalities. Lower seminal plasma 3-NT level and its significant association with sperm parameters, found in pathologies, strongly indicates that protein nitration is important for spermatozoa function and that failure to establish this post-translational protein modification might be involved in etiology of sperm abnormalities. According to our results, NO measurement can discriminate infertile men with sperm pathologies from infertile normospermic men but is not indicative of a specific type of sperm pathology

Correlation between Sperm Parameters and Protein Expression of Antioxidative Defense Enzymes in Seminal Plasma: A Pilot Study

Background. Semen analysis is the cornerstone in the evaluation of male (in) fertility. However, there are men with normal semen tests but with impaired fertilizing ability, as well as fertile men with poor sperm characteristics. Thus, there is rising interest to find novel parameters that will help to predict and define the functional capacity of spermatozoa. Methods. We examined whether there is a correlation between semen parameters (count, progressive motility, and morphology) and protein expression/activity of antioxidative defense enzymes in seminal plasma from 10 normospermic subjects. Results. Sperm progressive motility was in positive correlation with seminal plasma protein expression of both superoxide dismutase (SOD) isoforms (MnSOD and CuZnSOD) and catalase. Also, positive correlation was observed between sperm count and MnSOD protein expression, as well as between sperm morphology and protein expression of catalase in seminal plasma. In contrast, protein expression of glutathione peroxidase was not in correlation with any sperm parameter, while its activity negatively correlated with sperm morphology and motility. Conclusions. These data suggest that evaluation of protein expression of antioxidative defense enzymes in seminal plasma might be of importance in the evaluation of male fertility status and that could be used as an additional biomarker along with classic semen analysis in assessment of semen quality.Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173054]; COST {[}BM1203

Evaluation of the antioxidative enzymes in the seminal plasma of infertile men: Contribution to classic semen quality analysis.

Protein expression/activity of antioxidative defense enzymes (AD) in seminal plasma of fertile men might be used as biomarkers of male fertility status. To test this concept, the present study examined the semen parameters of males among 14 normal idiopathic (normozoospermia) and 84 subnormal (teratozoospermia, oligoteratozoospermia, oligoasthenoteratozoospermia) infertile individuals\. We investigated levels of protein expression/activity of Cu, Zn superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase and glutathione peroxidase (GSH-Px), their association with functional sperm parameters, as well as their potential to serve as biomarkers of specific sperm pathologies. Although the activity of CuZnSOD and protein expression of catalase were significantly correlated with several sperm parameters, underlying their potential role in etiology of various sperm abnormalities, investigation of their potential usefulness as a biomarker of semen quality showed that these AD enzymes could not distinguish subtle differences between various sperm pathologies. In contrast, GSH-Px activity was decreased in all groups with sperm pathologies and was a very good indicator of aberrations in functional sperm parameters, explaining up to 94.6% of infertility cases where functional sperm parameters were affected. Therefore, assessment of GSH-Px activity showed the potential to discriminate between infertile males with normal and subnormal semen characteristics and may prove useful in the evaluation of male (in)fertility. Abbreviations: AD: antioxidative defense; Cu, Zn SOD: copper, zinc superoxide dismutase; GSH-Px: glutathione peroxidase; MnSOD: manganese superoxide dismutase; NS: normospermia; OATS: oligoasthenoteratozoospermia; OTS: oligoteratozoospermia; ROC: receiver operating characteristic; ROS: reactive oxygen species; TS: teratozoospermia; WHO: world health organization

Collagen-induced arthritis in Dark Agouti rats as a model for study of immunological sexual dimorphisms in the human disease

Collagen-induced arthritis (CIA) is a frequently used animal model of rheumatoid arthritis, human autoimmune disease that exhibits clear sex bias in incidence and clinical course. Female Dark Agouti rats immunized for CIA showed also greater incidence and higher arthritic score than their male counterparts. The study investigated sex differences in mechanisms controlling the primary immune responses in draining lymph nodes (dLNs), as a factor contributing to this dimorphism. The higher frequencies of CD4 + CD25 + Foxp3- cells, presumably activated effector T (Teff) cells, and IL-17+, IFN-gamma + and IL-17 + IFN-gamma + T cells were found in female compared with male rat dLNs. However, the frequency of CD4 + CD25 + Foxp3 + T regulatory cells (Treg) did not differ between sexes. Thus, CD4 + Teff cells/Treg ratio, and IL-17 + T cells/Treg and IFN-gamma + T cells/Treg ratios were higher in female than in male rats, and among them was found lower frequency of PD-1+ cells. This suggested less efficient control of (auto)immune Th1/Th17 cell responses in female rat dLNs. On the contrary, the frequency of IL-4 + T cells was lower in female than in male rat dLNs. Consistently, the ratio of serum levels of collagen-specific IgG2a (IFN-gamma-dependent, with an important pathogenic role in CIA) and IgG1 (IL-4-dependent) was shifted towards IgG2a in female compared with male rats. As a whole, the study suggests that sexual dimorphism in the control of T cell activation/polarization could contribute to sex bias in the susceptibility to CIA. Moreover, the study advises the use of animals of both sexes in the preclinical testing of new drugs for rheumatoid arthritis

Manganese (II) pentaazamacrocyclic SOD mimic improves functional status of human sperm mitochondria: involvement of iNO

nul

Manganese (II) pentaazamacrocyclic SOD mimic improves functional status of human sperm mitochondria: involvement of iNO

nul

Is Manganese (II) Pentaazamacrocyclic Superoxide Dismutase Mimic Beneficial for Human Sperm Mitochondria Function and Motility?

Mitochondria play an important role in sperm cell maturation and function. Here, we examined whether (and how) changes in sperm redox milieu affect the functional status of sperm mitochondria, that is, sperm functionality. Compared with the control, incubation in Tyrode's medium for 3 h, under noncapacitating conditions, decreased sperm motility, the amount of nitric oxide ((NO)-N-center dot), the number of MitoTracker (R) Green FM (MT-G) positive mitochondria, and the expression of complexes I and IV of the mitochondrial respiratory chain. In turn, superoxide dismutase (SOD) mimic (M40403) treatment restored/increased these parameters, as well as the expression of endothelial nitric oxide synthase, manganese SOD, and catalase. These data lead to the hypothesis that M40403 improves mitochondrial functional state and motility of spermatozoa, as well as (NO)-N-center dot might be involved in the observed effects of the mimic. Antioxid. Redox Signal. 18, 170-178.Ministry of Education and Science of the Republic of Serbia [173054]; University of Erlangen-Nurember

Is Manganese (II) Pentaazamacrocyclic Superoxide Dismutase Mimic Beneficial for Human Sperm Mitochondria Function and Motility?

Mitochondria play an important role in sperm cell maturation and function. Here, we examined whether (and how) changes in sperm redox milieu affect the functional status of sperm mitochondria, that is, sperm functionality. Compared with the control, incubation in Tyrode's medium for 3 h, under noncapacitating conditions, decreased sperm motility, the amount of nitric oxide ((NO)-N-center dot), the number of MitoTracker (R) Green FM (MT-G) positive mitochondria, and the expression of complexes I and IV of the mitochondrial respiratory chain. In turn, superoxide dismutase (SOD) mimic (M40403) treatment restored/increased these parameters, as well as the expression of endothelial nitric oxide synthase, manganese SOD, and catalase. These data lead to the hypothesis that M40403 improves mitochondrial functional state and motility of spermatozoa, as well as (NO)-N-center dot might be involved in the observed effects of the mimic. Antioxid. Redox Signal. 18, 170-178.Ministry of Education and Science of the Republic of Serbia [173054]; University of Erlangen-Nurember