Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H2DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.

• Klíčová slova
• H3K4me2, MKRN1, PAWP, SPTRX-3, biomarkers, flow cytometry, native breeds, ram, semen, ubiquitin,
• MeSH
• analýza spermatu MeSH
• biologické markery MeSH
• chromatin MeSH
• fertilita MeSH
• kryoprezervace metody   MeSH
• motilita spermií * MeSH
• ovce MeSH
• průtoková cytometrie MeSH
• reaktivní formy kyslíku MeSH
• savci MeSH
• spermie MeSH
• uchování spermatu * metody   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• chromatin MeSH
• reaktivní formy kyslíku MeSH

Hypoxia is involved in the regulation of stem cell fate, and hypoxia-inducible factor 1 (HIF-1) is the master regulator of hypoxic response. Here, we focus on the effect of hypoxia on intracellular signaling pathways responsible for mouse embryonic stem (ES) cell maintenance. We employed wild-type and HIF-1α-deficient ES cells to investigate hypoxic response in the ERK, Akt, and STAT3 pathways. Cultivation in 1% O2 for 24 h resulted in the strong dephosphorylation of ERK and its upstream kinases and to a lesser extent of Akt in an HIF-1-independent manner, while STAT3 phosphorylation remained unaffected. Downregulation of ERK could not be mimicked either by pharmacologically induced hypoxia or by the overexpression. Dual-specificity phosphatases (DUSP) 1, 5, and 6 are hypoxia-sensitive MAPK-specific phosphatases involved in ERK downregulation, and protein phosphatase 2A (PP2A) regulates both ERK and Akt. However, combining multiple approaches, we revealed the limited significance of DUSPs and PP2A in the hypoxia-mediated attenuation of ERK signaling. Interestingly, we observed a decreased reactive oxygen species (ROS) level in hypoxia and a similar phosphorylation pattern for ERK when the cells were supplemented with glutathione. Therefore, we suggest a potential role for the ROS-dependent attenuation of ERK signaling in hypoxia, without the involvement of HIF-1.

• MeSH
• down regulace MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa metabolismus   MeSH
• mitogenem aktivované proteinkinasy kinas metabolismus   MeSH
• myší embryonální kmenové buňky metabolismus   MeSH
• myši MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• faktor 1 indukovatelný hypoxií - podjednotka alfa MeSH
• mitogenem aktivované proteinkinasy kinas MeSH
• reaktivní formy kyslíku MeSH

Reactive oxygen species (ROS) are important regulators of cellular functions. In embryonic stem cells, ROS are suggested to influence differentiation status. Regulated ROS formation is catalyzed primarily by NADPH-dependent oxidases (NOXs). Apocynin and diphenyleneiodonium are frequently used inhibitors of NOXs; however, both exhibit uncharacterized effects not related to NOXs inhibition. Interestingly, in our model of mouse embryonic stem cells we demonstrate low expression of NOXs. Therefore we aimed to clarify potential side effects of these drugs. Both apocynin and diphenyleneiodonium impaired proliferation of cells. Surprisingly, we observed prooxidant activity of these drugs determined by hydroethidine. Further, we revealed that apocynin inhibits PI3K/Akt pathway with its downstream transcriptional factor Nanog. Opposite to this, apocynin augmented activity of canonical Wnt signaling. On the contrary, diphenyleneiodonium activated both PI3K/Akt and Erk signaling pathways without affecting Wnt. Our data indicates limits and possible unexpected interactions of NOXs inhibitors with intracellular signaling pathways.

• MeSH
• acetofenony farmakologie   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosforylace účinky léků   MeSH
• myší embryonální kmenové buňky účinky léků   metabolismus   MeSH
• myši MeSH
• NADPH-oxidasy genetika   metabolismus   MeSH
• oniové sloučeniny farmakologie   MeSH
• oxidační stres účinky léků   MeSH
• proliferace buněk účinky léků   MeSH
• proteiny Wnt metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• synergismus léků MeSH
• transkripční faktor STAT3 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetofenony MeSH
• acetovanillone MeSH Prohlížeč
• diphenyleneiodonium MeSH Prohlížeč
• extracelulárním signálem regulované MAP kinasy MeSH
• fosfatidylinositol-3-kinasy MeSH
• NADPH-oxidasy MeSH
• oniové sloučeniny MeSH
• proteiny Wnt MeSH
• protoonkogenní proteiny c-akt MeSH
• reaktivní formy kyslíku MeSH
• transkripční faktor STAT3 MeSH

Oxidative stress after birth led us to localize reactive oxygen and nitrogen species (RONS) production in the developing rat brain. Brains were assessed a day prenatally and on postnatal days 1, 2, 4, 8, 14, 30, and 60. Oxidation of dihydroethidium detected superoxide; 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate revealed hydrogen peroxide; immunohistochemical proof of nitrotyrosine and carboxyethyllysine detected peroxynitrite formation and lipid peroxidation, respectively. Blue autofluorescence detected protein oxidation. The foetuses showed moderate RONS production, which changed cyclically during further development. The periods and sites of peak production of individual RONS differed, suggesting independent generation. On day 1, neuronal/glial RONS production decreased indicating that increased oxygen concentration after birth did not cause oxidative stress. Dramatic changes in the amount and the sites of RONS production occurred on day 4. Nitrotyrosine detection reached its maximum. Day 14 represented other vast alterations in RONS generation. Superoxide production in arachnoidal membrane reached its peak. From this day on, the internal elastic laminae of blood vessels revealed the blue autofluorescence. The adult animals produced moderate levels of superoxide; all other markers reached their minimum. There was a strong correlation between detection of nitrotyrosine and carboxyethyllysine probably caused by lipid peroxidation initiated with RONS.

• MeSH
• fluorescenční mikroskopie MeSH
• glykosylace MeSH
• kyselina peroxydusitá metabolismus   MeSH
• lysin analogy a deriváty   metabolismus   MeSH
• mozek růst a vývoj   metabolismus   MeSH
• novorozená zvířata MeSH
• oxidační stres * MeSH
• peroxid vodíku metabolismus   MeSH
• peroxidace lipidů MeSH
• posttranslační úpravy proteinů MeSH
• potkani Wistar MeSH
• reaktivní formy dusíku metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• superoxidy metabolismus   MeSH
• tyrosin analogy a deriváty   metabolismus   MeSH
• věkové faktory MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3-nitrotyrosine MeSH Prohlížeč
• kyselina peroxydusitá MeSH
• lysin MeSH
• N(6)-carboxyethyllysine MeSH Prohlížeč
• peroxid vodíku MeSH
• reaktivní formy dusíku MeSH
• reaktivní formy kyslíku MeSH
• superoxidy MeSH
• tyrosin MeSH

Reactive oxygen species (ROS) play several biological roles. We investigated the applicability of fluorescent probes for their detection (i) in rabbit lens epithelial cells during ageing in culture, and (ii) in thin sections of rat heart. We used dihydroethidium (DHE), dichlorofluorescin (DCFH), and dihydrorhodamine 123 (DHR) together with detection of autofluorescence both in cells and in chloroform extracts. Superoxide production was confirmed by a specific histochemical method using Mn(2+). All methods demonstrated higher production of ROS in older cells. All probes revealed different sites of ROS production in young and old cells and could be used for investigation of ROS generation during cell ageing. In the thin sections of rat heart DCFH was not suitable for intracellular ROS detection. The results indicate that the potential of fluorescent dyes in ROS detection is not usually fully exploited, and that blue autofluorescence is associated with oxidative damage.

• MeSH
• epitelové buňky metabolismus   MeSH
• ethidium analogy a deriváty   MeSH
• fluoresceiny MeSH
• králíci MeSH
• krysa rodu Rattus MeSH
• metody MeSH
• molekulární sondy - techniky * MeSH
• myokard metabolismus   MeSH
• oční čočka cytologie   MeSH
• reaktivní formy kyslíku analýza   MeSH
• rhodaminy MeSH
• volné radikály analýza   MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2',7'-dichlorofluorescein MeSH Prohlížeč
• dihydroethidium MeSH Prohlížeč
• dihydrorhodamine 123 MeSH Prohlížeč
• ethidium MeSH
• fluoresceiny MeSH
• reaktivní formy kyslíku MeSH
• rhodaminy MeSH
• volné radikály MeSH