Bisphenol A (BPA), a chemical component of plastics, is a widely distributed environmental pollutant and contaminant of water, air, and food that negatively impacts human health. Concerns regarding BPA have led to the use of BPA-free alternatives, one of which is bisphenol S (BPS). However, the effects of BPS are not well characterized, and its specific effects on reproduction and fertility remain unknown. It is therefore necessary to evaluate any effects of BPS on mammalian oocytes. The present study is the first to demonstrate the markedly negative effects of BPS on pig oocyte maturation in vitro, even at doses lower than those humans are exposed to in the environment. Our results demonstrate (1) an effect of BPS on the course of the meiotic cell cycle; (2) the failure of tubulin fibre formation, which controls proper chromosome movement; (3) changes in the supply of maternal mRNA; (4) changes in the protein amounts and distribution of oestrogen receptors α and β and of aromatase; and (5) disrupted cumulus cell expansion. Thus, these results confirm that BPS is an example of regrettable substitution because this substance exerts similar or even worse negative effects than those of the material it replaced.
- MeSH
- aromatasa genetika MeSH
- buněčná diferenciace účinky léků genetika MeSH
- fenoly farmakologie MeSH
- meióza účinky léků MeSH
- messenger RNA genetika MeSH
- oocyty cytologie účinky léků metabolismus MeSH
- prasata MeSH
- receptory pro estrogeny genetika MeSH
- sulfony farmakologie MeSH
- vývojová regulace genové exprese účinky léků MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hydrogen sulfide, one of three known gasotransmitters, is involved in physiological processes, including reproductive functions. Oocyte maturation and surrounding cumulus cell expansion play an essential role in female reproduction and subsequent embryonic development. Although the positive effects of exogenous hydrogen sulfide on maturing oocytes are well known, the role of endogenous hydrogen sulfide, which is physiologically released by enzymes, has not yet been described in oocytes. In this study, we observed the presence of Cystathionine β-Synthase (CBS), Cystathionine γ-Lyase (CTH) and 3-Mercaptopyruvate Sulfurtransferase (3-MPST), hydrogen sulfide-releasing enzymes, in porcine oocytes. Endogenous hydrogen sulfide production was detected in immature and matured oocytes as well as its requirement for meiotic maturation. Individual hydrogen sulfide-releasing enzymes seem to be capable of substituting for each other in hydrogen sulfide production. However, meiosis suppression by inhibition of all hydrogen sulfide-releasing enzymes is not irreversible and this effect is a result of M-Phase/Maturation Promoting Factor (MPF) and Mitogen-Activated Protein Kinase (MAPK) activity inhibition. Futhermore, cumulus expansion expressed by hyaluronic acid (HA) production is affected by the inhibition of hydrogen sulfide production. Moreover, quality changes of the expanded cumuli are indicated. These results demonstrate hydrogen sulfide involvement in oocyte maturation as well as cumulus expansion. As such, hydrogen sulfide appears to be an important cell messenger during mammalian oocyte meiosis and adequate cumulus expansion.
- MeSH
- imunohistochemie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- kyselina hyaluronová chemie MeSH
- oocyty enzymologie růst a vývoj MeSH
- prasata růst a vývoj fyziologie MeSH
- sulfan metabolismus MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Porcine oocytes that have matured in in vitro conditions undergo the process of aging during prolonged cultivation, which is manifested by spontaneous parthenogenetic activation, lysis or fragmentation of aged oocytes. This study focused on the role of hydrogen sulfide (H2S) in the process of porcine oocyte aging. H2S is a gaseous signaling molecule and is produced endogenously by the enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). We demonstrated that H2S-producing enzymes are active in porcine oocytes and that a statistically significant decline in endogenous H2S production occurs during the first day of aging. Inhibition of these enzymes accelerates signs of aging in oocytes and significantly increases the ratio of fragmented oocytes. The presence of exogenous H2S from a donor (Na2S.9H2O) significantly suppressed the manifestations of aging, reversed the effects of inhibitors and resulted in the complete suppression of oocyte fragmentation. Cultivation of aging oocytes in the presence of H2S donor positively affected their subsequent embryonic development following parthenogenetic activation. Although no unambiguous effects of exogenous H2S on MPF and MAPK activities were detected and the intracellular mechanism underlying H2S activity remains unclear, our study clearly demonstrates the role of H2S in the regulation of porcine oocyte aging.
- MeSH
- cystathionin-beta-synthasa metabolismus MeSH
- cystathionin-gama-lyasa metabolismus MeSH
- embryo savčí účinky léků MeSH
- inhibitory enzymů farmakologie MeSH
- kultivace embrya MeSH
- kultivované buňky MeSH
- oocyty účinky léků fyziologie MeSH
- partenogeneze účinky léků MeSH
- prasata MeSH
- stárnutí buněk účinky léků MeSH
- sulfan metabolismus farmakologie MeSH
- sulfurtransferasy metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Hydrogen sulfide (H2S) has been revealed to be a signal molecule with second messenger action in the somatic cells of many tissues, including the reproductive tract. The aim of this study was to address how exogenous H2S acts on the meiotic maturation of porcine oocytes, including key maturation factors such as MPF and MAPK, and cumulus expansion intensity of cumulus-oocyte complexes. We observed that the H2S donor, Na2S, accelerated oocyte in vitro maturation in a dose-dependent manner, following an increase of MPF activity around germinal vesicle breakdown. Concurrently, the H2S donor affected cumulus expansion, monitored by hyaluronic acid production. Our results suggest that the H2S donor influences oocyte maturation and thus also participates in the regulation of cumulus expansion. The exogenous H2S donor apparently affects key signal pathways of oocyte maturation and cumulus expansion, resulting in faster oocyte maturation with little need of cumulus expansion.
- MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- faktor podporující zrání metabolismus MeSH
- gasotransmitery farmakologie MeSH
- kokultivační techniky MeSH
- kultivované buňky MeSH
- kumulární buňky cytologie metabolismus MeSH
- meióza účinky léků MeSH
- oocyty cytologie metabolismus MeSH
- prasata MeSH
- sulfan farmakologie MeSH
- sulfidy farmakologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The processes of oocyte growth, acquisition of meiotic competence and meiotic maturation are regulated by a large number of molecules. One of them could be calcineurin consisting of catalytic subunit A (Aα, Aβ, Aγ isoforms) and regulatory subunit B (B1, B2 isoforms). Calcineurin is involved in the meiotic maturation of oocytes in invertebrates or in lower vertebrates. In the mammalian oocytes, the possible role of calcineurin in the regulation of oocyte meiosis has not been clarified to date. In this study, to investigate the role of calcineurin during porcine oocyte growth, acquisition of meiotic competence and meiotic maturation, we analysed the expression and localisation of calcineurin subunits and the mRNA expression of calcineurin isoforms. Calcineurin was expressed in growing porcine oocytes, in fully grown oocytes and during their in vitro meiotic maturation. We found both subunits of calcineurin. Calcineurin A and calcineurin B were localised mainly in the cortex in all porcine oocytes. The changes in the intracellular localisation of separate calcineurin subunits during meiotic maturation were determined. We detected mRNA for calcineurin isoforms Aβ, Aγ, B2 in oocytes and mRNA for calcineurin isoforms Aβ, Aγ, B1, and B2 in cumular cells. To our knowledge, this is the first confirmation of calcineurin presence in porcine oocytes.
- MeSH
- IVM techniky veterinární MeSH
- kalcineurin genetika metabolismus MeSH
- meióza fyziologie MeSH
- messenger RNA genetika metabolismus MeSH
- oocyty fyziologie MeSH
- prasata * MeSH
- regulace genové exprese fyziologie MeSH
- transport proteinů fyziologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The effect of different activation protocols on DNA integrity of porcine oocytes matured in vitro was analysed using the comet assay. The oocytes from ovaries of slaughtered gilts were cultured for 48 h in modified M199 medium. They were then freed of cumulus cells and treated continuously or intermittently with a nitric oxide (NO) donor for 6 h. Standard activation with calcium ions (Ca2+) and culture without any treatment served as positive and negative controls, respectively. The activation was assessed according to the formation of pronuclei. Exposure of oocytes to Ca2+ was associated with high activation efficiency, but decreased DNA integrity. The opposite, i.e. low activation efficiency but high DNA integrity was observed after continuous exposure to NO. Intermittent action of NO increased the activation rate, while the values of DNA damage remained at low levels. Our data suggest that an increased DNA instability could be the main reason compromising the further embryonic development of oocytes activated by the standard protocol. The intermittent treatment with NO thus represents a promising step to optimization of parthenogenetic activation of pig oocytes.
- MeSH
- buněčná diferenciace MeSH
- DNA genetika metabolismus MeSH
- kultivované buňky MeSH
- oocyty cytologie metabolismus MeSH
- poškození DNA MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this paper we assessed: (i) the change in nitric oxide synthase (NOS) isoforms' expression and intracellular localization and in NOS mRNA in porcine oocytes during meiotic maturation; (ii) the effect of NOS inhibition by N(omega)-nitro-l-arginine methyl ester (l-NAME) and aminoguanidine (AG) on meiotic maturation of cumulus-oocyte complexes (COC) as well as denuded oocytes (DO); and (iii) nitric oxide (NO) formation in COC. All three NOS isoforms (eNOS, iNOS and nNOS) and NOS mRNA (eNOS mRNA, iNOS mRNA and nNOS mRNA) were found in both porcine oocytes and their cumulus cells except for nNOS mRNA, which was not detected in the cumulus cells. NOS isoforms differed in their intracellular localization in the oocyte: while iNOS protein was dispersed in the oocyte cytoplasm, nNOS was localized in the oocyte cytoplasm and in germinal vesicles (GV) and eNOS was present in dots in the cytoplasm, GV and was associated with meiotic spindles. l-NAME inhibitor significantly suppressed metaphase (M)I to MII transition (5.0 mM experimental group: 34.9% MI, control group: 9.5% MI) and at the highest concentration (10.0 mM) also affected GV breakdown (GVBD); in contrast also AG inhibited primarily GVBD. The majority of the oocytes (10.0 mM experimental group: 60.8%, control group: 1.2%) was not able to resume meiosis. AG significantly inhibited GVBD in DO, but l-NAME had no significant effect on the GVBD of these cells. During meiotic maturation, NO is formed in COC and the NO formed by cumulus cells is necessary for the process of GVBD.
- MeSH
- konfokální mikroskopie MeSH
- kumulární buňky enzymologie MeSH
- meióza MeSH
- messenger RNA metabolismus MeSH
- NG-nitroargininmethylester farmakologie MeSH
- oocyty účinky léků enzymologie MeSH
- protein - isoformy antagonisté a inhibitory genetika metabolismus MeSH
- synthasa oxidu dusnatého antagonisté a inhibitory genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Background and Objectives Two-dimensional SDS PAGE (sodium dodecyl sulfate polyacrylamidegel electrophoresis) coupled with mass spectrometry is still a mainstream approach to analysing multiple protein expression levels. The requirement for some sophisticated devices and the lack of quantitative measurements for low-abundant proteins (e.g. cytokines) greatly limit its broad application. Cytokines present in the pg/ml levels in non-stimulated biological samples are traditionally detected by ELISA. We used a cytokine antibody array, a highly sensitive protein chip, for simultaneous detection of multiple cytokine expression levels in rat sarcoma lysates and serum samples . Material and methods We present here an optimized protocol for preparation and handling of tumour tissue lysates in protein chip detection. The sarcoma samples were processed at low temperatures to prevent cytokine degradation. Tumour cryosections (8?10 mm) were used for extraction of cytokines. The addition of NaN3 destroyed a high endogenous peroxidase activity, which may interfere with protein chip assay and decrease the signal/noise ratio. The data for the protein matrix effect from sandwich ELISA can also affect the protein chip detection. The optimal dilution of samples must be found to prevent pitfalls due to the non-optimal signal-to-noise ratio. This also enables recovery of low amounts of cytokines from difficult samples. Results We report optimized procedures for extraction, sample handling, inhibition of endogenous peroxidase activity and prevention of the protein matrix effect in serum and tumour lysates by detection of cytokine expression using the cytokine antibody array protein chip.
- MeSH
- aktivace makrofágů MeSH
- antigeny nádorové MeSH
- dendritické buňky imunologie MeSH
- financování organizované MeSH
- fúze buněk MeSH
- melanom imunologie terapie MeSH
- modely nemocí na zvířatech MeSH
- mutantní kmeny potkanů MeSH
- nádorové buňky kultivované imunologie MeSH
- prasata MeSH
- protinádorové vakcíny MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
Celiakia je považovaná za celoživotné ochorenie s precitlivenosťou na lepok. V klinickej praxi je bez kauzálnej liečby s terapiou spočívajúcou v prísnom dodržiavaní bezlepkovej diéty. Ochorenie poznali už v starovekom Grécku. V súčasnosti je v celosvetovej pozornosti odbornej verejnosti a v záujme mnohých vedeckých i výskumných pracovísk. Výsledkom je rad nových teoretických i praktických poznatkov. Vyplývajúc z toho sa aj charakter výskumu ochorenia neustále rozširuje. Toho času sa do diskusie môžu zapojiť lekári rôznej aprobácie, farmaceuti, prírodovedci, výskumní pracovníci na rôznych postoch, ale aj ekonómovia, právnici a manažéri. V spektre manifestácie ochorenia boli diagnostikované nové formy, včítane asociácií s inými ochoreniami. Tým došlo k nárastu prevalencie ochorenia aj v našej spoločnosti. Následne sa v domácej i zahraničnej literatúre objavila plejáda prehľadných článkov so snahou vyvolať pozornosť odbornej verejnosti na symptómy a prejavy tohto ochorenia. Ich spoločným cieľom bolo prispieť k odhaľovaniu ochorenia v populácii detí i dospelých. Napriek tomu je celiakia stále pod-diagnostikovaná. Vzhľadom k tomu je potrebné koncipovať nové úlohy na poli celiakie. Popisuje sa súčasná prax v diagnostike ochorenia a používané i potencionálne použiteľné diétne prostriedky k účinnej ochrane a k podpore terapie. Sú zvýraznené aspekty skorého screeningu, včítane ďalších úloh vedúcich k zaisteniu starostlivosti o chorých a novších prístupov k liečbe.
Celiac disease is a lifelong autoimmune intestinal disorder caused by sensitivity to gluten. In clinical practice, no causal treatment is currently available and the disease can only be treated by strict adherence to a gluten-free diet. The disease that was known in ancient Greece continues to draw attention of the professional public worldwide and has been studied by many researchers. As a result, a range of theoretical and practical knowledge has been obtained and thus further aspects of the disease can be addressed. At present the discussion is open not only to doctors, pharmacists, scientists and researchers at various posts, but also to economists, lawyers and managers. In the spectrum of disease manifestations, some new forms were diagnosed, including associations with other diseases. Therefore, a higher prevalence of this disease has been reported. Multiple review articles appeared in both the local and international literature to familiarize the professional public with the symptoms and signs of the disease. Their common aim was to contribute to the detection of the disease in children and adults. Despite that, the disease remains underdiagnosed. In this context, new challenges emerge in the field of celiac disease. The current practice in diagnosing celiac disease and the available and candidate dietary interventions for effective protection and support therapy are described. The importance of early screening and advanced approaches to disease management is underlined.
