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
Mammalian meiotic maturation is regulated by changes in the phosphorylation state of proteins involved in signalling pathways. The regulatory proteins include the family of Src tyrosine kinases. Src family kinases (SFKs) are required for meiotic maturation of mouse oocytes, and it remains to be elucidated whether they play the same role in porcine oocytes. To clarify the role of SFKs in the meiotic maturation of porcine oocytes we used inhibition of SFKs, western blotting and immunolocalisation to determine the presence of SFKs and localisation in the oocytes and assays to determine the activity of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). Inhibition of SFKs resulted in the disruption of oocyte maturation and led to a decline in MPF and MAPK activity. The fluorescence intensity of SFKs in the cytoplasm and membrane of MI oocytes decreased significantly compared with germinal vesicle oocytes. The highest fluorescence intensity for SFKs was detected on the membrane of MII oocytes. Only weak fluorescence was detected in the perichromosomal area of MI and MII oocytes. These results prove that SFKs play an active role in the meiotic maturation of porcine oocytes by regulating MPF and MAPK activity.
- MeSH
- buněčné jádro metabolismus MeSH
- faktor podporující zrání metabolismus MeSH
- meióza fyziologie MeSH
- mitogenem aktivované proteinkinasy metabolismus MeSH
- oocyty růst a vývoj metabolismus MeSH
- prasata MeSH
- signální transdukce fyziologie MeSH
- skupina kinas odvozených od src-genu metabolismus MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články 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
