Tetraspanins are multifunctional molecules located in specific microdomains on the plasma membrane. Thanks to their ability to form networks with other proteins they can participate in many cellular functions. Tetraspanins are part of the interactive network in gametes; however, their precise role in fertilization is not yet clear. The aim of this study was to compare the localization of CD9 and CD81 tetraspanins during oocyte maturation and early development of the embryos in bovine and porcine model. CD9 was detected on the oocyte plasma membrane and vesicles in the perivitelline space of bovine oocytes and embryos. We suggest that CD9 could be a component involved in transzonal projections. Based on the results of in vitro fertilization assay, CD9 and CD81 seem to be part of a more complex fusion network on the plasma membrane of bovine oocytes. On the other hand, both tetraspanins showed a clustered expression pattern on the plasma membrane and inner margin of zona pellucida (ZP) in porcine oocytes and embryos. We found a new species-specific pattern of CD9 and CD81 distribution in ZP which could reflect their specialized role in processes associated with cell adhesion and intercellular communication upon fertilization.
- MeSH
- antigeny CD81 metabolismus MeSH
- antigeny CD9 metabolismus MeSH
- buněčné linie MeSH
- embryo savčí cytologie metabolismus MeSH
- fertilizace in vitro účinky léků MeSH
- metafáze účinky léků MeSH
- myši inbrední BALB C MeSH
- oocyty cytologie metabolismus MeSH
- partenogeneze účinky léků MeSH
- prasata MeSH
- protilátky farmakologie MeSH
- skot MeSH
- stadium rýhování vajíčka cytologie účinky léků MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- 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
The aim of this work was to assess the FSH-stimulated expression of epidermal growth factor (EGF)-like peptides in cultured cumulus-oocyte complexes (COCs) and to find out the effect of the peptides on cumulus expansion, oocyte maturation, and acquisition of developmental competence in vitro. FSH promptly stimulated expression of amphiregulin (AREG) and epiregulin (EREG), but not betacellulin (BTC) in the cultured COCs. Expression of AREG and EREG reached maximum at 2 or 4 h after FSH addition respectively. FSH also significantly stimulated expression of expansion-related genes (PTGS2, TNFAIP6, and HAS2) in the COCs at 4 and 8 h of culture, with a significant decrease at 20 h of culture. Both AREG and EREG also increased expression of the expansion-related genes; however, the relative abundance of mRNA for each gene was much lower than in the FSH-stimulated COCs. In contrast to FSH, AREG and EREG neither stimulated expression of CYP11A1 in the COCs nor an increase in progesterone production by cumulus cells. AREG and EREG stimulated maturation of oocytes and expansion of cumulus cells, although the percentage of oocytes that had reached metaphase II was significantly lower when compared to FSH-induced maturation. Nevertheless, significantly more oocytes stimulated with AREG and/or EREG developed to blastocyst stage after parthenogenetic activation when compared to oocytes stimulated with FSH alone or combinations of FSH/LH or pregnant mares serum gonadotrophin/human chorionic gonadotrophin. We conclude that EGF-like peptides do not mimic all effects of FSH on the cultured COCs; nevertheless, they yield oocytes with superior developmental competence.
- MeSH
- buněčná diferenciace účinky léků genetika MeSH
- embryonální vývoj účinky léků genetika MeSH
- epidermální růstový faktor chemie farmakologie MeSH
- folikuly stimulující hormon farmakologie MeSH
- gonadotropiny farmakologie MeSH
- kultivace embrya MeSH
- kultivované buňky MeSH
- kumulární buňky účinky léků metabolismus fyziologie MeSH
- oocyty účinky léků metabolismus fyziologie MeSH
- oogeneze účinky léků genetika MeSH
- partenogeneze účinky léků genetika fyziologie MeSH
- peptidové fragmenty chemie farmakologie MeSH
- prasata genetika metabolismus fyziologie MeSH
- proliferace buněk účinky léků MeSH
- stanovení celkové genové exprese MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
The nitric oxide donor (+)-S-nitroso-N-acetylpenicillamine (SNAP) is capable of inducing parthenogenetic activation in pig oocytes matured in vitro. However, quite a long exposure to the nitric oxide donor, exceeding 10 h, is necessary for successful oocyte activation. Repeated short-term treatment with 2 mm SNAP significantly increased the activation rates despite the fact that the overall exposure time to the nitric oxide donor did not exceed 4 h. With regard to the activation rate, 12 repeated treatments lasting 10 min each were found to be the most efficient regimen (63.3%). The continuous exposure to the nitric oxide donor for the same overall time induced parthenogenetic activation in 12.5% oocytes (2-h continuous treatment with 2 mm SNAP). The development of parthenogenetic embryos increased after repeated short-term treatment with SNAP. After continuous treatment with 2 mm SNAP for 10 h, only 6.7% of the oocytes cleaved, and none developed beyond the 4-cell stage. Thirty-minute treatment repeated four times with 2 mm SNAP induced cleavage in 37.5% of the oocytes, 18.3% developed to the morula stage, and 6.7% reached the blastocyst stage. Based on the results, it is concluded that pulsatile treatment can significantly improve parthenogenetic activation rate when compared with the continuous treatment using nitric oxide donors.
- MeSH
- donory oxidu dusnatého aplikace a dávkování MeSH
- kultivace embrya veterinární MeSH
- kultivované buňky MeSH
- oocyty účinky léků fyziologie MeSH
- partenogeneze účinky léků fyziologie MeSH
- prasata MeSH
- S-nitroso-N-acetylpenicilamin aplikace a dávkování MeSH
- stadium rýhování vajíčka MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
