Oocyte meiotic maturation and embryogenesis are some of the most important physiological processes that occur in organisms, playing crucial roles in the preservation of life in all species. The post-transcriptional regulation of maternal messenger ribonucleic acids (mRNAs) and the post-translational regulation of proteins are critical in the control of oocyte maturation and early embryogenesis. Translational control affects the basic mechanism of protein synthesis, thus, knowledge of the key components included in this machinery is required in order to understand its regulation. Cytoplasmic polyadenylation element binding proteins (CPEBs) bind to the 3'-end of mRNAs to regulate their localization and translation and are necessary for proper development. In this study we examined the expression pattern of cytoplasmic polyadenylation element binding protein 2 (CPEB2) both on the mRNA (by real-time quantitative reverse transcription polymerase chain reaction, qRT-PCR) and protein (by Western blotting, WB) level, as well as its localization during the meiotic maturation of porcine oocytes and early embryonic development by immunocytochemistry (ICC). For the elucidation of its functions, CPEB2 knockdown by double-strand RNA (dsRNA) was used. We discovered that CPEB2 is expressed during all stages of porcine meiotic maturation and embryonic development. Moreover, we found that it is necessary to enable a high percentage of oocytes to reach the metaphase II (MII) stage, as well as for the production of good-quality parthenogenetic blastocysts.

• Klíčová slova
• CPEB2, CPEBs, embryonic development, oocyte maturation, translational control,
• MeSH
• 3' nepřekládaná oblast MeSH
• embryonální vývoj MeSH
• genový knockdown MeSH
• meióza * MeSH
• messenger RNA metabolismus   MeSH
• oocyty cytologie   metabolismus   MeSH
• partenogeneze MeSH
• prasata MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• těhotenství MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 3' nepřekládaná oblast MeSH
• messenger RNA MeSH
• proteiny vázající RNA MeSH

Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.

• MeSH
• Aurora kinasa A metabolismus   MeSH
• cyklin B1 genetika   MeSH
• faktory štěpení a polyadenylace mRNA chemie   metabolismus   MeSH
• fosforylace MeSH
• meióza * MeSH
• messenger RNA metabolismus   MeSH
• oocyty enzymologie   metabolismus   MeSH
• polyadenylace MeSH
• Sus scrofa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Aurora kinasa A MeSH
• cyklin B1 MeSH
• faktory štěpení a polyadenylace mRNA MeSH
• messenger RNA MeSH

The use of carrier ampholyte-free IEF (CAF-IEF) with ITP mobilization and conductivity detection in ITP mode for preconcentration and analysis of amino acids is demonstrated. The analytical procedure consists of three subsequent steps. In the first step, amino acids are continuously dosed from an infinite volume reservoir by electromigration to the column, where a sharp, stationary neutralization reaction boundary (NRB) is created in between acidic and basic primary electrolyte. Here, amino acids are selectively focused (trapped), if their pI falls to the pH difference on both sides of the NRB (pH gap). Amino acids create sharp rectangular zones, arranged according to their pI values. In the second step, focused zones are mobilized. After accumulation of the detectable amount of amino acids, dosing electrolyte in the infinite volume reservoir is changed for the mobilizing electrolyte. The migration mode is changed from CAF-IEF to ITP and substances start to migrate toward the analytical capillary. In the third step, analytes are transferred into the analytical column equipped with a conductivity detector and are detected in the new leading electrolyte in an ITP migration mode. The presented CAF-IEF-ITP-ITP with time-dependent accumulation of the large-volume sample enables to achieve in a reasonable time a 100 times lower c-LOD (here in orders of nmol/L), than can be reached by conventional hyphenated ITP-ITP.

• MeSH
• amfolytové směsi MeSH
• aminokyseliny analýza   MeSH
• arginin izolace a purifikace   MeSH
• beta-alanin izolace a purifikace   MeSH
• elektroforéza metody   MeSH
• histidin izolace a purifikace   MeSH
• isoelektrická fokusace metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• amfolytové směsi MeSH
• aminokyseliny MeSH
• arginin MeSH
• beta-alanin MeSH
• histidin MeSH