Global transcription silencing occurs in the oocyte during its final phase of growth. The particular mechanism of this silencing is not well understood. Here, we investigated the silencing of RNA polymerase II transcription in porcine oocytes. First, we investigated the transcriptional activity of germinal vesicle oocytes derived from stimulated and non-stimulated gilts, but no transcriptional activity was observed. Second, we focused on the fate of RNA polymerase II in growing and fully grown oocytes. Active and inactive forms of RNA polymerase II were detected in growing oocytes by immunofluorescence and Western blots. In contrast, only the inactive form of RNA polymerase II was detected in fully grown oocytes. To evaluate if the inactive form of RNA polymerase II is released from DNA, the oocytes were subsequently permeabilized and fixed in one step. After this modified fixation protocol, the immunofluorescent labeling was negative in fully grown oocytes, but remained unchanged (positive) in growing oocytes. These results indicate that the inactive form of RNA polymerase II is not bound to DNA during the oocyte growth. Finally, based on Western blot analysis of different stages of oocyte maturation, the inactive form of RNA polymerase II was detected in metaphase I but not in metaphase II. Our study confirmed the global transcription silencing of fully grown oocytes. Compared with other mammalian species (e.g., mouse), the mechanism of RNA polymerase II silencing in porcine oocytes seems to be similar, despite some differences in dynamics.

• MeSH
• adenosin chemie   metabolismus   MeSH
• autoradiografie MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• gonadotropiny metabolismus   MeSH
• imunohistochemie MeSH
• izotopové značení MeSH
• myši MeSH
• oocyty chemie   růst a vývoj   metabolismus   fyziologie   MeSH
• prasata MeSH
• RNA-polymerasa II chemie   genetika   metabolismus   MeSH
• umlčování genů * MeSH
• uridin chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Both the sperm and oocyte are terminally differentiated cells, but within a very short post-fertilization period, their genomes are converted into a totipotent zygote. The process of this transformation has been studied in a number of mammals as well as in the pig, for which very inconsistent results have been published. To clarify these inconsistencies, we have used the interspecies intracytoplasmic sperm injection technique for embryo production and subsequent paternal genome remodeling evaluation. First, we injected boar sperm heads into ovulated and in vitro matured mouse oocytes. The boar spermatozoa consistently decondense in ovulated oocytes and form fully developed pronuclei with demethylated DNA (5-methylcytosine; 5-MeC). Additional labeling against other histone modifications (H3/K9 dimethylation, H3/K4 trimethylation) and HP1 (Heterochromatin Protein 1) revealed similarity to those changes that are typical for natural mouse zygotes. On the other hand, no decondensation and formation of male pronuclei were observed, in spite of obvious oocyte activation, in in vitro matured oocytes. For this reason, we have evaluated the reprogramming parameters of in vitro matured mouse oocytes in more detail. In mouse zygotes (intraspecific), both pronuclei were consistently formed, but no sperm head chromatin demethylation was detected after 5-MeC labeling. Our observations suggest that porcine sperm heads are capable of undergoing active demethylation in in vivo matured mouse oocytes. On the other hand, in vitro matured oocytes possess much lower sperm remodeling capabilities.

• MeSH
• 5-methylcytosin metabolismus   MeSH
• chiméra genetika   MeSH
• chromatin metabolismus   MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• ektogeneze MeSH
• epigeneze genetická MeSH
• fertilizace fyziologie   MeSH
• histony metabolismus   MeSH
• hlavička spermie metabolismus   MeSH
• intracytoplazmatické injekce spermie MeSH
• metylace DNA MeSH
• metylace MeSH
• myši MeSH
• posttranslační úpravy proteinů MeSH
• přeprogramování buněk MeSH
• restrukturace chromatinu MeSH
• Sus scrofa MeSH
• zvířata MeSH
• zygota cytologie   metabolismus   MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH