This study was designed to specify chromatin and mitochondrial patterns in bovine oocytes with different meiotic competence in relation to maturation progress, resumption of meiosis, MII onset and completion of maturation. Oocytes with greater or lesser meiotic competence, recovered separately from medium (MF) and small follicles (SF), were categorized according to morphology. Four oocyte categories, healthy and light-atretic MF and healthy and light-atretic SF oocytes were matured and collected at 0, 3, 7, 16 and 24 h of maturation. Specific differences in terms of chromatin and mitochondrial patterns were found among the maturing oocyte categories. Resumption of meiosis was accelerated in light-atretic oocytes, as compared with healthy oocytes, regardless of their meiotic competence. More competent oocytes activated mitochondria twice during maturation, before resumption of meiosis and before completion of maturation, while less competent oocytes did it only once, before completion of maturation. Changes in mitochondrial activity differed in light-atretic compared with healthy in both more and less competent oocytes. Healthy meiotically more competent oocytes formed clusters and produced ATP for the whole time of maturation until its completion, while light-atretic more competent oocytes and healthy less competent oocytes reduced these activities earlier, at MII onset. Contrary to these oocyte categories, light-atretic less competent oocytes increased cluster formation significantly before resumption of meiosis. It can be concluded that bovine oocytes with different meiotic competence and health differed in the kinetics of mitochondrial patterns during maturation.
- MeSH
- chromatin ultrastruktura MeSH
- IVM techniky * MeSH
- konfokální mikroskopie MeSH
- meióza * MeSH
- mitochondrie ultrastruktura MeSH
- oocyty růst a vývoj ultrastruktura MeSH
- skot anatomie a histologie MeSH
- zvířata MeSH
- Check Tag
- skot anatomie a histologie MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although improvements in culture system have enhanced in vitro embryo production, success rates are still not adequate. The reasons for developmental arrest of a part of in vitro produced embryos are unknown, but are connected in part with low cytoplasmic competence of oocytes. The immaturity of cytoplasm can negatively influence fertilization efficiency and subsequent progression through embryonic genome activation (EGA), which are necessary steps in further pre-implantation development. A large number of studies have compared mRNA abundance among oocytes with different developmental competence with the aim to find markers of the normal embryo development. The amount of mitochondrial DNA (mtDNA) and mRNA for mitochondrial transcriptional factors directing oxidative phosphorylation belongs to such promising markers. Nevertheless, recently published studies revealed that the mammalian embryo is able to compensate for a reduced level of mtDNA in oocyte during subsequent pre-implantation development. The search for other molecular markers is in progress. Characterization of oocyte and embryonic mRNA expression patterns during the pre-implantation period, and their relationship to the successful in vitro and in vivo development will be essential for defining the optimized culture conditions or the nuclear transfer protocols. Microarrays technology enables us to reveal the differentially expressed genes during EGA, and to compare the expression profile of in vivo and in vitro produced embryos. Recent evidence indicates that the depletion of the pool of stored maternal mRNAs is critical for subsequent embryo development. All these experiments gradually offer a list of possible candidates for quality and developmental competence markers for mammalian oocytes and pre-implantation embryos.
- MeSH
- genetická transkripce * MeSH
- messenger RNA genetika metabolismus MeSH
- oocyty metabolismus MeSH
- skot embryologie MeSH
- transkriptom * MeSH
- vývojová regulace genové exprese fyziologie MeSH
- zvířata MeSH
- Check Tag
- skot embryologie MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The present study was designed to characterize bovine oocytes with different meiotic competence and atresia levels in terms of their mitochondrial status. Oocyte subpopulations were recovered either from medium (MF) or small (SF) follicles and categorized as healthy, light-atretic and mid-atretic according to oocyte morphology. Mitochondrial activity, morphology and distribution, adenosine triphosphate (ATP) content and expression of mitochondrial transcription factor A (TFAM) and nuclear respiratory factor 1 (NRF1) were assessed before (GV) and after (MII) maturation. The data were related to follicular size regardless of or with regard to oocyte atresia. Regardless of atresia, the MF subpopulation showed a significantly higher mitochondrial activity and frequency of oocytes with granulated mitochondria at GV and clustered mitochondria at MII than the SF subpopulation. With regard to atresia, mitochondrial activity decreased from healthy to mid-atretic oocytes in both MF and SF subpopulations at GV, but in the SF subpopulation at MII, the mitochondrial activity and frequency of oocytes with clustered mitochondria were significantly higher in light-atretic than in healthy oocytes. The light-atretic oocytes also produced more ATP than healthy ones in both SF and MF subpopulations. However, a significantly higher relative abundance of mRNA TFAM was found in SF than MF subpopulations at GV, and this difference remained in mid-atretic oocytes at MII. It can be concluded that meiotic competence and atresia level influence mitochondrial status of immature bovine oocytes. After maturation, healthy oocytes from medium follicles and light-atretic oocytes from small follicles were more developed in terms of mitochondrial status than the other oocytes.
- MeSH
- adenosintrifosfát analýza MeSH
- DNA vazebné proteiny analýza genetika MeSH
- folikulární atrézie fyziologie MeSH
- meióza * MeSH
- messenger RNA analýza MeSH
- mitochondriální proteiny analýza genetika MeSH
- mitochondrie fyziologie ultrastruktura MeSH
- oocyty metabolismus ultrastruktura MeSH
- ovariální folikul chemie MeSH
- skot * MeSH
- transkripční faktor NRF1 analýza genetika MeSH
- transkripční faktory analýza genetika MeSH
- zvířata MeSH
- Check Tag
- skot * MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pre-implantation embryos derived by in vitro fertilization differ in their developmental potential from embryos obtained in vivo. In order to characterize changes in gene expression profiles caused by in vitro culture environment, we employed microarray constructed from bovine oocyte and preimplantation embryo-specific cDNAs (BlueChip, Université Laval, Québec). The analysis revealed changes in the level of 134 transcripts between in vitro derived (cultured in COOK BVC/BVB media) and in vivo derived 4-cell stage embryos and 97 transcripts were differentially expressed between 8-cell stage in vitro and in vivo embryos. The expression profiles of 7 selected transcripts (BUB3, CUL1, FBL, NOLC1, PCAF, GABPA and CNOT4) were studied in detail. We have identified a switch from Cullin 1-like transcript variant 1 to Cullin 1 transcript variant 3 (UniGene IDs BT.36789 and BT.6490, respectively) expressions around the time of bovine major gene activation (8-cell stage). New fibrillarin protein was detected by immunofluorescence already in early 8-cell stage and this detection correlated with increased level of fibrillarin mRNA. The qRT-PCR analysis revealed significant differences in the level of BUB3, NOLC1, PCAF, GABPA and CNOT4 gene transcripts between in vivo derived (IVD) and in vitro produced (IVP) embryos in late 8-cell stage. The combination of these genes represents a suitable tool for addressing questions concerning normal IVD embryo development and can be potentially useful as a marker of embryo quality in future attempts to optimize in vitro culture conditions.
- MeSH
- blastocysta metabolismus MeSH
- embryonální vývoj genetika MeSH
- kulinové proteiny genetika metabolismus MeSH
- kultivace embrya veterinární MeSH
- kultivační média MeSH
- molekulární sekvence - údaje MeSH
- sekvence nukleotidů MeSH
- sekvenční seřazení MeSH
- skot MeSH
- stanovení celkové genové exprese MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- DNA virů izolace a purifikace MeSH
- dysplazie děložního hrdla virologie MeSH
- epidemiologické metody MeSH
- finanční podpora výzkumu jako téma MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádory děložního čípku virologie MeSH
- Papillomaviridae genetika izolace a purifikace MeSH
- vaginální stěr MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- MeSH
- buněčné jadérko fyziologie účinky léků ultrastruktura MeSH
- gama-butyrolakton farmakologie MeSH
- meióza účinky léků MeSH
- oocyty fyziologie účinky léků ultrastruktura MeSH
- ovariální folikul cytologie fyziologie MeSH
- RNA biosyntéza MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- techniky in vitro MeSH
- MeSH
- biomedicínský výzkum MeSH
- buněčné jadérko ultrasonografie MeSH
- buněčné jádro transplantace MeSH
- skot MeSH
- vývoj plodu MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- techniky in vitro MeSH
