Although our knowledge regarding oocyte quality and development has improved significantly, the molecular mechanisms that regulate and determine oocyte developmental competence are still unclear. Therefore, the objective of this study was to identify and analyze the transcriptome profiles of porcine oocytes derived from large or small follicles using RNA high-throughput sequencing technology. RNA libraries were constructed from oocytes of large (LO; 3-6 mm) or small (SO; 1.5-1.9 mm) ovarian follicles and then sequenced in an Illumina HiSeq4000. Transcriptome analysis showed a total of 14,557 genes were commonly detected in both oocyte groups. Genes related to the cell cycle, oocyte meiosis, and quality were among the top highly expressed genes in both groups. Differential expression analysis revealed 60 up- and 262 downregulated genes in the LO compared with the SO group. BRCA2, GPLD1, ZP3, ND3, and ND4L were among the highly abundant and highly significant differentially expressed genes (DEGs). The ontological classification of DEGs indicated that protein processing in endoplasmic reticulum was the top enriched pathway. In addition, biological processes related to cell growth and signaling, gene expression regulations, cytoskeleton, and extracellular matrix organization were among the highly enriched processes. In conclusion, this study provides new insights into the global transcriptome changes and the abundance of specific transcripts in porcine oocytes in correlation with follicle size.

• MeSH
• genové regulační sítě fyziologie   MeSH
• oocyty metabolismus   MeSH
• oogeneze genetika   MeSH
• ovariální folikul cytologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• prasata genetika   růst a vývoj   MeSH
• signální transdukce genetika   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom * MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

MicroRNAs (miRNAs) are ubiquitous small RNAs guiding post-transcriptional gene repression in countless biological processes. However, the miRNA pathway in mouse oocytes appears inactive and dispensable for development. We propose that marginalization of the miRNA pathway activity stems from the constraints and adaptations of RNA metabolism elicited by the diluting effects of oocyte growth. We report that miRNAs do not accumulate like mRNAs during the oocyte growth because miRNA turnover has not adapted to it. The most abundant miRNAs total tens of thousands of molecules in growing (∅ 40 μm) and fully grown (∅ 80 μm) oocytes, a number similar to that observed in much smaller fibroblasts. The lack of miRNA accumulation results in a 100-fold lower miRNA concentration in fully grown oocytes than in somatic cells. This brings a knock-down-like effect, where diluted miRNAs engage targets but are not abundant enough for significant repression. Low-miRNA concentrations were observed in rat, hamster, porcine and bovine oocytes, arguing that miRNA inactivity is not mouse-specific but a common mammalian oocyte feature. Injection of 250,000 miRNA molecules was sufficient to restore reporter repression in mouse and porcine oocytes, suggesting that miRNA inactivity comes from low-miRNA abundance and not from some suppressor of the pathway.

• MeSH
• buňky 3T3 MeSH
• druhová specificita MeSH
• křečci praví MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• messenger RNA genetika   metabolismus   MeSH
• mikro RNA genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• oocyty cytologie   metabolismus   MeSH
• oogeneze * MeSH
• prasata MeSH
• skot MeSH
• teoretické modely MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• krysa rodu rattus MeSH
• myši MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The degradation of maternally provided molecules is a very important process during early embryogenesis. However, the vast majority of studies deals with mRNA degradation and protein degradation is only a very little explored process yet. The aim of this article was to summarize current knowledge about the protein degradation during embryogenesis of mammals. In addition to resuming of known data concerning mammalian embryogenesis, we tried to fill the gaps in knowledge by comparison with facts known about protein degradation in early embryos of non-mammalian species. Maternal protein degradation seems to be driven by very strict rules in terms of specificity and timing. The degradation of some maternal proteins is certainly necessary for the normal course of embryonic genome activation (EGA) and several concrete proteins that need to be degraded before major EGA have been already found. Nevertheless, the most important period seems to take place even before preimplantation development-during oocyte maturation. The defects arisen during this period seems to be later irreparable.

• MeSH
• embryo nesavčí metabolismus   fyziologie   MeSH
• embryo savčí metabolismus   fyziologie   MeSH
• embryonální vývoj fyziologie   MeSH
• genom fyziologie   MeSH
• lidé MeSH
• oocyty metabolismus   fyziologie   MeSH
• proteiny metabolismus   MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

SummaryWe report here the existence of bands of higher molecular weight after western blot analysis in three proteins - Skp1, p27 and IκBα in bovine preimplantation embryos. This finding is specific to preimplantation embryos (from the 2-cell stage to the blastocyst stage) and not differentiated fibroblast cells in which these bands were of expected molecular weight. We suggest that these bands of higher molecular weight represent a complex of proteins that are characteristic of preimplantation embryos.

• MeSH
• blastocysta cytologie   metabolismus   MeSH
• embryonální vývoj * MeSH
• inhibitor p27 cyklin-dependentní kinasy chemie   metabolismus   MeSH
• molekulová hmotnost MeSH
• NFKB inhibitor alfa chemie   metabolismus   MeSH
• proteiny asociované s kinázou S-fáze chemie   metabolismus   MeSH
• proteiny chemie   metabolismus   MeSH
• skot MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The main goal of this study was to characterize the expression patterns of genes which play a role in mitochondrial DNA biogenesis and metabolism during the maturation of bovine oocytes with different meiotic competence and health. Meiotically more and less competent oocytes were obtained separately either from medium (MF) or small (SF) follicles and categorized according to oocyte morphology into healthy and light-atretic. The four oocyte categories were matured and collected after 0, 3, 7, 16 and 24 h of maturation. Either total RNA or poly(A) RNA were extracted from oocytes and the expression of selected mitochondrial translational factors (TFAM, TFB1M, and TFB2M), MATER, and Luciferase as external standard was assessed using a real-time RT-PCR. The level of TFAM, TFB1M and MATER poly(A) RNA transcripts significantly decreased during maturation in both healthy and light-atretic MF and SF oocytes. On the other hand, the level of TFB2M poly(A) increased during maturation in healthy and light-atretic SF oocytes, in contrast to MF oocytes. The abundance of TFAM total RNA was significantly higher after maturation than that before maturation in all oocyte categories. However, no differences in TFB1M and TFB2M total RNA were found in any oocyte categories. It can be concluded that the gene expression patterns differ in maturing bovine oocytes in dependence on their meiotic competence and health. The TFAM and TFB1M poly(A) RNAs are actively deadenylated at different meiotic stages but TFB2M poly(A) RNA remains elevated in light-atretic less competent oocytes until the completion of meiosis.

• MeSH
• IVM techniky veterinární   MeSH
• mitochondriální DNA biosyntéza   MeSH
• mitochondriální geny * MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• oocyty metabolismus   MeSH
• skot fyziologie   MeSH
• zvířata MeSH
• Check Tag
• skot fyziologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The mechanism of maternal protein degradation during preimplantation development has not been clarified yet. It is thought that a lot of maternal proteins are degraded by the ubiquitin-proteasome system. In this study, we focused on the role of the SCF (Skp1-Cullin-F-box) complexes during early bovine embryogenesis. We inhibited them using MLN4924, an inhibitor of SCF complex ligases controlled by neddylation. Oocytes maturated in MLN4924 could be fertilized, but we found no cumulus cell expansion and a high number of polyspermy after in vitro fertilization. We also found a statistically significant deterioration of development after MLN4924 treatment. After treatment with MLN4924 from the four-cell to late eight-cell stage, we found a statistically significant delay in their development; some of the treated embryos were, however, able to reach the blastocyst stage later. We found reduced levels of mRNA of EGA markers PAPOLA and U2AF1A, which can be related to this developmental delay. The cultivation with MLN4924 caused a significant increase in protein levels in MLN4924-treated oocytes and embryos; no such change was found in cumulus cells. To detect the proteins affected by MLN4924 treatment, we performed a Western blot analysis of selected proteins (SMAD4, ribosomal protein S6, centromeric protein E, P27, NFKB inhibitor alpha, RNA-binding motif protein 19). No statistically significant increase in protein levels was detected in either treated embryos or oocytes. In summary, our study shows that SCF ligases are necessary for the correct maturation of oocytes, cumulus cell expansion, fertilization, and early preimplantation development of cattle.

• MeSH
• blastocysta cytologie   účinky léků   fyziologie   MeSH
• časové faktory MeSH
• cyklopentany farmakologie   MeSH
• embryo savčí MeSH
• embryonální vývoj účinky léků   MeSH
• IVM techniky metody   veterinární   MeSH
• kultivované buňky MeSH
• multiproteinové komplexy antagonisté a inhibitory   metabolismus   MeSH
• oocyty cytologie   účinky léků   fyziologie   MeSH
• oogeneze účinky léků   MeSH
• proteinligasy komplexu SCF antagonisté a inhibitory   metabolismus   fyziologie   MeSH
• pyrimidiny farmakologie   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Oocyte developmental competence is acquired during folliculogenesis and regulated by complex molecular mechanisms. Several molecules are involved in these mechanisms, including microRNAs (miRNAs) that are essential for oocyte-specific processes throughout the development. The objective of this study was to identify the expression profile of miRNAs in porcine oocytes derived from follicles of different sizes using RNA deep sequencing. Oocytes were aspirated from large (LO; 3-6 mm) or small (SO; 1.5-1.9 mm) follicles and tested for developmental competence and chromatin configurations. Small RNA libraries were constructed from both groups and then sequenced in an Illumina NextSeq. 500. Oocytes from the LO group exhibited higher developmental competence and different chromatin configuration compared with oocytes from the SO group. In total, 167 and 162 known miRNAs were detected in the LO and SO groups, respectively. MiR-205, miR-16, miR-148a-3p, and miR-125b were among the top 10 highly expressed miRNAs in both groups. Eight miRNAs were differentially expressed (DE) between both groups. Target gene prediction and pathway analysis revealed 46 pathways that were enriched with miRNA-target genes. The oocyte meiosis pathway and signaling pathways including FoxO, PI3K-Akt, and cAMP were predictably targeted by DE miRNAs. These results give more insights into the potential role of miRNAs in regulating the oocyte development.

• MeSH
• chromatin genetika   metabolismus   MeSH
• mikro RNA biosyntéza   MeSH
• oocyty cytologie   metabolismus   MeSH
• oogeneze fyziologie   MeSH
• prasata MeSH
• sekvenční analýza RNA * MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Proper timing of degradation of maternal protein reserves is important for early embryonic development. The major modification that triggers proteins to degradation is ubiquitination, mediated by ubiquitin-proteolytic system. We focus here on Skp 1-Cul 1-F-box complex (SCF-complex), E3 ubiquitin-ligase, a part of ubiquitin-proteolytic system, which transfer ubiquitin to the substrate protein. We describe in this chapter the methods for the characterization of the expression profile of mRNA and protein of invariant members of SCF-complex and for the definition of SCF-complex activity.

• MeSH
• aktivace transkripce MeSH
• embryonální vývoj * MeSH
• proteinligasy komplexu SCF metabolismus   MeSH
• proteolýza MeSH
• skot MeSH
• stanovení celkové genové exprese MeSH
• ubikvitinligasy metabolismus   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The degradation of maternal proteins is one of the most important events during early development, and it is presumed to be essential for embryonic genome activation (EGA), but the precise mechanism is still not known. It is thought that a large proportion of the degradation of maternal proteins is mediated by the ubiquitin-proteolytic system. In this study we focused on the expression of the Skp1-Cullin1-F-box (SCF) complex, a modular RING-type E3 ubiquitin-ligase, during bovine preimplantation development. The complex consists of three invariable components--Cul1, Skp1, Rbx1 and F-box protein, which determines the substrate specificity. The protein level and mRNA expression of all three invariable members were determined. Cul1 and Skp1 mRNA synthesis was activated at early embryonic stages, at the 4c and early 8c stage, respectively, which suggests that these transcripts are necessary for preparing the embryo for EGA. CUL1 protein level increased from MII to the morula stage, with a significant difference between MII and L8c, and between MII and the morula. The CUL1 protein was localized primarily to nuclei and to a lesser extent to the cytoplasm, with a lower signal in the inner cell mass (ICM) compared to the trophectoderm (TE) at the blastocyst stage. The level of SKP1 protein significantly increased from MII oocytes to 4c embryos, but then significantly decreased again. The localization of the SKP1 protein was analysed throughout the cell and similarly to CUL1 at the blastocyst stage, the staining was less intensive in the ICM. There were no statistical differences in RBX1 protein level and localization. The active SCF-complex, which is determined by the interaction of Cul1 and Skp1, was found throughout the whole embryo during preimplantation development, but there was a difference at the blastocyst stage, which exhibits a much stronger signal in the TE than in the ICM. These results suggest that all these genes could play an important role during preimplantation development. This paper reveals comprehensive expression profile, the basic but important knowledge necessary for further studying.

• MeSH
• blastocysta metabolismus   ultrastruktura   MeSH
• embryonální vývoj genetika   MeSH
• F-box proteiny genetika   metabolismus   MeSH
• fertilizace in vitro MeSH
• genetická transkripce MeSH
• kulinové proteiny genetika   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• oocyty cytologie   růst a vývoj   metabolismus   MeSH
• proteinligasy komplexu SCF genetika   metabolismus   MeSH
• proteiny asociované s kinázou S-fáze genetika   metabolismus   MeSH
• signální transdukce MeSH
• skot MeSH
• spermie cytologie   metabolismus   MeSH
• substrátová specifita MeSH
• vývojová regulace genové exprese MeSH
• zinkové prsty genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The developmental competence of oocytes is acquired progressively during folliculogenesis and is linked to follicular size. It has been documented that oocytes originating from larger follicles exhibit a greater ability to develop to the blastocyst stage. The differences in cytoplasmic factors such as mRNA transcripts could explain the differences in oocyte developmental potential. We used bovine oligonucleotide microarrays to characterize differences between the gene expression profiles of germinal vesicle stage (GV) oocytes with greater developmental competence from medium follicles (MF) and those with less developmental competence from small follicles (SF). After normalizing the microarray data, our analysis found differences in the level of 60 transcripts (≥1.4 fold), corresponding to 49 upregulated and 11 downregulated transcripts in MF oocytes compared to SF oocytes. The gene expression data were classified according to gene ontology, the majority of the genes were associated with the regulation of transcription, translation, the cell cycle, and mitochondrial activity. A subset of 16 selected genes was validated for GV oocytes by quantitative real-time RT-PCR; significant differences (P˂0.01) were found in the level of TAF1A, MTRF1L, ATP5C1, UBL5 and MAP3K13 between the MF and SF oocytes. After maturation the transcript level remained stable for ATP5F1, BRD7, and UBL5 in both oocyte categories. The transcript level of another 13 genes substantially dropped in the MF and/or SF oocytes. It can be concluded that the developmental competence of bovine oocytes and embryos may be a quantitative trait dependent on small changes in the transcription profiles of many genes.

• MeSH
• embryonální vývoj genetika   MeSH
• fertilizace in vitro MeSH
• oocyty metabolismus   fyziologie   MeSH
• oogeneze genetika   MeSH
• ovariální folikul metabolismus   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• skot genetika   fyziologie   MeSH
• stanovení celkové genové exprese MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• skot genetika   fyziologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH