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 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

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 fully grown mammalian oocyte is transcriptionally quiescent and utilizes only transcripts synthesized and stored during early development. However, we find that an abundant RNA population is retained in the oocyte nucleus and contains specific mRNAs important for meiotic progression. Here we show that during the first meiotic division, shortly after nuclear envelope breakdown, translational hotspots develop in the chromosomal area and in a region that was previously surrounded the nucleus. These distinct translational hotspots are separated by endoplasmic reticulum and Lamin, and disappear following polar body extrusion. Chromosomal translational hotspots are controlled by the activity of the mTOR-eIF4F pathway. Here we reveal a mechanism that-following the resumption of meiosis-controls the temporal and spatial translation of a specific set of transcripts required for normal spindle assembly, chromosome alignment and segregation.

• MeSH
• časové faktory MeSH
• down regulace MeSH
• eukaryotický iniciační faktor 4F metabolismus   MeSH
• fertilizace MeSH
• jaderný obal metabolismus   MeSH
• lidé MeSH
• meióza MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši MeSH
• nestabilita genomu MeSH
• oocyty metabolismus   MeSH
• proteosyntéza * MeSH
• RNA čepičky metabolismus   MeSH
• savčí chromozomy metabolismus   MeSH
• savci metabolismus   MeSH
• signální transdukce * MeSH
• TOR serin-threoninkinasy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

This study was conducted to investigate the effect of silencing nucleophosmin in the development of in vitro-produced bovine embryos. Nucleophosmin is an abundant multifunctional nucleolar phosphoprotein that participates, for example, in ribosome biogenesis or centrosome duplication control. We showed that although the transcription of embryonic nucleophosmin started already at late eight-cell stage, maternal protein was stored throughout the whole preimplantation development and was sufficient for the progression to the blastocyst stage. At the beginning of embryogenesis, translation occurs on maternally derived ribosomes, the functionally active nucleoli emerge during the fourth cell cycle in bovines. We found that nucleophosmin localisation reflected the nucleolar formation during bovine preimplantation development. The protein was detectable from the beginning of embryonic development. Before embryonic genome activation, it was dispersed throughout the nucleoplasm. The typical nucleolar localisation emerged with the formation of active nucleoli. At the blastocyst stage, nucleophosmin tended to localise especially to the trophectoderm. To see for how long is maternal nucleophosmin preserved, we silenced the nucleophosmin mRNA using RNA interference approach. Although a large portion of nucleophosmin was degraded in embryos with silenced nucleophosmin mRNA, an amount sufficient for normal development was preserved and we detected only a temporal delay in nucleophosmin relocalisation to nucleoli. Moreover, we observed no defects in nuclear shape or cytoskeleton previously found in somatic cells and only a non-significant decrease in embryonic developmental competence. Thus, our results show that the preserved amount of maternal nucleophosmin is sufficient for preimplantation development of bovine embryo.

• MeSH
• blastocysta fyziologie   ultrastruktura   MeSH
• buněčné jadérko chemie   MeSH
• embryonální vývoj fyziologie   MeSH
• exprese genu MeSH
• jaderné proteiny analýza   genetika   fyziologie   MeSH
• messenger RNA genetika   MeSH
• skot embryologie   MeSH
• umlčování genů * MeSH
• zvířata MeSH
• Check Tag
• skot embryologie   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

• MeSH
• fertilizace in vitro MeSH
• fertilizace MeSH
• oocyty enzymologie   MeSH
• polyubikvitin analýza   MeSH
• skot MeSH
• thiolesterasa ubikvitinu antagonisté a inhibitory   metabolismus   MeSH
• ubikvitin C analýza   MeSH
• zvířata MeSH
• zygota enzymologie   MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Identification of genes that are important for normal preimplantation development is essential for understanding the basics of early mammalian embryogenesis. In our previous study, we have shown that CENPF (mitosin) is differentially expressed during preimplantation development of bovine embryos. CENPF is a centromere-kinetochore complex protein that plays a crucial role in the cell division of somatic cells. To our best knowledge, no study has yet been done on either bovine model, or oocytes and preimplantation embryos. In this study, we focused on the fate of bovine embryos after injection of CENPF double-stranded RNA (dsRNA) into the zygotes. An average decrease of CENPF mRNA abundance by 94.9% or more and an extensive decline in immunofluorescence staining intensity was detected relative to controls. There was no disparity between individual groups in the developmental competence before the 8-cell stage. However, the developmental competence rapidly decreased then and only 28.1% of CENPF dsRNA injected 8-cell embryos were able to develop further (uninjected control: 71.8%; green fluorescent protein dsRNA injected control: 72.0%). In conclusion, these results show that depletion of CENPF mRNA in preimplantation bovine embryos leads to dramatic decrease of developmental competence after embryonic genome activation.

• MeSH
• blastocysta cytologie   metabolismus   fyziologie   MeSH
• buněčné dělení genetika   fyziologie   MeSH
• chromozomální proteiny, nehistonové antagonisté a inhibitory   genetika   metabolismus   MeSH
• embryonální vývoj genetika   fyziologie   MeSH
• fertilizace in vitro veterinární   MeSH
• kontrolní body buněčného cyklu genetika   fyziologie   MeSH
• kultivované buňky MeSH
• mikrofilamentové proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• skot embryologie   genetika   fyziologie   MeSH
• stadium rýhování vajíčka metabolismus   fyziologie   MeSH
• umlčování genů fyziologie   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• skot embryologie   genetika   fyziologie   MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH