BACKGROUND: Oocytes of large animal species isolated from small ovarian follicles (< 2 mm) are less competent to support early embryonic development after in vitro maturation and fertilization than their counterparts isolated from medium-sized and preovulatory follicles. This study aimed to assess the effect of a new maturation medium containing FGF2, LIF, and IGF1 (FLI medium) on the meiotic and developmental competence of pig cumulus-oocytes complexes (COCs) derived from the small and medium-sized follicles. METHODS: The growing oocytes were isolated from 1 to 2 (small follicle; SF) and the fully-grown ones from 3 to 6 (large follicle; LF) mm follicles and matured in a control M199 medium with gonadotropins and EGF and the FLI medium enriched by the triplet of growth factors. The matured oocytes were parthenogenetically activated and cultured to the blastocyst stage. Chromatin configuration before and during the culture and MAP kinase activity were assessed in the oocytes. Finally, the expression of cumulus cell genes previously identified as markers of oocyte quality was assessed. RESULTS: The maturation and blastocyst rates of oocytes gained from LF were significantly higher than that from SF in the control medium. In contrast, similar proportions of oocytes from LF and SF completed meiosis and developed to blastocysts when cultured in FLI. Most of the oocytes freshly isolated from SF possessed germinal vesicles with fine filaments of chromatin (GV0) or chromatin surrounding the nucleolus (GVI; 30%); the oocytes from LF were mainly in GVI (or GVII) exhibiting a few small lumps of chromatin beneath the nuclear membrane. When cultured in the FLI medium for 16 h, an acceleration of the course of maturation in oocytes both from SF and LF compared to the control medium was observed and a remarkable synchrony in the course of chromatin remodeling was noticed in oocytes from SF and LF. CONCLUSIONS: This work demonstrates that the enrichment of culture medium by FGF2, LIF, and IGF1 can enhance the meiotic and developmental competence of not only fully-grown, but also growing pig oocytes and significantly thus expanding the number of oocytes available for various assisted reproductive technology applications.

• Klíčová slova
• Chromatin configuration, Developmental potential, Follicle size, Gene expression, Growth factors, MAPK activation, Oocyte maturation,
• MeSH
• chromatin metabolismus   MeSH
• fibroblastový růstový faktor 2 * farmakologie   metabolismus   MeSH
• IVM techniky * MeSH
• meióza MeSH
• oocyty metabolismus   MeSH
• ovariální folikul MeSH
• prasata MeSH
• těhotenství MeSH
• zvířata MeSH
• Check Tag
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chromatin MeSH
• fibroblastový růstový faktor 2 * MeSH

SCF-dependent proteolysis was first discovered via genetic screening of budding yeast almost 25 years ago. In recent years, more and more functions of SCF (Skp1-Cullin 1-F-box) ligases have been described, and we can expect the number of studies on this topic to increase. SCF ligases, which are E3 ubiquitin multi-protein enzymes, catalyse protein ubiquitination and thus allow protein degradation mediated by the 26S proteasome. They play a crucial role in the degradation of cell cycle regulators, regulation of the DNA repair and centrosome cycle and play an important role in several diseases. SCF ligases seem to be needed during all phases of development, from oocyte formation through fertilization, activation of the embryonic genome to embryo implantation. In this review, we summarize known data on SCF ligase-mediated degradation during oogenesis and embryogenesis. In particular, SCFβTrCP and SCFSEL-10/FBXW7 are among the most important and best researched ligases during early development. SCFβTrCP is crucial for the oogenesis of Xenopus and mouse and also in Xenopus and Drosophila embryogenesis. SCFSEL-10/FBXW7 participates in the degradation of several RNA-binding proteins and thereby affects the regulation of gene expression during the meiosis of C. elegans. Nevertheless, a large number of SCF ligases that are primarily involved in embryogenesis remain to be elucidated.

• Klíčová slova
• SCF ligases, embryogenesis, oogenesis, ubiquitin, ubiquitin-proteasome system,
• MeSH
• biologické modely MeSH
• embryonální vývoj * MeSH
• oocyty cytologie   metabolismus   MeSH
• oogeneze * MeSH
• proteinligasy komplexu SCF metabolismus   MeSH
• substrátová specifita MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• proteinligasy komplexu SCF 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.

• Klíčová slova
• Autophagy, Embryonic genome activation, Maternal to zygotic transition, Proteasome system, Ubiquitin, Ubiquitin ligase,
• 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
• Názvy látek
• proteiny 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.

• Klíčová slova
• IκBα, Molecular weight, Preimplantation embryo, Skp1, p27,
• 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
• Názvy látek
• inhibitor p27 cyklin-dependentní kinasy MeSH
• NFKB inhibitor alfa MeSH
• proteiny asociované s kinázou S-fáze MeSH
• proteiny 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.

• Klíčová slova
• MLN4924, SCF complexes, cumulus cells, early development, oocyte, ubiquitin–proteasome system,
• 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
• Názvy látek
• cyklopentany MeSH
• multiproteinové komplexy MeSH
• pevonedistat MeSH Prohlížeč
• proteinligasy komplexu SCF MeSH
• pyrimidiny 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.

• Klíčová slova
• Immunofluorescence, In situ proximity ligation assay, Preimplantation development, SCF-complex, Western blot, qRT-PCR,
• 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
• práce podpořená grantem MeSH
• Názvy látek
• proteinligasy komplexu SCF MeSH
• ubikvitinligasy 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
• Názvy látek
• Cullin 1 MeSH Prohlížeč
• F-box proteiny MeSH
• kulinové proteiny MeSH
• messenger RNA MeSH
• proteinligasy komplexu SCF MeSH
• proteiny asociované s kinázou S-fáze 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
• Názvy látek
• eukaryotický iniciační faktor 4F MeSH
• messenger RNA MeSH
• RNA čepičky MeSH
• TOR serin-threoninkinasy 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
• nukleofosmin 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
• Názvy látek
• jaderné proteiny MeSH
• messenger RNA MeSH
• nukleofosmin 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
• Názvy látek
• polyubikvitin MeSH
• thiolesterasa ubikvitinu MeSH
• ubikvitin C MeSH