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.

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.

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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.

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.

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