SCF Ligases and Their Functions in Oogenesis and Embryogenesis-Summary of the Most Important Findings throughout the Animal Kingdom.

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, SCFßTrCP and SCFSEL-10/FBXW7 are among the most important and best researched ligases during early development. SCFßTrCP 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.

Physiologically relevant miRNAs in mammalian oocytes are rare and highly abundant.

miRNAs, ~22nt small RNAs associated with Argonaute (AGO) proteins, are important negative regulators of gene expression in mammalian cells. However, mammalian maternal miRNAs show negligible repressive activity and the miRNA pathway is dispensable for oocytes and maternal-to-zygotic transition. The stoichiometric hypothesis proposed that this is caused by dilution of maternal miRNAs during oocyte growth. As the dilution affects miRNAs but not mRNAs, it creates unfavorable miRNA:mRNA stoichiometry for efficient repression of cognate mRNAs. Here, we report that porcine ssc-miR-205 and bovine bta-miR-10b are exceptional miRNAs, which resist the diluting effect of oocyte growth and can efficiently suppress gene expression. Additional analysis of ssc-miR-205 shows that it has higher stability, reduces expression of endogenous targets, and contributes to the porcine oocyte-to-embryo transition. Consistent with the stoichiometric hypothesis, our results show that the endogenous miRNA pathway in mammalian oocytes is intact and that maternal miRNAs can efficiently suppress gene expression when a favorable miRNA:mRNA stoichiometry is established.

MicroRNA dilution during oocyte growth disables the microRNA pathway in mammalian oocytes.

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.

The neglected part of early embryonic development: maternal protein degradation.

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.

Global transcriptome analysis of porcine oocytes in correlation with follicle size.

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.

Finding of bands of higher molecular weight than expected in three proteins in bovine preimplantation embryos.

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.

Expression of selected mitochondrial genes during in vitro maturation of bovine oocytes related to their meiotic competence.

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.

microRNA expression profile in porcine oocytes with different developmental competence derived from large or small follicles.

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.

Inhibition of Skp1-Cullin-F-box complexes during bovine oocyte maturation and preimplantation development leads to delayed development of embryos†.

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.

Potential Involvement of SCF-Complex in Zygotic Genome Activation During Early Bovine Embryo Development.

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.

Strategy and method for nanoporous cladding formation on silica optical fiber.

We demonstrate the proof of an innovative concept of fabricating nanostructured aluminum oxide cladding on silica optical fiber. Our fabrication strategy entails freeze-coating aluminum on silica fiber and its subsequent anodization, resulting in the formation of anodized aluminum oxide (AAO) cladding with highly organized nanopore channels vertically aligned to the fiber axis. We show that the structure (diameter of pore channels and the porosity) of AAO cladding can be controlled by varying anodization conditions such as the type and concentration of electrolyte solutions and applied voltage. The versatility of AAO as a cladding with tunable structural and optical characteristics and/or a host of other functional nanostructures within the pore channels has the potential to enable a new class of specialty optical fiber for new sensor architecture and applications.

Characterization of SCF-Complex during Bovine Preimplantation Development.

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.

Detection of genes associated with developmental competence of bovine oocytes.

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.

Lab-on-fiber optofluidic platform for in situ monitoring of drug release from therapeutic eluting polyelectrolyte multilayers.

A lab-on-fiber (LOF) optofluidic platform that provides physiologically relevant microenvironment was developed by integrating a long period grating (LPG) coupled with high order cladding mode to achieve high index sensitivity and a liquid-tight capillary tube assembly as a microfluidic chamber for LPG to mimic physiologically relevant microenvironment. We demonstrate the utility of LOF for in situ monitoring the construction of the [chitosan (CHI)/poly (acrylic acid) (PAA)/gentamicin sulfate (GS)/PAA]n multilayers at monolayer resolution as well as evaluating the rate of GS release at a flow rate of 0.127 mL/min at 37 °C in real time. We reveal that GS is released at a faster rate under the dynamic flow condition than in a static medium. Our findings underscore the importance of conducting drug release studies in physiologically relevant conditions.

Long-period gratings inscribed in photonic crystal fiber by symmetric CO(2) laser irradiation: erratum.

This erratum amends the wrongly cited NSF grant number in acknowledgment section in our publication.

Advantage of multi-mode sapphire optical fiber for evanescent-field SERS sensing.

An unclad, multi-mode single crystal sapphire fiber was used as a platform, and immobilized colloidal Ag nanoparticles (NPs) were used as enabler, for evanescent-field fiber-optic sensing via surface-enhanced Raman scattering (SERS) of Rhodamine 6G (R6G) solution. The dependence of the measured Raman intensity on NP coverage density (to a maximum of 120 particles/µm²) as well as the coverage length (to a maximum of 6 cm) was investigated. We demonstrate the utility of SERS-active sapphire fibers for sensitive measurements (10⁻8 M R6G). We further reveal, with the aid of theoretical analysis, that multi-mode fiber offers a significant advantage compared to its single-mode counterpart because the former allows two orders of magnitude higher particle coverage density than the latter to maximize SERS benefit, while maintaining the dominance of Raman gain despite the competitive interplay of NP-induced absorption and scattering loss along the interaction path length.

Long-period gratings inscribed in photonic crystal fiber by symmetric CO2 laser irradiation.

Long-period gratings (LPGs) inscribed in endlessly single mode (ESM) photonic crystal fibers (PCFs) with symmetric and asymmetric CO2 laser irradiation are investigated both numerically and experimentally. Parallel results from conventional single mode fibers (SMFs) are presented for comparison. Theoretical predictions, transmission measurements, and near-field imaging indicate that, regardless of the fiber type, symmetric index perturbation induced by laser irradiation with the aid of a 120° gold-coated reflecting mirror results in LP(0n) symmetric mode coupling, while asymmetric irradiation without using the mirror leads to LP(1n) asymmetric mode coupling. Our results show that, because of the azimuthally anisotropic hexagonal cladding structure, symmetric irradiation yields far more reproducible LPGs in PCFs than asymmetric irradiation. On the other hand, the irradiation symmetry has little effect on the reproducibility of LPGs inscribed in SMFs due to the isotropy of its all-solid cladding structure.

Long-period grating and its cascaded counterpart in photonic crystal fiber for gas phase measurement.

Regular and cascaded long period gratings (LPG, C-LPG) of periods ranging from 460 to 590 µm were inscribed in an endlessly single mode photonic crystal fiber (PCF) using CO(2) laser for sensing measurements of helium, argon and acetylene. High index sensitivities in excess of 1700 nm/RIU were achieved in both grating schemes with a period of 460 µm. The sharp interference fringes in the transmission spectrum of C-PCF-LPG afforded not only greatly enhanced sensing resolution, but also accuracy when the phase-shift of the fringe pattern is determined through spectral processing. Comparative numerical and experimental studies indicated LP(01) to LP(03) mode coupling as the principal coupling step for both PCF-LPG and C-PCF-LPG with emergence of multi-mode coupling at shorter grating periods or longer resonance wavelengths.

Photonic crystal fiber for layer-by-layer assembly and measurements of polyelectrolyte thin films.

The cladding air channels of an endlessly single-mode photonic crystal fiber (PCF) and the high-index sensitivity of its long-period gratings (LPG) inscribed by CO(2) laser have been exploited to deposit poly(vinyl pyrrolidone) (PVPON)/poly(methacrylic acid) (PMAA) polyelectrolyte thin films via layer-by-layer assembly (LbL) and to measure the deposition process. We show that LbL can be controllably carried out within the axially aligned air channels. PCF-LPG is highly sensitive to the LbL process as reflected by ~1.625 nm shift in the resonance wavelength per polyelectrolyte layer incorporated. PCF-LPG is also very robust for in situ monitoring of the release of PVPON from cross-linked polyelectrolytes, which results in the formation of pH-responsive PMAA hydrogel. PCF-LPG containing the hydrogel exhibits well-behaved response to changes in solution pH over 2 to 7.5. We demonstrate that PCF-LPG is 2 orders of magnitude more sensitive than its traditional all-solid counterpart through parallel investigation.

Bovine preimplantation embryos with silenced nucleophosmin mRNA are able to develop until the blastocyst stage.

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.