Jiří Kaňka

Nucleolar ultrastructure in bovine nuclear transfer embryos.

Nuclear transfer experiments in mammals have attempted to reprogram a donor nucleus to a state equivalent to the zygotic one. Reprogramming of the donor nucleus is, among other features, indicated by a synthesis of ribosomal RNA (rRNA). The initiation of rRNA synthesis is simultaneously reflected in nuclear morphology as a transformation of the nucleolus precursor body into a functional rRNA synthesising nucleolus with a characteristic ultrastructure. We examined nucleolar ultrastructure in bovine in vitro produced (control) embryos and in nuclear transfer embryos reconstructed from a MII phase (nonactivated) or S phase (activated) cytoplasts. Control embryos were fixed at the two‐, four‐, early eight‐ and late eight‐cell stages; nuclear transfer embryos were fixed at 1 and 3 hr post fusion and at the two‐, four‐, and eight‐cell stages. Control embryos possessed a nucleolar precursor body throughout all three cell cycles. In the eight‐cell stage embryo, a primary vacuole appeared as an electron lucid area originating in the centre of the nucleolar precursor body. In nuclear transfer embryos reconstructed from nonactivated cytoplasts, the nuclear envelope was fragmented or completely broken down at 1 hr after fusion and, by 3 hr after fusion, it was restored again. At this time, the reticulated fibrillo‐granular nucleolus had an almost round shape. The nucleolar precursor body seen in the two‐cell stage nuclear transfer embryos consisted of intermingled filamentous components and secondary vacuoles. A nucleolar precursor body typical for the two‐cell stage control embryos was never observed. None of the reconstructed embryos of this group reached the eight‐cell stage. Nuclear transfer embryos reconstructed from activated cytoplasts, in contrast, exhibited a complete nuclear envelope at all time intervals after fusion. In the two‐cell stage nuclear transfer embryo, the originally reticulated nucleolus of the donor blastomere had changed into a typical nucleolar precursor body consisting of a homogeneous fibrillar structure. A primary vacuole appeared in the four‐cell stage nuclear transfer embryos, which was one cell cycle earlier than in control embryos. Only nuclear transfer embryos reconstructed from activated cytoplasts underwent complete remodelling of the nucleolus. The reorganisation of the donor nucleolar architecture into a functionally active nucleolus was observed as early as in the four‐cell stage nuclear transfer embryo. These ultrastructural observations were correlated with our autoradiographic data on the initiation of RNA synthesis in nuclear transfer embryos. Mol. Reprod. Dev. 52:253–263, 1999.

Gene expression during minor genome activation in preimplantation bovine development

The main goal of this study was to identify mRNA transcripts whose content increases during bovine minor embryonic genome activation. We compared the gene expression profile of the bovine 4-cell-stage embryo and MII oocyte using the technique of suppression subtractive hybridization. Differentially expressed amplicons were subcloned, and 60 of them were sequenced. The resulting DNA sequences were compared with GenBank databases using BLAST search. The expression of five differentially expressed genes with an apparent function in cell cycle progression, chromatin remodeling, and splicing or translation initiation was further characterized by a real-time RT-PCR. Centromere protein F, 350/400ka (CENPF), and splicing factor arginine/serine-rich 3 (SRFS3) show an increase in mRNA content during the 2- to 4-cell and late 8-cell stages. For the high mobility group nucleosomal binding domain 2 (HMGN2), the level of mRNA increases in 2- to 4-cell and morula embryos. The transcription of splicing factor SRFS3 is alpha-amanitin sensitive both during 4-cell and late 8-cell stages. The transcription of CENPF and HMGN2 is alpha-amanitin sensitive only at late 8-cell stage and morula, respectively. SRFS3 represents the first described gene with an important function in preimplantation development, which is also expressed during bovine minor genome activation, and it is alpha-amanitin sensitive during this period. All described genes can play an important role in the preimplantation development of bovine embryos.

Silencing CENPF in bovine preimplantation embryo induces arrest at 8-cell stage

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.

Core-cladding mode coupling and recoupling in photonic crystal fiber for enhanced overlap of evanescent field using long-period gratings

Excitation of cladding modes has been achieved using long-period (LPGs) inscribed in an endlessly single-mode photonic crystal fiber (ESM PCF) by CO(2) laser irradiation. Core-cladding mode coupling and recoupling has resulted in marked improvement in the evanescent field overlap throughout the cladding air channels in the PCF-LPG, compared to the PCF alone. Our numerical simulation has shown that design optimization of the PCF-LPG configuration can lead to a field power overlap as high as 22% with a confinement loss of less than 1 dB/m in the cladding mode.

Sol-Gel Fabrication and Properties of Silica Cores of Optical Fibers Doped with Yb3+, Er3+, Al2 O3 or TiO2

Optical cores of preforms for drawing optical fibers doped with Er3+ and Yb3+ were fabricated by the sol-gel method with the aim of increasing the thickness of glass layers coated in a single coating cycle and to determine the relation between the preparation conditions and optical properties of the fibers. Al2O3-P2O5-SiO2 and TiO2-P2O5-SiO2 glasses have been studied as matrices for entrapping the rare-earth elements. Input sols have been prepared from silicon and titanium alkoxides, AlCl3, ErCl3, YbCl3, POCl3, water and a modifier under acidic catalysis of HCl. The sols were coated on the inner wall of a silica substrate tube and the gel layers were sintered at high temperatures up to 2000°C after which the tube was collapsed into the preform. Continuous and homogenous glass films with the maximum thickness of about 8 μm were fabricated. The influence of high-temperature heat treatment of the layers on their composition and optical attenuation was observed. The amplified stimulated emission of Er3+ around 1.55 μm was measured under the excitation of the fibers by an Nd : YAG laser at 1.064 μm.

Workplace for manufacturing devices based on optical fiber tapers

Many important optical fiber components are based on tapered optical fibers. A taper made from a single-mode optical fiber can be used, e.g., as a chemical sensor, bio-chemical sensor, or beam expander. A fused pair of tapers can be used as a fiber directional coupler. Fiber tapers can be fabricated in several simple ways. However, a tapering apparatus is required for more sophisticated fabrication of fiber tapers. The paper deals with fabrication and characterization of fiber tapers made from a single-mode optical fiber. A tapering apparatus was built for producing devices based on fiber tapers. The apparatus is universal and enables one to taper optical fibers of different types by a method utilizing stretching a flame-heated section of a silica fiber. Fiber tapers with constant waist length and different waist diameters were fabricated. The transition region of each fiber taper monotonically decreased in diameter along its length from the untapered fiber to the taper waist. The fiber tapers were fabricated with a constant drawing velocity, while the central zone of the original single-mode fiber was heated along a constant length. The spectral transmissions of the manufactured fiber tapers with different parameters were measured by the cut-back method.

Time-domain simulation of power transients in Raman fibre amplifiers: Research Articles

In this paper, a numerical technique suitable for characterizing a wide variety of interconnection configurations printed on cylindrical surfaces, is presented. The interconnection lines may have either finite or infinitesimal cross-sections. To model cylindrical interfaces, suitable space-domain integral equations are formulated to represent the potential on conductors and electric field at dielectric interfaces. The solution of the integral equations is then obtained numerically by applying the method of moments (MOM). The objective of this approach is to determine the capacitance matrix of cylindrical interconnection systems with different configurations. From the capacitance matrix, other quantities such as characteristic impedance, coupling coefficient and effective permittivity can be determined. The numerical technique described in this paper is implemented as a general computer program. Various circular cylindrical as well as elliptical cylindrical structures have been solved including microstrip lines and coplanar waveguide lines. The results obtained compare very well with other published data. Copyright

Er-DOPED TWIN-CORE FIBRE COUPLER AS A SATURABLE-ABSORBER-BASED NARROW-BAND FILTER FOR FIBRE LASERS*)

We have proposed and demonstrated experimentally that an Er-doped twin-core fibre (ErTCF) can be employed as a tracking narrow-band filter in fibre ring lasers for line narrowing and enhancement of the spectral stability. The device mimics a saturable absorber with inhomogeneous broadening. The slow erbium ion saturation dynamics in the ErTCF prevents wavelength hopping whilst it enables the filter to track the operation wavelength on its tuning. We have improved the device operation by utilizing the inherent filtering effect of a twin-core fibre coupler.

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.

Microstructure optical fibres for detection of gaseous analytes

This paper deals with a steering-wheel microstructure optical fibre designed prepared and tested for detection of gaseous analytes. The inner structure of the steering-wheel microstructure fibre consists of a thin silica core that is surrounded by three cladding holes. Numerical simulations showed that the evanescent wave of the guided fundamental mode at a wavelength of 1550 nm penetrates into the cladding holes. The calculated overlap of the evanescent wave of guided mode with the cladding holes of 0.78% can suitably be employed for gaseous analytes detection. The prepared steeringwheel microstructure fibre was experimentally tested for detection of toluene vapors flowing in the cladding holes of the fibre. It has been proved that this type of microstructure fibre can be used for detection of gaseous analytes such as toluene in nitrogen or in air in concentrations of about 0.1 mol.%.