Influence of nitric oxide during maturation on bovine oocyte meiosis and embryo development in vitro

The effect of s-nitroso-N-acetyl-1,1-penicillamine (SNAP, a nitric oxide donor) during in vitro maturation (IVM) on nuclear maturation and embryo development was investigated. The effect of increasing nitric oxide (NO) during prematuration or maturation, or both, on embryo development was also assessed. 10(-3) M SNAP nearly blocked oocytes reaching metaphase II (MII) (7%, P < 0.05) while 10(-5) M SNAP showed intermediate proportions (55%). For 10(-7) M SNAP and controls (without SNAP), MII percentages were similar (72% for both, P > 0.05), but superior to the other treatment groups (P < 0.05). Blastocyst development, however, was not affected (38% for all treatments, P < 0.05). TUNEL-positive cells in hatched blastocysts (Day 9) increased when IVM included 10(-5) M SNAP (8 v. 3 to 4 cells in the other treatments, P > 0.05), without affecting total cell numbers (240 to 291 cells, P > 0.05). When oocytes were prematured followed by IVM with or without 10(-7) M SNAP, during either culture period or both, blastocyst development was similar (26 to 40%, P > 0.05). When SNAP was included during both prematuration and IVM, the proportion of Day 9 hatched embryos increased (28% v. 14 to 19% in the other treatments, P < 0.05). Apoptotic cells, however, increased when SNAP was included (6 to 10 cells) in comparison to prematuration and maturation without SNAP (3 cells, P < 0.05). NO may be involved in meiotic progression and apoptosis during embryo development.

Control of oocyte maturation

Oocyte maturation is a complex process involving nuclear and cytoplasmic maturation. The nuclear maturation is a chromosomal segregation and the cytoplasmic maturation involves the reorganization of the cytoplasmic organelles, mRNA transcription and storage of proteins to be used during fertilization and early embryo development. The mechanism of oocyte maturation in vivo and in vitro still are not totally understood. However it is generally accepted that the second messenger cyclic adenosine monophosphate (cAMP) plays a critical role in the maintenance of meiotic blockage of mammalian oocytes. A relative increase in the level of cAMP within the oocyte is essential for maintaining meiosis block, while a decrease in cAMP oocyte concentration allows the resumption of meiosis. The oocyte cAMP concentration is regulated by a balance of two types of enzymes: adenylate cyclase (AC) and phosphodiesterases (PDEs), which are responsible for the synthesis and degradation of cAMP, respectively. After being synthesized by AC in cumulus cells, cAMP are transferred to the oocyte through gap junctions. Thus, specific subtypes PDEs are able to inhibit or attenuate the spontaneous meiotic maturation of oocytes with PDE4 primarily involved in the metabolism of cAMP in granulosa cells and PDE3 in the oocyte. Although the immature oocytes can resume meiosis in vitro, after being removed from antral follicles, cytoplasmic maturation seems to occur asynchronously with nuclear maturation. Therefore, knowledge of the oocyte maturation process is fundamental for the development of methodologies to increase the success of in vitro embryo production and to develop treatments for various forms of infertility. This review will present current knowledge about the maintenance of the oocyte in prophase arrest, and the resumption of meiosis during oocyte maturation, focusing mainly on the changes that take place in the oocyte.

The Drosophila cdc25 homolog twine is required for meiosis.

We have identified a second cdc25 homolog in Drosophila. In contrast to string (the first homolog identified in Drosophila) this second homolog, twine, does not function in the mitotic cell cycle, but is specialized for meiosis. Expression of twine was observed exclusively in male and female gonads. twine transcripts are present in germ cells during meiosis, and appear only late during gametogenesis, well after the end of the mitotic germ cell divisions. The sterile Drosophila mutant, mat(2)synHB5, which had previously been isolated and mapped to the same genomic region as twine (35F), was found to carry a missense mutation in the twine gene. This missense mutation in twine abolished its ability to complement a mutation in Schizosaccharomyces pombe cdc25. Phenotypic analysis of mat(2)synHB5 mutant flies revealed a complete block of meiosis in males and severe meiotic defects in females.

Meiotic inhibition of bovine oocytes in medium supplemented with a serum replacer and hormones: effects on meiosis progression and developmental capacity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

SPO21 Is Required for Meiosis-specific Modification of the Spindle Pole Body in Yeast

During meiosis II in the yeast Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body changes from a site of microtubule initiation to a site of de novo membrane formation. These membranes are required to package the haploid meiotic products into spores. This functional change in the spindle pole body involves the expansion and modification of its cytoplasmic face, termed the outer plaque. We report here that SPO21 is required for this modification. The Spo21 protein localizes to the spindle pole in meiotic cells. In the absence of SPO21 the structure of the outer plaque is abnormal, and prospore membranes do not form. Further, decreased dosage of SPO21 leaves only two of the four spindle pole bodies competent to generate membranes. Mutation of CNM67, encoding a known component of the mitotic outer plaque, also results in a meiotic outer plaque defect but does not block membrane formation, suggesting that Spo21p may play a direct role in initiating membrane formation.

Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I.

Synthesis and structural characterization of block and random low molecular weight copolymers composed of L-lactic acid and isosorbide succinate moieties

Isosorbide succinate moieties were incorporated into poly(L-lactide) (PLLA) backbone in order to obtain a new class of biodegradable polymer with enhanced properties. This paper describes the synthesis and characterization of four types of low molecular weight copolymers. Copolymer I was obtained from monomer mixtures of L-lactide, isosorbide, and succinic anhydride; II from oligo(L-lactide) (PLLA), isosorbide, and succinic anhydride; III from oligo(isosorbide succinate) (PIS) and L-lactide; and IV from transesterification reactions between PLLA and PIS. MALDI-TOFMS and 13C-NMR analyses gave evidence that co-oligomerization was successfully attained in all cases. The data suggested that the product I is a random co-oligomer and the products II-IV are block co-oligomers.

Universal and nonuniversal contributions to block-block entanglement in many-fermion systems

We calculate the entanglement entropy of blocks of size x embedded in a larger system of size L, by means of a combination of analytical and numerical techniques. The complete entanglement entropy in this case is a sum of three terms. One is a universal x- and L-dependent term, first predicted by Calabrese and Cardy, the second is a nonuniversal term arising from the thermodynamic limit, and the third is a finite size correction. We give an explicit expression for the second, nonuniversal, term for the one-dimensional Hubbard model, and numerically assess the importance of all three contributions by comparing to the entropy obtained from fully numerical diagonalization of the many-body Hamiltonian. We find that finite-size corrections are very small. The universal Calabrese-Cardy term is equally small for small blocks, but becomes larger for x > 1. In all investigated situations, however, the by far dominating contribution is the nonuniversal term stemming from the thermodynamic limit.

Novel blue emitters based on pi-conjugated block copolymers

A series of new phenyl-based conjugated copolymers has been synthesized and investigated by vibrational and photoluminescence spectroscopy (PL). The materials are: poly( 1,4-phenylene-alt-3,6-pyridazine) (COP-PIR), poly(9,9-dioctylfluorene)-co-quaterphenylene (COP-PPP) and poly[(1,4-phenylene-alt-3,6-pyridazine)-co-(1,4-phenylene-alt-9,9-dioctylfluorene)] (COP-PIR-FLUOR), with 3.5% of fluorene. COP-PPP and COP-PIR-FLUOR have high fluorescence quantum yields in solution. Infrared and Raman spectra were used to check the chemical structure of the compounds. The copolymers exhibit blue emission ranging front 2.8 to 3.6 eV when excited at E(exc)=4.13 eV. Stokes-shift Values were estimated on pristine samples in their condensed state from steady-state PL-emission and PL-excitation spectra. They suggest a difference in the torsional angle between the molecular configuration of the polymer blocks at the absorption and PL transitions and also in the photoexcitation diffusion. Additionally, the time-resolved PL of these materials has been investigated by using 100 fs laser pulses at E(exc)=4.64 eV and a streak camera. Results show very fast biexponential kinetics for the two fluorene-based polymers with decay times below 300 ps indicating both intramolecular, fast radiative recombination and migration of photogenerated electron-hole pairs. By contrast, the PL of COP-PIR is less intense and longer lived, indicating that excitons are confined to the chains in this polymer. (C) 2008 Elsevier B.V. All rights reserved.

Linear viscoelasticity of styrenic block copolymers-clay nanocomposites

In this work, the rheological behavior of block copolymers with different morphologies (lamellar, cylindrical, spherical, and disordered) and their clay-containing nanocomposites was studied using small amplitude oscillatory shear. The copolymers studied were one asymmetric starblock styrene-butadiene-styrene copolymer and four styrene-ethylene/butylenes-styrene copolymers with different molecular architectures, one of them being modified with maleic anhydride. The nanocomposites of those copolymers were prepared by adding organophilic clay using three different preparation techniques: melt mixing, solution casting, and a hybrid melt mixing-solution technique. The nanocomposites were characterized by X-ray diffraction and transmission electron microscopy, and their viscoelastic properties were evaluated and compared to the ones of the pure copolymers. The influence of copolymer morphology and presence of clay on the storage modulus (G`) curves was studied by the evaluation of the changes in the low frequency slope of log G` x log omega (omega: frequency) curves upon variation of temperature and clay addition. This slope may be related to the degree of liquid- or solid-like behavior of a material. It was observed that at temperatures corresponding to the ordered state, the rheological behavior of the nanocomposites depended mainly on the viscoelasticity of each type of ordered phase and the variation of the slope due to the addition of clay was small. For temperatures corresponding to the disordered state, however, the rheological behavior of the copolymer nanocomposites was dictated mostly by the degree of clay dispersion: When the clay was well dispersed, a strong solid-like behavior corresponding to large G` slope variations was observed.

WATER ACTIVITY OF AQUEOUS SOLUTIONS OF ETHYLENE OXIDE-PROPYLENE OXIDE BLOCK COPOLYMERS AND MALTODEXTRINS

The water activity of aqueous solutions of EO-PO block copolymers of six different molar masses and EO/PO ratios and of maltodextrins of three different molar masses was determined at 298.15 K. The results showed that these aqueous solutions present a negative deviation from Raoult`s law. The Flory-Huggins and UNIFAC excess Gibbs energy models were employed to model the experimental data. While a good agreement was obtained with the Flory-Huggins equation, discrepancies were observed when predicting the experimental behavior with the UNIFAC model. The water activities of ternary systems formed by a synthetic polymer, maltodextrin and water were also measured and used to test the predictive capability of both models.

Removal of selected metals from drinking water using modified powdered block carbon

This paper presents the possible alternative removal options for the development of safe drinking water supply in the trace elements affected areas. Arsenic and chromium are two of the most toxic pollutants, introduced into natural waters from a variety of sources and causes various adverse effects on living bodies. Performance of three filter bed method was evaluated in the laboratory. Experiments have been conducted to investigate the sorption of arsenic and chromium on carbon steel and removal of trace elements from drinking water with a household filtration process. The affinity of the arsenic and chromium species for Fe/Fe(3)C (iron/iron carbide) sites is the key factor controlling the removal of the elements. The method is based on the use of powdered block carbon (PBC), powder carbon steel and ball ceramic in the ion-sorption columns as a cleaning process. The PBC modified is a satisfactory and practical sorbent for trace elements (arsenite and chromate) dissolved in water.

The sorption of toxic elements onto natural zeolite, synthetic goethite and modified powdered block carbon

Inorganic elements analyses of Carapicuiba lake reveal that As, Cr, Pb and Mn are above the recommended drinking water standards. The mean total concentrations of toxic elements in surface water decrease in the order Mn > Cr > Pb > As. At elevated concentrations, toxic elements like Cr can accumulate in soils and enter the food chain, leading to serious health hazards and threatening the long-term sustainability of the local ecosystem. Absorbing materials has often been used to improve water quality. In this investigation three types of material were studied: the natural zeolite (mordenite); synthetic goethite and the powdered block carbon modified. The adsorption of Pb(2+) and Mn(2+) onto natural zeolite as a function of their concentrations was studied at 24 degrees C by varying the metal concentration from 100 to 400 mg L(-1) while keeping all other parameters constant. The low-cost zeolites removed Pb from water without any pretreatment at pH values <6. The maximum adsorption attained was as follows: Pb(2+) 78.7% and Mn(2+) 19.6%. The modified powdered block carbon effectively removed As(V) and Cr(VI) while goethite removed more chromate than arsenate in the pH range 5-6. Results of this study will be used to evaluate the application these materials for the treatment of the Carapicuiba lake`s water.

Enhanced fibroblast adhesion and proliferation on electrospun fibers obtained from poly(isosorbide succinate-b-L-lactide) block copolymers

Block copolymers containing isosorbide succinate and L-lactic acid repeating units with different mass compositions were synthesized in two steps: bulk ring-opening copolymerization from L-lactide and poli(isosorbide succinate) (PIS) preoligomer, in the presence of tin(II) 2-ethylhexanoate as catalyst. followed by chain extension in solution by using hexamethylene diisocyanate. Poly(L-lactide) (PLLA) and a chain extension product from PIS were also obtained, for comparison. SEC, (1)H and (13)C NMR, MALDI-TOFMS, WAXD, DSC, TG, and contact angle measurements were used in their characterization. The incorporation of isosorbide succinate into PLLA main backbone had minor effect on the thermal stability and the T(g) of the products. However, it reduced the crystallinity and increased the surface energy in relation to PLLA. Nonwoven mats of the block copolymers and PLLA obtained by electrospinning technique were submitted to fibroblasts 3T3-L1 cell culture. The copolymers presented enhanced cell adhesion and proliferation rate as revealed by MTT assay and SEM images. (C) 2009 Elsevier Ltd. All rights reserved.

The line structure of balanced ternary designs with block size three, index three and rho(2) = 1, 2

In this paper necessary and sufficient conditions for a vector to be the fine structure of a balanced ternary design with block size 3, index 3 and rho(2) = 1 and 2 are determined with one unresolved case.