Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.     

Prediction of cutting forces and machining error in ball end milling of curved surfaces -II experimental verification

This paper presents the results of a series of experiments performed to examine the validity of a theoretical model for evaluation of cutting forces and machining error in ball end milling of curved surfaces. The experiments are carried out at various cutting conditions, for both contouring and ramping of convex and concave surfaces. A high precision machining center is used in the cutting tests. In contouring, the machining error is measured with an electric micrometer, while in ramping it is measured on a 3-coordinate measuring machine. The results show that in contouring, the cutting force component that influences the machining error decreases with an increase in milling position angle, while in ramping, the two force components that influence the machining error are hardly affected by the milling position angle. Moreover, in contouring, high machining accuracy is achieved in “Up cross-feed, Up cut” and “Down cross-feed, Down cut” modes, while in ramping, high machining accuracy is achieved in “Left cross-feed, Downward cut” and “Right cross-feed, Upward cut” modes. The theoretical and experimental results show reasonably good agreement.     

A useful method for observing intracellular structures of free and cultured cells by scanning electron microscopy

Scanning electron microscopy (SEM) using osmium-maceration methods has been used for analyzing the three-dimensional structure of cell organelles in tissue samples, but it has been quite difficult to observe free and cultured cells with this technique. The present study was performed to develop a method that can be applied to free and cultured cells for SEM studies of intracellular structures after osmium maceration. The method was also applied to light microscopy (LM) and to transmission electron microscopy (TEM). HeLa cells and human leukocytes were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde followed by an additional fixation with 1% osmium tetroxide. These cells were embedded in low-melting-point agarose. A temperature-responsive dish was also used for collection of cultured cells before embedding. For LM and TEM, the cell-embedded agarose was further embedded in epoxy resin, and semi- and ultrathin sections were examined conventionally. For SEM, the agarose was freeze-fractured in 50% dimethyl sulfoxide, processed for osmium maceration and observed in a high-resolution SEM. Low-melting-point agarose was useful as an embedding medium for SEM, because it was well preserved during prolonged osmication for SEM. Thus, the fine structure of cell organelles was clearly analyzed by SEM after osmium-maceration treatment. These SEM images could also be compared with those of LM and TEM of the agarose-embedded tissues.     

Ordered Mesoporous Silica Derived from Layered Silicates

Here, the development of ordered mesoporous silica prepared by the reaction of layered silicates with organoammonium surfactants is reviewed. The specific features of mesoporous silica are discussed with relation to the probable formation mechanisms. The recent understanding of the unusual structural changes from the 2D structure to periodic 3D mesostructures is presented. The formation of mesophase silicates from layered silicates with single silicate sheets depends on combined factors including the reactivity of layered silicates, the presence of layered intermediates, the variation of the silicate sheets, and the assemblies of surfactant molecules in the interlayer spaces. FSM‐16‐type (p6mm) mesoporous silica is formed via layered intermediates composed of fragmented silicate sheets and alkyltrimethylammonium (C_nTMA) cations. KSW‐2‐type (c2mm) mesoporous silica can be prepared through the bending of the individual silicate sheets with intralayer and interlayer condensation. Although the structure of the silicate sheets changes during the reactions with C_nTMA cations in a complex manner, the structural units caused by kanemite in the frameworks are retained. Recent development of the structural design in the silicate framework is very important for obtaining KSW‐2‐based mesoporous silica with molecularly ordered frameworks. The structural units originating from layered silicates are chemically designed and structurally stabilized by direct silylation of as‐synthesized KSW‐2. Some proposed applications using these mesoporous silica are also summarized with some remarks on the uniqueness of the use of layered silicates by comparison with MCM‐type mesoporous silica.     

Stable and Metastable Phase Relationships in the System ZrO2–ErO1.5

Stable and metastable phase relationships in the system ZrO₂–ErO_1.5 were investigated using homogeneous samples prepared by rapid quenching of melts and by arc melting. The rapidly quenched samples were annealed in air for 48 h at 1690°C or for 8 months at 1315°C. Two tetragonal phases ( t - and t '-phases) were observed after quenching samples heated at 1690°C to a room temperature, whereas one t -phase and cubic ( c -) phase were found in those treated at 1315°C. Since the t '-phase is obtained through a diffusionless transformation during cooling from a high-temperature c -phase, t - and c -phases can coexist at high temperature. The t - and c -phases field spans from 4 to 10 mol% ErO_1.5 at 1690°C and from 3 to 15 mol% ErO_1.5 at 1315°C. The equilibrium temperature T ^t-m ₀ between the t - and monoclinic ( m -) phases estimated from A_s and M_s temperatures decreased with increasing ErO_1.5 contents.     

Application of Macromonomers for Pressure Sensitive Adhesives I. Adhesive and Surface Properties

Adhesive and surface-chemical properties of poly(styrene macromer-butyl acrylate) copolymers, poly (styrene-butyl acrylate macromer) copolymers and poly (styrene-butyl acrylate) random copolymers were investigated. The results were as follows:

(1) The adhesive properties of the copolymers which were prepared by the macromer-technique were different from the random copolymers.

(2) It was found that there were differences in the surface and molecular structure between the copotymers which were prepared by the macromer technique and the random copolymers. These differences were evident from the results of measurements of the surface free energy of the copolymers and of the interfacial tension of water/toluene in the presence of the copolymers.

(3) The reason why there were differences in the surface and molecular structure seemed to be that the copolymers which were prepared by the macromer technique had graft chains and a different inner-structure from the random copolymers. Therefore, it was suggested that the graft copolymers which were prepared by the macromer technique had a phase-separated structure.     

Removal of nonionic surfactant by systems based on chloromethylated polystyrene–poly(ethylene glycol)

For the purpose of obtaining compounds which can remove nonionic surfactants in water, chloromethylated polystyrene (CMPS) was allowed to react with triethylene glycol monomethyl ether (3EGMME), tetraethylene glycol (4EG), poly(ethylene glycol) (PEG) 200, 400, 600, 1000, and 1500. The amount of PEG groups combined with CMPS decreased with an increase in the molecular weight of PEG. The ability of the product to remove polyethylene glycol mono-p-nonyl phenyl ether (NP, n = 10), a nonionic surfactant, solutes in water was greater in the product with PEG of greater MW than that with PEG of smaller MW, and in the product with more PEG groups (mol/g prod.) than in that with less PEG groups. The removal behavior of the products conformed to Freundlich's adsorption formula. Constants of the formula, the effect of temperature on the constants, the effect of combined PEG groups on the removed amount, and the removal manner were studied.     

Establishment and characterization of rat dental epithelial derived ameloblast-lineage clones

Teeth are the hardest tissues covered with enamel produced by ameloblasts. The ameloblast differentiation is controlled by sequential epithelial-mesenchymal interactions during tooth morphogenesis. However, the molecular mechanism of ameloblast differentiation remains unclear. To address this question, we developed an in vitro assay system to evaluate the molecular mechanism of amelogenesis. First, we established dental epithelium-derived clones from 6-day-old rat incisors and established that cells of the clone SRE-G5 were the largest producers of amelogenin mRNA. Next, we analyzed the effects of several chemicals on the amelogenin expression in SRE-G5 cells. Only mitogen-activated protein kinase (MAPK) activators enhanced amelogenin mRNA expression. This finding corresponded to the immunohistochemical data showing the presence of phosphorylated forms of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) during ameloblast differentiation. To examine the roles of MAPK signals, we compared the effects of anisomycin and sodium salicylate on the expression of tooth-related differentiation markers. Both anisomycin and sodium salicylate induced amelogenin, Abcg2, and Bmp4 mRNA and down-regulated p75NGFR mRNA. On the other hand, ALP, ectodin, Bmp2 and Fgf8 mRNA were up-regulated only by anisomycin. These results indicate that MAPK signaling functions, at least in part, as the inducer of ameloblast differentiation.