Synergistic Effect of Porcine Follicular Fluid and Dibutyryl Cyclic Adenosine Monophosphate on Development of Parthenogenetically Activated Oocytes from Pre-Pubertal Gilts

This study investigated the effect of porcine follicular fluid (PFF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) during in vitro maturation (IVM) of porcine oocytes on meiotic maturation, fertilization and embryo development, and compared the effect of supplementing the embryo culture media with PFF or foetal bovine serum (FBS) on embryo development. Oocytes from pre-pubertal gilts were IVM for 44 h, and parthenogenetically activated or in vitro-fertilized. Embryos were cultured in porcine zygote medium (PZM3) for 7 days. Cleavage and blastocyst rates were evaluated at 48 h and 7 days of culture. The supplementation of the IVM medium with 25% PFF and 1 mm dbcAMP for the first 22 h resulted in more (p < 0.05) embryos developing to the blastocyst stage as compared with the inclusion of dbcAMP alone. The dbcAMP + PFF combination increased (p < 0.05) the average number of nuclei per blastocyst as compared with either of these components alone or in its absence. A synergistic effect of dbcAMP + PFF during IVM was also reflected in the capacity of oocytes to regulate sperm penetration and prevent polyspermy, as twice as many oocytes from the control group were penetrated by more than one sperm as compared with those matured in the presence of both dbcAMP and PFF. The supplementation of PZM3 with 10% FBS from days 5 to 7 of culture significantly improved the total cell quantity in embryos derived either from control or dbcAMP + PFF matured oocytes. There was no effect on the total cell quantity when FBS was replaced by the same concentration of PFF. These studies showed that dbcAMP, PFF and FBS can improve both the quantity (57.3% vs 41.5%) and quality (74.8 vs 33.3 nuclei) of porcine blastocysts derived from oocytes recovered of pre-pubertal gilts.

Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo Development

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 mu M beta-mercaptoethanol (beta-ME) and 50 mu M cysteamine (Cyst)] or a pro-oxidant (5 mm buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for beta-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.

Coordinated regulation of follicle development by germ and somatic cells

The continuum of folliculogenesis begins in the fetal ovary with the differentiation of the oogonia and their isolation within the primordial follicles. Primordial follicle activation is an enigmatic process, whereby some follicles enter the growing pool to become primary follicles, thereby embarking on an irreversible progression towards ovulation or atresia. This process is under the coordinated regulation of factors from the oocyte itself, as well as from the somatic cells of the ovary, in particular the theca and granulosa cells, which are structural components of the follicle. These two influences provide the principal stimuli for the growth of the follicle to the late preantral or early antral stage of development. The endocrine effects of the gonadotrophins FSH and LH are essential to the continued progression of the follicle and most atresia can be attributed to the failure to receive or process the gonadotrophin signals. The peri-ovulatory state has received intensive investigation recently, demonstrating a coordinated role for gonadotrophins, steroids, epidermal growth factor family proteins and prostaglandins. Thus, a complex programme of coordinated interaction of governing elements from both germ and somatic cell sources is required for successful follicle development.

Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm-Egg Binding, Fertilization and Embryo Development

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus-associated protein, osteopontin (OPN), lipocalin-type prostaglandin D synthase (L-PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg-associated OPN and L-PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre-incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L-PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 x 10(5) frozen-thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre-treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L-PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.

Morphological and Molecular Pathology of CCl(4)-Induced Hepatic Fibrosis in Connexin43-Deficient Mice

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4)-induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis. Microsc. Res. Tech. 74:421-429, 2011. (C) 2010 Wiley-Liss, Inc.

Influence of matrix composition on polymerization stress development of experimental composites

Objective. Stress development at the tooth/restoration interface is one of the most important reasons for failure of adhesive restorations. The aim of this study was to evaluate the influence of BisGMA/TEGDMA (B/T) and UDMA/TEGDMA (U/T) ratios on polymerization stress (PS) and on the variables related to its development: degree of conversion (DC), polymerization maximum rate (Rp(max)), volumetric shrinkage (VS), elastic modulus (E), stress relaxation (SR) and viscosity of experimental composites. Method. Composites were formulated containing B/T or U/T in mol% ratios of 2: 8, 3: 7, 4: 6, 5: 5, 6: 4, 7: 3 and 8: 2, and 15 wt% of fumed silica. PS was determined with a universal testing machine. VS was measured with a linometer. E and SR were obtained in three-point bending. DC and Rp(max) were determined by real time NIR spectroscopy and viscosity was measured in viscometer. Data were submitted to one-way ANOVA, Tukey test (alpha = 0.05%) and regression analyses. Results. PS, VS, E and DC decreased and viscosity and Rp(max) increased with base monomer content in both series. PS showed strong correlation with VS, DC and viscosity. PS, VS and DC were higher and viscosity was lower for UDMA-based materials. Significance. Reduced viscosity, kinetics parameters and molecular characteristics led UDMA-based composites to elevated conversion and relatively lower PS at lower TEGDMA contents, compared to B/T composites. (C) 2010 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Immunohistochemical approach reveals involvement of inducible nitric oxide synthase in rat late development

To better understand the role of nitric oxide (NO) in mammal development, specifically in the transition of the fetal stages at birth, we studied the timing of cell-specific expression of inducible NO synthase (iNOS) isoform during gestational periods of rats, mainly at the late stages of intra-uterine development. Before experimentation, the samples were collected (from 17th to 21st gestational days), fixed in 10% buffered formalin and embedded in paraffin for histological procedures. Hereafter, the sections (5 mu m thickness) obtained from different embryos were immunostained by avidin-biotin-immunoperoxidase technique, by using antibody against iNOS isoform. The most of cell immunopositive was suggestive of granulocyte-like cells and those cells were resident close to the blood vessels in different organs, such as: lung, liver or bone marrow environment. Sometimes we noted immunopositive cells in the blood flow, as reported in the thymus. In agreement, iNOS expression, obtained by western blotting analysis, showed the same profile. Together, our data shows that iNOS expression increased gradually during the late stages of rat development (from E17 to E21) and it was executed by cells close to blood vessels. Thus, we can clearly to predict that this expression was finely modulated and it contributes for time-line dependent NO production during rat late development.

Reproductive development and function of female rats exposed to di-eta-butyl-phthalate (DBP) in utero and during lactation

Phthalates are environmental contaminants used in the production of plastics, cosmetics and medical devices. Studies on the effects of phthalates on female reproductive health are particularly sparse and mostly restricted to high-dose exposure in rats. In the present study, pregnant rats were treated with 100 mg/kg-d of di-eta-butyl-phthalate (DBP) or only the vehicle (control group), from GD 12 to GD 20 for evaluation of reproductive outcomes and fetal gonads analysis (F0), and from GD 12 to PND 21 to evaluate reproductive development and function on F1 female offspring. Results showed that all parameters were comparable between groups, although there was a significant increase in the fetal weight after DBP exposure. However, the body weight at birth was normal. Based on these data we can conclude that, in these experimental conditions, DBP did not disturb the reproductive development or function of female rats. (C) 2009 Elsevier Inc. All rights reserved.

Development of ligase-assisted spacer addition for the measurement of microsatellites

Conventional methods for detecting differences in microsatellite repeat lengths rely on electrophoretic fractionation on long denaturing polyacrylamide gels, a time-consuming and labor-intensive method. Therefore, there is a need for the development of new and rapid approaches to routinely detect such length polymorphisms. The advent of techniques allowing the coupling of DNA molecules to solid surfaces has provided new prospects in the area of mutation. We describe here the development and optimization of the ligase-assisted spacer addition (LASA) method, a novel and rapid procedure based on an ELISA format to measure microsatellite repeat lengths. The LASA assay was successfully applied to a set of 11 bird samples to assess its capability as a genotyping method.

The evolution of controlled multitasked gene networks: The role of introns and other noncoding RNAs in the development of complex organisms

Eukaryotic phenotypic diversity arises from multitasking of a core proteome of limited size. Multitasking is routine in computers, as well as in other sophisticated information systems, and requires multiple inputs and outputs to control and integrate network activity. Higher eukaryotes have a mosaic gene structure with a dual output, mRNA (protein-coding) sequences and introns, which are released from the pre-mRNA by posttranscriptional processing. Introns have been enormously successful as a class of sequences and comprise up to 95% of the primary transcripts of protein-coding genes in mammals. In addition, many other transcripts (perhaps more than half) do not encode proteins at all, but appear both to be developmentally regulated and to have genetic function. We suggest that these RNAs (eRNAs) have evolved to function as endogenous network control molecules which enable direct gene-gene communication and multitasking of eukaryotic genomes. Analysis of a range of complex genetic phenomena in which RNA is involved or implicated, including co-suppression, transgene silencing, RNA interference, imprinting, methylation, and transvection, suggests that a higher-order regulatory system based on RNA signals operates in the higher eukaryotes and involves chromatin remodeling as well as other RNA-DNA, RNA-RNA, and RNA-protein interactions. The evolution of densely connected gene networks would be expected to result in a relatively stable core proteome due to the multiple reuse of components, implying,that cellular differentiation and phenotypic variation in the higher eukaryotes results primarily from variation in the control architecture. Thus, network integration and multitasking using trans-acting RNA molecules produced in parallel with protein-coding sequences may underpin both the evolution of developmentally sophisticated multicellular organisms and the rapid expansion of phenotypic complexity into uncontested environments such as those initiated in the Cambrian radiation and those seen after major extinction events.

Characterization of mouse Frizzled-3 expression in hair follicle development and identification of the human homolog in keratinocytes

Frizzled genes encode a family of Wnt ligand receptors, which have a conserved cysteine-rich Wnt binding domain and include both transmembrane and secreted forms. Work by others has shown that experimental perturbation of Wnt signaling results in aberrant hair formation, hair growth, and hair structure. To date, however, there is no information on the contribution of individual Frizzled proteins to hair development. We now report that Frizzled-3 expression in skin is restricted to the epidermis and to the developing hair follicle. Northern analysis on total mouse skin mRNA revealed a single Frizzled-3 transcript of 3.7 kb. Reverse transcription-polymerase chain reaction and in situ hybridization analysis revealed Frizzled-3 expression in epidermal and hair follicle keratinocytes. Frizzled-3 transcripts are first detected in discrete foci in the developing epidermis of 13 d embryos and later in the hair follicle placodes of 15 d embryos, suggesting a role for this Frizzled isoform in follicle development. In 17 d embryos and id old newborn mice Frizzled-3 expression is limited to suprabasal keratinocytes and is not seen in pelage follicles until 3 d postpartum. In 7 d old neonatal skin, Frizzled-3 is expressed throughout the epidermis and in the outer cell layers of hair follicles. We have also identified the mRNA encoding human Frizzled-3 in epidermal keratinocytes and in the HaCaT keratinocyte cell line. Human Frizzled-3 mRNA encodes a 666 amino acid protein with 97.8% identity to the mouse protein. The human Frizzled-3 gene was mapped using a radiation-hybrid cell line panel to the short arm of chromosome 8 between the markers WI-1172 and WI-8496 near the loci for the Hypotrichosis of Marie Unna and Hairless genes.

Molecular basis by which garlic suppresses atherosclerosis

The aim of this study was to determine the mechanism by which the aged garlic extract Kyolic has a protective effect against atherosclerosis. Plasma cholesterol of rabbits fed a 1% cholesterol-enriched diet for 6 wk was not reduced by supplementation with 800 muL Kyolic/(kg body . d). In spite of this, Kyolic reduced by 64% (P < 0.05) the surface area of the thoracic aorta covered by fatty streaks and significantly reduced aortic arch cholesterol. Kyolic also significantly inhibited by 50% the development of thickened, lipid-filled lesions in preformed neointimas produced by Fogarty 2F balloon catheter injury of the right carotid artery in cholesterol-fed rabbits. In vitro studies found that Kyolic completely prevented vascular smooth muscle phenotypic change from the contractile. high volume fraction of filament (V(v)myo) state, and inhibited proliferation of smooth muscle cells in the synthetic state with a 50% effective dose (ED50) of 0.2%. Kyolic also slightly inhibited the accumulation of lipid in cultured macrophages but not smooth muscle, and had no effect an the expression of adhesion molecules on the surface of the endothelium or the adherence of leukocytes. It is concluded that Kyolic exerts antiatherogenic effects through inhibition of smooth muscle phenotypic change and proliferation, and by another (unclarified) effect on lipid accumulation in the artery wall.

Characterisation of templated xerogels for molecular sieve application

This paper presents the results of the characterisation of templated silica xerogels as precursor material for molecular sieve silica membranes for gas separation. The template agent integrated in the xerogel matrix is a methyl ligand covalently bended to the siloxane network in the form of methyltriethoxysilane (MTES). Several surface and microstructural characterisation techniques such as TGA, FTIR, NMR, and nitrogen adsorption have been employed to obtain information on the reaction mechanisms involved in the sol-gel processing of such molecular sieves. The characterisation results show the effects of processing parameters such as heat treatment temperature, and the concentration of the covalently bonded template on the development of the pore structure. It was found that calcination temperature significantly enhanced the condensation reactions thus resulted in more Si-O-Si groups being formed. This was also confirmed with the data of FTIR characterisation showing enhanced silicon bands at higher heat treatment temperatures. As a result of the promoted densification and shrinkable pore network the micropore volume also reduced with increasing methyl ligand molar ratio. However, the mean pore diameter does not change significantly with calcination temperature. While the contribution of the templates towards controlling pore size is less precise, increasing the methyl ligand molar ratio results in the broadening of the pore size distribution and lower pore volume. Higher template concentration induces the collapse of the xerogel matrix due to capillary stress promoting dense xerogels with low pore volume (C) 2001 Elsevier Science B.V. All rights reserved.

The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

Development of response-selective agonists of human C5a anaphylatoxin: Conformational, biological, and therapeutic considerations

Numerous studies on the relationship between the structure and function of peptide agonists derived from the biologically active, C-terminal region of human C5a anaphylatoxin have been reported over the past decade. These studies have been performed with the objective of parlaying this structure-function information into the design of peptide/peptidomimetic modulators of C5a receptor (C5aR)-mediated function. In this review, we describe a rational approach for the development of conformationally biased, decapeptide agonists of C5a and described how these stabilized and specific conformational features relate to the expression of specific C5a-like activities in vitro and in vivo. The therapeutic potential of such response-selective C5a agonists is discussed and underscored by the results of one such response-selective C5a agonist that was used in vivo as an effective molecular adjuvant capable of generating antigen-specific humoral and cellular immune responses. Finally, we describe the synthesis of a new generation of highly response-selective, conformationally biased C5a agonist and discuss the in vitro and in vivo biologic results that so indicate this biologic selectivity.