Glucose and pyruvate metabolism of preimplantation goat blastocysts following in vitro fertilization and parthenogenetic activation

The energy metabolism of preimplantation embryos can be used to predict viability and postimplantation development. Although preimplantation development and mean blastocyst cell numbers of goat in vitro-fertilized (IVF) embryos and chemically activated parthenogenotes are comparable, mammalian parthenogenotes are not viable, with most dying shortly after implantation. The objective of this study was to compare glucose and pyruvate metabolism of IVF goat blastocysts with that of parthenogenetic blastocysts developing from chemically activated oocytes. Embryos derived from IVF and parthenogenotes produced by exposing oocytes to either ionomycin or ethanol followed by 6-dimethylaminopurine (6-DMAP) were cultured in G1.2/G2.2 sequential culture media. Metabolism was determined for individual blastocysts using [5-3H]glucose and [2-14C]pyruvate to determine glycolytic and Kreb's cycle activity, respectively. Data were analyzed by ANOVA. A significantly higher percentage of activated oocytes underwent cleavage and developed to the blastocyst stage compared to IVF oocytes (p < 0.05). There was no significant difference in glucose or pyruvate metabolism between IVF and parthenogenetically activated blastocysts. Mean glucose metabolism through glycolysis was 154.9 +/- 29.1, 130.3 +/- 17.1, and 129 +/- 16.5 pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively. Mean pyruvate metabolism through the Kreb's cycle was 28.1 +/- 8.0, 15.8 +/- 4.2, and 24.4 +/- 4.4 in pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively. Our results suggest that known differences in postimplantation development observed in IVF versus parthenogenetic embryos cannot be attributed to differences in pyruvate or glucose metabolism in the preimplantation blastocysts. Thus, these activation protocols result in embryos capable of appropriate regulation of key metabolic enzymes.     

Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture media.

Development of in vitro-produced bovine embryos was studied in 3 two-step culture media: synthetic oviduct fluid (SOF), Gardner's G1/G2, and control (hamster embryo culture medium with 11 amino acids [HECM-6] followed by tissue culture medium 199 + 10% bovine calf serum). Modifications were made to reduce or eliminate protein. Glycolysis and Krebs cycle activity of morulae and blastocysts developed from selected immature oocytes were measured. There were no differences in development to the morula and blastocyst stages between SOF, G1/G2, or control (41%, 36%, and 46%, respectively), although more blastocysts developed in control medium than in G1/G2 (46%, 30%, respectively). Reducing or removing BSA during the initial culture period did not significantly reduce development to blastocyst (31%, 33%, respectively), although development was reduced in SOF with BSA removed from the final culture period (19%). There were no differences in development to the blastocyst stage between SOF, SOF with BSA removed during the initial culture period, and control (44%, 32%, 49%, respectively), but development was reduced in chemically defined protein-free medium throughout the culture period (21%). Krebs cycle activity did not differ between treatments; however, glycolysis was highest in the control embryos and lowest in embryos cultured in protein-free medium. Embryos that developed in the presence of serum appeared dark and granular and had elevated glycolytic rates compared to embryos developed in completely defined medium. This study shows that both metabolism and blastocyst development of embryos are altered by different culture media, implying a functional linkage between these two indicators of successful embryogenesis.     

Hormonally regulated programmed cell death in barley aleurone cells

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.     

Expression of the alpha 1-proteinase inhibitor gene family during evolution of the genus Mus

alpha 1-Proteinase inhibitors (alpha 1-PIs) are members of the serpin superfamily of proteinase inhibitors, and are important in the maintenance of homeostasis in a wide variety of animal taxa. Previous studies have shown that in mice (genus Mus), evolution of alpha 1-PIs is characterized by gene amplification, region-specific concerted evolution, and rapid accumulation of amino acid substitutions. The latter occurs primarily in the reactive center, which is the region of the alpha 1-PI molecule that determines the inhibitor's specificity for target proteinases. The P1 residue within the reactive center, which is methionine in so-called orthodox alpha 1-PIs and an amino acid other than methionine in unorthodox alpha 1-PIs, is a primary determinant of inhibitor specificity. In the present study, we find that the expression of mRNAs encoding unorthodox alpha 1-PIs is polymorphic within Mus species, i.e., among individuals or inbred strains. This is in striking contrast to mRNAs that encode orthodox alpha 1-PIs, whose concentrations are relatively invariant. The intraspecies variations in mRNA expression represent polymorphisms in the structure of the alpha 1- PI gene family. The results, taken together with previously described aspects of alpha 1-PI evolution, indicate that the dissimilar levels of polymorphism exhibited by orthodox and unorthodox alpha 1-PIs, which likely have distinct physiological functions, may reflect different levels of selective constraint. The significance of this finding to the evolution of gene families is discussed.      

Aberrant protein expression is associated with decreased developmental potential in porcine cumulus–oocyte complexes

Oocyte developmental competence is progressively obtained during pubertal development in females. Poor developmental potential in oocytes derived from prepubertal females suggests that essential processes required for oocyte development have not been fulfilled. The objective of this experiment was to analyze the protein profiles of porcine cumulus–oocyte complexes (COC) derived from cyclic and prepubertal females to identify alterations in protein abundance that correlate with developmental potential. COC complexes, aspirated from prepubertal and cyclic ovaries, were pooled into three replicates of 400 COCs each per treatment in ～100 µl SOF‐HEPES medium. Protein samples were extracted and analyzed by two‐dimensional differential in gel electrophoresis (2D‐DIGE). Over 1,600 proteins were resolved on each of the three replicate gels. Sixteen protein spots were identified by mass spectrometry, representing 14 unique, differentially expressed proteins (volume ratio greater than 1.3). Glutathione‐S‐transferase and pyruvate kinase 3 were more abundant in COCs derived from cyclic females, whereas soluble epoxide hydrolase and transferrin were more abundant in prepubertal derived COCs. Abundance of several glycolytic enzymes (enolase 1, pyruvate kinase 3, and phosphoglycerate kinase) was increased in COCs derived from cyclic females, suggesting glucose metabolism is decreased in prepubertal derived COCs. We conclude that the abundance of proteins involved in metabolism and oxidative stress regulation is significantly altered in prepubertal derived COCs and may play a role in the mechanisms resulting in developmental competence. Mol. Reprod. Dev. 77: 51–58, 2010. © 2009 Wiley‐Liss, Inc.     

Antibodies designed as effective cancer vaccines

Antigen/antibody complexes can efficiently target antigen presenting cells to allow stimulation of the cellular immune response. Due to the difficulty of manufacture and their inherent instability complexes have proved inefficient cancer vaccines. However, anti-idiotypic antibodies mimicking antigens have been shown to stimulate both antibody and T cell responses. The latter are due to T cell mimotopes expressed within the complementarity-determining regions (CDRs) of antibodies that are efficiently presented to dendritic cells in vivo. Based on this observation we have designed a DNA vaccine platform called ImmunoBody™, where cytotoxic T lymphocyte (CTL) and helper T cell epitopes replace CDR regions within the framework of a human IgG1 antibody. The ImmunoBody™ expression system has a number of design features which allow for rapid production of a wide range of vaccines. The CDR regions of the heavy and light chain have been engineered to contain unique restriction endonuclease sites, which can be easily opened, and oligonucleotides encoding the T cell epitopes inserted. The variable and constant regions of the ImmunoBody™ are also flanked by restriction sites, which permit easy exchange of other IgG subtypes. Here we show a range of T cell epitopes can be inserted into the ImmunoBody™ vector and upon immunization these T cell epitopes are efficiently processed and presented to stimulate high frequency helper and CTL responses capable of anti-tumor activity.Key words: DNA vaccines, cancer vaccines, melanoma, CTL, helper T cells     

Intracellular adenosine triphosphate and glutathione concentrations in oocytes from first estrous,multi-estrous,and testosterone-treated gilts

Cytoplasmic maturation refers to a variety of cellular changes that must occur in the oocyte in order to progress through subsequent fertilization and embryonic development. Intracellular concentrations of ATP (ATPi) or glutathione (GSHi), indicative of metabolic activity or the ability of the oocyte to form a male pronucleus and cope with cellular stress, respectively, have been used as markers of cytoplasmic maturation in vitro. In the current study, our objective was to determine if concentrations of ATPi and GSHi in oocytes recovered from three groups of gilts were associated with known differences in developmental competence within these populations. In vivo matured oocytes were surgically recovered 36-38 h after the onset of estrus from first estrous gilts, multi-estrous gilts, and multi-estrous gilts receiving testosterone (1mg/2 ml per day; day 13 to estrus, day 0=day of estrus). Concentrations of ATPi and GSHi were determined using a bioluminescent somatic cell assay kit (luciferin-luciferase reaction) and the dithiobisnitrobenzoic acid (DTNB)-glutathione reductase recycling reaction, respectively. There were no differences (P>0.05) between ATPi concentrations in oocytes from the three groups (1.52 +/- 0.10, 1.51 +/- 0.11, 1.56 +/- 0.11pmol per oocyte). In contrast, oocytes from multi-estrous gilts had higher (P<0.05) concentrations of GSHi (31.53 +/- 1.66 to 33.67 +/- 2.30 pmol per oocyte) than oocytes from first estrous gilts (25.07 +/- 0.82). Administration of testosterone did not affect (P>0.05) GSHi concentrations in oocytes from multi-estrous gilts. Differences in developmental potential between the three groups of gilts were apparently not due to different concentrations of ATPi. However, GSHi concentrations were higher in oocytes from multi-estrous gilts, suggesting that reduced developmental potential of oocytes from first-estrus gilts may be related to insufficient amounts of GSHi. The beneficial effect of exogenous testosterone on the percentage of embryos surviving early gestation does not appear to be due to increased GSHi. Of the numerous potential markers of developmental potential, two were examined in the current study, and GSHi appeared to be useful for assessing porcine oocytes.     

Do orthologous gene phylogenies really support tree-thinking?

Background  

Since Darwin's Origin of Species, reconstructing the Tree of Life has been a goal of evolutionists, and tree-thinking has become a major concept of evolutionary biology. Practically, building the Tree of Life has proven to be tedious. Too few morphological characters are useful for conducting conclusive phylogenetic analyses at the highest taxonomic level. Consequently, molecular sequences (genes, proteins, and genomes) likely constitute the only useful characters for constructing a phylogeny of all life. For this reason, tree-makers expect a lot from gene comparisons. The simultaneous study of the largest number of molecular markers possible is sometimes considered to be one of the best solutions in reconstructing the genealogy of organisms. This conclusion is a direct consequence of tree-thinking: if gene inheritance conforms to a tree-like model of evolution, sampling more of these molecules will provide enough phylogenetic signal to build the Tree of Life. The selection of congruent markers is thus a fundamental step in simultaneous analysis of many genes.