Production of nuclear transfer horse embryos by Piezo-driven injection of somatic cell nuclei and activation with stallion sperm cytosolic extract

We investigated the use of direct nuclear injection using the Piezo drill and activation by injection of stallion sperm cytosolic extract for production of cloned equine embryos. When metaphase II horse oocytes were injected with either of two dosages of sperm extract and cultured 20 h, similar activation rates (88% vs. 90%) and cleavage rates (49% vs. 46%) were obtained. The successful reconstruction rate of horse oocytes with horse somatic cell donor nuclei after direct injection using the Piezo drill was 82%. Four dosages of sperm extract (containing 59, 176, 293, or 1375 microg/ml protein) and two activation times (1.5-2 vs. 8-10 h after nuclear transfer) were examined. Cleavage and activation (pseudopronucleus formation) rates of oocytes injected with sperm extract containing 59 microg/ml protein were significantly (P < 0.05) lower than any other dosage. The percentage of embryos cleaving with normal nuclei in oocytes injected with the 1375 microg/ml preparation 1.5-2 h after donor injection was significantly (P < 0.05) higher than that of the 293 microg/ml preparation 8-10 h after donor injection (22 vs. 6%). Embryos developed to a maximum of 10 nuclei. Interspecies nuclear transfer was performed by direct injection of horse nuclei into enucleated bovine oocytes, followed by chemical activation. This resulted in 81% reconstruction (successful injection of the donor cell), 88% cleavage, and 73% cleavage with normal nuclei. These results indicate that direct nuclear injection using the Piezo drill is an efficient method for nuclear transfer in horse and cattle oocytes and that sperm extract can efficiently activate horse oocytes both parthenogenetically and after nuclear transfer     

Class-specific restrictions define primase interactions with DNA template and replicative helicase

Bacterial primase is stimulated by replicative helicase to produce RNA primers that are essential for DNA replication. To identify mechanisms regulating primase activity, we characterized primase initiation specificity and interactions with the replicative helicase for gram-positive Firmicutes (Staphylococcus, Bacillus and Geobacillus) and gram-negative Proteobacteria (Escherichia, Yersinia and Pseudomonas). Contributions of the primase zinc-binding domain, RNA polymerase domain and helicase-binding domain on de novo primer synthesis were determined using mutated, truncated, chimeric and wild-type primases. Key residues in the β4 strand of the primase zinc-binding domain defined class-associated trinucleotide recognition and substitution of these amino acids transferred specificity across classes. A change in template recognition provided functional evidence for interaction in trans between the zinc-binding domain and RNA polymerase domain of two separate primases. Helicase binding to the primase C-terminal helicase-binding domain modulated RNA primer length in a species-specific manner and productive interactions paralleled genetic relatedness. Results demonstrated that primase template specificity is conserved within a bacterial class, whereas the primase–helicase interaction has co-evolved within each species.     

Structure of the Ni(II) complex of Escherichia coli peptide deformylase and suggestions on deformylase activities depending on different metal(II) centres

Crystal structures of polypeptide deformylase (PDF) of Escherichia coli with nickel(II) replacing the native iron(II) have been solved with chloride and formate as metal ligands. The chloro complex is a model for the correct protonation state of the hydrolytic hydroxo ligand and the protonated status of the Glu133 side chain as part of the hydrolytic mechanism. The ambiguity that recently some PDFs have been identified with Zn²⁺ ion as the active-site centre whereas others are only active with Fe²⁺ (or Co²⁺, Ni²⁺) is discussed with respect to Lewis acid criteria of the metal ion and substrate activation by the CD loop.     

Ejaculate and type of freezing extender affect rates of fertilization of horse oocytes in vitro

In vitro fertilization (IVF) was performed on in vitro-matured equine oocytes in three experiments. Frozen-thawed sperm were prepared using swim-up separation and heparin treatment. In Experiment 1, fertilization was achieved with sperm from only one frozen ejaculate of four obtained from the same stallion. Within this ejaculate, fertilization rates were higher with fresh media, as compared to media held for 6-8 days before use (39.6% versus 7.3%, respectively; P<0.001). The type of bovine serum albumin used affected fertilization rates (4% versus 39.6%; P<0.001). To determine if IVF rates were influenced by factors associated with the freezing process (Experiment 2), a single ejaculate from a second stallion was frozen using eight variations in timing of steps in the freezing protocol. There were no differences among treatments in fertilization rates (range, 0-3%). In Experiment 3, fertilization rates of semen frozen in an extender containing 21.5% egg yolk were lower than fertilization rates of semen from the same ejaculate but frozen with a 3% egg-yolk extender (0% versus 15%, respectively; P<0.01). We inferred that rates of equine IVF with frozen-thawed sperm were influenced by ejaculate, the composition and age of the media used, and freezing extender. To our knowledge, this is the first report of ejaculate or extender differences affecting in vitro fertilization in this species. These factors may help to explain the great variability in fertilization rates reported with equine IVF, both among and within laboratories.     

Classical Xenopus laevis progesterone receptor associates to the plasma membrane through its ligand-binding domain

During the last decade, considerable evidence is accumulating that supports the view that the classic progesterone receptor (xPR-1) is mediating Xenopus laevis oocyte maturation through a non-genomic mechanism. Overexpression and depletion of oocyte xPR-1 have been shown to accelerate and to block progesterone-induced oocyte maturation, respectively. In addition, rapid inhibition of plasma membrane adenylyl cyclase (AC) by the steroid hormone, supports the idea that xPR-1 should be localized at the oocyte plasma membrane. To test this hypothesis, we transiently transfected xPR-1 cDNA into Cos-7 cells and analyzed its subcellular distribution. Through Western blot and immunofluorescence analysis, we were able to detect xPR-1 associated to the plasma membrane of transfected Cos-7 cells. Additionally, using Progesterone-BSA-FITC, we identified specific progesterone-binding sites at the cell surface of xPR-1 expressing cells. Finally, we found that the receptor ligand-binding domain displayed membrane localization, in contrast to the N-terminal domain, which expressed in similar levels, remained cytosolic. Overall, these results indicate that a fraction of xPR-1 expressed in Cos-7 cells, associates to the plasma membrane through its LBD.     

Phylogenetic and Metabolic Diversity of Planctomycetes from Anaerobic, Sulfide- and Sulfur-Rich Zodletone Spring, Oklahoma

We investigated the phylogenetic diversity and metabolic capabilities of members of the phylum Planctomycetes in the anaerobic, sulfide-saturated sediments of a mesophilic spring (Zodletone Spring) in southwestern Oklahoma. Culture-independent analyses of 16S rRNA gene sequences generated using Planctomycetes-biased primer pairs suggested that an extremely diverse community of Planctomycetes is present at the spring. Although sequences that are phylogenetically affiliated with cultured heterotrophic Planctomycetes were identified, the majority of the sequences belonged to several globally distributed, as-yet-uncultured Planctomycetes lineages. Using complex organic media (aqueous extracts of the spring sediments and rumen fluid), we isolated two novel strains that belonged to the Pirellula-Rhodopirellula-Blastopirellula clade within the Planctomycetes. The two strains had identical 16S rRNA gene sequences, and their closest relatives were isolates from Kiel Fjord (Germany), Keauhou Beach (HI), a marine aquarium, and tissues of marine organisms (Aplysina sp. sponges and postlarvae of the giant tiger prawn Penaeus monodon). The closest recognized cultured relative of strain Zi62 was Blastopirellula marina (93.9% sequence similarity). Detailed characterization of strain Zi62 revealed its ability to reduce elemental sulfur to sulfide under anaerobic conditions, as well as its ability to produce acids from sugars; both characteristics may potentially allow strain Zi62 to survive and grow in the anaerobic, sulfide- and sulfur-rich environment at the spring source. Overall, this work indicates that anaerobic metabolic abilities are widely distributed among all major Planctomycetes lineages and suggests carbohydrate fermentation and sulfur reduction as possible mechanisms employed by heterotrophic Planctomycetes for growth and survival under anaerobic conditions.     

Update on equine ICSI and cloning

Intracytoplasmic sperm injection (ICSI) has recently become efficient enough to be considered for clinical use. With ICSI, one spermatozoa is injected into a mature oocyte. Harvesting of an oocyte ex vivo, followed by ICSI and transfer of the fertilized oocyte to the oviduct, may be applicable when semen quality is insufficient for standard insemination. Sperm injection, followed by in vitro embryo culture to the blastocyst stage, may be used in cases where multiple oocytes are to be fertilized (e.g. when oocytes are collected post-mortem). Nuclear transfer (cloning) of horses is possible but still inefficient; however, commercial companies currently will culture and store cells from privately owned animals for a reasonable fee. Horse owners are beginning to realize the potential of cloning for salvaging valuable equine genetics that may otherwise be lost.