demecolcine and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

The present study aimed to facilitate widespread application of a previously described manual method of somatic cell nuclear transfer (SCNT) by investigating the effects of demecolcine (a microtubule-depolymerizing chemical), cytochalasin-B (a microfilament-depolymerizing chemical: 2.5μg/ml for 15min) and MG-132 (a proteasome inhibitor chemical) on the (i) incidence of cytoplasmic protrusion of MII chromosomes, (ii) improvement of manual oocyte enucleation, and (iii) in vitro and in vivo developmental competence of SCNT embryos in the goat. Following in vitro maturation, around 65% of goat oocytes contained a characteristic cytoplasmic protrusion of MII-chromosomes. Treatment with demecolcine (0.4μg/ml for 30min) significantly increased this rate to 92.2±4.5%. Treatment with MG-132 (2μM for 30min) could not improve this rate when used alone (61.4±11.5%), but when combined with demecolcine (86.4±8.1%). Treatment with cytochalasin-B completely suppressed this rate whenever used, either alone (7.7±5.1%) or in combination with demecolcine (3.9±1.3%). In a direct comparison, there was no significant difference in quantity and quality of embryos propagated by the manual vs. micromanipulation-based methods of SCNT (cleavage: 85.3±4.5 vs. 89.5±8.9%, blastocyst: 19.5±4.3 vs. 24.3±4.4%, grade 1 and 2 blastocyst: 33.8±7.1 vs. 29.5±6.3%, total cell count: 125±11.1 vs. 122±10.5, respectively). Furthermore, development to live kids at term was not significant between the two SCNT methods. From both technical and economical points of view, the overall in vitro and in vivo efficiency of this manual method of SCNT proved it a simple, fast and efficient alternative for large scale production of cloned goats.

Topics: Animals; Cell Nucleus; Cloning, Organism; Cysteine Proteinase Inhibitors; Cytochalasin B; Demecolcine; Goats; Leupeptins; Nuclear Transfer Techniques; Oocytes; Tubulin Modulators

The removal of chromosomes from recipient oocytes is one of the key steps in nuclear transfer cloning. Although microtubule interrupters have been successfully used for oocyte enucleation, their potential side effect on oocyte developmental potential should be considered, and less harmful drugs should be explored for chemical-assisted enucleation. Based on our previous findings that any maturation promoting factor-activating agent induces ooplasmic protrusion without disrupting microtubules, we have studied the feasibility to use caffeine or MG132 for chemical-assisted enucleation. Experiments using goat oocytes showed that treatments for 30 min with 1-mM caffeine or 5-μM MG132-induced ooplasmic protrusions in about 85% of the oocytes, a percentage similar to that achieved with optimal demecolcine treatment. Rates of enucleation, cell fusion and in vitro blastulation were similar among caffeine, MG132, and demecolcine enucleation but significantly higher than blind aspiration. Furthermore, neither rates of pregnancy on days 90 and 120 nor the general rate of live births/embryos transferred differed significantly (p > 0.05) between caffeine and demecolcine enucleation. Although oocytes treated with caffeine did not retract protrusions until 2 h, many oocytes treated with MG132 withdrew protrusions as early as 0.5 h after treatment. The optimal treatment to induce ooplasmic protrusion in 75% pig oocytes was 8-mM caffeine for 60 min. Mouse oocytes responded poorly to demecolcine or caffeine with less than 40% forming inconspicuous protrusions following optimal treatments. It is concluded that caffeine can be used for enucleation of goat and pig oocytes with similar results as demecolcine, and live kids were born after caffeine-assisted enucleation.

Topics: Animals; Caffeine; Cell Nucleus; Cysteine Proteinase Inhibitors; Demecolcine; Dose-Response Relationship, Drug; Female; Goats; Leupeptins; Mice; Microtubules; Models, Animal; Nuclear Transfer Techniques; Oocytes; Pregnancy; Pregnancy Rate; Swine; Tubulin Modulators

The correlation between stress-induced nucleolar disruption and abrogation of p53 degradation is evident after a wide variety of cellular stresses. This link may be caused by steps in p53 regulation occurring in nucleoli, as suggested by some biochemical evidence. Alternatively, nucleolar disruption also causes redistribution of nucleolar proteins, potentially altering their interactions with p53 and/or MDM2. This raises the fundamental question of whether the nucleolus controls p53 directly, i.e., as a site where p53 regulatory processes occur, or indirectly, i.e., by determining the cellular localization of p53/MDM2-interacting factors. In this work, transport experiments based on heterokaryons, photobleaching, and micronucleation demonstrate that p53 regulatory events are directly regulated by nucleoli and are dependent on intact nucleolar structure and function. Subcellular fractionation and nucleolar isolation revealed a distribution of ubiquitylated p53 that supports these findings. In addition, our results indicate that p53 is exported by two pathways: one stress sensitive and one stress insensitive, the latter being regulated by activities present in the nucleolus.

Topics: 3T3 Cells; Active Transport, Cell Nucleus; Animals; Cell Fusion; Cell Line, Tumor; Cell Nucleolus; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p21; Cycloheximide; Cytoplasm; Demecolcine; DNA Damage; Fatty Acids, Unsaturated; Green Fluorescent Proteins; Humans; Intranuclear Space; Leupeptins; Mice; Models, Biological; Nucleolus Organizer Region; Photobleaching; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-mdm2; Recombinant Fusion Proteins; Ribosomal Proteins; Subcellular Fractions; Tumor Suppressor Protein p53; Ubiquitin; Ubiquitination

We examined the optimal conditions for somatic cell nuclear transfer (SCNT) in rat oocytes. First, we compared the effects of two types of inhibitors of spontaneous activation, MG132 and demecolcine, on the developmental potential of parthenogenetic oocytes. The potential of activated oocytes to develop into blastocysts significantly decreased 2 h after oocyte recovery (77% vs. 7%). The developmental potential of oocytes preserved in MG132-supplemented medium for 1 to 4 h was high (62% to 77%), but the potential of those preserved in demecolcine-supplemented medium for 3 and 4 h was low (77% vs. 41% and 37%, respectively). Second, the effect of the duration of parthenogenetic activation on the developmental potential was examined. When oocytes preserved in MG132 for 4 h were treated with 10 mM strontium for 5 or 6 h, the potential of activated oocytes to develop into blastocysts was high (78% and 70%, respectively). Using the optimal conditions for parthenogenetic activation, we examined the potential of rat enucleated oocytes receiving cumulus cells to develop into blastocysts. In contrast to parthenogenotes, the potential of SCNT rat oocytes to develop into blastocysts was low (2%) even if then oocytes were treated with the histone deacetylation inhibitor trichostatin A. The reason for the low developmental potential of rat SCNT oocytes is discussed.

Topics: Animals; Blastomeres; Cloning, Organism; Cysteine Proteinase Inhibitors; Demecolcine; Embryonic Development; Female; Hydroxamic Acids; Leupeptins; Nuclear Transfer Techniques; Oocytes; Parthenogenesis; Rats; Rats, Sprague-Dawley; Tubulin Modulators