Regulation Of Chromatin Condensation In Mouse Oocytes And Application Of Caffeine In Goat Somatic Cell Cloning

A common feature in the configuration of germinal vesicle (GV) chromatin in most species was that diffuse chromatin condenses into perinucleolar rings or other corresponding condensed configurations with or without the perinucleolar rings, depending on species. Previous investigations focused on the correlation between different chromatin configuration and the acquisition of meiotic as well as developmental competence of oocytes, little was known concerning the cellular and signaling pathways regulating chromatin condensation.Recent studies have revealed that epigenetic modifications,such as DNA methylation and histone modifications,played important roles in the regulation of chromatin structure and gene expression. In the present study, using immunofluorescence and distinct specific inhibitors, we conducted a comprehensive analysis of the DNA methylation,histone phosphorylation and acetylation during chromatin and chromosome condensation in mouse oocytes. Results as follows:1.GV oocytes treated with inhibitors of MPF and MAPK underwent chromatin condensation, suggested that chromatin condensation was not correlated with MPF and MAPK activities.2. GV chromatin and MII chromosome condensation were associated with increased levels of DNA methylation, however, condensations remained occurred after treating oocytes with 5-aza-cytidine, a specific inhibitor of DNA methylation. These results indicated that DNA methylation was not required for chromatin and chromosome condensation.3. GV chromatin and MII chromosome condensation were associated with increased levels of histone phosphorylation, however, condensations remained occurred after treating oocytes with ZM447439, a specific inhibitor of histone phosphorylation. These results indicated that histone phosphorylation was not required for chromatin and chromosome condensation.4. GV chromatin condensation was associated with increased levels of histone acetylation, however,chromatin condensation did not occur while levels of acetylation remained increased after treatment with TSA,a inhibitor of HDACs. These evidences provided for a role of HDACs in the control of GV chromatin condensation,but not take place via histone acetylation/deacetylation-dependent mechanism.5. MII chromosome condensation was associated with histone deacetylation, chromatin condensation remained continued while levels of acetylation increased after treatment with TSA. These results indicated that histone deacetylation was not required for chromosome condensation.6. Dynamic equilibrium between HATs and HDACs activitie were presented in GV and MII oocytes, by which, to regulate the acetylation/deacetylation state. The preparation of recipient cytoplasm was one of the key steps of somatic cell nuclear transfer,the removal of chromosomes from recipient oocytes was named enucleation. Improper measures used in enucleation resulted in problems as aneuploidy or polyploidy with subsequent detrimental effects on development.Chemically assisted enucleation was an approach of choice, oocytes were exposed to the drug, and as a result of treatment, a cortical protrusion formed on the surface of the oocyte containing all the oocyte chromosomes, this protrusion can be easily removed by micromanipulation techniques.Chemically assisted enucleation seemed to be a very attractive procedure that could simplify conventional enucleation methods with a minimal decrease of the cytoplasmic volume and without reducing the viability of the resulting cytoplast. Chemically assisted enucleation previously reported mainly focused on anti-microtubule drugs.Exposured to these drugs impaired spindle rotation, altered chromosome migration, and thereby, resulted in the generation of enucleated oocytes.Caffeine and MG132 were used to increase MPF activity, however,no study about these drugs has been reported in chemically assisted enucleation procedure. In the present study, effects of supplementation of cysteamine and cystine on the developmental capacity for parthenogenetic and SCNT embryos were investigated, and then, using the improved system of IVM, the protocol for caffeine and MG132 assisted enucleation of goat oocytes was first applied. Results as follows:1. Simultaneous supplementation of 100μM cysteamine and 200μM cystine to maturation medium improved parthenogenetic blastulation of goat oocytes.2. Simultaneous supplementation of 100μM cysteamine and 200μM cystine to maturation medium improved SCNT blastulation of goat oocytes. 3. A 30-min treatment with 1mM caffeine or 5μM MG132-induced cytoplasmic protrusions in over 80% of the MII oocytes.4. Treatment with caffeine or MG132 did not impair the structure of oocyte spindle compared with demecolcine.5. Treatment with caffeine or MG132 did not compromise the capacity of parthenogenetic development.6. Rates of enucleation and blastocyst formation were significantly higher after caffeine or MG132-assisted than after blind aspiration enucleation.7. After withdraw exposured to drugs, caffeine had longer persistence of the cytoplasmic protrusions than MG132, and equivalent to demecolcine.and so,caffeine was more suitable to application.8. Production of live cloned goat from SCNT embryos using caffeine-enucleated cytoplasts demonstrated the potential of this new agent for nuclear transfer practice.