Studies On Telomerase Activity In Buffalo Oocytes And Early Embryos

Telomeres are the specialized DNA-protein structures at the ends of eukaryotic chromosomes, allowing the stable maintenance of chromosome during multiple cell divisions, while telomerase is a specialized cellular ribonucleoprotein (RNP) reverse transcriptase, with core enzyme containing the protein subunit and the RNA component. Telomeres lost during DNA replication can be added back by the activity of telomerase, regulated in the mammalian development. The telomere rebuilding may be attributed to the reprogramming of telomerase activity at the different stages in the early embryo development. Therefore, investigating telomere and telomerase reprogramming during early embryo development is of great importance. Here, an overview of structure and function in telomere and telomerase, and related study on embryo development in mammalian cloned animal, will be represented. This experiment detect and analysis the telomerase activity in different cell lines and early embryos from buffalo, which will be essential and useful for the studies on embryogenesis and somatic cell nuclear transfer.1. The objective of this study was to modify the polymerase chain reaction-based telomeric repeat amplification protocol to detect telomerase activity in the different cell lines and embryos from buffalo. The telomerase activity of different dilution of 293-cell and different cell lines such as oocytes, sperms, granule cells, fibroblast and embryos were assayed using Telomerase TRAPeze Detection Kit with silver staining. The result showed that the sensitivity decreased with dilution from 10 000 to 10-cell equivalents and a correlating trend was noted in telomerase activity, which is to verify the TRAP silver-staining method suitable for evaluating telomerase activity in single cell. Telomerase activity in buffalo embryoes was higher than that in sperms and oocytes, and significiantly diclined in the granule cells and fibroblast (P<0.05 ).2. The objective of this study was to examine the telomerase activity in swamp buffalo oocytes and early embryos derived from in vitro fertilization(IVF) and parthenogenetic activation (PA) . The telomerase activity of immature and matured oocytes, IVF and PA embryos at the 2～4 cell, 8～16 cell, morula and blastocyst stages was assayed using a TRAP silver-staining protocol. The telomerase activity was higher in the immature oocytes in comparison with the matured oocytes (P < 0.05 ) . After in vitro fertilization and parthenogenetic activation, the telomerase activity decreased as the zygotes cleaved to the 2～4 cell and 8～16 cell stages, increased again when the embryos developed to the morula stage (P < 0.05 ) , and reached the highest level at the blastocyst stage. When the telomerase activity was calculated on the individual cell, telomerase activity declined gradually as the immature oocytes matured to Metaphase II and developed to the blastocyst stage after in vitro fertilization and parthenogenetic activation.These results indicate that TRAP silver-staining protocol is suitable for the detecting and analyzing the different cell lines and early embryos from buffalo with its high sensibility and reliable safety, and the changes of telomerase activity in the buffalo oocytes and early embryos are related to their maturation, embryonic developmental block and totipotency decline during the embryogenesis.