277 PLASMID MEDIATED EXPERIMENTAL TELOMERE EXTENSION IN BOVINE EMBRYOS BY ECTOPIC EXPRESSION OF HUMAN TERT

Abstract

Telomeres are composed of repetitive hexanucleotide sequences, (TTAGGG)n, encompassing several kilobase pairs, and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage and up to the morula stage, telomerase is not active or is found only at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) has 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from a local abattoir, followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. Two plasmid encoding CMV promoter-driven sequences of hTERT and green fluorescent protein (GFP) were coinjected in bovine zygotes, and GFP driven by a muscle specific promoter was injected for mock experiments. The hTERT and GFP were co-injected to allow live separation of embryos. A total of 768 bovine embryos were injected; 468 (61%) of the treated embryos showed specific GFP-fluorescence. Of a total of 132 blastocysts (17%), 45 showed GFP fluorescence (34%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic hTERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. The mRNA level of the ectopic hTERT began to increase from the 4- to the 8-cell stage and remained high up to the morula stage. Embryos at the morula and blastocyst stages were spread on slides and analyzed by quantitative fluorescence in situ hybridization (qFISH). A Cy3-labeled 18-mer peptide nucleic acid (PNA) probe was used to hybridize the telomeres. The resulting spot intensities were quantified by using TFL-Telo software and were statistically analyzed. A modest increase in telomere length was observed in hTERT injected [775 ± 69 fluorescence unit (fu)] group compared to uninjected control (679 ± 75 fu) group at blastocyst stage. In conclusion, this study demonstrates that the ectopic expression of hTERT in embryos results in telomere elongation; overexpression of TERT may facilitate the derivation of bovine embryonic stem cells.

Supported by DFG and Goyaike SAACIYF.