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Recently, we have demonstrated a stage-specific increase of telomere length at morula-blastocyst transition in bovine and murine embryos (Schaetzlein et al. 2004 PNAS 101, 8034-8038). Telomeres are composed of repetitive hexanucleotide sequences (TTAGGG) encompassing several kilobasepairs 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 up to the morula stage, telomerase is not active or only found 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) was selected as the target molecule due to its 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from 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. In preliminary tests, it was shown that co-injection of green fluorescent protein (GFP) and red fluorescent protein (dsRed) constructs resulted in the simultaneous expression of the 2 proteins. The onset of fluorescent protein expression was recorded 30 to 40 hours after injection by fluorescence microscopy. Because all cDNA were driven by the cytomegalovirus (CMV) promoter, it was assumed that hTERT is expressed in parallel. In the main experiment, 2 constructs encoding human TERT and GFP were co-injected to allow live separation of embryos. A total of 400 bovine embryos were injected, 209 (53%) of the treated embryos showed specific GFP fluorescence; out of a total of 104 blastocysts (26%), 55 showed GFP fluorescence (53%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic human TERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. Surprisingly, expression pattern of the endogenous TERT revealed a transient increase at the 2-4 cell stage and a major increase at the blastocyst stage. The mRNA level of the ectopic hTERT started to rise from 4 to 8 cell stage and remained high up to the morula stage. Currently, qFISH and TRAP techniques are being employed to assess enzymatic activity of hTERT and to quantitatively determine telomere length. In conclusion, this study demonstrates the ectopic expression of TERT and fluorescent proteins in early embryos; overexpression of TERT may facilitate the derivation of bovine ES cells.<fn_group><fn>Supported by DFG and Goyaike S.A.A.C.I. Y.F.</fn></fn_group> |
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