111 CHARACTERIZATION OF BOVINE TELOMERASE REVERSE TRANSCRIPTASE DURING PREIMPLANTATION EMBRYOGENESIS

Abstract

Telomerase is a specialized reverse transcriptase that extends telomeric DNA of eukaryotic chromosomes and is composed of 2 essential subunits, the telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT). Together, this ribonucleoprotein complex is normally highly active in germ cells and stem/progenitor cells but not in normal somatic cells. In humans, this regulation of telomerase activity is primarily coordinated at the level of transcription of the TERT gene, whereas TERC is virtually ubiquitously expressed. Previous studies from our laboratory have detected telomerase activity in embryos from all stages of early bovine development. However, the regulation of the telomerase subunits remains poorly understood. Therefore, the objective of our study was to characterize the expression of bovine TERT (bTERT) during bovine preimplantation embryogenesis. UsingRT-PCR and immunofluorescence staining procedures (n = 20 embryos at timed stages of development; r = 3), we demonstrate that mRNA transcripts and protein for bTERT were detected in preimplantation bovine embryos from 1-cell to the blastocyst stage. The specificity of bTERT PCR products was confirmed by sequencing and demonstrated 94% sequence homology to the human hTERT cDNA sequence. In the immature oocyte, bTERT protein was localized within the germinal vesicle, and after 18–20 h of in vitro maturation, bTERT was observed as doublet foci co-localized with the condensed metaphase II meiotic stage chromosomes. Post-fertilization, the expression of bTERT was observed within the pronuclei. Through the initial cleavage stages, the expression of bTERT was variable, with some blastomeres showing punctuate staining and others exhibiting a uniform staining pattern. By the 8-16 cell stage, the embryos demonstrated a peri-nuclear presence of bTERT. However, in morula and blastocysts, bTERT was localized to the nuclei as demonstrated by co-localization with 42,6-diamidino-2-phenylindole staining. Based on this pattern of expression, it is tempting to speculate that bTERT could be playing an important role in regulating telomerase activity during early embryo development. The translocation of bTERT protein from the cytoplasm to the nucleus in morulae supports the telomere elongation event that is known to occur in mammalian embryos during the transition from the morula to blastocyst stages.