91 INHIBITION OF TELOMERASE REVERSE TRANSCRIPTASE (TERT) INDUCES EMBRYO ARREST IN BOVINE PRE-IMPLANTATION EMBRYOS

Abstract

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. The objective of this study was to characterize the expression and function of the bovine telomerase reverse transcriptase (bTERT) subunit during bovine pre-implantation embryogenesis. Using RT-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 pre-implantation bovine embryos from 1-cell to the blastocyst stage. The specificity of bTERT PCR products was confirmed by sequencing and exhibited 94% sequence homology to the human hTERT cDNA sequence. In immature oocytes, the bTERT protein was localized within the germinal vesicle and after 18 to 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 punctate staining, whereas others exhibited a more-uniform staining pattern within the cytoplasm. By the 8- to 16-cell stage, the embryos demonstrated a peri-nuclear presence of bTERT. However, while in morulae and blastocysts, bTERT was localized to the nuclei as demonstrated by co-localization with 4′,6-diamidino-2-phenylindole (DAPI) staining. Treatment of zygotes with Telomerase Inhibitor III (telomere mimic that inhibits telomerase activity; Calbiochem, San Diego, CA) significantly induced embryos to permanently arrest in a senescence-like state at the 2- to 4-cell cleavage stage compared with nontreated controls. These arrested embryos showed abnormal nuclear phenotypes (elongated chromosomes, anaphase bridging) and phosphorylated histone γ-H2A.X foci suggesting a critical role(s) of TERT in chromosomal segregation and telomere integrity in early embryos. The translocation of bTERT protein from the cytoplasm to the nucleus in morulae supports the known telomere elongation event, whereas low levels of TERT at the 2- to 4-cell stage are associated with the previously documented state of permanent embryo arrest. Future studies would be directed toward understanding the relationship between TERT expression, telomere dysfunction, and permanent embryo arrest.

Operating grants from the Canadian Institutes of Health Research (CIHR) to D.H.B. and W.A.K. supported this research.