Date of Award

Winter 2000

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

Susan E. Lanzendorf

Committee Member

William Gibbons

Committee Member

Mary Mahony

Committee Member

Sergio Oehninger

Abstract

Telomerase, a ribonucleoprotein, has been described as an essential component of highly proliferative cells, which stabilizes the telomeres and avoids cellular senescence. Telomerase has been identified in various embryonic cell stages, hematopoietic cells, and in >85% of tumor tissue biopsies analyzed. The ability to measure the potential to proliferate successfully could provide an objective measure of an embryo's quality. The objectives of this study were to modify the telomeric repeat amplification protocol (TRAP) assay system for increased sensitivity to allow detection of telomerase activity in the single cell of an oocyte and embryo, obtain telomerase activity levels for the oocyte through blastocyst, and finally, evaluate the use of measuring telomerase activity within biopsied blastomeres to predict blastocyst development. Telomerase positive (DU145 and PMEF) and negative (Hs27 and Detroit 551) cell lines were used to evaluate the assay system followed by the use of discard and donated oocytes and embryos for the comparative evaluation of telomerase activity and maturation level. Immature oocytes, mature oocytes, zygotes, 2–3 cell, 4–5 cell, 6–7 cell, 8–16 cell embryos, morulae, and blastocysts were evaluated individually for telomerase activity. Thawed zygotes cultured to day three were biopsied, by removing 1–2 cells and the biopsied embryos followed through culture to blastocyst. Telomerase activity was detected in positive cell lines and none measured in negative cell lines. Analysis of 60 single DU-145 cells showed detectable levels of telomerase activity in all cells. Of discard oocytes and embryos analyzed, 97.6% had measurable levels of telomerase activity. Telomerase activity was detected in all developmental stages. Immature oocytes and blastocysts had similar levels of telomerase activity, however both groups had significantly higher activity than the zygote through pre-morula stage embryo. There was no difference in telomerase activity of cells biopsied from embryos that reached the blastocyst stage or those that arrested in growth. Human oocytes through blastocyst stage embryos express telomerase activity, however the level of telomerase activity in a single blastomere of the day 3 cleavage stage embryo was not able to predict the growth potential of an embryo.

Comments

A Dissertation Submitted to the Faculties of Old Dominion University and Eastern Virginia Medical School in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.

DOI

10.25777/n8gn-y288

ISBN

9780599965959

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