Date of Award

Winter 2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Committee Director

R. James Swanson

Committee Member

Christopher J. Osgood

Committee Member

Robert E. Ratzlaff

Committee Member

Silvina Bocca

Abstract

Vemurafenib is a selective B-RafV600E inhibitor in melanoma targeted therapy which also inhibits the wild type B- and C-Raf. In oocyte maturation, the C-Raf/MAPK pathway acts as an important self-enhancing and promoting system, w hereas in embryo development, the C-Raf/M APK pathway participates in pre- and post- implantation embryo proliferation and differentiation.

The hypothesis: Vemurafenib has detrimental effects on oocyte maturation and/or embryo development. Mouse oocytes and one cell (1C) mouse embryos were tested by ex vivo culturing with Vemurafenib in serial dilution. Oocytes were evaluated by cell cycle morphology, spindle formation and chromosomal alignment by im munofluorescence (IF) staining as well as DNA integrity by Terminal Transferase dUTP Nick End Labeling. Embryo development was evaluated by morphological changes and the C -R af bioactivity via a p-M ek 1/2 IF assay. In vivo exposure and subsequent morphological evaluation of ex vivo two-cell (2C) embryo development was conducted by pre-injections of Vemurafenib (10 mg/kg).

Ten μM or above produced oocyte toxicity, spindle formation disturbance, chromosome misalignm ent and DNA damage, whereas the therapeutic Vemurafenib concentration 1 μM did not display significant oocyte morphological toxicity to ex vivo oocyte maturation, spindle formation, chromosom e alignment and DNA integrity.

Ten μM and 100 μM showed phosphorylation inhibition o f embryo Mek 1/2. decreased C -R af activity and obvious toxicity to 1C to 2C transition. However, the 1 μM Vemurafenib was not shown to have significant ex vivo preimplantation 1C embryo development toxicity on the 1C to 2C transition and no effect on C -R af activity.

Ten mg/kg Vemurafenib pre-injections demonstrated only a mild delayed toxic influence throughout the embryo development. However, the majority of embryos progressed to normal blastocysts and did not show significant in vivo degenerative preimplantation embryotoxieity.

Thus Vemurafenib, at its therapeutic concentration, was free of ex vivo oocyte and embryo morphological toxicity, but in vivo toxicity needs further clarification on mechanisms and survivability of collapsed unhatched embryos.

DOI

10.25777/9ckm-dv79

ISBN

9781321562309

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