Date of Award

Summer 2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

R. James Swanson

Committee Member

Frank J. Castora

Committee Member

Christopher Osgood

Committee Member

David T. Gauthier

Abstract

This study's objective was to verify age-related mitochondrial changes in oocytes from old hamsters compared with those from young hamsters. We used autologous platelet mitochondrial microinjection to improve the mitochondrial quality and quantity of aged oocytes to improve their pregnancy potential. Metaphase II oocytes were collected from super-ovulated female hamsters and prepared for biochemical and morphological analysis. Adenosine triphosphate (ATP) levels, mitochondrial DNA (mtDNA) number, reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) were determined in individual oocytes from young and old hamsters. Transmission electron microscopy (TEM) allowed evaluation of oocyte ultra-structure. In oocytes from old hamsters, ATP levels and mtDNA number were reduced an average of 35.4% and 51.8% respectively compared to young oocytes. Lower MMP and higher ROS levels were observed in oocytes from old hamsters compared with those from young hamsters. TEM analysis supported low mitochondrial quantity and increased mitochondrial electron density in old oocytes, along with collapsed cytoplasmic lamellae. When comparing platelet mitochondria between young and old hamsters, no significant difference was found in ATP level per mitochondrion or ultrastructure. After autologous platelet mitochondrial microinjection, ATP production increased significantly in the microinjected group, however mtDNA number and embryo development to blastocyst stage were not improved. MitoTracker Green FM (MGF) was used to help quantify mitochondria, but the results conflicted with mtDNA and TEM data, which suggests that MGF, as a MMP dependent reagent, is not an ideal method to reflect mitochondrial number. Our data suggest that (1) age-related morphological and functional mitochondrial changes occur in oocytes from old hamsters compared with young hamsters; (2) platelets can be used as a source for mitochondrial transfer; and (3) autologous platelet mitochondrial microinjection improves mitochondrial ATP production in old oocytes. The fact that no change in blastocyst development after microinjection was observed does not rule out that there might not be improvement of post-implantation embryo development. Therefore, further study will focus on effect of autologous platelet mitochondrial microinjection on post-implantation embryo development.

DOI

10.25777/my8q-fg37

ISBN

9781124990705

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