Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Nursing

Date of Defense

12-7-2010

Graduate Advisor

Roberta K Lee

Committee

Roberta K. Lee

Lori L Paul

Kelle H. Moley

M. Christina Hines

Christine Kasper,

Abstract

Studies link paternal environmental exposures to pesticides, herbicides, and radiation to adverse reproductive outcomes such as developmental delays, malignancies, and structural birth defects. To date, 900 children of Vietnam veterans with presumed Agent Orange exposure receive VA presumptive service connected benefits because these children have Spina Bifida. To date, there are limited data to explain the underlying causal mechanisms whereby paternal environmental exposures result in adverse reproductive events. Laser microdissection (LMD) is the only technology that allows the isolation of cell subpopulations from complex tissues such as the testes without disturbing the cell’s bimolecular signature. LMD is essential in the comparison of the molecular signatures of diseased and non diseased cells. As a result, LMD will translate to toxicology protocols that seek to elucidate gene expression data from exposed and non exposed populations. The purpose of this study was to a) demonstrate the reliability of the sequential methods of LMD and the selected downstream applications through the establishment of qualitative and quantitative checkpoints upon which each subsequent step was based, b) ensure that the expected differences in gene expression during spermatogenesis can be characterized with LMD, and c) validate the LMD methods prior to proceeding to animal exposures. This study used the Leica LMD 6000 combined with qRT-PCR to generate stage specific gene expression data during spermatogenesis. Cluster LMD was used to maximize RNA yield and obtain pure stage specific cell populations. qRT-PCR was used to validate stage specific LMD success. With the exception of non specific mMagea4 amplification across all cell stages, the 10 fold rise in HspA2 in spermatocytes, and the 12 fold increase in Tnp1 and the 10 fold increase in prm2 in elongated spermatids supported stage specific LMD success. Cyp1A1 was found in spermatogonia, but was absent in spermatocytes and spermatids. These expression patterns paralleled the published data and coincided with the physiologic functions of these genes and the blood testes barrier.

Included in

Nursing Commons

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