Document Type

Thesis - University Access Only

Award Date

2003

Degree Name

Master of Science (MS)

Department / School

Chemistry

First Advisor

Donald P. Evenson

Abstract

The advent of assisted reproduction techniques such as in-vitro fertilization and intracytoplasmic sperm injection has occurred swiftly and with a certain lack of caution. Not enough attention has been paid to their possible genetic risks and the possibility exists that indiscriminate use of these procedures could lead to an increased risk of congenitally abnormal fetuses. Therefore, the male genome should be tested for its genetic integrity to participate in healthy embryogenesis. The Sperm Chromatin Structure Assay (SCSA) is an accurate means to detect male genome damage in the form of DNA strand breaks and chromatin structural defects. The SCSA is a flow cytometric assay utilizing the properties of the fluorescent dye acridine orange (AO) which fluoresces red in the presence of denatured single-strand DNA and green in the presence of double-stranded DNA. A scatter plot is generated showing each cell measured by the assay plotted according to their amounts of red vs green fluorescence. This plot allows 4 distinct populations of sperm cells to be discriminated: 1) normal cells exhibiting AO fluorescence characteristics typical of fertile sperm, 2) cells exhibiting higher than normal green fluorescence, and finally cells exhibiting 3 ) moderate and 4) high increases in the ratio of red/red+green fluorescence DNA fragmentation index (DFI) in comparison to the normal cells. To test the hypothesis that these populations are distinct from each other, they were physically sorted and collected by flow cytometry. Sperm nuclei were then characterized light microscopically by Feulgen staining for roundness, roughness, DNA stainability, and size. DNA fragmentation levels were also determined for each cell population by fluorescence microscopy using the Comet assay. The High DNA Stainability (HDS) cells were found to exhibit rounder, larger and more DNA stainable chromatin masses. Taken together with other studies, this suggested the HDS sperm were immature due to lack of full developmental chromatin condensation. The moderate and normal DFI populations were not significantly different from one another by Feulgen staining but the moderate DFI population had greatly elevated levels of DNA damage as seen by the Comet assay. There was degradation in the DNA stainability and shape characters of the chromatin mass in the high DFI population as well as a significant increase in DNA fragmentation compared to the moderate population. These results confirm other studies which have suggested that the SCSA is able to detect both immature sperm and sperm with fragmented DNA.

Library of Congress Subject Headings

Spermatozoa.
Chromatin.
Germ cells -- Testing.
DNA damage.

Publisher

South Dakota State University

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Rights Statement

In Copyright