Document Type

Dissertation - University Access Only

Award Date

1996

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology and Microbiology

Abstract

Studies have shown that a procedure developed in our laboratory, the sperm chromatin structure assay (SCSA), is a sensitive measure of sperm chromatin structure related to reproductive toxicology and fertility. Abnormal sperm chromatin structure is defined as chromatin with an increased susceptibility of DNA to acid or head induced denaturation in situ. Studies with a variety of chemicals have indicated that the SCSA is a highly sensitive monitor of external stresses. Experiments have now been performed in this thesis research to examine the effects of physical stresses on sperm chromatin structure, to study factors underlying abnormal chromatin structure, and to relate abnormal sperm chromatin structure with digital measurements of sperm head morphology. The testicular regions of male mice were exposed to x-ray doses ranging from Oto 400 rads. Forty days after exposure the mice were killed and flow cytometric measurement of acridine orange stained testicular samples indicated a repopulation of testicular cells following the x-ray killing of stem cells. Cauda epididymal sperm, analyzed b the SCSA showed increased susceptibility to DNA denaturation following doses of radiation as low as 12.5 rads with significant increases following 25 rads. Abnormal sperm head morphology was not significantly increased until 60 rads of radiation or higher, suggesting that the SCSA is currently the most sensitive, non-invasive method of detecting x-ray damage to testicular stem spennatogonia. Sperm from four mammalian species were analyzed by the SCSA and the terminal deoxynucleotidyl transferase assay (TdTA) to correlate the susceptibility of sperm DNA to in situ denaturation with the presence of endogenous DNA strand breaks. Significant correlations were seen for samples from humans, rams, bulls and stallions. No differences were seen when using either fresh or frozen semen samples for either assay. These results suggest that sperm ce11s which are more susceptible to in situ DNA denaturation have a greater number of accessible endogenous DNA strand breaks. Studies were performed to determine if a relationship existed between morphologically abnormal sperm, sperm chromatin structure and fertility potential. Semen samples, obtained from thirteen bulls, were analyzed by the SCSA. Feulgen stained sperm head morphology was performed on a computerized digital image analysis system (ONCOR-lmage): sixteen parameters of size, shape and texture were measured for each of 200 nuclei per sample. Fertility estimates based on non-return rates were available for nine of the thirteen bulls. The SCSA variable SDa1 was correlated with fertility rankings. No correlations were seen between the means of the imaging variables and the SCSA variables %COMPa1 and SDa1. Significant correlations were seen between SDa1 and the variation of the imaging variables eccentricity width and light blobs. Significant correlations were seen between %COMPa1 and the variation of the imaging variables area, perimeter, perimeter/area ratio (p2a), bending energy, normalized mean absolute curvature, sphericity, eccentricity, length and width. A regression model describing fertility potential incorporating the variation of the imaging variables area, bending energy, nmac, eccentricity condensity, light blobs and dark blobs was highly significant. These results indicated that the variation of morphometry may be a sensitive biomarker related to fertility potential and abnormal chromatin structure. To justify our digital measurements of sperm morphometry, repeatabilities were calculated. Semen from four bulls and four stallions were used, with four slides being prepared for each subject. Two independent sets of digital measurements were made per slide. Sixteen parameters were measured for each of the 200 stained sperm nuclei per slide and repeatabilities were calculated for each parameter within a subject. Measurements for both bulls and stallions proved to be highly repeatable across the majority of the imaging measurements. One subgroup of slides from two bulls and three stallions was used to study the effects of storage prior to staining. All smears for a subject were prepared on the same day, but two slides per subject were stained on one day, and the other two stained after a two month storage at room temp. Comparisons between staining days showed significant differences between size and shape parameters, indicating that smeared cells are altered by storage conditions. These results help support the use of digital morphometry analysis for the study of fertility and reproductive toxicology.

Library of Congress Subject Headings

Mammals -- Spermatozoa -- Morphology Spermatogenesis in animals Flow cytometry

Format

application/pdf

Number of Pages

126

Publisher

South Dakota State University

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