Class
Article
College
College of Agriculture and Applied Sciences
Department
Animal, Dairy, and Veterinary Sciences Department
Faculty Mentor
Ralph Meyer
Presentation Type
Poster Presentation
Abstract
Declining body-wide levels of nicotinamide adenine dinucleotide (NAD) that are present in normal aging men are linked to decreased sperm quality and DNA integrity. When NAD is low, the amount of oxidative stress on the DNA appears to be increased, due to the limited regeneration of the antioxidant glutathione, which is NAD-dependent. We therefore hypothesize that the increased sperm DNA damage in the aging male is caused by an elevated formation of 8-oxoguanine in the DNA, caused by increased oxidative stress when NAD levels are low. To test this hypothesis, a transgenic mouse model (ANDY mouse) was used to create low NAD levels typical of aging men. A direct method for detecting 8-oxoguanine using fluorescent-labeled antibodies in situ was tested on HeLa cells, which was not successful. Therefore, an indirect method was developed, where 8-oxoguanine is excised by treatment with the glycosylase-endonuclease FPG, which leaves a DNA single strand break in place of the oxidized base. Subsequent elongation of the cleaved DNA strand at the 3’ end with fluorescent-labeled nucleotides, using the enzyme terminal deoxynucleotidyl transferase (TdT), was then employed to quantify the resulting DNA strand breaks. The new method will be used to screen sperm samples from the ANDY mouse model to detect oxidized guanine produced by the increased oxidative stress from low levels of NAD linked to aging.Presentation Time: Wednesday, 10-11 a.m.
Location
Logan, UT
Start Date
4-12-2021 12:00 AM
Included in
Detecting DNA Oxidation in Sperm
Logan, UT
Declining body-wide levels of nicotinamide adenine dinucleotide (NAD) that are present in normal aging men are linked to decreased sperm quality and DNA integrity. When NAD is low, the amount of oxidative stress on the DNA appears to be increased, due to the limited regeneration of the antioxidant glutathione, which is NAD-dependent. We therefore hypothesize that the increased sperm DNA damage in the aging male is caused by an elevated formation of 8-oxoguanine in the DNA, caused by increased oxidative stress when NAD levels are low. To test this hypothesis, a transgenic mouse model (ANDY mouse) was used to create low NAD levels typical of aging men. A direct method for detecting 8-oxoguanine using fluorescent-labeled antibodies in situ was tested on HeLa cells, which was not successful. Therefore, an indirect method was developed, where 8-oxoguanine is excised by treatment with the glycosylase-endonuclease FPG, which leaves a DNA single strand break in place of the oxidized base. Subsequent elongation of the cleaved DNA strand at the 3’ end with fluorescent-labeled nucleotides, using the enzyme terminal deoxynucleotidyl transferase (TdT), was then employed to quantify the resulting DNA strand breaks. The new method will be used to screen sperm samples from the ANDY mouse model to detect oxidized guanine produced by the increased oxidative stress from low levels of NAD linked to aging.Presentation Time: Wednesday, 10-11 a.m.