Reproduction is a fundamental process for all living organisms and understanding this processes is essential for increasing successful pregnancy outcomes. Mitochondrial deoxyribonucleic acid (mtDNA) copy number has been implicated as a biomarker for infertility. The aim of these studies is to develop a method to determine mtDNA copy number and microdeletions in stallion sperm and to then use these endpoints to determine age-related changes to mtDNA. A quantitative polymerase chain reaction (qPCR) technique was used to calculate an average mtDNA copies for a population of sperm and to screen for microdeletions on the following three mitochondrial genes: NADH dehydrogenase subunit 1 (ND1), NADH dehydrogenase subunit 4 (ND4), and cytochrome B (CytB); along with either amyloid precursor protein (APP) or β-actin (Bact) as a nuclear reference gene. This adapted qPCR assay was validated by testing its sensitivity and technical variation of sampling. We also attempted to detect mtDNA replication in sperm. Sensitivity was tested by measuring mtDNA copy numbers in sperm, liver, and pancreas tissues. Liver cells had the highest level of average copies per cell in ND1, ND4, and CytB genes; with copies of 164.9±52.5, 99.0±31.4, and 139.6±35.3 respectively. Pancreas had fewer average mtDNA copies per cell for ND1, ND4, and CytB at 99.2±19.4, 54.2±13.1, and 60.8±12.6 respectively. The sperm sample was significantly lower (P < 0.05) than both of the other tissues for ND1, ND4, and CytB with 0.5±0.1, 0.5±0.1, and 0.4±0.1 average mtDNA copies per cell respectively. Replication of mtDNA was tested using glucose and fructose as metabolic stimulators with and without the presence of polymerase gamma inhibitors: 0.5µg/µl of ethidium bromide (EtBr), 100µM 2’,3’-dideoxycytidine (ddC), 20mM phosphonoformate (PF), and 0.1mM 2’,3’-dideoxythymidine 5’-triphosphate (ddTTP). The results did not show any change between treatment groups, thus replication of mtDNA in sperm was unsupported. The qPCR assay was then used for determining if mtDNA copies increase with advancing age in stallions after validation. Samples from three stallion groups, varying in age, were analyzed for mtDNA copy number and correlated with the stallion’s age. We observed a significant (P < 0.05) positive relationship between mtDNA copy number and increasing stallion age with an r value of 0.54 for the ND1 gene in a subset of stallions from Thoroughbred stallions collected with an artificial vagina (AV). When all mitochondrial genes were averaged, mtDNA copy number also showed a positive trend with an r value of 0.48, although this was not statistically significant. For the first time, a qPCR technique was developed to quantitate mtDNA copy number in stallion sperm. Further research is necessary for using mtDNA copy number as a biomarker for fertility in stallions.
|Advisor:||Meyers, Stuart A.|
|Commitee:||Cherr, Gary, Ross, Pablo|
|School:||University of California, Davis|
|School Location:||United States -- California|
|Source:||DAI-B 78/03(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Animal sciences|
|Keywords:||Equine, Mitochondria, Sperm|
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