Reply of the Authors

Abstract

We thank Dr. Kurt and his colleagues for their interest in our paper and their observations about our assessment of the number of mitochondria in the individual oocytes we examined. We agree that this estimate of mitochondrial number based on real-time polymerase chain reaction (PCR) assays of mitochondrial (mt) DNA is imprecise. As noted by Dr. Kurt and colleagues, aside from any imprecision associated with the real-time PCR methodology, there is the uncertainty associated with the current accepted estimate of mtDNA molecules per mitochondrion in oocytes. Is it one? Is it two?We agree that estimating the number of mitochondria from a measured number of mtDNA molecules is not precise. And, quite possibly, using accurate measures of citrate cynthase activity might provide a more accurate measure. However, a major factor in our decision to estimate mitochondrial number from mtDNA numbers was due to our analysis of individual oocytes. Because of this constraint, we chose to use real-time PCR, a technique capable of determining the number of DNA molecules in a single oocyte. With that said, we used a value of one mtDNA molecule per mitochondrion to calculate our number of mitochondria. Clearly, we could be wrong by a factor of two or more if the number of mtDNA molecules per mitochondrion were really two or more.We are unaware of a standardized assay for citrate synthase activity that is sensitive down to a single cell. If or when there is such a sensitive assay, that might prove to be a better measure of mitochondrial number than real-time PCR. Until then, we must use the tools at our disposal, as accurate or as imprecise as they may be. We thank Dr. Kurt and his colleagues for their interest in our paper and their observations about our assessment of the number of mitochondria in the individual oocytes we examined. We agree that this estimate of mitochondrial number based on real-time polymerase chain reaction (PCR) assays of mitochondrial (mt) DNA is imprecise. As noted by Dr. Kurt and colleagues, aside from any imprecision associated with the real-time PCR methodology, there is the uncertainty associated with the current accepted estimate of mtDNA molecules per mitochondrion in oocytes. Is it one? Is it two? We agree that estimating the number of mitochondria from a measured number of mtDNA molecules is not precise. And, quite possibly, using accurate measures of citrate cynthase activity might provide a more accurate measure. However, a major factor in our decision to estimate mitochondrial number from mtDNA numbers was due to our analysis of individual oocytes. Because of this constraint, we chose to use real-time PCR, a technique capable of determining the number of DNA molecules in a single oocyte. With that said, we used a value of one mtDNA molecule per mitochondrion to calculate our number of mitochondria. Clearly, we could be wrong by a factor of two or more if the number of mtDNA molecules per mitochondrion were really two or more. We are unaware of a standardized assay for citrate synthase activity that is sensitive down to a single cell. If or when there is such a sensitive assay, that might prove to be a better measure of mitochondrial number than real-time PCR. Until then, we must use the tools at our disposal, as accurate or as imprecise as they may be. Quantification of mitochondrial abundanceFertility and SterilityVol. 97Issue 5PreviewWe read the article by Duran et al. titled “The association of reproductive senescence with mitochondrial quantity, function, and DNA integrity in human oocytes at different stages of maturation” (1) with great interest. In that article, the authors aimed to investigate the contribution of mitochondrial aging to female reproductive senescence in humans. To test this hypothesis, an attempt was made to estimate the number of mitochondria by the quantification of mitochondrial (mt) DNA copy number, which is measured via real-time polymerase chain reaction. Full-Text PDF