Quantification of mitochondrial abundance

28 02 2012

To the Editor:

We read the article by Duran et. al. entitled “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 this article, authors aimed to investigate the contribution of mitochondrial aging to female reproductive senescence in humans. In order to test this hypothesis, an attempt was made to estimate the number of mitochondria by the quantification of mtDNA copy number which is measured via real-time polymerase chain reaction (PCR). They also studied some other biochemical and molecular parameters. They found that the number of mitochondria in the oocytes from various maturational stages did not show any statistically significant differences. They hypothesized that reproductive aging may lead to decreased numbers of mitochondria in human oocytes, which are interpolated by using mtDNA copy number. In their discussion, they concluded that inherently imperfect ovarian reserve indicators, such as follicle stimulating hormone (FSH) level and total number of oocytes retrieved by controlled ovarian hyperstimulation, may also predict the number of mitochondria in individual human oocytes.

It seems that “the number of mitochondria” has been utilized as one of the main parameters for their study. Some of the conclusions have been built solely on the “number of mitochondria.” However, the method for the quantification of mitochondria they used is quite “inferential,” although the idea behind it is reasonable and well described. To our knowledge, the copy number of mtDNA is not always a reliable predictor for number of mitochondria. In other words, it is known that the copy number of mtDNA does not necessarily correlate to the number of mitochondria. Some mitochondrial diseases are known to have increased mitochondria while the mtDNA copy number is decreased (2). Alternatively, citrate synthase, a mitochodrial matrix enzyme, is one of the best indicators for mitochondrial abundance in tissues (2). We think that citrate synthase enzyme activity might have been utilized to estimate the abundance of mitochondria. This approach could have affected some of the results they found as “statistically insignificant.”

Tuncer Cayci, M.D.
Bulent Kurt, M.D.
Emin Ozgur Akgul, M.D.
Yasemin Gulcan Kurt, M.D.
Gulhane Military Medical Academy and Medical School
Ankara, Turkey

References
1. Duran HE, Simsek-Duran F, Oehninger SC, Jones HW Jr, Castora FJ. The association of reproductive senescence with mitochondrial quantity, function, and DNA integrity in human oocytes at different stages of maturation. Fertil Steril. 2011 Aug;96(2):384-8.

2. DiMauro S, Bonilla E. Mitochondrial encephalomyopathies. In: Engel AG, Franzini-Armstrong C, eds. Myology. Vol. II, Philadelphia, Mc Grav Hill, 2004, 1623-1676.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.12.053

The Authors Respond:

We would like to thank Dr. Kurt and his colleagues for their interest in our paper and their observations concerning our assessment of the number of mitochondria in the individual oocytes we examined. We agree that this estimate of mitochondrial number based upon real time PCR assays of mtDNA 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 2 or more if the number of mtDNA molecules per mitochondrion were really 2 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 CS 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 imprecise as they may be.

Frank Joseph Castora, Ph.D.
Department of Obstetrics and Gynecology
Jones Institute for Reproductive Medicine

Eyup H Duran, M.D.
Department of Physiological Sciences
Eastern Virginia Medical School
Norfolk, Virginia

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.12.053

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