Abstract
To sustain energy-demanding developmental processes, oocytes must accumulate adequate stores of metabolic substrates and mitochondrial numbers prior to the initiation of maturation. In the past, researchers have utilized pooled samples to study oocyte metabolism, and studies that related multiple metabolic outcomes in single oocytes, such as ATP concentration and mitochondrial DNA copy number, were not possible. Such scenarios decreased sensitivity to intraoocyte metabolic relationships and made it difficult to obtain adequate sample numbers during studies with limited oocyte availability. Therefore, we developed and validated procedures to measure both mitochondrial DNA (mtDNA) copy number and ATP quantity in single oocytes. Validation of our procedures revealed that we could successfully divide oocyte lysates into quarters and measure consistent results from each of the aliquots for both ATP and mtDNA copy number. Coefficient of variation between the values retrieved for mtDNA copy number and ATP quantity quadruplicates were 4.72 ± 0.98 and 1.61 ± 1.19, respectively. We then utilized our methodology to concurrently measure mtDNA copy number and ATP quantity in germinal vesicle (GV) and metaphase two (MII) stage oocytes. Our methods revealed a significant increase in ATP levels (GV = 628.02 ± 199.53 pg, MII = 1326.24 ± 199.86 pg, p < 0.001) and mtDNA copy number (GV = 490,799.4 ± 544,745.9 copies, MII = 1,087,126.9 ± 902,202.8 copies, p = 0.035) in MII compared to GV stage oocytes. This finding is consistent with published literature and provides further validation of the accuracy of our methods. The ability to produce consistent readings and expected results from aliquots of the lysate from a single oocyte reveals the sensitivity and feasibility of using this method.
Highlights
Published: 7 December 2021The accumulation of adequate stores of metabolic substrates as well as an increase in mitochondrial number within the oocyte are important components of the acquisition of developmental competence
This transfer of metabolic substrates occurs throughout oogenesis until the onset of oocyte maturation leads to gap junction breakdown
A 260/280 ratio of approximately 1.8 is generally accepted as “pure” for DNA, so we confidently used this polymerase chain reaction (PCR) product to generate the standards for mitochondrial DNA (mtDNA) copy number [26,27]
Summary
The accumulation of adequate stores of metabolic substrates as well as an increase in mitochondrial number within the oocyte are important components of the acquisition of developmental competence. Substrates and co-factors such as pyruvate, acetyl CoA, NADH, CO2 , and FADH are produced from glycolytic activity of the cumulus cells and transferred to the oocyte via gap junctions to support oxidative phosphorylation [12,15,16]. This transfer of metabolic substrates occurs throughout oogenesis until the onset of oocyte maturation leads to gap junction breakdown. The oocyte must support energy demanding developmental processes like fertilization and embryonic cell division with the mitochondria and metabolic substrates accumulated prior to oocyte maturation
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