O-203 Live real-time determination of mitochondrial bioenergetics as a reliable tool to evaluate mitochondrial activity in mammalian oocytes
Authors: Di Emidio G , Capece D , Flati I , Vergara T , Placidi M , Virmani A , D'Alessandro A M , Tatone C 
Published: June 2022

Abstract

Study question
Can live real-time determination of mitochondrial bioenergetics represent a reliable assay of the mitochondrial activity and bioenergetic metabolism in mouse oocytes?

Summary answer
We demonstrated that real-time determination of mitochondrial oxidative phosphorylation is a feasible method for the characterization of mitochondrial activity and adaptation capacity of mammalian oocytes.

What is known already
Mammalian oocytes possess about 300.000 mitochondria and their alterations represent the main factors underlying reduced oocyte competence. Mitochondrial oxidative phosphorylation, measured as the oxygen consumption rate (OCR) is the largest contributor to cellular ATP demand. Fine-tuning mitochondrial activity is required to sustain bioenergetics homeostasis and maintain physiological levels of ROS production in the female gamete. Despite numerous techniques have been used to measure OCR, the application in oocytes have remained infeasible. Recently the development of a non-invasive, real-time assay has provided a more comprehensive picture of OCR in terms of the components of mitochondrial oxygen flux.

Study design, size, duration
Mitochondrial bioenergetic profile was determined in GV oocytes from young unprimed and primed mice; MII oocytes from young hCG-primed mice; oocytes from aged hCG-primed mice; young MII oocytes from IVM; aged MII oocytes from IVM; MII oocytes from IVM following oxidative stress in the presence or absence of the NAD+ booster p7C3 or propionyl-L-carnitine (PLC). Mean values of basal oxygen consumption rate, maximal capacity, reserve capacity and non-mitochondrial sources on oxygen consumption were evaluated.

Participants/materials, setting, methods
Young (4-8 weeks) and aged (36-40weeks) CD-1 mice were employed. GV oocytes were isolated from PMSG-primed and unprimed mice. MII oocytes were retrieved from hCG-primed mice. Oxidative stress was induced by exposure to 25-100µM H2O2 for 10-30’. IVM was performed in M2 medium with/out 1-5 mM P7C3 or 0.2mg/ml PLC. SeahorseXFp96 Agilent was employed to measure OCR (pmol/min/oocytes/wells) in pools of 8 oocytes after establishment of mitochondrial inhibitors concentrations. SigmaStat was used for statistical analysis.

Main results and the role of chance
From the analysis of the parameters above described, GV oocytes from primed and unprimed mice showed different bioenergetic profiles revealing lower basal OCR in unprimed GV. When exposed to oxidative stress, GV oocytes exhibited of redox state alteration evidenced by the response to the different inhibitors. These effects were prevented by exposure to P7C3 or PLC. A similar profile was present in GV oocytes from aged oocytes. All classes of MII displayed an OCR lower than GV. Ovulated MII oocytes showed an OCR lower than MII from IVM oocytes. Moreover, the latter displayed an increased non-mitochondrial respiration as evidence of oxidative stress. IVM oocytes exposed to oxidative insult revealed increased proton leak associated with reduced ATP levels. The presence of P7C3 in the IVM medium reduced non-mitochondrial respiration and proton leak with positive effects on ATP production and IVM rate. MII oocytes from aged mice when compared with the young group showed a different bioenergetic profile revealing lower OCR in the aged MII along with reduced spare respiratory capacity and increased proton leak. These changes suggest decreased mitochondrial mass and/or altered integrity of ETC components. The presence of P7C3 during IVM allowed a partial recovery of these dysfunctions.

Limitations, reasons for caution
Results from animal studies should be extrapolated to humans with caution.

Wider implications of the findings
Live real-time analysis of mitochondrial bioenergetics can be a reliable approach for the study of mitochondrial dysfunctions linked with reduced oocyte competence associated with aging and other oxidative stress conditions. Thus, it could be employed to set up specific recovery strategies based on metabolic boosters to improve human IVF.

Trial registration number
not applicable