Metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) accurately detects mitochondrial dysfunction in mouse oocytes

Mitochondrial dysfunction caused by reproductive aging (mild) and Clpp deficiency (severe) was accurately detected with the use of fluorescence lifetime imaging microscopy (FLIM).

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Volume 110, Issue 7, Pages 1387–1397

Authors:

Tim Sanchez, Ph.D., Tianren Wang, M.D., Ph.D., Marta Venturas Pedro, M.Sc., Man Zhang, M.D., Ph.D, Ecem Esencan, M.D., Denny Sakkas, Ph.D., Dan Needleman, Ph.D., Emre Seli, M.D.

Abstract:

Objective

To determine whether metabolic imaging with the use of fluorescence lifetime imaging microscopy (FLIM) identifies metabolic differences between normal oocytes and those with metabolic dysfunction.

Design

Experimental study.

Setting

Academic research laboratories.

Patient(s)

None.

Intervention(s)

Oocytes from mice with global knockout of Clpp (caseinolytic peptidase P; n = 52) were compared with wild-type (WT) oocytes (n = 55) as a model of severe oocyte dysfunction. Oocytes from old mice (1 year old; n = 29) were compared with oocytes from young mice (12 weeks old; n = 35) as a model of mild oocyte dysfunction.

Main Outcome Measure(s)

FLIM was used to measure the naturally occurring nicotinamide adenine dinucleotide dehydrogenase (NADH) and flavin adenine dinucleotide (FAD) autofluorescence in individual oocytes. Eight metabolic parameters were obtained from each measurement (4 per fluorophore): short (τ1) and long (τ2) fluorescence lifetime, fluorescence intensity (I), and fraction of the molecule engaged with enzyme (F). Reactive oxygen species (ROS) levels and blastocyst development rates were measured to assess illumination safety.

Result(s)

In Clpp-knockout oocytes compared with WT, FAD τ1 and τ2 were longer and I was higher, NADH τ2 was longer, and F was lower. In old oocytes compared with young ones, FAD τ1 was longer and I was lower, NADH τ1 and τ2were shorter, and I and F were lower. FLIM did not affect ROS levels or blastocyst development rates.

Conclusion(s)

FLIM parameters exhibit strong differentiation between Clpp-knockout versus WT, and old versus young oocytes. FLIM could potentially be used as a noninvasive tool to assess mitochondrial function in oocytes.


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Fertility and Sterility

Editorial Office, American Society for Reproductive Medicine

Fertility and Sterility® is an international journal for obstetricians, gynecologists, reproductive endocrinologists, urologists, basic scientists and others who treat and investigate problems of infertility and human reproductive disorders. The journal publishes juried original scientific articles in clinical and laboratory research relevant to reproductive endocrinology, urology, andrology, physiology, immunology, genetics, contraception, and menopause. Fertility and Sterility® encourages and supports meaningful basic and clinical research, and facilitates and promotes excellence in professional education, in the field of reproductive medicine.

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