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

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 τ2 were 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.

Keywords: CLPP; FLIM; Mitochondria; aging; fluorescence lifetime imaging microscopy; mitochondrial unfolded protein response; oocyte.

Copyright © 2018 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1:

(Upper Left) NADH intensity image of four Clpp+/+ (wildtype) oocytes in one microwell of a 9-well dish. The cytoplasm of the oocytes was segmented (blue line boundaries), and oocytes were individually identified (here labelled o1, o2, o3, and o4). 50um scale bar. (Inserted Histograms) Histograms of photon counts vs fluorescence photon arrival time were constructed for each oocyte (blue curves) and fit with a model containing two fluorescent species (black lines). (Right) These fits allow four parameters to be measured. Similar fits were also performed for FAD fluorescence, allowing an additional four parameters to be measured.

Figure 2:

Comparative assessments of Clpp+/+ and Clpp−/− oocytes. A) Brightfield images show gross morphological features of oocytes, while NADH FLIM intensity images reflect the mitochondrial distribution. B) Obvious defects in Clpp−/− oocytes were not observed with brightfield, but fluorescence imaging revealed aberrant mitochondrial distributions in many oocytes. C) Metabolic imaging of Clpp+/+ (n=55) and Clpp−/− oocytes (n=52) detected highly significant differences in five of the eight parameters measured. Parameters are plotted in order of decreasing separation, with asterisks indicating the following p-values: (*: p<10−11) and (**: p<10−26) D) If the three most sensitive metabolic parameters (NADH long lifetime, NADH fraction engaged, and FAD long lifetime) are represented in a 3D plot, we can fit a plane that perfectly separates the two data sets. E) Clpp+/+ oocytes (n=46) and Clpp−/− oocytes (n=43) were lysed to obtain individual mtDNA copy number measurements. T-tests on mtDNA measurements showed only a marginally significant difference with a p-value of 0.042. Error bars represent standard errors.

Figure 3:

Comparative assessments of oocytes from old (12-month) and young (3-month) mice. A,B) Neither brightfield images nor FLIM intensity images revealed obvious differences in morphology or mitochondrial distribution. 20um bar. C) Conversely, metabolic imaging measurements on the same oocytes effectively differentiated young (n=35) and old (n=29) groups, with four of the eight parameters showing significant differences. Parameters are plotted in order of decreasing separation, with asterisks indicating the following p-values: (*:p−3). P-values were not as low as with Clpp, the more severe case of metabolic dysfunction. D) If the three most sensitive metabolic parameters (NADH intensity NADH short lifetime, and NADH long lifetime) are represented in a 3D plot, we can draw a plane that effectively separates the two data sets; however, there is more overlap between the distributions than in the more extreme case of Clpp. E) mtDNA copy number measurements were taken on young (n=35) and old (n=29) oocytes, and no significant difference between the two groups was observed (p=0.14). Error bars represent standard errors.

Figure 4:

Evaluation of safety for varying photodoses of FLIM illumination. Top: Reactive oxygen species levels were measured via HC-DCFDA fluorescence (custom units). Significant differences between illuminated and non-illuminated embryos were not observed for any of the photodoses studied. Embryos exposed to 30mM H2O2 were measured as a positive control. Error standard error bars represent variation between individual embryo measurements. Bottom: Embryos were cultured on the microscope, and blastocyst development rates of illuminated embryos were compared to non-illuminated embryos in the same dish. Embryos cultured in a standard incubator were used as a control. FLIM illumination did not have any significant impact on blastocyst development rates. Standard error bars represent variation between experiment batches.