Maternal diet-induced obesity alters mitochondrial activity and redox status in mouse oocytes and zygotes

Abstract

The negative impact of obesity on reproductive success is well documented but the stages at which development of the conceptus is compromised and the mechanisms responsible for the developmental failure still remain unclear. Recent findings suggest that mitochondria may be a contributing factor. However to date no studies have directly addressed the consequences of maternal obesity on mitochondria in early embryogenesis.Using an established murine model of maternal diet induced obesity and a live cell dynamic fluorescence imaging techniques coupled with molecular biology we have investigated the underlying mechanisms of obesity-induced reduced fertility. Our study is the first to show that maternal obesity prior to conception is associated with altered mitochondria in mouse oocytes and zygotes. Specifically, maternal diet-induced obesity in mice led to an increase in mitochondrial potential, mitochondrial DNA content and biogenesis. Generation of reactive oxygen species (ROS) was raised while glutathione was depleted and the redox state became more oxidised, suggestive of oxidative stress. These altered mitochondrial properties were associated with significant developmental impairment as shown by the increased number of obese mothers who failed to support blastocyst formation compared to lean dams. We propose that compromised oocyte and early embryo mitochondrial metabolism, resulting from excessive nutrient exposure prior to and during conception, may underlie poor reproductive outcomes frequently reported in obese women.

Figure 1. Maternal diet-induced obesity leads to increased mitochondrial activity in the oocytes and zygotes.

The measurements of inner mitochondrial membrane potential (Δψ_m) were made using confocal imaging of TMRM fluorescence. The signal intensity was quantified per pixel in a confocal slice after thresholding to remove background signal. (A) – oocytes and (B) zygotes derived from lean (n = 15 cells/group) and obese females (n = 15 cells/group). Relative intensity of TMRM fluorescence is expressed as a percentage of the signal from oocytes of lean mice. (C) Representative confocal images of mitochondria distribution in oocytes from lean and obese mice. * p<0.05. Data are mean ± SEM.

Figure 2. Maternal diet-induced obesity is associated with an oxidised intracellular redox state in oocytes and zygotes.

The redox state of single oocytes and zygotes from lean (n = 15 cells/group) and obese (n = 15 cells/group) mice was estimated through measurements of NAD(P)H and FAD²⁺ autofluorescence intensity. The resting redox state is defined as a ratio of the maximally oxidised (response to 1 mM FCCP) and maximally reduced (response to 1 mM NaCN) signals. The fluorescence signals are normalised between 100 and 0. For NAD(P)H autofluorescence (A): 0 - maximally oxidised state; 100 – maximally reduced state. This scale is reversed for FAD²⁺ fluorescence (B). 0 – maximally reduced state; 100 – maximally oxidised state. * p<0.05 relative lean group. Data are mean ± SEM.

Figure 3. Maternal diet induced obesity increases rates of ROS generation and depletes glutathione in oocytes and zygotes.

Cytosolic ROS production in oocytes and zygotes was measured by rate of oxidation of HEt. The traces represent changes of HEt fluorescence in oocytes (A) and zygotes (B) from lean (n = 15 cells/group) and obese (n = 15 cells/group) mice as a function of time. These data are summarised in histograms (C, D), in which the mean rates of ROS production are shown as the mean rate of HEt fluorescence change per minute. Results are expressed as percentage changes from HEt fluorescence in lean oocytes. Intracellular glutathione staining with MCB in oocytes (E) and zygotes (F) recovered from lean and obese female mice. Relative intensity of MCB fluorescence is expressed as a percentage of the signal from oocytes of lean mice. (G) Representative confocal images of GSH staining in oocytes. *p<0.05, ** p<0.01. Data are mean ± SEM.

Figure 4. Mitochondrial biogenesis is up-regulated in oocytes from obese mice.

(A) mtDNA copy number in oocytes from lean (n = 2 oocytes/8 females/group) and obese (n = 2 oocytes/8 females/group) mice. Relative abundance of TFAM mRNA (B) and NR1mRNA (C) in oocytes from lean (n = 20 oocytes/8 females/group) and obese (n = 20 oocytes/8 females/group) mice. qPCR was used to determine the absolute mtDNA copy number and the amount of specific transcripts relative to the H2AmRNA.* p<0.05. Data are mean ± SEM.