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. 2022 Jun 13;106(6):1218-1231.
doi: 10.1093/biolre/ioac032.

Glutathione deficiency decreases lipid droplet stores and increases reactive oxygen species in mouse oocytes†

Kelli F Malott  1   2   3 , Samantha Reshel  4 , Laura Ortiz  3 , Ulrike Luderer  1   2   3   4
Affiliations

Glutathione deficiency decreases lipid droplet stores and increases reactive oxygen species in mouse oocytes†

Kelli F Malott et al. Biol Reprod. .

Abstract

Glutathione (GSH) is a tripeptide thiol antioxidant that has been shown to be important to overall reproductive health. Glutamate cysteine ligase, the rate-limiting enzyme in GSH synthesis consists of a catalytic and a modifier (GCLM) subunit. We previously showed that oxidative stress in the ovary and oocytes of Gclm-/- mice is associated with accelerated age-related decline in ovarian follicles and decreased female fertility due to preimplantation embryonic mortality. Mammalian preimplantation development is a highly regulated and energy-intensive process that primarily relies on coordination between lipid droplets (LDs) and mitochondria to maintain cellular homeostasis. In this study, we hypothesized that GSH deficiency in oocytes increases oxidative stress, leading to increased mitochondrial dysfunction and decreased LD consumption, thereby decreasing oocyte developmental competence. We observed that Gclm-/- oocytes have increased oxidative stress, primarily in the form of mitochondrial superoxide and decreased subcortical mitochondrial clusters. Further, Gclm-/- oocytes have decreased mitochondrial membrane potential (ΔΨm) compared with Gclm+/+. We surmise this is likely due to the decreased availability of LDs, as we observed a significant decrease in LD content in Gclm-/- oocytes compared with Gclm+/+. The decreased oocyte LD content is likely related to an altered serum lipidome, with Gclm-/- serum having relatively lower unsaturated fatty acids and triglycerides than that of Gclm+/+ and Gclm+/- females. Altogether these data support that decreased LDs and increased oxidative stress are primary drivers of decreased oocyte developmental competence in GSH-deficient oocytes.

Keywords: glutathione; lipid droplets; lipidomics; mitochondria; oocyte; oxidative stress.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1
Figure 1
Gclm−/− oocytes have increased levels of ROS compared to Gclm+/+ oocytes. (A) There is no difference in the number of morphologically healthy oocytes ovulated per female of each genotype (N = 13 = 31 females/genotype). (B) Green DCF fluorescence of experimental oocytes relative to mean fluorescence of 0.003% hydrogen peroxide-treated, positive control oocytes of same genotype on the same experimental day is elevated in Gclm−/− oocytes (**P = 0.008, t-test, N = 6 females/genotype; superimposed filled circles, squares and triangles show values for individual oocytes in this and subsequent figures). (C) Gclm−/− oocytes have increased levels of mitochondrial superoxide, as estimated by fold Gclm−/− fluorescence of oocytes analyzed on the same day (*P = 0.045, GEE, N = 5–8 females/genotype). (D) There is no statistically significant difference in telomere length between genotypes (N = 3 pools of 20 oocytes each from 1–3 females/genotype).
Figure 2
Figure 2
Gclm−/− oocytes have decreased ΔΨm but no difference in lipid peroxidation compared with Gclm+/+ oocytes. (A) Representative confocal images of JC-1 ΔΨm measurement (red = high ΔΨm, green = low ΔΨm). (B) Gclm−/− and Gclm+/− oocytes have lower mitochondrial membrane potential measured as total mean red:green fluorescence ratio using JC-1 (*P < 0.05, GEE, N = 5–11 females/genotype). The mitochondrial uncoupler CCCP was used as a positive control. (C) There is no Gclm genotype-related difference in lipid oxidation measured as total mean green:red fluorescence ratio using BODIPY 581/591 C11, excitation at 581 nm and emission at 591 nm (red), which will shift to 510 nm (green) when oxidized (N = 4–9 females/genotype). (D) Total volume of mitochondrial clusters per whole oocyte normalized to volume in Gclm−/− oocytes imaged on same day does not differ by Gclm genotype (N = 3–7 females/genotype). (E) Ratio of mitochondrial volume clustered in the subcortical region of the oocyte to total mitochondrial volume is significantly decreased in Gclm−/− and Gclm+/− compared to Gclm+/+ oocytes (N = 3–7 females/genotype, *P < 0.05, GEE). Superimposed filled symbols show values for individual oocytes in all graphs in this figure.
Figure 3
Figure 3
Serum lipidomics demonstrate altered lipid profile in superovulated Gclm−/− mice compared to superovulated Gclm+/− and Gclm+/+ mice and unsuperovulated Gclm+/− mice. (A) Hierarchical cluster analysis of serum lipid concentrations shows distinct clusters of Gclm−/− and unsuperovulated Gclm+/−. (B) Principal component analysis similarly shows that Gclm−/− females have distinct serum lipid profile compared with other genotypes. (C) Random Forest classification shows the cumulative classification error rates for each experimental group, as well as the overall error rate, as the group of classification trees is grown by random feature selection from a bootstrap sample. The analysis shows that Gclm−/− and unsuperovulated mice have distinct serum lipid profiles, which allow them to be reliably separated with zero error rates from the other experimental groups, while Gclm+/− and Gclm+/+ mice do not. (N = 4–9 females/group).
Figure 4
Figure 4
Representative differences in serum fatty acids, triacylglycerols, phospholipids, and sphingomyelins among Gclm genoytypes. (A–D) Representative box-and-whisker plots from serum lipidomics analysis described in Figure 3 showing significantly different concentrations of representative (A) fatty acid (FDR P-value = 0.015), (B) triacylglycerol (FDR P-value = 0.004), (C) phospholipid (FDR P-value = 0.0001), and (D) sphingomyelin (FDR P-value = 7.43E−05) among experimental groups. *Significantly different (P < 0.05) from Gclm−/− by Fisher’s LSD test; # significantly different (P < 0.05) from unsuperovulated by Fisher’s LSD test. (N = 4–9 females/group).
Figure 5
Figure 5
Gclm−/− oocytes have fewer LDs than Gclm+/+ oocytes. (A) Neutral lipids were stained using BODIPY 493/503. Green fluorescence intensity, expressed as the fold mean intensity of Gclm−/− oocytes imaged on the same day, was higher in Gclm+/+ and Gclm+/− compared to Gclm−/− oocytes (#P-value = 0.083, **P-value = 0.007, by generalized estimating equation, N = 3–6 females/genotype). (B) There were no Gclm-genotype-related differences in percentage of mitochondria colocalized with LDs in oocytes measured using Mitotracker Deep Red and BODIPY 493/503 (N = 5–6 females/genotype). (C) Volume measurements, rendered in the Imaris imaging software using BODIPY 493/503 fluorescence, show Gclm−/− oocytes have smaller LDs on average compared with Gclm+/+ (***P-value<0.001, generalized estimating equation N = 5–6 females/genotype). (D) Representative original confocal images and same images after Imaris processing of LDs and mitochondria in oocytes of Gclm+/+, Gclm+/−, and Gclm−/− mice. LDs are stained using BODIPY 493/503 and mitochondria are stained using Mitotracker Deep Red. Superimposed filled symbols show values for individual oocytes in all graphs in this figure.
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