Developmental Sex Differences in the Metabolism of Cardiolipin in Mouse Cerebral Cortex Mitochondria

Abstract

Cardiolipin (CL) is a mitochondrial-specific phospholipid. CL content and acyl chain composition are crucial for energy production. Given that estradiol induces CL synthesis in neurons, we aimed to assess CL metabolism in the cerebral cortex (CC) of male and female mice during early postnatal life, when sex steroids induce sex-dimorphic maturation of the brain. Despite the fact that total amount of CL was similar, its fatty acid composition differed between males and females at birth. In males, CL was more mature (lower saturation ratio) and the expression of the enzymes involved in synthetic and remodeling pathways was higher, compared to females. Importantly, the sex differences found in CL metabolism were due to the testosterone peak that male mice experience perinatally. These changes were associated with a higher expression of UCP-2 and its activators in the CC of males. Overall, our results suggest that the perinatal testosterone surge in male mice regulates CL biosynthesis and remodeling in the CC, inducing a sex-dimorphic fatty acid composition. In male's CC, CL is more susceptible to peroxidation, likely explaining the testosterone-dependent induction of neuroprotective molecules such as UCP-2. These differences may account for the sex-dependent mitochondrial susceptibility after perinatal hypoxia/ischemia.

Figure 1. CL content and CL saturation ratio in mitochondria from the cerebral cortex of postnatal male and female mice.

(a) CL content in mitochondria from cerebral cortex of male and female mouse pups. (b) CL composition in mitochondria from the cerebral cortex of male and female mouse pups, expressed as the saturation ratio (∑ Saturated (16:0 + 18:0)/∑ Polyunsaturated (18:2 + 18:3 + 20:4 + 22:5 + 22:6)). Sample size N ≥ 4. *Significant differences (p < 0.05) vs. Females for each time point. ^&Significant differences (p < 0.05) vs. Females at the previous time point. ^#Significant differences (p < 0.05) vs. Males at the previous time point. ^@Significant difference (p < 0.05) vs. Females at PND 0.

Figure 2. Androgenization of female pups abolishes sex differences in CL saturation ratio in mitochondria from the developing cerebral cortex.

(a) CL composition in the cerebral cortex of male and female mouse pups treated with vehicle and females treated with TP at PND 1, 2 and 3, expressed as the saturation ratio (∑ Saturated (16:0 + 18:0)/∑ Polyunsaturated (18:2 + 18:3 + 20:4 + 22:5 + 22:6)). (b) Relative amount of CL species in the cerebral cortex of Males Veh, Females Veh and Females TP at PND 1–3. Sample size N ≥ 3. *Significant differences (p < 0.05) vs. Females Veh for each time point.

Figure 3. Sex differences and effect of neonatal testosterone on CL biosynthetic pathways.

(a) Scheme of CL synthesis. The de novo synthesis is catalized by Phosphatidylglycerolphosphate synthase (PGS-1) which transforms cytidinediphosphate CDP-diacylglycerol (CDP-DAG) into phosphatidylglycerolphosphate (PGP), which is later desphosphorylated. CL synthase (CLS) forms immature CL from phosphatidylglycerol and another molecule of CDP-DAG. The remodeling of CL is initiated by the calcium-independent phospholipase A2 gamma (iPLA2-γ), which removes the acyl chains from CL and generates the intermediate monolysocardiolipin (MLCL). Taffazin (Taz), or alternatively lysocardiolipin acyltransferase-1 (LCLAT-1), reacylate MLCL to CL. (b–f) mRNA expression of the main enzymes involved in CL synthesis: PGS-1 (b), CLS (c), iPLA2-γ (d), TAZ (e) and LCLAT-1 (f). Sample size N ≥ 4. **^, ***Significant differences (p < 0.01 and p < 0.001) between Males Veh and Females Veh at each time point. ^{%, %%, %%%}Significant differences (p < 0.05, p < 0.01 and p < 0.001) between Females Veh and Females TP at each time point. ^$Significant difference (p < 0.05) vs. Males Veh PND 1. ^&&Significant differences (p < 0.01) vs. Females TP PND 1. ^@Significant (p < 0.05) differences between Females TP and Males Veh.

Figure 4. Sex differences and effect of neonatal testosterone on desaturases.

mRNA expression of FADS-1 (Fatty acid desaturase-1) (a) and FADS-2 (Fatty acid desaturase-2) (b). Sample size N ≥ 4. *^, ***Significant differences (p < 0.05 and p < 0.001) between Males Veh and Females Veh at each time point. ^{%%, %%%}Significant differences (p < 0.01 and p < 0.001) between Females Veh and Females TP at each time point. ^$$$Significant difference (p < 0.001) vs. Males Veh PND 1. ^@@Significant (p < 0.01) differences between Females TP and Males Veh.

Figure 5. Sex differences and effect of neonatal testosterone on UCP2 and PPAR-α expression.

mRNA expression of UCP2 (a) and PPAR-α (b). Sample size N ≥ 4. *^, **Significant differences (p < 0.05 and p < 0.01) between Males Veh and Females Veh at each time point. ^{%, %%, %%%}Significant differences (p < 0.05, p < 0.01 and p < 0.001) between Females Veh and Females TP at each time point.

Figure 6. Correlation of UCP2 mRNA expression with PPAR-α(A) and iPLA2-γ(B) mRNA levels.

Number of XY pairs = 62 for global analyses, 19 for Males Veh and Females TP groups and 24 for Females Veh group.