Intracellular cholesterol deposits associate with mitochondrial dysfunction and lipid storage abnormalities in infertile SR-B1 KO mice

Abstract

Objectives: Female mice deficient in Scavenger Receptor Class B Type I (SR-B1 KO), the main high-density lipoprotein receptor, are infertile. Their oocytes show unesterified cholesterol (UC) excess, high spontaneous activation, and diminished viability. Cholesterol accumulation in different organelles from various cell types impacts their lipid content, viability, and function. Our aim was to characterize cholesterol deposits and global lipid content in eggs from SR-B1 KO females. Method(s): We used eggs from superovulated SR-B1 KO and WT females. Filipin staining (UC marker) was combined with specific organelle markers for mitochondria, lipid droplets (LD), plasma membrane, and lysosomes, and co-localization was determined using Manders' coefficient M2 (n=6 WT and KO oocytes). Mitochondrial membrane potential (MMP) was evaluated using JC-1 dye (n=23 WT and 25 KO oocytes). Lipids were characterized by shotgun lipidomics (3 pools of 100 oocytes/genotype) (Lipotype, Germany). Result(s): In SR-B1 KO eggs, filipin fluorescence in deposits was brighter than in WT eggs [2.3X; p<0.0001] and cholesterol co-localized higher with lysosomes (41.0+/-2.8% vs 21.7+/-1.7% WT; p=0.0002), mitochondria (10.5+/-0.8% vs 4.3+/-0.6% WT; p=0.0001) and LD (14.8 +/-1.8% vs 7.2+/-2.1% WT; p=0.0373). Filipin staining was particularly higher in KO eggs lysosomes (2.6X, p=0.0001), mitochondria (2.1X, p=0.0001) and LD (1.5X, p=0.01). A lower red/green ratio in SR-B1 KO eggs (0.56+/-0.03 vs. 1.11+/-0.07 WT; p<0.0001) was suggestive of a lower MMP. Results of global lipidomics showed that KO eggs had higher cholesterol ester (CE) (8.6+/-2.8%mol/sample vs non detected (ND) in WT; p=0.039) and 0.6X lower TAG levels (p=0.045). Also, some phosphatidylserine subspecies were only detected in SR-B1 KO and not in WT eggs (0.04+/-0.01%mol/sample PS16:0;0_20:4;0 and 0.013+/-0.003%mol/sample PS18:2;0_18:2;0). Conclusion(s): In SR-B1 KO eggs, UC is accumulated in lysosomes, LD, and mitochondria. The latter may contribute to the altered MMP. Besides, the association of cholesterol excess with altered CE and TAG proportions suggests abnormal lipid storage in those eggs.Copyright © 2022