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Background <p>Ovarian aging-induced decline in oocyte quality has been a main issue in women of advanced maternal age. However, the potential mechanism remains elusive, and there are no effective strategies to ameliorate aged oocyte quality. The lipid metabolism of oocytes has drawn great attention, but the intrinsic regulation of oocyte quality by metabolites, metabolic enzymes, and intracellular mediators is less well-characterized.</p> Methods <p>Targeted lipidomics was employed to detect the neutral lipids in oocytes during maturation. We used 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3<i>a</i>,4<i>a</i>-diaza-<i>s</i>-indacene (BODIPY&#xa0;493/503) and Filipin to stain cholesteryl ester and free cholesterol, respectively. The Cholesterol/Cholesteryl Ester Quantification Assay kit was used further to quantify cholesterol-related metabolites. Western blotting was performed to evaluate acyl-coenzyme A: cholesterol acyltransferase 1/2 (ACAT1/2) expression. Immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to validate the knockdown efficiency of ACAT1. Avasimibe treatment and ACAT1 small interfering RNA (siRNA) microinjection were performed to investigate the effect of impaired cholesterol–cholesteryl ester metabolism on oocyte quality. Single-oocyte RNA sequencing was conducted to explore the mechanism. Mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) production, reactive oxygen species (ROS), and mitochondrial autophagosomes were detected to evaluate mitochondrial function and mitophagy.</p> Results <p>There is a profound increase in the conversion of cholesterol to cholesteryl ester in oocytes during maturation, which depends on ACAT1. Conversely, disturbing the homeostasis of cholesterol–cholesteryl ester metabolism by manipulating ACAT1 impairs oocyte quality, primarily manifested as decreased polar body extrusion (PBE), increased meiotic defects, and abnormal early embryonic development. Mechanistically, the impaired conversion of cholesterol to cholesteryl ester reduces oocyte mitophagy, leading to mitochondrial dysfunction, including reduced MMP and ATP production, and excessive accumulation of ROS. Notably, we also reveal that this metabolic homeostasis is impaired in aged oocytes, accompanied by decreased ACAT1 levels. Moreover, cholesteryl ester supplementation via cholesterol conjugated to methyl-β-cyclodextrin (CCM) can effectively ameliorate aged oocyte quality by enhancing mitophagy.</p> Conclusions <p>This study reveals the mechanism by which cholesterol–cholesteryl ester metabolism regulates oocyte quality and thus participates in the process of oocyte aging by influencing mitophagy and mitochondrial function.</p> Graphical Abstract <p></p>

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Abnormal cholesterol–cholesteryl ester metabolism impairs mouse oocyte quality during ovarian aging

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月24日

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  • Sainan Zhang,
  • Bichun Guo,
  • Junshun Fang,
  • Shanshan Wang,
  • Yicen Liu,
  • Die Wu,
  • Nannan Kang,
  • Yang Zhang,
  • Xin Zhen,
  • Guijun Yan,
  • Lijun Ding,
  • Haixiang Sun,
  • Chuanming Liu

摘要

Background

Ovarian aging-induced decline in oocyte quality has been a main issue in women of advanced maternal age. However, the potential mechanism remains elusive, and there are no effective strategies to ameliorate aged oocyte quality. The lipid metabolism of oocytes has drawn great attention, but the intrinsic regulation of oocyte quality by metabolites, metabolic enzymes, and intracellular mediators is less well-characterized.

Methods

Targeted lipidomics was employed to detect the neutral lipids in oocytes during maturation. We used 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) and Filipin to stain cholesteryl ester and free cholesterol, respectively. The Cholesterol/Cholesteryl Ester Quantification Assay kit was used further to quantify cholesterol-related metabolites. Western blotting was performed to evaluate acyl-coenzyme A: cholesterol acyltransferase 1/2 (ACAT1/2) expression. Immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR) were conducted to validate the knockdown efficiency of ACAT1. Avasimibe treatment and ACAT1 small interfering RNA (siRNA) microinjection were performed to investigate the effect of impaired cholesterol–cholesteryl ester metabolism on oocyte quality. Single-oocyte RNA sequencing was conducted to explore the mechanism. Mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) production, reactive oxygen species (ROS), and mitochondrial autophagosomes were detected to evaluate mitochondrial function and mitophagy.

Results

There is a profound increase in the conversion of cholesterol to cholesteryl ester in oocytes during maturation, which depends on ACAT1. Conversely, disturbing the homeostasis of cholesterol–cholesteryl ester metabolism by manipulating ACAT1 impairs oocyte quality, primarily manifested as decreased polar body extrusion (PBE), increased meiotic defects, and abnormal early embryonic development. Mechanistically, the impaired conversion of cholesterol to cholesteryl ester reduces oocyte mitophagy, leading to mitochondrial dysfunction, including reduced MMP and ATP production, and excessive accumulation of ROS. Notably, we also reveal that this metabolic homeostasis is impaired in aged oocytes, accompanied by decreased ACAT1 levels. Moreover, cholesteryl ester supplementation via cholesterol conjugated to methyl-β-cyclodextrin (CCM) can effectively ameliorate aged oocyte quality by enhancing mitophagy.

Conclusions

This study reveals the mechanism by which cholesterol–cholesteryl ester metabolism regulates oocyte quality and thus participates in the process of oocyte aging by influencing mitophagy and mitochondrial function.

Graphical Abstract