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Deep Profiling of Oocyte Aging Enabled by Simple One-Step Vial-Based Pretreatment and Single-Cell Proteomics

ABSTRACT: Single-cell proteomics is a pivotal technology for studying cellular phenotypes, offering unparalleled insights into cellular heterogeneity and dynamic functions. Technical improvement in mass spectrometry instruments and sample preparation has made proteomics profiling of single mouse oocytes or early embryos feasible in recent years. Yet, developing a simple and robust sample preparation method to enable deep proteomics profiling of single germline cells remains a significant challenge. Herein, we developed a simple one-step vial-based pretreatment (SOViP) for deep label-free single-cell proteomics of germline cells. SOViP integrates all sample preparation procedures into a single step in autosampler vials, yet it is highly efficient and high-throughput in comparison to reported multistep methods. SOViP can be finished within ∼2 h with hands-on time limited to merely a few minutes. On average, over 6500 protein groups can be quantified from a single mouse oocyte using SOViP. In total, 6983 protein groups were identified from single mouse oocytes across an entire reproductive lifespan, offering a valuable proteomics resource for oocyte aging. Unique molecular characteristics of oocytes at different ages were revealed, and a classifier consisting of ten proteins demonstrated accurate age-group classification and fertility-level prediction. Although demonstrated using mouse oocytes in this study, SOViP is adaptable to rare cell samples and other large cells including follicles and preimplantation embryo cells, among others.

ORGANISM(S): Homo Sapiens Mus Musculus

SUBMITTER: Yun Yang

PROVIDER: PXD063686 | iProX | Fri May 02 00:00:00 BST 2025

REPOSITORIES: iProX

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Project description:Background: Non-genetic disease inheritance and offspring phenotype is substantially influenced by germline epigenetic programming, including genomic imprinting. Loss of Polycomb Repressive Complex 2 (PRC2) function in oocytes causes non-genetically inherited effects on offspring, including embryonic growth restriction followed by post-natal offspring overgrowth. While PRC2 dependent non-canonical imprinting is likely to contribute, less is known about germline epigenetic programming of non-imprinted genes during oocyte growth. In addition, de novo germline mutations in genes encoding PRC2 lead to overgrowth syndromes in human patients, but the extent to which PRC2 activity is conserved in human oocytes is poorly understood. Results: In this study we identify a discrete period of early oocyte growth during which PRC2 is expressed in mouse growing oocytes. Deletion of Eed during this window led to the de-repression of 343 genes. A high proportion of these were developmental regulators, and the vast majority were not imprinted genes. Many of the de-repressed genes were also marked by the PRC2-dependent epigenetic modification histone 3 lysine 27 trimethylation (H3K27me3) in primary-secondary mouse oocytes, at a time concurrent with PRC2 expression. In addition, we found H3K27me3 was also enriched on many of these genes by the germinal vesicle (GV) stage in human oocytes, strongly indicating that this PRC2 function is conserved in the human germline. However, while the 343 genes were de-repressed in mouse oocytes lacking EED, they were not de-repressed in pre-implantation embryos and lost H3K27me3 during pre-implantation development. This implies that H3K27me3 is a transient feature that represses a wide range of genes in oocytes. Conclusions: Together, these data indicate that EED has spatially and temporally distinct functions in the female germline to repress a wide range of developmentally important genes, and that this activity is conserved in the mouse and human germlines.

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Deep Profiling of Oocyte Aging Enabled by Simple One-Step Vial-Based Pretreatment and Single-Cell Proteomics

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