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Human oocyte proteome and secretome

ABSTRACT: The proteome and secretome of human oocytes was studied by shotgun LCMSMS, starting from pools of 100 cells. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ~100ng per cell, we consistently identified ~450 proteins from individual oocytes. When comparing proteomes of individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage by label-free quantification, we found several proteins preferentially expressed in matuare and immature cells. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and pre-implantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Jeroen Krijgsveld

PROVIDER: MSV000088387 | MassIVE | Mon Nov 15 19:54:00 GMT 2021

SECONDARY ACCESSION(S): PXD003691

REPOSITORIES: MassIVE

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Identification of Maturation-Specific Proteins by Single-Cell Proteomics of Human Oocytes.

Virant-Klun Irma I Leicht Stefan S Hughes Christopher C Krijgsveld Jeroen J

Molecular & cellular proteomics : MCP 20160523 8

Oocytes undergo a range of complex processes via oogenesis, maturation, fertilization, and early embryonic development, eventually giving rise to a fully functioning organism. To understand proteome composition and diversity during maturation of human oocytes, here we have addressed crucial aspects of oocyte collection and proteome analysis, resulting in the first proteome and secretome maps of human oocytes. Starting from 100 oocytes collected via a novel serum-free hanging drop culture system, ...[more]

PMID: 27215607

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Human oocyte proteome and secretome

Project description:The proteome and secretome of human oocytes was studied by shotgun LCMSMS, starting from pools of 100 cells. Exploiting SP3, a novel technology for proteomic sample preparation using magnetic beads, we scaled down proteome analysis to single cells. Despite the low protein content of only ~100ng per cell, we consistently identified ~450 proteins from individual oocytes. When comparing proteomes of individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage by label-free quantification, we found several proteins preferentially expressed in matuare and immature cells. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and pre-implantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types.

2016-05-26 | PXD003691 | Pride

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Transcription profiling of mouse pre-implantation development over twelve time points from the germinal vesicle (GV) stage oocyte to the late (expanded) blastocyst

Project description:The goal of the experiments was to profile and analyze gene activity during murine pre-implantation development. Samples were collected at twelve time points from the germinal vesicle (GV) stage oocyte to the late (expanded) blastocyst.

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Single-cell multi-omics profiling reveals key regulatory mechanisms that poise germinal vesicle oocytes for maturation in pigs

Project description:The molecular mechanisms controlling the transition from meiotic arrest (germinal vesicle stage) to meiotic resumption (germinal vesicle breakdown stage) in mammalian oocytes have not been fully elucidated. Single-cell omics technology provides a new opportunity to decipher the early molecular events of oocyte maturation in mammals. Here we focused on analyzing oocytes that are collected from antral follicles of porcine puberty ovary. We used single-cell M&T-seq technology to analyze the nuclear DNA methylome and cytoplasmic transcriptome in parallel for 62 oocytes. We developed a package of pipelines for single-cell methylomics analysis, namely methyConcerto, to specifically and comprehensively characterize the methylation state and allele-specific methylation events for a single cell. We also performed 10X Genomics single-cell transcriptomic analyses to explore the bi-directional cell-cell communications within antral follicles. We characterized the gene expression and DNA methylation programs of individual oocytes in porcine antral follicle, thereby enabled defining two distinct types of oocytes, one of which is significantly more poised for maturation. Significantly differentially expressed or allele-specifically methylated genes were enriched in “RNA metabolism”, “oocyte meiosis” important signaling pathways, e.g., HIF-1/Ras/mTOR/Phospholipase D/ErbB signaling pathways etc. These results are in concert with the cellular communication results. We further confirmed Insulin Receptor Substrate-1 (IRS-1) in insulin signaling pathway is a key regulator of germinal vesicle stage oocyte maturation by in vitro maturation experiments. Our study provides new insights into the regulatory mechanisms between meiotic arrest and meiotic resumption in mammalian oocytes. We also provide a new analytical package for future single-cell methylomics study.

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Implantation Failure of Blastocysts Derived from Oocyte-directed Connexin 43 depleted Mice is Associated with Impaired Ribosomal and Translational Machinery Gene Expression

Project description:Oocyte quality is a well- established determinant of embryonic fate. However, the molecular participants and biological markers that affect and predict adequate embryonic development are largely elusive. We have previously reported that oocyte- directed Connexin 43 (Cx43) depletion leads to embryo implantation defects, although both the morphology of the oocyte and processes presiding embryo implantation appear to undergo normally. In the context of previous data determining Cx43 indispensability to oocyte and embryonic development, we show here that the timing of Cx43 depletion from the oocyte and the ovarian follicle is crucial in determining the severity of subsequent embryonic defects. Specifically, we show that the implantation defects of blastocysts resulting from oocyte- directed Cx43- depleted follicles (depletion occurs at day 3 postnatal), is not due to maternal luteal insufficiency but rather depends solely on the defective blastocysts. Gene expression microarray analysis revealed global defects in the expression of ribosomal proteins, translation initiation factors and other genes associated with cellular biosynthetic and metabolic processes in these defective oocytes and specifically blastocysts. We therefore propose that timely expression of Cx43 in the oocyte and ovarian follicles is a major determinant of oocyte developmental competence, by determining the ability of the resulting blastocyst to facilitate biomass expansion and undergo adequate embryo implantation To study the effect of CX43 on the transcriptom of the pre implantation stages, we compared CX43 KO oocytes to the WT oocytes in three different stages of the very early development. First comparison MII oocytes, second comparison blastocysts, third comparison implantation site.

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Project description:We aimed to reveal gene expression profiles of human oocytes at each maturation stage by single cell RNA-seq analyses. We investigated transcriptomes of immature oocytes at the germinal vesicle (GV) stage, maturating oocytes at the metaphase I (MI) stage and in vitro matured oocytes at the metaphase II (MII) stage.

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The transcriptome of ejaculates used for successful/failed in vitro-fertilization

Project description:We investigated the transcriptome obtained from microarray analysis of human ejaculates used in an IVF programme. The gene expression profiles are to be correlated with successful/failed induction of oocyte fertilisation and/or pregnancy. Since human ejaculates are often contaminated with so-called 'round cells' (mostly somatic cells such as leukocytes and macrophages, but also immature germ cells), a considerable part of the gene expression pertains not to spermatozoa. Rigorous statistical analysis of the data is to be used to extract genes that correlate with fertilization success. A set of 25 human ejaculates (12 pregnancy; 3 biochemical pregnancy; 7 oocyte fertilization; 3 no oocyte fertilization) from an IVF procedure were used for RNA extraction and microarray hybridization.

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