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

Abstract

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, we identified 2,154 proteins, whose function indicate that oocytes are largely resting cells with a proteome that is tailored for homeostasis, cellular attachment, and interaction with its environment via secretory factors. In addition, we have identified 158 oocyte-enriched proteins (such as ECAT1, PIWIL3, NLRP7)(1) not observed in high-coverage proteomics studies of other human cell lines or tissues. 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 ∼100 ng per cell, we consistently identified ∼450 proteins from individual oocytes. When comparing individual oocytes at the germinal vesicle (GV) and metaphase II (MII) stage, we found that the Tudor and KH domain-containing protein (TDRKH) is preferentially expressed in immature oocytes, while Wee2, PCNA, and DNMT1 were enriched in mature cells, collectively indicating that maintenance of genome integrity is crucial during oocyte maturation. This study demonstrates that an innovative proteomics workflow facilitates analysis of single human oocytes to investigate human oocyte biology and preimplantation development. The approach presented here paves the way for quantitative proteomics in other quantity-limited tissues and cell types. Data associated with this study are available via ProteomeXchange with identifier PXD004142.

Fig. 1.

Proteome analysis of oocytes from two different culture systems. (A) Conventional IVF culture system with oocyte culturing in a closed system–conventional IVF medium in covered four-well dishes (0.5 ml of medium per well). (B) New hanging drop culture system culturing oocytes in small, 8 μl droplets of DMEM/F12 medium. After culturing, both oocytes and droplets of corresponding medium were sampled to analyze the proteome and secretome. (C) Estimated abundance by iBAQ value of proteins identified in oocytes grown in IVF media and in hanging drops. Blue symbols: proteins identified both in IVF and hanging drops. Red symbols: proteins identified in hanging drops only.

Fig. 2.

Gene ontology terms of proteins exclusively identified in oocytes (blue) and in the reference proteome obtained from 11 cell lines and embryonic stem cells (red).

Fig. 3.

Proteins and their gene ontology classification identified in human oocytes but absent in the proteomes of 11 human cell lines, human embryonic stem cells, and sperm.

Fig. 4.

Proteome comparison of mature and immature oocytes. (A) Label-free quantification (LFQ) of proteins in pools of 100 mature (MII) and 100 immature (GV) oocytes maintained in hanging drops. (B) Same as in A but oocytes maintained in IVF media. (C) Label-free quantification of proteins in single oocytes. The absence of red or blue bars indicates that the respective proteins were not identified in MII oocytes (TDRKH) or GV oocytes (DNMT1, Wee2, PCNA). (D) Protein quantification by stable-isotope-containing dimethyl labeling in pools of 10 mature (MII) and immature (GV) oocytes.

Fig. 5.

Immunofluorescence microscopy of PCNA (A) and DNMT1 (B) in oocytes at the germinal vesicle (GV) state, in metaphase II (MII) and in parthenogenetic embryos (PE).