Single-cell proteomics reveals decreased abundance of proteostasis and meiosis proteins in advanced maternal age oocytes

Abstract

Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed data-independent acquisition, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman's age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.

Keywords: advanced maternal age; aging; human oocytes; meiosis; oocyte quality; proteostasis; single-cell proteomics.

Figure 1.

Proteomic changes during the final steps of human oocyte maturation. DAPs were identified during the meiotic maturation of human oocytes. Volcano plots show DAPs identified by plexDIA between MII and GV oocytes from the (a) Young and (b) AMA groups. Protein name is indicated for statistically significant DAPs (P.adj ≤ 0.05, Wilcoxon test, ǀfold changeǀ> 1.5). Dot colour indicates protein abundance in MII oocytes compared to GV: purple (more abundant), green (less abundant), and grey (stable levels). (c) Heatmap showing the Log₂ levels of DAPs for all oocyte groups, red (high levels), blue (low levels). (d) Ontology terms representing the pathways that statistically differ between GV and MII oocytes in Young and AMA groups (Wilcoxon test, P.adj ≤ 0.05). Dot size reflects the ratio of identified proteins that change during GV to MII transition to the total proteins within the respective pathway. Dot color represents the P. adjusted value (Wilcoxon test), red (high values), blue (low values). AMA, advanced maternal age; DAPs, differentially abundant proteins; GV, germinal vesicle; MII, metaphase II; Young, 18–30 years; AMA, 37–43 years.

Figure 2.

Age effect on the proteasome complex levels in human oocytes. (a–e) Scatter plots showing the levels of the proteasome complex subunits which were found to be strongly correlated (P.adj ≤ 0.02, P ≤ 0.05, ǀRǀ ≥ 0.5 with age or to have moderate correlation (P ≤ 0.05, ǀR ǀ ≥ 0.3) in GV oocytes. (f) Distribution of proteasome subunits mean correlation coefficient (R_mean) of GV oocytes with age in 10⁴ times randomized data; the proteasome subunits mean correlation coefficient in the original data is represented by the dashed line. GV, germinal vesicle; age range: 18–43 years.

Figure 3.

Age effect on TRiC complex levels in human oocytes. (a) Box plots showing the levels of the TRiC complex subunits in GV oocytes from the Young (black box) and AMA (red box) groups. (b) Scatter plot showing the levels of the TRiC complex subunit TCPH which was found to be strongly correlated with age in GV oocytes (P.adj ≤ 0.02, P ≤0.05, ǀRǀ ≥ 0.5). (c) Distribution of TRiC subunits mean correlation coefficient (R_mean) of GV oocytes with age in 10⁴ times randomized data; the TRiC subunits mean correlation coefficient in the original data is represented by the dashed line. (d) Box plots showing the levels of the TRiC complex subunits in MII oocytes from the Young (black box) and AMA (red box) groups. (e) Scatter plot showing the levels of the TRiC complex subunit TCPD which shown a moderate correlation with age in MII oocytes (P ≤ 0.05, ǀRǀ ≥ 0.3). (f) Distribution of TRiC subunits mean correlation coefficient (R_mean) of MII oocytes with age in 10⁴ times randomized data; the TRiC subunits mean correlation coefficient in the original data is represented by the dashed line. AMA, advanced maternal age; GV, germinal vesicle; MII, metaphase II; Young, 18–30 years; AMA: 37–43 years.

Figure 4.

Functional analysis of proteasome complex during the last step of human oocyte maturation. (a) Immunofluorescence staining of the proteasome and chromosomes in human GV oocytes. The proteasome complex was stained with an antibody against 20S alpha and beta subunits (red), and DNA were counterstained with Hoechst 33342 (pink). Left panel, DNA; middle panel, proteasome; right panel, merged image. Scale = 10 µm. (b) rIVM rates (%) for GV and MI oocytes cultured in presence or absence of 10 µM of the proteasome inhibitor MG-132, for up to 48 h. (c) Immunofluorescence staining of the spindle with an antibody against TUBA (yellow) and chromosomes (DNA, Hoechst 33342) (pink) of human oocytes after rIVM in the presence or absence of 10 µM of MG-132. Scale = 10 µm. (d) Percentages of rIVM-MII oocytes with correct alignment (pink bar) or misalignment (black bar) of their chromosomes. AMA, advanced maternal age; GV, germinal vesicle; MI, metaphase I; MII, metaphase II; rIVM, rescue IVM; TUBA, tubulin alpha isoform.