Proteomic quantification of native and ECM-enriched mouse ovaries reveals an age-dependent fibro-inflammatory signature

Abstract

The ovarian microenvironment becomes fibrotic and stiff with age, in part due to increased collagen and decreased hyaluronan. However, the extracellular matrix (ECM) is a complex network of hundreds of proteins, glycoproteins, and glycans which are highly tissue specific and undergo pronounced changes with age. To obtain an unbiased and comprehensive profile of age-associated alterations to the murine ovarian proteome and ECM, we used a label-free quantitative proteomic methodology. We validated conditions to enrich for the ECM prior to proteomic analysis. Following analysis by data-independent acquisition (DIA) and quantitative data processing, we observed that both native and ECM-enriched ovaries clustered separately based on age, indicating distinct age-dependent proteomic signatures. We identified a total of 4,721 proteins from both native and ECM-enriched ovaries, of which 383 proteins were significantly altered with advanced age, including 58 ECM proteins. Several ECM proteins upregulated with age have been associated with fibrosis in other organs, but to date their roles in ovarian fibrosis are unknown. Pathways regulating DNA metabolism and translation were downregulated with age, whereas pathways involved in ECM remodeling and immune response were upregulated. Interestingly, immune-related pathways were upregulated with age even in ECM-enriched ovaries, suggesting a novel interplay between the ECM and the immune system. Moreover, we identified putative markers of unique immune cell populations present in the ovary with age. These findings provide evidence from a proteomic perspective that the aging ovary provides a fibroinflammatory milieu, and our study suggests target proteins which may drive these age-associated phenotypes for future investigation.

Reproductive Ageing: Inflammation, immune cells, and cellular senescence in the aging ovary

“…Pro-inflammatory cytokine (IL-6, IL-1β, IL-8, interferon-γ [Infg], TNF-α) and chemokine (monocyte chemoattractant protein-1 [Ccl2], C-C motif chemokine ligand [Ccl] 5, and C-X-C motif chemokine ligand [Cxcl] 2) levels also increase within the aged ovary (Briley et al, 2016, Lliberos et al, 2021, Umehara et al, 2022. A recent proteomics study in murine ovary also demonstrated an age-related increase in several markers of pro-inflammatory stress and immune responses, suggesting increased chemotaxis and lymphocyte-mediated immunity (Dipali et al, 2023). Although the contributions of individual ovarian cell types and immune cells to these phenotypic changes remain unclear, the overall changes are aligned with what is reported in a recent single-cell transcriptomic and FACS analyses (Isola et al, 2024).…”

“…For instance, transforming growth factor-β (TGF-β) signaling is important for theca and granulosa cell communication during follicular growth (Knight and Glister, 2006) and also provoked during aging. Enhanced TGF-β activity has been linked to lymphocyte chemotaxis and activation (McCloskey et al, 2020, Li et al, 2006, which increases in the aging ovary (Dipali et al, 2023, Isola et al, 2024. Moreover, TGF-β activity plays a well-established role in driving collagen accumulation and fibrosis during aging (Ren et al, 2023).…”

“…However, collagenase pathway activity decreases in the aged ovary (Isola et al, 2024), likely contributing to age-related ovarian fibrosis and impairments in ovulation (Umehara et al, 2022). Age-related ovarian fibrosis is also closely associated with proteomic changes indicative of a pro-inflammatory environment within the ovary (Dipali et al, 2023). Interestingly, pharmacological interventions (e.g., metformin, BGP-15) that attenuate ovarian fibrosis also curtail ovarian pro-inflammatory stress (Umehara et al, 2022, McCloskey et al, 2020, Landry et al, 2022, further supporting their interactive role in accelerating ovarian aging.…”

Shear wave elastography to assess stiffness of the human ovary and other reproductive tissues across the reproductive lifespan in health and disease

“…With age, mouse ovaries exhibit increased expression of pro-inflammatory molecules as well as the presence of a unique immune cell population associated with chronic inflammation [ 13 , 15 , 16 , 19 ]. Moreover, ovaries from reproductively old mice contain high levels of collagen I and III, are enriched in fibro-inflammatory proteins, and have reduced hyaluronan content [ 11 , 13 , 24 , 25 ] Many of these phenotypes are conserved in the human ovary as well [ 26 , 27 ]. This age-dependent increase in ovarian fibrosis is directly associated with increased ovarian stiffness [ 24 ].…”