Abstract
The ovarian stroma, the microenvironment in which female gametes grow and mature, becomes inflamed and fibrotic with age. Hyaluronan is a major component of the ovarian extracellular matrix (ECM), and in other aging tissues, accumulation of low molecular weight (LMW) hyaluronan fragments can drive inflammation. Thus, we hypothesized that LMW hyaluronan fragments contribute to female reproductive aging by stimulating an inflammatory response in the ovarian stroma and impairing gamete quality. To test this hypothesis, isolated mouse ovarian stromal cells or secondary stage ovarian follicles were treated with physiologically relevant (10 or 100 μg/mL) concentrations of 200 kDa LMW hyaluronan. In ovarian stromal cells, acute LMW hyaluronan exposure, at both doses, resulted in the secretion of a predominantly type 2 (Th2) inflammatory cytokine profile as revealed by a cytokine antibody array of conditioned media. Additional qPCR analyses of ovarian stromal cells demonstrated a notable up-regulation of the eotaxin receptor Ccr3 and activation of genes involved in eosinophil recruitment through the IL5-CCR3 signaling pathway. These findings were consistent with an age-dependent increase in ovarian stromal expression of Ccl11, a major CCR3 ligand. When ovarian follicles were cultured in 10 or 100 μg/mL LMW hyaluronan for 12 days, gametes with compromised morphology and impaired meiotic competence were produced. In the 100 μg/mL condition, LMW hyaluronan induced premature meiotic resumption, ultimately leading to in vitro aging of the resulting eggs. Further, follicles cultured in this LMW hyaluronan concentration produced significantly less estradiol, suggesting compromised granulosa cell function. Taken together, these data demonstrate that bioactive LMW hyaluronan fragments may contribute to reproductive aging by driving an inflammatory stromal milieu, potentially through eosinophils, and by directly compromising gamete quality through impaired granulosa cell function.
Highlights
Fibrosis and inflammation are key hallmarks of the aging ovarian stroma [1,2]
Given the known inflammatory properties of low molecular weight (LMW) hyaluronan fragments in other tissue types, we investigated whether these fragments induced inflammatory cytokine secretion and gene expression in ovarian stromal cells
Using two controlled in vitro systems, we have demonstrated that physiologically relevant concentrations of LMW hyaluronan exert an adverse effect on both major compartments of the ovary
Summary
Fibrosis and inflammation are key hallmarks of the aging ovarian stroma [1,2]. The stroma is the extra-follicular sub-compartment of the ovary and is comprised of a heterogeneous mix of cell types including fibroblasts, smooth muscle cells, endothelial cells, theca-interstitial cells, and immune cells in addition to abundant extracellular matrix (ECM) components [3]. The age-dependent increase in stromal inflammation and fibrosis likely influences gamete quantity and quality, either directly or indirectly, and resembles aging in other tissues which is associated with a chronic pro-inflammatory state, referred to as “inflammaging” [4,5,6,7,8,9]. This phenomenon contributes to most, if not all, age-related pathologies across organ systems. Reproductive aging begins in women as early as their mid-thirties and is characterized by a significant decline in oocyte quantity and quality in the ovary, which contributes to adverse reproductive outcomes [2]
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