Low Molecular Weight Hyaluronan Induces an Inflammatory Response in Ovarian Stromal Cells and Impairs Gamete Development In Vitro

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.

Ovarian stiffness increases with age in the mammalian ovary and depends on collagen and hyaluronan matrices

“…Decreased Has3 and increased Hyal1 expression in the ovarian stroma with age may be indicative of reduced synthesis coupled to increased fragmentation of HMW‐HA into LMW‐HA, contributing to ovarian HA loss with age. We previously demonstrated that LMW‐HA fragments can trigger a Th2 inflammatory response in ovarian stromal cells which is predicted to increase fibrogenic pathways (Rowley, et al, 2020). Thus, we evaluated HA mass distribution in individual ovaries from reproductively young and old mice using solid‐state nanopore sensor technology (Rivas et al, 2018).…”

The ovarian stroma as a new frontier

“…If dysregulated, ECM degradation may also trigger pathogen-free inflammation. For example, hyaluronan is a glycosaminoglycan that forms low molecular weight fragments during turnover, which have been shown in cultured murine stromal cells to increase the secretion of type 2 inflammatory cytokines and activate genes involved in eosinophil recruitment, while also leading to adverse effects on cultured follicles (Rowley et al 2020).…”

The Inflammasome Contributes to Depletion of the Ovarian Reserve During Aging in Mice

“…In this publication, Urzua et al demonstrated an excessive accumulation of incompletely digested cell debris in the form of lipofuscin in reproductively aged mouse ovaries (Urzua et al, 2018 ). More recently, Rowley et al reported that age-related DAMPs, such as low molecular weight (LMW) hyaluronan fragments, may be a potential driver of the age-associated inflammation characteristic of ovaries from reproductively old mice (Rowley et al, 2020 ). However, whether accumulation of LMW hyaluronan fragments occurs in advanced reproductively aged ovaries is still under investigation.…”