Chase R. Hubbart

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Female humans experience a sharp decline in fertility around 35 years of age, which corresponds to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging mouse ovary to identify early drivers of ovarian decline. To fill this gap we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress-response, immunogenic and fibrotic signaling pathway inductions with aging. This report provides critical insights into mechanisms responsible for ovarian aging phenotypes. The data can be explored interactively via a Shiny-based web application.

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling, and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Around 35 years old, women experience a sharp decline in fertility, corresponding to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging ovary to identify early drivers of ovarian decline. To fill this gap, we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress response, immunogenic, and fibrotic signaling pathway inductions with aging. This report raises provides critical insights into mechanisms responsible for ovarian aging phenotypes.

Abstract Ovaries exhibit accelerated aging phenotypes, displaying aging hallmarks earlier than other organs. During pre-menopause, endocrine function declines, and the ovarian microenvironment becomes pro-inflammatory and fibrotic. However, the cellular mechanisms driving ovarian aging remain unclear, hindering the development of therapies to delay this process for overall health benefits. Macrophages are crucial in ovarian functions like folliculogenesis, ovulation, and corpus luteum formation. Ovarian macrophages have two origins: tissue-resident macrophages (TRMs) from yolk sac and fetal liver progenitors, and monocyte-derived macrophages (MDMs) from bone marrow. In this study, we examined the transcriptomic profiles of ovarian TRMs in young (5-6 months) and older (12-14 months) Cx3cr1-NuTRAP mice. This mouse model uses a tamoxifen-inducible, cre-lox system to label nuclei and ribosomes, enabling DNA/RNA isolation without cell sorting. Flow cytometry confirmed the identity of eGFP-labeled cells, and translating ribosome affinity purification (TRAP) was used to assess age-related transcriptomic changes in TRMs. We found that TRMs exhibit inflammatory (e.g., IL1B) and senescent (e.g., CDKN2A) transcriptomic signatures, likely contributing to aging hallmarks such as T cell accumulation, multinucleated giant cell formation, and fibrosis. Translatability was established by comparing our data with publicly available human data. These findings suggest that ovarian TRMs play a critical role in ovarian aging, warranting future mechanistic studies.