Biology subjects

Duncan, F. E.

Publications and source records attributed to Duncan, F. E..

12 recordsLinked to original sources

bioRxiv2026

Matched Plasma and Tissue Proteomics Reveal the Selective Nature of Circulating Biomarkers of Human Aging

Aging is systemic, yet how circulating proteins reflect molecular aging within solid tissues remains unclear. We profiled 5,415 proteins using Olink Explore HT in 178 adults aged 20-87 years, including matched plasma-muscle (n = 56) and plasma-ovary (n = 24) pairs. Plasma-tissue concordance was limited, with 50 muscle and 17 ovarian proteins significantly correlated, indicating that plasma selectively reflects tissue protein abundance. Age-correlated proteins and pathways differed markedly across sex and tissue strata, Including immune/inflammatory signatures in female plasma, vascular and extracellular-matrix pathways in male plasma, and collagen remodeling in the ovary. Expression Variation Analysis further revealed predominantly context-specific age-related changes in protein coordination, including FAS/FADD/TRAIL death-receptor networks in plasma and a TP53-CDKN1A senescence-associated axis in muscle. Exploratory multivariate models identified additional plasma associations with subsets of tissue proteins, including tissue-restricted muscle proteins. Together, these findings define plasma as a selective, context-dependent window into human tissue aging.

systems biology↗

bioRxiv2026

Age-dependent dysregulated post-ovulatory debris clearance promotes ovarian fibroinflammation

The ovary is one of the first organs to age, with declines in oocyte quantity and quality leading to infertility and loss of endocrine function. Why the ovary ages earlier than somatic tissues has remained enigmatic but may relate to its highly dynamic physiology and substantial demand for cellular debris clearance. Each ovulation forms a corpus luteum (CL), a transient endocrine structure which supports pregnancy but regresses in its absence. Because CLs have a short functional lifespan but prolonged structural regression, they represent a substantial source of ovarian debris. We hypothesized that post-ovulatory debris clearance becomes impaired with age and contributes to ovarian fibroinflammation. Using a CL-labeling system, we demonstrated that CLs from reproductively old mice show dysregulated regression, resulting in an accumulation of persistent CLs (pCLs) which appeared concurrently with ovarian multinucleated giant cells (MNGCs). Laser capture microdissection, RNA sequencing, and histological evaluation of CLs, pCLs, and MNGCs demonstrated that pCLs were focal hotspots of proinflammatory and debris-associated signatures enriched in lipids, extracellular matrix, and immune cells. Although pCLs retained luteal features, they were transcriptionally more similar to MNGCs than CLs. Consistent with this, an ex vivo CL model acquired MNGC-associated molecular features during extended culture, suggesting that pCLs may represent sites of MNGC formation. Culture of isolated follicles or ovarian stromal organoids with factors from MNGC-enriched explants induced compartment-specific fibroinflammatory responses. These findings provide novel mechanistic insight into ovarian aging, whereby post-ovulatory debris clearance is compromised with age, leading to pCL accumulation, MNGC formation, and a fibroinflammatory milieu.

physiology↗

bioRxiv2026

Proprotein convertase activity regulates cumulus-oocyte-complex matrix integrity and cumulus cell migration during ovulation via a GDF9-dependent mechanism

Cumulus cells have well-established roles early in ovulation but the key molecules that drive their behavior in later stages, leading to follicle rupture, remain underexplored. Here, we observed that inhibition of proprotein convertases (PCSKs) via a pan-inhibitor (PCI) impaired follicular rupture and disrupted the cumulus matrix integrity within intact follicles. Reduced cumulus cell adherence to the cumulus-oocyte-complex (COC) matrix was also observed in isolated COCs and notably occurred late during the maturation window without affecting oocyte maturation. Visualization of PCSK transcript and protein expression, as well as selective inhibition of specific PCSKs, determined that the observed phenotype in COCs is likely attributed to PCSK5A inhibition. We conducted bulk RNA-sequencing and proteomics of PCI-treated COCs which revealed that PCSK inhibition caused dysregulation of extracellular matrix organization, cell migration/adhesion, and TGF-{beta} signaling pathways. Subsequent validation showed that this inhibition translated to disrupted matrix organization and altered migratory and adhesive behaviors in cumulus cells. The TGF-{beta} ligand GDF9 has a predicted PCSK cleavage site, and supplementation with GDF9 rescued matrix integrity suggesting its role as a downstream substrate of PCSKs to regulate matrix organization. Altogether, this study identified PCSK5A and GDF9 as key regulators of COC matrix integrity and cumulus cell migration during late ovulation. These findings highlight novel factors required for follicle rupture which can be leveraged for the development of fertility therapeutics and contraceptives.

cell biology↗

bioRxiv2025

Senescence-Linked Fibrosis in the Aging Human Ovary Revealed by p16-Based Histological Profiling and Spatial Transcriptomics

Cellular senescence is implicated as a driver of ovarian aging, but senescent cells in the human postmenopausal ovary remain poorly defined. Using spatially resolved p16INK4a protein expression, a canonical senescence marker, we identified and mapped senescent cells in postmenopausal ovaries. We integrated p16 immunohistochemistry, multiplexed immunofluorescence, spatial transcriptomics, and AI-guided digital pathology to map senescent microenvironments. p16-positive cells formed discrete stromal, vascular, and cyst-associated clusters that increased with age and were enriched for macrophages and myofibroblast-like cells. Wholetranscriptome profiling of 92 spatial regions uncovered a 32-gene p16-associated signature, BuckSenOvary, that distinguished p16-positive regions across cortex and medulla. BuckSenOvary is characterized by suppression of cell-cycle regulators and activation of inflammatory and extracellular-matrix remodelling genes. AI-based collagen matrix analysis confirmed that p16-positive regions exhibit more architecturally complex collagen, demonstrating that focal senescent microenvironments are fibro-inflammatory. These findings position senescent ovarian niches as therapeutic targets to preserve ovarian function.

cell biology↗

bioRxiv2025

Inorganic profiles of preimplantation embryos reveal a role for zinc in blastocyst development

Elements such as iron, copper and zinc play essential roles in the mammalian oocyte, egg, and embryo, however among these metals, zinc plays unique regulatory roles. Temporal fluctuations in zinc concentrations drive reproductive milestones such as meiotic resumption, egg activation, and initiation of the mitotic cell cycle. Roles for zinc in late preimplantation embryo development, have not been well characterized. Using a quantitative element approach we report the inorganic profiles of mouse embryos progressing through the late blastocyst stage. We find that blastocysts, like oocytes and eggs, and distinct to somatic cells, maintain higher levels of zinc than copper and iron. All three of these essential metals are more abundant in the inner cell mass, which contains the population of pluripotent stem cells that give rise to the fetus, relative to the trophectoderm which gives rise to the placenta and extraembryonic tissues. To test whether zinc abundance was associated with mitotic progress and cell fate lineage, we perturbed zinc homeostasis during blastocyst formation by artificially raising intracellular zinc concentrations with zinc pyrithione. This treatment during the morula-to-blastocyst transition when cell fate lineages emerge resulted in an elevation of zinc in the ICM. This treatment did not impact cell number but did increase expression of the pluripotency and epiblast marker, Nanog. These results demonstrate that the inorganic profiles of the late preimplantation embryo retain elemental hallmarks of earlier developmental stages and perturbation of zinc levels alters pluripotency gene expression in the blastocyst. In BriefZinc plays important regulatory roles during oocyte maturation, fertilization and early embryo development; however, the role of zinc in the late preimplantation embryo is unknown. In this study, inorganic profiling of mouse embryos reveal zinc levels are higher than copper and iron in the late blastocyst and zinc supplementation during the morula-to-blastocyst alters expression of pluripotency marker, Nanog.

developmental biology↗

bioRxiv2025

Transcriptomic analysis of whole staged ovarian follicles reveals stage-specific folliculogenesis signatures in mice

Activation and maturation of ovarian follicles are essential for female reproduction, yet the underlying molecular and transcriptional mechanisms that govern these processes remain poorly understood. In this study, we used single follicle RNA-sequencing (RNA-seq) to identify transcriptional signatures of staged ovarian follicles, from primordial to secondary stages, to uncover the genes and pathways involved in early folliculogenesis. Our findings demonstrate that primordial follicles are transcriptionally distinct from growing follicles, with enrichment in DNA integrity and RNA processing pathways, which may play a role in preserving oocyte genomic stability and cell state during dormancy. Additionally, our analysis reveals minimal transcriptomic differences between primary and secondary follicles using traditional differential expression analysis. To better distinguish growing follicle stages, we introduce unsupervised approaches, including discrete-variable predictors of follicle stage and weighted gene co-expression analysis. We identified pathways involved in DNA integrity, meiotic arrest, and cellular metabolism that drive the transition from dormant to active follicle states, as well as pathways related to cellular growth, ECM organization, and biosynthesis in growing follicle stages. Our study offers novel insights into the molecular mechanisms governing early follicle activation and growth, providing a foundation for future research with applications in reproductive biology, contraception, and fertility preservation. Author SummaryThe development of ovarian follicles is essential for female fertility, but the molecular signals that control their growth remain unclear. In this study, we used advanced gene sequencing techniques to analyze the genetic activity of individual ovarian follicles at different stages of early development. We found that dormant follicles have unique gene expression patterns that help protect the genetic material of the egg and maintain their inactive state. In contrast, follicles that have begun to grow show increased activity in genes related to cell growth, communication, and structural changes. Interestingly, we observed that early growing follicles are more similar to each other than previously thought, prompting us to apply new analytical methods to better distinguish their developmental stages. Our findings highlight key biological pathways that regulate the transition from dormant to active follicles and uncover new genes that may play a role in this process. Understanding these mechanisms provides valuable insights into ovarian biology and could inform future research on fertility treatments, contraception, and reproductive health.

bioinformatics↗

bioRxiv2025

Regulation of Female Reproductive Aging by the Spag17 Gene

Reproductive aging in females is characterized by a decline in oocyte quantity and quality, as well as uterine and cervical dysfunction that contributes to infertility and pregnancy complications. To investigate mechanisms underlying reproductive aging, we explored the contribution of Spag17, a cilia-related gene associated with tissue homeostasis and fibrosis. Spag17 was expressed throughout the female reproductive tract; however, its expression declined with age in ovarian tissue, while high expression levels were observed in the cervix of young females during cervical tissue remodeling in the pre- and post-parturition periods. Loss of Spag17 in mice resulted in impaired fertility, obstructed labor, and maternal death. This phenotype was associated with accelerated ovarian aging, increased fibrosis, and cervical stiffness, further complicating parturition. At the molecular level, Spag17 loss activated key aging-associated pathways, including proinflammatory, profibrotic, and senescence signaling, suggesting that SPAG17 may be a critical player in female reproductive aging. TEASERSpag17 is a key modulator of female reproductive aging.

cell biology↗

bioRxiv2024

A comprehensive multi-omics signature of doxorubicin-induced cellular senescence in the postmenopausal human ovary.

A major aging hallmark is the accumulation of cellular senescence burden. Over time senescent cells contribute to tissue deterioration through chronic inflammation and fibrosis driven by the Senescence-Associated Secretory Phenotype (SASP). The human ovary is one of the first organs to age, and prominent age-related fibroinflammation within the ovarian microenvironment is consistent with the presence of senescent cells, but these cells have not been characterized in the human ovary. We thus established a doxorubicin-induced model of cellular senescence to establish a "senotype" (gene/protein signature of a senescence cell state) for ovarian senescent cells. Explants of human postmenopausal ovarian cortex and medulla were treated with doxorubicin for 24 hours followed by culture for up to 10 days in a doxorubicin-free medium. Tissue viability was confirmed by histology, lack of apoptosis, and continued glucose consumption by explants. Single nuclei sequencing and proteomics revealed an unbiased signature of ovarian senescence. We identified distinct senescence profiles for the cortex and medulla, driven predominantly by epithelial and stromal cells. Proteomics uncovered subregional differences in addition to 120 proteins common to the cortex and medulla SASP. Integration of transcriptomic and proteomic analyses revealed 26 shared markers, defining a senotype of doxorubicin-induced senescence unique to the postmenopausal ovary. A subset of these proteins: Lumican, SOD2, MYH9, and Periostin were mapped onto native tissue to reveal compartment-specific localization. This senotype will help determine the role of cellular senescence in ovarian aging, inform biomarker development to identify, and therapeutic applications to slow or reverse ovarian aging, senescence, and cancer.

molecular biology↗

bioRxiv2024

Self-organizing ovarian somatic organoids preserve cellular heterogeneity and reveal cellular contributions to ovarian aging

Ovarian somatic cells are essential for reproductive function, but no existing ex vivo models recapitulate the cellular heterogeneity or interactions within this compartment. We engineered a novel ovarian somatic organoid model by culturing a stroma-enriched fraction of mouse ovaries in scaffold-free agarose micromolds. Ovarian somatic organoids self-organized, maintained diverse cell populations, produced extracellular matrix, and secreted hormones. Organoids generated from reproductively old mice exhibited reduced aggregation and growth compared to young counterparts, as well as differences in cellular composition. Interestingly, matrix fibroblasts from old mice demonstrated upregulation of pathways associated with the actin cytoskeleton and downregulation of cell adhesion pathways, indicative of increased cellular stiffness which may impair organoid aggregation. Cellular morphology, which is regulated by the cytoskeleton, significantly changed with age and in response to actin depolymerization. Moreover, actin depolymerization rescued age-associated organoid aggregation deficiency. Overall, ovarian somatic organoids have advanced fundamental knowledge of cellular contributions to ovarian aging.

cell biology↗

bioRxiv2024

Single-cell and spatiotemporal profile of ovulation in the mouse ovary

Ovulation is a spatiotemporally coordinated process that involves several tightly controlled events, including oocyte meiotic maturation, cumulus expansion, follicle wall rupture and repair, and ovarian stroma remodeling. To date, no studies have detailed the precise window of ovulation at single-cell resolution. Here, we performed parallel single-cell RNA-seq and spatial transcriptomics on paired mouse ovaries across an ovulation time course to map the spatiotemporal profile of ovarian cell types. We show that major ovarian cell types exhibit time-dependent transcriptional states enriched for distinct functions and have specific localization profiles within the ovary. We also identified gene markers for ovulation-dependent cell states and validated these using orthogonal methods. Finally, we performed cell-cell interaction analyses to identify ligand-receptor pairs that may drive ovulation, revealing previously unappreciated interactions. Taken together, our data provides a rich and comprehensive resource of murine ovulation that can be mined for discovery by the scientific community.

bioinformatics↗

bioRxiv2024

The oocyte microenvironment is altered in adolescents compared to oocyte donors.

Study questionAre the molecular signatures of cumulus cells (CCs) and follicular fluid (FF) of adolescents undergoing fertility preservation differ from that of reproductively adult oocyte donors? Summary answerThe microenvironment immediately surrounding the oocyte, including the CCs and FF, is altered in adolescents undergoing fertility preservation compared to oocyte donors. What is known alreadyAdolescents experience a period of subfecundity following menarche. Recent evidence suggests that this may be at least partially due to increased oocyte aneuploidy. Reproductive juvenescence in mammals is associated with suboptimal oocyte quality. Study design, size, durationThis was a prospective cohort study. Adolescents (10-19 years old, N=23) and oocyte donors (22-30 years old, N=31) undergoing ovarian stimulation and oocyte retrieval at the Northwestern Fertility and Reproductive Medicine Center between November 1, 2020 and May 1, 2023 were enrolled in this study. Participants/materials, setting, methodsPatient demographics, ovarian stimulation, and oocyte retrieval outcomes were collected for all participants. The transcriptome of CCs associated with mature oocytes was compared between adolescents (10-19 years old, n=19), and oocyte donors (22-30 years old, n=19) using bulk RNA-sequencing. FF cytokine profiles (10-19 years old, n=18 vs. 25-30 years old, n=16) were compared using cytokine arrays. Main results and the role of chanceRNA-seq analysis revealed 581 differentially expressed genes (DEGs) in cumulus cells of adolescents relative to oocyte donors, with 361 genes downregulated and 220 upregulated. Genes enriched in pathways involved in cell cycle and cell division (e.g., GO:1903047, p= 3.5 x 10-43; GO:0051983, p= 4.1 x 10- 30; GO:0000281, p= 7.7 x 10-15; GO:0044839, p= 5.3 x 10-13) were significantly downregulated, while genes enriched in several pathways involved in cellular and vesicle organization (e.g., GO:0010256, p= 1.2 x 10-8; GO:0051129, p= 6.8 x 10-7; GO:0016050, p= 7.4 x 10-7; GO:0051640, p= 8.1 x 10-7) were upregulated in CCs of adolescents compared to oocyte donors. The levels of 9 cytokines were significantly increased in FF of adolescents compared to oocyte donors: IL-1 alpha (2-fold), IL-1 beta (1.7-fold), I-309 (2-fold), IL-15 (1.6-fold), TARC (1.9-fold), TPO (2.1-fold), IGFBP-4 (2-fold), IL-12-p40 (1.7-fold) and ENA-78 (1.4-fold). Interestingly, 7 of these cytokines have known pro-inflammatory roles. Importantly, neither the CC transcriptomes or FF cytokine profiles were different in adolescents with or without cancer. Large scale dataOriginal high-throughput sequencing data will be deposited in Gene Expression Omnibus (GEO) before publication, and the GEO accession number will be provided here. Limitations, reasons for cautionThis study aims to gain insights into the associated gamete quality by studying the immediate oocyte microenvironment. The direct study of oocytes is more challenging due to sample scarcity, as they are cryopreserved for future use, but will provide a more accurate assessment of oocyte reproductive potential. Wider implications of the findingsUnderstanding the underpinnings of altered immediate oocyte microenvironment of adolescent patients may provide insights into the reproductive potential of the associated gametes in the younger end of the age spectrum. This has implications for the fertility preservation cycles for very young patients. Study funding/competing interest(s)This project was supported by Friends of Prentice organization SP0061324 (M.M.L and E.B.), Gesualdo Family Foundation (Research Scholar: M.M.L.), and NIH/NICHD K12 HD050121 (E.B.). The authors have declared that no conflict of interest exists.

developmental biology↗

bioRxiv2023

CD38 regulates ovarian function and fecundity via NAD+ metabolism

Mammalian female reproductive lifespan is typically significantly shorter than life expectancy and is associated with a decrease in ovarian NAD+ levels. However, the mechanisms underlying this loss of ovarian NAD+ are unclear. Here, we show that CD38, a NAD+ consuming enzyme, is expressed in the ovarian extrafollicular space, primarily in immune cells, and its levels increase with reproductive age. Reproductively young mice lacking CD38 exhibit larger primordial follicle pools, elevated ovarian NAD+ levels, and increased fecundity relative to wild type controls. This larger ovarian reserve results from a prolonged window of follicle formation during early development. However, the beneficial effect of CD38 loss on reproductive function is not maintained at advanced age. Our results demonstrate a novel role of CD38 in regulating ovarian NAD+ metabolism and establishing the ovarian reserve, a critical process that dictates a females reproductive lifespan.

developmental biology↗