Reprogramming the aging ovarian microenvironment via mitochondrial sharing and structural remodeling

Abstract

Rationale: Mitochondrial dysfunction in ovarian granulosa cells (GCs) and cumulus cells (CCs) is a defining feature of reproductive aging, contributing to impaired oocyte quality and reduced fertility. This study investigates whether enhancing cytoskeletal dynamics or promoting structural contact between cells can restore mitochondrial function and mitigate ovarian aging. Methods: Mitochondrial exchange was assessed using co-culture systems, live-cell imaging, and mitochondrial labeling in human ovarian somatic cells. Cytoskeletal modulation was achieved using FTY720, and cell-cell contact was enhanced through soft 3D extracellular matrix (ECM) scaffolds. Functional outcomes were evaluated through ATP assays, mitochondrial membrane potential, Seahorse bioenergetics profiling, and transcriptomic analysis. In vivo validation was conducted in aged mice treated with FTY720. Results: Granulosa and cumulus cells exchanged mitochondria via tunneling nanotubes (TNTs), a process significantly reduced with age. Mitochondrial transfer was contact-dependent and not mediated by paracrine signaling. FTY720 enhanced TNT formation and mitochondrial delivery, restoring ATP levels, membrane potential, and oxidative phosphorylation in aged cells. 3D ECM culture promoted spheroid formation, activated YAP signaling, and improved mitochondrial function without pharmacological agents. In aged mice, FTY720 treatment increased follicle numbers, improved oocyte mitochondrial quality, and elevated serum AMH levels. Conclusions: These findings demonstrate that somatic cell contact is essential for mitochondrial complementation in aging ovaries. By promoting intercellular connectivity through cytoskeletal or microenvironmental remodeling, endogenous mitochondrial sharing can be reactivated to restore bioenergetic function. This approach offers a novel regenerative strategy to counteract reproductive aging.

Keywords: Cell-cell communication; Mitochondrial transfer; Ovarian aging; Reproductive microenvironment.

Figure 1

Human cumulus cell aggregation preserves mitochondrial bioenergetics and enables intercellular mitochondrial transfer. (A) Schematic representation of mitochondrial energy differences in aggregated versus separated human CCs and GCs. (B) Representative fluorescence images showing mitochondrial morphology (MitoTracker green), ATP signal (red), and pseudocolor ATP intensity heatmaps (blue-red) in CCs from women <34 years and >38 years under two morphological states: mitochondrial aggregation and separation. (C) Quantification of ATP fluorescence intensity (arbitrary units, a.u.) under aggregation (Agg.) and separation (Sep.) states. (D) 3D live-cell imaging using NanoLive in CCs from a patient >36 years old shows long protrusions (boxed region, left) with fragmented mitochondrial ends (yellow arrowheads). Panels 1-4 are sequential time-lapse frames showing dynamic process. (E) MitoTracker (red) and DIC overlay images show intercellular transfer of mitochondria in TNTs (yellow arrows). Scale bar = 20 µm. **p < 0.01, ***p < 0.001.

Figure 2

Mitochondrial transfer from young to aged granulosa cells restores mitochondrial structure and bioenergetics. (A) Experimental design: young and aged HGL5 cells were labeled with MitoTracker Green and Red, respectively, and co-cultured for 24 h. (B) Representative images of mitochondrial transfer in Y/A and A/A co-cultures (C) Classification of mitochondrial subtypes in recipient cells. Color-coded traces represent distinct mitochondrial morphologies. (D) Quantification of mitochondrial subtypes in recipient aGCs. (E) Western blot analysis of mitochondrial dynamics-related proteins in recipient aGCs. Increased OPA1 and MFN1, and reduced DRP1/pDRP1 are observed in Y/A versus A/A. (F) Workflow for identifying recipient aGCs using CFDA staining and cell sorting. (G) Flow cytometric identification of recipient aGCs and measurement of intracellular ATP intensity (right histogram). Y/A cells display increased ATP signal compared to A/A. (H) Quantification of intracellular ATP levels. (I) TMRE staining indicates improved mitochondrial membrane potential in Y/A group. (J) OCR tracing in recipient aGCs using Seahorse analysis. (K-N) Quantification of basal respiration (K), maximal respiration (L), ATP production (M), and spare respiratory capacity (N). Scale bar = 20 µm. **p < 0.01, ***p < 0.001.

Figure 3

Co-culture with young granulosa cells reverses metabolic decline in aged cells. (A) mRNA expression levels of glycolysis and TCA cycle-related genes in aged granulosa cells co-cultured with young cells (Y/A) or aged cells alone (A/A). Genes include HK2, GPI, ENO1, PKM1, LDHA/B/C, PDHA, CS, ACO1/2, IDH1, FH, and MDH1. (B) Western blot analysis of glycolysis and TCA cycle enzymes confirms reduced expression in A/A recipient cells. α-tubulin serves as the loading control. (C) Measurement of extracellular lactate levels in Y/A and A/A cells. (D) Heatmap of targeted metabolomics analysis showing relative abundance of central carbon metabolites, including glycolytic intermediates, TCA metabolites, and associated energy substrates. Color scale indicates Z-score normalization. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Cytoskeletal enhancement in aged granulosa cells promotes TNT formation and facilitates mitochondrial energy transfer. (A) Representative images showing TNT formation between cells in Y/A, A/A, and A+FTY720/A co-cultures. Heatmaps display intensity of TNT structures. (B-C) Quantification of TNT and branch per two cells across groups. (D) Proportions of cell-cell connection types in co-culture settings. (E) ELISA measurement of AMH in co-culture media. (F) Time-lapse imaging of mitochondrial trafficking along TNTs in A+FTY720/A co-cultures. Yellow and blue arrowheads indicate movement of mitochondria. (G-H) Immunofluorescence and 3D reconstruction of mitochondrial transfer from FTY720-pretreated donor cells (green) to untreated aGC recipients (red). Quantification of MitoTracker intensity (I) and ATP levels (J) in donor aGCs after co-culture with FTY720-pretreated donors. (K) Western blotting of cytoskeletal proteins in yGC, aGC, and FTY720-treated aGCs. MitoTracker intensity (L) and ATP levels (M) in recipient cells following donation from FTY-treated cells. (N) Cytoskeletal protein levels in recipient aGCs post-FTY720 transfer. Scale bar = 20 µm. *p<0.05, **p<0.01, ***p<0.001.

Figure 5

Three-dimensional culture environments promote cell-cell interactions and support enhanced mitochondrial function. (A) Schematic of hydrogel-based 3D culture with tunable stiffness. (B) Brightfield images showing HGL5 spheroid formation under soft matrix conditions. (C) Western blot of cytoskeletal markers across 2D, stiff, and soft conditions. (D-E) YAP/pYAP levels in different matrix conditions by western blot and quantification. (F) Western blot of YAP and TAZ levels. (G-H) Brightfield imaging and quantification of spheroid formation rate after YAP inhibition. (I) Immunostaining of pYAP distribution after YAP inhibition. (J) Western blot of cytoskeletal markers after YAP inhibition. (K-L) Mitochondrial mass and ATP measured by flow cytometry. (M-Q) OCR curves and respiratory parameter quantification in 2D and 3D-soft cultured aGCs. (R-S) Mitochondrial mass and ATP measured by flow cytometry. (T-U) Brightfield and merged fluorescence images with LUT-based fluorescence intensity analysis of patient-derived cumulus cells in 3D culture. (V-W) Western blot and ELISA quantification of AMH protein and secreted AMH in aGCs. (X) RT-qPCR analysis of hormone-related gene expression in aGCs cultured under 2D or 3D conditions. Scale bar = 20 µm. **p < 0.01 and ***p < 0.001.

Figure 6

Three-dimensional ECM environments promote metabolic reprogramming in aGCs. (A) Gene expression of glycolytic and mitochondrial metabolic enzymes in aGCs cultured on 2D vs. 3D ECM conditions. Genes include HK2, GPI, ENO1, PKM1, PDHA, CS, ACO1, IDH1, FH, MDH1, and LDH isoforms. (B) Western blot analysis of HK2, PGAM1, LDHC, PDHA, CS, ACO1, IDH1, and MDH1 protein levels under 2D and 3D conditions. (C) Extracellular lactate concentration in culture medium from 2D versus 3D aGCs. (D) Heatmap representing relative metabolite abundance of central carbon metabolism intermediates in aGCs under 2D or 3D conditions. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

FTY720 improves folliculogenesis, oocyte quality, and mitochondrial function in aged female mice. (A) Schematic of FTY720 administration in reproductively aged mice. (B) H&E staining of ovarian sections from vehicle- and FTY720-treated mice. (C-F) Quantification of follicle numbers at different stages: primordial (C), primary (D), secondary (E), and antral (F). (G-H) Immunohistochemical staining and quantitative analysis of AMH and BMP15 expression in ovarian tissue sections. (I-J) Immunofluorescence staining and quantitative analysis of oxidative stress markers 8-OHdG and 4-HNE. (K) Fluorescence imaging of ovulated MII oocytes stained with DCFDA and MitoTracker. (L-N) Quantification of (L) polar body extrusion number, (M) DCFDA fluorescence intensity, and (N) MitoTracker fluorescence. (O) RNA-sequencing and pathway enrichment analysis of ovarian tissues. (P) Western blot analysis of mitochondrial trafficking and biogenesis markers in ovaries from aged mice treated with vehicle or FTY720. (Q) Representative immunofluorescence images of ovarian sections stained for Miro1 and DAPI in tertiary follicles. Pseudocolor LUT was applied to quantify Miro1 intensity. (R) Quantification of Miro1 fluorescence intensity in oocytes and cumulus cells from (Q). (S) OCR tracing of whole ovaries measured by Seahorse assay. (T-W) Quantification of basal respiration (T), maximal respiration (U), ATP-linked respiration (V), and spare respiratory capacity (W). (X) Serum AMH levels measured by ELISA. (K) Scale bar = 20 µm. (B, I, Q) Scale bar = 200 µm. *p < 0.05, **p < 0.01, ***p < 0.001.