DOI: 10.5061/dryad.kh18932j4
title: Histological analyses data for: Multinucleated giant cells are hallmarks of ovarian aging with unique immune and degradation-associated molecular signatures

description: The ovary is one of the first organs to exhibit signs of aging,
 characterized by reduced tissue function, chronic inflammation, and
 fibrosis. Multinucleated giant cells (MNGCs), formed by macrophage fusion,
 typically occur in chronic immune pathologies, including infectious and
 non-infectious granulomas and the foreign body response, but are also
 observed in the aging ovary. The function and consequence of ovarian MNGCs
 remain unknown as their biological activity is highly context-dependent,
 and their large size has limited their isolation and analysis through
 technologies such as single-cell RNA sequencing. In this study, we define
 ovarian MNGCs through a deep analysis of their presence across age and
 species using advanced imaging technologies as well as their unique
 transcriptome using laser capture microdissection. MNGCs form complex
 interconnected networks that increase with age in both mouse and nonhuman
 primate ovaries. MNGCs are characterized by high Gpnmb expression, a
 putative marker of ovarian and non-ovarian MNGCs. Pathway analysis
 highlighted functions in apoptotic cell clearance, lipid metabolism,
 proteolysis, immune processes, and increased oxidative phosphorylation and
 antioxidant activity. Thus, MNGCs have signatures related to degradative
 processes, immune function, and high metabolic activity. These processes
 were enriched in MNGCs compared to primary ovarian macrophages, suggesting
 discrete functionality. MNGCs express CD4 and colocalize with T-cells,
 which were enriched in regions of MNGCs, indicative of a close interaction
 between these immune cell types. These findings implicate MNGCs in
 modulation of the ovarian immune landscape during aging given their high
 penetrance and unique molecular signature that supports degradative and
 immune functions.
description: # Histological analyses data for: Multinucleated giant cells are hallmarks
 of ovarian aging with unique immune and degradation-associated molecular
 signatures Dataset DOI: [10.5061/dryad.kh18932j4](10.5061/dryad.kh18932j4)
 ## Description of the data and file structure Multinucleated giant cells
 are hallmarks of ovarian aging with unique immune and
 degradation-associated molecular signatures
 [https://doi.org/10.5061/dryad.kh18932j4](https://doi.org/10.5061/dryad.kh18932j4) **Description of the data and file structure** Includes data used to generate graphs related to transcriptomic data as well as histological quantification of MNGCs and postitive antibody staining was performed on tissue sections from mouse and non-human primate ovaries using ImageJ. Paper_raw_data.xlsx represents data from bar and line graphs (Fig 1 and Fig 4). For these data, to quantify the amount of ovarian area taken up by MNGCs (figure 1), the Trainable Weka Segmentation plugin in Fiji (NIH) was used to train the model on regions of non-ovary, non-MNGC ovary, and MNGCs. For figure 4, percent positive CD4 or CD3ε area was determined as DAB positive area over the total area of an ROI that included area of MNGCs, corpora lutea, or stroma. **Version changes** 18 May 2025: Files related to transcriptomic data analyses in all figures have now been included. This includes top gene lists, DEGs, and the count values for heatmap generation, pathway analysis files, and gene comparison between group files. ### Files and variables #### **File: Paper\_raw\_data.xlsx** **Description:** Raw data from ImageJ analysis of Figures 1C, 1D, 4F, 4G, 4H. **Variables** * Figure 1C and 1D * Age in months (mouse) and age in years (non-human primate, NHP); RY refers to reproductively young 6-12 wks * % multinucleated giant cell (MNGC) area * Figure 4F * % CD4 positive area * Animal number, mouse * Figure 4G and 4H * % CD3 (4G) and % CD4 (4H) postitive area * Ovarian structure (corpora lutea, CL; stroma; multinucleated giant cell, MNGC) **File: MNGC_vs_Mac_DEGs_sig.xlsx** **Description:** Significant differentially expressed genes (DEGs) between MNGC (multinucleated giant cell) and Mac (ovarian macrophage) sample groups. Data was generated from raw gene counts through DESeq2. This data was used to determine up and down regulated genes that were used for pathway analysis displayed in Fig. 3 F-G, and volcano plot shown in Fig. 3E. Log2FoldChange represents MNGC vs Mac. **Variables** * MNGC (multinucleated Giant cells) and Mac (ovarian macrophages) **File: Pathway_Analysis_MNGC_top_1000genes.csv** **Description:** Pathway analysis of top 1000 MNGC genes. Data table was obtained from Metascape ([https://metascape.org/gp/index.html#/main/step1](https://metascape.org/gp/index.html#/main/step1)). Gene list for input was obtained from **Top_gene_lists.xlsx**, with the top 1000 genes from the MNGC group with the highest average expression serving as input. Data used to generate Fig 2D. **File: Pathway_Analysis_Mac_Unique.csv** **Description:** Pathway analysis of protein-coding genes that are expressed in MNGC samples but not Mac samples. Data table was obtained from Metascape ([https://metascape.org/gp/index.html#/main/step1](https://metascape.org/gp/index.html#/main/step1)). Gene list (MNGC subset) for input was obtained from **MNGC_Mac_shared_gene_comparison.xlsx**. Data used to generate Fig. 4A. **File: Pathway_Analysis_MNGC_vs_Mac_upreg.csv** **Description:** Pathway analysis of genes that are upregulated in MNGC samples compared to Mac (Log2FoldChange&gt;2, p&lt;0.05). Data table was obtained from Metascape ([https://metascape.org/gp/index.html#/main/step1](https://metascape.org/gp/index.html#/main/step1)). Gene list for input was obtained from **MNGC_vs_Mac_DEGs_sig.xlsx**, using the cutoffs stated above. Data used to generate Fig. 3F. **File: Top_gene_lists.xlsx** **Description:** Normalized gene count lists for all genes found in MNGC and Mac groups. Used to generate Fig. 2C (MNGC). **File: Pathway_Analysis_MNGC_vs_Mac_downreg.csv** **Description:** Pathway analysis of genes that are downregulated in MNGC samples compared to Mac (Log2FoldChange&gt;2, p&lt;0.05). Data table was obtained from Metascape ([https://metascape.org/gp/index.html#/main/step1](https://metascape.org/gp/index.html#/main/step1)). Gene list for input was obtained from **MNGC_vs_Mac_DEGs_sig.xlsx**, using the cutoffs stated above. Data used to generate Fig. 3G. **File: Pathway_Analysis_MNGC_Unique.csv** **Description:** Pathway analysis of protein-coding genes that are expressed in Mac samples but not MNGC samples. Data table was obtained from Metascape ([https://metascape.org/gp/index.html#/main/step1](https://metascape.org/gp/index.html#/main/step1)). Gene list (Mac subset) for input was obtained from **MNGC_Mac_shared_gene_comparison.xlsx**. Data used to generate Fig. 4B. **File: mac_genes_heatmap.xlsx** **Description:** Gene counts used to generate Fig. 3D. Gene counts were used as input to generate a heatmap in SRplot ([https://www.bioinformatics.com.cn/srplot](https://www.bioinformatics.com.cn/srplot)). **Variables** * MNGC (multinucleated giant cells), Mac (ovarian macrophages), Stroma (non-MNGC ovarian stroma) **File: Aerobic_electron_transport_chain_heatmap.xlsx** **Description:** Gene counts used to generate Aerobic electron transport chain panel in Fig. 3H. Gene counts were used as input to generate a heatmap in SRplot ([https://www.bioinformatics.com.cn/srplot](https://www.bioinformatics.com.cn/srplot)). **Variables** * MNGC (multinucleated giant cells), Mac (ovarian macrophages) **File: apoptotic_cell_clearance_heatmap.xlsx** **Description:** Gene counts used to generate apoptotic cell clearance panel in Fig. 3H. Gene counts were used as input to generate a heatmap in SRplot ([https://www.bioinformatics.com.cn/srplot](https://www.bioinformatics.com.cn/srplot)). **Variables** * MNGC (multinucleated giant cells), Mac (ovarian macrophages) **File: Immune_processes_heatmap.xlsx** **Description:** Gene counts used to generate immune processes panel in Fig. 3H. Gene counts were used as input to generate a heatmap in SRplot ([https://www.bioinformatics.com.cn/srplot](https://www.bioinformatics.com.cn/srplot)). **Variables** * MNGC (multinucleated giant cells), Mac (ovarian macrophages) **File: Proteolysis_heatmap.xlsx** **Description:** Gene counts used to generate proteolysis panel in Fig. 3H. Gene counts were used as input to generate a heatmap in SRplot ([https://www.bioinformatics.com.cn/srplot](https://www.bioinformatics.com.cn/srplot)). **Variables** * MNGC (multinucleated giant cells), Mac (ovarian macrophages) **File: Regulation_of_lipid_metabolic_processes_heatmap.xlsx** **Description:** Gene counts used to generate lipid metabolic processes panel in Fig. 3H. Gene counts were used as input to generate a heatmap in SRplot ([https://www.bioinformatics.com.cn/srplot](https://www.bioinformatics.com.cn/srplot)). **Variables** * MNGC (multinucleated giant cells), Mac (ovarian macrophages) **File: MNGC_Mac_shared_gene_comparison.xlsx** **Description:** Shared gene list used to determine MNGC and Mac unique genes for Fig 4 (A-C). Subset indicates if genes are "shared" by both MNGC and Mac samples, or only present in one sample type ("MNGC" or "Mac"). **Variables** * MNGC (multinucleated giant cells), Mac (ovarian macrophages) **File: tcell_marker_heatmap.xlsx** **Description:** Gene counts used to generate Fig. 4D. Gene counts were used as input to generate a heatmap in SRplot ([https://www.bioinformatics.com.cn/srplot](https://www.bioinformatics.com.cn/srplot)). **Variables** * MNGC (multinucleated giant cells), Mac (ovarian macrophages), Stroma (non-MNGC ovarian stroma) #### File: Venn\_Supplemental\_F1.xls **Description:** Shared gene list used to determine how the top 100 compare between ovarian MNGCs and in vitro induced foreign body giant cells (FBGC), Osteoclasts (Osteo), and Langhans giant cells (LGC). Subset indicates if genes are "shared" by both all groups, or only present in one sample type ("Ovarian MNGC," ("FBGC", "Osteo", or "LGC"). Comparison shown in Venn diagram in Supplemental Fig. 1. FBGC, Osteo, and LGC data was obtained from [https://doi.org/10.15252/embr.202256310.](https://doi.org/10.15252/embr.202256310) ##### Variables * Ovarian MNGC, foreign body giant cells (FBGC), osteoclasts (Osteo), and Langhans giant cells (LGC) ## Access information Other publicly accessible locations of the data: * [https://www.ncbi.nlm.nih.gov/bioproject/?term=GSE283393](https://www.ncbi.nlm.nih.gov/bioproject/?term=GSE283393 "Go to dataset") Data was derived from the following sources: * N/A