Single-cell reconstruction of follicular remodeling in the human adult ovary

Abstract

The ovary is perhaps the most dynamic organ in the human body, only rivaled by the uterus. The molecular mechanisms that regulate follicular growth and regression, ensuring ovarian tissue homeostasis, remain elusive. We have performed single-cell RNA-sequencing using human adult ovaries to provide a map of the molecular signature of growing and regressing follicular populations. We have identified different types of granulosa and theca cells and detected local production of components of the complement system by (atretic) theca cells and stromal cells. We also have detected a mixture of adaptive and innate immune cells, as well as several types of endothelial and smooth muscle cells to aid the remodeling process. Our results highlight the relevance of mapping whole adult organs at the single-cell level and reflect ongoing efforts to map the human body. The association between complement system and follicular remodeling may provide key insights in reproductive biology and (in)fertility.

Fig. 1

Morphology of different follicles in adult ovaries. a, b Immunofluorescence of several healthy (a) and atretic follicles (b) of different sizes present in the ovaries immunostained for DDX4. Green arrows point to (DDX4-positive) oocytes. Panels in the bottom row show magnifications of the boxed areas with corresponding roman numbers. Tissue was counterstained with DAPI. Scale bars are 1 mm in two top rows and 100 μm in bottom row (panels i-vii). c, d Immunofluorescence of atretic (c) and healthy follicles (d) of different sizes present in the ovaries analyzed for KI67 and TUNEL. Insets in d show a different area of the follicle showing mural GC with same magnification. Single channel images were converted to an intensity map. Scale bars are 100 μm

Fig. 2

Transcriptome map of human adult ovaries analyzed. a Schematic representation of human ovarian tissue preparation for single cell transcriptome analysis. b tSNE cluster map revealing 19 specific clusters representing the major ovarian somatic cell types. c Violin plots showing expression of one representative differential expressed gene for each cluster. d Heatmap and hierarchical clustering based on expression of top 50 most variable genes. e tSNE cluster map showing expression of genes characteristic the major ovarian somatic cell types. Red dashed lines give the boundaries of the main clusters of interest

Fig. 3

Vascular remodeling in the ovaries analyzed. a, b Immunofluorescence of ovarian stroma for PECAM1 a, VWF b, and the respective single channel images. Slides were counterstained with DAPI. Scale bars are 100 μm. c tSNE cluster maps showing expression of selected endothelial marker genes. Red dashed lines give the boundaries of the endothelial-clusters of interest. d Venn diagram showing the intersection of 200 differential expressed genes (DEGs) of the three endothelial cell clusters (CL7, CL9, and CL16); and three selected enriched terms obtained for the unique DEGs. e Immunofluorescence of ovarian stroma for ACTA2, and the respective single channel image. Slides were counterstained with DAPI. Scale bars are 100 μm. f tSNE cluster map showing expression of selected smooth muscle marker genes. Red dashed lines give the boundaries of the smooth muscle-clusters. g Venn diagram showing the intersection of 200 differential expressed genes (DEGs) of the two smooth muscle cell clusters (CL14 and CL17) and genes from four-cell death and apoptosis-related GO terms (GO:0043068 positive regulation of programmed cell death, GO:0010942 positive regulation of cell death, GO:0043067 regulation of programmed cell death, GO:0042981 regulation of apoptotic process); and three selected enriched terms obtained for the unique DEGs

Fig. 4

Divergent populations of granulosa cells in different follicles. a Distribution of single cells from different-sized follicles on the tSNE. Black dashed lines give the boundaries of several clusters of GC (common progenitor GC (pGC), mural GC, cumulus GC, atretic GC (atrGC), theca cells (TC), and atretic TC (atrTC)). b tSNE cluster map showing expression of selected marker genes differentially expressed by GC and pGC (top row), cumulus GC (middle row), and mural GC and pGC (bottom row). Red dashed lines give the boundaries of the GC-clusters of interest. c, d Immunostaining of follicles (ø, diameter) growing (c) and atretic (d) for WT1 and pan-KRT (pKRT). Inset shows mural GC of the same follicle with the same magnification. Single channel images were converted to an intensity map. Scale bars are 100 μm

Fig. 5

Granulosa cells in early atretic follicles. a tSNE cluster map showing expression of selected genes downregulated in CL10, but not on the other granulosa cell (GC) clusters. Red dashed lines give the boundaries of expression. b Violin plots showing expression levels of GJA1 and CDH2 in the different clusters of GC. c Immunostaining of follicles (ø, diameter) growing (top two rows) and atretic (bottom two rows) for IFITM3, GJA1, and ZP3. Inset shows mural GC of the same follicle with same magnification. Single channel images were converted to an intensity map. White dotted line marks the basement membrane. Scale bars are 100 μm. d Immunostaining of follicles (ø, diameter) growing (top two rows) and atretic (bottom two rows) for CDH2, COLIV, and DDX4. Inset shows mural GC of the same follicle with same magnification. Single channel images were converted to an intensity map. Scale bars are 100 μm

Fig. 6

Cell trajectory analysis and characterization of granulosa cells. a, b Analysis of cell trajectories of granulosa cells (GC) (CL3, CL8, CL11, CL15) by Monocle a and Diffusion maps b. Individual cells (dots) are colored by cluster, follicle, patient, and pseudotime (Monocle). c tSNE cluster map showing selected genes expressed by GC. Red dashed lines give the boundaries of the GC-clusters. d Immunostaining of follicles (ø, diameter) growing (top two rows) and atretic (bottom two rows) for TNNI3, AMH, and DDX4. Higher magnification of inner and basement GC is shown on the right side. Single channel images for TNNI3 converted to an intensity map are shown. White arrowheads depict basement GC, yellow arrowheads depict inner GC. Scale bars are 100 μm

Fig. 7

Divergent populations of theca cells in different follicles. a tSNE cluster map showing expression of selected theca cells (TC) genes. Red dashed lines give the boundaries of the expression. b tSNE cluster map revealing sub-clusters of CL5 representing ovarian TC types. Black dashed lines give the boundaries of several sub-clusters of TC: common progenitor TC (pTC), externa TC (exTC), and interna TC (inTC). c Expression of marker genes in sub-clusters of CL5. d, e Analysis of cell trajectories of TC (CL5) by Monocle a and Diffusion maps b. Individual cells (dots) are colored by cluster, follicle, patient, and pseudotime (Monocle). f Immunostaining of follicles (ø, diameter) growing (top two rows) and atretic (bottom two rows) for STAR, ACTA2, and ZP3. Inset shows mural GC of the same follicle with same magnification. Single channel images were converted to an intensity map. White dotted line marks the basement membrane. Scale bars are 100 μm

Fig. 8

Characteristics of theca and stromal cells. a tSNE cluster map showing expression of selected marker genes healthy and atretic theca cells (TC). Red dashed lines give the boundaries of the TC-clusters of interest. b, c Immunostaining of follicles (ø, diameter) growing (top row) and atretic (bottom row) for cFOS and 8OHdG (b) and for IGFBP5 and STAR c. Inset shows mural GC and TC of the same follicle with same magnification. Single channel images were converted to an intensity map. Scale bars are 100 μm. d, f tSNE cluster map showing expression of selected marker genes in the ovarian stroma d, healthy stroma e and stressed stroma (f). Red dashed lines give the boundaries of the stromal clusters of interest

Fig. 9

Complement and immune system in the adult ovaries. a tSNE cluster map showing expression of selected complement genes. b Gene network of C1S in TC and stroma. The color of the circles represents function and the color of the edges represent networks. c Immunostaining of follicles (ø, diameter) growing (top row), atretic (middle row), and degenerated (bottom row) for C1S, C1Q, and CD68. Single channel images were converted to an intensity map. Scale bars are 100 μm. d Immunostaining of follicles (ø, diameter) growing (top row), atretic (middle row), and degenerated (bottom row) for C1Q and STAR. Scale bars are 100 μm. e Concentration of secreted C1Q and C3 produced by pieces of human ovarian stroma (N = 3 samples) and atretic follicle walls (N = 6 samples) after 1 and 5 days of culture. Median and sample distribution (dots) are shown. f tSNE cluster map showing expression of C1QA and C3. g–i tSNE cluster map showing expression of selected immune marker genes for innate immune cells g, B lymphocytes h and T lymphocytes and NK cells i. Red dashed lines give the boundaries of the specific immune-clusters