Fig 2: Procr+ cells contribute to OSE repair after ovulation. a Illustration of lineage tracing and superovulation-inducing strategy using Procr-CreER;Rosa26-mTmG mice. b, c At 4pi (pre-ovulation stage), ovary whole-mount confocal imaging showing dispersed GFP+ cells in the OSE (b). n = 4 mice and eight ovaries images. Section imaging confirming the appearance of a few GFP+ OSE cells on both sides of the pre-ovulation follicle (c). Scale bars, 100 μm. n = 4 mice and 16 images. d–f At 4.5pi (ovulation stage), ovary whole-mount confocal image demonstrating zones of concentrated GFP+ cells surrounding the rupture site (R.S.) (d). Section imaging confirming the appearance of abundant GFP+ OSE cells at both edges of the rupture site (e). Scale bars, 100 μm. Quantification of GFP+ cell numbers (per 0.04 mm2 area boxed by the dashed line) showing that GFP+ cells by rupture sites are significantly more than those in randomly selected non-R.S. (f). n = 4 mice, > 40 areas were counted in each group. Unpaired two-tailed t test is used for comparison. ***P < 0.001. g–i At 7pi (repair completed stage), ovary whole-mount confocal imaging showing large GFP+ patches located at corpus luteum (C.L.) area that can be distinguished by the residual protrusion (g). Section imaging demonstrating large patch of GFP+ cells in the OSE covering corpus luteum (C.L.), whereas GFP+ cells are fewer in areas where the OSE covers the follicle (F) (h). Scale bars, 100 μm. Quantification of GFP+ cells (per 0.04 mm2 area) showing that GFP+ cells in the C.L. area are significantly more than those in randomly selected non-C.L. sites (i). n = 4 mice, > 40 areas were counted in each group. Unpaired two-tailed t test is used for comparison. ***P < 0.001. j Quantification of GFP+ cell numbers showing the continuous expansion of GFP+ cells during tracing. At 4d pi, GFP+ cells in the pre-ovulation follicle (P.F.) area were counted. At 4.5d pi, GFP+ cells flanking the rupture sites (R.S.) were counted. At 7d pi, GFP+ cells covering the C.L area were counted. Each view was 0.04 mm2 in size. n = 4 mice, > 40 areas were counted for each time point. One-way ANOVA with Tukey test is used for comparison of multiple groups. *P < 0.05, ***P < 0.001

Fig 3: Procr+ cells expand immediately upon ovulation. a Illustration of superovulation and analysis strategy as indicated using wildtype mice. b, c FACS analysis (b) and quantification (c) of the percentage of Procr+ OSE cells at three different stages as indicated. n = 3 mice. One-way ANOVA with Tukey test is used for comparison of multiple groups. ***p < 0.001. d Illustration of the visualization and tracing strategy for Procr+ cells using Procr-rtTA;TetO-H2B-GFP mice. e, f At 4 days after Dox feeding (pre-ovulation), confocal images showing peak level of nuclear GFP+ cells in the OSE close to pre-ovulation follicle. Scale bars, 100 μm. n = 3 mice and 15 images. More than 30 pre-ovulation follicles were scored. f, g At 4.5d pi (ovulation), confocal images showing increasing numbers of peak levels of nuclear GFP+ cells in the OSE on both edges of the rupture. Scale bars, 100 μm. n = 3 mice and 15 images. More than 30 rupture sites were scored. Data are presented as mean±s.e.m. Unpaired two-tailed t test is used for comparison. ***p < 0.001. h At 7d pi (repair completed), confocal images showing GFP+ cells with various diluted signal intensities in the OSE covering the newly formed corpus luteum. Scale bars, 100 μm. n = 3 mice and 15 images. More than 30 corpus luteum were scored. i Percentages of GFP+ cells of all levels in the OSE covering pre-ovulation follicle, ovulating follicle, and corpus luteum were quantified, showing the increasing number of GFP+ cells over time. n = 3 mice in each time point. Data are presented as mean±s.e.m. One-way ANOVA with Tukey test is used for comparison of multiple groups. ***p < 0.001. j After 10 hrs EdU incorporation, Procr+ and Procr− cells were FACS-isolated and underwent cytospin and EdU and K19 staining. Representative images are shown on the right, quantification of EdU+ cells is shown on the left. n = 3 mice in each time point. Data are presented as mean±s.e.m. One-way ANOVA with Tukey test is used for comparison of multiple groups. **p < 0.01, ***p < 0.001. k At 4.5d pi (ovulation), confocal images of Procr-rtTA;TetO-H2B-GFP ovary sections showing EdU staining in GFP+ (Procr+) cells at the edges of ruptured site. Scale bars, 100 μm. n = 3 mice and 15 images

Fig 4: Proposed model of Procr+ progenitor cell activity during ovulatory rupture repair. Procr+ progenitor cells (dark green) are distributed in all regions of the ovarian surface (beige). Upon ovulation, Procr+ progenitors near the rupture site expand immediately, facilitating rapid expansion of progeny cells. When repair is completed, Procr+ cells return to their original numbers that are required for their role in maintaining homeostasis. During the process, the progeny of Procr+ progenitors (light green) are the major contributors for replenishing the OSE

Fig 5: Procr+ cell exhibit distinct lineage tracing behaviors compared with Lgr5+ cells. a FACS analysis of 8-week-old Lgr5-CreER-IRES-EGFP mice ovary showing that the OSE cells, consisting of 40.58 ± 4.20% GFP+ (Lgr5+) cells, and Lgr5+ and Procr+ cells are two distinct OSE cell populations. n = 3 mice. b, c Lgr5+ cells (b) and Procr+ cells (c) were traced for 2d and 1 month using (8-week-old) Lgr5-CreER;R26-tdtomato mice (b) and Procr-CreER;R26-tdtomato mice (c). Ovary whole-mount confocal images showing the distribution pattern of labeled (tdTom+) cells in the OSE (K8+). Scale bar, 100 μm. n = 3 mice and six ovaries in each time point. d FACS quantification of tdTomato+ OSE cells (Lin−, EpCAM+, tdTomato+) after 2d and 1mth tracing, in Lgr5-traced and Procr-traced mouse models. Data are displayed as mean±s.e.m. Unpaired two-tailed t test is used for comparison. ***P < 0.001. n.s, not significant. n = at least three mice in each time point. e–f Strategy of ablating Procr+ cells and tracing Lgr5+ cells using Procr-rtTA;TetO-DTA;Lgr5-CreER;R26-tdtomato mice. Dox was fed into 8-week-old adult mice to ablate Procr+ cells, and 7 days pi, TAM was administered to initiate tracing of Lgr5+ cells. In the ablation group, ovary whole-mount confocal images showing loss of OSE (absence of K8 staining, star). Lgr5+ cell-derived progeny (tdTom+ cells) displayed dispersed pattern, suggesting the loss of OSE injury did not stimulate more robust Lgr5+ cell division (e). In control group (Procr-rtTA;Lgr5-CreER;R26-tdtomato), ovary whole-mount confocal images with K8 staining indicating the Lgr5+ cell-derived progeny cell pattern in normal homeostasis (f). Scale bar, 100 μm. n = 3 mice and six ovaries