Fig 2: RSPO1 interference partly reverses circ_0000396 overexpression-induced effects in RASFs. A Western blot assay was conducted to determine the protein expression of RSPO1 in RASFs transfected with si-NC or si-RSPO1. (B-J) RASFs were transfected with circ_0000396 plasmid alone or together with si-RSPO1. B Cell proliferation was analyzed by MTT assay. C Cell proliferation was analyzed by colony formation assay. D EDU assay was conducted to assess the proliferation ability of RASFs. E The protein levels of PCNA, Cyclin D1 and Cyclin E1 were determined by Western blot assay. F The apoptosis rate of RASFs was assessed by flow cytometry. G The protein levels of Bax and Bcl2 were examined by Western blot assay. H and I Cell inflammation was analyzed by ELISA assay. J The protein levels of IL-1β and TNF-α were examined by Western blot assay. **P < 0.01, ***P < 0.001

Fig 3: (A–C) Immunolocalization of RSPO1 protein in tammar ovary on Day 85 pp. ovary. RSPO1 immunofluorescence (red) and nuclei stained with DAPI (blue). The white dotted box in figure A is magnified in figure B. (B) Day 85 pp. ovary showing RSPO1 staining in cytoplasm of granulosa cells (yellow arrows) and germ cells (arrowheads) and oocytes (white arrows) in primary and primordial follicles scattered throughout the ovary. (D–F) Immunolocalization of RSPO1 protein in tammar ovary in Day 154 pp. ovary. RSPO1 immunofluorescence (red) and nuclei stained with DAPI (blue). The white dotted box in figure D is magnified in figure E. (D) Membrane‐bound RSPO1 still seen around germ cells in germ cell nests in the cortex of ovary (arrows). (E) Cytoplasmic RSPO1 in cuboidal granulosa cells (arrows) of primary follicles surrounding central oocytes that are faintly positive for RSPO1 as well. (G–I) Immunolocalization of RSPO1 protein in tammar ovary in adult ovary. RSPO1 immunofluorescence (red) and nuclei stained with DAPI (blue). (H) Cytoplasmic RSPO1 seen in granulosa cell layer (arrows) surrounding central oocyte. Some interstitial cells also stained positive for RSPO1.

Fig 4: Phylogenetic tree analysis of (A) RSPO1, (B) FOXL2, and (C) RUNX1. Evolutionary history was inferred by using the Maximum Likelihood method based on Ref. 64 . The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

Fig 5: (A–C) Immunolocalization of RSPO1 protein in tammar ovary on Day 18 pp. ovary. RSPO1 immunofluorescence (red) and nuclei stained with DAPI (blue). The white dotted box in figure A is magnified in figure B. (B) Diffuse RSPO1 staining in cytoplasm of granulosa cells (arrowheads) and germ cells (arrows). (D–F) RSPO1 immunofluorescence (red) and nuclei stained with DAPI (blue). The white dotted box in figure D is magnified in figure E (E) rounded germ cells (arrows), germ cells in leptotene (arrowhead). (G–I) Immunolocalization of RSPO1 protein in tammar ovary on Day 40 pp. ovary. RSPO1 immunofluorescence (red) and nuclei stained with DAPI (blue). (G) Day 40 pp. ovary showing elongated rete cords (arrows) extending outwards from central medulla of ovary. White dotted box in figure G is magnified in figure H. (H) Day 40 pp. ovary close up showing elongated somatic cells of the intra‐ovarian rete positive for RSPO1 (arrowheads) and rounded germ cells associated with the rete (arrows) positive for RSPO1, granulosa cells are also stained positive for RSPO1 (blue arrowheads).(J–L) Immunolocalization of RSPO1 protein in tammar ovary in Day 64 pp. ovary. RSPO1 immunofluorescence (red) and nuclei stained with DAPI (blue). (J) germ cell nests in outer cortex (c) (arrowheads) and primordial follicles (arrow) in medulla (m) positive for RSPO1 (K) RSPO1 localized to the cell membrane of germ cells (arrows) surrounded by RSPO1‐positive granulosa cells with cytoplasmic staining (arrowheads).