(A) UMAP clustering shows the granulosa cells. Ube2c-negative mural granulosa cells (dotted area) were isolated from the total granulosa cell population and used for t-SNE clustering and cell trajectory.
(B) Cell trajectories by pseudotime in mural granulosa cells of WT and Esr2-PgrKO show two different states (Branch-1 and −2) of mural granulosa cells at the end of pseudotime. Changes in expression of Amh, Lyve1, Pgr, Ptgs2, and Snap25 in Branch-1 and −2 are shown by pseudotime plot. Red circles in the pseudotime plot indicate when the Pgr gene begins to be expressed and when it reaches its highest expression level, respectively. Ovulatory granulosa cells (cells in Branch-1) express Pgr, whereas non-ovulatory granulosa cells (cells in Branch-2) do not express Pgr until the Pgr expression level in Branch-1 reaches the highest level.
(C) Localization of PGR and PTGS2 protein in preovulatory follicles at 9 h after hCG injection. An, Antrum; mGC, mural granulosa cells; Th, theca cells (scale bar, 0.1 mm).
(D) Ptgs2 mRNA level in isolated granulosa cells from WT and Esr2-PgrKO ovaries at 6 h and 9 h after hCG injection. Error bars, SD (n = 4); **p<0.01 (Student’s t test).
(E) Ovarian PTGS2 protein level in WT and Esr2-PgrKO mice at 9 h after hCG injection. Error bars, SD (n = 4); *p < 0.05 (Student’s t test).
(F) PGE2 concentration in WT and Esr2-PgrKO ovaries at 0, 6, 9, and 12 h after hCG injection. Error bars, SD (n = 3–4); *p < 0.05 (Student’s t test).