Fig. 4

pcMSC-CM and E2-CM restore ovarian circadian rhythm in CTX-induced POI animal model. A Regulatory expression loops of Per2-Bmal1 (Loop1), Cyp19a1 (Loop 2), and Cyp11a1 (Loop 3). Clock-controlled elements (CCEs) of Cyp19a1 and Cyp11a1 are RRE and D box, respectively. RORA and REV-ERBα competitively bind to RRE (Cyp19a1), and DBP and E4BP4 competitively bind to D box (Cyp11a1). Binding of RORA and DBP to their targeted CCEs induces upregulation of their targeted genes; this effect is reversed for REV-ERBα and E4BP4. B Experimental conditions are illustrated. Four experimental groups were involved, namely the control, POI, CM (POI plus CM), and E2-CM (POI plus E2-CM) groups. C Loop 1 for Per2 and Bmal1 expression. D Loop 2 for expression of Cyp19a1-Rora-Rev-erbα. E Loop 3 for expression of Cyp11a1-E4bp4-Dbp. a Gene expression levels detected by RT-qPCR, b quantitative and statistical analysis of gene expression (n = 3 each time point), and c protein levels detected through western blotting at specific ZTs were shown. Curves were created using Fit Spline/LOWESS (medium smoothing). *p < 0.05, **p < 0.01, ***p < 0.001. Comparisons between groups at a given time point (p < 0.05), specifically, a between control and POI groups, b between CM and POI groups, c between E2-CM and POI groups, and d between CM and E-CM groups. Two-way ANOVA with Tukey’s test. Light and dark backgrounds represent light and dark phases of the day, respectively. ZT0 indicates the time when the light is switched on, and ZT12 indicates the time when the light is switched off. F Protein expression localization and statistical analysis of CYP19A1 (a and a’), REV-ERBα (b and b’) and RORA (c and c’) in ovary tissues of mice in each experimental group (n = 5). Results obtained through immunohistochemical staining. G and H Nuclear expressions of REV-ERBα and E4BP4 in KGN cells were shown. Protein expression levels detected by immunofluorescence staining (n = 4). F, G One-way ANOVA with Tukey’s test