Fig 2: Hypobaric hypoxia-induced disorder of circadian clock protein expression in the ovaries of female mice. (A–D) Female mice were treated as shown in Figure 1A. Ovaries were collected 48 h after hypoxic exposure, and the expression levels of circadian clock proteins were detected. The Western blot quantitative results of CLOCK, BMAL1, and E4BP4 (relative to ß-actin) are shown (B–D) (n = 6, **p < 0.01; *p < 0.05). The RT-PCR results of BMAL1, CLOCK, and E4BP4 (relative to ß-actin) are shown (E–G) (n = 3, *p < 0.05). (H) Potential CLOCK:BMAL1- and E4BP4-binding sites (E-box and D-box) were identified within mouse LHCGR promoter regions.

Fig 3: Hypoxia-induced dysregulation of clock-controlled LHCGR reduction in HIF1?-dependent and ?-independent manners. (A–B) Female mice were treated as shown in Figure 1A. Ovaries were collected 48 h after hypoxic exposure, and the expression level of HIF1? was detected. The quantitative results of HIF1? expression (relative to ß-actin) are shown (B) (n = 6, **p < 0.01). (C–D) Primary GCs (C) and KK1 cells (D) were cultured under normoxic or hypoxic conditions for 6 h. Then, the expression level of HIF1? was detected. (E) Primary GCs were transfected with HIF1? siRNA or control siRNA and then left untreated or subjected to hypoxic exposure. Cells were collected 6 h later, and the expression level of HIF1? was detected. (F) KK1 cells were transfected with the LHCGR promoter-driven luciferase reporter in combination with siRNA targeting HIF1? or control siRNA. The luciferase activity was detected 36 h after transfection ( *p < 0.05). (G) Primary GCs were transfected and treated as described for (E), and the expression levels of circadian clock proteins were detected. (H) Potential HIF1?-binding sites (HRE) were identified within mouse E4BP4 promoter regions. (I) ChIP assays were performed on the hypoxia-treated KK1 cells to detect the functional HRE within the mouse E4BP4 promoter region.

Fig 4: CLOCK, BMAL1, and E4BP4 were involved in LHCGR transcription in the granulosa cells. (A) Primary granulosa cells (GCs) were prepared and cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 6 h. Cells were then collected to detect LHCGR expression. (B) KK1 cells cultured under the same conditions and LHCGR levels detected. (C–E) Primary GCs were transfected with CLOCK, BMAL1, E4BP4 siRNA, or control siRNA. Changes in LHCGR mRNA and protein levels were detected. KK1 cells were transfected as described for the primary GCs, and LHCGR levels were detected using Western blot (F–H) and in situ immunofluorescence staining (I). (J–K) ChIP assays were performed in KK1 cells to detect the binding site of CLOCK: BMAL1 heterodimer and E4BP4 within the mouse LHCGR promoter region. (L–N) KK1 cells were transfected with the LHCGR promoter-driven luciferase reporter in combination with siRNA targeting CLOCK, BMAL1 or E4BP4 or control siRNAs. Luciferase activity was detected at 36 h after transfection (**p < 0.01).