Enhanced glycolysis in granulosa cells promotes the activation of primordial follicles through mTOR signaling

Abstract

In mammals, nonrenewable primordial follicles are activated in an orderly manner to maintain the longevity of reproductive life. Mammalian target of rapamycin (mTOR)-KIT ligand (KITL) signaling in pre-granulosa cells and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-forkhead Box O3a (FOXO3a) signaling in oocytes are important for primordial follicle activation. The activation process is accompanied by the enhancement of energy metabolism, but the causal relationship is unclear. In the present study, the levels of glycolysis-related proteins GLUT4, HK1, PFKL, and PKM2 were significantly increased in granulosa cells but were decreased in oocytes during the mouse primordial-to-primary follicle transition. Both short-term pyruvate deprivation in vitro and acute fasting in vivo increased the glycolysis-related gene and protein levels, decreased AMPK activity, and increased mTOR activity in mouse ovaries. The downstream pathways Akt and FOXO3a were phosphorylated, resulting in mouse primordial follicle activation. The blockade of glycolysis by 2-deoxyglucose (2-DG), but not the blockade of the communication network between pre-granulosa cells and oocyte by KIT inhibitor ISCK03, decreased short-term pyruvate deprivation-promoted mTOR activity. Glycolysis was also increased in human granulosa cells during the primordial-to-primary follicle transition, and short-term pyruvate deprivation promoted the activation of human primordial follicles by increasing the glycolysis-related protein levels and mTOR activity in ovarian tissues. Taken together, the enhanced glycolysis in granulosa cells promotes the activation of primordial follicles through mTOR signaling. These findings provide new insight into the relationship between glycolytic disorders and POI/PCOS.

Fig. 1. The expression pattern of glycolysis-related genes and proteins in neonatal mouse ovaries.

a The mRNA levels of the isoforms in each family of Glut, Hk, Pfk, Aldo, Eno, and Ldh in mouse ovaries at 4 dpp. The mRNA values of Glut3, Hk3, Pfkp, Aldoc, Eno4, and Ldhd were set as 1. (n = 3 independent experiments). Bars indicate the mean ± SD. **P < 0.01 and ***P < 0.001. b The mRNA levels of Glut4, Hk1, Pfkl, Aldoa, Eno1, Tpi, Pkm2, and Ldhb in the ovaries at 1, 4 and 7 dpp. (n = 3 independent experiments). Bars indicate the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. 1 dpp group. c, d The protein levels of GLUT4, HK1, PFKL, and PKM2 in the ovaries at 1, 4, and 7 dpp. (n = 3 independent experiments). β-actin was used as an internal control. Bars indicate the mean ± SD. **P < 0.01 and ***P < 0.001 vs. 1 dpp group. e Immunofluorescence stain of GLUT4, HK1, PFKL, and PKM2 (green) in the ovaries at 1, 4, and 7 dpp. (n = 3 independent experiments). The cytoplasm of oocytes was stained with the oocyte-specific marker DDX4 (red), and the nuclei were counterstained by DAPI (blue). The arrowheads and the arrows show the primordial and primary follicles, respectively. Scale bars: 50 μm. f–i The intensity of GLUT4 (f), HK1 (g), PFKL (h), and PKM2 (i) fluorescent signals in granulosa cells (GC) and oocytes (OO) of primordial follicles (PF) and primary follicles (PrF). (n = 9 sections from 7 dpp ovaries. The total number of 45 follicles was scored in each group). Bars indicate the mean ± SD. **P < 0.01 and ***P < 0.001. The representative images are shown.

Fig. 2. Effect of short-term pyruvate deprivation on mouse primordial follicle activation in vitro.

Ovaries at 2 dpp were cultured in standard (control) or pyruvate-free (pyr-free) medium for 1 day (e–j) or 2 days (a–d). a, b Morphological comparison of the ovaries (a) and the number of primordial and growing follicles (arrows, b) in the control and pyruvate-free group. Nuclei were stained by hematoxylin. Scale bars: 50 μm. c The mRNA levels of Gdf9 and Zp3 in the control and pyruvate-free group. d DDX4 protein levels in the control and pyruvate-free group. e The mRNA levels of Pcna, Ki-67, Bax/Bcl-2, and Caspase-3 in the control and pyruvate-free group. f The protein levels of PCNA, BAX, BCL-2, and Cleaved Caspase-3 in the control and pyruvate-free group. g Immunofluorescence stain of PCNA, Ki-67, BrdU, and TUNEL (green) in the control and pyruvate-free group. DAPI, blue. Scale bars: 50 μm. h–j The percentage of granulosa cells (h) and primordial follicles (i) with PCNA-, Ki-67-, or BrdU-positive signals, and the number of cells with TUNEL-positive signals (j) in the control and pyruvate-free group. All the experiments were repeated three times, and the representative images are shown. In western blot results, β-actin was used as an internal control. Bars indicate the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

Fig. 3. Effect of short-term pyruvate deprivation on glycolysis and mTOR signaling in cultured mouse ovaries.

Ovaries at 2 dpp were cultured in standard (control) or pyruvate-free (pyr-free) medium for 6 h (a), 12 h (b), 24 h (c, e) and 48 h (d, f, g), or cultured in pyruvate-free medium supplemented with 10 mM 2-DG (pyr-free + 2-DG) or 5 μM ISCK03 (pyr-free + ISCK03) for 48 h (f–l). a The protein levels of p-AMPK in the control and pyruvate-free group. b The mRNA levels of Glut4, Hk1, Pfkl, Aldoa, Eno1, Tpi, Pkm2, and Ldhb in the control and pyruvate-free group. c The protein levels of GLUT4, HK1, PFKL, and PKM2 in the control and pyruvate-free group. d The protein levels of p-Akt and p-FOXO3a in the control and pyruvate-free group. e The protein levels of p-AMPK, p-mTOR, p-TSC2, p-S6K, p-rpS6, and KITL in the control and pyruvate-free group. f, g The localization of FOXO3a in oocyte nuclear (arrowheads) and cytoplasm (arrows, f) and the percentage of oocytes with FOXO3a nuclear export (g) in each section in the control, pyruvate-free and pyruvate-free + 2-DG groups. FOXO3a, green; DDX4, red; DAPI, blue. Scale bars: 50 μm. h, i The protein levels of p-AMPK, p-mTOR, p-Akt, and p-FOXO3a in the pyruvate-free (as control) and pyruvate-free + 2-DG group. 2-DG, 2-deoxyglucose. j, k Morphological comparison of the ovaries (j) and the number of primordial and growing follicles (arrows. k) in the control, pyruvate-free and pyruvate-free + ISCK03 groups. Nuclei were stained by hematoxylin. Scale bars: 50 μm. l The protein levels of p-mTOR, p-Akt and p-FOXO3a in the control, pyruvate-free and pyruvate-free + ISCK03 groups. All the experiments were repeated three times, and the representative images are shown. In western blot results, total AMPK, mTOR, TSC2, S6K, rpS6, Akt, and FOXO3a were used as the corresponding internal control for p-AMPK, p-mTOR, p-TSC2, p-S6K, and p-rpS6, p-Akt, and p-FOXO3a, respectively, and β-actin was used as the internal control for GLUT4, HK1, PFKL, PKM2, and KITL. Bars indicate the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4. Effect of short-term pyruvate deprivation on the development of mouse follicles.

a The timeline of the experiment was shown. Ovaries at 2 dpp were cultured in standard (control) or pyruvate-free (pyr-free) medium for 6 days, or cultured in pyruvate-free medium for 2 days and then in standard medium for 4 days (recovery). b, c Morphological comparison of the ovaries (b) and the number of primordial, growing (red arrows, c) and atretic follicles (yellow arrows, c) in the control, pyruvate-free and recovery groups. Nuclei were stained by hematoxylin. Scale bars: 50 μm. d DDX4 protein levels in the control, pyruvate-free, and recovery groups. e The mRNA levels of Pcna, Ki-67, Bax/Bcl-2, and Caspase-3 in the control, pyruvate-free and recovery groups. f The protein levels of PCNA, BAX, BCL-2, and Cleaved Caspase-3 in the control, pyruvate-free and recovery groups. g Immunofluorescence stain of PCNA, Ki-67, TUNEL and Cleaved Caspase-3 (green) in the control, pyruvate-free and recovery groups. DAPI, blue. Arrows show the cells with positive signals. Scale bars: 50 μm. h The percentage of granulosa cells with PCNA- and Ki-67-positive signals, and the number of cells with TUNEL- and Cleaved Caspase-3-positive signals in each section in the control, pyruvate-free and recovery groups. i The protein levels of GLUT4, HK1, PFKL, and PKM2 in the control, pyruvate-free and recovery groups. All the experiments were repeated three times, and the representative images are shown. In western blot results, β-actin was used as an internal control. Bars indicate the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

Fig. 5. Effect of acute fasting on glycolysis and mouse primordial follicle activation in vivo.

Two-day-old female mice were kept with their mother (control), or were separated from their mother for 18 h and then returned to their mother (acute fasting). The ovaries were collected at 24 h (c–f) and 48 h (a, b, g, h) of treatment, respectively. a, b Morphological comparison of the ovaries (a) and the number of primordial and growing follicles (arrows, b) in the control and acute fasting group. Nuclei were stained by hematoxylin. Scale bars: 50 μm. c The mRNA levels of Glut4, Hk1, Pfkl, Aldoa, Eno1, Tpi, Pkm2, and Ldhb in the control and acute fasting group. d The protein levels of GLUT4, HK1, PFKL, and PKM2 in the control and acute fasting group. e, f The protein levels of p-AMPK, p-mTOR (e), p-Akt, and p-FOXO3a (f) in the control and acute fasting group. g, h The localization of FOXO3a in oocyte nuclear (arrowheads) and cytoplasm (arrows, g) and the percentage of oocytes with FOXO3a nuclear export (h) in each section in the control and acute fasting group. FOXO3a, green; DDX4, red; DAPI, blue. Scale bars: 50 μm. Fasting, acute fasting. All the experiments were repeated three times, and the representative images are shown. In western blot results, the levels of total AMPK, mTOR, Akt, and FOXO3a were used as the corresponding internal control for p-AMPK, p-mTOR, p-Akt, and p-FOXO3a, respectively, and β-actin was used as the internal control for GLUT4, HK1, PFKL, and PKM2. Bars indicate the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

Fig. 6. Effect of short-term pyruvate deprivation on human primordial follicle activation in vitro.

a Immunofluorescence stain of GLUT4, HK1, PFKL, and PKM2 (green) in human primordial and growing follicles. FOXL2, purple; DAPI, blue. Scale bars: 20 μm. b, c Log₂FPKM values were extracted from previously published data (GSE107746). The expression levels of isoforms in each family of HK, PFK, ALDO, ENO, and LDH in human granulosa cells of primary follicles (n = 15 follicles, b), and the expression levels of HK1, PFKL, ALDOA, ENO1, PKM, and LDHB in human granulosa cells of primordial (n = 8 follicles) and primary follicles (n = 15 follicles, c). GCs granulosa cells, pre-GCs pre-granulosa cells. d–g Human ovarian fragments were directly fixed in 4% PFA (noncultured), or cultured in standard medium (control), or cultured in the pyruvate-free medium for 2 days and then in standard medium for indicated days (pyruvate-free group). The fragments were collected after 3 days (e, g) and 6 days (d, f) of treatment, respectively. d, f Morphological comparison of human ovarian tissue fragments (d) and the proportion of primordial (arrowheads, f) and growing follicles (arrows, f) in the control and pyruvate-free group. (n = 5 independent experiments). Nuclei were stained by hematoxylin. Scale bars: 50 μm. e The protein levels of GLUT4, HK1, PFKL, and PKM2 in the noncultured, control, and pyruvate-free groups. g The protein levels of p-AMPK, p-mTOR, p-Akt, and p-FOXO3a in the noncultured, control, and pyruvate-free groups. The representative images are shown. In western blot results, the levels of total AMPK, mTOR, Akt, and FOXO3a were used as the corresponding internal control for p-AMPK, p-mTOR, p-Akt, and p-FOXO3a, respectively, and β-actin was used as the internal control for GLUT4, HK1, PFKL, and PKM2. Bars indicate the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 7. The proposed model of glycolysis in primordial follicle activation.

Enhanced glycolysis in granulosa cells activates mTOR signaling by decreasing AMPK activity. This process triggers the transduction of KITL signaling in pre-granulosa cells to PI3K-Akt-FOXO3a signaling in oocytes, resulting in the activation of dormant primordial follicles.