Fig 1.
Ggpps is primarily expressed in the oocyte and associated with early follicular development.
(A) Immunohistochemistry assays of Ggpps expression in ovary sections from PD 4 (primordial and primary follicle) and PD 23 (preantral and antral follicle) mice. Negative control involved exception of the primary antibody stage. Arrowheads indicated different stages of follicles. Scale bar, 100 μm. (B) Western blot analysis of ovaries from PD 2 (indicating primordial follicles), PD 6 (indicating primary follicles) and PD 12 (indicating secondary follicles) mice. Actin and GAPDH were used as internal loading controls. (C) Western blot analysis of Ggpps protein levels in the oocytes that were larger or smaller than 25 μm, respectively. Actin was used as internal loading controls. Data were presented as the mean ± SEM. * p<0.05, **p<0.01.
Fig 2.
Oocyte-specific GGPP depletion impairs follicular development and female fertility.
(A) A comparison of the cumulative number of pups generated by female Ggppsfl/fl Ddx4-Cre and CTL mice. (B and C) The morphology, size and weight of 6-week-old ovaries in Ggppsfl/fl Ddx4-Cre and CTL mice. Scale bar, 250 μm. (D) H&E staining of 6-week-old ovaries. Scale bar, 250 μm. Data were presented as the mean ± SEM. **p<0.01.
Fig 3.
GGPP depletion in oocytes inhibits ovarian primary-secondary follicle transition.
(A) Ovary weight at the indicated time points (n = 3–6). (B) The images of the ovaries at different time points were captured using a light microscope. (C and D) MVH immunofluorescence and primordial follicle numbers (n = 4) in PD 3 and PD 23 ovaries from Ggppsfl/fl Ddx4-Cre and CTL mice. DAPI (blue) indicates the cell nuclei. (E-G) H&E staining of ovaries at the indicated time points and quantification of the different types of follicles observed in ovaries from PD 8, PD 10, and PD 13 mice, including primordial (Pri), primary (Pr), type 4 (T4), and type 5 (T5) follicles. The number of follicles per ovary was quantified as described in Materials and Methods (n = 4). Arrowheads indicate abnormal contact between the oocyte and granulosa cells. Data were presented as the mean ± SEM. * p<0.05, **p<0.01. Scale bar, 200 μm.
Fig 4.
GGPP depletion in oocytes arrests granulosa cell proliferation by impairing secretion of oocyte factors.
(A and B) Ki67 immunofluorescence assays and the quantification of Ki67-positive granulosa cells in the ovaries of PD 13 Ggppsfl/fl, Ddx4-Cre and CTL mice. Scale bar, 25 μm. (C and D) PCNA immunohistochemistry and the quantification of PCNA-positive granulosa cells in PD 13 ovaries. Scale bar, 25 μm. The number of Ki67-positive and PCNA-positive granulosa cells per primary follicle was quantified as described in Materials and Methods (n = 4). (E) Western blot analysis of activated Smad2 in isolated granulosa cells from PD 12–14 ovaries. (F) Quantitative PCR (qPCR) analysis of Gdf9 and Bmp15 expression in PD 8 ovaries. (G) Western blot analysis of Gdf9 protein in PD 8 ovaries. Actin was used as internal loading controls. The control growing oocytes were co-cultured with PD 12–14 Ggppsfl/fl, Ddx4-Cre granulosa cells. After 24 h in culture, p-Smad2 and Smad2 levels in the granulosa cell lysates were evaluated using western blot (H), Ki67 immunofluorescence was measured in isolated granulosa cells (I). Scale bar, 50 μm. (J) The prenylation of Rab27a in PD 12–14 ovaries. (K) Subcellular fractionation of Rab27a in PD 12–14 ovaries was conducted using the Triton X-114 partition method. The aqueous upper phase (up) contained the water-soluble small GTPases, and the lower organic phase (down) contained the lipid-soluble small GTPases. The data were presented as the mean ± SEM. **p<0.01.
Fig 5.
GGPP depletion in oocytes inhibits the physical connection between oocyte and granulosa cells.
(A) Transmission electron microscopy analysis of PD 13 ovaries. Zona pellucida between a granulosa cell (gc) and oocyte (o) is indicated by red arrowheads in the primary follicles. Black arrowheads indicated the gap junctions. White arrowhead indicated the autophagosomes. Mi, Mitochondria. (B) Western blot analysis of N-cadherin, E-cadherin, connexin 37, and connexin 43 using lysates from PD 8 ovaries. Actin and GAPDH were used as the internal loading controls. (C-E) β-catenin, and N-cadherin immunofluorescence and the quantification of adhesion between oocyte and granulosa cells in the primordial and primary follicles of PD 13 ovaries. The cell adhesion was assessed as described in Materials and Methods (n = 4). Data were presented as the mean ± SEM. *p<0.05, **p<0.01. Scale bar, 25 μm.
Fig 6.
GGPP depletion disrupts cadherin-mediated cell contact by inhibiting Rho GTPase geranylgeranylation and GTPase activity.
(A) The enzymatic activity of Rac1, RhoA, and Cdc42 in PD 12–14 ovaries. (B) The prenylation of Rac1 and RhoA in PD 12–14 ovaries. (C) Subcellular fractionation of Rac1 and RhoA in PD 12–14 ovaries was conducted using the Triton X-114 partition method. The aqueous upper phase contained the water-soluble small GTPases, and the lower organic phase contained the lipid-soluble small GTPases. (D) Rac1 and RhoA membrane association, as determined using ultracentrifugation, in PD 12–14 ovaries. (E) Rac1 prenylation in organ-cultured PD 12–14 ovaries by GGPP treatment (20 μM, 24 h). (F) β-catenin immunofluorescence in the primary follicles of PD 13 ovaries 5 days after daily intraperitoneal injections of GGPP (2 mg/kg). (G) Ki67 immunofluorescence, H&E staining (H), quantification of the T4 follicles (I) and ovary weight (J) in PD 13 ovaries after 5 days of daily intraperitoneal injections of GGPP (2 mg/kg). Data were presented as the mean ± SEM. *p<0.05, **p<0.01.
Fig 7.
Schematic of GGPP-mediated protein geranylgeranylation involving in the regulation of primary-secondary follicle transition via remodeling oocyte-granulosa cell communication.
Cholesterol was biosynthesized from farnesyl diphosphate (FPP), a metabolic intermediate of the mevalonate pathway. FPP could be catalyzed into another metabolite, geranylgeranyl diphosphate (GGPP), by geranylgeranyl diphosphate synthase (GGPPS). GGPP then was used for the geranylgeranylation and activation of Rho GTPase and Rab GTPase. The activated Rho GTPase was responsible for the localization of cell junction proteins in the oocyte membrane to maintain physical connection between oocyte and granulosa cells. The activated Rab GTPase might account for the secretion of oocyte materials such as Gdf9. The two processes were probably important for the proliferation of granulosa cells from one layer to multiple layers and ultimately promote the primary-secondary follicle transition. The pathway in the box might mainly occur in granulosa cells. Dashed arrows depicted possible mechanism of GGPP-regulated small GTPase on oocyte-granulosa cell communication.