Fig 1.
Immunohistochemical analysis of follistatin (FST) in prenatal and neonatal mouse ovaries.
Weak FST staining was observed in the germ cell cytoplasm and strong staining in many germ cell nuclei (arrowheads) in 17.5 days post coitus (dpc) and 19.5 dpc mouse ovaries (A and B). FST continued to be expressed in the oocyte cytoplasm of nests and some primordial follicles (arrows) in 1 day post parturition (dpp) mouse ovaries; intense staining was observed in a portion of oocyte nuclei (arrowheads) (C). FST immunostaining in 4 and 7 days post parturition (dpp) mouse ovaries was prominent in the oocyte cytoplasm of primordial (arrows) and primary (double arrows) follicles as well as cuboidal granulosa cells (double arrowheads) of primary and growing follicles, but not in the flattened pre-granulosa cells (arrowheads) of primordial follicles (D and E). 5 dpp mouse ovaries were immunohistochemically stained with rabbit IgG as a negative control for FST (F). Scale bars: 40 μm (A-F).
Fig 2.
FST expression in the mouse ovary.
Western blotting was performed to examine the mouse ovary FST protein levels from 17.5 dpc to 7 dpp, which were normalized to β-actin (A and B). Significant differences are indicated by * (P < 0.05).
Fig 3.
Phenotypes of ovary cultures treated with FST288.
Ovaries at 17.5 dpc were cultured for 7 days without treatment (as a control) or with 500 ng/mL FST288. After culture, ovaries were fixed and sectioned, and then the shape and number of total germ cells and growing follicles were examined. Control ovaries had mostly primordial follicles containing small oocytes surrounded by flattened pre-granulosa cells (A and B, arrows, enlarged in C), while FST288-treated ovaries had a greater number of germ cells within nests (D and E, black boundaries, enlarged in F) compared to control ovaries. Follicle populations in the largest cross-section of control and FST288-treated ovaries were quantified (G). Western blot analysis revealed a dramatic decrease of the p-smad2 level in FST288-treated ovaries relative to control group (H). Scale bars: 40 μm (A, B, D and E). Smad2 was measured as internal control. Significant differences between control and FST288-treated ovaries are indicated by * (P < 0.05).
Fig 4.
Proliferation of granulosa cells in FST288-treated ovaries.
Ovaries at 17.5 dpc were cultured alone (as a control) or with 500 ng/mL FST288 for BrdU incorporation assays. After 4 days of culture, when compared to granulosa cells of control ovaries (A-C, arrows; BrdU-positive granulosa cells), BrdU incorporation into granulosa cells of FST288-treated ovaries was reduced (D-F, arrows; BrdU-positive granulosa cells), particularly near the ovarian cortex. Similarly, BrdU incorporation into granulosa cells near the periphery of FST288-treated ovaries (J-L) was reduced relative to granulosa cells of control ovaries after 7 days of culture (G-I, arrows; BrdU-positive granulosa cells). Almost all of the somatic cells near the ovarian cortex were BrdU-negative (J-L). Scale bars: 40 μm (A-L).
Fig 5.
Germ cell apoptosis in FST288-treated mice ovaries.
Ovaries at 17.5 dpc were cultured alone (as a control) or with 500 ng/mL FST288 for 4 days prior to apoptosis assay (TUNEL). After culture ovaries were fixed and sectioned, a TUNEL assay was performed on the sections. No significant difference in oocyte apoptosis was observed between control and FST288-treated ovaries (A-D, arrows; TUNEL-positive oocytes). Populations of apoptotic oocytes and total oocytes per section in each treatment group were quantified (E and F). Scale bars: 40 μm (A-D).
Fig 6.
Effect of FST288 treatment on Notch signaling in ovaries cultured for 4 or 7 days.
Ovaries at 17.5 dpc were cultured without treatment (as a control) or with 500 ng/mL FST288 for 4 or 7 days. The protein levels of Notch1, Notch1 intracellular domain (NICD1), and hairy/enhancer-of-split related with YRPW motif protein 2 (Hey2) were analyzed by Western blotting. Levels of Notch1, NICD1, and Hey2 were not altered in FST288-treated ovaries relative to control ovaries after 4 days of culture (A) and were significantly reduced after 7 days of culture (B). The protein level of β-actin was used as a loading control. Representative Western blot images are shown on the left and quantification of at least 3 independent Western blot results are shown on the right. Significant differences are indicated by * (P < 0.05).
Fig 7.
Phenotypes of ovary cultures treated with ACT A.
Ovaries at 17.5 dpc were cultured for 7 days without treatment (as a control) or with 100 ng/mL ACT A. Compared to control ovaries (A, black boundary enlarged in B), there were significantly more primordial follicles in the periphery of ovaries treated with ACT A (C, black boundary enlarged in D). The total number of ovarian follicles in the largest cross-section of control and ACT A-treated ovaries were quantified (E). Western blot analysis revealed an obvious increase of the p-smad2 level in Activin A-treated ovaries relative to control group (F). Scale bars: 40 μm (A-D). Smad2 was measured as internal control. Significant differences are indicated by * (P < 0.05).