Transforming growth factor-β signaling participates in the maintenance of the primordial follicle pool in the mouse ovary

Abstract

Physiologically, only a few primordial follicles are activated to enter the growing follicle pool each wave. Recent studies in knock-out mice show that early follicular activation depends on signaling from the tuberous sclerosis complex, the mammalian target of rapamycin complex 1 (mTORC1), phosphatase and tensin homolog deleted on chromosome 10, and phosphatidylinositol 3-kinase (PI3K) pathways. However, the manner in which these pathways are normally regulated, and whether or not TGF-β acts on them are poorly understood. So, this study aims to identify whether or not TGF-β acts on the process. Ovary organ culture experiments showed that the culture of 18.5 days post-coitus (dpc) ovaries with TGF-β1 reduced the total population of oocytes and activated follicles, accelerated oocyte growth was observed in ovaries treated with TGF-βR1 inhibitor 2-(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-yl-amine (SD208) compared with control ovaries, the down-regulation of TGF-βR1 gene expression also activated early primordial follicle oocyte growth. We further showed that there was dramatically more proliferation of granulosa cells in SD208-treated ovaries and less proliferation in TGF-β1-treated ovaries. Western blot and morphological analyses indicated that TGF-β signaling manipulated primordial follicle growth through tuberous sclerosis complex/mTORC1 signaling in oocytes, and the mTORC1-specific inhibitor rapamycin could partially reverse the stimulated effect of SD208 on the oocyte growth and decreased the numbers of growing follicles. In conclusion, our results suggest that TGF-β signaling plays an important physiological role in the maintenance of the dormant pool of primordial follicles, which functions through activation of p70 S6 kinase 1 (S6K1)/ribosomal protein S6 (rpS6) signaling in mouse ovaries.

Keywords: Cell Growth; Mouse; Oocyte; Ovary; Transforming Growth Factor-β (TGFβ).

FIGURE 1.

Immunohistochemical and Western blot analyses of TGF-β1 and TGF-βR1 in prenatal and neonatal mouse ovaries. Germ cells with cyst structure in 18.5-dpc mouse ovaries exhibited positive staining for TGF-β1 and TGF-βR1 in contrast to somatic cells, which exhibited negative staining (A and D, rings). Both TGF-β1 and TGF-βR1 were expressed in the oocytes of primordial and primary follicles in 1-dpp (B and E, arrows) and 4-dpp (C and F, arrows) mouse ovaries and were beginning to express in the cuboidal granulosa cells (C and F, arrowheads) of primary follicles. Western blot analysis of the expression patterns of TGF-β1 and TGF-βR1 in the mouse ovary from 17.5 to 7 dpp normalized to β-actin (G). Rings indicate germ cell cysts, arrows indicate primordial follicles, and arrowheads indicate cuboidal granulosa cells. Scale bars: 40 μm (A–F). Bars with different letters are significantly different (p < 0.05).

FIGURE 2.

Phenotypes of ovary cultures treated with TGF-β1 and SD208. Ovaries at 18.5 dpc were cultured for 7 days without treatment (as a control), with 10 ng/ml of TGF-β1, or with 1 μm SD208. Following culture, ovaries were fixed, sectioned, and the shape and number of total germ cells and growing follicles were evaluated. All cultured ovaries exhibited mostly primordial follicles and some activated follicles. However, the primordial follicle oocytes in TGF-β1-treated ovaries seemed to grow more slowly (D–F), whereas those in the SD208-treated ovaries appeared to grow faster (G–I, arrowheads) compared with control ovaries (A–C). The numbers of total germ cells and growing follicles were quantified (J); germ cell data were obtained from at least six ovaries. The images in B, C, E, F, H, and I are enlargements of A, B, D, E, G, and H. Arrows indicate primordial follicles and arrowheads indicate growing follicles. Scale bars: 40 μm (A–I). Bars with different letters are significantly different (p < 0.05).

FIGURE 3.

Down-regulation of TGF-βR1 gene expression activates early primordial follicle growth. Ovaries at 18.5 dpc were transfected with scrambled or TGF-βR1 siRNA for 7 days and transfection efficiency and ovarian histology were evaluated. Real-time PCR and Western blot analyses revealed an obvious decrease in TGF-βR1 gene and protein expressions following transfection with TGF-βR1 siRNA (A). C–F, morphological analysis of scrambled and TGF-βR1 siRNA-transfected ovaries. Primordial follicle oocyte growth was accelerated (E and F, arrows) and more primordial follicles were activated into the growing follicle pool in TGF-βR1 siRNA-treated ovaries compared with scrambled siRNA-treated ovaries (C and D, arrows). Numbers of germ cells and growing follicles were counted (B). Arrows indicate growing follicles. Scale bars: 40 μm (C–F). Bars with different letters are significantly different (p < 0.05).

FIGURE 4.

TGF-β1 partially reverses the effect of SD208 on primordial follicle oocyte growth. Ovaries at 14.5 dpc were cultured in medium containing 4% FBS for 4 days (equivalent to 18.5 dpc) to make them adhere to the dish and then cultured in fresh medium without treatment as a control (E), with 10 ng/ml of TGF-β1, 1 μm SD208 (F), 1 μm SD208 plus 10 ng/ml of TGF-β1, 1 μm SD208 plus 20 ng/ml of TGF-β1, 1 μm SD208 plus 50 ng/ml of TGF-β1, or 1 μm SD208 plus 100 ng/ml of TGF-β1 (G) for 7 days. Ovaries at 18.5 dpc were directly cultured with the same treatments as described above for 7 days. After culture, ovaries were fixed, sectioned, and the shape (A–D) and the number of growing follicles (H) was determined. Data were obtained from at least six ovaries. Arrows indicate growing follicles. Scale bars: 40 μm (A–D) and 100 μm (E–G). Bars with different letters are significantly different (p < 0.05).

FIGURE 5.

Proliferation and apoptosis in TGF-β1- and SD208-treated ovaries. Ovaries at 18.5 dpc were cultured alone as a control, with 10 ng/ml of TGF-β1, or with 1 μm SD208 for the staining of PCNA, a BrdU incorporation assay, and the TUNEL assay. Incorporation of BrdU into the granulosa cells was decreased in TGF-β1-treated ovaries and increased in SD208-treated ovaries compared with control ovaries (A–C, arrows: BrdU positive granulosa cells) after 7 days of culture. Immunostaining of PCNA expression in ovarian sections from TGF-β1- and SD208-treated ovaries (D–F, arrows, PCNA negative granulosa cells; arrowheads, PCNA positive granulosa cells) after 7 days of culture. TUNEL assays for apoptosis were performed in control, TGF-β1-, and SD208-treated ovaries (G–I, arrows, TUNEL positive oocytes) after 5 days of culture and the number of apoptotic germ cells was analyzed in the largest cross-section of all treated ovaries (K). Western blot was performed using anti-cleaved caspase-3 and anti-GAPDH antibodies after 5 days of culture (J). Data were obtained from at least six ovaries. Scale bars: 40 μm (A–I). Bars with different letters are significantly different (p < 0.05).

FIGURE 6.

The molecular mechanisms underlying the accelerated enlargement of oocytes in TGF-β1- and SD208-treated ovaries. Ovaries at 18.5 dpc were cultured without treatment (as a control), or with 10 ng/ml of TGF-β1 or 1 μm SD208. The expression levels of Gdf9 and Inhibin α were analyzed by real-time PCR after 7 days of culture (A and B). After 5, 7, or 9 days of culture, levels of activated Akt (C, Ser-473), total Akt (D), activated FOXO3a (E, Thr-32), total FOXO3a (F), activated S6K1 (G, Thr-389), total S6K1 (H), activated rpS6 (I, Ser-235/Ser-236), activated rpS6 (J, Ser-240/Ser-244), total rpS6 (K), and β-actin (L) were analyzed by Western blotting. The levels of p-Akt (C) and p-FOXO3a (E) in SD208-treated ovaries were similar to those of control and TGF-β1-treated ovaries. The phosphorylation of S6K1 at Thr-389 (G), rpS6 at Ser-235/Ser-236 (I), and Ser-240/Ser-244 (J) were elevated in SD208-treated ovaries but reduced in TGF-β1-treated ovaries compared with control ovaries. Levels of total Akt, FOXO3a, S6K1, rpS6, and β-actin were measured as internal controls. The experiments were repeated at least three times and representative results are shown. Bars with different letters are significantly different (p < 0.05).

FIGURE 7.

Immunohistochemistry of p-rpS6 (Ser-235/Ser-236) in TGF-β1- and SD208-treated ovaries. Ovaries at 18.5 dpc were cultured alone (as a control), with 10 ng/ml of TGF-β1, or 1 μm SD208 for 5, 7, or 9 days, respectively, and then collected for immunohistochemistry of p-rpS6 (Ser-235/Ser-236). In 1-dpp (A, arrows) and 4-dpp (B, arrows) mouse ovaries, p-rpS6 (Ser-235/Ser-236) was only expressed in the oocytes of growing follicles but not in the oocytes of primordial follicles. In 18.5-dpc ovaries after 5, 7, or 9 days of culture, respectively, a greater number of growing follicle oocytes exhibited positive staining for p-rpS6 (Ser-235/Ser-236) in SD208-treated ovaries and fewer in TGF-β1-treated ovaries compared with control ovaries (C–K, arrows). More rapid growth of primordial follicles and increased staining of p-rpS6 (Ser-235/Ser-236) in growing follicle oocytes were observed in SD208-treated ovaries (E, H, and K, arrows), whereas slower growth of primordial follicles and decreased staining were observed in TGF-β1-treated ovaries (D, G, and J, arrows) compared with control ovaries (C, F, and I, arrows). Scale bars: 40 μm (A–K). The experiments were repeated at least three times and representative results are shown.

FIGURE 8.

Rapamycin partially prevents the accelerated primordial follicle oocyte growth in SD208-treated ovaries. A–F, the accelerated primordial follicle oocyte growth in SD208-treated ovaries can be reversed by treatment with the PI3K-specific inhibitor LY294002 (LY, 25 μm) or the mTORC1-specific inhibitor rapamycin (Rap, 50 nm). Ovaries at 3 dpp were cultured for 5 days without treatment (as a control), with 1 μm SD208, 25 μm LY294002, 50 nm rapamycin, 1 μm SD208 plus 25 μm LY294002, or with 1 μm SD208 plus 50 nm rapamycin. Following culture, ovaries were fixed, sectioned, and the morphological analyses and average numbers of growing follicles were evaluated. Clusters of primordial follicles were seen in all cultured ovaries, whereas more primordial follicles were activated into growing follicles in SD208-treated ovaries (B and B′, arrows) as compared with control ovaries (A and A′). In SD208-treated ovaries that had also been treated with LY294002 or rapamycin, the ovaries showed decreased numbers of growing follicles compared with SD208-treated ovaries but still had more enlarged growing follicles as compared with control ovaries (E, E′, F, and F′, arrows). Average numbers of growing follicles were quantified (G). H, 3-dpp mouse ovaries cultured with the same treatments as described above were collected to evaluate the rpS6 and Akt phosphorylation levels by Western blotting. Ovaries treated with LY294002 or SD208 plus LY294002 largely suppressed levels of p-Akt (Ser-473) and p-rpS6 (Ser-240/Ser-244), whereas treatment with rapamycin or SD208 plus rapamycin only suppressed the level of p-rpS6 (Ser-240/Ser-244), but not the level of p-Akt (Ser-473), treatment with SD208 only elevated the level of p-rpS6 (Ser-240/Ser-244), but did not altere the level of p-Akt (Ser-473). Levels of total Akt, rpS6, and β-actin were used as internal controls. Scale bars: 40 μm (A–F and A′–F′). Bars with different letters are significantly different (p < 0.05).