A novel action of follicle-stimulating hormone in the ovary promotes estradiol production without inducing excessive follicular growth before puberty

Abstract

In cyclic females, FSH stimulates ovarian estradiol (E2) production and follicular growth up to the terminal stage. A transient elevation in circulating FSH and E2 levels occurs shortly after birth. But what could be the action of FSH on the ovary during this period, and in particular how it stimulates ovarian steroidogenesis without supporting terminal follicular maturation is intriguing. By experimentally manipulating FSH levels, we demonstrate in mice that the mid-infantile elevation in FSH is mandatory for E2 production by the immature ovary, but that it does not stimulate follicle growth. Importantly, FSH increases aromatase expression to stimulate E2 synthesis, however it becomes unable to induce cyclin D2, a major driver of granulosa cell proliferation. Besides, although FSH prematurely induces luteinizing hormone (LH) receptor expression in granulosa cells, LH pathway is not functional in these cells to induce their terminal differentiation. In line with these results, supplying infantile mice with a superovulation regimen exacerbates E2 production, but it does not stimulate the growth of follicles and it does not induce ovulation. Overall, our findings unveil a regulation whereby high postnatal FSH concentrations ensure the supply of E2 required for programming adult reproductive function without inducing follicular maturation before puberty.

Figure 1. High activity of the gonadotrope axis during the postnatal period.

(A,B) Serum levels of LH (A) and FSH (B) between 7–21 dpn and in cyclic mice at proestrus (PE), estrus (E) and diestrus 2 (D). LH and FSH levels were simultaneously assayed by Luminex assay in serum samples from 5 to 15 females/group. (C) Circulating levels of estradiol between 7–21 dpn and in cycling females at proestrus (PE), estrus (E) and diestrus 2 (D) as determined by GC-MS in 4–6 pooled (postnatal) or 3–6 (adult) mouse serum. Error bars indicate SEM. Different letters indicate significant differences (P < 0.05) between ages during the postnatal period, as analyzed by one-way ANOVA. Statistical difference by Student-t test with *P < 0.05, **P < 0.01, ***P < 0.001 between 14 dpn females and a given stage of the estrous cycle. NS, not significant. (D,E) Assessment of folliculogenesis and steroidogenesis in a 14-dpn mouse ovary by in situ hybridization of Amh or Cyp19a1 mRNAs (purple color digitally converted into green), respectively, and immunodetection of fibronectin (FN, in red) on the same ovarian section. (E) Immunohistochemistry for AMH or aromatase (brown staining) in a human ovary at 2 months counterstained by hematoxylin (blue color). Stars (*) show large follicular cysts. Rectangles within left panels are enlarged to show AMH- or aromatase-positive preantral/antral follicles (middle panels) and negative follicular cysts (right panels). Scale bars: 100 μm.

Figure 2. The high levels of gonadotropins stimulate E2 biosynthesis but have no effect on follicle growth during the infantile period.

(A) Schematic representation of the procedure used to experimentally decrease FSH levels between 12 and 14 dpn in mice. (B and C) Circulating LH (B) and FSH (C) levels in females treated with saline (−), Ganirelix (+) or Ganirelix plus eCG (eCG). Gonadotropins were simultaneously assayed in the serum from 9 females/group. (D) Circulating E2 levels in 5–6 pooled serum in females treated with saline (−), Ganirelix (+) or Ganirelix plus eCG (eCG), as determined by GC-MS. (E,F) Morphometric analyses of preantral/early antral follicle number (left) and size (right) (E) performed on histological sections of hematoxylin/eosin stained ovarian sections (F) of females treated with saline (−), Ganirelix (+) or Ganirelix plus eCG (eCG) from 3–4 ovaries/group. (G) Schematic view of the superovulation procedure. (H) Effect of the superovulation treatment (eCG/hCG) on circulating E2 levels. (I,J) Effects of the superovulation treatment on follicular growth as shown by fibronectin immunodetection (red) to visualize follicle morphology (I) and morphometric analyses of antral follicle number and size (J) from at least 3 ovaries/group. In F and I, micrographs are representative of the observations performed on 3 ovaries/group. Scale bars: 100 μm. In E and J, morphometric analyses were performed by ImageJ, as described in Materials and Methods.

Figure 3. Distinct regulatory action of FSH on Cyp19a1 and Ccnd2 expression in infantile ovaries.

(A) Relative intra-ovarian contents of both Cyp19a1 and Ccnd2 mRNAs and proteins (aromatase and cyclin D2, respectively) in 7–21 dpn mice. The relative abundance of mRNAs was determined by RT-qPCR and normalized to the mRNA levels of Hprt (5–7 ovaries/group). Aromatase and CCND2 protein abundance was analyzed by Western blots, with GAPDH as a loading control. Representative immunoblots are shown. Stars show non-specific bands. (B) Merged images of in situ hybridization assays showing Cyp19a1 mRNAs (purple color digitally converted into green color), and immunolabeled fibronectin (FN, red) and immunofluorescence study of CCND2 in the ovaries of females treated with saline (−), Ganirelix (+) or Ganirelix plus eCG (eCG). Dashed lines represent the outlines of growing follicles located in the core of the ovary. (C) Relative intra-ovarian contents of both Cyp19a1 and Ccnd2 mRNAs and proteins (aromatase and cyclin D2, respectively) in females treated with saline (−), Ganirelix (+) or Ganirelix plus eCG (eCG). Data are the mean ± SEM. The relative abundance of mRNAs was determined by RT-qPCR after normalization to the levels of Hprt mRNAs (5–8 ovaries/group). Aromatase and CCND2 protein abundance was analyzed by Western blots with GAPDH as a loading control. Representative immunoblots are shown. (D) Ovarian immuno-detection of phosphorylated histone H3 (p-HH3) and of BrdU, and quantification of the mean number of p-HH3 positive granulosa cells per cross-sectioned preantral/early antral follicles of females treated with saline (−), Ganirelix (+) or Ganirelix plus eCG (eCG). Dashed lines represent outlines of follicles. Arrowheads show positive granulosa cells. (E) Schematic representation of the experimental set-up for ovarian cultures. (F) Relative intra-ovarian content of both Cyp19a1 and Ccnd2 mRNAs in cultured postnatal ovaries (3–13 ovaries/group) in response to different concentrations of purified FSH. Data were obtained by RT-qPCR and normalized to the mRNA levels of Hprt. In graphs of (A,C,D and F) data are the mean ± SEM. Data were analyzed by one-way ANOVA, with distinct letters indicating significant differences between ages or groups (P < 0.05). Scale bars: 100 μm.

Figure 4. The elevated FSH levels of the infantile period stimulate the expression of LHCGR but probably not the activation of LHCGR signaling in granulosa cells.

(A) Merged images of in situ hybridization assay detecting Lhcgr mRNAs (purple color digitally converted into green color) with immunolabelled fibronectin (FN, red) in preantral/early antral follicles of females treated with saline (−), Ganirelix (+) or Ganirelix plus eCG (eCG). (B) Relative intra-ovarian contents of Lhcgr mRNAs in the ovaries of females treated with saline (−), Ganirelix (+) or Ganirelix plus eCG (eCG) (8–9 ovaries/group). (C) Relative intra-ovarian contents of Lhcgr mRNAs in cultured postnatal ovaries (3–10 ovaries/group) in response to different concentrations of purified FSH. (D) Relative intra-ovarian contents of Cyp19a1 mRNAs in the ovaries of females treated with Ganirelix (+), Ganirelix plus hCG (hCG), Ganirelix plus eCG (eCG), or Ganirelix plus hCG and eCG (hCG/eCG) (7–9 ovaries/group). (E) Circulating E2 levels in 5–6 pooled serum in females treated with Ganirelix (+), Ganirelix plus hCG (hCG), Ganirelix plus eCG (eCG), or Ganirelix plus hCG and eCG (hCG/eCG), as determined by GC-MS. Data in B, D and E were analyzed by one-way ANOVA, with distinct letters indicating significant differences between groups. Data in C were analyzed by Student t-test with **P < 0.01 in treated samples versus control group. Scale bars: 100 μm.

Figure 5. Proposed model of the effect of sustained postnatal FSH elevation on the infantile ovary.

FSH increases the expression of Cyp19a1 to activate E2 biosynthesis. However, at those high concentrations, FSH is unable to induce Ccnd2 expression in granulosa cells, and thereby it loses its stimulatory action on follicular growth. In addition, FSH stimulates Lhcgr expression in granulosa cells but LH signaling pathway remains inactive in contrast with that in preovulatory follicles from cyclic females. Thus, during postnatal life, the dramatic elevation in FSH ensures the supply in E2 for reproductive function programming without inducing excessive follicular growth and premature follicular maturation.