FSH inhibits AMH to support ovarian estradiol synthesis in infantile mice

in Journal of Endocrinology
Correspondence should be addressed to C J Guigon: celine.guigon@univ-paris-diderot.fr

Anti-Müllerian hormone (AMH) regulates ovarian function in cyclic females, notably by preventing premature follicle-stimulating hormone (FSH)-mediated follicular growth and steroidogenesis. Its expression in growing follicles is controlled by FSH and by estradiol (E2). In infantile females, there is a transient increase in the activity of the gonadotrope axis, as reflected by elevated levels of both gonadotropins and E2. We previously demonstrated in mice that elevated FSH concentrations are necessary to induce E2 production by preantral/early antral follicles through the stimulation of aromatase expression without supporting their growth. However, whether this action of FSH could involve AMH is unknown. Here, we show that Amh mRNA and protein abundance and serum AMH levels are elevated in infantile mouse females, compared with those in adults. By experimentally manipulating FSH and E2 levels in infantile mice, we demonstrate that high FSH concentrations lower Amh expression specifically in preantral/early antral follicles, whereas E2 has no effect. Importantly, treatment of infantile ovaries in organotypic cultures with AMH decreases FSH-mediated expression of Cyp19a1 aromatase, but it does not alter the expression of cyclin D2-mediating granulosa cell proliferation. Overall, our data indicate that the infantile elevation in FSH levels suppresses Amh expression in preantral/early antral follicles, thereby favoring Cyp19a1 aromatase expression and E2 production. Together with recent discoveries that AMH can act on both the hypothalamus and the pituitary to increase gonadotropin levels, this work suggests that AMH is a critical regulator of the gonadotrope axis during the infantile period, thereby contributing to adult reproductive function programming.

 

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    Ontogenesis of AMH expression in prepubertal female mice. (A) The relative intra-ovarian abundance of Amh transcripts in infantile (7–21 dpn), juvenile (27 dpn) and adult cycling females was determined by quantitative real-time RT-PCR and normalized to the mRNA levels of Hprt. The number of samples is indicated under parentheses. (B) AMH protein expression was analyzed in immature (7–27 dpn) and adult (A) ovaries by Western blot, with GAPDH used as a loading control. Representative immunoblots are shown. The star shows a non-specific upper band. (C) Serum AMH levels were measured by ELISA assay during the infantile (7–21 dpn) and the juvenile periods (27 dpn), and in adult cycling females (P, proestrus; E, estrus; D, diestrus) with the number of serum samples for each group shown under parentheses. (D) In situ hybridization with Amh probes (purple color digitally converted into green) and detection of fibronectin by immunofluorescence (red color) showing the distribution of Amh transcripts in the ovary during the infantile (8, 14 and 21 dpn) and the juvenile periods (27 dpn). Pictures in a’, b’, c’ and d’ are enlargement of the rectangles located on pictures a, b, c and d, respectively. Arrows show Amh-positive preantral/early antral follicles, whereas stars show Amh-negative preantral/antral or medium/large antral follicles. Triangles show large antral follicles expressing Amh in cumulus cells. Due to the large size of the ovaries at 21 and 27 dpn, the pictures in c and d are reconstitution obtained from microphotographs taken in different parts of the same ovarian section. (E) Co-detection of apoptotic cells by TUNEL assay (green color digitally converted into blue), Amh transcripts by in situ hybridization (purple color digitally converted into green) and fibronectin by immunofluorescence (red color) at 14 (a), 21 (b) and 27 (c and d) dpn. Arrows show apoptotic cells located either outside (a, b) or inside (c) follicles. In graphs, bars are the means ± s.e.m. Data were analyzed by one-way ANOVA (A) or Kruskal–Wallis (C) tests. Distinct letters indicate significant differences between ages. In (D) and (E), scale bars: 100 μm.

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    High FSH concentrations repress Amh expression in the first follicular waves. (A) Schematic representation of the procedure used to experimentally manipulate FSH levels between 12 and 14 dpn in mice. (B) Relative intra-ovarian contents of Fshr mRNA in the ovaries of infantile females treated with saline (controls), Ganirelix (+) or Ganirelix + eCG (+/eCG) determined by quantitative real-time RT-PCR after normalization by the levels of Hprt mRNAs. The number of used samples is indicated under parentheses. (C) Relative intra-ovarian contents of Amh mRNA in the ovaries of infantile females treated with saline (controls), Ganirelix (+) or Ganirelix + eCG (+/eCG) determined by quantitative real-time RT-PCR after normalization by the levels of Hprt mRNAs. The number of used samples is indicated under parentheses. (D) Distribution pattern of Amh mRNA by in situ hybridization with specific probes (purple color digitally converted into green) and fibronectin immunofluorescence (red color) in the ovaries of control or Ganirelix-treated mice supplemented or not with eCG in vivo. The area containing follicles of the first follicular waves is delimited by dotted lines. (E) Schematic representation of the procedure used for ovarian culture. (F) Effect of increasing FSH concentrations (50–500 ng/mL) on the relative intra-ovarian levels of Fshr mRNA in organotypic culture. Data were obtained by quantitative real-time RT-PCR and normalized by the levels of Hprt mRNAs. The number of used samples is indicated under parentheses. (G) Effect of increasing FSH concentrations (50–500 ng/mL) on the relative intra-ovarian levels of Amh mRNA in organotypic culture. Data were obtained by quantitative real-time RT-PCR and normalized by the levels of Hprt mRNAs. The number of used samples is indicated under parentheses. (H) Representative histological sections of ovaries cultured in control (CTR) or FSH (500 ng/mL)-supplemented medium. Right panels show enlargement of the largest follicle seen on the left panels. In graphs, bars are the means ± s.e.m. Data were analyzed by one-way ANOVA (B and G) or Kruskal–Wallis (C and F) tests. Distinct letters indicate significant differences between ages. In (D) and (H), scale bars: 100 μm.

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    Estradiol does not repress Amh expression in infantile ovaries. (A) Schematic representation of the procedure used to experimentally alter E2 levels between 12 and 14 dpn in mice. (B) Uterus weights in controls, Ganirelix (+) and Ganirelix + E2-treated (+/E2) female mice. The number of samples for each group is shown under parentheses. (C) Relative intra-ovarian abundance of Amh transcripts in controls, Ganirelix (+) and Ganirelix + E2-treated (+/E2) female mice determined by quantitative real-time RT-PCR after normalization by the levels of Hprt mRNAs. The number of samples for each group is shown under parentheses. (D) Distribution pattern of Amh mRNA by in situ hybridization with specific probes (purple color digitally converted into green) and fibronectin immunofluorescence (red color) in the ovaries of control or Ganirelix-treated mice supplemented or not with estradiol (E2) in vivo. The area containing follicles of the first follicular waves is delimited by dotted lines. (E) Relative intra-ovarian abundance of E2-target genes, i.e., Cyp19a1, Foxo1 and Glut1 transcripts, in controls, Ganirelix (+) and Ganirelix + E2-treated (+/E2) female mice, determined by quantitative real-time RT-PCR after normalization by the levels of Hprt mRNAs. The number of samples for each group is shown under parentheses. In graphs, bars are the means ± s.e.m. Data in (B), (C), and (E) were analyzed by Kruskal–Wallis non-parametric test, with distinct letters indicating significant differences between groups (P < 0.05). In (D), scale bars: 100 μm.

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    AMH represses the relative abundance of Cyp19a1 mRNA but not that of either Fshr or Ccnd2. (A) Schematic representation of the procedure used for ovarian culture. (B) Representative histological sections of ovaries cultured in FSH (500 ng/mL) or FSH+AMH-supplemented medium. The right panels show enlarged magnification of the largest follicles seen on the left panels. Scale bars: 100 μm. (C) Effect of FSH (500 ng/mL) and AMH (400 ng/mL) alone or combined, on Cyp19a1 (a), Ccnd2 (b), and Fshr (c) mRNA levels in cultured ovaries. Transcript levels were analyzed by quantitative real-time RT-PCR, and normalized by those of Hprt mRNAs. The number of samples for each group is shown under parentheses. (D) Aromatase and Cyclin D2 protein expression was analyzed in ovarian explants by Western blot, with GAPDH used as a loading control. Representative immunoblots are shown. The star shows an upper non-specific band. In graphs, bars are the means ± s.e.m. Data were analyzed by Kruskal–Wallis non-parametric test with *, P < 0.05; **, P < 0.01. NS, not significant.

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    Proposed model for the regulatory loop between FSH and AMH during the infantile period in the mouse. The high FSH levels would enhance FSH receptivity in preantral/small antral follicles of the first waves located in the center of the ovary, and decrease their expression of Amh. The loss of Amh expression in these follicles may facilitate FSH-induction of Cyp19a1 aromatase and thus the synthesis of E2 that is required for programming adult reproductive function. The production of AMH by subsequent follicular waves located in the ovarian periphery may target the hypothalamus and the pituitary (Cimino et al. 2016, Garrel et al. 2016) to increase LH and FSH levels, thereby leading to increased ovarian activity (see text for more explanations). A full-colour version of this figure is available at https://doi.org/10.1530/JOE-18-0313.

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