Anti-Müllerian hormone: a new actor of sexual dimorphism in pituitary gonadotrope activity before puberty

Abstract

Anti-Müllerian hormone (AMH) contributes to male sexual differentiation and acts on gonads of both sexes. Identification of AMH receptivity in both pituitary and brain has led to the intriguing idea that AMH participates to the hypothalamic-pituitary control of reproduction, however in vivo experimental evidence is still lacking. We show that AMH stimulates secretion and pituitary gene expression of the gonadotropin FSH in vivo in rats. AMH action is sex-dependent, being restricted to females and occurring before puberty. Accordingly, we report higher levels of pituitary AMH receptor transcripts in immature females. We show that AMH is functionally coupled to the Smad pathway in LβT2 gonadotrope cells and dose-dependently increases Fshb transcript levels. Furthermore, AMH was shown to establish complex interrelations with canonical FSH regulators as it cooperates with activin to induce Fshb expression whereas it reduces BMP2 action. We report that GnRH interferes with AMH by decreasing AMH receptivity in vivo in females. Moreover, AMH specifically regulates FSH and not LH, indicating that AMH is a factor contributing to the differential regulation of gonadotropins. Overall, our study uncovers a new role for AMH in regulating gonadotrope function and suggests that AMH participates in the postnatal elevation of FSH secretion in females.

Figure 1. Amhr2 is highly expressed in gonadotrope cell lineage and AMH recruits the Smad signaling pathway in LβT2 cells.

Quantification of Amhr2 transcripts in representative cell lines of the different anterior pituitary cell types. Levels of Amhr2 mRNA were measured by Taqman real-time qPCR in the gonadotrope αT3-1 and LβT2, thyrotrope TαT-1, corticotrope AtT20 and somatolactotrope GH3 cell lines. Data are the mean ± SEM of 3 independent cultures. (b) Time course of AMH precursor (AMHp) and cleaved AMH (AMH) effects on Smad1/5/8 phosphorylation in the gonadotrope LβT2 cell line. Cells were treated for 4 or 24 h with 2.5 μg/ml (17.5 nM) AMHp or AMH (concentrations conventionally used in AMH in vitro studies) and Phospho-Smad1/5/8 (P-Smad1/5/8) protein level was evaluated by immunoblotting. Total Smad1 was used as a loading control. (c) No effect of AMH on Smad1/5/8 phosphorylation in the corticotrope AtT20 and the somatotrope GH3 cell lines. Cells were treated for 4 h with 2.5 μg/ml AMH and Phospho-Smad1/5/8 (P-Smad1/5/8) protein level was evaluated as described above. The LβT2 cell line is used as a positive control.

Figure 2. Fshb and Inhbb are two target genes of AMH in gonadotrope LβT2 cells.

(a) AMH selectively stimulates Fshb and Inhbb expression in LβT2 cells. Cells were stimulated with 2.5 μg/ml AMH for 4 h and transcripts levels were determined by real time qPCR. (b) Time course of AMH precursor and AMH effects on Fshb and inhbb transcripts levels. LβT2 cells were treated for 4 or 24 h with 2.5 μg/ml AMH precursor or AMH. (c) Dose-response of AMH on Fshb transcript levels. LβT2 cells were treated for 4 h with increasing concentrations (0.06 to 10 μg/ml) of AMH precursor or AMH. Fshb mRNA levels were expressed as percentage of control. Data are the mean ± SEM of 3 to 10 experiments. *P ≤ 0.05; **P ≤ 0.001 compared with untreated cells (control).

Figure 3. AMH potentiates activin signaling and activin effect on Fshb expression in LβT2 cells.

LβT2 cells were stimulated for 4 h with 2.5 μg/ml AMH alone or combined with 10 ng/ml activin A or 20 ng/ml BMP2. (a) AMH enhances activin and counteracts BMP2 stimulation of Fshb expression. Fshb and Lhb mRNA levels were determined by real-time qPCR and expressed as the mean ± SEM of at least 3 independent experiments. (b) AMH improves activin coupling to the Smad2/3 pathway. P-Smad2 protein level was evaluated by immunoblotting and normalized with total Smad2. *P ≤ 0.05; **P ≤ 0.01 compared with control cells. a, P ≤ 0.05 between AMH and AMH+activin treatments. b, P ≤ 0.05 between AMH and AMH+BMP2 treatments.

Figure 4. GnRH down-regulates AMH receptivity in vitro in LβT2 cells and in vivo in pituitary of female rats at pnd 18.

(a) GnRH inhibits AMH signaling. LβT2 cells were stimulated for 24 h with 2.5 μg/ml AMH, 10 nM GnRHa or with a combination of both hormones. P-Smad1/5/8 protein level was evaluated by immunoblotting and normalized with total Smad1. *P ≤ 0.05 compared with AMH-stimulated cells. (b) Combined effects of GnRH and AMH on Fshb expression. Cells were incubated with 2.5 μg/ml AMH, 10 nM GnRHa or with both hormones for 24 h. Fshb mRNA levels were analyzed by real-time qPCR and expressed as percentage of control levels. *P ≤ 0.05 ; **P ≤ 0.01 compared with control cells. (c) GnRH down-regulates Amhr2 expression. Cells were incubated with 10 nM GnRHa, 10 ng/ml activin A or 20 ng/ml BMP2 for 4 and 24 h. Amhr2 mRNA levels were determined by real-time qPCR and expressed as percentage of control cells. Results are the mean ± SEM of 8 independent experiments. *P ≤ 0.05 compared with control cells. (d) GnRH decreases AMHR2 protein level. Cells were treated for 24 h with 10 nM GnRHa and AMHR2 protein level was determined by immunoblotting after normalization with vinculin. Results are the mean ± SEM of 4 independent experiments. *P ≤ 0.05 compared with control cells. (e) GnRH down-regulates Amhr2 expression in pituitary of female rats at pnd 18. Male and female rats were injected subcutaneously at pnd 17 with 100 μl of saline solution containing or not 0.1 μg of GnRHa. Anterior pituitary Amhr2 expression was determined 24 h after injection by Taqman real time qPCR. Each value is a mean ± SEM of 8 to 14 rats. *P ≤ 0.05 compared to control rats; a, P ≤ 0.01 compared to female control rats.

Figure 5. Postnatal ontogeny of Amh and Amhr2 expression in anterior pituitary of male and female rats.

Anterior pituitary of male and female Wistar rats were collected at pnd 4, 8, 11, 18, 32 or 70. (a) Ontogeny of Amh and Amhr2 expression in rat pituitary. Transcript levels were measured by Taqman real-time qPCR and expressed as the number of Amh or Amhr2 mRNA copies/μg total RNA. ND: non detectable. (b) Concomitant pituitary expression of gonadotropin subunit transcripts. Levels of Fshb, Lhb and Cga mRNA (arbitrary units) were measured throughout the same developmental period. Each value is a mean ± SEM of 6 to 13 rats. *P ≤ 0.05; **P ≤ 0.01 compared to age-matched male rats.

Figure 6. AMH stimulates FSH secretion and pituitary Fshb expression, in vivo, in female but not in male rats at postnatal day 18.

Male and female rats were injected intraperitoneally at pnd 17 with 100 μl of saline solution containing or not 10 μg of AMH precursor (a conventionally used concentration in AMH in vivo studies). Serum LH and FSH secretion (a) and gonadotropin subunits gene expression (b), were determined 18 h after injection. Each value is a mean ± SEM of 13 rats. *P ≤ 0.05; **P ≤ 0.01 compared to control rats.