Novel role for anti-Müllerian hormone in the regulation of GnRH neuron excitability and hormone secretion

Irene Cimino, Filippo Casoni, Xinhuai Liu, Andrea Messina, Jyoti Parkash, Soazik P Jamin, Sophie Catteau-Jonard, Francis Collier, Marc Baroncini, Didier Dewailly, Pascal Pigny, Mel Prescott, Rebecca Campbell, Allan E Herbison, Vincent Prevot, Paolo Giacobini, Irene Cimino, Filippo Casoni, Xinhuai Liu, Andrea Messina, Jyoti Parkash, Soazik P Jamin, Sophie Catteau-Jonard, Francis Collier, Marc Baroncini, Didier Dewailly, Pascal Pigny, Mel Prescott, Rebecca Campbell, Allan E Herbison, Vincent Prevot, Paolo Giacobini

Abstract

Anti-Müllerian hormone (AMH) plays crucial roles in sexual differentiation and gonadal functions. However, the possible extragonadal effects of AMH on the hypothalamic-pituitary-gonadal axis remain unexplored. Here we demonstrate that a significant subset of GnRH neurons both in mice and humans express the AMH receptor, and that AMH potently activates the GnRH neuron firing in mice. Combining in vivo and in vitro experiments, we show that AMH increases GnRH-dependent LH pulsatility and secretion, supporting a central action of AMH on GnRH neurons. Increased LH pulsatility is an important pathophysiological feature in many cases of polycystic ovary syndrome (PCOS), the most common cause of female infertility, in which circulating AMH levels are also often elevated. However, the origin of this dysregulation remains unknown. Our findings raise the intriguing hypothesis that AMH-dependent regulation of GnRH release could be involved in the pathophysiology of fertility and could hold therapeutic potential for treating PCOS.

Figures

Figure 1. Specificity tests of anti-AMHR2 antibody…
Figure 1. Specificity tests of anti-AMHR2 antibody and identification of AMHR2-expressing cells in the adult mouse brain.
(a–p) Coronal sections immunolabelled with the indicated antibodies, from AMHR2::Cre+/+ (AMHR2-wt, number of immunostained P120 female brains, n=5) and AMHR2::Cre −/− (AMHR2-null, number of immunostained P120 female brains, n=5). AMHR2 expression was also analysed in AMHR2::Cre+/−;Z/EG reporter line (h; number of immunostained P60 female brains, n=3). The vector of Z/EG mouse line was designed to provide lacZ expression before Cre excision and EGFP expression after Cre excision and is referred to as Z/EG (lacZ/EGFP). AMHR2 expression was found in the cortex (a,i,m), dentate gyrus of the hippocampus (a,k,o), organum vasculosum of the lamina terminalis (OVLT; c) hypothalamic median eminence (me) and dorsomedial hypothamaus (DMH) arcuate nucleus (Arc; e,f,h). Moreover, AMHR2-expressing cells were found in hypothalamic tanycytes (t) lining the third ventricle (3V) and endothelial (EC) cells (f,h). Brains of AMHR2-null mice were totally depleted of AMHR2 immunoreactivity when using an antibody against an intracellular epitope of the AMHR2 (b,d,g,j,l) or an antibody against the extracellular region of the receptor (n,p). Experiments were replicated at least three times. Scale bars, (a,b) 200 μm; (c–e,g) 100 μm; (f,h) 25 μm; (i–p) 40 μm (inset in k and o). DAPI, 4′,6-diamidino-2-phenylindole.
Figure 2. AMHR2 is expressed in mouse…
Figure 2. AMHR2 is expressed in mouse and human GnRH neurons.
(a–c) Confocal representative photomicrographs showing GnRH and AMHR2 immunoreactivity in sagittal sections of the E12.5 embryonic nasal compartment (number of immunostained E12.5, n=5). Dashed line indicates the boundary between the vomeronasal organ (vno) and the nasal mesenchyme (nm). GnRH neurons migrating out of the vno express AMHR2. (d–i) Confocal photomicrographs showing GnRH (d,g) and AMHR2 (e,h) immunoreactivity in coronal sections through the hypothalamus of adult female mice (P90–120; number of immunostained brains, n=5). Images show widespread expression of AMHR2 at the level of the organum vasculosum of the lamina terminalis (OVLT). (g–i) High-magnifications images of boxed area in d. Arrows point to GnRH neurons expressing AMHR2. (j–m) Representative sagittal section of a human fetus at 9 weeks LMP, immunolabelled for GnRH and AMHR2 (number of immunostained fetuses, n=3). At this developmental stage, the majority of GnRH neurons are still located in the nasal region, at the beginning of their migratory path. Double immunofluorescence shows co-expression of these antigens in the same migratory neurons. Experiments were replicated at least three times. cp, cribriform plate; fb, forebrain; oe, olfactory epithelium; vfb, ventral forebrain. (n–p) Representative coronal section of an adult hypothalamus double labelled for GnRH and AMHR2. Human hypothalami were obtained between 24 and 36 h post mortem from two autopsied individuals: a 20-year-old female and a 72-year-old male subject; 17 out of 29 and 20 out of 35 GnRH neurons exhibited AMHR2 immunoreactivity, respectively (analyses have been performed in eight consecutive 16-μm thick coronal sections for each individual). Scale bars, (a–c) 10 μm; (d–f) 100 μm; (g–i) 20 μm, (j) 100 μm, (k–m) 40 μm, (n–p) 5 μm.
Figure 3. AMH receptor transcript expression in…
Figure 3. AMH receptor transcript expression in GnRH neurons.
(a) Schematic summarizing the isolation of GnRH-GFP cells. GFP-positive GnRH neurons were isolated by fluorescence-activated cell sorting from the nasal region of E12.5 embryos (n=3) and from the hypothalamic preoptic area of postnatal (females P12, n=3) and adult (females P90, n=3) female mice. (b) Real-time PCR analysis of expression levels of AMHR2 messenger RNA (mRNA) in GnRH cells sorted at E12.5, P12 and P90. AMHR2 transcript expression was detectable in GnRH neurons at all stages, although it reached its highest level at P90. Values are expressed relative to values at E12.5, set at 1, and shown as means±s.e.m. One-way analysis of variance, F(2,8)=7.6, P=0.02. Fisher's least significant difference post hoc test, *: P<0.05 between P90–P120 and E12.5 and P90–P120 versus P12. (c) Relative mRNA expression of AMH type-I receptors (Alk2, Alk3 and Alk6) in GnRH neurons isolated from the preoptic region of adult female mice (females P90, n=3). All receptor transcripts were detectable in adult GnRH neurons. (d–f) Nasal explants were generated from E11.5 GnRH-GFP embryos (n=7) and maintained in serum-free media for 7 days before immunohistochemistry procedures. Immunostaining for AMHR2 show co-localization with fully differentiated GnRH-GFP neurons. Experiments were replicated three times. LS, lateral septum; MS, medial septum; DBB, diagonal band of Broca; LSI, lateral septal nucleus, intermediate part; LSV, lateral septal nucleus, ventral part; OVLT, organum vasculosum of the lamina terminalis. Scale bars, (d–f) 10 μm.
Figure 4. AMH increases GnRH neuron firing…
Figure 4. AMH increases GnRH neuron firing and hormone secretion.
(a,b) Cell-attached voltage recordings of GnRH neurons from GnRH::GFP male mice stimulated with AMH. (c,d) Cell-attached voltage recordings of GnRH neurons from GnRH::GFP diestrous mice. AAB (AP5, CNQX and GABAzin) application shows that AMH excitation of GnRH neurons is not dependent upon amino-acid transmission. (e) Whole-cell current recording of a GnRH neuron in the presence of AAB and tetrodotoxin (TTX). **Spontaneous synaptic currents blocked by AAB+TTX. Dots indicate truncated currents induced by the ramp voltage protocol. (f) Current–voltage plots before (black) and during AMH response (AMH, red). The ‘AMH current' (IAMH, green) is obtained by the subtraction of control from AMH current at ramp potentials (−120 to −20 mV for 1.5 s). AMH current with a reversal potential around −30 mV is replotted as an inset with an expanded current axis. (g) Schematics illustrating ME dissection and explant preparation. Hypothalamic MEs were microdissected from adult diestrous or ovariectomized (OVX) female rats. (h) Quantification of GnRH secretion from ME explants of P120 diestrous rats stimulated or not with 125 nM AMH (Die, n=4; Die+AMH, n=4). GnRH mean concentration±s.e.m. Unpaired Student's t-test, t(6)=−6.01, ***P<0.001. (i) MEs dissected from OVX rats (n=4 each group). One-way analysis of variance, F(3,15)=30.9, P<0.0001. ***P<0.0001, **P<0.001 Fisher's least significant difference post hoc test. All the experiments were replicated at least three times.
Figure 5. Intracerebroventricular (i.c.v.) administration of AMH…
Figure 5. Intracerebroventricular (i.c.v.) administration of AMH in vivo increases the LH secretion.
(a) Schematic depicting AMH injection into the lateral cerebral ventricle of diestrous mice. Trunk blood was collected from 3–4-month-old mice 15 or 30 min after the injection. (b) Following AMH administration (AMH 0.5 μM, n=3; AMH 1 μM, n=4; AMH 3 μM, n=6; saline, n=5), LH was measured. Values are expressed as means±s.e.m. One-way analysis of variance, F (3,19)=6.6, P=0.004. *P:<0.01, **P:<0.001, Fisher's least significant difference post hoc test. (c) Adult diestrous mice were injected with AMH 3 μM (n=20), or saline (n=7). Plasma LH secretion peaked at 15 min after injection and returned to basal levels 30 min later (n=7). AMH was injected i.c.v 2 h after an intraperitoneal injection of the GnRH antagonist (n=7). Treatment with an ALK receptor inhibitor prevented the AMH-dependent increase in LH secretion (n=6). One-way analysis of variance, F(4,47)=11.1, P<0.0001. *P<0.01, ***P<0.0001, Fisher's least significant difference post hoc test. (d) Schematic representation of AMH injection into the lateral cerebral ventricle of diestrous (3–4-month old) mice with 50 nM AMH or saline. Tail blood was collected every 10 min for 2 h and LH measured (n=7 control, n=8 AMH; e–g). Asterisks in e and f indicate LH pulses in two -representative saline- and AMH-treated mice. Values are represented as means±s.e.m. Unpaired Student's t-test, t(13)=−2.56, *P<0.05. Experiments were replicated three times. MS, medial septum; LSI, lateral septal nucleus, intermediate part; LSV, lateral septal nucleus, ventral part; OVLT, organum vasculosum of the lamina terminalis.
Figure 6. Schematic representation of the proposed…
Figure 6. Schematic representation of the proposed mechanism of action of AMH on GnRH neurons in normal and PCOS women.
In normal women of reproductive age, the levels of circulating AMH are low and do not significantly fluctuate over the menstrual cycle. GnRH neurons express AMHR2 as well as type-I AMH receptors (ALK 2/3/6). In women with PCOS, circulating AMH levels are two to three times higher than in normal women. We hypothesize that AMH could bypass the blood–brain barrier by passing through the fenestrated capillaries at the level of the organum vasculosum of the lamina terminalis (OVLT) and the median eminence and act directly on GnRH dendrites and terminals, respectively. In the median eminence, AMH could also act indirectly on GnRH neurons via tanycytes and vascular endothelial cells, which also express AMHR2, possibly contributing with other dysregulated factors to the increase in GnRH and LH pulsatilities. The altered ratio of LH to FSH is known to be responsible for the ovarian androgen production, partly explaining the two central diagnostic features of PCOS: hyperandrogenemia and hyperandrogenism (hirsutism). FSH, follicle-stimulating hormone; LH, luteinizing hormone.

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