# Preventing and correcting polycystic ovary syndrome by targeting anti-Müllerian hormone signaling in minipuberty and adulthood in mice.

Ludovica Cotellessa, V. Sobrino, Silva Mauro, Maxime Delit, Hélène Maitre, Emilie Caron, Gaetan Ternier, N. da Silva Lima, Tori Lhomme, F. Giton, et al.

► To cite this version:

Ludovica Cotellessa, V. Sobrino, Silva Mauro, Maxime Delit, Hélène Maitre, et al. Preventing and correcting polycystic ovary syndrome by targeting anti-Müllerian hormone signaling in minipuberty and adulthood in mice. Cell Metabolism, 2025, Cell Metabolism, 37 (6), pp.1260-1276. 10.1016/j.cmet.2025.03.013. hal-05216353

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# Cell Metabolism

Preventing and correcting polycystic ovary syndrome by targeting anti-Müllerian hormone signaling in minipuberty and adulthood in mice

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Ludovica Cotellessa,
Veronica Sobrino, Mauro S.B. Silva, ...,
Manuel Tena-Sempere, Vincent Prevot,
Paolo Giacobini

## Correspondence

paolo.giacobini@inserm.fr

## In brief

Polycystic ovary syndrome (PCOS) is the most common female reproductive and metabolic disorder. PCOS treatment is limited to symptom management. Here, Cotellessa et al. showed that high levels of anti-Müllerian hormone (AMH) during minipuberty may contribute to PCOS development and discovered that blocking AMH's effects in mice prevented PCOS-like symptoms.

## Highlights

* PCOS-like mice have elevated levels of AMH during minipuberty

* Exposure to high AMH during minipuberty in mice causes PCOS-like defects

* An AMHR2-neutralizing antibody prevents PCOS-like defects when given at minipuberty

* An AMHR2-neutralizing antibody alleviates PCOS-like defects when given at adulthood

<figure><img src="image_3.png" /></figure>
Article

# Preventing and correcting polycystic ovary syndrome by targeting anti-Müllerian hormone signaling in minipuberty and adulthood in mice

Ludovica Cotellessa,<fnref n="1" /> Veronica Sobrino,<fnref n="2" /> Mauro S.B. Silva,<fnref n="1" /> Maxime Delit,<fnref n="1" /> Hélène Maitre,<fnref n="1" /> Emilie Caron,<fnref n="1" />

Gaëtan Ternier,<fnref n="1" /> Natalia da Silva Lima,<fnref n="3" /> Tori Lhomme,<fnref n="1" /> Frank Giton,<fnref n="4" /> Andrea Sorrentino,<fnref n="5" />,<fnref n="6" /> Laura Carraresi,<fnref n="5" />,<fnref n="7" />

Giovanna Di Nardo,<fnref n="8" /> Ruben Nogueiras,<fnref n="3" /> Manuel Tena-Sempere,<fnref n="2" /> Vincent Prevot,<fnref n="1" /> and Paolo Giacobini<fnref n="1" />,<fnref n="9" />,\*

## SUMMARY

Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women, causes significant reproductive and metabolic comorbidities, with no current cure. Gestational androgen and anti-Müllerian hormone (AMH) excess are linked to PCOS, and prenatal aberrant exposure to these hormones induces PCOS-like traits in animal models. However, whether the AMH effects on PCOS programming could extend to early postnatal life remains unknown. Clinical observations showed higher AMH levels during minipuberty in infants of mothers with PCOS, but whether this contributes to PCOS development is uncertain. Here, we show that exposure to high AMH levels during minipuberty in mice causes PCOS-like reproductive and metabolic defects in both sexes. A neutralizing antibody targeting AMH receptor 2 (AMHR2) prevented these defects when administered during minipuberty and alleviated symptoms when given in adulthood. These findings highlight the causal role of elevated AMH in PCOS and suggest AMHR2-targeting therapy as a potential preventive or curative approach.

## INTRODUCTION

Polycystic ovary syndrome (PCOS) is a highly prevalent endocrine condition, affecting up to 13% of women of reproductive age worldwide, and is characterized by a range of endocrine, reproductive, metabolic, and psychological abnormalities.<fnref n="1" />,<fnref n="2" />

The origin of PCOS is largely unknown, leaving treatment limited to symptom management without a cure.<fnref n="3" /> Thus, there is an urgent need to design effective therapeutic strategies aimed at preventing, curing, and/or alleviating the long-term health-related consequences of PCOS. The international PCOS guideline<fnref n="2" /> endorses the use of the Rotterdam criteria for diagnosis, requiring two of these features: hyperandrogenism, irregular cycles (oligo-anovulation), and polycystic ovarian morphology or elevated anti-Müllerian hormone (AMH). Hyperandrogenism is a common feature among women with PCOS, affecting nearly 80% of women diagnosed using the Rotterdam definition.<fnref n="4" /> The aberrant PCOS hormonal milieu inhibits follicular maturation,

causing an excess of small antral follicles and ovulatory disturbance.<fnref n="5" /> Pre-antral and small antral production of AMH by granulosa cells is enhanced in the PCOS ovary, with serum concentrations of AMH two to three times above normal.<fnref n="6" />,<fnref n="7" /> Indeed, circulating levels of AMH are elevated in >90% of women with PCOS, reliably predicting PCOM.<fnref n="8" />

AMH, a transforming growth factor (TGF)-beta family member,<fnref n="9" /> is produced by ovarian granulosa cells and released into circulation, suggesting hormonal functions in tissues expressing the AMH receptor 2 (AMHR2).¹⁰⁻¹²

PCOS has a strong heritable component,¹³⁻¹⁷ which might relate to interactions between genetic and epigenetic factors,¹⁸ the latter induced by the aberrant maternal-fetal environment in PCOS. Among factors that have been proposed to predispose to PCOS adult manifestation is exposure during fetal life to excess of maternal androgens and/or AMH.¹⁷,¹⁹ Consistently, women with PCOS retain higher circulating levels of both hormones during pregnancy than pregnant women without

<footnote n="1">¹University Lille, Inserm, CHU Lille, Lille Neuroscience & Cognition, UMR-S 1172, Lille, France</footnote>

<footnote n="2">²Maimonides Biomedical Research Institute of Cordoba (IMIBIC) and Department of Cell Biology, Physiology and Immunology, University of Cordoba, 14004 Cordoba, Spain</footnote>

<footnote n="3">³CIMUS, University of Santiago de Compostela, Instituto de Investigación Sanitaria, Santiago de Compostela, Spain</footnote>

<footnote n="4">⁴IMRB - Inserm U955, Faculté de Santé, Créteil, France</footnote>

<footnote n="5">⁵DIVAL Toscana Srl, Sesto Fiorentino, Florence, Italy</footnote>

<footnote n="6">⁶Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy</footnote>

<footnote n="7">⁷Department of Neurosciences, Psychology, Drug Research and Child Health, University of Florence, Florence, Italy</footnote>

<footnote n="8">⁸Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy</footnote>

<footnote n="9">⁹Lead contact</footnote>

<footnote>\*Correspondence: paolo.giacobini@inserm.fr</footnote>

<footnote>https://doi.org/10.1016/j.cmet.2025.03.013</footnote>
PCOS.<sup>5,20</sup> To model these clinical findings, we previously developed a preclinical PCOS mouse model (prenatal AMH [PAMH]) based on late-gestation AMH exposure,<sup>21</sup> replicating key reproductive, neuroendocrine, and metabolic traits of human PCOS.<sup>19,21,22</sup> However, the impact of AMH on early postnatal PCOS programming remains unknown. Interestingly, clinical studies and recent meta-analyses have shown that AMH levels are higher in daughters and sons born to mothers with PCOS from minipuberty until the peripubertal period compared with daughters and sons born to women without PCOS.<sup>13,23-25</sup> Minipuberty refers to the transient gonad-independent activity of the hypothalamic-pituitary-gonadal (HPG) axis that occurs in both boys and girls shortly after birth.<sup>26</sup> According to preclinical data, minipuberty could play an important role in the maturation of the HPG axis and reproductive function in both sexes.<sup>27,28</sup>

Herein, we developed an animal model mimicking PCOS-related reproductive and metabolic abnormalities. Excess AMH exposure during minipuberty programmed females to develop PCOS-like phenotypes and males to exhibit metabolic features in adulthood. Blocking AMH signaling with an anti-AMHR2 antibody during this period prevented the manifestation of these defects and their transmission. Chronic anti-AMHR2 treatment in adulthood corrected the neuroendocrine and reproductive alterations linked to PCOS.

## RESULTS

### AMH treatment during minipuberty in mice triggers the neuroendocrine and reproductive disturbances of PCOS

We first assessed whether an established PCOS-like mouse model, created by prenatal treatment with high AMH (PAMH model),<sup>19,21</sup> which displays the major cardinal neuroendocrine,<sup>12,21</sup> reproductive,<sup>21</sup> and metabolic PCOS defects,<sup>19</sup> could also mimic the elevated AMH levels observed in daughters of women with PCOS during minipuberty.<sup>13,23,24</sup> In female rodents, minipuberty starts the second week of postnatal life,<sup>27-29</sup> peaking at P12 and concluding before the emergence of the first external signs of sexual maturation (around P30).<sup>28</sup> We thus measured AMH in female PAMH mice and controls at minipuberty. Our findings showed that PAMH mice had elevated AMH levels compared with controls during this critical early developmental stage (Figures S1A and S1B). We also assessed plasma luteinizing hormone (LH) levels, and we did not observe any differences between the two groups (Figure S1B).

To determine whether elevated AMH levels during infancy in offspring of women with PCOS are a bystander condition or a driving force behind the condition, we generated another animal model by exposing female mice to exogenous AMH during minipuberty and by assessing whether this exposure could lead to PCOS defects later in life. We administered phosphate-buffered saline (PBS: minipuberty-control mice [mCNTR]) or human recombinant AMHC (minipuberty-AMH mice [mAMH]) to infantile mice via intraperitoneal (i.p.) daily injection from P12 to P14, and we examined the neuroendocrine reproductive and metabolic characteristics of these animals at adulthood (Figure 1A).

At P21, we collected the blood from mCNTR and mAMH animals, and we used mass spectrometry coupled with gas chromatography (GC-MS) to measure gonadal steroids in these animals. At this postnatal stage, circulating androgens (testos-

terone and androstenedione [delta4-Dione]), estradiol (E2), and progesterone (P4) levels did not display any difference between the two groups of animals (Figure S2A). Since AMH levels during minipuberty have been documented to be higher also in boys born to mothers with PCOS as compared with boys born to women without PCOS,<sup>25</sup> we also assessed gonadal steroid levels in males treated with AMH or PBS during minipuberty. We found that mAMH P21 males did not show any difference in circulating testosterone, dihydrotestosterone (DHT), or delta4-Dione as compared with controls (Figure S2A). However, at this stage, mAMH males had significantly higher P4 levels than in mCNTR males (Figure S2A).

Vaginal opening is initiated by minipuberty-induced gonadal estrogen production, and the first estrus correlates with the first ovulation at puberty in rodents. Vaginal opening was significantly delayed by ~5 days in mAMH female mice compared with control females (Figure S2B). Similarly, the first estrus was significantly delayed in mAMH vs. mCNTR mice (Figure S2C). Male mAMH mice showed normal puberty timing (Figure S2D). Interestingly, mAMH mice of both sexes displayed a significant increase in body weight as compared with control animals from P21 to adulthood (P120; Figures S2E and S2F).

Next, we characterized the neuroendocrine and reproductive features of adult females exposed either to PBS or AMH during minipuberty. Similar to the clinical condition, mAMH mice exhibited signs of oligo-anovulation (Figures 1B and 1C), displaying prolonged time in diestrus and significantly less time in proestrus as compared with mCNTR females (Figure 1C), together with a significant reduction in the number of completed ovulatory cycles observed for 15 days as compared with controls (Figure 1D). In addition, similarly to the human clinical condition, mAMH female mice also experienced impaired fertility, as indicated by a significantly reduced litter size (Figure 1E). Ovarian histology of mAMH animals showed abnormalities consistent with their oligo-anovulatory phenotype, with the presence of fewer post-ovulation corpora lutea as compared with controls (Figures 1F and 1G). Circulating androgens (testosterone and delta4-Dione), measured at diestrus, were significantly elevated in mAMH mice compared with controls, whereas estradiol (E2) and progesterone (P4) levels did not display any difference between the two groups (Figure 1H). We then evaluated LH pulsatility by serial blood sampling in diestrous mice (Figure 1I) and found that mAMH animals had significantly higher mean LH levels (Figure 1J) and LH pulse frequency (Figure 1K), followed by a shortened pulse interval time (Figure 1L) than in controls.

We did not observe any difference in testicular morphology (Figure S2G), relative testis weight (normalized by body weight; Figure S2H), or circulating testosterone levels (Figure S2I) between control and mAMH males.

### AMH treatment during minipuberty triggers PCOS-like metabolic disturbances

We have previously shown that exposure to high PAMH leads to PCOS-like metabolic dysfunctions in female offspring, which progressively manifest with age (~5-6 months of postnatal life).<sup>19</sup> Herein, we investigated whether AMH exposure during minipuberty also promotes metabolic alterations in adulthood in both sexes. 6-month-old mAMH females displayed higher body weight and fat mass (Figures 2A and 2B) and lower lean mass
<figure><img src="image_4.png" /><figcaption>Figure 1. Exposure to high AMH during minipuberty induces PCOS-like reproductive features in female mice</figcaption></figure>

(A) Illustration illustrating the minipuberty-AMH model.

(B) Representative estrous cyclicity of mCNTR and mAMH mice during 15 consecutive days. D, diestrus; P, proestrus; E, estrus.

(C) Analysis of the percentage of the time spent in each estrous cycle stage over 15 days (mCNTR, *n* = 7; mAMH, *n* = 7; age: P60-P80 for both groups).

(D) Number of complete estrous cycles during 15 days (mCNTR, *n* = 7; mAMH, *n* = 7; age: P60-P80 for both groups).

(E) Number of pups per litter in mCNTR, *n* = 8 and mAMH, *n* = 7; age: P60-P120.

(F) Representative photomicrographs of ovaries stained with hematoxylin and eosin from adult (P60) mCNTR and mAMH mice (*n* = 3 each group).

(G) Mean number of corpora lutea (CL) in mCNTR, *n* = 5, and mAMH, *n* = 6 (age: P180 for both groups).

(H) Mean circulating levels of gonadal sex steroids in diestrous female mice measured by mass spectrometry coupled with gas chromatography (GC-MS). Testosterone, androstenedione (delta4-Dione), estradiol (E2), and progesterone (P4) were measured in P90 mCNTR and mAMH females (*n* = 6 for each animal group).

(I) LH pulsatility of diestrous mCNTR and mAMH mice (P60; *n* = 5 biological replicates). Histograms indicate the number of LH pulses per 2-h interval.

(J-L) (J) Mean LH, (K) number of LH pulses/hour, and (L) time (min) of LH pulse interval in adult (P60) diestrous females (L) (mCNTR, *n* = 5; mAMH, *n* = 5).

Data are presented as mean ± SD, ns, not significant, \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.005, \*\*\*\*p < 0.0001 (two-way ANOVA in (C), two-sided paired Mann-Whitney U test in (D), paired Student's t test in (E) and (J)-(L), or unpaired Student's t test in (G) and (H).

(Figure 2C) compared with controls. The percentage of free body fluids was comparable between all groups (Figure 2D). Glucose tolerance was lower in mAMH females than in controls following i.p. glucose administration (Figure 2E). Consistently, this was followed by exacerbated insulin release after glucose injection in mAMH animals (Figure 2F) and increased homeostasis model

assessment of insulin resistance (HOMA-IR) compared with control mice (Figure 2G). Using an indirect calorimetric system, we found that mAMH females displayed modest modifications in their feeding patterns, with a significant increase in food intake observed only during the first hours of the nocturnal active phase (Figure S3A). In addition, no difference was observed in
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Exposure to high AMH during minipuberty drives PCOS-like metabolic defects in female mice</figcaption></figure>

(A) Body weight of mCNTR (n = 10; 6 months old) and mAMH adult diestrous females (n = 10; 6 months old).

(B–D) Body composition of mCNTR (n = 7; 6 months old) and mAMH adult females (n = 7; 6 months old).

(E) Curve representing glycaemia (glucose levels) during a glucose tolerance test (GTT) in mCNTR (n = 6) and mAMH (n = 4) 6-month-old females.

(F) Serum insulin concentrations during the first 30 min of a GTT in mCNTR (n = 6) and mAMH (n = 4) 6-month-old females.

(G) Homeostasis model assessment of insulin resistance (HOMA-IR) index in mCNTR (n = 6) and mAMH (n = 4) 6-month-old females.

(H) Coronal section of the MBH containing the ARC and the median eminence (ME) in an adult female mouse (P90) showing *Pomc*, *Agrp1*, and *Esr1* transcripts, representative of 3 replicates.

(legend continued on next page)
respiratory exchange ratio (RER), locomotor activity, energy expenditure (EE), or in the Z rearing between the two groups of animals (Figures S3B and S3C). In males, we found that food intake was comparable in mAMH and mCNTR animals (Figure S3D). However, we found that weight gain observed in male mAMH mice (Figure S2F) was associated with an increased RER during the second part of the dark phase (Figure S3E) and decreased EE (Figure S3F). Locomotor activity and Z rearing were comparable between mAMH and control males (Figure S3F). Given the increased RER in mAMH male mice, indicative of increased consumption of carbohydrates over lipids to meet energy requirements,³⁰ we next examined lipid metabolism in these animals, and we found that the changes in RER and food-intake-independent body-weight gain in mAMH males appeared to be due to decreased fatty acid (FA) oxidation (Figure S3G). Comparable alterations as those detected in females, in terms of glucose tolerance, insulin release, and insulin resistance, were also detected in mAMH males (Figures S4A–S4C).

Because anorexigenic proopiomelanocortin (POMC) and orexi-
genic agouti-related peptide (AgRP) neurons located in the hypo-
thalamic arcuate nucleus (ARC) are key neuronal regulators of
food intake and energy homeostasis,³¹ we evaluated the expres-
sion of *Pomc* and *Argp1* in the ARC/mediobasal hypothalamus
(MBH) of control and mAMH females. We did not observe any dif-
ference between the groups of animals in *Pomc* and *Argp1* gene
expression in the ARC/MBH as revealed by RNAscope analyses
(Figure 2H) and by RT-qPCR experiments (Figure 2I).

We then investigated the expression of estrogen receptor alpha (*Esr1*) mRNA in the ARC/MBH in these animals, together with other genes whose expression might be altered in PCOS, namely kisspeptin (*Kiss1*), progesterone receptor (*Pgr*), and androgen receptor (*Ar*). Our results showed that mAMH females have significantly lower levels of *Esr1* expression in the ARC/MBH as compared with mCNTR females (Figures 2H and 2I). By contrast, *Kiss1*, *Pgr*, and *Ar* transcript expression was similar between the two animal groups (Figure 2I).

Since hyperandrogenism in PCOS patients often leads to obesity, characterized by increased subcutaneous and visceral adipose tissue,¹ we next focused on the morphology of the white adipose tissue (WAT) of these animals. mAMH females presented larger white adipocytes in the visceral fat (Figures 2J and 2K) and increased visceral fat mass compared with controls (Figure 2L), suggesting lipid accumulation in white adipocytes. Like mAMH females, also mAMH males presented increased visceral fat mass (Figure S4D) and larger white adipocytes in the visceral fat (Figures S4E and S4F) compared with controls.

Development and characterization of an AMHR2
blocking-function antibody

Considering the data presented, along with previous preclini-
cal¹² and clinical studies,⁶⁻⁸ it is biologically and clinically logical

to explore AMH inhibition as a potential preventive or therapeutic
strategy for PCOS. Here, we report the generation, structure,
and function of an epitope-specific AMHR2-blocking antibody
(Figure 3A).

Initially, a research investigation was undertaken to identify the amino acid sequences within the N-terminal region of AMHR2, exhibiting antigenic properties and significant homology across various mammalian species. Based on the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2,³² we identified one peptide sequence, GCESLHC DPSPRAHPSPGSTLFTCSCGT, as one of the most promising to produce antibodies with blocking-function activity. Indeed, this peptide presents 100% homology with the human and private sequences and 92% and 89% homology, respectively, for the rat and mouse AMHR2 sequences (Figure 3B). Moreover, by using Alphafold2, the peptide is predicted to fold in a 3D structure almost superimposable with the one adopted by the same region in the entire protein, with a root-mean-square deviation (RMSD) of 0.933 Å (Figures S5A and S5B). We then conducted a detailed mapping of the atomic interactions between the human peptide sequence and the ligand AMH. Using the crystal structure of human AMH as a template for our modeling (Figure 3C), we found it to have high homology with the structural model of murine AMH-AMHR2 (Figure 3D). In both human and murine AMHR2, the residues involved in hydrophobic contacts with the ligand are conserved (F62, I64, M76, and L106; Figures 3E and 3F). The residues forming hydrogen bonds and salt bridges, including R97, crucial non-covalent interactions that stabilize the binding of a ligand to its receptor, are also identical between the two species (Figures S5C–S5E). Next, we modeled the interaction of the antigenic AMHR2 peptide with human and murine AMH (Figures 3G and 3H). The modeled human peptide-AMH complex largely mirrors the binding mode of the murine AMH. These models indicate that the interactions stabilizing the binding between the ligand and the antigenic peptide occur at residues L106 and R97 of the peptide-AMH complex (Figures 3G and 3H).

The synthesized peptide was used to immunize mouse fe-
males and to generate a mouse monoclonal IgM anti-AMHR2
antibody, which we named Ha13, with a K<sub>D</sub> of 37.1 nM, suggest-
ing a relatively strong binding affinity between the ligand and its
receptor (Figure 3I). The protein sequences of variable domains
of the antibody are represented in Table S1.

Next, we performed RT-qPCR experiments to assess *Amhr2*
expression in the ovary, visceral WAT (vWAT), pancreas, and
liver of adult diestrous female mice. We confirmed expression
of *Amhr2* transcript in the ovarian tissue as well as in the
vWAT, with negligible levels in the liver and pancreas (Figure 3J).
As a prelude to the testing of AMHR2-Ab in mice, we then deter-
mined the specificity of Ha13 for the murine AMHR2 using west-
ern blot and immunofluorescence. Immunoblotting with Ha13

(I) Relative expression of *Esr1*, *Pomc*, *Argp1*, *Kiss1*, *Pgr*, and *Ar* mRNAs in the MBH of mCNTR and mAMH adult females (n = 4 animals per group, 6 months old).

(J) Representative images of histological haematoxylin and eosin staining of adipose tissue of the vWAT (intra-abdominal adipose tissue) in mCNTR and mAMH adult diestrous females.

(K) Mean adipocyte area in mCNTR and mAMH adult females (n = 8 animals per group, 6 months old).

(L) Visceral fat mass (in mg) in mCNTR (n = 8 females, 6 months old) and mAMH adult diestrous females (n = 4 females, 6 months old).

Data are presented as mean ± SD, ns, not significant, \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.005, \*\*\*\*p < 0.0001 (paired Student's t test in A–D, two-way ANOVA in E and F, unpaired Student's t test [area under the curve, AUC] in E–G, K, and L, or a two-sided paired Mann-Whitney U test in I).
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>Figure 3. Generation and functional validation of an anti-AMHR2-neutralizing antibody</figcaption></figure>

(A) Schematic of the AMH-AMHR2 signaling pathway and anti-AMHR2 antibody targets.

(B) Sequence of the human AMHR2 N-terminal immunizing peptide with % homology across species. Conserved residues are green, and mismatches are red.

(legend continued on next page)
revealed a ~70-kDa specific band in protein extracts of the ovaries, while a very faint band was detectable in the liver and absent in the pancreas (Figure 3K). Immunofluorescence experiments using the Ha13 antibody confirmed the spatial distribution of AMHR2 in the granulosa cell layer of the ovarian follicles and in the ovarian stroma (Figures S6A and S6B), as well as in the vWAT (Figures S6C–S6E).

In order to further establish whether Ha13 antibody binds to AMHR2, we then used an immortalized *in vitro* model of murine ovarian granulosa cells, KK1, previously shown to be a suitable model to study AMH signaling as these cells retain AMHR2 expression as well as endogenous secretion of AMH.³³,³⁴ We immunostained KK1 cells using a previously validated anti-AMHR2 antibody¹⁰ and the Ha13 antibody directly conjugated with a fluorescent dye (ATTO-red). These experiments confirmed the co-localization between AMHR2 and Ha13 (Figures S6F–S6K).

After establishing the specificity of the antibody and its capacity to bind to AMHR2, we next performed *in vitro* functional experiments to assess the blocking-function capability of Ha13. Treatment of KK1 cells with two concentrations of Ha13 antibody, 50 and 100 µg/mL, significantly reduced phosphorylation of the transcription factor SMAD1/5 by 40% as compared with untreated cells (control, CNTR) or cells treated with the same concentrations of an IgM mock antibody (Figures 3L and 3M). These experiments revealed that the antibody successfully inhibited the autocrine stimulation of the specific AMH-AMHR2 signaling pathway.

We have previously shown that AMH potently induces the neuronal electrical activity of gonadotropin-releasing hormone (GnRH) neurons, which express AMHR2,¹⁰ and that PCOS-like mice generated by prenatal exposure to high AMH (PAMH mice) have a persistent hyperactivation of GnRH neurons as compared with controls.²¹ Therefore, to validate the efficacy of Ha13 to inhibit hypophysiotropic GnRH neurons, we first generated PAMH;Gnrh1-green fluorescent protein (gfp) mice.²¹ Subsequently, brain coronal slices were prepared for *in vitro* electrophysiological recordings of GnRH*rostral preoptic area (rPOA)* neurons from CNTR;Gnrh1-gfp and PAMH;Gnrh1-gfp adult females (Figures 3N and 3O). Ha13 administration significantly reduced GnRH neuronal firing rate in PAMH mice hypothalamic slices (Figures 3O and 3P). PAMH mice initially showed increased spontaneous action potential firing compared with controls,

which was normalized to control levels by Ha13 administration (Figure 3P). After washing out Ha13, firing rates returned to initial levels, confirming the antibody's specific inhibitory effect on GnRH neuronal activity (Figure 3P).

Overall, these data show that the Ha13 antibody specifically binds and inhibits AMHR2 in cell-based assays, both *in vitro* and *ex vivo*, a prelude to further preclinical testing.

## Ha13 treatment during minipuberty prevents PCOS-like reproductive and neuroendocrine defects

We then tested Ha13's therapeutic potential in PCOS-like animals. We administered daily i.p. injections of Ha13 or mouse IgM to infantile PAMH female mice from P10 to P15, aiming to prevent the development of PCOS traits in adulthood (Figure 4A).

We first analyzed estrous cyclicity of adult female mice over 15 days in control + vehicle, PAMH + vehicle, PAMH + IgM, and PAMH + Ha13 animals. The disrupted estrous cycle of PAMH animals was normalized by Ha13 treatment (Figures 4B–4D). Ovarian histology of PAMH animals showed abnormalities consistent with their oligo-anovulatory phenotype, with the presence of fewer post-ovulation corpora lutea as compared with control animals, whereas Ha13 treatment at minipuberty prevented these defects in adulthood (Figure 4E). In addition, Ha13 rescued the hyperandrogenic phenotype (Figures 4F and 4G), while it did not affect circulating levels of E2 and progesterone (Figures 4H and 4I). Finally, the AMHR2-Ab treatment over minipuberty prevented the PCOS-like pulsatile LH hypersecretion and the elevation in mean LH levels in adulthood (Figures 4J and 4K).

## Ha13 treatment during minipuberty prevents PCOS-like metabolic defects

We next characterized the effects of the Ha13 treatment on the metabolic phenotype of PAMH female mice. We previously reported that PAMH females manifest PCOS-like metabolic deformations by the age of P180.¹⁹ Here, we showed that Ha13 administration to PAMH mice during minipuberty prevented the appearance at P180 of exacerbated body weight and adiposity (Figure 5A), as well as glucose intolerance (Figure 5B) and insulin insensitivity (Figure 5C).

Previous evidence suggested that impaired brown adipose tissue (BAT)/WAT function and mass may contribute to the link between metabolic disturbances and reproductive issues

(C) Crystal structure of human AMH (cyan) bound to AMHR2 extracellular domain (yellow; PDB: 7L0J).

(D) Structural model of murine AMH (coral) bound to AMHR2 extracellular domain (purple).

(E and F) Close views of AMH-AMHR2 interaction regions highlighting conserved hydrophobic contacts in human and murine complexes.

(G) Close view of the interaction between the AMHR2 peptide (green) used for antibody production with human AMH.

(H) Equivalent interaction with murine AMH, highlighting key residues involved in complex formation.

(I) Schematic of the experimental workflow for generating the anti-AMHR2 antibody (Ha13).

(J) Relative Amhr2 mRNA expression in indicated tissues (n = 3 female mice, P90).

(K) Western blot experiments for Ha13 and actin were performed in ovaries, liver, and pancreas, dissected from three adult diestrous females (P90).

(L) KK1 mouse granulosa cell line was treated with or without IgM antibodies and Ha13 antibody concentrated at 50 and 100 µg/mL. Western blot experiments were performed using anti-phospho-Smad-1-5 and β-actin antibodies.

(M) Quantification of the ratio P-Smad-1-5 over β-actin.

(N) Illustration representing the whole-cell current-clamp recording set-up.

(O) Whole-cell current-clamp recording showing the spontaneous burst firing of a GnRH*POA* neuron recorded from a PAMH;GnRH-GFP female mouse (P60) before, during, and after bath application of Ha13 antibody. Bottom traces show expansions of the recording at the indicated time points 1 (min 1), 2 (min 11), and 3 (min 22).

(P) Firing rate (Hz) of GnRH*POA* neurons (n = 8–11 cells recorded from n = 3–5 mice per experimental group; P60).

Data are presented as mean ± SEM; ns, not significant; \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.0001; one-way ANOVA.
<figure><img src="image_8.png" /><figcaption>Figure 4. Ha13 treatment during minipuberty prevents PCOS-like reproductive and neuroendocrine defects</figcaption></figure>

(A) Illustration of the experimental design.

(B) Representative estrous cycle profiles of the 4 groups of animals (P60).

(C) Number of complete estrous cycles during 15 days (CNTR + veh, *n* = 11; PAMH + vehicle, *n* = 11; PAMH + IgM, *n* = 11; PAMH + Ha13, *n* = 8; age: P60 for both groups).

(D) Analysis of the percentage of the time spent in each estrous cycle stage over 15 days (CNTR + veh, *n* = 11; PAMH + vehicle, *n* = 11; PAMH + IgM, *n* = 11; PAMH + Ha13, *n* = 8; age: P60 for both groups).

(E) Representative photomicrographs of ovaries stained with hematoxylin and eosin from adult (P60) mice (*n* = 3 each group).

(F-I) Mean circulating levels of gonadal sex steroids in diestrous adult females measured by GC-MS. Testosterone, androstenedione (delta4-Dione), estradiol (E2), and progesterone (P4) were measured in P90 females (CNTR + veh, *n* = 10; PAMH + vehicle, *n* = 6; PAMH + IgM, *n* = 6; PAMH + Ha13, *n* = 10-11; age: P90 for both groups).

(J and K) Number of LH pulses/hour and (K) mean LH in adult (P90) diestrous females (CNTR + veh, *n* = 9; PAMH + vehicle, *n* = 6; PAMH + IgM, *n* = 6; PAMH + Ha13, *n* = 6).

Data are presented as mean ± SEM; ns, not significant, \*p < 0.05, \*\*p < 0.005, \*\*\*p < 0.0005, \*\*\*\*p < 0.0001 (two-way ANOVA in C and D, or one-way ANOVA in F-K).

in PCOS.³⁵⁻³⁸ We evaluated the histology of BAT and vWAT in control + vehicle, PAMH + vehicle, PAMH + IgM, and PAMH + Ha13 animals. PAMH females had more lipid accumulation in the BAT than in controls (Figures 5D and 5E). Because BAT whitening may induce tissue dysfunction and impaired thermogenesis, we measured the internal body temperature of these animals (Figure 5F). PAMH animals treated with either vehicle or IgM at minipuberty displayed a significant decrease in

body temperature at thermoneutrality as compared with control animals (Figure 5F). The rectal temperature of PAMH animals treated with Ha13 at minipuberty was normalized to control levels (Figure 5F).

PAMH females presented larger white adipocytes in the visceral fat and increased visceral fat mass compared with controls (Figures 5D, 5G, and 5H), suggesting that PAMH mice have hyperlipidemia and lipid accumulation in white adipocytes.
<figure><img src="image_9.png" /><figcaption>Figure 5. Ha13 treatment during minipuberty prevents PCOS-like metabolic defects</figcaption></figure>

(A) Body weight and body composition in CNTR, PAMH + vehicle, PAMH + IgM, PAMH + Ha13 mice (P180 females).

(B) GTT curve with corresponding AUC analysis (CNTR, n = 6; PAMH + vehicle, n = 5; PAMH + IgM, n = 8; PAMH + Ha13, n = 6).

(legend continued on next page)
Interestingly, these defects were also prevented by Ha13 treatment (Figures 5D, 5G, and 5H).

To shed some light on the adipose tissue dysfunctions detected in PCOS-like mice, we next investigated the expression of proteins involved in lipolysis or *de novo* lipogenesis in the visceral fat. Because PAMH animals injected with either saline or IgM did not show significant differences in any of the reproductive and metabolic parameters described above, for the subsequent analyses we only used three animal groups: CNTR (control + vehicle), PAMH (PAMH + vehicle), and PAMH + Ha13.

In agreement with increased adiposity and lipid accumulation in white adipocytes, PAMH mice showed a marked increase in protein levels of phosphorylated acetyl-coenzyme A (CoA) carboxylase (ACC) and FA synthase (FAS), enzymes crucial for *de novo* lipogenesis (Figures 5I and 5J). These defects were prevented by Ha13 (Figures 5I and 5J). The expression of the key lipolytic enzyme, hormone-sensitive lipase (HSL), as well as the phosphorylated form of HSL, did not change between the different groups (Figures 5I and 5J).

Overall, these results suggest that exposure to high PAMH promotes lipid accumulation by enhancing lipogenesis in the WAT of female offspring and increases lipid droplet depot in the BAT, ultimately affecting thermogenesis. Notably, administering Ha13 during minipuberty can counteract all PCOS-like metabolic defects.

## Ha13 treatment during minipuberty prevents intergenerational transmission of PCOS-like traits to female offspring

Given the strong heritability of PCOS and the well-documented transmission of the cardinal neuroendocrine and metabolic features observed in daughters and sons of women with PCOS,¹⁷,³⁹ as well as in PCOS-like animals,¹⁷,¹⁹,³⁹ we sought to test whether the AMHR2-Ab treatment of PAMH offspring during minipuberty could prevent the transmission of PCOS-like traits to the subsequent generation. Female PAMH offspring (F1) of gestating mice prenatally exposed to high AMH (F0) were injected intraperitoneally with either Ha13 or PBS from P10 to P15 and then mated at P60 with CNTR males to produce PAMH F2 offspring (Figure 6A). CNTR F2 female offspring was generated by breeding CNTR F1 females with CNTR F1 unrelated males (Figure 6A). F2 female offspring were then subjected to phenotypic testing. In agreement with our previous findings,¹⁹ at P60, PAMH F2 female offspring displayed hyperandrogenism (Figure 6B), oligo-anovulation

(Figures 6C and 6D), and a significant increase in LH pulsatility as compared with CNTR F2 females (Figure 6E). In addition, at P150 PAMH F2 females showed a significant elevation in body weight in comparison with CNTR F2 females (Figure 6F) and altered insulin sensitivity (Figure 6G). Treating PAMH F1 females with Ha13 during minipuberty prevented the transmission of all PCOS-like traits to the next generation (Figures 6B–6G).

## Biodistribution of Ha13

To identify the possible target tissues of Ha13 treatment during minipuberty, we investigated the biodistribution of the neutralizing antibody administered intravenously to female mice at P16. To this purpose, the antibody was labeled with a fluorescent molecule and retro-orbitally injected (Figure S7A). The animals were euthanized after 5 min, and the ovaries, visceral fat, pancreas, and brain were dissected and processed for immunostaining and confocal microscopy imaging. We detected a significant presence of fluorescently labeled Ha13 within the developing ovarian follicles and the stroma (Figures S7B and S7C) as well as in the adipocytes of visceral fat (Figure S7D). Consistent with the low expression levels of *Amhr2* mRNA (Figure 3J) and protein (Figure 3K) in the murine pancreas, the Ha13-ATTO fluorescence was found only in the vessels and not in the insulin-immunoreactive islets of Langerhans (Figure S7E), thus excluding the endocrine pancreas as a possible target of the Ha13 treatment at minipuberty. In the medio-basal hypothalamus (Figures S7F–S7H), the antibody specifically bound GnRH terminals projecting to median eminence (ME; Figure S7H), ME tanycytes (Figure S7G), and the endothelial cells of the brain vasculature (Figure S7H). Within the rostral POA (Figures S7I–S7L), the antibody-bound GnRH neurons (Figure S7L) and endothelial cells of the brain vasculature (Figure 7L).

Collectively, these results show that Ha13 binds the ovaries and the adipocytes of the visceral fat in the periphery and GnRH neurons and tanycytes in the central nervous system.

## Ha13 treatment rectifies ovulation and hyperandrogenism in PCOS-like animals

Our results support the view that Ha13 may be an effective pharmacological intervention to prevent PCOS defects when administered during the infantile period. We then wondered whether Ha13 might also hold therapeutic potential to treat PCOS in adulthood. To test that, PAMH adult females were injected intraperitoneally with either Ha13 or mouse IgM three

<footnote>(C) Insulin tolerance test (ITT) curve after 4-h fasting with corresponding AUC analysis. CNTR, *n* = 5; PAMH + vehicle, *n* = 6; PAMH + IgM, *n* = 5; PAMH + Ha13, *n* = 5.</footnote>

<footnote>(D) Representative images of histological haematoxylin and eosin staining of the BAT and of the vWAT at diestrus in the four animal groups indicated in the figure (*n* = 3 biological replicates).</footnote>

<footnote>(E) Mean lipid droplets area in the BAT (CNTR, *n* = 6; PAMH, *n* = 7; PAMH + IgM, *n* = 8; PAMH + Ha13, *n* = 6; 6-month-old females).</footnote>

<footnote>(F) Mean rectal temperature, expressed in °C, in CNTR, *n* = 13; PAMH, *n* = 4; PAMH + IgM, *n* = 4; PAMH + Ha13, *n* = 10 (6-month-old females).</footnote>

<footnote>(G) Mean visceral fat weight (in grams) in 6-month-old females CNTR (*n* = 8), PAMH (*n* = 7), PAMH + IgM (*n* = 7), PAMH + Ha13 (*n* = 7).</footnote>

<footnote>(H) Mean adipocyte area in the vWAT of 6-month-old females CNTR (*n* = 8), PAMH (*n* = 8), PAMH + IgM (*n* = 8), PAMH + Ha13 (*n* = 8).</footnote>

<footnote>(I) Representative immunoblots of proteins in the vWAT of CNTR, PAMH, and PAMH + Ha13 6-month-old females (*n* = 3 biological replicates).</footnote>

<footnote>(J) Relative densitometric bar graphs of protein levels in the WAT of 6-month-old females CNTR (*n* = 5; gray column), PAMH (*n* = 8; red column), PAMH + Ha13 (*n* = 5; blue column). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize protein levels.</footnote>

<footnote>Scale bar in (D): 50 µm. Data are presented as mean ± SEM; ns, not significant; \* *p* < 0.05, \*\* *p* < 0.005, \*\*\* *p* < 0.0005, \*\*\*\* *p* < 0.0001; one-way ANOVA (A, in AUC of B and C, and in E-H and J), or a two-way ANOVA (B and C). In (B), letters define statistical differences (A: CNTR vs. PAMH, B: CNTR vs. PAMH + IgM, C: CNTR vs. all groups). In (C), letters define statistical differences (A: CNTR vs. PAMH; B: CNTR vs. PAMH + Ha13).</footnote>
<figure><img src="image_10.png" /><figcaption>Figure 6. Ha13 treatment during minipuberty prevents intergenerational transmission of PCOS-like traits to female offspring</figcaption></figure>

(A) Schematic illustrating the experimental design.

(B) Mean circulating levels of testosterone and androstenedione (delta4-Dione) measured by GC-MS in P60 females (CNTR F2, *n* = 7; PAMH F2, *n* = 7; PAMH F2+Ha13 (F1), *n* = 10; age: P60 for all groups).

(C) Percentage of time spent in each estrous cycle stage over 15 days in the three groups (CNTR F2, *n* = 6; PAMH F2, *n* = 6; PAMH F2 + Ha13 [F1], *n* = 6; age: P60 for all groups).

(D) Number of complete estrous cycles during 15 days (CNTR F2, *n* = 6; PAMH F2, *n* = 6; PAMH F2 + Ha13 [F1], *n* = 6; age: P60 for all groups).

(E) Number of LH pulses per hour in P60 diestrous females (CNTR F2, *n* = 11; PAMH F2, *n* = 17; PAMH F2 + Ha13 [F1], *n* = 19).

(F) Body weight at P150 (CNTR F2, *n* = 6; PAMH F2, *n* = 9; PAMH F2 + Ha13 (F1), *n* = 9).

(G) ITT after 4-h fasting at P150 (CNTR F2, *n* = 6; PAMH F2, *n* = 7; PAMH F2 + Ha13 [F1], *n* = 7).

Data are presented as mean ± SEM; ns, not significant; \*p < 0.05, \*\*p < 0.005, \*\*\*p < 0.0005, \*\*\*\*p < 0.0001 (one-way ANOVA in B and D–F or a two-way ANOVA in C and G). In (G), statistical differences: (A) CNTR F2 vs. PAMH F2, *p* < 0.01; (B) PAMH F2 vs. PAMH F2 + Ha13, *p* < 0.05.

times a week for 1 month (Figure 7A). Estrous cyclicity was examined in control and PAMH mice injected or not with IgM or Ha13. Consistent with our data presented above, PAMH mice displayed an oligo-ovulatory phenotype (Figure 7B), characterized by sporadic ovulations, resulting in a significant reduction in the number of complete estrous cycles in 15 days compared with control animals (Figure 7C). Notably, Ha13 administration fully restored the ovulatory pattern of PCOS-like animals back to control conditions (Figures 7B and 7C), and it rescued the hyperandrogenic phenotype (Figure 7D). Ovarian histology of PCOS-like animals showed abnormalities

consistent with their oligo-anovulatory phenotype (Figure 7E), with the presence of fewer post-ovulation corpora lutea as compared with control animals, whereas Ha13 treatment corrected these defects (Figure 7F).

DISCUSSION

Previous studies have demonstrated that fetal exposure to elevated levels of androgens and/or AMH during prenatal development may significantly contribute to predisposing the offspring to PCOS.<sup>17,19,21</sup> However, it remains unclear whether
<figure><img src="image_11.png" /><figcaption>**Figure 7.** Ha13 chronic treatment during adulthood corrects PCOS-like neuroendocrine and ovulatory defects</figcaption></figure>

(A) Schematic illustrating the experimental design.

(B) Analysis of the percentage of the time spent in each estrous cycle stage over 15 days (CNTR, *n* = 9; PAMH, *n* = 9; PAMH + IgM, *n* = 10; PAMH + Ha13, *n* = 8; age: P120).

(C) Number of complete estrous cycles during 15 days (CNTR, *n* = 9; PAMH, *n* = 10; PAMH + IgM, *n* = 10; PAMH + Ha13, *n* = 8; age: P120).

(D) Mean circulating levels of testosterone and androstenedione (delta4-Dione) measured by GC-MS in diestrous females (CNTR, *n* = 9; PAMH, *n* = 10; PAMH + IgM, *n* = 10; PAMH + Ha13, *n* = 8; age: P120).

(E) Representative photomicrographs of ovaries from the indicated treatment groups stained with hematoxylin and eosin from adult.

(F) Mean number of CL in diestrous females (*n* = 5 for each group; age: P120).

Data are presented as mean ± SEM; ns, not significant; \*p < 0.05, \*\*p < 0.005, \*\*\*p < 0.0001 (two-way ANOVA in B, one-way ANOVA in C and D, or Kruskal-Wallis in F). CL, corpus luteum; AF, atretic follicle.

the developmental window for PCOS programming extends into early postnatal life. Here, we show that the PAMH mouse model has higher circulating AMH levels during minipuberty compared with controls, and we demonstrate that aberrant AMH exposure during this period in otherwise healthy pups drives key reproductive and metabolic PCOS features in female mice. These data are consistent with recent clinical data showing that both females and males born to mothers with PCOS have significantly higher serum levels of AMH during minipuberty compared with those

born to mothers without PCOS.<sup>13,23-25</sup> In addition, previous studies have proposed the existence of a male metabolic phenotype of PCOS,<sup>40-42</sup> and a recent investigation reported that sons of women affected by PCOS are frequently obese and exhibit dyslipidemia.<sup>39</sup> In line with this evidence, we show that elevated AMH exposure during minipuberty in male mice contributes to long-term metabolic issues that emulate traits found in the male close relatives of women with PCOS.<sup>39-42</sup> However, in contrast with the exposed females, mAMH males do not show
gross gonadal alterations nor changes in circulating levels of testosterone.

Our results also highlight that mAMH females have lower *Esr1* expression in the ARC/MBH than controls, potentially causing LH hypersecretion, hyperandrogenism, and metabolic issues. Indeed, hyperandrogenemia, acting through the *Ar*, has been shown to reduce estrogen receptor alpha (ERα) expression in the ARC/MBH.<sup>43</sup> This contributes to diminished estradiol-mediated hypothalamic negative feedback, perpetuating LH hypersecretion and ovarian hyperandrogenism in both women with PCOS and animal models alike.<sup>44-47</sup> Additionally, decreased estradiol-mediated ERα activity in the MBH has been linked to metabolic dysfunctions.<sup>48,49</sup> These findings suggest that disruptions in sex hormone signaling and ERα expression in the hypothalamus may contribute to reproductive and metabolic dysfunctions in PCOS.

We have previously shown that AMHR2 is expressed by GnRH neurons throughout the lifespan in both rodents and humans as well as in other neuronal and non-neuronal populations of the hypothalamus.<sup>10,12</sup> In addition, AMH acts as a potent stimulator of GnRH neuronal activity, driving increased LH secretion, which in turn exacerbates ovarian androgen production.<sup>10</sup> Hence, AMH overexposure during minipuberty may create a feedforward loop to GnRH neurons, leading to ovarian hyperandrogenism and metabolic dysfunction later in life. This supports a causal role for elevated AMH levels during early life in the pathophysiology of PCOS and offers a window for testing pharmacological interventions. To this purpose, we designed an anti-AMHR2-blocking antibody (Ha13) aiming to reduce AMH action by preventing its binding. Treatment with the Ha13 antibody during minipuberty prevented the manifestation of reproductive and metabolic cardinal defects of PCOS in adult PAMH females, and it also eliminated transmission of these traits to the F2 generation, highlighting the potential of early intervention to reduce long-term PCOS-related health impacts over multiple generations.

Importantly, Ha13 was observed to accumulate in the hypothalamus, ovaries, and visceral fat, suggesting that the beneficial effects of Ha13 could operate through both central and peripheral mechanisms. Within the brain, we detected fluorescently labeled Ha13 in various brain cell types, including GnRH neurons, endothelial cells of the brain vasculature, and tanycytes of the ME. These findings align with prior research on AMHR2 expression in these cells, confirming the targeting specificity of the antibody.<sup>10,12</sup>

Previously, we demonstrated that circulating AMH can reach GnRH terminals through the fenestrated vessels of the ME.<sup>21</sup> In this study, we peripherally injected the Ha13 antibody, which, classified as an IgM, possesses a pentameric structure approximately 35 nm in size. Due to its dimensions, we hypothesize that it can traverse the trans-endothelial pores (range: 30–80 nm in diameter) of the blood vessels found in circumventricular organs<sup>50</sup> such as the ME.<sup>51,52</sup> By binding to GnRH cell bodies and terminals, the antibody may counteract the excitatory effect of AMH on GnRH neuronal activity and GnRH release, a mechanism observed in PCOS-like animals.<sup>21</sup> In addition, we recently showed that AMH promotes profound micro-structural changes in the murine hypothalamic ME, inducing the retraction of AMHR2-expressing tanycytes and thus creating a permissive

environment for GnRH secretion.<sup>12</sup> These data, together with recent clinical trial research point to the GnRH neuroendocrine system as a promising therapeutic target in PCOS.<sup>53-55</sup> While our study found no changes in *Kiss1* mRNA expression in PCOS-like models, this does not discard the possibility of alteration at subsequent steps of kisspeptin biosynthesis or release, not captured by mRNA levels. In fact, inhibiting neurokinin B (NKB) signaling, which regulates pulsatile GnRH release, has shown therapeutic promise to some extent.<sup>53,54,56</sup>

In addition to acting on the hypothalamic GnRH neural system, the Ha13 antibody could exert at least part of its beneficiary effects by acting directly on the ovaries, where it may counteract the aberrant AMH signaling. Indeed, ovarian AMH over-secretion, along with elevated testosterone, promotes pre-antral follicle survival and inhibits follicle-stimulating hormone (FSH)-induced antral follicle growth,<sup>57</sup> contributing to oligo-anovulation and type-2 diabetes in PCOS.<sup>58</sup>

Moreover, our study provides evidence for AMHR2 expression in the mouse visceral adipocytes at both transcriptional and protein levels. Consistent with these data, the systemically delivered fluorescently tagged Ha13 antibody also successfully binds to adipocytes in the visceral fat.

Enlarged adipocytes contribute to PCOS metabolic issues by driving insulin resistance,<sup>37</sup> with adipocytes from women with PCOS having higher lipid content.<sup>59,60</sup> Androgen excess promotes *de novo* lipogenesis, reduces β-oxidation and lipolysis, and causes lipotoxicity,<sup>61</sup> leading to lipid accumulation in tissues like muscle and liver,<sup>1</sup> which contributes to insulin resistance. Accordingly, in PAMH animals, increased body weight, greater adiposity, enlarged adipocytes, and elevated lipogenic enzyme expression were observed. Previous studies have also shown that androgen excess hinders adipose stem cell differentiation into mature adipocytes,<sup>38,62,63</sup> which triggers compensatory adipocyte hypertrophy, contributing to dyslipidemia in PCOS.<sup>64</sup> It is plausible that AMH could play a role in adipocyte cellular differentiation, although further research is needed to elucidate how AMH might influence adipose tissue biology under both physiological and pathological conditions.

Taken together, these findings suggest that Ha13 protects against PCOS by inhibiting ovarian androgen production, restoring ovulation, and also mitigating hyperandrogenism's impact on adipose tissue. This dual action, along with its central effects, highlights Ha13's therapeutic potential for both reproductive and metabolic aspects of PCOS.

Gestational exposure to elevated levels of AMH in mice has been shown to induce PCOS-like traits in female offspring, with elevated AMH levels persisting across multiple generations.<sup>19,21</sup> Here, we show that abnormal hormonal exposure *in utero* also leads to increased circulating AMH levels during minipuberty. Based on the minipuberty AMHR-2-blocking strategy applied in PAMH animals, it thus appears that the key endocrine change driving adult PCOS-like traits could be the rise in AMH during this postnatal window. Our study, together with previous work,<sup>27</sup> suggests that minipuberty is a critical developmental stage contributing to both reproductive and metabolic traits of adult females. This is in agreement with recent studies revealing a minipuberty in infant girls characterized by two peaks of elevated AMH,<sup>65</sup> ovarian hormones, and gonadotrophins<sup>66</sup> at 1 and 4–6 months after birth. Furthermore, during this period,
higher AMH levels correlate with larger ovaries and larger follicles.⁶⁵,⁶⁷

Overall, we show that the Ha13 antibody shows benefits in preventing PCOS traits when administered during minipuberty and alleviates reproductive symptoms when given in adulthood, thus suggesting AMHR2-targeting therapy as a potential preventive or curative approach for PCOS.

## Limitations of the study

It is important to recognize that changes in circulating gonadal hormones over development might differ between mice and humans. In humans, the HPG axis experiences two periods of transient activity before puberty onset: during mid-gestational fetal life and in the first postnatal months (minipuberty).⁶⁸ The functionality of the HPG axis in rodents develops gradually during postnatal development, with minipuberty peaking at P12 in female mice.²⁷⁻²⁹

Additionally, the most recent international guidelines² state that PCOS should not be diagnosed until after menarche, which limits the use of Ha13 treatment in young women prior to diagnosis. Clinical trials are needed to confirm potential therapeutic effects of Ha13 in adult women with PCOS. Finally, further preclinical investigations are required to test the effect of Ha13 in control healthy mice.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Paolo Giacobini (paolo.giacobini@inserm.fr).

### Materials availability

This study generated a novel reagent: neutralizing anti-AMHR2 antibody. Requests for such reagents should be directed to the lead contact, Paolo Giacobini (paolo.giacobini@inserm.fr).

### Data and code availability

Uncropped scans of all western blots and all raw data used to generate graphs are included in Data S1. This paper does not report original code.

## ACKNOWLEDGMENTS

We thank all staff of the Lille Neuroscience & Cognition (LilINCog) research center for their constant assistance. The authors thank Meryem Tardivel and Antoninon Bongiovanni (microscopy core facility; The Biomaging Center Lille, BICel; PLBS UAR 2014 – US41), Sarah Gabut (histology core facility; PLBS UAR 2014 – US41), Julien Devassine, and all the staff of the Animal facility (PLBS UAR 2014 – US41) for expert technical assistance. We also thank Dr. Virginie Mattot, Inserm U1172 of Lille, for helping with the retro-orbital injections and Dr. Beatriz Rodriguez-Cortes, Inserm U1172 of Lille, for her help in blood collection from P14 animals. This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (ERC-2016-CoG to P.G., grant agreement no. 725149/REPRODAMH, ERC-2018-Synergy to V.P. and R.N., and grant agreement no. 810331/WATCH and European Union Horizon 2020 research and innovation program no. 847941 miniNO to V.P.), by a French Government Grant managed by the National Research Agency (ANR) under the action France 2030 with the reference ANR-24-CHBS-0002 (to P.G.), and by the British Society for Neuroendocrinology, United Kingdom (BSN project support grant 2024 to L. Cotellessa). Schemes and drawings were created with BioRender.com.

## AUTHOR CONTRIBUTIONS

Conceptualization, writing, and preparation of the figures, L. Cotellessa and P.G.; methodology and formal analysis, L. Cotellessa, V.S., M.S.B.S., M.D., H.M., E.C., G.T., N.d.S.L., T.L., F.G., A.S., L. Carraresi, and G.D.N.; writing/review/editing, P.G., L. Cotellessa, R.N., M.T.-S., and V.P.; and funding acquisition, P.G., V.P., and R.N.

## DECLARATION OF INTERESTS

P.G., L. Cotellessa, and V.P. disclose that they are inventors of a submitted patent application by the Institut National de la Santé et de la Recherche Médicale (INSERM) covering methods for the prevention and treatment of PCOS.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Animals

* Postnatal anti-Müllerian hormone mouse model (minipuberty: mAMH)

* Prenatal anti-Müllerian hormone (PAMH) mouse model

* METHOD DETAILS

* Assessment of reproductive status and blood sampling

* Hormone measurements and analysis

* Measurement of mouse steroid levels in serum by GC/MS

* Ovarian histology

* Physiological measurements

* Immunoblots

* Adipose tissue histomorphology

* Structural modelling of AMH-AMHR2 complex

* Immunization and generation of hybridomas

* Monoclonal anti-AMHR2 neutralizing antibody production

* Monoclonal anti-AMHR2 neutralizing antibody purification

* Determination of kinetics and affinity constants of anti-AMHR2

* *In vitro* studies using anti-AMHR2 neutralizing antibody

* Electrophysiology

* *In vivo* studies using anti-AMHR2 neutralizing antibody

* RNA extraction and RT-qPCR

* *In vivo* biodistribution of Ha13

* RNAscope fluorescence *in situ* hybridization

* Ethics statement

* Statistics and data analysis

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cmet.2025.03.013.

Received: September 23, 2024

Revised: February 3, 2025

Accepted: March 19, 2025

Published: April 11, 2025

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79. Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402-408. https://doi.org/10.1006/meth.2001.1262.

80. Manfredi-Lozano, M., Leysen, V., Adamo, M., Paiva, I., Rovera, R., Pignat, J.M., Timzoura, F.E., Candlish, M., Eddarkaoui, S., Malone, S.A., et al. (2022). GnRH replacement rescues cognition in Down syndrome. Science 377, eabq4515. https://doi.org/10.1126/science.abq4515.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Anti-Bovine Lhβ subunity</td><td>Janet Roser, University of California</td><td>Cat#: 518B7; RRID: AB_2756886</td></tr><tr><td>Rabbit LH antiserum</td><td>National Hormone and Peptide Program (NHPP)</td><td>Cat#: AFP240580Rb; RRID: AB_2784499</td></tr><tr><td>Anti-mouse monoclonal LH antibody 5303 SPRN</td><td>Medix Biochemica</td><td>Cat#: 100588; RRID: AB_2784503</td></tr><tr><td>Polyclonal rabbit anti-AMH-R2</td><td>Custom-made, CASLO, Denmark</td><td>RRID:AB_2909430</td></tr><tr><td>Ha13, anti-AMHR2 mouse monoclonal antibody</td><td>This paper</td><td>N/A</td></tr><tr><td>Anti-Hormone sensitive lipase/HSL antibody</td><td>Abcam</td><td>Cat#: ab45422; RRID: AB_2135367</td></tr><tr><td>Phospho-HSL (Ser660) Antibody</td><td>Cell Signaling</td><td>Cat#: 4126; RRID: AB_490997</td></tr><tr><td>Anti-acetyl CoA Carboxylase Antibody</td><td>Millipore</td><td>Cat#: 04-322; RRID: AB_673047</td></tr><tr><td>Phospho-Acetyl-CoA Carboxylase (Ser79) Antibody</td><td>Cell Signaling</td><td>Cat#: 3661; RRID: AB_330337</td></tr><tr><td>LPL Antibody (H-53)</td><td>Santa Cruz</td><td>Cat#: sc-32885; RRID: AB_2088681</td></tr><tr><td>Anti-Fatty Acid Synthase antibody [EPR7466]</td><td>Abcam</td><td>Cat#: ab128870; RRID: AB_11143436</td></tr><tr><td>Anti-GAPDH Antibody</td><td>Sigma-Aldrich</td><td>Cat#: G9545; RRID: AB_796208</td></tr><tr><td>Monoclonal Anti-β-Actin antibody</td><td>Sigma-Aldrich</td><td>Cat#: A2228; RRID: AB_476697</td></tr><tr><td>Phospho-SMAD1/5 (Ser463/465) (41D10)</td><td>Cell Signaling</td><td>Cat#: 9516; RRID: AB_491015</td></tr><tr><td>Goat anti-rabbit Immunoglobulins/HRP</td><td>DAKO</td><td>Cat#: P0448; RRID: AB_2617138</td></tr><tr><td>Horse anti-mouse Immunoglobulins/HRP</td><td>DAKO</td><td>Cat#: P1-2000; RRID: AB_2336177</td></tr><tr><td>Anti-Laminin 2 alpha</td><td>Abcam</td><td>Cat#: ab11576; RRID: AB_298180</td></tr><tr><td>GNRH1 Polyclonal antibody</td><td>Proteintech</td><td>Cat#: 26950; RRID: AB_2880698</td></tr><tr><td>Anti-PLVAP/PV-1 antibody</td><td>Abcam</td><td>Cat#: ab81719; RRID: AB_1658370</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488</td><td>Thermo-Fisher</td><td>Cat#: A-21206; RRID: AB_2340620</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568</td><td>Thermo-Fisher</td><td>Cat#: A10042; RRID: AB_3095472</td></tr><tr><td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488</td><td>Thermo-Fisher</td><td>Cat#: A-21202; RRID: AB_141607</td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>RNAScope probe Pomc</td><td>Acd biotechne</td><td>Cat#: 314081</td></tr><tr><td>RNAScope probe Agrp</td><td>Acd biotechne</td><td>Cat#: 400711</td></tr><tr><td>RNAScope probe Esr1</td><td>Acd biotechne</td><td>Cat#: 478201</td></tr><tr><td>RNAScope 3-plex negative control probes</td><td>Acd biotechne</td><td>Cat#: 320871</td></tr><tr><td>Rn18S primers</td><td>Thermo-Fisher</td><td>Cat#: Mm03928990_g1</td></tr><tr><td>Beta Actin primers</td><td>Thermo-Fisher</td><td>Cat#: Mm00607939_s1</td></tr><tr><td>Esr1 primers</td><td>Thermo-Fisher</td><td>Cat#: Mm00433149_m1</td></tr><tr><td>Pomc primers</td><td>Thermo-Fisher</td><td>Cat#: Mm00435874_m1</td></tr><tr><td>Agrp primers</td><td>Thermo-Fisher</td><td>Cat#: Mm00475829_g1</td></tr><tr><td>Kiss1 primers</td><td>Thermo-Fisher</td><td>Cat#: Mm03058560_m1</td></tr><tr><td>Pgr primers</td><td>Thermo-Fisher</td><td>Cat#: Mm00435628_m1</td></tr><tr><td>Amhr2 primers</td><td>Thermo-Fisher</td><td>Cat#: Mm00513847_m1</td></tr><tr><td colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td></tr><tr><td>Dulbecco Phosphate Buffered Saline</td><td>Glibco</td><td>Cat#: 14190-094</td></tr><tr><td>E coli-derived human MIS/AMH protein</td><td>R&D Systems a Bio-technne</td><td>Cat#: 1737-MS-010/CF</td></tr><tr><td>TMB</td><td>Thermo Fisher</td><td>Cat#: 34028</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>Cat#: 1379</td></tr><tr><td>Hematoxylin-eosin</td><td>Sigma Aldrich</td><td>Cat#: GHS132</td></tr></tbody></table>

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Continued

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Insulin</td><td>Eli Lilly and Company</td><td>Cat#: HI0210</td></tr><tr><td>RIPA Lysis Buffer 10X</td><td>Merck</td><td>Cat#: 20-188</td></tr><tr><td>Substrat chemiluminescent SuperSignal™ West Pico PLUS</td><td>Thermo Scientific™</td><td>Cat#: 34580</td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>Cat#: D6429</td></tr><tr><td>HAT Media Supplement (50x) Hybri-Max™</td><td>Sigma-Aldrich</td><td>Cat#: H0262</td></tr><tr><td>BSA</td><td>Merck</td><td>Cat#: A7906</td></tr><tr><td>3,3,5,5-Tetramethylbenzidine (TMB)</td><td>Merck</td><td>Cat#: T8665</td></tr><tr><td>L-glutamine</td><td>Thermo-Fisher</td><td>Cat#: 25030081</td></tr><tr><td>Penicillin-streptomycin</td><td>Thermo-Fisher</td><td>Cat#: 15140122</td></tr><tr><td>Fetal Bovine Serum (FBS), Premium, One Shot™</td><td>Thermo-Fisher</td><td>Cat#: A5670401</td></tr><tr><td>HEPES</td><td>Gibco</td><td>Cat#: 11560496</td></tr><tr><td>Complete Freund's Adjuvant</td><td>Sigma-Aldrich</td><td>Cat#: F5881</td></tr><tr><td>Incomplete Freund's Adjuvant</td><td>Sigma-Aldrich</td><td>Cat#: F5506</td></tr><tr><td>Poly(éthylène glycol)</td><td>Sigma-Aldrich</td><td>Cat#: 202398</td></tr><tr><td>ATTO 565 NHS ester</td><td>Supelco</td><td>Cat#: 72464</td></tr><tr><td>Fluoromount GTM with DAPI</td><td>Invitrogen</td><td>Cat#: 00-4959-52</td></tr></tbody></table>

Critical commercial assays

<table><tr><td>Rat and Mouse AMH ELISA</td><td>Ansh-Labs</td><td>Cat#: AL-113</td></tr><tr><td>Mouse Insulin ELISA Kit</td><td>Mercodia</td><td>Cat#: 10-1247-01</td></tr><tr><td>Pierce™ BCA Protein Assay Kits</td><td>Thermo-Fisher</td><td>Cat#: 23225</td></tr><tr><td>EZNA total RNA kit II</td><td>Omega</td><td>Cat#: R6934-02</td></tr><tr><td>RNA-to-cDNA kit</td><td>Applied Biosystems</td><td>Cat#: 4387406</td></tr><tr><td>RNAScope™ Multiplex Fluorescent V2 Assay</td><td>Acd biotechne</td><td>Cat#: 323270</td></tr></table>

Experimental models: Cell lines

<table><tr><td>Mouse granulosa cell line KK-1</td><td>Kananen et al.<sup>69</sup></td><td>N/A</td></tr></table>

Experimental models: Organisms/strains

<table><tr><td>Mus musculus: C57BI/6J</td><td>Charles River</td><td>RRID:MGI:5657312</td></tr><tr><td>Mus musculus: BALB/c</td><td>Charles River</td><td>RRID:MGI:2161072</td></tr><tr><td>Gnrh::gfp mice</td><td>Developed in the Catherine Dulac Lab (Harvard University, MA, USA)</td><td>Yoon et al.<sup>70</sup></td></tr></table>

Software and algorithms

<table><tr><td>ImageJ</td><td>National Institutes of Health</td><td>RRID: SCR_003070</td></tr><tr><td>AlphaFold2</td><td>Google DeepMind</td><td>https://deepmind.google/technologies/alphafold/</td></tr><tr><td>Clampfit 10.2 pClamp</td><td>Molecular Devices</td><td>N/A</td></tr><tr><td>Adobe Photoshop 22.1.0</td><td>Adobe Systems</td><td>RRID: SCR_014199</td></tr><tr><td>Graphpad Prim 10.0.2insulin</td><td>GraphPad Prism</td><td>http://www(Graphpad.com)</td></tr></table>

Other

<table><tr><td>MiniSpec mq 7.5, Nuclear Magnetic Resonance</td><td>Bruker</td><td>N/A</td></tr><tr><td>Glucometer</td><td>Accu-Chek® Guide</td><td>Roche</td></tr><tr><td>Precellys 24 Touch Homogenizer</td><td>Bertin technologies</td><td>N/A</td></tr><tr><td>Percellys Lysing Kit</td><td>Bertin technologies</td><td>Cat#: P000912-LYSK1-A</td></tr><tr><td>Provis AX70 microscope</td><td>Olympus</td><td>N/A</td></tr><tr><td>ÄKTA pure™ chromatography system</td><td>Cytiva</td><td>N/A</td></tr><tr><td>HiPrep Desalting column</td><td>Cytiva</td><td>N/A</td></tr><tr><td>HiTrap Protein A HP antibody purification columns</td><td>Cytiva</td><td>N/A</td></tr><tr><td>T200 Biacore instrument</td><td>Ge Healthcare Bio-science</td><td>Piscataway, NJ</td></tr><tr><td>NuPage 4-12% Bis-Tris Gel</td><td>Thermo-Fisher</td><td>Cat#: NW04120BOX</td></tr><tr><td>Nitrocellulose membrane 0,45µm</td><td>Biorad</td><td>Cat#: 1620115</td></tr></table>

(Continued on next page)
**Continued**

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>VT1200 Vibratome</td><td>Leica</td><td>N/A</td></tr><tr><td>DM-LFSA microscope</td><td>Leica</td><td>N/A</td></tr><tr><td>ORCA-Flash4.0 digital CMOS camera</td><td>Hamamatsu</td><td>N/A</td></tr><tr><td>Confocal microscope (LSM 710)</td><td>Zeiss</td><td>N/A</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Animals

C57BL/6J mice (Charles River, USA) were housed at 21–22°C with a 12 h light/dark cycle and provided *ad libitum* access to water and standard laboratory chow (9.5 mm Pelleted RM3, Special Diets Services; Competence Centre for Lab. Animal Science of SAFE®; France). Mice were randomly assigned to groups of six animals per cage at the time of purchase or weaning with litters within the same treatment allocation group mixed (pseudo-random mixing based on weight) to minimize any potential bias. No data were excluded from analyses.

### Postnatal anti-Müllerian hormone mouse model (minipuberty: mAMH)

C57BL/6J mice (Charles River, USA) were injected daily intraperitoneally (i.p.) from postnatal day 12 to 14 with either 50 µL of 0.01 M phosphate-buffered saline (PBS) (control group) or 0.26 µg/mouse/day human recombinant AMH<sub>C</sub> (R&D Systems, rhMIS, catalogue n°1737-MS-10) diluted in PBS (mAMH group).

### Prenatal anti-Müllerian hormone (PAMH) mouse model

PAMH mice were generated according to previous studies.<sup>35</sup> Pregnant adult (3–4 months) female mice were injected daily intraperitoneally (i.p.) at embryonic day (E) 16.5, 17.5, and 18.5 with either 200 µL of 0.01 M phosphate-buffered saline (PBS) only (control group) or 0.12 mg/Kg/day human recombinant AMH<sub>C</sub> (R&D Systems, rhMIS 1737-MS-10) diluted in PBS (PAMH group). Mouse pregnancy was timed considering the detection of vaginal plugs as GD 0.5. Six PAMH female mice were bred with *Gnrh-Gfp*<sup>70</sup> homozygous mice to generate PAMH;GnRH-GFP<sup>+/-</sup> mice that were used for electrophysiology experiments.

## METHOD DETAILS

### Assessment of reproductive status and blood sampling

Reproductive cycles were followed and characterized according to well-described guidelines of the mouse estrous cycle according to well-established guidelines.<sup>36</sup> Briefly, vaginal smears were collected using 10 µL of sterile saline, transferred to glass slides, and evaluated during the morning period at 8:00-9:00h (room lights were on at 7:00h). Vaginal cytology was performed with freshly collected samples using an inverted microscope with maximum microscope condenser distance allowing a better contrast for proper visualization. Estrous cyclicity was followed for 15-17 consecutive days. Blood sampling for LH and testosterone levels was conducted when mice were in diestrus. For LH-level profile studies, mice were trained to interact with the investigator and allow restraint of the tail for at least two weeks before the commencement of each set of experiments. A 4 µL of the whole blood sample from the tail tip was taken every 5 min (LH profile) for 2-3h. Samples were collected into Eppendorf tubes pre-loaded with 50 µL of 0.1M phosphate-buffered saline (PBS)-0.05% Tween (pH = 7.4), thoroughly mixed, and snap-frozen with dry ice and stored at -80 °C. Plasma testosterone samples were collected from the cheek at a single point for each treatment. Samples were kept in ice for one hour followed by centrifugation at 6,8 rcf for 15 minutes at 4 °C. Plasma supernatant was transferred to sterile 200-µL Eppendorf tubes and stored at -80 °C.

### Hormone measurements and analysis

For this study, we used an ultrasensitive ELISA method to measure circulating LH levels in the whole blood in mice as validated previously to assess pulsatile LH release in female mice over the estrous cycle.<sup>37,38</sup> Briefly, this ELISA uses a 96-well high-affinity binding microplate (Corning) coated with 50 µL of primary capture antibody (bovine LHβ subunit, 518B7; L. Sibley; University of California, UC Davis) at a dilution of 1:1000. The mouse LH-RP reference used for this assay was provided by Dr. Albert F. Parlow (National Hormone and Pituitary Program, Torrance, California, USA) and used to generate a standard curve with a twofold serial dilution from a start standard of 4 ng/mL to 0.0019 ng/mL LH-RP reference diluted in 0.2% bovine serum albumin (BSA)-0.1M PBS-0.05% Tween solution. Whole blood samples were transferred to the coated as singlets for each point of sampling and incubated for 2 h under agitation at room temperature. A rabbit LH antiserum primary antibody (AFP240580Rb; NIDDK- NHPP) was used at 1:10,000 dilution and a secondary horseradish peroxidase-conjugated antibody (Streptavidin, Thermo Fisher, catalogue n N200) was used at 1:8000 dilution. We used 100 µL of 1-Step Ultra TMB-Elisa Substrate Solution (ThermoFisher Scientific, catalogue n° 34028) for the final revelation step followed by a stop solution with 50 µL of 3M HCl per well. The assay sensitivity of this LH ELISA was 0.04 ng/ml and intra-assay coefficient of variation was 3.9% and the inter-assay coefficient of variation was 8.3%. LH pulse analysis was analysed based
on previous studies.<sup>38-40</sup> Briefly, mean LH levels were considered as the average of all measured values within the 2-h blood sampling protocol. LH pulse peaks were determined as a single point with a value more than 20% from 1 of the 2 previous points followed by a decrease by >10% in 1 of the next 2 subsequent points.<sup>71</sup> Next, LH pulse frequency was determined by counting the number of identified pulses per hour. The average time distance (in min) between each LH pulse peak was used to calculate the LH pulse interval. The LH pulse amplitude was calculated considering the peak of each LH pulse minus its preceding zenith values followed by a final average calculation of all amplitudes within the 2-h blood sampling protocol. Basal LH level analysis considered the average of all LH nadir and non-peak values. The area under the curve (AUC) analysis is considered the total area summed over the 2-h blood sampling protocol.

Plasma AMH levels were measured using a commercial rat and mouse ELISA kit following the manufacturer's instructions (AnshLabs®, catalogue n° AL-113). The assay sensitivity of this ELISA was 0.011 ng/mL as reported by the manufacturer, and the intra-assay coefficient of variation was 5.2%. Quality controls were also provided by the manufacturer.

## Measurement of mouse steroid levels in serum by GC/MS

### Hormonal parameters

Testosterone (T), Dihydrotestosterone (DHT), Estradiol (E2), Δ4-androstenedione (4-dione), and Progesterone (Prog) were measured by GC/MS. Twice charcoal dextran-stripped aged female rabbit serum was used as matrix for calibrators and quality control (QC) standards.

### Sample extraction and purification

Briefly, each sample (200 µl of serum per mouse, calibration standards, quality controls, and blank matrix) was collected in a 4 ml borosilicate tube. A spiking solution of deuterated steroid internal standards (IS) (10 µl containing 0.5 ng of T-d5, 0.5 ng of DHT-d3, 50 pg of E2-d4, 2 ng of 4-dione-d7, and 3 ng of Prog-d9, except for blank matrix) (CDN Isotopes, Inc., Point-Claire, Canada), 200 µl of saline water, and 2.8 ml of 1-chlorobutane were added to each sample. After a 2 min vortex-step followed by rapid centrifugation, the upper organic phase was collected on a conditioned Hypersep SI 500 mg SPE mini-column (Thermo Scientific). The column and adsorbed material were then washed with ethyl acetate/hexane (6 ml; 1/9). The second fraction containing the steroids of interest was eluted with ethyl acetate/hexane (4 ml; 1/1), then evaporated at 60 °C to dryness.

### Derivatization reactions and determination of steroid levels in the supernatant

T, DHT, and E2 were derivatized as previously described<sup>72</sup> with pentafluorobenzoyl chloride (PFBC) (Sigma-Aldrich, catalogue n° 103772-1G). Final extracts were reconstituted in 20 µl of isooctane, then transferred into conical vials for GC injection.

4-dione, and Progesterone were derivatized with 50 µl of heptafluorobutyric anhydride (Sigma-Aldrich) and anhydrous acetone (1/1) mixture. Final extracts were reconstituted in 20 µl of anhydrous n-hexane, then transferred in conical vials for injection into the GC system (GC-2010 Plus, Shimadzu) using a 50 % phenylmethylpolysiloxane VF-17MS capillary column (Agilent Technologies). A TQ8050 triple quadrupole mass spectrometer (Shimadzu) equipped with a chemical ionization source (NCI), and operating in Q3 single ion monitoring (SIM) mode, was used for the detection of DHT, T, and E2, and with an electron impact source (EI) operating in multiple reaction monitoring (MRM) mode for the detection of 4-dione, and Progesterone. For NCI detection, the reagent gas was methane, and the GC was performed in pulsed splitless mode with a 1 min pulsed splitless-time. The oven temperature was initially 150°C for 0.50 min, further increased to 305°C at 20°C/min and held at 305°C for 3.60 min, and then to 335°C at 30°C/min and held at 335°C for 1.7 min. The injection port and transfer line temperatures were respectively 290 and 280°C. The flow rate of helium (carrier gas) was maintained constant at 0.96 ml/min. The mass spectrometer CI source temperature was 220°C. For EI detection, GC was performed in the splitless mode with a 1-min splitless-time. The temperature in the oven was initially 70°C for 1 min, further increased to 238°C at 25°C/min, and then to 261°C at 5°C/min. The injection port and transfer line temperatures were respectively 290 and 280°C. The flow rate of carrier gas was maintained constant at 0.70 ml/min. The mass spectrometer EI source was 230°C.

The linearity of steroid measurement was confirmed by plotting the ratio of the steroid peak response / internal standard (IS) peak response to the concentration of steroid for each calibration standard. Accuracy, target ions, corresponding deuterated internal control, range of detection, low limit of quantification (LLOQ), and intra & inter assay CVs of the quality control are reported in Table S2.

## Ovarian histology

Ovaries were collected from 3- to 4-month-old diestrus mice, immersion-fixed in 4% PFA solution and stored at 4 °C. Paraffin-embedded ovaries were sectioned at a thickness of 5 µm (histology facility, University of Lille 2, France) and stained with hematoxylin-eosin (Sigma Aldrich, catalogue n° GHS132, HT1103128). Sections were examined throughout the ovary. Corpora lutea (CL) were classified and quantified as previously reported.<sup>73</sup> To avoid repetitive counting, CL were counted every 100 µm by comparing the section with the preceding and following sections. CL were characterized by a central cavity filled with blood and follicular fluid remnants or by prominent polyhedral to round luteal cells.

## Physiological measurements

### Body composition

Body composition was measured weekly over several experiments using a Minispec LF Series (Bruker Corporation). Fat and lean mass data are expressed as percentage body weight.
**Analysis of basal metabolism**

Mice were analyzed for total energy expenditure, oxygen consumption, CO2 production, food intake and ambulatory movements (total beam breaks h-1) using calorimetric cages (TSE Systems) and standard procedures. Mice were individually housed and acclimatized to cages for 48 h before measurements. Pair-fed animals received the mean weight of food eaten by the control group during the previous 24 h. RER and energy expenditure (EE) were calculated as reported previously,⁷⁴ and FA oxidation as reported by Bruss and colleagues⁷⁵: FA oxidation (kcal h⁻¹) = EE × (1 - RER/0.3).

**Glucose tolerance**

After overnight fasting (12 h), tail blood samples for insulin measurement were taken using glass capillaries before and 15 and 30 min after glucose administration. Blood samples were kept on ice before centrifugation (4 °C, 6.8 rcf, 15 min) and plasma was frozen at -80 °C until an ultrasensitive mouse insulin ELISA (Mercodia, catalogue n° 10-1247-01) was performed on 12.5 µL of plasma per time point. Basal blood glucose levels were measured before i.p. glucose administration (2 g glucose kg⁻¹ body weight) and 15, 30, 45, 60, 90 and 120 min after glucose administration using a Accu-Chek Guide glucometer.

**Insulin response measurement**

Basal blood glucose levels were measured after a 6-h fast for the ITT before i.p. insulin administration (0.5 UI kg⁻¹ body weight, Eli Lilly and Company, catalogue n° HI0210) and 15, 30, 45, 60, 90 and 120 min after glucose administration using a Accu-Chek Guide glucometer.

**Immunoblots**

Tissues, all dissected during the diestrous stage, were homogenized using Parcellys 24 touch homogenizer (Bertin Technologies) in cold RIPA buffer (200 mM Tris/HCl pH 7.4, 130 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) SDS, 1% (v/v) Triton X-100, 10 mM MgCl2) with anti-proteases and anti-phosphatases (Sigma-Aldrich), and lysates were centrifuged for 30 min at 18,000g and 4 °C. Liver, white adipose tissue (intra-abdominal adipose tissue), ovaries and pancreas total protein lysates were separated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred to polyvinylidene difluoride membranes and probed successively with the following antibodies: hormone-sensitive lipase/HSL (1:1,000; Abcam, catalogue n° ab45422); phospho-HSL (Ser660) (1:1000; Cell Signaling Technology, catalogue no. 4126); ACC (1:1000; Millipore, catalogue n° 04-322); phospho-Acc (Ser79) (1:1000; Cell Signaling Technology, catalogue n° 3661); lipoprotein lipase (1:1000; H-53, Santa Cruz Biotechnology, catalogue n° sc-32885); FAS antibody (1:5000; Abcam, catalogue n° ab128870); and β-actin (1:5000; Sigma-Aldrich, catalogue n° A2228) and GAPDH (1:5000: Sigma-Aldrich, Catalogue n° G9545-100UL), after incubation of membranes with 5% BSA blocking buffer. Proteins were detected using horseradish peroxidase-conjugated secondary antibodies (Dako, catalogue n° PI-2000). Specific immunolabelling was visualized using chemiluminescence following the manufacturer's instructions (Thermo-Fisher, catalogue n° 34580), and values expressed relative to β-actin.

**Adipose tissue histomorphology**

Visceral (intra-abdominal) white adipose tissue and intrascapular brown adipose tissue were collected during the diestrous stage, fixed in 10% formalin for 24 h, dehydrated and paraffin-embedded. Sections of 5-µm thickness were cut on a microtome, stained using standard alcoholic hematoxylin and eosin (BioOptica), and observed and photographed using a Provis AX70 microscope (Olympus). Two 20x pictures per sample/mouse/treatment were analysed. 8-bit images of adipose tissue sections were processed with Fiji (https://doi.org/10.1038/nmeth.2019) as follows. Automatic thresholding was first performed using the Triangle method. To correct for damaged and open membranes, two rounds of erosion were applied to the resulting binary image, before using the particle analysis tool to segment adipocytes and extract their pixel area. To exclude potential artifacts, partial adipocytes on the edges of the images, as well as particles of small size (< 2300 px) were excluded from the detection.

**Structural modelling of AMH-AMHR2 complex**

The 3D fold of the peptide sequence GCESLHCDPSPRAHPSPGSTLFTCSCGT was predicted by using Alphafold2. Out of the 5 models generated, the best one was used for further analysis. The overall pLDDT was 67.2 with the regions of the peptide predicted to form β-strands with a pLDDT > 70 (high confidence of prediction).

The structural models of murine AMH and AMHR, generated by Alphafold2, are available on Uniprot database. In particular, the domains involved in the complex formation have a high or very high model confidence with pLDDT >70 and, for some regions, >90. Structural superimposition was generated using UCSF Chimera and the crystal structure of the human complex (PDB: 7L0J) as a template. From the superimposition, a structural alignment was generated by UCSF Chimera and the structural conservation of the amino acids involved in the AMH-AMHR interaction in the human and murine proteins were analyzed.

**Immunization and generation of hybridomas**

A 28 amino acid peptide (GCESLHCDPSPRAHPSPGSTLFTCSCGT), spanning a terminal region of AMHR2 involved in the interaction with AMH and showing high homology among different mammalian species (human, mouse, and rats), identified by Di.V.A.L. Toscana S.r.l. (Italy), was synthesized and conjugated to KLH carrier molecule (Primm Srl). Hybridomas were produced according to Köhler and Milstein's standard protocol.⁷⁶ Briefly, six-week-old female Balb/c mice, purchased from Envigo (Envigo-Harlan Laboratories, Italy) were immunized by intraperitoneal injection of an emulsion of 100 µg KLH-conjugated peptide in PBS and Complete Freund's Adjuvant (Sigma-Aldrich) by Di.V.A.L. Toscana S.r.l. (Italy). Three more intraperitoneal injections of 50 µg KLH-conjugated
peptide emulsionated with Incomplete Freund's Adjuvant (Sigma-Aldrich) were performed every 2-3 weeks and a final immunization with 100 µg peptide was administered intravenously. Three days after the last immunization, spleen cells from immunized mice were fused with the aminopterin-sensitive myeloma cells (NS0) by the polyethylene glycol (PEG; Sigma-Aldrich) method.⁷⁷ Hybridomas were cultured in DMEM with 20% Fetal Clone I serum medium supplemented with hypoxanthine-aminopterin-thymidine (HAT; Sigma-Aldrich) in humidified air at 37° C with 5% CO₂ and supernatants were analyzed by ELISA assay.

## Monoclonal anti-AMHR2 neutralizing antibody production

The best hybridoma population underwent clonal isolation by Di.V.A.L. Toscana S.r.l. (Italy) using the soft agar cloning method. Different numbers of hybridoma cells (10000, 5000, 2500, 1250 cells) were plated in soft agar 60mm dishes composed of a Base Layer (0.5% low melting point agarose in DMEM added 20% Fetal Clone I serum and 1x HAT) and a Top Layer (cells in 0.25% low melting point agarose in DMEM added with 20% Fetal Clone I serum and 1x HAT). About 15 days after seeding, clones were picked up, amplified and gradually adapted to grow in DMEM with Fetal Clone I serum 5%. The supernatants of hybridomas were added to 96-well plates pre-coated with 10 µg/ml of the uncoupled form of the peptide used for immunization followed by blocking with 3% BSA (Sigma-Aldrich). After a two-hour supernatants' incubation, 10 µg/ml anti-mouse secondary antibody, conjugated with horseradish peroxidase (Sigma-Aldrich) was added to each well for 1 hour. Finally, TMB (3,3',5,5'-Tetramethylbenzidine) substrate (Sigma-Aldrich) was added and optical density was determined by absorbance at 405 nm.

## Monoclonal anti-AMHR2 neutralizing antibody purification

Anti-AMHR2 monoclonal antibody was purified by Affinity Chromatography, using ÄKTA Pure Protein Purification System (Cytiva). Clone supernatant was exchanged with Phosphate buffer 20mM pH 8,0 by HiPrep 26/10 Desalting column (Cytiva). The purification was hence performed with HiTrap Protein A HP column (Cytiva). The monoclonal antibody was eluted with a 0,1M citric acid buffer pH 4,0 immediately neutralized with 1M Tris-HCl pH 9,0. The fractions were collected and dialyzed in PBS.

## Determination of kinetics and affinity constants of anti-AMHR2

The equilibrium dissociation and affinity constant ($K_D$ and $K_a$) between the Ha13 mAb and rhAMHR2 (Human MIS RII (AMHR2) Protein, His Tag; Acrobiosystems #MII-H52H6, batch 5726-21CQF1-ZL) was determined using a T200 Biacore instrument (GE Healthcare Bio-Sciences, Piscataway, NJ). Capture antibodies (anti-His) were immobilized on a CM5 sensor chip surface and the kinetics and affinity constants were determined by performing a single cycle kinetics (SCK) of anti-AMHR2 antibodies at 2.5 nM, 7.4 nM, 22.2 nM, 66.7 Mn and 200 nM over captured anti-His antibodies. For each determination, a first cycle was performed with injection of buffer instead of antibody (blank cycle). The response curve of this blank cycle was subtracted from one of the cycles with the antibody. The kinetics and affinity constants were calculated using a Langmuir 1:1 fitting model.

## In vitro studies using anti-AMHR2 neutralizing antibody

### Cell lines

The mouse granulosa cell line KK-1⁶⁹ has previously been shown to be a suitable model to study AMH signaling.³³ Cells were cultured in DMEM (Sigma-Aldrich, catalogue n° D6429) containing 1mM Pyruvate, 2mM L-Glutamine (Thermo-Fisher, catalogue n° 25030081), 100 µg/ml streptomycin, 100 U/ml penicillin, supplemented with 10% v/v fetal bovine serum (FBS, Thermo-Fisher) in a 10% CO₂ humidified atmosphere at 37 °C.

### Western blot analysis

KK-1 cells were plated at 10⁶ cells/well in 6-well plates and treated with either IgM or Ha13 neutralizing antibody at 50 and 100 µg/ml, respectively for 2 hours. Cells were then scraped into ice-cold PBS, centrifuged at 4°C for 30 s at 16 000g, and cell pellets re-suspended in RIPA buffer with protease and phosphatase inhibitors (10mM Tris-HCl pH 7.5, 500 mM NaCl, 0.1% SDS, 1% NP40, 1% sodium deoxycholate, 2 mM EDTA, 2 mM Na₂VO₄, 2 mM Na4P2O7, 2mM NaF) and stored at -80°C. Protein content was assayed by the BCA protein Assay Kit (Thermo-Fisher, catalogue n° 23225). Total proteins were fractionated by SDS electrophoresis on NuPage 4-12% Bis-Tris gel and transferred onto nitrocellulose membranes (0.45µm, Biorad, catalogue n° NW04120BOX). Western blotting was performed using equal amounts of protein from each treatment group with the relevant antibodies against pSMAD proteins 1/5 (Rb polyclonal, Cell Signaling, catalogue n° 41D10, 1:1000). Actin (anti-β-actin, 1:5000; Sigma-Aldrich, catalogue n° A2228) was used as the reference protein and loading control. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Gt anti-rabbit HRP, DAKO, catalogue n° P0448) diluted 1:2000 in 5% non-fat milk TBST for 1 hr at RT.

## Electrophysiology

Electrophysiological recordings were performed on living brain slices from 8 to 12-week-old PAMH;Gnrh-GFP+/- and CNTR;Gnrh-GFP+/- mice. Mice were put under isoflurane anesthesia and killed by decapitation. The brain was dissected and rapidly placed in ice-cold aCSF containing: 120 mM NaCl, 3.2 mM KCl, 1 mM NaH₂PO₄, 26 mM NaHCO₃, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose (300 mOsm, pH 7.4) and bubbled with 95% O₂ to 5% CO₂. 200 µm coronal slices containing the rostral preoptic area were cut using a VT1200 vibratome (Leica). Slices were incubated at 34°C in oxygenated aCSF for a recovery period of 1 hour and then placed at room temperature until patch-clamp recording. Individual brain slices were placed in a submerged recording chamber (Warner Instruments) and continuously perfused at a rate of 3mL/min with oxygenated aCSF maintained at 32.8°C by a heater controller (TC-344C-Warner Instrument). GnRH neurons were visualized under x10 and x40 magnification using an upright fluorescence
microscope with infrared differential interference contrast (DM-LFSA, Leica) and an ORCA-Flash4.0 digital CMOS camera (Hama-
matsu). Recording pipettes were pulled from borosilicate glass capillaries (1.5 mm outer diameter, 1.12 mm inner diameter; World
Precision Instruments) using a P1000 Flaming Brown puller (Sutter Instruments) and had a resistance of 7-9 MΩ when filled with
an internal solution containing 140 mM K-gluconate, 10 mM KCl, 1 mM EGTA, 2 mM Na₂-ATP, and 10 mM HEPES, pH 7.3, with
KOH. Whole-cell patch-clamp recordings were performed in current-clamp mode using a Multiclamp700B Amplifier, digitized
with the Digidata 1322A interface, and acquired with pClamp 10.2 software (Molecular Devices). For antibody functional character-
ization, 25 µg/mL of Ha13 (anti-AMHR2 IgM) was added to the aCSF bathing medium by the use of the perfusion system after stable
baseline recording. Recordings were analyzed using Clampfit 10.2 pClamp software (Molecular Devices). For each recording, the
membrane potential and mean firing rate were determined before and during the bath application of Ha13. Only cells that showed
less than 20% change in access resistance throughout the recording were included in this study. The junction potential was corrected
in the data analysis.

In vivo studies using anti-AMHR2 neutralizing antibody

For *in vivo* Ha13 functional validation, we administered daily intraperitoneal injections of either Ha13 or mouse IgM (4 mg/Kg/injection) to infantile PAMH female mice from P10 to P15. This dose of antibody was chosen based on previous works using another anti-AMHR2 neutralizing antibody to suppress AMH proliferative effect on ovarian adenocarcinoma in mice.⁷⁸ We next characterized the effects of the Ha13 treatment applied during minipuberty on the neuroendocrine/reproductive and metabolic outcome of the adult animals, respectively at 3 months and 6 months.

For the intergenerational study, PAMH female offspring (F1) were administered daily with intraperitoneal injections of either Ha13 (4 mg/Kg/injection) or PBS from P10 to P15 and then mated at P60 with CNTR males (generated by prenatally treating gestating mice with PBS from E16.5 to E18.5 as described above) to generate PAMH F2 offspring. CNTR F2 female offspring were generated by breeding CNTR females (generated by prenatally treating gestating mice with PBS from E16.5 to E18.5 and treated at minipuberty with PBS) with CNTR unrelated males (generated by prenatally treating gestating mice with PBS from E16.5 to E18.5). F2 female offspring were subjected to phenotypic testing as described above. The exact number of mice used for each procedure and their sex and age are given in the figure legends.

RNA extraction and RT-qPCR

Ovaries, perigonadal fat, liver and pancreas were harvested from diestrous female mice and snap-frozen in liquid nitrogen. Frozen tissues were homogenized using 1 ml of Lysis Buffer from the EZNA total RNA kit II (Omega, catalogue n°R6934-02) with a tissue homogenizer and total RNA was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen; catalogue n° 74804) following the manufacturer's instructions. For gene expression analyses, cDNA was synthesized from 1000 ng of total RNA using the High-Capacity RNA-to-cDNA kit (Applied Biosystems, catalogue n° 4387406) using the manufacturer's recommended cycling conditions. Real-time PCR was carried out on Applied Biosystems QuantStudio3 7900HT Standard Real-Time PCR system using exon-boundary-specific TaqMan Gene Expression Assays (Termo-Fisher Scientific). Data were analyzed by using the 2^ΔΔCT^ method⁷⁹ and normalized to housekeeping gene *Rn18S* (Thermo-fisher #Mm03928990) *ActB* (Thermo-Fisher #Mm00607939-s1) levels. The other primers were directed against *Esr1* (Thermo-Fisher #Mm00433149_m1); *Pomc* (Thermo-Fisher #Mm00435874_m1); *Agrp* (Thermo-Fisher #Mm00475829_g1); *Kiss1* (Thermo-Fisher #Mm03058560_m1); *Pgr* (Thermo-Fisher #Mm00435628_m1); *Ar* (Thermo-Fisher #Mm00442688_m1); *Amhr2* (Thermo-Fisher #Mm00513847_m1). Values are expressed relative to control values, as appropriate, set at 1.

In vivo biodistribution of Ha13

To image the biodistribution of Ha13 *in vivo*, anesthetized P16 female mice were intravenously injected (orbital venous sinus) with 30 µl of anti-AMHR2-BF antibody (Ha13, 4mg/Kg) conjugated to a fluorophore (ATTO-565). Animals were sacrificed 5 min or 30 mins later. The brain, ovaries, visceral adipose tissue and pancreas were dissected and immersion-fixed in 4% PFA solution overnight at 4°C, cryoprotected overnight in PBS/sucrose 30% at 4°C, embedded in OCT embedding medium (Tissue-Tek), and frozen and stored at - 80 °C until cryosectioning. Tissues were cut at 35-µm-thick sections and processed as free-floating sections for immunofluorescence. At the end of the immunofluorescence experiments, sections were coverslipped with Fluoromount-GTM with DAPI (Invitrogen, Cat: 00-4959-52) and stored at 4°C.

RNAscope fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was performed on fresh-frozen brain sections of the ME of adult female mice using the RNA-
scope Multiplex Fluorescent Kit v.2, according to the manufacturer's instructions (ACD, Bio-techne, catalogue n° 323270). Specific
probes were used to detect *Pomc* (ACD, Bio-techne, catalogue n° 314081), *Agrp1* (ACD, Bio-techne, catalogue n° 400711), and *Esr1*
(ACD, Bio-techne, catalogue n° 478201) mRNAs. Hybridization with a probe against the Bacillus subtilis dapB gene (ACD, Bio-
techne, catalogue n° 320871) was used as a negative control. Image acquisition was performed using an inverted confocal micro-
scope (LSM 710, Zeiss).
**Ethics statement**

Animal studies were approved by the Institutional Ethics Committees of Care and Use of Experimental Animals of the Universities of Lille 2 (France). All experiments were performed in accordance with the guidelines for animal use specified by the European Union Council Directive of September 22, 2010 (2010/63/EU) and the approved protocol (APAFIS# 29172-2020121811279767 v5) by the Ethical Committee of the French Ministry of Education and Research. All efforts were made to minimize animal suffering and animal care was supervised by veterinarians and animal technicians skilled in rodent healthcare and housing.

**Statistics and data analysis**

All analyses were performed using 10.0.2 GraphPad Prism software (GraphPad; San Diego, CA, USA). The normality of each group was determined by Shapiro-Wilk test. We did not assume equal variances. Statistically significant *P* values were considered when *P* < 0.05. No statistical methods were used to pre-determine sample sizes but our sample sizes were similar to those reported in previous publications.<sup>22,27,80</sup> Animals were randomized to experimental groups and investigators were blinded to group allocation during data collection and analyses. For each experiment, replicates are described in the figure legends. No samples were excluded from the analyses. For the normal distribution, data were compared using unpaired two-sided Student's *t*-test and one-way and two-way ANOVA followed by Tukey's or Holm-Sídák's multiple comparisons test tests. For non-normally distributed values, Mann-Whitney U and Kruskal-Wallis tests were used. The numbers of biologically independent experiments, sample size, *P* values, and ages and sexes of the animals together with the details of the statistical tests used for the analyses are all indicated in the main text or figure legends.