DOI: 10.6076/d1xw21
title: Supplementary data for: Reproductive deficits induced by prenatal anti-Mullerian hormone exposure require androgen receptor in kisspeptin cells

description: Polycystic ovary syndrome (PCOS) is a common reproductive disorder
 characterized by elevated androgens and anti-Mullerian hormone (AMH).
 These hormones remain elevated throughout pregnancy, and potential effects
 of hormone exposure on offspring from women with PCOS remain largely
 unexplored. Expanding on recent reports of prenatal AMH exposure in mice,
 we have fully characterized the reproductive consequences of prenatal AMH
 (pAMH) exposure throughout the lifespan of first- and second-generation
 offspring of both sexes. We also sought to elucidate mechanisms underlying
 pAMH-induced reproductive effects. There is a known reciprocal
 relationship between AMH and androgens, and in PCOS and PCOS-like animal
 models, androgen feedback is dysregulated at the level the hypothalamus.
 Kisspeptin neurons express androgen receptors and play a critical role in
 sexual development and function. We therefore hypothesized that
 pAMH-induced reproductive phenotypes would be mediated by androgen
 signaling at the level of kisspeptin cells. We tested the pAMH model in
 kisspeptin-specific androgen receptor knockout (KARKO) mice and found that
 virtually all pAMH-induced phenotypes assayed are eliminated in KARKO
 offspring compared to littermate controls. By demonstrating the necessity
 of androgen receptor in kisspeptin cells to induce pAMH phenotypes, we
 have advanced understanding of the interactions between AMH and androgens
 in the context of prenatal exposure, which could have significant
 implications for children of women with PCOS.
description: Mice: Mice
 were group-housed on a 12:12 light/dark cycle with ad libitum standard
 chow diet and water in a temperature-controlled room. All experimental
 procedures were approved by the University of California, San Diego
 Institutional Animal Care and Use Committee. Initial characterization of
 first (F<sub>1</sub>) and second (F<sub>2</sub>)
 generation pAMH offspring was completed using PER2::LUC transgenic mice
 (RRID:IMSR_JAX:006852) maintained in our local colony on a C57BL/6
 background. Mice with the PER2::LUC transgene are viable and fertile with
 no developmental or morphological differences compared to wild-type
 littermates. To
 generate kisspeptin-specific AR knockout mice, we crossed together a
 previously established <i>Ar</i>-flox mouse (RRID:
 IMSR_NM-CKO-0010) with a validated <i>Kiss1</i>-Cre mouse
 (RRID: IMSR_JAX:023426) to generate a kisspeptin-specific AR knockout
 (KARKO) mouse.
 <i>Ar</i><sup>flox/flox</sup>;<i>Kiss1</i><sup>Cre+</sup> females were bred to <i>Ar</i><sup>flox/Y</sup> to produce <i>Ar</i><sup>flox/flox</sup>;<i>Kiss1</i><sup>Cre+</sup> (KARKO) conditional knockouts or <i>Ar</i><sup>flox/flox</sup>;<i>Kiss1</i><sup>Cre-</sup> control littermates (Ctrl). KARKO Rosa-tdT reporter mice were generated by crossing KARKO or <i>Kiss1</i>-Cre mice with Ai9 Rosa-tdTomato mice (RRID: IMSR_JAX:007909), to create mice in which <i>Kiss1</i>-Cre expressing cells were identifiable by tdTomato expression. Mice used for these experiments were either <i>Ar</i><sup>flox/flox</sup>;<i>Kiss1</i><sup>Cre+</sup>;<i>RosatdT</i><sup>+/- </sup>or <i>Ar<sup>wt/wt</sup></i>;<i>Kiss1</i><sup>Cre+</sup>;<i>RosatdT</i><sup>+/- </sup>(Ctrl). Generation of pAMH offspring: To generate pAMH offspring, virgin adult females (3-4 months old) were paired with adult males (3-4 months old) and checked for copulatory plug as indication of embryonic day (E) 0.5. Timed-pregnant dams were randomly assigned to receive daily intraperitoneal injections from E16.5-E18.5 of either vehicle (VEH) or 0.12 mg/kg/day AMH (recombinant human MIS/AMH protein, R&D Systems, #1737-MS), as previously reported. AMH was reconstituted in stocks of 100 μg/ml according to manufacturer’s instructions (4 mM HCl + 0.1% BSA) and diluted into 0.01 M PBS pH 7.4. Dams received different volume injections based on weight (100 μl/10 g). Experimenters were blinded to experimental groups. F<sub>1</sub> offspring of both sexes were born into experimental pAMH or VEH control groups, then weaned at postnatal day (P) 21 to begin reproductive phenotyping. Minimum sample sizes were determined based on previous publications of reductive phenotypes following prenatal hormone exposure, and two separate cohorts pAMH and VEH offspring were generated to confirm reproducibility of results. F<sub>2</sub> pAMH and VEH offspring were the product of the F<sub>1</sub> fertility assay, in which F<sub>1</sub> pAMH and VEH females were paired with experimentally naïve wild-type C57BL/6 males (Envigo). Litters from a random subset (n=4) of pAMH and VEH pairings in the last month of the assay (when F<sub>1</sub> dams were around 5 months old) were kept after weaning and phenotyped.             Virgin adult KARKO (<i>Ar</i><sup>flox/flox</sup>;<i>Kiss1</i><sup>Cre+</sup>) females (3-4 months old) were set up in timed matings with adult control (<i>Ar</i><sup>flox/Y</sup>;<i>Kiss1</i><sup>Cre-</sup>) males (3-4 months old), and received AMH or VEH injections as described above. F<sub>1</sub> offspring of both sexes and genotypes were born into experimental pAMH or VEH control groups, then weaned at P21 to begin phenotyping. Germline-recombined mice were identified and removed from the experiment. Body weight (g) was measured at various ages throughout postnatal development. Fluorescent in situ hybridization: <em>Ar </em>and <em>Kiss1 </em>were analyzed in wildtype gonads using RNAScope according to our previous procedure (Lavelle et al. 2021, PMCID: PMC8319105). Briefly, 20  μm serial coronal sections were collected from fresh frozen testis and ovary.  Sections were fixed in chilled 4% PFA, washed two times with 1X PBS, and dehydrated through a series of ethanol washes ranging from 50% to 100% ethanol. RNAscope Multiplex Fluorescent v2 Assay (Advanced Cell Diagnostic, 323100) was performed according to manufacturer's instructions with the following probes: Mm-Kiss1 (500141) and Mm-FosAR-C2 (316991). Sections were counterstained with DAPI and coverslipped with ProLong Gold (Invitrogen). .nd2 files can be opened in Fiji (ImageJ) or similar software.