Alain N'guessan

Elias Fattal

David Chapron

Claire Gueutin

Armand Koffi

Nicolas Tsapis
email: nicolas.tsapis@u-psud.fr

Dexamethasone palmitate large porous particles: A controlled release formulation for lung delivery of corticosteroids

Keywords: Dexamethasone palmitate, Dexamethasone, large porous particles, spray drying, lung delivery, pharmacokinetics

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Asthma is a chronic inflammatory disorder of the airways, usually associated with airway hyper-responsiveness and variable airflow obstruction, that can be reversed either spontaneously or under treatment [START_REF] Asthma | Global Initiative for Asthma. Global strategy for asthma management and prevention[END_REF]. Allergen sensitization is an important risk factor for asthma [START_REF] Bateman | Global strategy for asthma management and prevention: GINA executive summary[END_REF] and for the past 40 years, the prevalence of asthma has increased in all countries in parallel with that of allergy. It is estimated that as many as 300 million people of all ages in the world suffer from asthma [START_REF] Asthma | Global Initiative for Asthma. Global strategy for asthma management and prevention[END_REF]. Anti-asthmatic treatments can be administered via different routes of administration such as parenteral or oral (prednisone) but the pulmonary route is often preferred, as it allows delivering the medication right to the site of action where it is needed. Anti-asthmatic treatments vary depending on the extent of the disease but for quick relief they mostly consist in a combination of beta2 adrenergic agonists and corticosteroids. Beta2 adrenergic agonists such as albuterol, levalbuterol, metaproterenol or terbutaline are used as bronchodilators helping to relax airway muscles within 5 minutes [START_REF] Spina | Current and novel bronchodilators in respiratory disease[END_REF]. They lead to an increase of the airflow, facilitating patient breathing. Beta2 adrenergic agonists help relieving asthma symptoms for 3 to 6 hours. Corticosteroids such as beclomethasone propionate, fluticasone furoate or propionate, budesonide, ciclesonide, or flunisolide are used for their anti-inflammatory effects and required several administrations per day. Inhaled corticosteroids are effective in the treatment of asthma because of their ability to interfere with multiple inflammatory processes involved in the asthmatic pathology [START_REF] Allen | Inhaled corticosteroids: past lessons and future issues[END_REF][START_REF] Barnes | Efficacy and Safety of Inhaled Corticosteroids in Asthma[END_REF][START_REF] Crim | A review of the pharmacology and pharmacokinetics of inhaled fluticasone propionate and mometasone furoate[END_REF]. Inhalation offers advantages for the treatment of asthma using corticosteroids as compared with the systemic route. It helps, with efficiency and optimal tolerance; to locally treat a condition that requires the use of high doses of active product by systemic route. The inhaled corticosteroids allow a rapid onset of action and induce fewer side effects than does administration by other routes [START_REF] Beck-Broichsitter | Pulmonary drug delivery with aerosolizable nanoparticles in an ex vivo lung model[END_REF][START_REF] Boisson | Comparison of Intrapulmonary and Systemic Pharmacokinetics of Colistin Methanesulfonate (CMS) and Colistin after Aerosol Delivery and Intravenous Administration of CMS in Critically Ill Patients[END_REF]. However, these benefits are often associated with limited lung deposition and short duration of action because of respiratory protection mechanisms [START_REF] Hanania | Adverse effects of inhaled corticosteroids[END_REF].

Thus, the ideal inhaled corticosteroid should have: long residence time in the lung; intrinsic activity; low oral bioavailability and high systemic clearance resulting in negligible systemic side effects. However, most currently marketed inhaled corticosteroids still require several daily administrations [START_REF] Burgt | Efficacy and safety overview of a new inhaled corticosteroid, QVAR (hydrofluoroalkane-beclomethasone extrafine inhalation aerosol), in asthma[END_REF][START_REF] Czock | Pharmacokinetics and Pharmacodynamics of Systemically Administered Glucocorticoids[END_REF][START_REF] Hübner | Comparative Pharmacology, Bioavailability, Pharmacokinetics, and Pharmacodynamics of Inhaled Glucocorticosteroids[END_REF]. To modify corticoids residence time in the lungs, an appropriate formulation can be developed to deliver these molecules.

Lung delivery can be achieved by nebulization of drug solutions [START_REF] Rodrigo | Advances in acute asthma[END_REF] or suspensions of nanoparticles [START_REF] Sahib | Rehydrated sterically stabilized phospholipid nanomicelles of budesonide for nebulization: physicochemical characterization and in vitro, in vivo evaluations[END_REF], or using either metered dose inhalers [START_REF] Berger | Bronchodilation with mometasone furoate/formoterol fumarate administered by metered-dose inhaler with and without a spacer in children with persistent asthma[END_REF] or dry powders for inhalation [START_REF] Chawes | Systemic exposure to inhaled beclometasone/formoterol DPI is age and body size dependent[END_REF]. Current formulation approaches of corticosteroid dry powders consist in micronizing the active pharmaceutical ingredient and blend it with lactose as a carrier, which characteristics can be optimized [START_REF] Buttini | Back to basics: the development of a simple, homogenous, two-component dry-powder inhaler formulation for the delivery of budesonide using miscible vinyl polymers[END_REF][START_REF] Donovan | Influence of size and surface roughness of large lactose carrier particles in dry powder inhaler formulations[END_REF][START_REF] Hoppentocht | Technological and practical challenges of dry powder inhalers and formulations[END_REF]. Alternatively, large porous particles (LPPs), characterized by geometric sizes greater than 4-5 µm and mass densities lower than 0.4 g/cm 3 , have been introduced for both local and systemic applications by the pulmonary route to the lungs [START_REF] Cruz | Formulation and in vivo evaluation of sodium alendronate spray-dried microparticles intended for lung delivery[END_REF][START_REF] Edwards | Bioengineering of Therapeutic Aerosols[END_REF][START_REF] Edwards | Large porous particles for pulmonary drug delivery[END_REF][START_REF] Gervelas | Direct lung delivery of a dry powder formulation of DTPA with improved aerosolization properties: Effect on lung and systemic decorporation of plutonium[END_REF][START_REF] Pham | Pulmonary delivery of pyrazinamide-loaded large porous particles[END_REF]. A major advantage of LPPs relative to conventional inhaled therapeutic aerosol particles is their aerosolization efficiency [START_REF] Dunbar | Dispersion and Characterization of Pharmaceutical Dry Powder Aerosols[END_REF][START_REF] Edwards | Bioengineering of Therapeutic Aerosols[END_REF]. This allows the supply of large drug masses using a simple inhalation device [START_REF] Edwards | Delivery of biological agents by aerosols[END_REF]. These characteristics suggest that inhalation of LPPs may be beneficial in the treatment of asthma by delivering the corticosteroids directly to the primary site of inflammation to achieve therapeutic local drug concentrations with low systemic exposure.

To favor prolonged efficacy of the corticosteroid it appeared interesting to combine LPPs with a corticosteroid prodrug. We have chosen to use a prodrug of dexamethasone since this corticosteroid is often used a reference to evaluate the efficacy of other molecules [START_REF] Kelly | Comparison of inhaled corticosteroids: an update[END_REF]. Dexamethasone palmitate is of particular interest since its aliphatic chain may help modulating its release from lipid-based LPPs. As excipients,dipalmitoyl-snglycero-3phosphatidylcholine (DPPC) and hyaluronic acid (HA) have been chosen due to their biocompatibility, biodegradability and ability to yield porous particles [START_REF] Gomez-Gaete | Supramolecular organization and release properties of phospholipid-hyaluronan microparticles encapsulating dexamethasone[END_REF]. DXP encapsulation into DPPC-HA microparticles was optimized by spray drying, varying DXP concentration with respect to the lipid content. Physico-chemical characterization of the powders will be presented as well as aerosolization efficacy using a multistage liquid impiger and drug release in sink conditions. Once the formulation optimized, pharmacokinetics of DXP and its active metablite dexamthasone (DXM) were determined in plasma and epithelial lung fluid after intratracheal administration of the DXP powder by insufflation.

The aim of the work is therefore to optimize the formulation of DXP by spray-drying into large porous particles and to evaluate their pharmacokinetics and lung distribution in vivo in rats.

We expect the delivery of a lipidic corticosteroid prodrug will allow to prolong drug residence in the lungs.

Materials and methods

Materials

Dexamethasone palmitate (DXP) was provided by Interchim (France), Dexamethasone (DXM) and Dexamethasone acetate (DXA) were provided by ChemosGmbH (Germany) , Testosterone decanoate (TC) was provided by Sigma-Aldrich (France), 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC) by Corden Pharma (Switzerland) and hyaluronic acid, sodium salt 95% (HA) (MW = 1 000 kDa) by Acros Organics. Acetonitrile was of high-performance liquid chromatography (HPLC) grade. All chemicals used were of analytical grade. Organic solvents were provided by Carlo Erba (Italy) and were of analytical grade when not specified. Water was purified using a RIOS/MilliQ system from Millipore (France).

Microparticles preparation

DXP-loaded microparticles were prepared by spray drying using a mini spray-dryer BÜCHI B-290 (France) equipped with a 0.7 mm diameter two-fluid nozzle, which operates in a cocurrent mode according to conditions detailed in Table 1. An aqueous solution of HA was prepared by dissolving 200 mg of HA into 150 ml of water upon magnetic stirring at room temperature. An ethanolic solution was prepared by dissolving DPPC and DXP into 350 ml of ethanol absolute to obtain a final amount of 800mg of lipophilic compounds. The weight percent of DXP was varied between 0 and 15%. Ethanolic and aqueous solutions were then mixed at a ratio of 70/30 (v/v) prior to spray-drying and the mixture maintained under moderate stirring while fed into the spray-dryer. The yield was calculated as a percentage of the mass of the powder collected divided by the initial mass of solids in the solution prior to spray-drying. (Eq. 1)

Tap density and aerodynamic diameter

Powder tap density (ρ) was determined using a tapping apparatus (Pharma test PT-TD1).

Accurately weighed powder samples were filled into a 5 ml graduated cylinder and the height measured following 1000 taps which allowed the density to plateau [START_REF] Pharmacopoeia | European Pharmacopoeia[END_REF].

Assuming an efficient packing, the tap density of monodisperse spheres is approximately a 21% underestimate of the true particle density due to the void spaces between particles.

Although polydispersity may reduce the void volume between particles, this is probably counterbalanced by an imperfect packing [START_REF] Vanbever | Formulation and physical characterization of large porous particles for inhalation[END_REF]. Measurements were performed in duplicate.

Scanning electron microscopy

Scanning electron microscopy (SEM) was performed using a LEO1530 microscope (LEO Electron Microscopy Inc., Thornwood, USA) operating between 1 and 3 kV with a filament current of about 0.5 mA. Powder samples were deposited on carbon conductive double-sided tape (Euromedex, France) and were coated with a palladium-platinum layer of about 4 nm using a Cressington sputter-coater 208HR with a rotary planetary-tilt stage, equipped with a MTM-20 thickness controller.

Differential scanning calorimetry (DSC)

The DSC analysis was performed on hydrated samples (0 to 15% DXP) on a differential scanning calorimeter (DSC7, Perkin-Elmer, USA). About 10 mg of powder was hydrated with 100 µl of water 12 h before experiments. Then about 15 mg of hydrated samples were loaded into 40 µl aluminum pans and analyzed. The DSC runs were conducted from 20 to 80 °C at a rate of 5 °C/min. Calibration was achieved using Indium (T onset = 156.60 °C) as well as n-decane (T onset = -29.66 °C). Experiments were performed at least in duplicate. Enthalpies were normalized with respect to DPPC weight in the sample.

Aerosolization characterization

The aerodynamic particle size distribution was determined using a Multi-Stage Liquid Impinger (MSLI). A dry powder inhalation device (Aerolizer®, Novartis, Switzerland) was filled with a size 3 capsule made of hydroxypropylmethyl cellulose (HPMC, LGA France) containing the powder. The test was carried out at 60 L/min for 4 s with a ratio of pressure P3/P2 < 0.5.

Five capsules loaded with an average of 10 mg powder were taken for each test. Drug deposition in the device, the piece mimicking the throat, the four stages and the filter was determined by HPLC analysis. For accuracy, each test was repeated three times. The particles with aerodynamic diameters smaller than 5 µm and 3.1 µm were determined by interpolation from the cumulative amount of respective stages and considered as the fine particle fraction (FPF) (%) and as the alveolar particle fraction (AF) (%) expressed as a percentage of the loaded/nominal dose [START_REF] Hinds | Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles[END_REF][START_REF] Pharmacopoeia | European Pharmacopoeia[END_REF].

DXP release from microparticles

Drug release from microparticles prepared with 5% DXP was carried out the following way: 25 mg of microparticles were accurately weighted, resuspended in 25 mL of PBS (corresponding to a final DXP concentration of 50 µg/mL) and the preparation was protected from light and placed at 37 °C under magnetic stirring. At each time point, the suspension was vortexed and 1 mL was collected. 100 µL of the vortexed sample was diluted to the tenth (1/10) in acetonitrile, in order to determine total DXP concentration. The remaining 900 µL were centrifuged (13.000 rpm, 15 min) (Mini Spin Eppendorf centrifuge) and 100 µL of the supernatant were diluted to the tenth in acetonitrile, to determine DXP concentration in the supernatant. After filtration (0.45 μm PVDF filter), samples were stored at 4°C until analysis by HPLC as described below. Experiments were performed in duplicate.

Pharmacokinetics and bronchoalveolar distribution study

Sprague Dawley male rats with average weight of 300g were obtained from Envigo (Gannat, Rats were anesthetized with an intraperitoneal injection of a mixture of ketamine (100 mg.kg - 1 ) and xylazine (10 mg.kg -1 ) and then intratracheally administered with 3mg of DXP powder corresponding to 150g of DXP using a Microsprayer (PennCentury, Philadelphia, PA) [START_REF] Kohno | Antedrug budesonide by intrapulmonary treatment attenuates bleomycin-induced lung injury in rats with minimal systemic adverse effects[END_REF][START_REF] Neuhaus | Comparison of metabolites in exhaled breath and bronchoalveolar lavage fluid samples in a mouse model of asthma[END_REF][START_REF] Wijagkanalan | Enhanced Anti-Inflammation of Inhaled Dexamethasone Palmitate Using Mannosylated Liposomes in an Endotoxin-Induced Lung Inflammation Model[END_REF]. Powder was delivered through the insufflation device by rapidly pushing a 2 cm 3 bolus of air through the device. After administration, rats were supported vertically for 1 min. Blood and bronchoalveolar fluid were collected at t=30min, 1h, 2h, 3h, 4h, 6h, 18h and 24h after administration. For each rat, blood was taken at 3 different time points via the jugular vein [START_REF] Khoder | Removal of residual colonic ciprofloxacin in the rat by activated charcoal entrapped within zinc-pectinate beads[END_REF][START_REF] Nave | Deposition and metabolism of inhaled ciclesonide in the human lung[END_REF]. At least 3 different rats were used for each time point. Before taking the blood, the rats were anesthetized by isoflurane inhalation. Blood samples (1mL) were collected in citrated tubes and immediately separated at 4000 rpm during 5 min at room temperature (20°C) using a microcentrifuge (Minispin Eppendorf). Plasma was aspirated and frozen at -80°C until further analysis. Bronchalveolar lavage fluid (BALF) was collected by lavaging lungs three times with 1mL of PBS per intratracheal lavage. The lungs were massaged and the fluid withdrawn immediately and collected in centrifuge tube kept on ice. The rats were euthanized by cardiac puncture under deep isoflurane anesthesia before BALF collection. The trachea was cannulated using an 18 gauge needle adaptor for subsequent injection and retrieval of BALF.

Then BALF collected was frozen at -80°C until further analysis. PK of DXP and DXM in plasma and ELF were evaluated according to a non compartmental model. PK parameters were calculated using GraphPad Prism (Version 5 software for windows, Inc.). The AUC was calculated from time zero to the last blood collection time (24h).

Extraction of DXP or DXM from plasma and BALF.

Extraction of DXP and DXM from either plasma or BALF was performed as follows (Alhareth et al., 2012). In an amber glass tube to protect DXP and DXM from light (REF), 100µL of plasma or BALF and 100µL of internal standards (testosterone decanoate (TD) for DXP and dexamethasone acetate (DXA) for DXM, both at 4g/mL in acetonitrile were added and vortexed during 30 seconds. Then 3mL of a chloroform:methanol mixture (9:1, v:v) were added in the tube. Sample was vortexed during 3 min to obtain protein precipitation and then was centrifuged during 10 min at 10,000 rpm using a min centrifuge (minispin eppendorf, France). The organic phase in the bottom was transferred into a clean amber vial and evaporated to dryness under a stream of nitrogen at 30°C. The residue was then reconstituted into 100µL of acetonitrile and vortexed prior to analysis using HPLC conditions described below.

HPLC method

Quantification of DXM or DXP concentration in microparticles, in PBS (release), in plasma and Laser diffraction granulometry confirmed SEM observations with a median volume geometric diameter D50 between 12.3 and 13.7 µm, and a span between 2 and 3, with no obvious trend as DXP content increases (Figure 2A and2B). The tap density varies between 0.053 and 0.066g/cm 3 , independently of DXP content (Figure 2C). The low tap density indicates microparticles present some porosity as observed previously with DXM using the same excipients [START_REF] Gomez-Gaete | Supramolecular organization and release properties of phospholipid-hyaluronan microparticles encapsulating dexamethasone[END_REF]. The powder yield however showed a decrease from 34% to 26% as DXP concentration increased from 0 to 15%. This yield decrease is associated with an increase in powder cohesion. This result is in agreement with previous ones obtained when incorporating dexamethasone in a similar formulation (Gomez-Gaete et al., 2008): the higher the drug content, the more cohesive the powder. The aerodynamic behavior of the powder was then evaluated in terms of emitted fraction, fine particle fraction and alveolar fraction. The emitted fraction was 98±2% independently of the formulation. Contrary to microparticle median size, span or tap density, a clear trend can be observed for both the FPF and the AF that decrease as DXP weight percent increases (Figure 3). The FPF and AF varies from 40±7% to 14±1% and from 30±6% to 7±1%, respectively. This decrease is most probably related to powder cohesion as described above. Given these results, we decided to focus on microparticles formulated with 5% DXP which lead to the highest FPF and AF with a good yield.

In vitro release of DXP

DXP release from 5% DXP microparticles was followed during several days. About 2% of DXP was immediately released after vortexing. This burst effect corresponds to a concentration of 1µg/mL. Then a gradual and rather slow release was observed for 21 days from 2% to about 5% (Figure 4). It should be noted that no dexamethasone was detected during this experiment. Although the release conditions are very different from what would happen in vivo, results indicate a sustained release from the microparticles. In vivo, we expect that esterases and lipases would accelerate considerably the release, as observed by others [START_REF] Lu | Nanoparticles containing anti-inflammatory agents as chemotherapy adjuvants II: role of plasma esterases in drug release[END_REF]. In addition, one should note that the solubility of DXP in PBS was not measurable.

The fact that we detect DXP in our release experiment might be favoured by the presence of DPPC. Although this specific phospholipid is very hydrophobic, it possesses a critical micellar concentration of 0.46nM at 20°C [START_REF] Smith | The critical micelle concentration of L-dipalmitoylphosphatidylcholine in water and water-methanol solutions[END_REF]. The very few micelles probably help solubilising DXP.

Fig 4:

In vitro DXP release from 5% DXP microparticles in PBS at 37°C.

Differential scanning calorimetry (DSC)

To better understand DXP release, DSC was performed on hydrated samples to assess the possible interactions between DPPC and DXP (0% to 15%). For hydrated microparticles made without DXP, the DSC thermogram exhibit a very flat pretransition of DPPC due to the interaction with HA and the typical main phase transition of DPPC at Tonset= 42.3°C with an enthalpy ΔH=45.8J/g (Figure 5). This result is in agreement with those obtained for similar microparticles [START_REF] Gomez-Gaete | Supramolecular organization and release properties of phospholipid-hyaluronan microparticles encapsulating dexamethasone[END_REF]. When hydrated, lipid bilayers swell forming typical liquid crystalline phases of DPPC [START_REF] Gomez-Gaete | Supramolecular organization and release properties of phospholipid-hyaluronan microparticles encapsulating dexamethasone[END_REF]. As DXP content increases, the pretransition does not appear anymore. The onset temperature of the main DPPC transition shifts down to 39°C and the enthalpy of DPPC decreases (Figure 5). At 15% DXP, one can also observe the appearance of a thermal event with a T onset of 55.7ºC. We do believe this thermal event corresponds to a phase transition of DXP, although nothing can be found in the literature. Indeed when DSC experiments were conducted with higher DXP content, the peak became more important (data not shown). The combination of the decrease of the onset temperature of DPPC and of its enthalpy (Fig 6) is the signature of the insertion of DXP within the DPPC bilayer as observed for cholesterol [START_REF] Kessel | Interactions of Cholesterol with Lipid Bilayers: The Preferred Configuration and Fluctuations[END_REF]. This insertion is rather logical since DXP possesses an aliphatic chain of 16 carbons, the exact same length as DPPC aliphatic chains. DXP insertion within bilayers further explains the slow release observed upon incubation.

In vivo experiments

The extraction method for biological samples was satisfactory with extraction yields for DXP of 84 ± 2% for extraction from BALF, and 66 ± 7% for extraction from plasma, as well as for DXM with 87 ± 4% extraction from BALF and 83 ± 7% extraction from plasma. Limits of detection (LOD) and quantification (LOQ) for each molecule are presented in Table 2. The results of DXP and DXM obtained in BALF were converted to epithelial lining fluid (ELF) concentrations according to the theoretical volume of ELF reported in the literature (30 L/kg) and to the volume of PBS used for bronchoalveolar lavage (1mL). Thus, for a rat of 300g, the dilution factor determined by calculation was 333 [START_REF] Gontijo | Biopharmaceutical Characterization of Nebulized Antimicrobial Agents in Rats: 1. Ciprofloxacin, Moxifloxacin, and Grepafloxacin[END_REF]. All PK parameters are presented in Table 3 for both DXP and DXM. The results of DXP and DXM concentrations obtained in ELF are presented in figure 6 as semi-log plots. The maximal concentration of DXP in the ELF is obtained 30 min after administration with a value C DXP-ELF- max = 10794 µg/mL. It corresponds to about 65% of the theoretical initial concentration given the administered dose. Then the DXP concentration decreases and can be described by three different phases. The first phase occurs between 30min and 3h corresponding to a slow decrease from 10794 µg/mL (65% of initial theoretical concentration) to 5657µg/mL (34% of initial theoretical concentration). This first phase can be fitted by a single exponential decrease y= y A exp(-k 1 /t) with k 1 = 0.247 h -1 and a characteristic half-time t 1/2A = = 2.8±0.4 h.

The second phase corresponds to a faster decrease between 3h and 6h from 5657 µg/mL to 204 µg/mL, which corresponds to 1.2% of the initial theoretical concentration. This second decrease can be fitted by a single exponential decrease y= y B exp(-k 2 /t) with k 2 =1.09h -1 and a characteristic half-time t 1/2B = = 0.63±0.01 h. The last phase suggests a slow elimination of DXP from ELF although it cannot be modeled since the last two concentrations at 18h and 24h

were below the LOQ of DXP in ELF. For DXM in ELF, one can observe a rapid decrease of concentration between 30 min and 4h, then the concentration remains stable but very low below the LOQ. The decrease can be fitted by a single exponential decrease y= y A exp(-k 1 /t) with k 1 =0.743h -1 and a characteristic half-time t 1/2 = = 0.93±0.20 h. The area under the curve (AUC) of DXP in ELF was 26 703 µg.h/mL whereas for DXM the exposure was much lower with an AUC around 674 µg.h/mL (Table 3). The concentrations found in the plasma were very low for DXP with values between the LOQ and the LOD (Figure 7). The systemic exposure to DXP in the plasma therefore cannot be determined. For DXM, the concentrations found in plasma are low but above the LOQ and seem to follow a two-step decrease (Figure 7). The first decrease takes place between 30min and 4h and can be fitted by a single exponential decrease y= y A exp(-k 1 /t) with k 1 = 0.207 h -1 and a characteristic half-time t 1/2A = = 3.34±0.33 h. The second phase corresponds to a slower decrease between 4h and 24h and can be fitted by a single exponential decrease y= y B exp(-k 2 /t) with k 2 =0.0182h -1 and a characteristic half-time t 1/2B = = 38h±10h. The plasmatic exposure to DXM can be quantified by calculating the AUC and one finds 4.89 µg.h/mL (Table 3). The ELF/plasma AUC ratio is around 138 for DXM and could not be calculated for DXP. This suggests a very pronounced local delivery and a rather low passage of molecules from the lungs to the blood stream. Experimental concentrations could be explained by three main phenomena as described in Figure 8: powder solubilization, DXP hydrolysis into DXM, DXP/DXM distribution in lung tissue and DXP/DXM passage in the blood stream. Indeed, the first part of the DXP curve in ELF may correspond to the solubilization of particles to release DXP molecules along with hydrolysis of DXP into DXM occuring due to the presence of esterases in the ELF [START_REF] Basu | Isolation and characterization of a fatty acyl esterase from rat lung[END_REF][START_REF] Deimling | The non-specific esterases of mouse lung[END_REF]. The fast decrease of DXP concentration in ELF probably corresponds to DXP distribution in lung tissue. This distribution is favored by DXP lipophilicity. DXP lipophilicity also explains the very low DXP concentration detected in the bloodstream [START_REF] Derendorf | Pharmacokinetics and pharmacodynamics of inhaled corticosteroids[END_REF][START_REF] Edsbäcker | Airway Selectivity: An Update of Pharmacokinetic Factors Affecting Local and Systemic Disposition of Inhaled Steroids[END_REF][START_REF] Georgitis | The 1997 Asthma Management Guidelines and therapeutic issues relating to the treatment of asthma[END_REF]. One can notice that DXM is cleared faster from ELF (t 1/2A of about 1h) than from the plasma (t 1/2A of about 3h) due to the route of administration and the physicochemical properties of the molecule. DXM is absorbed in the blood more efficiently than DXP since it is less lipophilic. The second decrease of DXM concentration observed in the plasma with a very long half-life of 38h most probably corresponds to the elimination phase of the drug. One can hypothesize that the lung tissue contains a very high content of DXP that is slowly converted into DXM which in turn is absorbed in the bloodstream [START_REF] Johnson | Pharmacodynamics and pharmacokinetics of inhaled glucocorticoids[END_REF].

Conclusion

We have optimized a formulation of a prodrug of dexamethasone, dexamethasone palmitate for pulmonary delivery as a dry powder. In vitro drug release is rather slow with less than 5% of DXP in the supernatant after 21 days, and arises from DXP insertion within the DPPC bilayers once powders were hydrated. Although a thorough model of what is happening after DXP microparticle lung administration cannot be proposed, one can notice that the prodrug (DXP) remains for up to 6h in the ELF at very high concentration, that the drug itself (DXM) can be detected at rather important concentration in ELF up to 24h. The passage of DXP from the lungs to the bloodstream is very poor whereas DXM seems to be absorbed in the blood more easily. Overall however the systemic exposure seems low compared to the lung exposure which suggests limited side effects. Therapeutic efficacy of this formulation remains to be evaluated in the future by using asthmatic animal models. The strategy of using a lipophilic prodrug could be extended to other molecules to promote long residence time in the lungs while reducing drug systemic exposure.

  France). All animal experiments were carried out in accordance with the Principles of Laboratory Animal Care as adopted and propagated by the EU guidelines for Animal Experiments (86/609/EEC and 2010/63/EU) and Legislation in force in France (Decree No. 2013-118 of February 1, 2013). They were housed 4 per cage while on study in accordance to EEC guidelines. The light/dark cycle was 12 h/12 h. The temperature in the animal room was ambient room temperature of approximately 25 °C and the ambient humidity was in the range of approximately 35-60%. Animals were allowed access to food and water ad libitum throughout the duration of the study.

  in BALF was performed by HPLC. A Water TM 2707 autosampler chromatographic system was employed equipped with a Waters TM 1525 binary HPLC pump, a Waters TM 2998 photodiode array detector, and a Waters TM Breeze software. The analysis was performed at 240 nm using a SymmetryShield TM RP18 column (5 µm, 250×4.6 mm; Waters, Saint-Quentin-en-Yvelines, France). Column temperature was maintained at 40 °C using a column Heater (Model 1500, Waters Corporation). The mobile phase was composed by a mixture of acetonitrile and milliQ water: 85/15 v/v for DXP and 35/65 v/v for DXM. The mobile phase flow rate was 1.2mL/min, the injection volume was 50L and the run time was 30min. Retention times were 24min and 9min for DXP and DXM, respectively, 21min for TD and 26min for DXA. Each sample was analyzed twice, once with DXP quantification method, second with DXM quantification method. For quantification from in vitro samples, the developed method showed good linearity between 0.5 and 250 µg/mL (y = 50896x -1239; r 2 = 0.998) for DXP and 0.3 and 260 µg/mL (y = 43920x + 9731; r 2 = 0.9995) for DXM. For quantification from in vivo samples, limits of detection and quantifications were determined. obtained by spray-drying by varying the DXP content up to 15% (w/w), We obtained white and fluffy powders. Polydisperse aggregated spherical microparticles exhibiting some roughness were observed by SEM, with a tendency to aggregation as DXP content increased (Figure1).

Figure 1 :

 1 Figure 1: SEM images of typical microparticles obtained by spray drying. Image A corresponds to DPPC-HA microparticles exempt of DXP, Images B to D correspond to 5, 10 and 15% DXP (w/w), respectively.

Fig 2 :

 2 Fig 2 : Microparticles characteristics in terms of median volume geometric diameter (D50, A), span (B), tap density (C) and yield (D) as a function of the weight percent of DXP in the formulation. Results are presented as mean±SD (n=3).

Fig 3 :

 3 Fig 3: Fine particle fractions (FPF) and Alveolar fractions (AF) of DXP microparticles determined using an MSLI as a function of DXP weight percent in the formulation. Values are presented as the mean± SD (n=3).

Fig 5 :

 5 Fig 5: DSC thermograms obtained from hydrated powders upon heating at 5°C/min with different concentrations of DXP (0 to 15%). The arrow indicates the position of the DPPC main transition around 40°C, whereas the star indicates what we believe corresponds to a transition associated to DXP (Panel A). Variation of the onset temperature (B) and of the enthalpy (C) of DPPC main transition as a function of DXP concentration in the powder. Values are presented as mean±SD (n=3). Error bars are smaller than the size of the symbols.

Figure 6 :

 6 Figure 6: Concentration of DXP (top) and DXM (bottom) in ELF after intratracheal insufflation of 3mg of DXP microparticles (DXP dose=150 g). LOQs are represented as dashed curves and LOD as full curves. The LOD is 10 g/mL. Data are presented as mean±SEM (Standard error of the mean), n=3.

Figure 7 :

 7 Figure 7: Concentration of DXP (top) and DXM (bottom) in plasma after intratracheal insufflation of 3mg of DXP microparticles (DXP dose=150 g). LOQs are represented as dashed curves and LOD as full curves. The LOD of DXM in plasma was not presented as all values were above the LOQ. Data are presented as mean±SEM (Standard error of the mean), n=3.

Figure 8 :

 8 Figure 8: Schematic description the different phenomena occuring after DXP microparticles are delivered to the lungs.

Table 1 .

 1 Operational conditions used for spray-drying.

	Feed flow rate (mL/min)	17
	Inlet temperature (ºC)	150 ± 2
	Outlet temperature (ºC)	55 ± 4
	Aspiration setting (%)	100 (35m 3 /h)
	Air-flow rate (L/h)	414
	2.3. Particle size distribution
	Powders size distributions were measured by light diffraction using a Mastersizer 2000
	equipped with a Scirocco dry disperser (Malvern Instruments, France) at a dispersing pressure
	of 3 bars. The refractive index used was 1.5. Data obtained were expressed in terms of the
	particle diameter at 10%, 50% and 90% of the volume distribution (D10, D50 and D90
	respectively). The span of the volume distribution, a measure of the width of the distribution
		Span		50 D 90 D 10 D 

relative to the median diameter was calculated according to Eq 1. A large span is indicative of a more heterogeneous size distribution. Values presented are the average of at least 3 determinations.

Table 2 :

 2 Limits of detection and quantification of the different molecules after extraction from plasma or BALF.

	Parameters	Plasma	BALF	ELF
	DXM	LOQ (µg/mL) LOD (µg/mL)	0.133 0.04	0.1 0.03	33 10
	DXP	LOQ (µg/mL) LOD (µg/mL)	0.17 0.06	0.1 0.03	33 10

Table 3 :

 3 PK parameters (mean  SEM) after intratracheal insufflations of 3mg of DXP powder corresponding to a dose of 150 g of DXP (93 g of DXM).

	Parameters		ELF	Plasma
		C max (µg/mL)	191	0.37
		t max (h)	0.5	0.5
		AUC (µg.h/mL)	674	4.89
	DXM	AUC/Dose (h/mL)	7.22	0.053
		t 1/2A (h)	0.93±0.20	3.34±0.3
		t 1/2B (h)	NA	38±10
		AUC ELF /AUC Plasma	138
		C max (µg/mL)	10 794	NA
		t max (h)	0.5	NA
	DXP	t 1/2A (h) t 1/2B (h)	2.8±0.4 0.63±0.01	NA NA
		AUC (g.h/mL)	26 703	NA
		AUC/Dose (h/mL)	178	NA

max , maximal concentration in ELF and in plasma; t max , time of maximal concentration in ELF and in plasma ; AUC, area under the curve of ELF and plasma concentrations ; t 1/2 , terminal half-life in ELF and in plasma.

Acknowledgments:

A. N'Guessan PhD fellowship was supported by University Félix Houphouet-Boigny, Côte d'Ivoire, Univ Paris-Sud, Châtenay-Malabry and Institut Galien Paris-Sud CNRS UMR 8612. Authors would like to thank V. Domergue-Dupont, L. Aragão-Santiago, C. Ruge and D.D. Pham for support with animal experiments. Institut Galien Paris-Sud is a member of the Laboratory of Excellence LERMIT supported by a grant from ANR (ANR-10-LABX-33). The authors would also like to acknowledge the financial support provided by COST-European Cooperation in Science and Technology, to the COST Action MP1404: Simulation and pharmaceutical technologies for advanced patient-tailored inhaled medicines (Siminhale).


Gonzalo Quiroga Artigas

Pascal Lapébie

Lucas Leclère

Philip Bauknecht

Julie Uveira

Sandra Chevalier

Gáspár Jékely

Tsuyoshi Momose

Evelyn Houliston
email: houliston@obs-vlfr.fr

Gonzalo Quiroga Artigas

A G-protein-coupled receptor mediates neuropeptide-induced oocyte maturation in the jellyfish Clytia

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Oocyte meiotic maturation is an essential process for animal sexual reproduction. It transforms the tetraploid, fully-grown, ovarian oocyte into a haploid female gamete [START_REF] Voronina | The regulation of oocyte maturation[END_REF]. The core biochemical and cellular pathways operating within the oocyte during maturation are highly conserved between animals across the phylogenetic spectrum. Activation of the CyclinB-Cdk1 kinase complex assures meiotic progression from prophase I arrest into M phase, while parallel Mos-MAP kinase activation steers polar body formation as well as cytostatic arrest once maturation is complete [START_REF] Verlhac | Oocyte Maturation and Development[END_REF]3]. In contrast the upstream physiological processes vary widely, as does the molecular nature of the maturation inducing hormones (MIHs) that act on the oocyte to trigger maturation [START_REF] Voronina | The regulation of oocyte maturation[END_REF][START_REF] Stetina | Developmental control of oocyte maturation and egg activation in metazoan models[END_REF]. This reflects clade-specific acquisition of endocrine tissues such as ovarian follicles, the corpus cardiacum/corpus allatum in insects, or the pituitary in vertebrates [START_REF] Hartenstein | The neuroendocrine system of invertebrates: a developmental and evolutionary perspective[END_REF][START_REF] Deguchi | Comparative biology of cAMP-induced germinal vesicle breakdown in marine invertebrate oocytes[END_REF]. These tissues act downstream of other neuroendocrine sites such as the vertebrate hypothalamus to integrate environmental, behavioural and physiological information in order to achieve optimal conditions for gamete development and release [START_REF] Tissier | An updated view of hypothalamic-vascular-pituitary unit function and plasticity[END_REF]. The complex evolutionary history of hormonal reproductive regulation has made it challenging to unravel the crucial regulatory events operating within the oocyte at the onset of maturation.

G-protein coupled receptors (GPCRs), the largest superfamily of integral transmembrane receptors [START_REF] Kristiansen | Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function[END_REF], are good candidates to serve as MIH receptors. These 7-transmembrane domain proteins activate a variety of cytoplasmic signalling pathways via Gα and/or Gβγ subunits that become released from receptor-associated heterotrimeric Gαβγ protein upon ligand binding [START_REF] Neves | G protein pathways[END_REF][START_REF] Oldham | Heterotrimeric G protein activation by G-protein-coupled receptors[END_REF][START_REF] Chakravorty | G protein subunit phosphorylation as a regulatory mechanism in heterotrimeric G protein signaling in mammals, yeast, and plants[END_REF]. Members of the four main Gα subunits classes, Gα s , Gα i Gα q and Gα 12/13 , associate variously with members of the vast GPCR family. In vertebrates, constitutively active GPCRs are coupled to Gα s , which stimulates adenylate cyclase to maintain high cytoplasmic cAMP concentrations in ovarian oocytes [START_REF] Kalinowski | Maintenance of meiotic prophase arrest in vertebrate oocytes by a Gs protein-mediated pathway[END_REF][13][START_REF] Nader | Role for endocytosis of a constitutively active GPCR (GPR185) in releasing vertebrate oocyte meiotic arrest[END_REF].

These high cAMP levels help hold the oocyte in an immature state, with the cell cycle arrested in meiotic prophase. The mechanisms by which MIHs override this arrest and initiate meiotic maturation vary between species and are not fully understood (see Discussion). In marked contrast, oocytes of many invertebrate species show a rise in cytoplasmic cAMP concentration upon MIH stimulation that is required for meiotic maturation [START_REF] Deguchi | Comparative biology of cAMP-induced germinal vesicle breakdown in marine invertebrate oocytes[END_REF]. In these species, GPCRs working through Gα s are thus good candidates to trigger oocyte maturation, rather than to inhibit it as in vertebrates. The identification of such receptors could help to understand the origin of this diversity by providing an evolutionary perspective.

Here we identify the MIH receptor (MIHR) in the hydrozoan jellyfish Clytia hemisphaerica as a GPCR likely working through Gα s , and determine its in vivo function. In Clytia and other hydrozoan species, MIH consists of PRPamide and related tetrapeptides that are released upon light stimulation from opsin-expressing gonad ectoderm cells, and act at the plasma membrane [START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF][START_REF] Artigas | A gonadexpressed opsin mediates light-induced spawning in the jellyfish[END_REF]. Neuropeptides such as these are of major importance in cnidarian biology, acting both as "neuroendocrine" mediators of physiological transitions such as metamorphosis, as well as in fast neuromuscular transmission regulating swimming and feeding mediated by ligand-gated ion channels [reviewed by [START_REF] Takahashi | Insight into the Molecular and Functional Diversity of Cnidarian Neuropeptides[END_REF][START_REF] Bosch | Back to the Basics: Cnidarians Start to Fire[END_REF]. We uncovered an oocyte-expressed Class A GPCR in Clytia (MIHR) activated by MIH peptides, which is to our knowledge the first characterized cnidarian neuropeptide GPCR [START_REF] Grimmelikhuijzen | Mini-review: the evolution of neuropeptide signaling[END_REF][START_REF] Elphick | Evolution of neuropeptide signalling systems[END_REF]. Antibody inhibition experiments further provided evidence that Gα s links MIHR activation to cAMP production to trigger maturation [START_REF] Freeman | The role of cAMP in oocyte maturation and the role of the germinal vesicle contents in mediating maturation and subsequent developmental events in hydrozoans[END_REF][START_REF] Takeda | Increase in intracellular cAMP is a prerequisite signal for initiation of physiological oocyte meiotic maturation in the hydrozoan Cytaeis uchidae[END_REF]. CRISPR-Cas9 mediated mutation of the MIHR gene revealed an essential in vivo function of MIHR in initiating oocyte maturation. Phylogenetic analysis of the MIHR sequence identified an evolutionary link to a subset of bilaterian neuropeptide-hormone GPCR families, including several upstream regulators of reproduction. These results allow us to propose a new scenario for the evolution of hormonal signalling pathways regulating oocyte maturation.

Results

Selection of candidate MIH GPCRs

Amidated neuropeptides like MIH commonly signal through GPCRs, although other receptor types can also be used [START_REF] Jékely | The long and the short of it -a perspective on peptidergic regulation of circuits and behaviour[END_REF]. As the first step to select candidate MIH receptors, we compiled a comprehensive catalogue of Clytia GPCRs from a Clytia reference transcriptome covering all life-cycle stages. Our bioinformatics pipelines first retrieved all sequences predicted to code for 7 transmembrane domain (7TM domain) proteins and bearing GPCR-related Pfam tags. An initial list of 761 sequences was then assigned by Pfam to the three main GPCR classes: A (rhodopsin-like), B (secretin-like) and C (metabotropic glutamate-like) [START_REF] Kristiansen | Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function[END_REF][START_REF] Audet | Restructuring G-protein-coupled receptor activation[END_REF] or to an "other" category (which included for instance sweet-taste receptors and cAMP-like receptors). The final dataset of 536 class-sorted putative Clytia GPCRs obtained after removal of duplicates (File S1) may be a slight overestimate due to some incorrectly identified or incomplete sequences. We focussed on the 377 class-A GPCRs, since most neuropeptide GPCRs belong to this class [START_REF] Kristiansen | Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and mutagenesis approaches to receptor structure and function[END_REF][START_REF] Jékely | Global view of the evolution and diversity of metazoan neuropeptide signaling[END_REF].

An important criterion for the selection of MIH-receptor candidates from the class-A GPCR list was enrichment in oocytes (Fig. 1A). We mapped Illumina HiSeq mRNA reads previously obtained from Clytia gonad tissues [START_REF] Artigas | A gonadexpressed opsin mediates light-induced spawning in the jellyfish[END_REF] and life cycle stage [START_REF] Leclère | The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle[END_REF] against all putative GPCR sequences. Profile clustering revealed three groups of sequences with oocyte-enriched expression (Fig. S1). We further narrowed down the number of potential MIHRs from these expression groups to 96 sequences, taking into account also Pfam indicators and sequence similarity with a set of bilaterian GPCRs [25; see Figure 6 for clustering of a subset of sequences closest to the Clytia MIHR]. Finally we compiled a short-list of 16 candidates for functional testing, using high expression level in oocytes as the final selection criterion (Fig. 1A, File S2).

Identification of the Clytia MIH receptor by GPCR deorphanization assay

We used a cell culture-based GPCR deorphanization assay to identify the MIH receptor from our candidate shortlist. cDNAs for each candidate were transfected into CHO-K1 cells along with an aequorin-GFP luminescence reporter that measures Ca2+ mobilization downstream of a promiscuous Gα protein [START_REF] Tunaru | Characterization of Determinants of Ligand Binding to the Nicotinic Acid Receptor GPR109A (HM74A/PUMA-G)[END_REF][START_REF] Bauknecht | Large-Scale Combinatorial Deorphanization of Platynereis Neuropeptide GPCRs[END_REF]. We first screened the 16 candidate Clytia GPCRs against a mixture of 33 synthetic amidated peptides (including MIHs) predicted to be generated from previously identified putative Clytia neuropeptide precursors [START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF] and one additional one identified from transcriptome data (File S4).

Given our imperfect knowledge of pro-peptide processing in cnidarians, some of the synthetic peptides may not correspond to endogenous peptides [START_REF] Nielsen | De novo transcriptome assembly of the cubomedusa Tripedalia cystophora, including the analysis of a set of genes involved in peptidergic neurotransmission[END_REF][START_REF] Hayakawa | A combined strategy of neuropeptide prediction and tandem mass spectrometry identifies evolutionarily conserved ancient neuropeptides in the sea anemone Nematostella vectensis[END_REF]. Only one of the 16 GPCRs was activated by this peptide mixture. This receptor responded well to all 4 synthetic Clytia MIH tetrapeptides, as well as to related Cladonema MIH peptides that can also activate Clytia oocyte maturation, but not to other Clytia

Slow growth, gametogenesis disruption and spawning failure in MIHR mutant jellyfish

To determine the function of MIHR in vivo we generated Clytia MIHR knockout (KO) polyp colonies using CRISPR/Cas9 mutagenesis (see Methods). This gene-editing technique is very effective in Clytia, allowing extensive bi-allelic mutation of target genes already in F0 polyp colonies [START_REF] Artigas | A gonadexpressed opsin mediates light-induced spawning in the jellyfish[END_REF][START_REF] Offermanns | G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C[END_REF].

Three guide RNAs designed to target the 3 rd transmembrane domain of the MIHR protein were tested by genotyping at the planula stage, and the most effective one used to generate polyp colonies. After genotyping around the target site, we selected and propagated for phenotypic analysis six colonies showing no detectable wild type sequence and carrying mainly frame-shift mutations (Table 1). All six mutant polyp colonies expanded slowly compared to wild type colonies. They displayed variable morphologies, with two of them rambling pronouncedly away from the glass substrate (Fig. 2A; Table 1), but all produced active gonozooids that budded baby medusae. Growth of these mutant baby medusae was slower for the six mutants than for wild types, but they were able to reach the full adult size of about 1cm in diameter. The adult jellyfish all swam less vigorously than wild types. The slow growth of mutant polyps and medusae was not due to any obvious feeding problems: both forms could capture Artemia nauplii without difficulty.

Of the six MIHR mutant colonies, three produced male jellyfish and three females. In addition to the shared defects in growth and swimming behaviour, they showed one of two distinct phenotypes affecting gonad development. For one female and one male MIHR mutant (n5-8 and n5-24 respectively), the gonads of the adult jellyfish developed poorly, failing to accumulate gametes as occurs during wild type adult growth (Fig. 2B). Examination of the gonads by confocal microscopy revealed the presence of small oocytes for n5-8 and a thin spermatogenic zone for n5-24. This suggests that mutation of the MIHR gene can compromise gametogenesis in male and female jellyfish. One possible explanation is that gamete development can stall completely when overall growth of the jellyfish is poor. In marked contrast, jellyfish from the four other MIHR mutant polyp colonies underwent gametogenesis to produce large numbers of fully-grown oocytes (n5-13, n5-23; Fig. 2C) or spermatozoids (n5-6, n5-10; Fig. 2D). In the female medusae, the gonads were grossly inflated by accumulation of fully-grown immature oocytes, due to failure in light-induced oocyte maturation and subsequent release of the unfertilised egg (see below). In males, the gonads were markedly deformed and irregular. Following dark-light transitions that induced spawning in wild type males, some local sperm release was observed from rupture sites of the gonad epithelium, but the sperm remained concentrated at the gonad surface and failed to disperse.

Overall this initial phenotype analysis of six independent MIHR mutant F0 colonies indicated that MIHR has non-essential functions in medusa growth and in regulating medusa swimming. These could relate to expression of MIH and MIHR at other sites in the jellyfish, notably the manubrium (mouth/stomach structure) and tentacles (see below). In addition, two distinct gonad defects were observed: failure of gamete development or over-accumulation of mature gametes. These had no obvious relationship to particular genotypes of the corresponding F0 polyp colonies (Table 1) and so may be the consequence of MIHR mutation in different genetic or epigenetic backgrounds. In any case, the accumulation of immature, fully grown oocytes in medusae from two independent MIHR mutants strongly supported a key role for this GPCR in oocyte maturation, as characterized further below.

Clytia MIHR is expressed in oocytes and also in tentacle cells

The defects observed in behaviour and growth as well as in gametogenesis in MIHR mutant jellyfish suggested that this receptor may have roles both in the gonads and other sites. In situ hybridization detection of MIHR mRNA in adult jellyfish (medusae) and in isolated gonads (Fig. 3A,B) supported this idea. Strong expression was expressed in developing oocytes and in male gametes, consistent with the ability of MIH to induce male spawning [START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF]. Expression was also detected in clusters of small somatic cells located within each tentacle bulb. Individual cells from these clusters extended in a line along each tentacle. The location of these cells suggests that they belong to a neural subtype potentially involved in regulating tentacle contraction. The MIH receptor in these tentacle cells could potentially bind MIH neuropeptides produced by neural/neuroendocrine cells of the endodermal gastrovascular system, detectable using an antibody recognising the PRPamide peptides produced from both Che-pp1 and Che-pp4 [anti-PRPa; 15; Fig. 3C]. These two precursors are expressed respectively exclusively in the manubrium and tentacle, as well as being co-expressed in scattered cells of the gonad [START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF]. As well as the cells in the gonad ectoderm that mediate spawning, the anti-PRPa antibody decorates cells in the tentacle especially at its junction with the circular gastrovascular canal, and in the endoderm of the manubrium feeding/digestive organ [START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF][START_REF] Artigas | A gonadexpressed opsin mediates light-induced spawning in the jellyfish[END_REF] (Fig. 3C). Comparison of in situ hybridization patterns for MIHR and MIH showed that the ligand and receptor-expressing cells in the tentacles have distinct distributions: the single file of MIHR cells lies on the rounded oral side [see 31], whereas the MIH cells form two flanking lines (Fig. 3B). The position and morphology of these cells suggest that they correspond to two sub-populations of neural cells positioned between the tentacle endoderm and ectoderm in hydrozoans [START_REF] Condamine | Molecular characterisation of a cellular conveyor belt in Clytia medusae[END_REF][START_REF] Sinigaglia | A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species[END_REF]. No MIHR-expressing cells were detected in the manubrium, only nonspecific staining of the manubrium floor. Our inability to detect MIHR expression close to these manubrium PP1-expressing cells may be due to the limitations of the in situ hybridization technique, with low expression in the manubrium and/or other tissues not sufficient to provide a clear signal.

Another possibility is that a second receptor is expressed in the manubrium and more sensitive to RPRGa/WPRPa peptides. Alternatively, the MIHR-expressing cells at the base of the tentacles might respond to endocrine MIH tetrapeptides circulating in the gastrovascular system, for example secreted in response to nutritional status. Regulation by these cells of tentacle contractions, and directly or indirectly of swimming behaviour, could account for the sluggish movement and poor growth of MIHR mutant medusae. Specific assays should be devised in the future to examine this hypothesis in detail.

Clytia MIHR is the essential oocyte receptor for oocyte maturation

We used jellyfish from the two MIHR female mutant colonies showing the swollen-gonad phenotype (n5-13 and n5-23) to characterize the role of this GPCR in oocyte maturation. For n5-23 the predominant genotypes were a 4 base deletion which introduces a premature STOP codon, and a 55 base insertion, but a small proportion of wild type cells was also detected. Colony n5-13 contained a majority 4 base deletion but also a high proportion of a 3 base deletion. We performed a series of oocyte maturation and spawning assays, comparing the responses of wild type and MIHR KO isolated gonads and oocytes to different stimuli (Figure 4). Isolated wild type gonads underwent oocyte maturation and spawning in response to light stimulation or treatment with the synthetic MIH peptide WPRPamide (100nM) as previously shown [START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF], but MIHR mutant gonads did not respond (Fig. 4A,B). In contrast, treatment with 4mM 5Br-cAMP, a cell-permeable analogue of cAMP which induces hydrozoan oocyte maturation by mimicking the early cytoplasmic cAMP rise [3,[START_REF] Freeman | The role of cAMP in oocyte maturation and the role of the germinal vesicle contents in mediating maturation and subsequent developmental events in hydrozoans[END_REF], rescued the phenotype of MIHR mutant gonads, efficiently triggering oocyte maturation and spawning. 5-Br-cAMP treatments but not MIH peptides also promoted maturation of isolated fully grown MIHR mutant oocytes (Fig. 4C). Br-cAMP-matured MIHR mutant oocytes could be fertilized to develop into planula larvae, although had lower development and metamorphosis rates than wild type oocytes. This demonstration of a cAMP-reversible maturation initiation failure in mutant female jellyfish confirmed that the Clytia MIHR has an essential in vivo function as the oocyte receptor for MIH.

MIH-induced oocyte maturation blocked by an inhibitory Gα s antibody

GPCR activation can lead to a cytoplasmic cAMP concentration rise in the responding cell as a result of adenylate cyclase stimulation via S type Gα subunits of heterotrimeric G proteins. We tested the role of Gα s in Clytia oocyte maturation by injecting isolated oocytes with a specific inhibitory antibody previously shown to cause meiotic maturation of mouse, Xenopus and zebrafish oocytes [START_REF] Kalinowski | Maintenance of meiotic prophase arrest in vertebrate oocytes by a Gs protein-mediated pathway[END_REF][START_REF] Gallo | Stimulation of Xenopus oocyte maturation by inhibition of the G-protein alpha S subunit, a component of the plasma membrane and yolk platelet membranes[END_REF][START_REF] Mehlmann | Meiotic arrest in the mouse follicle maintained by a Gs protein in the oocyte[END_REF]. All oocytes were tested for maturation competence by incubation at the end of the experiment in 5'-Br-cAMP. Any that failed to mature when treated with cAMP were discounted in the analyses. Oocytes injected with anti-Gα s responded less efficiently than oocytes injected with PBS or a control anti-GST antibody when treated with synthetic MIH at low and then high doses (10nM then 100nM WPRPamide; Kinetics observed in one of three equivalent experiments shown in Fig. 5A). These results strongly suggest that MIHR acts mainly through Gα s .

Based on these findings we can now propose a model for maturation initiation in Clytia oocytes through MIH, MIHR, Gα s and cAMP (Fig. 5B). A light cue triggers MIH release from neuroendocrinetype cells in the gonad ectoderm via an essential opsin protein [START_REF] Artigas | A gonadexpressed opsin mediates light-induced spawning in the jellyfish[END_REF]. The MIH peptides act on MIHR at the oocyte surface to promote Gα s activation, stimulating adenylate cyclase and thus promoting an increase in cytoplasmic cAMP concentration, essential for the transition into meiotic M phase.

Clytia MIHR is related to a bilaterian superfamily of neuropeptide hormone receptors

Much progress has been made in understanding the relationships between GPCR-neuropeptide families between protostomes and deuterostomes [START_REF] Elphick | Evolution of neuropeptide signalling systems[END_REF][START_REF] Jékely | Global view of the evolution and diversity of metazoan neuropeptide signaling[END_REF][START_REF] Bauknecht | Large-Scale Combinatorial Deorphanization of Platynereis Neuropeptide GPCRs[END_REF][START_REF] Mirabeau | Molecular evolution of peptidergic signaling systems in bilaterians[END_REF][START_REF] Zandawala | Discovery of novel representatives of bilaterian neuropeptide families and reconstruction of neuropeptide precursor evolution in ophiuroid echinoderms[END_REF], and more recently with those in acoels and cnidarians [START_REF] Thiel | Xenacoelomorph Neuropeptidomes Reveal a Major Expansion of Neuropeptide Systems during Early Bilaterian Evolution[END_REF]. We performed sequence-similarity based clustering to explore the relationship of the Clytia MIHR sequence with known GPCR families in Bilateria using established datasets from human and the annelid Platynereis [25,28 Fig. 6A]. The Clytia MIHR fell within the majority group of cnidarian GPCRs that cluster with a superfamily of peptide hormone receptors including human neuropeptide Y, neuropeptide FF, Tachykinin, Orexin/allastropin, Elevenin, and EFLGa/Thyrotropin releasing hormone, as well as Luqin from Platynereis (Fig. 6B). Adding to this analysis sequences recovered from the anthozoan cnidarian Nematostella highlighted the extensive independent expansion of GPCR-A sequence since cnidarian and bilaterian neuropeptide systems split [START_REF] Thiel | Xenacoelomorph Neuropeptidomes Reveal a Major Expansion of Neuropeptide Systems during Early Bilaterian Evolution[END_REF]. This analysis demonstrated that the MIHR group does not contain any of the GPCRs directly involved in controlling oocyte maturation in vertebrates, including the constitutively active GPR3, gonadotropin releasing hormone (GnRH) receptor or Luteinizing/Follicle-stimulating hormone (LH/FSH) receptor (see Introduction and Figure 7). A GPCR from Nematostella annotated as a GnRH receptor [START_REF] Anctil | Chemical transmission in the sea anemone Nematostella vectensis: A genomic perspective[END_REF] belongs as sister to distinct 'superfamily' including the bilaterian GnRH, vasotocin, CCAP corazonin and achatin receptor families.

Maximum Likelihood phylogenetic analyses of the large GPCR cluster containing MIHR confirmed phylogenetic support for a GPCR 'superfamily' associating two distinct cnidarian groups (A and B in Fig. 6C,D) with a set of bilaterian neuropeptide hormone receptors [START_REF] Thiel | Xenacoelomorph Neuropeptidomes Reveal a Major Expansion of Neuropeptide Systems during Early Bilaterian Evolution[END_REF]. Cnidarian GPCR group A contained Clytia MIHR, six other Clytia GPCRs and a large number of Nematostella receptors. It showed weakly-supported association with a set of bilaterian peptide hormone receptor families including those for neuropeptide Y/neuropeptide F, GnIH/neuropeptide FF, RYa/Luqin, Tachykinin and pyroglutamylated RFamide peptide (QRFP) [START_REF] Jékely | Global view of the evolution and diversity of metazoan neuropeptide signaling[END_REF][START_REF] Bauknecht | Large-Scale Combinatorial Deorphanization of Platynereis Neuropeptide GPCRs[END_REF][START_REF] Thiel | Xenacoelomorph Neuropeptidomes Reveal a Major Expansion of Neuropeptide Systems during Early Bilaterian Evolution[END_REF]. Group B included two Clytia GPCRs and associated with further bilaterian neurohormonal receptor families including Elevenin, EFLGa/TRH and Orexin/Allatotropin receptors (Fig. 6C,D). The low support values for many deep branch relationships within this superfamily makes it difficult to determine definitive evolutionary relationships between the neuropeptide hormone receptors and cnidarian group A and B GPCRs. We can nevertheless conclude that Clytia MIHR along with eight other Clytia GPCRs and a very large group of Nematostella GPCRs have a common ancestor with a subset of bilaterian peptide hormone receptor families that includes many involved in regulating sexual reproduction and feeding (see Discussion).

Discussion

GPCR deorphanization followed by targeted gene mutation allowed us to identify Clytia MIHR as the neuropeptide receptor responsible for initiating oocyte meiotic maturation. MIHR female mutant medusae accumulated fully-grown oocytes that failed to mature unless treated with cAMP analogues to bypass the receptor. We further provided evidence that a Gα s subunit links MIHR activation to the increase in cytoplasmic cAMP concentration in the oocyte that initiates maturation. MIHR belongs to a superfamily of neuroendocrine GPCRs involved in regulating reproduction but also nutrition, as well as diverse other physiological processes. Consistent with a wider role for Clytia MIH-MIHR, MIHR expression was detected in jellyfish tentacle cells that may respond to MIH neuropeptides produced in the gastrovascular system, while MIHR mutants showed sluggish behaviour and slow growth, with gamete development also stalled in jellyfish from some mutants. These findings thus open doors to an improved understanding of the vital animal process of oocyte maturation and to its evolution. More widely they shed light on the evolution of the neurohormonal regulation of sexual reproduction in the animal kingdom.

An oocyte receptor for maturation initiation

Our work shows that Clytia MIHR is entirely responsible for the oocyte response to MIH, allowing us to fill in the main molecular actors leading to oocyte meiotic maturation (Fig. 5B): MIH is released from specialized gonad ectoderm cells in response to a dark-light transition at dawn via the essential photoresponsive GPCR Opsin9 [START_REF] Artigas | A gonadexpressed opsin mediates light-induced spawning in the jellyfish[END_REF]. Binding of MIH to MIHR at the oocyte surface is likely swiftly followed by a rise in cytoplasmic cAMP levels in the oocyte to activate cAMP-dependent Protein Kinase (PKA), as has been shown in other hydrozoan species [START_REF] Takeda | Increase in intracellular cAMP is a prerequisite signal for initiation of physiological oocyte meiotic maturation in the hydrozoan Cytaeis uchidae[END_REF]. Our antibody inhibition experiments strongly implicate Gα s -stimulated adenylate cyclase activity in driving this cAMP rise.

PKA leads to activation of the Cdk1-CyclinB and Mos-MAP kinase systems, that have highly conserved roles in oocyte maturation [START_REF] Haccard | Oocyte Maturation, Mos and Cyclins-A Matter of Synthesis: Two Functionally Redundant Ways to Induce Meiotic Maturation[END_REF][START_REF] Nagahama | Regulation of oocyte maturation in fish[END_REF]. To complete the full chain of events between MIH secretion and meiotic maturation will require the identification of PKA substrates from Clytia oocytes that interact with regulators of Mos translation and/or Cdk1-CyclinB autoactivation. cAMP rises initiate oocyte maturation not only in hydrozoans but also in a diverse range of protostome and deuterostome species [START_REF] Deguchi | Comparative biology of cAMP-induced germinal vesicle breakdown in marine invertebrate oocytes[END_REF], so it is tempting to speculate that the straightforward pathway linking MIH secretion to meiotic maturation in Clytia may have retained its main features from a distant cnidarian-bilaterian ancestor. A useful step towards testing this idea would be to search for MIHR superfamily GPCRs in oocytes of nemertean, ascidian, bivalves or ophiuroid species that depend on cAMP signalling for maturation. If confirmed as the ancestral state, the Clytia MIH-MIHR system could provide a paradigm for the molecular dissection of a common, cAMP-stimulated, mechanism for oocyte meiotic resumption.

Evolution of reproductive regulation

The role of cAMP in initiating oocyte maturation is not universal. In some species such as starfish it has no significant role [START_REF] Kishimoto | MPF-based meiotic cell cycle control: Half a century of lessons from starfish oocytes[END_REF], while in vertebrates, high cAMP levels in the ovarian oocyte maintain the prophase arrest. Clues to understanding how these marked differences arose during evolution may be found by considering the GPCRs involved in regulating gamete maturation and release across species.

In vertebrates, GPCRs are involved in triggering oocyte maturation at several levels along the H-P-G axis (Figure 7). In ovarian oocytes, constitutively active GPCRs of the GPR3/6/12 family help maintain prophase arrest via Gα s -cAMP [START_REF] Kalinowski | Maintenance of meiotic prophase arrest in vertebrate oocytes by a Gs protein-mediated pathway[END_REF][13][START_REF] Nader | Role for endocytosis of a constitutively active GPCR (GPR185) in releasing vertebrate oocyte meiotic arrest[END_REF]. Luteinizing Hormone receptors in the surrounding follicle cells provide upstream signalling also via Gα s -cAMP. The follicle cells signal is transmitted to the oocyte in mouse via a cGMP decrease through gap-junctions [START_REF] Jaffe | Regulation of Mammalian Oocyte Meiosis by Intercellular Communication Within the Ovarian Follicle[END_REF], or in fish and amphibians via steroid hormone cocktails that act on one or more types of membrane receptor, potentially including GPCR progestin receptors linked to Gαi that contribute to lowering cAMP levels [START_REF] Haccard | Oocyte Maturation, Mos and Cyclins-A Matter of Synthesis: Two Functionally Redundant Ways to Induce Meiotic Maturation[END_REF][START_REF] Nagahama | Regulation of oocyte maturation in fish[END_REF][START_REF] Zhu | Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes[END_REF][START_REF] Ben-Yehoshua | The Role of Xenopus Membrane Progesterone Receptor β in Mediating the Effect of Progesterone on Oocyte Maturation[END_REF]. Upstream of the vertebrate follicle, LH production from the pituitary is under control of the master reproductive regulator GnRH receptor acting via Gα q and cytoplasmic Ca ++ release. Many other hormone-GPCR pairs influence production of GnRH by the hypothalamus, as well as the gonadotropins LH and FSH by the pituitary. Molecular phylogeny showed that Clytia MIHR is not closely to receptors of the core H-P-G GPCRs, but rather forms a superfamily with receptors of these "upstream" neuropeptide hormones, notably GnIH, QRFP, Neuropeptide Y and NkB. Production of GnRH and gonadotropins along the H-P-G axis are inhibited by GnIH and stimulated by QRFPs [START_REF] Patel | Pyroglutamylated RFamide Peptide 43 Stimulates the Hypothalamic-Pituitary-Gonadal Axis via Gonadotropin-Releasing Hormone in Rats[END_REF][START_REF] Tsutsui | Discovery and Evolutionary History of GnIH and Kisspeptin: New Key Neuropeptides Controlling Reproduction [Internet[END_REF], while the Tachykinin family hormone NkB acts in a group of hypothalamus neurons together with Kisspeptin and Dynorphin A in the generation of cyclic GnRH pulses [START_REF] Hu | Neurokinin B and reproductive functions: "KNDy neuron" model in mammals and the emerging story in fish[END_REF]. The ancestral cnidarian-bilaterian GPCR for MIHR and the receptors to all these vertebrate hormones may thus already had a role in regulating gamete production and/or release. In this case, vertebrate GPCRs in the hypothalamus and the pituitary, rather than those in gonad follicle cells or gametes, would have retained the trace of this ancestor. Likewise, protostome members of the MIHR GPCR group regulate sexual reproduction indirectly from sites far from the gonad. The planarian NeuropeptideY NPY-8 regulates gametogenesis through its GPCR expressed in CNS neuroendocrine hormone cells [START_REF] Saberi | GPCRs Direct Germline Development and Somatic Gonad Function in Planarians[END_REF], while nematode Luqin peptides produced by pharyngeal neurons regulate egg laying via serotonergic RIH interneurons [START_REF]Luqin-like RYamide peptides regulate food-evoked responses in C. elegans[END_REF].

An ancient regulatory system linking reproduction and nutrition?

It is striking that many bilaterian neuropeptide GPCRs closely related to Clytia MIHR are known for roles in regulating feeding and nutritional balance. In mammals these include GPR83/PEN [START_REF] Gomes | Identification of GPR83 as the receptor for the neuroendocrine peptide PEN[END_REF] as well as QRFP receptors and Neuropeptide Y receptors, which provide important links between metabolic state and reproductive regulation [START_REF] Chartrel | The Neuropeptide 26RFa (QRFP) and Its Role in the Regulation of Energy Homeostasis: A Mini-Review[END_REF][START_REF] Navarro | Novel role of 26RFa, a hypothalamic RFamide orexigenic peptide, as putative regulator of the gonadotropic axis[END_REF][START_REF] Wójcik-Gładysz | Neuropeptide Y--a neuromodulatory link between nutrition and reproduction at the central nervous system level[END_REF], in conjunction with GnIH signalling [START_REF] Tsutsui | GnIH Control of Feeding and Reproductive Behaviors[END_REF]. Related hormone receptor families from protostomes are also known for regulating feeding, including those for Drosophila Leucokinin [START_REF] Al-Anzi | The leucokinin pathway and its neurons regulate meal size in Drosophila[END_REF] and Luqins in arthropods (RYamides) and nematodes [START_REF]Luqin-like RYamide peptides regulate food-evoked responses in C. elegans[END_REF][START_REF]Suppressive effects of dRYamides on feeding behavior of the blowfly, Phormia regina[END_REF]. It is thus tempting to propose that the ancestral GPCR of the Clytia MIHR and these bilaterian hormone receptor families was involved in integrating sexual reproduction with nutritional status. It should be noted, however, that bilaterian neuropeptide GPCRs in this superfamily also function in many other physiological processes. Conversely, many neuropeptide hormone systems involved in integrating sexual reproduction with feeding, notably including Kisspeptin, involve GPCRs only distantly related to MIHR [START_REF] Mirabeau | Molecular evolution of peptidergic signaling systems in bilaterians[END_REF][START_REF] Thiel | Xenacoelomorph Neuropeptidomes Reveal a Major Expansion of Neuropeptide Systems during Early Bilaterian Evolution[END_REF][START_REF] Shahjahan | Central pathways integrating metabolism and reproduction in teleosts[END_REF]. To unravel further the evolutionary history of hormonal regulation of gamete production in relation to nutrition it will be of great interest to examine in detail the function of MIHR in Clytia relating to the sluggish movement and poor growth of MIHR mutants, but also to determine the functions of other "group A" GPCRs from Clytia and other cnidarian species.

Methods

Animals

Sexually mature jellyfish generated from laboratory maintained Clytia hemisphaerica polyp colonies ("Z strains") [START_REF] Leclère | The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle[END_REF] were fed regularly with Artemia nauplii and cultured under light-dark cycles to allow daily spawning. Red Sea Salt brand artificial seawater (ASW) was used for all culture and experiments.

Selection of candidate Clytia MIH Receptors

From a comprehensive Clytia reference transcriptome (86,606 contigs) derived from mixed larva, polyp and jellyfish samples,we generated a list of predicted protein sequences from complete and incomplete ORFs using a homemade script [START_REF] Lapébie | Differential responses to Wnt and PCP disruption predict expression and developmental function of conserved and novel genes in a cnidarian[END_REF]. This dataset was screened using TMHMM 2.0c to produce a list of Clytia complete protein sequences coding for 7 transmembrane domain proteins (7TMD) including putative incomplete sequences containing 2 to 7 transmembrane domains. Scanning with Interproscan 5.22 was used to generate a list of 761 potential Clytia GPCRs with Pfam tags related to 7TMD receptors were retained, sorted by class. CD-HIT [START_REF] Fu | CD-HIT: accelerated for clustering the next-generation sequencing data[END_REF] was run with 95% identity to eliminate sequence duplicates, obtaining a final dataset of 536 Clytia GPCRs (sequences in File S1). Illlumina HiSeq 50nt reads from mRNA isolated from manually dissected gonad ectoderm, endoderm, growing oocytes and fully-grown oocytes [START_REF] Artigas | A gonadexpressed opsin mediates light-induced spawning in the jellyfish[END_REF] and from other Clytia life cycle stages including polyp and planula larvae [START_REF] Leclère | The genome of the jellyfish Clytia hemisphaerica and the evolution of the cnidarian life-cycle[END_REF] were mapped against all candidate GPCR sequences using Bowtie2 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF]. The counts for each contig were normalized per total of reads of each sample and contig length to allow expression comparisons between genes and samples.

Presumptive GPCR sequences were separated in groups using a hierarchical pipeline based on the correlation of their expressions (gplots package in R). Z-scores were obtained after standardization of the counts in the different sequenced samples for each GPCR candidate and plotted in a heat map using R (Fig. S1). For clustering, a previous collection of GPCRs [START_REF] Jékely | Global view of the evolution and diversity of metazoan neuropeptide signaling[END_REF] was complemented with several bilaterian class C GPCRs retrieved from UniProtKB and all putative Clytia GPCRs. Clustering analysis was performed using CLANS2 [START_REF] Frickey | CLANS: a Java application for visualizing protein families based on pairwise similarity[END_REF] with a BLOSUM62 matrix and a p-value cutoff of 1.e-30. Based on this information, Pfam signatures, reciprocal BLASTs and high expression level in oocytes, a subset of 16 top candidates was manually selected (sequences and accession numbers in FileS2).

Candidate receptor cloning

The selected Clytia GPCRs were cloned from gonad-extracted cDNA into pcDNA3.1(+) (Thermo Fisher Scientific, Waltham, MA, USA) using the Gibson Assembly® Cloning Kit (New England Biolabs) [START_REF] Frickey | CLANS: a Java application for visualizing protein families based on pairwise similarity[END_REF]. pcDNA3.1(+) vector was linearized with BamHI and NotI restriction enzymes. Primers were designed using the Gibson Cloning option in Geneious v8. Forward primers consisted of the overhang left after BamHI vector linearization followed by the Kozak consensus sequence (CGCCACC), a start codon (ATG), and a sequence corresponding to the target sequence. Reverse primers consisted of the overhang left after NotI vector linearization followed by a STOP codon, and a reverse complementary sequence to the target sequence. The primers for all cloned GPCRs are listed in File S3. Polymerase chain reaction was performed using Phusion polymerase (New England Biolabs).

Cloned GPCRs were sequenced using primers for T7: TAATACGACTCACTATAGGG and BGHrev: TAGAAGGCACAGTCGAGG.

Receptor deorphanization

Cell culture GPCR ligand response assays were performed as described [START_REF] Bauknecht | Large-Scale Combinatorial Deorphanization of Platynereis Neuropeptide GPCRs[END_REF]. Briefly, CHO-K1 cells were cultured in Ham's F12 Nut Mix medium (Thermo Fisher Scientific) with 10% foetal bovine serum. Cells were seeded in 96-well plates at approximately 10.000 cells/well and transfected the following day with pcDNA3.1(+) plasmids encoding each of the 16 candidate Clytia GPCRs (File S2), the promiscuous Gα-16 protein [START_REF] Offermanns | G alpha 15 and G alpha 16 couple a wide variety of receptors to phospholipase C[END_REF], and a reporter construct GFP-apoaequorin [START_REF] Baubet | Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level[END_REF] (60 ng each) using the transfection reagent TurboFect (Thermo Fisher Scientific). After 2 days of expression, the medium was removed and replaced with Hank's Balanced Salt Solution (HBSS) supplemented with 1.8 mM Ca2+, 10 mM glucose, and 1 mM coelenterazine h (Promega, Madison, WI, USA). After incubation at 37 °C for 2 hours, cells were tested by adding synthetic peptides (GenScript) in HBSS supplemented with 1.8 mM Ca2+ and 10 mM glucose. A list of all synthetic peptides used is provided in File S4.

Luminescence was recorded for 45 s in a plate reader (BioTek Synergy Mx or Synergy H4; BioTek, Winooski, VT, USA). Data were integrated over the 45-s measurement period and recorded as technical triplicates in each case. Data were normalized using the response of Platynereis FLamide receptor to 1 µM AKYFL-NH2 [START_REF] Bauknecht | Large-Scale Combinatorial Deorphanization of Platynereis Neuropeptide GPCRs[END_REF]. Dose-response curves were obtained using concentrations between 0.01 nM and 100 µM for each peptide. Data for dose-response curves were recorded in triplicate for each concentration and the experiment was repeated independently 3 times. Dose-response curves were fitted with a four-parameter curve using Prism 6 (GraphPad, La Jolla, CA, USA) and were normalized to the calculated upper plateau values (100% activation).

Generation of CRISPR-Cas9 mutant Clytia polyp colonies

Following our established protocol [START_REF] Momose | High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion[END_REF] MIHR small guide RNA (sgRNA) was assembled by hybridising crRNA and tracrRNA synthesized at IDT (Integrated DNA Technologies), obtaining a final concentration of 50 µM. sgRNA was kept at -80ºC until use. The crRNA MIHR sequence is shown in File S5. We avoided off-target matches by scanning the Clytia genome assembly at http://crispor.tefor.net. Purified Cas9 protein in Cas9 buffer (10 mM Hepes, 150 mM KCl) provided by J-P Concordet (MNHN Paris) was diluted to 10 µM. sgRNA was added to Cas9 protein in excess (~2:1) prior to injection and incubated for 10 minutes at room temperature. The final Cas9 concentration was adjusted to 4 µM and for sgRNA to 10 µM. The mixture was centrifuged at 14,000 rpm for 10 minutes at room temperature before injection (2-3% of egg volume) into unfertilized eggs within 1 hour after spawning, prior to fertilization.

Injected embryos were cultured for 3 days in Millipore-filtered sea water (MFSW) at 18-20°C.

Metamorphosis of planula larvae into polyps was induced about 72 hours after fertilization by placing larvae (20-80/slide) on 75 x 50mm glass slides in drops of 3-4 ml MFSW containing 1 µg/ml synthetic metamorphosis peptide (GNPPGLW-amide), followed by overnight incubation. Slides with fixed primary polyps were transferred to small aquariums kept at 24°C, a temperature which favours the establishment of female colonies [START_REF] Carré | Origin of germ cells, sex determination, and sex inversion in medusae of the genus Clytia (Hydrozoa, leptomedusae): the influence of temperature[END_REF]. Primary polyps and young polyp colonies were fed twice a day with smashed Artemia nauplii until they were grown enough to be fed with swimming nauplii.

Following colony vegetative expansion, a single well-growing colony on each slide was maintained as a founder. After several weeks of growth, polyp colonies were genotyped to assess mutation efficiency and mosaicism, and medusae were collected from the most strongly mutant colony (MIHR KO) for further experimentation.

Genotyping

Genomic DNA from Clytia polyps was purified using DNeasy blood/tissue extraction kit (Qiagen). The MIHR target site was amplified by PCR using Phusion DNA polymerase. Primers used for genotyping are listed in File S5. PCR products were sequenced and mutation efficiency was assessed using TIDE analyses [START_REF] Baubet | Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level[END_REF]. In cases where Agarose gel analysis of the PCR product revealed large insertions or deletions, this was cloned into pGEM easy vector and clones randomly selected for individual sequencing.

Gonad spawning assays

Sexually mature MIHR mutant medusae from colonies n5-13 and n5-23 and wild type medusae were cultured on the same day-night cycle. Individual gonads were dissected in the evening after afternoon spawning. To test the light response, one group of each was transferred to 100 µl MFSW in wells of 96well plastic plates, covered overnight and re-exposed to white light the following day. To test responses to MIH and cAMP, other groups of dissected gonads were cultured overnight in constant light, then transferred to 96-well plastic plates for two hours. MIH was added to wells as an equal volume of 200 nM WPRPamide [GenScript; 15] stock in MFSW was added to give a final concentration of 100 nM.

Alternatively, a stock of 20 mM bromo adenosine 3′5′cyclic MonoPhosphate (5-Br-cAMP -Fluka; Sigma Aldrich) in distilled water was added to give a final concentration of 4 mM. Gonads were washed after 5 minutes incubation in 5-Br-cAMP. Oocyte maturation was scored after 30 minutes as Germinal Vesicle breakdown (GVBD), i.e. visible dissolution of the oocyte nuclear membrane upon entry into M phase, which in all cases was followed by spawning about 1 hour later. Gonads that showed premature maturation or spawning due to manipulation stress were excluded from analysis.

Oocyte maturation assays

Fully grown oocytes were isolated manually from dissected gonads of wild type and MIHR KO mutant jellyfish prepared as above. Maturation assays were performed in wells of 96-well plates or small plastic petri dishes lined with 2% agarose in MFSW. Spontaneous maturation occurs at a low frequency in control oocytes. To test MIH-induced maturation, 10 µl WPRPamide from 1 µM solution in MFSW were added to a final concentration of 100 nM. In some experiments, 5-Br-cAMP from a 20 mM stock in H 2 O was used at a final concentration of 4 mM. GVBD was scored at 30 minutes, every 5 minutes, depending on experiments.

Antibody injection into isolated oocytes was performed using Nanoject or Narisige compressed air microinjection systems [START_REF] Momose | Two oppositely localised frizzled RNAs as axis determinants in a cnidarian embryo[END_REF][START_REF] Yasuo | Practical Guide for Ascidian Microinjection: Phallusia mammillata[END_REF]. Solutions were centrifuged at 14000 rpm at 4°C for 5 minutes before use, and approximately 2% oocyte volume injected. An inhibitory anti-GS antibody [START_REF] Gallo | Stimulation of Xenopus oocyte maturation by inhibition of the G-protein alpha S subunit, a component of the plasma membrane and yolk platelet membranes[END_REF] was concentrated to 8 mg/ml in PBS by three passages through a ULTRAFREE spin column (Millipore-UFV5BQK25). A purified anti-GST antibody (Sigma 67781) at 8.3 mg/mL was used as a control.

GPCR molecular phylogeny

Clustering analysis was performed using CLANS2 [START_REF] Fu | CD-HIT: accelerated for clustering the next-generation sequencing data[END_REF] with a BLOSUM62 matrix and a p-value cutoff of 1e-40 or 1e-50. Sequences were retrieved from NCBI (Homo and Nematostella) and from [START_REF] Bauknecht | Large-Scale Combinatorial Deorphanization of Platynereis Neuropeptide GPCRs[END_REF] for the deorphanized Platynereis GPCRs. Nematostella GPCRs were identified using HMMER [START_REF] Potter | HMMER web server: 2018 update[END_REF].

Identifiers for the sequences used are given in Files S6 and S7. Multiple alignments were generated using Muscle [START_REF] Edgar | MUSCLE: a multiple sequence alignment method with reduced time and space complexity[END_REF] with default parameters. Positions containing more than 80% gaps were excluded from the alignment, provided in File S8. Phylogenetic analyses were performed using RaxML v8.2.9 [START_REF] Stamatakis | RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models[END_REF] and the model PROTGAMMAGTR with Bootstrap support calculated from 500 replicates. The resulting tree file was visualized with FigTree (http://tree.bio.ed.ac.uk/software/figtree/).

In situ hybridization

A urea-based protocol for in situ hybridization was used as previously [START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF][START_REF] Artigas | A gonadexpressed opsin mediates light-induced spawning in the jellyfish[END_REF][START_REF] Sinigaglia | A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species[END_REF].

Immunofluorescence

For co-staining of neuropeptides and tyrosinated tubulin, dissected Clytia gonads, whole medusae were fixed overnight at 18ºC in HEM buffer (0.1 M HEPES pH 6.9, 50 mM EGTA, 10 mM MgSO 4 ) containing 3.7% formaldehyde, then washed five times in PBS containing 0.1% Tween20 (PBS-T).

Treatment on ice with 50% methanol/PBS-T then 100% methanol plus storage in methanol at -20ºC improved visualization of microtubules in neural cells. Samples were rehydrated, washed several times in PBS-0.02% Triton X-100, then one time in PBS-0.2% Triton X-100 for 20 minutes, and again several times in PBS-0.02% Triton X-100. After overnight incubation at 4ºC in PBS with 3% BSA they were incubated in a rabbit anti-PRPa antibody [START_REF] Takeda | Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones[END_REF] and a rat monoclonal anti-Tyr tubulin (YL1/2, Thermo Fisher Scientific) in PBS/BSA at room temperature for 2 h. After washes, the specimens were incubated with secondary antibodies (Rhodamine goat anti-rabbit and Cy5 donkey anti-rat-IgG; Jackson ImmunoResearch, West Grove, PA) overnight in PBS at 4ºC, and nuclei stained using Hoechst dye 33258. Images were acquired using a Leica SP8 confocal microscope and maximum intensity projections of z-stacks prepared using ImageJ software. All MIHR mutant colonies contained gastrozooid and gonozooid polyps (pink and yellow arrows respectively), however the connecting stolons in some mutant colonies (see Table 1) were convoluted and frequently detached from the glass substrate, while stolons of wild type colonies were straight and adhered tightly. B) Fully grown mutant jellyfish n5-8 and n5-24 (bottom row) compared to wild type (top row) showed poorly developed gonads (white arrows) and sluggish behaviour but are otherwise indistinguishable. Centre (females) and right (males) panels show confocal microscope images through the gonads of adult medusae; Nuclei are stained with Hoechst 33342 (cyan) and F-actin with Phalloidin (Red). In the n5-8 female gonad, small oocytes (arrows) can be detected between the endodermal (en) and ectodermal (ec) layers, but no large growing oocytes (oo) are present compared to the wild type female gonad. In the n5-24 male gonad, the spermatogenic zone (asterisk) between endoderm and ectoderm is much thinner than in the wild type male gonad.

Statistics

  peptide mixtures or poorly-active MIH penta-/tripeptides. We termed this Clytia GPCR the Maturation Inducing Hormone Receptor (MIHR). The activity of individual peptides at 1 µM to stimulate the MIHR closely matched their in vivo ability to induce oocyte maturation [15; Fig.1B]. The Clytia MIH amidated peptides are produced from two precursor genes Che-pp4 (generating multiple copies of WPRAa, WPRYa and WPRPa) and Che-pp1 (multiple copies of WPRPa and RPRGa) [15; File S4]. Their predicted structural similarity suggests they bind the same site on the receptor with different affinities. Dose-response curves for each of the 4 Clytia MIH neuropeptides, generated from three independent experiments, showed half-maximal effective concentration (EC50) values in the high-nanomolar or low-micromolar range for all 4 MIHs, with RPRYamide showing the highest activity and RPRGamide the lowest (Fig. 1C). To our knowledge, Clytia MIH and MIHR are the first neuropeptide ligand-GPCR pair demonstrated in a cnidarian.

Figure 1 .

 1 Figure 1. Identification of the Clytia MIH receptor (MIHR)

Figure 2 .

 2 Figure 2. Phenotypes of Clytia MIHR mutants

C)

  Comparison of mutant n5-23 and n5-13 (bottom row) female medusae gonads (white arrows) swollen by an accumulation of large oocytes to wild type (Z11) female medusae (top row). Right panels show gonads dissected from 3-week old n5-23 medusae 10 hours after a light cue that induced spawning in the wild type but not the mutant gonad. D) Fully grown mutant n5-10 and n5-6 male medusa (bottom row) compared to wild type (Z13) male medusae (top row) have deformed gonads (white arrows). Right panels show isolated gonads, illustrating the thickened and irregular spermatogenic layer (arrowheads) in the mutant. Scale bars 1mm for light microscope jellyfish and polyp images; 200µm for isolated gonad images; 50µm for confocal images.

Figure 3 .

 3 Figure 3. Sites of MIHR expression in the Clytia medusa

Figure 4 .

 4 Figure 4. Oocyte maturation failure in MIHR mutant medusae

Figure 5 .

 5 Figure 5. MIH-induced oocyte maturation blocked by an inhibitory Gα s antibody

Figure 6 .

 6 Figure 6. Relationship of Clytia MIHR to bilaterian neuropeptide hormone GPCRs

Figure 7 .

 7 Figure 7. Schematic comparison of GPCR regulation of Clytia and vertebrate oocyte maturation

  Fisher exact tests were performed at http://www.socscistatistics.com. of inhibition mechanism for cell cycle progression. J Biol Chem. 2000;275: 41512-41520. 74. Nader N, Courjaret R, Dib M, Kulkarni RP, Machaca K. Release from Xenopus oocyte prophase I meiotic arrest is independent of a decrease in cAMP levels or PKA activity. Development. 2016;143: 1926-1936.

Table 1 . Characteristics of MIHR mutant colonies Colony name Predominant mutations

 1

			Medusa sex Polyp	Medusa gonads
				colony*
	n5-23	-4nt , +74nt	F	normal	Enlarged and deformed
	n5-8	-7nt	F	rambling	Poorly developed
	n5-13	-4nt , -3nt	F	rambling	Enlarged and deformed
	n5-24	-34nt ,+59nt	M	normal	Poorly developed
	n5-10	-4nt	M	normal	Enlarged and deformed
	n5-6	-4nt , -3nt	M	normal	Enlarged and deformed

*see Fig.

2A

Acknowledgements

We thank our LBDV colleagues Philippe Dru for Bioinformatics support, Maeva Goulais for assistance with immunofluorescence and Céline Hebras for antibody injections. Thanks also to J-P Concordet (MNHN Paris) for generously providing Cas9 protein, Noriyo Takeda (Hiroshima University) for the anti-PRPamide antibody and Laurinda Jaffe (University of Connecticut Health Centre) for the anti-Gα S antibody. Funding was provided by the Marie Curie ITN NEPTUNE and French ANR grant OOCAMP-ANR-13-BSV2-0008, as well as core CNRS and Sorbonne University funding to the LBDV. For animal care and microscopy we thank of the Service Aquariologie (especially Alexandre Jan) and the Villefranche-sur-mer Imaging platform of the Institut de la Mer de Villefranche (IMEV) /EMBRC-France (ANR-10-INBS-02).

List of Supplementary Figures and Files