Avian uterine fluid proteome: exosomes and biological processes potentially involved in sperm survival Cindy Riou, Aurélien Brionne, Luiz-Augusto Cordeiro, Grégoire Harichaux, Audrey Gargaros-Ratajczak, Valérie Labas, Joël Gautron, Nadine Gérard

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Cindy Riou, Aurélien Brionne, Luiz-Augusto Cordeiro, Grégoire Harichaux, Audrey Gargaros- Ratajczak, et al.. Avian uterine fluid proteome: exosomes and biological processes potentially involved in sperm survival. Molecular Reproduction and Development, Wiley, 2020, acceptée (4), Acceptée. ￿10.1002/mrd.23333￿. ￿hal-01605894￿

HAL Id: hal-01605894 https://hal.archives-ouvertes.fr/hal-01605894 Submitted on 26 May 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Version postprint 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., SponsorGrant Keywords 6 Nouzilly, F-37380 (PAIB), France et l’EnseignementRecherche Pôleet d’Imagerie d’Analyse (CIRE), des Biomolécules 5 4 3 2 1 Avian uterinefluid and proteome: exosomes biological potentially processes involved in Corresponding author. Email: author. Corresponding [email protected] University ofSemiFederal Mossoro, Arid Region, 59625-900, Rio Grande doNorte, Brazil Université BOA, de Tours,INRA, 37380,Nouzilly, France Station de Phénotypage, Perroi,ALLICE, 37380Nouzilly, Lieu-Dit FranceLe CNRS, Tours, PRC, de IFCE, 37380, Nouzilly, Université France INRA, INRA, Université de Université Tours,Tours, Plate-forme de Chirurgiela CHU de INRA, et pour Imagerie Riou Cindy : uterine fluid, exosomes, sperm, proteome avian, 1,2 , Brionne Aurélien , Brionne : Conseil duCentre régional Val de : namegrant OVISPERM. Loire, Labas Valérie Labas Comment citer cedocument: 3 , Cordeiro Luiz

5 , Gautron , Gautron Joël sperm survivalsperm 1 1

1,4 , Harichaux Grégoire, Harichaux 3 , Gérard Nadine , Gérard 1,6

5 , Gargaros Audrey, Gargaros

5 , Version postprint 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., the between female and sperm. tract genital in the sperm presence Their storage tract. genital female tubules the within functions sperm may preserving in represent role key a play an may important mechanism regarding interaction These exosomes. contains fluid uterine avian that time first the for demonstrated we conclusion, with one study describing the proteomic content of fluid,and henuterine of proteomic on published studies twopreviously to were compared data rooster seminal plasma and sperm. In Exosomes observedwere both at the surface epithelium and inside sperm storage tubules. Our used to analyze uterovaginal junctions for the exosomal proteins ANXA4, VCP and PARK7. MDH1) using immunoblotting. Electron microscopy and immunohistochemistry targeted were also several exosomes markers (ANXA1/2/4/5, VCP, HSP90A, isolated HSPA8, exosomes from PARK7, avian and uterine fluid, analyzed them identified, using160 are known electron to be microscopy secreted and and proteins 640 913 are the Among referenced ProteomeXchange. in to submitted exosomes were databases. data proteomic We The mechanisms. performed to improve understanding of was fluid uterine hen of this analysis bioinformatic and strategy proteomic up bottom fluid a study this In and its potential role in sperm survival fluid is Uterine an aqueous milieu sperm to which exposed their are during andstorage ascent. Abstract Comment citer cedocument:

2 2 Version postprint 76 75 74 73 72 71 70 69 68 67 66 65 64 63 62 61 60 59 58 57 56 55 54 53 52 51 50 49 48 47 46 45 44 43 42 41 40 39 38 37 36 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., still understood.are poorly preserve sperm function and/or regulate their release. Nevertheless, the mechanisms involved Then these may adhere to the epithelium in order in to the lumen enhance of the SST sperm may originate retentionfrom other regions within of the genital SST, tract such as by UF. Furthermore, we can hypothesize that some key proteins localized at the surface epithelium or has been postulated that UF constituents are essential for sperm storage efficiency hens inexhibit a birds. longer period of fertile egg production (Brillard et al., 1987). Consequently, it activitysecretory of the increasesuterus and the is highlyUF secreted. Under theseconditions efficiently stored when inseminated at the beginning ovulation) of than the the eggshell formation, calcifying when phase the (Ahammad et insemination is performed during initialization al., of eggshell mineralization (i.e. 2013). 5-6 hours after Thus, sperm may be sperm storage more efficiency. They showed that the filling of UVJ-SST is that the more stage of the secretory activityeffective of the female reproductive whentract has an incidence on the (Ahammad et al., 2013; Brillard et al., 1987). Actually, Ahammad et al. (2013) demonstrated formation, UF has been shown to promote the maintenance of sperm Additionally to the motility fact that the secretory and activity of the viability reproductive tract depends to the egg al., 2007;2019; 2006; Gautron et et al., Silphaduang al., Wellman-Labadie et 2008). of bactericidal factors, providing an aseptic package composed for also embryo developmentis (Gautron It et 2013). al., al., et Sun 2015; al., et (Marie ovulation after hours 5 from occurs that proteins mineralization involved process key inepithelialcontainseggshell cells. the several It also contains locally produced factors, mainly related relation to the egg formation (Gautron et al., to2019). is aIt selective transudate of serum which the metabolic activity of uterine to the infundibulum. Uterine vagina fluid the from has ascent its beenduring exposed is widely sperm which in investigated environment aqueous over the represents the past few years in tract.It reproductive avian the in described yet fluid reproductive only the is (UF) fluid Uterine 2013). al., et (Sasanami fertilization of site the is which infundibulum, the to UVJ-SST the from and are stored in the uterovaginal SST (UVJ-SST) for several days. Then, the sperm mating, at the same timetransit as the daily process of egg formation, sperm pass through the vagina After laid. is and vagina the through transits eggthe ovulation, after hours 24 Finally, 1. Figure contiguous with those of the uterine tissue (Bakst & Akuffo, 2009), as shown schematically in compresses the UVJ folds that are positioned towards the uterus, resulting in its mucosa being takes place in the uterus within an 18 hours period. At calcification eggshell Then that ovulation). post time, hours 5 to the (3.5 synthesized egg are membranes mass eggshell in the uterus isthmus, the ovulation). In post hours 3.5 (0.5to isproduced white theegg where magnum, the ensures the deposition of the vitelline membrane outer layer. The developing egg then enters isthmus, uterus and vagina, which are associated with the egg components. The infundibulum The female avian reproductive tract (oviduct) consists of the infundibulum, magnum, white junctionof the utero-vaginal called are sperm (UVJ), tubules (SST) 2011). storage (Bakst, located Moller, 1993). Sperm mainly in & the al., mucosa 1994; reservoirs, Birkhead et (Bakst Avian species have the capacity to store sperm for several weeks in the female genital tract Introduction Comment citer cedocument:

3 3 Version postprint 92 91 90 89 88 87 86 85 84 83 82 81 80 79 78 77 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., a mechanismrepresent genital interaction tract-sperm of involved in sperm survival. preservation of sperm functions during storage and ascent. Their inthe role a play may which particularlyexosomes, EV, presencecontained UF avian that demonstrated in the SST may better the potential underlying mechanisms involved in composition of UF the using proteomic analysis, to sustained examine its EV sperm content, and thus understand survival. We their storage and ascent in the In this study, female we hypothesized that genital UF contained molecules tract. that may preserve We sperm during aimed to determine the exosomes protein produce cells SST that showed (2017) al et Huang recently more and 2015), Bauchan, & (Bakst demonstratedpresenceUVJ-SST (2015) of30-130turkey Bauchan the nm EV in the of lumen described the presence of EV in the fluid from of the female genital tract. In birds, Baskt and in cell-to-cell signaling (Valadi et al., 2007). To date, in mammals, only a few involved in studies the transfer of molecules from haveone cell to another, and thus play an important role forms, but also engulfed in cell-derived membranous extracellular vesicles (EV). These Many are biological fluids have been shown to vehicle molecules not only as soluble/secreted in vitro in the presence of sperm (Huang etsperm (Huang presence of al., 2017). in the Comment citer cedocument:

4 4 Version postprint 129 128 127 126 125 124 123 122 121 120 119 118 117 116 115 114 113 112 111 110 109 108 107 106 105 104 103 102 101 100 130 99 98 97 96 95 94 93 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., In-gel and liquid digestion pieces. mm3 1 approximately into cut was which slices, 30 into sectioned was lane Each Fermentas®). Solution, Staining Protein (PageBlue™ staining Blue Coomassie with stained and (10%) PAGE from individual samples. Fifty µg of proteins from the pool of UF were fractionated by SDS- proteins of same amount the mixing pooledby were above) (see buffer in Laemmli diluted and Thermofisher kit, The protein concentrations of samples were determined using a Pierce SDS-PAGE fractionationwith hen processed. and each uterovaginal junction. Samples of UVJ mucosa containing the UVJ-SST were collected from to vagina was excised uterus from section tract as genital The dislocation.cervical euthanizedby were Hens microcopy. one segment. Connective tissue Avian UVJ were was collected from 6 removedhens for immunohistochemistry and transmission to electron expose the Tissue Collection and to measureMS concentration. analysis, protein to limit calcium carbonate and proteins precipitation, in order to perform in solution digestion (1v:1v) PBS dilutedwith were hen per fluid of aliquots other Two -20°C. at storage before min % SDS, 12.5 % β-mercaptoethanol, 50 % glycerol, bromophenol blue), and then boiled for 5 UF was immediately diluted with 5x Laemmli buffer (5v:1v) (312.5 mM Tris-HCl pH 6.8, 10 vagina, from 10 virgin hens as described previously (Gautron et al., 1997). An aliquot of each During egg expulsion UF was collected in a plastic tube placed at the entrance 50µg/hen. of at the F2A everted prostaglandin of injection intravenous by induced was expulsion Egg utero. in collectionUF was scheduledoviposition 10hafter and after theconfirming presence of an egg Uterine fluid collection MinistryFrench and the Research underof agreement number #443. n°19) Loire de Val de d’éthique (Comité committee ethics local the by approved was collection 8D photoperiod, and fed 16L: a a under kept were They oviposition. layer of time the record to mash devices automatic with equipped ad libitum. The protocol of bird management (B27-175-1 and dated 28/08/2012). At 50 weeks of age, birds euthanize and rear authorizedtoofficially are 1295, Agronomique,UE-PEAT Recherche hens were placed in individual cages authorized scientist (Authorization # 37035). The the French facilities Ministry of at Agriculture on the animal experimentation, Institut of under practices use, and care animal National of the practices regarding Directives Council Union European the supervision de of an la in this study. Breeding procedures and handling protocols were carried out in accordance with used were 1992) al., et (Beaumont strain broiler-type light a from hens domestic female Mature Birds andMaterial Methods TM . For exhaustive identification, the aliquots of UF collected from 10 females 10 from collected UF of aliquots the identification, exhaustive For . Comment citer cedocument:

5 5 TM BCA protein assay Version postprint 170 169 168 167 166 165 164 163 162 161 160 159 158 157 156 155 154 153 152 151 150 149 148 147 146 145 144 143 142 141 140 139 138 137 136 135 134 133 132 131 171 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., noise to signal and 350–5000 Da of range mass precursor A USA). Jose, San Scientific, Fischer Thermo 1.4; (version software Discoverer Proteome with MSF to converted were files data Raw Protein identification data validation and accurate massfor measurements. enabledmass was 1. The lock count of for 30s with a repeat Dynamic exclusion was activated scanned at the “normal scan rate” with quantity for FT full MS was 1e6 and for MS ion forCID-MS/MS.Target ms 25 and fullscans msfor 500 allowedtimeswere accumulation (CID). The ion selection threshold (isolation was width, 2 500 m/z; 1 counts microscan) and for fragmented using MS/MS, Collision 300-1800, Induced the and Dissociation 20 most the intense peptide ions maximum with charge states ion ≥2 were sequentially isolated dependent mode. Resolution in the Orbitrap was set at R data- in = mode positive60,000. in operated In was the instrument The scan CA). range Jose, San of Scientific, m/zFisher Thermo 2.1; software (version usingXcalibur acquired were Data nl/min. 300 at set was rate nanoflow The runs. between B 4% at min 15 for re-equilibrated was column The min. 1 in B 4% to return gradient consisted of 4-55%B 90min, for 55 to 99% B for 1min, constant 20 99% B min and (Acclaim PepMap C with 4% solvent B. The peptide separation was conducted using a LCPackings nano-column 5µL/min at 10 min for preconcentrated and desalted were Peptides (v/v/v). ACN 84% water, % 15.9 FA, 0.1% (B) and (v/v/v) ACN 2% water, 97.9% FA, 0.1% (A) of consisted phases Mobile (Acclaim PepMap 100 C Netherlands). Five microliters of each sample were The Amsterdam, Dionex, ; loaded SR11 6.8 (version Software on Chromeleon by controlled Netherlands) an LCPackings trap column 3000 RSLC Ultra High mass spectrometer (Thermo Pressure Fisher Scientific, Bremen, Liquid Germany) coupled to Chromatographer an spectrometry (nanoLC-MS/MS). Ultimate® All experiments (Dionex, were performed on a Amsterdam, LTQ Orbitrap Velos The Peptide mixtures were analyzed NanoLC-MS/MS by nanoflow liquid chromatography-tandem ACN, 2% sonicated10 minFA, and MS analysis. for before mass and in ACN for 5 min. The two extracts were pooled, dried, reconstituted with 13µL of 0.1% peptides were extracted from gel by successive incubations in 0.1% FA/ACN (1:1) for 10 min 2% ACN, and sonicated for 10 min before FA, 0.1% of 13µL MS with reconstituted dried, was solution analysis. peptides resulting the digestion, For liquid in-gel digestion, the resulting overnight in 25 mM NH NH temperature in the dark. For in-gel digestion, gel slices were washed by incubation in 50 mM NH and alkylation were performed by successive incubations in in water/ACN (1:1) for 10 5 min and in mM ACN for 10 dithiothreitol/50 min. For both digestions, mM cystein reduction individual samples collected from 10 females. For in-gel digestion each gel slice was washed from proteins of amount same the mixing by pooled was sample UF of µg 5 digestion liquid For 4 4 HCO HCO 3 3 /ACN (1:1) for 10 min and by incubation in ACN for 15 min. Proteins were digested were Proteins min. 15 for ACN in incubation by and min 10 for (1:1) /ACN NH mM iodoacetamide/50 mM 55 and 56°C at min 30 for 18 , 75µm inner diameter x 50 cm long, 3µm particles, 100Å pores). The Comment citer cedocument: 4 HCO 18 , 100 µm inner diameter x 2cm long, 3µm particles, 100Å pores). 3 with 12.5 ng/µl trypsin (Sequencing Grade, Roche, Paris). For

q = 0.25 activation and activation time of 10 ms. 2 6 6 it was 1e4. The resulting fragment ions were 4 HCO 3 for 20 min at room at min 20 for Version postprint 213 212 211 210 209 208 207 206 205 204 203 202 201 200 199 198 197 196 195 194 193 192 191 190 189 188 187 186 185 184 183 182 181 180 179 178 177 176 175 174 173 172 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., 2011) al., et (Petersen 4.1 P Signal the as well as (http://www.cbs.dtu.dk/services/SecretomeP/), identified match, i.e. number proteins. of mismatches and gaps) were considered orthologous to than the 91% previously (in terms of their blast results, The including the size of peptides and the profile of the non-redundant proteome presented in the present study. Only proteins that display SIsc higher SecretomeP result did not share any similar name or protein description. The resulting file constituted the 2.0 unknown as considered b) and result, Scaffold Chordata (Bendtsen gallus strictly identified in the et gallus al., a with =100%) (IDsc match perfect a Only (IDsc). identity and (SIsc) similarity of score 2004) et al., 2009). The alignment result of each peptide for each protein was expressed as a global database limited to strength for the next step of analysis. Longer peptide sequences were blasted against nr NCBI (reconstituted peptide), in order to remove the peptidic redundancy and to generate a higher continuous peptides from the same sequence were concatenated to description of create the a annotated longer gene peptide were extracted from nr NCBI resulted database. from the Overlapping digestion process, and respectively. For each protein the identifier, symbol and (http:/cran.r-project.org) after elimination of keratin and trypsin, as they were contaminants or Proteomic data were extracted from Scaffold software miningStatistical, data and bioinformatics analysis and analyzed using R language to clarifyscaffold groups.in order the similar and Omega Clustal with constructed was tree phylogenic A software. Scaffold using emPAI the calculating by estimated was proteins identified of abundance The level. protein or peptide the at 1% < as calculated was rate discovery false A peptides. identified twoleast at contained and algorithm Prophet Protein accepted if they could be established at greater than 95.0% Peptides probability were as considered specified distinct by if the they differed in sequence. established Protein at greater identifications than were 95.0% probability as specified by the Peptide Prophet algorithm. Proteome Software, Portland, USA). Peptide identifications were accepted if they fragment could ion matches. be Mascot results were incorporated in Scaffold 4 software (version 4.3, variable modifications. The tolerance of the ions was set at 5 ppm for parent and 0.8 Da for carbamidomethylcysteine, oxidation of methionine and for database N-terminal searches included trypsin as protein a protease with two acetylation missed cleavages allowed, and as Daemon and search engine (version 2.3; Matrix Science, London, UK). The parameters used of nrNCBI (1601319 sequences). MS/MS copy maintained locally a of section Chordata the against automatically ion matched were obtained searches were performed using MASCOT dataset identifier [ (http://proteomecentral.proteomexchange.org) via the PRIDE 2019) partner repository with the converted using a PRIDE Converter (version 2.5.3) (Deutsch et al., 2017; Perez-Riverol et al., and ratio of 1.5 were the criteria used for generation of peak lists. The MS proteomics data were deposited with the ProteomeXchange Consortium proteins when the blastp result indicated the same protein description and name as the protein allowed removal of redundancy inside protein groups. Proteins which were not submitted Gallus gallus Comment citer cedocument: Gallus gallus ] and project [ Gallus gallus Gallus taxon, using the blastp program (BLAST+ suite) (Camacho

database were: a) considered as ortholougous to proteins were searched in nr databases using blast blast using databases nr in searched were proteins submitted 7 7 ]. The peptide and fragment masses Gallus gallus Gallus proteins when the blastp the when proteins Gallus Gallus Version postprint 252 251 250 249 248 247 246 245 244 243 242 241 240 239 238 237 236 235 234 233 232 231 230 229 228 227 226 225 224 223 222 221 220 219 218 217 216 215 214 253 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., for 5 minutes were loaded and separated on a 10% SDS-PAGE, before being and transferred isolated-EV samples that to were added with 5x Laemmli buffer (5v:1v) and boiled at 95°C samples, (EV-depleted) UF ultra-centrifuged samples, UF the from proteins of 10µg of Aliquots instructions. manufacturer’s the to according and standard protein the as albumin serum bovine BiCinchoninic Acid protein assay kit (Life Technologies SAS, Saint The protein Aubin, concentration France) was determined using in each sample of UF and EV using the Pierce® Western blotting proteins of EV using a Megaviewacquired camera coupled withsolf III imaging software.system AnaySIS lead were citrate and uranyl acetate using 80Kv. and a observed CM10 Philips TEM at Images using counterstained were Sections ultramicrotome. Leica Ultracut an with prepared were 70nm embedded in epon resin. Polymerisation was performed at 60°C for 48 h. Ultrathin sections of and 100%) to (70 ethanol of baths graded in dehydrated then were Samples 20°C. at h 2 for 2% Following fixation, samples were transferred to cacodylate 0.1M buffer containing osmic acid with 4% glutaraldehyde in 0.1 M Na cacodylate buffer pH 7.2, for 4 h at room temperature. charge-coupled device camera (AMT). Tissue samples from uterovaginal junctions were fixed electron microscope operated at 80kV (Elexience – France), and images were acquired with a and contrasted for 10s in 1% uranyl acetate. Grids were EV were analyzed as whole-mounted vesicles examined deposited on EM copper/carbon grids for 5min, with a Hitachi HT7700 analysisTransmissionEV microscopy electron and of uterovaginal junction were supernatant also kept at -20°C. and stored at -20°C until analysis. An with SW41T1 aliquot rotor) provided an exosome pellet. of The pellet was resuspended each in 50µl of PBS initial pool and ultracentrifugation and cell debris. Two successive ultracentrifugations at 100,000g for 90min (Beckman L8-M before being centrifuged at 100 g for 15 min, followed by 12,000g for 15min to remove cells pooled 2 to were UF (n= completed5 animals/replicate) and the volume was to 5ml with PBS Briefly, 2006). al., et (Thery described previously as centrifugations serial by obtained were EV preparation EV BLAST+ seminal suite, andusing usingrooster’s and plasma sperm from protein ID. quantitative proteome lists from avian UF and eggshell (Marie et al., 2015; Sun et al., 2013) compared using Fisher’s exact test. Our identified peptides were also compared to published immunity and sperm survival. The molecular function of total UF and exosomal proteins were proteins were categorized in three term main annotation from putative Uniprot and functions Genecards (chicken and of GO- mammals), the to and function, interest molecular their to to According the (http://www.ensembl.org/biomart/martview). as literature, mineralization, protein sequences using (chicken) databases BioMart (personal data). Extraction allow of conserved to and constituted functional by domains classify merging from Exocarta proteins (human, obtained in the study.present Our rat, data were then tocompared an exosome whichdatabase we by mouse), UniprotKB molecular (chicken) proteins each for exosomes) via or and independent or peptide-dependent (signal pathway function secretion KEGG (http://www.cbs.dtu.dk/services/SignalP) servers were used as a prediction method of the Comment citer cedocument:

8 8 Version postprint 295 294 293 292 291 290 289 288 287 286 285 284 283 282 281 280 279 278 277 276 275 274 273 272 271 270 269 268 267 266 265 264 263 262 261 260 259 258 257 256 255 254 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., 30 min with the secondary antibody ImmPRESS non-fat dry milk. The slides were then washed three times in TBS (5 min), and incubated for CSB-PA001845ESR2HU, Cusabio, Clinisciences, Nanterre, France) diluted in TBS with 5% 270 Novus biologicals, Bio-Techne, Lille, France) or anti-ANXA4 (1:500; rabbit polyclonal; monoclonal; ab11433, Abcam, Paris, France), anti-PARK7 (1:500; rabbit polyclonal; NB300- were incubated overnight at 4°C placed in witha bath with normal the horse serum blocking primary solution (Vector) for antibodies, 20 min. The recommended anti-VCP slides by the manufacturer. (1:500; The slides were mouse rinsed once in TBS for 5 min based and then unmasking solution of excess H3300 ethanol by water baths. (Vector After removal rehydration, and bathes,) ethanol the 70% and 80% slides 95%, Laboratories, 100%, of were each min (2 treated rehydration progressive with 1% Burlingame, citrate CA) diluted The staining as procedures consisted in deparaffinization 60°C. at overnight incubated then and hours 12 for temperature room at stored overnight, dried for 5 min in toluene, followed air- water, in poly-L-Lysine 0.01% with treated slides onto sequence in collected were bysections thick 7-μm six to Four IL). Richmond, Inc., Richmond, Microsystems Leica Center, Embedding Processor). The samples were then embedded in to paraffin an (Leica EG1150 automated Modular tissue Tissue processor system weretransferredmucosa) to two successive baths of 70% ethanol (2x30 min), and then moved (Leica TP1020 Semi-enclosed Benchtop Tissue Following fixation for 24 hours in 4% saline buffered (PBS) formalin, tissue samples (UVJ Immunohistochemistry on uterovaginal tissue ImageMaster bio VDS-CL system (Amersham imaging Biosciences/GE Healthcare). (GE Healthcare, Velizy-Villacoublay, France) peroxydase activity was detected with the and ECL select the signal Interchim), diluted was 1/5000 in captured the using blocking solutionIgG (A4416 the and Sigma-Aldrich) finally or washed goat with anti-rat TBS-T. IgG anti-rabbit (112-036-003 The IgG (A6154, Jackson Sigma-Aldrich, Saint ImmunoResearch, Quentin Fallavier, France) or goat anti-mouse goat i.e. antibody, secondary peroxidase-conjugated with hour 1 for thensolution, blocking the Gautron. The membranes were then sequentially washed with TBS-T, incubated for 1 hour in BPIFB3/OCX36 (1/5000), or anti-OC17 (1/1000) both rabbit kindlypolyclonal provided by J. rabbit polyclonal; CSB-PA001845ESR2HU, Cusabio, Clinisciences, Nanterre, France), anti- (1:1000; anti-ANXA4 Germany), Heidelberg, biotechnology, cruz Santa sc-11387, polyclonal; PA06384A0Rb, Cusabio, Clinisciences, Nanterre, Clinisciences, France), anti-ANXA1 Nanterre, (1:1000; rabbit Lille, France), anti-ANXA2 France), (1:1000; rabbit polyclonal; CSB-PA001840HA01HU, Cusabio, anti-ANXA5 France), anti-PARK7 (1:1000; rabbit polyclonal; NB300-270 Novus biologicals, Bio-Techne, (1:1000; (1:1000; rabbit rabbit polyclonal; polyclonal; (1:500; CSB-PA013621ESR1HU, rabbit CSB- Cusabio, polyclonal; Clinisciences, bs-5117R, rat Bioss, Nanterre, monoclonal; Interchim, ADI-SPA-840-D, Montluçon, Enzo (1:1000; anti-HSP90A life France), France), Paris, Abcam, ab11433, monoclonal; mouse sciences, (1:1000; anti-VCP anti-MDH1 Villeurbanne, France), anti-HSPA8 (5% (w/v) non-fat dry milk in TBS-T), and then overnight in the blocking solution containing 7.4) containing 0.1% (v/v) Tween-20 (TBS-T), incubated for 1 hour in the blocking solution nitrocellulose filters. The membranes were washed with TBS (10mM Tris, 150mM NaCl, pH Comment citer cedocument:

9 9 TM HRP Anti-Rabbit/Mouse IgG (Vector). TM Western Blotting Detection Reagent Version postprint 306 305 304 303 302 301 300 299 298 297 296 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., observationsseveral performed hens onthreeconditions. per isotypic-specific immunoglobulin at the same concentration. Figures immunohistochemistry are were performed representative by of the substitution of Instruments) the primary antibody coupled by an with using amicroscope. were digital Images (Spot-Flex, acquired camera monochrome Diagnostic the SPOT were 5.2 incubated in toluene for 1 min. imaging Sections were examined using an software. Axioplan dehydration Carl through graded Zeiss alcohol baths (70%, 80% 95%, 100%; 20s Negative each) and the sections controls Papanicolaou for stain (Sigma). Coverslips were applied with aqueous mounting medium and after finally rinsed in They were rinsed in TBS distilled (5 min), incubated with peroxidase water ImmPACT NovaRED (Vector) (5 min). The sections were counterstained with Comment citer cedocument:

10 10 Version postprint 346 345 344 343 342 341 340 339 338 337 336 335 334 333 332 331 330 329 328 327 326 325 324 323 322 321 320 319 318 317 316 315 314 313 312 311 310 309 308 307 347 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., (supplementary data 1). Of these 437 proteins, 324 (74.1%) have previously been observed in (47.9%) have not been either These contained peptidenota the (155) or (321). To date, signal remaining 437proteins referenced as potentially a total secreted of 476/913 proteins (52.1%) proteins that have already demonstrated tools bioinformaticbeen SignalP and in SecretomeP categorized of use The as 3. Figure in secreted presented are proteins. either database study present the in identified proteins UF 913 the of localization extracellular and Intracellular Analysisproteins in fluid of uterine relation to cellular localization emPAI displayed they UF; values to from 21.667 (rank 16) 0.015(rank 908). remaining 586 proteins (64.1%) revealed in our study had previously been identified in avian listed in the a possessed 10 the in listed protein a to corresponded 312 proteins, shared 327 these of Out studies.previous two the of withsharedone were ouridentified proteins of 174 Moreover, than 91%) (Figure 2B). Their emPAI values ranged from 1,000 (rank 1) to 0.037 (rank 900). proteins common to the studiesthree (i.e. with a protein sequence similarity coefficient higher proteins. Of to the 913 UF proteins described in the current study, we identified 153 (16.8%) in these two previous studies were combined resulting in the identification of 1,230 avian UF and study current the in described proteome The 2013). al., et Sun 2015; al., et (Marie approach for laying hens (White Leghorn and ISA-Hendrix, respectively) using a bottom-up proteomic A few years ago, Sun et al., 2013 and Marie et al., 2015 revealed the UF protein composition Uterine fluid proteins overlap with which laying-type from hens those sequenced already avian proteins. Falco peregrinus n=1). One of these 18 proteins 90.7% displayed similarity with thatALDOA was annotated in proteins), or to a neighboring order (Passeriformes, n=9; Psittaciformes, n=1; Falconiformes, as family same the to related are some species, Actinopterygii (n=2), or Amphibian (n=1) species. Among the 18 proteins identified in Aves identified in Aves, whereas others in either wereidentified identified in Mammalia (n=15), (n=3), Reptilia Amphibian (n=3), (n=1) or Cephalochordata Aves (n=1) species, compared (Figure to 38 2A). proteins in Of Mammalia (n=26), theReptilia (n=7), database (supplementary data Actinopterygii1 part 3). 39 A total of 875 proteins have already been identified in proteins not (supplementary data 1 part 2), 39 corresponded to the proteins not listed in the emPAI value of 1000 and 999.99, respectively. Of the 913 proteins, 837 are already listed in calculation.emPAI Thetwo main components were Lysozyme Albuminand that displayed an the using determined was sample the within abundance Their 1). data (supplementary identified were proteins UF 913 of total a redundancies, of elimination After proteomic. shotgun then and broiler-type hens was performed using GeLC-MS/MS followed by SDS-PAGE fractionation light- of UF the in present proteins soluble of inventory proteomic global a study, present the In Results Gallus gallus Gallus gallus Gallus gallus Gallus database (supplementary data 1 part 1), 37 presented an orthologous protein , whereas all the others displayed between 40.6% and 90% similarity with Gallus gallus Gallus Comment citer cedocument: database yet (supplementary data 1). None of the peptides from the orthologous protein and five corresponded to proteins that are not are that proteins to corresponded five and protein orthologous or orthologous to orthologous or

11 11 Gallus gallusGallus Gallus gallus Gallus such as as such proteins, 18 have already been already have 18 proteins, Meleagris gallopavo Meleagris Gallus gallus Gallus Gallus gallus database, (n=7 (n=7

Version postprint 385 384 383 382 381 380 379 378 377 376 375 374 373 372 371 370 369 368 367 366 365 364 363 362 361 360 359 358 357 356 355 354 353 352 351 350 349 348 386 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., determined using InterPro, to extract the functional domains The of molecular functions each of protein, the followed 913 by avian UF proteins identified in the present study were Analysisproteins in fluid of uterine molecular relation functionsto observed onthe apicalmainly part of the epithelium 6D). (Figure visualized within epithelial cells and lumen ofSST 6B-D). A (Figure high signalANXA4 was (Figure 6A). VCP, PARK7 and ANXA4, which are immunohistochemistry all demonstrated known as the exosome absence markers, of were non-specific for controls signals Negative sperm. store to is role whose UVJ inthe localized glands tubular SSTare for all antibodies Immunohisto-localization PARK7 of and ANXA4VCP, in SST 5C)SST.nm) (Figure in the lumen of (<100 nm) (Figure 5B). We also detected apocrine blebs containing microvesicles (100-1000 epithelium (Figure 5A). Within SST, we observed intracellular MVB TEM containing analysis exosomes of UVJ revealed the presence of exosomes (30-100 nm) at the surface of the observed were in EV-depletedPARK7 UF. signals the avian EV fraction, whereas BPIFB3 and OC-17 were not. Moreover, HSPA8, MDH1 and HSP90A, HSPA8, ANXA2, MDH1, ANXA5, ANXA1, ANXA4 and PARK7 were present in VCP, that demonstrated proteins non-exosomal and exosomal of 4C) (Figure Immunodetection from 30 to 100 4A). contained It several vesicles of various sizes (20.695 - 244.628 nm). majority rangedThe nm and (Figure microscopy electron transmission wasusing analyzed was UF from purified morphologically fraction EV The consistent with exosomes (Figure 4B). Analysis from of EV/exosomes UF and in SST MAPRE3). and (Loc102114577 (Immunoglobulin A heavy chain variable region), but are and already listed 3/113 in the Aves correspond class to in the Aves class and possess a proteins that are of these, 108/113 proteins are listed in the not listed Out 2013). al., et Sun 2015; al., et (Marie studies proteomics UF avian two the of one least in at in the identified been already have 12 secreted, as identified not yet as proteins 113 these of Out 907). (rank 0.01 and 3) (rank 170.55 between ranged values emPAI Their shown). not (data databases when considering the GO-term enrichment in localized in FunRich cellular (human) components such or as cytosol, Genomatix lysosomes, (chicken) cytoskeleton have and not centrosome, been categorized as secreted (12.4%) (Figure proteins 113/913 only date to 3; Finally, rank=24). (emPAI=12.56; HSPA2 and supplementary rank=17) data 1). These are mostly rank=578), and several proteins from the (MVB) biogenesis such HSP70 as Alix (PDCD6IP; emPAI=1.27; rank=162), TSG101 family (emPAI=0.24; such as HSPA8 (emPAI=22.06; 0.36 (rank 458). Moreover, we identified some proteins markers (Exocarta DB) involved have been found. Their in emPAI values varied from multivesicular 999.62 (rank 2) to bodies EV/exosome the of 25 top the of 18 study, present the in identified proteins UF 913 the Among biological fluids within EV named exosomes (UniprotKB, Exocarta and KEGG databases). Comment citer cedocument: Gallus gallus

Gallus gallus orthologous protein orthologous (ADPRHL1 and RPS6KA3) 12 12 database, 2/113 have been identified Gallus Gallus gallus database Version postprint 424 423 422 421 420 419 418 417 416 415 414 413 412 411 410 409 408 407 406 405 404 403 402 401 400 399 398 397 396 395 394 393 392 391 390 389 388 387 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., bothor sperm (64/99; 8). (Figure 10%) or 20%) (141/154; 22%) (125/143; plasma seminal to and cluster, exosomethe to belong proteins330/396 Furthermore, 2015). al., et Labas 24%, both(154; or 15%), (99; sperm or 16%) (143; plasma seminal rooster It is noteworthy that 396/913 proteins that we described in avian UF have been identified in Uterine fluid proteins overlap with which semen avian proteins binding and/or activity, calcium properties. ANXA2, ANXA8, ANXA5 and ANXA1, ALB, which PRDX1, all PRDX6, GSTA3, possess GSTO1, and phospholipase GSTO1, A2 and comprising proteins antioxidant included it Moreover, VCP. and annexins.HSP90AA1 HSPA2,HSPA8, inhibitor These latter include or chaperone binding proteins including chaperones contained also subunits It pathway. ubiquitin the from in involved proteins comprises and the proteins TCP1-ring complex as well as (Figure 7B). This exosome cluster was significantly (p <0.001) The third enriched group contained with proteins that proteasomal potentially mediate sperm survival via the exosomes response. belonged to the immunoglobulin superfamily and bioavailability of one iron and is vitamins, known 27 displayed to microbial protease mediate inhibiting which the activity, five 10 immune contained microbial degrading components, The second five group concerned molecular were defense (Table 2). involved It was made in up of decreasing 48 proteins of between 0.03 emPAI displayed and (ROS1). 1,000(LYZ) were included in collagen fiber (ECM group possessed components). either calcium They binding or were proteoglycan/proteoglycans major binding domains, thisfrom mineralization. UF 31proteins otherto The relatedphosphatases chaperons proteases, or or proteins that of which 28 may be involved in the regulation of protein activity since they corresponded to proteins,59 composedof was mineralization1). It concerned (Table functional group first The mineralization,defense molecular pathogens against survival via and sperm exosomes. Potential functions of the 913 proteins were examined with particular emphasis on eggshell Analysisproteins in fluid of uterine relation processesto biological biogenesis trafficking, and membrane(MVB) respectively. body multivesicularwith associated were proteins 27 and 29 that noteworthy is It etc.). 6, and 4 PDIA3/ TCP1, 1, protein T-complex of 8 and 7 6A/ 5/ 4/ 3/ 2/ subunits HSP90B1, HSP90AA1, family HSPA2/ 4/ (HSP70 proteins chaperon of 4L number large a of and presence the observed we 8, Moreover, etc.). GSN, DNAJA4/ B6/ B1/ B13 and C10, proteins mostly participating HYOU1, in cytoskeletal assembly SGT1, (12 actin isoforms, 6 STIP1, tubulin isoforms, metabolism (GSTA3, GSTM2 and 3, GPX1 and 3, etc.). etc.), The lipid UF homeostasis also (FASN, contained LYPLA1 structural and 2, PLA2G4A, PLCD1, metabolic etc.) processes, such and as glutathione glucose synthesis (MDH1, GPI, PGK, PKM, ALDOA, ENO1, (Figure 7A). The major molecular Uniprot description to attribute the molecular function. This showed a wide range of activities function encompassed enzymes involved in various Comment citer cedocument:

13 13 Version postprint 445 444 443 442 441 440 439 438 437 436 435 434 433 432 431 430 429 428 427 426 425 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., 36), PIT54, SPINK2 immunoglobulin(clone alphachain. and heavy or both (Figure 8). Among them, the six main proteins were HBG2, UBC, Moreover,Ig 66 non-exosomalgamma proteins were chain identified in UF and either seminal plasma, sperm HSPA2, GAPDH and HSP90AA1. YWHAE, ANXA2, ENO1, ALDOC, HBAA, PRDX1, CKB, ACTG1, ALB, were proteins abundant most proteins involved in metabolic pathways comprising 11 proteins taking part in glycolysis. The 31 antioxidantexhibitingproperties, proteins nine complex,TCP1-ring the subunitsfrom eight composed of 11 proteasomal proteins, 12 chaperons or chaperon binding proteins including The 141 proteins referenced in exosomes and in both the seminal plasma and the sperm were and TUBB4B. involved in fatty acid degradation. The most abundant proteins of this group included HSPA8 involved in metabolic process comprising six proteins from chaperon binding proteins, TCA nine proteins cycle,from proteasome or ubiquitin and pathways, 19 five proteins proteins The 64 proteins referenced in the exosomes and the sperm contained seven chaperones or ANXA5, AKR7A2 andANXA8, SERPINF1. most abundant proteins from this group were proteins properties. antioxidant The and presenting four coagulationcomplement LYZ, and cascades ACTB, PRDX6, GDI2, from proteins GC, eight domain, serpin a GSTO1, possessing proteins five pathways, metabolic from proteins proteins involved in vesicle-mediated transport, 25 The 125 proteins referenced proteins in both exosomes and involved the seminal plasma were composed in of 34 homeostasis, 24 Comment citer cedocument:

14 14 Version postprint 484 483 482 481 480 479 478 477 476 475 474 473 472 471 470 469 468 467 466 465 464 463 462 461 460 459 458 457 456 455 454 453 452 451 450 449 448 447 446 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., the improve communication female and sperm. between tract genital et al., 2016). These proteins, observed here in avian UF, could thus support the EV cargo and interact with exosomes when in the presence of heparan sulfate glycosaminoglycan (Dismuke Moreover, it has been demonstrated recently that some proteoglycan binding proteins (FN1) in mammals (Talevi & Gualtieri, 2001), this protein may have a similar role in avian species. release sperm trigger to and binding sperm-oviduct modulate to known are glycosaminoglycans knowing that MEPE is a protein Interestingly, TNXB). core MMP2, PLOD2, (PLOD1, fibers collagen of modulation the of in involved dermatan sulfate proteoglycan, and that sulfated SDCBP2), that may display antimicrobial with abilities functions.to bind proteoglycans (FN1, SDCBP, SERPINF1, HAPLN3, VTN, We SERPIND1, also identified proteins and HSPG2) GPC4, TSKU, (MEPE, proteoglycans of several presence the revealed study Our proteins an influence on the ability of sperm to stick to the extracellular matrix (Koehler et have al., may 1980). (FN1) protein binding proteoglycan and collagen like components ECM mammals, In the uterus.through in the constitution of shell organic matrix may be important (Brillard et al., 1987). Therefore, for it is reasonable to hypothesize that sperm UF components involved survival and ascent shown to be an optimal window for sperm motility artificial insemination regarding the fertility of hens during the initiation of mineralization (6-10 collected hours UF The 2004). al., post et Nys 2012; al., oviposition) et Hincke 2007; al., was et (Gautron formation shell in shown to improve role key a play to known are these All 1). Table proteins; modulating collagen proteins, binding proteins involved in the composition of the extracellular matrix (proteoglycans, proteoglycans proteases, protease inhibitors, chaperones, proteins possessing calcium binding domains, and dehydratases, hydrolases, and OCX36) and RARRES1, (LOC771972, ovocalyxins OC-17), and unique to the process of shell formation such as ovocleidins (MEPE, also known as OC-116 proteins matrixeggshell comprise proteins These 2015). al., et (Marie demonstrated previously Our study demonstrated that major proteins from the UF are involved in shell formation, as Proteins linked to formation shell that in are potentially involved sperm function al., 2015). et (Marie doubled beto listed recently proteins of number the allowed study Our proteome. UF avian of proteins 1,230 of list a revealing respectively), sequences, protein 308 and (n=550 hens identified proteins) to those of Sun et al. (2013) and Marie et al. (2015) both relating to laying We ourcompared dataset, obtained with domestic hens from a light broiler-type strain (n=913 survival within the SST. interaction that may play an essential role in the preservation of sperm functions and extracellularsperm vesicles, mainly exosomes. These EV represent a mechanism of oviduct-sperm classical proteomic approaches. We demonstrated for the first time usingUF inpresentavian proteins of in-depthinventory an we present performed study, the In that avian UF contains Discussion in vitro (Ahammad et al., 2013; Brillard et al., 1987). Moreover, this stage was Comment citer cedocument:

15 15 Version postprint 523 522 521 520 519 518 517 516 515 514 513 512 511 510 509 508 507 506 505 504 503 502 501 500 499 498 497 496 495 494 493 492 491 490 489 488 487 486 485 524 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., 2011; Slowinska et al., 2015). Therefore, sperm the in the epididymis, presence the testis, and the of seminal plasma (Jalkanen SPINKs et al., 2006; in Lee et al., the demonstrated to avian protect sperm from the proteolytic action of UF proteases from dead or damaged may others (LYZ, SPINKs, RARRES1, CST3, OC-17, SERPINs, OVSTL). calcium SPINKs binding haveproteins (OVOT), been ECM components (i.e. proteoglycan binding proteins) or Among these proteins, several are known to regulate the shell mineralization process, such as acceptance. sperm the to participate and tract, genital female the in transit and storage its during antimicrobial proteins most probably act to maintain a bacteria-free environment These 2011). al., et (Réhault-Godbert A2M, LOC418892) C5, C3, OVSTL, SERPINs, for OVAX, sperm and VTG1), or in the inhibition of proteases (RARRES1, SPINKs, CST3, AMBP, AVD VTG2, GC, OVALY, (OVOT, bioavailability vitamin and iron in decrease the in LOC102118097), involved in the degradation of microbial components (LYZ, OC-17, BPIFCB, are oviposition.They post 10h collected UF the in antimicrobial proteins several BPIFB3 identified We and Proteins antimicrobial activitiesexhibiting potentially involved in function sperm regulateconsequently sperm survival and/or motility in the female tract. genital membrane, calcium levels), luminal (pH, features EV), and interactions (cell-cell binding) and Finally, UF proteins involved in eggshell mineralization could impact sperm features (plasma duringthe storage. quiescence survival during its storage in the female genital tract by lowering of the pH in SST leading sperm to for interest major of be may thus It 1996). al., et Holm 1998; Ridderstrale, & (Holm quail hypothesized to be related to the modulation of pH in the lumen of SST in turkey, hens andCarbonic anhydrase 2 (CA2) that has been described in the present study, has been previously storage modulation and of spermbeforeascent fertilization. during proteins may participate, at least in part, 2006; Lee et al., 2011; to Slowinska et al., 2015) have membrane been identified in avian UF. There, changes these leading to the et al., functional 2012), or kazal domain proteins like SPINK5, SPINK7 and SPINK2 (Jalkanen proteinsOther involved in the remodeling of membrane events like PDIA3, HSP90B/B1et (Dun al., 2012; et al., etNakamura 1993). al., (Dun mammals in reaction acrosome and hyperactivation during sperm of calcium intracellular also demonstrated the presence of CALR protein that has binding sperm-egg of modulator key been a as considered described as a regulator of (Blesbois & Caffin, 1992), whereas exhibits MFGE8, antioxidant activity localized (Gum on et al., sperm 2004) deformation, plasma lipid and peroxidation and impacts maintains membrane, fertility fowl and motility is sperm motility demonstrated that calcium, used as an additive to 1984) Koga, & (Fujihara study sperm previous A GSN). and MFGE8 CALM, diluent, HPX, ANXA5, EDIL3, protects fowl sperm from OVAL, ANXA2, OVOT, (ALB, proteins binding calcium numerous identified we study present sperm motility is strongly linked to extracellular calcium (Froman, 2003). Moreover, that suggests model which Froman’s within accordance in Thisis sperm. for availability calcium the MEPE is assumed to be a calcium chelator (Hincke et al., 1999) and it thus could regulate Comment citer cedocument:

16 16 in vitro in (Raymond et al., 2009). Our work Our 2009). al., et (Raymond in vitro . Albumin (ALB) in vitro

Version postprint 563 562 561 560 559 558 557 556 555 554 553 552 551 550 549 548 547 546 545 544 543 542 541 540 539 538 537 536 535 534 533 532 531 530 529 528 527 526 525 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., tract. exosomes key components sperm are survivalfor long term storageduring in the hen’s genital sperm receptors (Ignotz et al., 2007; Talevi were localized intracellularly. & Considering that in Gualtieri, mammals annexins have 2010), PARK7 been and proposed VCP and as wecells epithelial SST of part apical can the at localized was hypothesize ANXA4 exosomes. that demonstrated that ANXA4, VCP and PARK7 were relevant markers that of they avian may genital sustain tract sperm storage in the Exosomes have SST been observed (Bakstwithin SST, fusing & with sperm Bauchan, plasma membrane, suggesting 2015). Moreover, we 2014).et al., Hiyama 2003; al., et (Breque species avian and 2015) Huang, & Yu 2012; al., et (Dun mammals in reviewed function,as sperm toregulate shown been already havethese components of Most All and MDH1. are known toexosomal in the referenced be proteins as GSTM3 GSTO1, GSTA3, LDHB, ALDOC, CKB, like pathways metabolic in involved proteins PRDX1, PRDX6, GSTA3 and SOD1, the chaperones HSPA8, HSPA2 and proteins antioxidantHSP90AA1, the identified and we UF the in Moreover, MDH1. and PARK7 annexins, VCP, HSP90A, containedHSPA8,they that demonstrated UF and avianfrom exosomes isolatedWe recipient cells. exosomes are known to deliver ubiquitous and specific genital tract proteins, and sperm lipids,cells during their RNA ascent and and storage in ions the female to genital tract sinceavian UF could provide an alternative mode of communication between epithelial cells of the from exosomes that hypothesize can one Therefore, 2017). al., et (Huang cells vaginal and UVJ by vitro in secreted be to demonstrated were and 2015), Bauchan, & (Bakst UVJ-SST of lumen et al., 2017; Burns et al., 2014; Ng et al., 2013). In avian species, they were observed in from the human and ovine uterine luminal fluid as well as from bovine oviductal fluid (Alminana isolateddescribed and been already exosomeshave tract, genitalfemale the milk. In and saliva et al., 2014). To date, they have been described in almost all body fluids, such as blood, urine, participate in several cellular processes and exhibit pleiotropic biological functions (Colombo that cells from released vesicles extracellular are Exosomes post-oviposition. hours 10 collected To our knowledge, our study is the first to demonstrate the presence of exosomes in avian UF Exosomal involvedproteins potentially in sperm survival storage insurvival during SST. et al., 2011; Long et al., 2003). Thus, both proteins may protein participate which supports long toterm sperm storage the in uterovaginal sustainedSST in turkey sperm (Foye-Jackson marker of fertility in roosters (Labas et al., 2015). We also identified avidin gamma chain in has recently hen’s been linked UF, to sperm a survival and has been proposed as a seminal Moreover, our study revealed several fragments of immunoglobulins. The immunoglobulin Ig and beforestorage ascent fertilization. during changes membrane sperm to participate and/or proteins, surface sperm the of processing contribute to the local proteases/proteases inhibitors homeostasis, regulate the proteolytic Comment citer cedocument:

17 17 Exocarta database. Version postprint 602 601 600 599 598 597 596 595 594 593 592 591 590 589 588 587 586 585 584 583 582 581 580 579 578 577 576 575 574 573 572 571 570 569 568 567 566 565 564 603 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., Declaration of interest of biologicalcollection the samples,financial supportmanuscript. and found corrected the samples. NG conceived the study and identification. participated JG participated to in the conception its of the study design, and collection of coordination the biological and the in manuscript. the GH, AG and participated VL to the collection of the biological samples, performed performed experiments, and corrected actively the LC data. proteomic the of nano analysis the to participated actively LC AB manuscript. the wrote MS/MS analysis and protein and data analyzed experiments, performed samples, biological the of collection and procedures experimental the to participated and coordinated study, the of conception the to participated CR Authors' contribution sperm as well as sperm acceptance. survival. The presence of antimicrobial proteins may ensure a bacteria-free environment for identified in the UF which are linked to shell formation may constitute a functions, support by for participating in sperm long-term sperm storage in chicken. Moreover, the proteins we approaches supported this result. Exosomes may play a key role in the preservation of sperm The latter were purified and proteomicsusing demonstrates forthe first time that avian UF contains EV, mainly exosomes. immunodetection associated and withUF avian of content protein the of knowledge our improves transmission work present the conclusion, In microscopy spermenable to produce ATP (Ronquist al., 2013). et may enzymes glycolytic plasma, seminal human In sperm. and plasma seminal UF, to common PGAM1, GSS, GPI, PFKP, GOT1, GSTM2, proteins LDHA, (CKB, MDH2 ALDOC, and ENO1, ALDH9A1) GAPDH, LDHB, were peroxidation GSTA3, of also sperm MDH1, lipids in PGK1, the hen’s TPI1, oviduct (Breque PKM, et al., spermseminal plasma and et (Labas al., 2015). GPX SOD and been have suggested to prevent 2003). Metabolic pathway antioxidant proteins (ALB, PRDX1, SOD1, GPX3 and TXNL1) were common between UF, al., 2015) in order to identify key proteins potentially We compared our data to proteomic involved analysis of rooster’s seminal plasma and sperm in (Labas et sperm survival. Several betweenOverlapping avian UF and proteins semen investigation. further componentsfunctions onsperm requires capabilities, and fecundity before fertilization. exosomes Nevertheless, to the stored impact sperm to of guarantee UF sperm Similarly, exosome membrane we properties, could motility, hypothesize antioxidant that exosome which are components insufficiently represented may or be have been transferred lost from from exosomes may sperm constitute UF a (Ronquist pool et of enzymes/proteins al., used to 2013). replenish the enzymes/proteins properties, and antioxidant capabilities motility maturation, (Sullivan their to relative function sperm ratio, et phospholipid cholesterol: sperm’s the al., 2005). It has altering thereby sperm, to molecules been transferring of capable are plasma seminal from proposed exosomes that in calcium signaling to sperm involved molecules (Park key of transfer et the ensure to al., and 2013), al., 2011). et (Piehl membrane As sperm the a of result of their fusogenic properties, fluidity regulate demonstratedto been have exosomes seminalplasma male, mammalian the In Comment citer cedocument:

18 18 Version postprint 645 644 643 642 641 640 639 638 637 636 635 634 633 632 631 630 629 628 627 626 625 624 623 622 621 620 619 618 617 616 615 614 613 612 611 610 609 608 607 606 605 604 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., C., Brillard, J.Beaumont, Reviers, P., De M. (1992). Comparison Millet, N., & of various M. R., Wishart,Bakst, G. J., J. selection, Brillard, sperm & P. (1994). Oviductal transport, M. R., Apical & Bauchan,Bakst, blebs G. (2015). tubule storage on sperm cell epithelial Akuffo, M. R., & Bakst, V. (2009). Morphology vagina oftheturkey with and without an egg M. R. (2011). Physiology endocrinologyBakst, and symposium: of the oviduct role in Alminana, C., Corbin, E., Tsikis, Alcantara-Neto, V., Reynaud, A. S., G., K., . Labas, Miyazato,Ahammad, M. U., N., Okamoto, T., Nishino, C., H., Okura, Tatemoto, S., . References Susan of the Edrich English. for correction thanked for the submission of the dataset to ProteomeXchange via the PRIDE database, and their helpful discussions during the writing of the manuscript. Lucie Combes-Soia is sincerely protocole for EV preparation. We thank Sophie Réhault-Godbert and Jean-Pierre Brillard for technical expertise in microscopy approach. We thank Carmen Alminana for of providing the laboratory the of animal infection expertise (ISP, INRA, Nouzilly) for providing help and INRA, Nouzilly, France), MIMA2 platform (MET-GABI, INRA, Jouy-en-Josas, France), and France) for the care of birds. We would like to thank the personnel of the PIC platform (PRC, We are grateful to the personnel of the Avian experimental unit (UE-PEAT, INRA, Nouzilly, Acknowledgments French and theNational Institute and MedicalHealth (Inserm). Research of (INRA) Research Agricultural for Institute National French the Centre, du Régional Conseil the financed (SMHART project n°3069) by the European Regional Development Fund (ERDF), was spectrometer mass resolution high The 200391/2014-3). number (grant program fellowship Council of Scientific National Brazilian CNPq, the by and supported was Cordeiro Luiz Dr. Technological ALLICE. Association French Development, on the Science Without French National Borders Institute for Agricultural Research. Dr. Cindy Riou This research was was financed supported by by the Conseil the Régional du Centre Val de Loire and INRA, the Funding declareTheno conflict authors of interest. doi:10.1080/00071669208417503 of durationcharacteristics fertility in hens. of in poultry. and storage Theriogenology, 83 microvilli:and interaction with resident release their sperm in hen the turkey oviduct. mass in the uterus. doi:10.2527/jas.2010-3663 sustainedmaintaining fertility in hens. 17-0054 oviduct-embryo cross-talk.during Mermillod,vesicles extracellular their P. (2017). Oviduct protein content and role 50 Journal of Poultry Science, Production in Hens, Survivability PenetrabilityEgg of and and SpermFowl in vitro. T. EffectsNakada, (2013). Secreted onDuration of Fluid from the Uterus of Fertile Comment citer cedocument: Poult Sci, 88 (9), 1438-1444.(9), doi:10.1016/j.theriogenology.2015.01.016 Poultry Reviews, 5 Science

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Curr ProtocCell Biol, Blood Cells Dis,Mol FEBS Lett, 582 Biol Biomed (5), (5), Version postprint 857 856 855 854 853 852 851 850 849 848 847 846 845 844 843 842 841 840 839 838 837 836 835 834 833 832 831 830 829 828 827 826 825 824 823 822 821 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., 640 proteins referenced in and the (B). MVB:exosome Multivesiculardatabases Bodies. 7 Figure epithelium from uterovaginal junction. numerous observations. sst: sperm storage was observed in tubules; the apical SST epithelium lu: and signal the positive UVJ ANXA4 cilia. (D) The lumen; epithelium. figure SST is and UVJ representative in of present UVJ was signal positive PARK7 (C) ep: pseudostratified of the primary antibody. (B) VCP positive signal was observed in UVJ insteadand immunoglobulins (right)SST rabbit epithelium. and (left) mouse with performed immunohistochemistry Figure 6 observations. of numerous Bleb; Bl: Lu: Lumen; MultivesicularMVB: Bodies. representative is figure The SST. the of lumen the in present were nm) (100-1000 microvesicles Intracellular MVB containing exosomes (< 100 nm) (arrowheads) were (< observed. 100 nm) (C) were Blebs observed at containing surface epithelium of uterovaginal junction. Figure (B) 5 of proteins. molecular weight (kDa) from a simultaneous protein standard run. All lanes contained 10 µg either previously observed in exosomes, or not. proteins against Numbers directed antibodies primary using on 3) (lane EV and the 2), (lane UF left EV-depleted 1), of each blot represent (lane UF the of blot western using analyzed were spectrometry mass by UF in observed proteins ANXA1, ANXA4, PARK7) and non-exosomal (BPIFB3, OC-17) proteins from UF. Selected (C) Immunodetection of exosomal EV. of size the of Distribution (B) (VCP, microscopy. electron transmission by observed (arrowheads) HSP90A, HSPA8, ANXA2, MDH1, ANXA5, Figure 4 databases).and KEGG (NCP, yellow, 321 proteins), and EV/exosomal proteins (4643 proteins, UniprotKB, Exocarta (orange, 437 proteins), or secreted containing a signal peptide (PS, pink, 155 proteins) or not Figure 3 from al.Marie (2015) and Sun et al. (2013). et relation to taxonomy. (B) Venn diagram of proteins identified in the present study and studies 2 Figure 10hours post collection, oviposition (B). Figure 1 functions. Table 2 togroups according their functions. Table 1 Legends . UF proteins linked to antimicrobial activities clustered in 5 groups according to their . UF proteins potentially linked to the process of shell mineralization clustered in 3 . Pie molecular representing functionschart the913 proteins from UF(A) of the avian . Proteomic analysis of avian UF. (A) Distribution of the 913 proteins from the UF in . Venn diagram of identified proteins in the UF categorized either as not secreted . Analysis of EV from the UF. (A) Purification of the EV containing exosomes . Transmission electron microscopy of the uterovaginal junction. (A) Exosomes . Schematic representation of the hen’s genital tract at ovulation (A) and fluid . Immunohistochemistry on UVJ and SST. (A) Negative controls for Comment citer cedocument:

24 24 Version postprint 862 861 860 859 858 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., Data provided by etroosters. al., (2015).Labas seminal plasma (SP, grey, 607 proteins) and spermatozoa (SPZ, yellow, 822 proteins) (blue, from 589 proteins), or exosomal (green, 640 proteins), compared 8 Figure to proteins identified in . Venn diagram of the 913 proteins identified in UF categorized either as non-exosomal non-exosomal as either categorized UF in identified proteins 913 the of diagram Venn . Comment citer cedocument:

25 25 Version postprint 864 863 865 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J., 1Figure Comment citer cedocument:

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Version postprint 866 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J.,

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Version postprint 870 869 868 867 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J.,

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Version postprint 874 873 872 871 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J.,

Figure 4Figure Comment citer cedocument:

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Version postprint 877 876 875 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J.,

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Version postprint 880 879 878 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J.,

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Version postprint 883 882 881 884 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J.,

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Version postprint 890 889 888 887 886 885 Gérard, N.(2020).Avian uterinefluid proteome:exosomes andbiological processes potentially involved in sperm survival. Molecular Reproduction and Development, acceptée, acceptée. Riou, C., Brionne,A., Cordeiro, L.-A., Harichaux,G., Gargaros,A., Labas,V.,Gautron, J.,

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