158 4

REPRODUCTIONRESEARCH

Proteomic analysis of uterine fluid of fertile and subfertile hens before and after insemination

Cindy Riou1,2, Aurélien Brionne3, Luiz Cordeiro1,4, Grégoire Harichaux5, Audrey Gargaros5, Valérie Labas5, Joël Gautron3 and Nadine Gérard1 1PRC, INRA, CNRS, IFCE, Université de Tours, Nouzilly, France, 2ALLICE, Station de Phénotypage, Lieu-Dit Le Perroi, Nouzilly, France, 3BOA, INRA, Université de Tours, Nouzilly, France, 4Federal University of Semi Arid Region, Mossoro, Rio Grande do Norte, Brazil and 5INRA, Université de Tours, CHU de Tours, Plate-forme de Chirurgie et Imagerie pour la Recherche et l’Enseignement (CIRE), Pôle d’Analyse et d’Imagerie des Biomolécules (PAIB), Nouzilly, France Correspondence should be addressed to N Gérard; Email: [email protected]

Abstract

Avian uterine fluid (UF) and uterovaginal sperm storage tubules (SST) are key components in accepting sperm in SST, maintaining sperm function for several weeks, releasing sperm from SST and their ascent through the uterus. To improve the understanding of sperm storage processes requires investigating UF and SST. This study aimed to identify proteins modulated by sperm in the hen’s genital tract and to highlight their role during sperm storage. Two genetic lines of hens exhibiting long (F+) or short (F−) sperm storage ability were used. GeLC MS/MS analysis was used to establish a quantitative inventory of proteins regulated after insemination in both lines. The proteomic data are available via ProteomeXchange with identifier PXD013514. Immunohistochemistry was used to identify high (ANXA4/ANXA5/OCX32) and low (HSPA8/PIGR) fertility markers in the uterovaginal junction. Our results demonstrated that sperm induced a significant and rapid change in the UF proteomic content and also in the SST epithelium. In F+ hens, mobilization of the ANXA4 protein in the apical part of SST cells after insemination was associated with increased levels of some proteoglycans and binding proteins, and also antimicrobial eggshell matrix protein (OCX32) in the UF. We also observed increased levels of lipid transporters involved in egg formation (VTG1-2, APOA1-4-H). In F− hens, insemination induced increased levels of PIGR in both UF and SST, of ANXA5 in SST, of UF enzymes exhibiting metallopeptidase activity and mucins. In conclusion, sperm induced significant changes in the UF proteomic content. This study also provides evidence that the SST immune system plays a major role in regulating sperm storage. Reproduction (2019) 158 335–356

Introduction the magnum, where the egg white is secreted (0.5–3.5 h post ovulation), the isthmus is the region where eggshell Avian species have the capacity to store spermatozoa membranes are synthesized (3.5–5 h post ovulation), and for several weeks in the female genital tract during eggshell calcification takes place in the uterus within an the period between mating and successive ovulations 18-h period. Finally, 24 hours after ovulation, the egg (Birkhead & Moller 1993, Bakst et al. 1994). Sperm transits through the vagina and is laid. After mating, at survival is believed to be preserved by local secretion of the same time as the daily process of egg formation, proteins and metabolites (Froman 2003, Bakst & Akuffo sperm pass through the vagina and are initially stored 2007) and also due to specific interactions between in the uterovaginal SST. The sperm then transit to sperm and epithelial cells within sperm reservoirs. the infundibulum, the site of fertilization (Sasanami Sperm reservoirs are called sperm storage tubules (SSTs) et al. 2013). in avian species (Tingari & Lake 1973, Das et al. 2008, Reproductive tract fluid represents the aqueous Bakst & Bauchan 2015). environment present in the avian female genital tract in The female avian reproductive tract (oviduct) consists which sperm bathe during their ascent from the vagina to of the infundibulum, magnum, white isthmus, uterus the infundibulum. The only fluid described in the avian and vagina. These specialized regions are associated reproductive tract is uterine fluid (UF), which bathes with the deposition of different components of the egg. the egg during its formation. Uterine fluid composition The infundibulum ensures the deposition of the vitelline has been widely investigated (Gautron et al. 2019), and membrane outer layer. The developing egg then enters it has been suggested that UF constituents and female

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-19-0079 336 C Riou and others reproductive tract secretions are key factors for sperm suspected either to enhance or to inhibit sperm storage storage in birds (Bakst & Bauchan 2015). The hen’s in the uterovaginal epithelium to obtain new insights UF has been shown to promote the maintenance of into their potential role in this process. sperm motility and viability in vitro (Brillard et al. 1987, Ahammad et al. 2013). Therefore, it is hypothesized that UF contains specific molecules that preserve sperm Materials and methods during their ascent of the female genital tract. Birds The uterovaginal junction (UVJ) which contains SST is considered as the main area of sperm storage We used sexually mature hens from two genetic lines and also ensures sperm selection (Brillard 1993). previously selected for their ability to produce a normal Inside SST, sperm remain alive and fertile before being (F+) or low (F−) number of hatched chicks and to preserve gradually released to fertilize ovulated eggs (Sasanami sperm in their SST for a long (F+) or short (F−) period (Brillard et al. 2013). The arrival of sperm in SST has been et al. 1998). These two lines diverge in the duration of their fertile period, the F line expressing a shorter time potential shown to induce a rapid change in the transcriptomic − to lay fertile eggs than the F+ line (Beaumont et al. 1992). profiles of both the UVJ and uterine tissue Long( et al. Breeding procedures and handling protocols were carried out 2003, Atikuzzaman et al. 2015). Recently, it has been in accordance with the European Union Council Directives suggested that local inhibition of the immune system regarding practices of animal care and use, practices of the in the sperm reservoir may enhance sperm storage French Ministry of Agriculture on animal experimentation, in avian species (Das et al. 2006a, Atikuzzaman under the supervision of an authorized scientist (Authorization et al. 2015, 2017). Other factors are also thought # 37035). The facilities at the Institut National de la Recherche to be secreted in the SST lumen to maintain sperm Agronomique, UE-PEAT, 1295 are officially authorized to rear survival. These include fatty acids (Huang et al. and kill birds (B27-175-1 dated 28/08/2012). At 47 weeks 2016) and extracellular vesicles (exosomes) that have of age, hens were placed in individual cages equipped with recently been identified binding to sperm plasma automatic devices to record the time of oviposition. They were membrane (Bakst & Bauchan 2015). Moreover, we can kept under a 16L:8D photoperiod, and fed a layer mash ad hypothesize that some key proteins on the surface of libitum. The protocol of bird management and collection was the SST epithelium or in the SST lumen may originate approved by the local ethics committee (Comité d’éthique de from other segments of the genital tract and adhere to Val de Loire no.19) and the French Ministry of Research under the epithelium in order to enhance sperm retention agreement number # 443. within SST, preserve sperm function and/or regulate their release. Nevertheless, the mechanisms involved are still poorly understood. Assessment of fertility The UF and uterovaginal SST are essential for The duration of the fertile period within each hen line was acceptance of sperm in uterovaginal storage sites, defined as effective duration (De), that is, the number of maintenance of sperm function for several weeks, days after artificial insemination (AI) during which a hen lays sperm release from the uterovaginal sites, and the 100% fertile eggs, and maximum duration (Dm), that is, the ascent of sperm cells through the uterus. Thus, enhanced number of days after AI until the hen lays its last fertile egg. knowledge of UF composition and uterovaginal SST cell Hens (50 weeks old) were inseminated on two consecutive biology will improve the understanding of sperm storage days with 200 × 106 sperm from mixed ejaculates collected processes in the hen’s genital tract. from six broiler breeder males on both days. All intravaginal Chicken UF composition has been widely investigated AI were performed by the same experienced operator from in layer hens in relation to shell biomineralization, as the avian experimental unit by introducing the semen in this fluid bathes the egg during its calcificationGautron ( the vagina (2 cm depth) using a yellow tip. To determine et al. 1997) and 644 uterine fluid proteins have already the laying rate (eggs laid/days × 100), eggs were identified individually and recorded daily from Day 2 to Day 22 been reported (Sun et al. 2013, Marie et al. 2015a). following the second AI. They were stored for a maximum of The present study was based on the hypothesis that UF 7 days prior to incubation. The fertility rate was calculated contains specific molecules, that SST are key structures as the percentage of fertile eggs (fertile eggs/incubated and that both are essential for sperm survival. Thus, we eggs × 100). It was determined after egg candling on Day 7 first identified UF proteins before and after insemination of incubation. To calculate the embryo mortality rate (dead of fertile (F+) and subfertile (F ) hens (Beaumont et al. − embryos/fertile eggs × 100), eggs initially classified as infertile 1992, Brillard et al. 1998) in order to characterize were broken for macroscopic examination of the germinal them functionally. These two lines of hens exhibit long disc to determine the presence or not of dead blastodiscs. (F+) or short (F−) duration of the fertility period, as a Comparisons of De, Dm, and fertility, laying and embryo consequence of good or poor sperm storage capacity, mortality rates between the two lines were computed using respectively (Brillard et al. 1998). We aimed firstly to a Mann–Whitney test (P < 0.05). Nineteen hens (10 F+ and 9 identify specific proteins related to the sperm storage F− hens) were then selected from the 60 hens according to process, and secondly to investigate key molecules their fertility rate.

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Uterine fluid collection 10% SDS-PAGE (minigel 10 cm × 8.3 cm × 1 mm). The gel was stained with Coomassie Blue (PageBlue™ Protein Staining Uterine fluid collection was scheduled 10 h after the previous Solution, Fermentas®) before sectioning each lane into 20 oviposition and after confirming the presence of an egg in slices. Each gel band was cut into approximately 1 mm3 pieces utero. Egg expulsion was induced by intravenous injection of and washed with a volume of 50% acetonitrile/50% water and prostaglandin F2A at 50 µg/hen. During egg expulsion uterine a second volume of 100% acetonitrile. Proteins were reduced fluid was collected in a plastic tube placed at the entrance of the with incubation of 10 mM dithiothreitol with 50 mM NH HCO everted vagina, from 5 F+ and 5 F hens before insemination 4 3 − for 30 min at 56°C and alkyled with 55 mM iodoacetamide and 5 F+ and 4 F hens 24 h after insemination as described − in 50 mM NH HCO , for 20 min at room temperature in the previously (Gautron et al. 1997). An aliquot of UF was 4 3 dark. They were washed with 50 mM NH HCO /acetonitrile immediately diluted with 5 Laemmli buffer (5v:1v) (312.5 mM 4 3 × (1:1) for 10 min, and with 100% acetonitrile for 15 min. After Tris–HCl pH 6.8, 10% SDS, 12.5% B-mercaptoethanol, 50% being dried in a vacuum centrifuge using a SPD1010 speedvac glycerol, bromphenol blue), and then boiled for 5 min before system (ThermoSavant, ThermoFisher Scientific), the gel pieces storage at 20°C. Another aliquot of fluid was diluted with − were incubated overnight in a solution of 6.25 ng/µL of trypsin PBS (1v:1v) to measure protein concentrations and to perform (Trypsin Sequencing Grade, Roche Diagnostics®) in 25 mM in solution digestion MS analysis. All aliquots were rapidly NH HCO to digest the proteins. Peptides were extracted from frozen in liquid nitrogen to limit any spontaneous precipitation 4 3 the gel through successive incubations under agitation with of calcium carbonate and proteins, and stored at 80°C − 5% formic acid for 10 min and with 50% acetonitrile/50% (Hincke et al. 1999). A total of 19 samples were collected water in the presence of 1% formic acid for 10 min and with and used for nanoLC-MS/MS proteomic analysis, five F hens − 100% acetonitrile for 5 min. All extracts were pooled and dried before AI, four F hens after AI, five F+ hens before AI and five − in a vacuum centrifuge, and then reconstituted with 30 µL of F+ hens after AI. The experimental design described below is 0.1% formic acid, 2% acetonitrile, and sonicated for 10 min. summarized in Fig. 1. The resulting peptide mixture was analyzed using nanoflow liquid chromatography coupled to tandem mass spectrometry Tissue collection (nano LC-MS/MS).

Hens were killed, either before AI (F+, n = 3; F−, n = 3) or 24 h after AI (F+, n = 3; F−, n = 3). The genital tract section from NanoLiquid chromatography coupled to tandem mass uterus to vagina was excised as one segment. Connective tissue spectrometry (nanoLC-MS/MS) was removed to expose the uterovaginal junction. Samples of All experiments were performed on a linear ion trap Fourier uterovaginal junction mucosa containing uterovaginal sperm Transform Mass Spectrometer (FT-MS) LTQ Orbitrap Velos storage tubules were collected from each hen and processed. coupled to an Ultimate® 3000 RSLC Ultra-High Pressure Liquid Chromatographer (Thermo Fisher Scientific). Five Protein concentration of uterine fluid samples microliters of each sample were loaded on a trap column and desalted. The peptide separation was conducted with The protein concentration was determined in each UF Chromeleon software (version 6.8 SR11; Dionex, Amsterdam, sample individually, using the Pierce® BiCinchoninic The Netherlands) using a nano-column (Acclaim PepMap Acid protein assay kit (Life Technologies SAS) using bovine C18, 75 μm inner diameter × 50 cm long, 3 μm particles, 100 Å serum albumin as the protein standard and according to the pores). The gradient consisted of 4–55% B for 90 min at a flow manufacturer’s instructions. rate of 300 nL/min. Data were acquired using Xcalibur software (version 2.1; ThermoFisher Scientific). The LTQ Orbitrap Velos Separation of UF proteins by SDS-PAGE and in-gel instrument was operated in a positive mode in data-dependent trypsin digestion mode. Resolution in the Orbitrap was set to R = 60,000. In a scan range of m/z 300–1800, the 20 most intense peptide ions All samples from each group were pooled according to with charged states ≥2 were fragmented using collision ion condition (F+ before AI, F+ after AI, F− before AI and F− after dissociation (CID). Dynamic exclusion was activated during AI) for GeLC-MS/MS analysis. Pooled samples were made of 30 s with a repeat count of 1. Polydimethylcyclosiloxane 50 µg of proteins by mixing the same amount of proteins from (m/z, 445.1200025) ions were used as lock mass for internal individual samples. Pooled samples were then separated using calibration. Each pooled sample was analyzed in triplicate.

Figure 1 Schematic representation of the experimental design. Uterine fluid was collected from F+ and F− hens before and 24 h after AI. The genetic effect between both lines was obtained by comparing both kinds of samples before or after AI. The AI effect within each line was obtained by comparing samples collected before and after AI.

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Database search and protein identification the Chordata Scaffold result and (b) considered as unknown Gallus gallus proteins when the blastp result did not share Raw data files were converted to MGF as previously any similar name or protein description. The resulting file described (Labas et al. 2015). The identification of proteins constituted the non-redundant proteome presented in the was conducted using the MASCOT search engine (v 2.3, present study. Our identified peptides were compared to Matrix Science, London, UK). The peptide and fragment published quantitative proteome lists from avian UF and masses obtained were matched automatically against the eggshell (Sun et al. 2013, Marie et al. 2015a) using BLAST+ chordata section of nr NCBI database (1601319 sequences, suite. Only proteins that display SIsc higher than 91% (in downloaded on 2014/01). Enzyme specificity was set to trypsin term of their blast results, including the size of peptides with two missed cleavages using carbamidomethylcysteine, and the profile of the match, that is, number of mismatches oxidation of methionine and N-terminal protein acetylation and gaps) were considered orthologous to the previously as variable modifications. The tolerance of the ions was set to identified proteins. The SecretomeP 2.0 (Bendtsen et al. 5 ppm for parent and 0.8 Da for fragment ion matches. Mascot 2004) (http://www.cbs.dtu.dk/services/SecretomeP/), as well results obtained from the target and decoy database searches as the Signal P 4.1 (Petersen et al. 2011) (http://www.cbs. were incorporated in Scaffold 4 software (version 4.3.4, dtu.dk/services/SignalP) servers, were used as a prediction Proteome Software, Portland, USA). Peptide and protein method of the secretion pathway (signal peptide-dependent or identification were accepted if they could be established at signal peptide-independent or via exosomes) for each protein a probability greater than 95% as specified by the Peptide obtained in the present study. Our data were then compared and Protein Prophet algorithms (Keller et al. 2002). Peptides to an exosome database which we constituted by merging were considered distinct if they differed in sequence. A Exocarta (human, rat, mouse), UniprotKB (chicken) and KEGG false discovery rate was calculated as 1% at the peptide < (chicken) databases (personal data). The mass spectrometry or protein level. The abundance of identified proteins was proteomics data have been deposited in the ProteomeXchange estimated by calculating the emPAI using Scaffold software. Consortium via PRIDE (Deutsch et al. 2017, Perez-Riverol A phylogenetic tree was constructed with Clustal Omega and et al. 2019) partner repository with the dataset identifier similar Gallus gallus proteins were searched in nr databases PXD013514 and project DOI 10.6019/PXD013514. The intra- using blast in order to clarify the scaffold groups. genital tract potential function of each differential protein was determined based on their molecular function extracted Label-free quantification using BioMart (http://www.ensembl.org/biomart/martview), their GO-term annotation from Uniprot and Genecards (chicken To determine the differences in proteomic composition, the and mammals) and the literature. two label-free quantitative methods employed were spectral counting (P < 0.05) and XIC (eXtraction Ion Chromatogram) P method ( < 0.05 and ratio >2) using Scaffold Q+ (v 4.3.4, Western blotting proteome Software, Portland, USA). A t-test was performed to differentiate the significantly changed proteins with a P <0.05. Aliquots of 10 µg of proteins from the UF pooled samples were added to 5× Laemmli buffer (5v:1v), boiled at 95°C Statistical, data mining and bioinformatics analysis for 5 min, then loaded and separated on a 10% SDS-PAGE, before being transferred to nitrocellulose filters. The Proteomic data were extracted from Scaffold software and membranes were washed with TBS (10 mM Tris, 150 mM analyzed using R language (http:/cran.r-project.org) after NaCl, pH 7.4) containing 0.1% (v/v) Tween-20 (TBS-T), elimination of keratin and trypsin, as they were contaminants incubated for 1 h in the blocking solution (5% (w/v) non-fat or resulted from the digestion process, respectively. For dry milk in TBS-T), and then overnight in the blocking each protein the identifier, symbol and description of the solution containing anti-HSPA8 (1:500; rabbit polyclonal; annotated gene were extracted from nr NCBI database bs-5117R, Bioss, Interchim, Montluçon, France), anti- (September, 2014). Overlapping and continuous peptides ANXA4 (1:1000; rabbit polyclonal; CSB-PA001845ESR2HU, from the same sequence were concatenated to create a Cusabio, Clinisciences, Nanterre, France), and anti-ANXA5 longer peptide (reconstituted peptide), in order to remove (1:1000; rabbit polyclonal; CSB-PA06384A0Rb, Cusabio). the peptidic redundancy and to generate a higher strength The membranes were then sequentially washed with TBS- for the next step of analysis. Longer peptide sequences were T, incubated for 1 h in the blocking solution, then for 1 h blasted against nr NCBI database limited to Gallus gallus with peroxidase-conjugated secondary antibody, that is, taxon, using the blastp program (BLAST+ suite) (Camacho goat anti-rabbit IgG (A6154, Sigma-Aldrich) diluted 1/5000 et al. 2009). The alignment result of each peptide for each in the blocking solution and finally washed with TBS-T. protein was expressed as a global score of similarity (SIsc) The peroxydase activity was detected with the ECL select™ and identity (IDsc). Only a perfect match (IDsc = 100%) Western Blotting Detection Reagent (GE Healthcare) and with a Gallus gallus protein allowed removal of redundancy the signal was captured using the ImageMaster VDS-CL bio inside protein groups. Proteins which were not strictly imaging system (Amersham Biosciences/GE Healthcare) and identified in theGallus gallus database were (a) considered quantified using Image Quant software. A Mann–Whitney as orthologous to Gallus gallus proteins when the blastp test was performed to differentiate the significantly changed result indicated the same protein description and name as proteins with a P <0.05.

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Immunohistochemistry on uterovaginal tissue Results Following fixation for 24 h in 4% saline buffered (PBS) formalin, Comparison of F+ and F− fertility tissue samples (uterovaginal junction) were transferred to two Two groups of hens from each genetic line were successive baths of 70% ethanol (2 × 30 min), and then moved to an automated tissue processor system (Leica TP1020 Semi- constituted, F+ (n = 8) and F− (n = 7). These lines differ enclosed Benchtop Tissue Processor). The samples were mainly in sperm storage ability, that is, their maximum then embedded in paraffin (Leica EG1150 Modular Tissue duration of fertility (Dm) (Table 1). The efficient (De) and Embedding Center, Leica Microsystems Richmond, Inc.). maximum (Dm) duration of fertility in the fertile F+ hens Four to six 7 μm thick sections were collected in sequence were 10.13 ± 1.55 and 15.63 ± 1.00 (days) respectively, onto slides treated with 0.01% poly-l-Lysine in water, air- nearly ten times and three times higher than in the dried overnight, stored at room temperature (RT) for 12 h and subfertile F− hens (1.29 ± 0.36 and 5.14 ± 0.96 days then incubated overnight at 60°C. The staining procedures respectively). The fertility rates were 62.88 ± 2.88 consisted in deparaffinization for 5 min in toluene, followed and 11.95 ± 2.33% for the two groups, respectively. by progressive rehydration (2 min each of 100, 95, 80 Thus, throughout this manuscript F− and F+ hens are and 70% ethanol baths) and removal of excess ethanol referred to as subfertile and fertile hens. The laying rate by water baths. After rehydration, the slides were treated and embryo mortality were not significantly different with 1% citrate based unmasking solution H3300 (Vector between the two genetic lines (Table 1). Laboratories, Burlingame, CA, USA) diluted as recommended by the manufacturer. The slides were rinsed once in TBS for 5 min and then placed in a bath with normal horse serum Genetic effect before AI blocking solution (Vector) for 20 min. The slides were incubated overnight at 4°C with the primary antibodies anti- GeLC–MS/MS analyses combined with two label-free HSPA8 (1:500; rabbit polyclonal; bs-5117R, Bioss, Interchim, quantitative analyses based on spectral counting (SC) Montluçon, France), anti-ANXA4 (1:500; rabbit polyclonal; and eXtraction Ion Chromatogram (XIC) quantitative CSB-PA001845ESR2HU, Cusabio), and anti-ANXA5 (1:500; methods performed on the same set of data were rabbit polyclonal; CSB-PA06384A0Rb, Cusabio), PIGR used to compare the respective abundance of distinct (1:500, rabbit polyclonal) kindly provided by M. Berri, and proteins in UF from subfertile (F−) and fertile (F+) hens anti-OCX32 (1:1000; rabbit polyclonal) kindly provided by before insemination. Gautron et al. (2001), diluted in TBS with 5% non-fat dry A total of 94 (XIC) and 148 (SC) proteins were milk. The slides were then washed three times in TBS (5 min), differentially abundant (P < 0.05) between the UF of and incubated for 30 min with the secondary antibody subfertile (F−) and fertile (F+) hens, and 52 of these were ImmPRESS™ HRP Anti-Rabbit/Mouse IgG (Vector) and common to the two label-free quantitative methods rinsed in TBS (5 min), incubated with peroxidase ImmPACT (Supplementary data 1, see section on supplementary NovaRED (Vector) and finally rinsed in distilled water (5 min). data given at the end of this article). The present study The sections were counterstained with Papanicolaou stain focused on the latter 52 of which 5/52 were unique to (Sigma). Coverslips were applied with aqueous mounting medium after dehydration through graded alcohol baths (70, F+ hens and 47/52 were common to both lines (Fig. 2, 80, 95, 100%; 20 s each) and the sections were incubated left). Moreover, seven out of these 47 proteins were two- in toluene for 1 min. Sections were examined using an fold higher and 40/47 were two-fold lower in subfertile Axioplan Carl Zeiss microscope. Images were acquired F− hens, (Table 2, over abundant in subfertile hens and using a digital monochrome camera (Spot-Flex, Diagnostic over abundant in fertile hens, respectively). Instruments) coupled with the SPOT 5.2 imaging software. The potential functions of the 52 differential proteins Negative controls for immunohistochemistry were performed identified were examined according to descriptions in by omitting the primary antibody and by its substitution by an the literature, data annotations and functional domain isotype-specific immunoglobulin at the same concentration. databases, with particular emphasis on their potential Figures are representative of several observations performed roles in sperm survival (Table 2). Their secretory pathways on three hens per conditions. were examined and revealed that the proteins were

Table 1 Comparison of efficient (De) and maximum (Dm) duration of fertility, fertility rate, laying rate and embryo mortality rate between two divergent lines of hens (F+, n = 8; F−, n = 7).

Trait F+ F− Level of significance De (days) 10.13 ± 1.55 1.29 ± 0.36 P <0.01 Dm (days) 15.63 ± 1.00 5.14 ± 0.96 P <0.01 Fertility rate (%) 62.88 ± 2.88 11.95 ± 2.33 P <0.001 Laying rate (%) 85.71 ± 3.12 61.90 ± 8.18 NS Embryo mortality rate (%) 5.60 ± 1.54 4.21 ± 1.58 NS Data were expressed as mean ± s.e.m. P <0.01 indicates a high significant difference (Mann–Whitney test).

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Figure 2 Quantified proteins in UF collected before and after insemination in subfertile (F−) and fertile (F+) hens. Extracted ion chromatograms (XIC) and spectral counting (SC) methods were used. A fold change (FC) >2 and P < 0.05 were chosen to indicate a differential quantification in abundance. (A) Numeric values. (B) Venn diagram. either referenced in exosomes (uniprotKB and exocarta F+ hens, and 78 of them were common to both dabases) (77%) or secreted with a peptide signal (9%) quantitative methods (Supplementary data 1). Among or secreted by non-classical pathways (40%) (Table 2). these 78 proteins, 26 were two-fold higher and 52 were Thirty (57.69%) of them have been previously identified two-fold lower in subfertile F− hens (Supplementary in avian UF (Sun et al. 2013, Marie et al. 2015a) as data 1). Moreover, 64/78 proteins were common to both shown in Table 2. A search for these 52 proteins was lines of hens, 11 were unique to F+ hens, and three were conducted in previously published proteomic data of unique to F− hens (Fig. 2, right). Of note is the fact that avian seminal plasma and spermatozoon (Labas et al. the eggshell matrix protein OCX32 (RARRES1), which 2015). As shown in Table 2, 21 have been identified in is unique to the mineralization process in the avian both male samples, 10 have been identified in seminal uterus, was 2-fold lower in the subfertile line (F−) after plasma only and 6 in spermatozoon only. insemination (Supplementary data 1). Three out of these 52 proteins were analyzed using When comparing post-AI data to those obtain before Western blotting: heat shock 70 kDa protein 8 (HSPA8) AI between the two genetic lines, ten proteins were and two annexins (ANXA4 and ANXA5). As shown in in common (Fig. 2, middle). These were OVALY that Fig. 3, Western blot analysis confirms our quantitative was two-fold higher, and LASP1, ATP6V1E1, CAPZB, proteomic results, that is, overabundance of HSPA8 in DHDH, ENKUR, GPI, IMPA1 and TALDO1 that were subfertile F− hens and overabundance of ANXA4 and two-fold lower in the subfertile F− line. It also included ANXA5 in subfertile F− hens. Ig gamma fragments which were two-fold higher and two-fold lower in subfertile F− hens before and after insemination, respectively. Genetic effect after AI GeLC–MS/MS combined with label-free quantitative AI effect analysis based on SC and XIC methods performed on the same set of data were used to compare the abundance In UF from F+ hens, a total of 80 (XIC) and 153 (SC) of distinct proteins in avian UF 24 h after insemination. proteins were differentially abundant (P < 0.05) in A total of 114 (XIC) and 164 (SC) proteins were response to AI, 52 of which were common to both differentially abundant (P < 0.05) between F− and quantitative methods. Out of these 52 proteins, 41 were

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Downloaded from Bioscientifica.com at 10/04/2021 06:51:59PM via free access Avian uterine fluid proteins and fertility 341 ) Continued ( Other potential function in genital tract Fertilization sperm survival Immunity, Sperm survival Inflammation Inflammation Immunity Immunity Cytoskeleton organization Acidification Cytoskeleton organization metabolism Carbohydrate metabolism Carbohydrate biosynthesis Phosphatidylinositol metabolism Carbohydrate fusion Membrane Previously Previously described in avian SPZ or SP UF, UF: (1, 2) SPZ: NPD SP: NPD UF: (1) SPZ: (3) SP: (3) UF: (2) SPZ: (3) SP: NPD UF: (2) SPZ: (3) SP: (3) UF: NPD SPZ: (3) SP: (3) UF: (1) SPZ: (3) SP: (3) UF: (1, 2) SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: NPD UF: (1) SPZ: (3) SP: (3) UF: (1, 2) SPZ: (3) SP: (3) UF: (1) SPZ: NPD SP: NPD UF: (1, 2) SPZ: (3) SP: (3) UF: (2) SPZ: (3) SP: (3) UF: (1) SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: (3) Secretory Secretory pathway NCP NCP EXO/NCP EXO EXO PS EXO/PS EXO/NCP EXO/NCP EXO/NCP – EXO EXO/NCP EXO EXO ) − 7 − (F+/F ∞ Ratio XIC: 0.3 SC: 0.2 XIC: 0.2 SC: 0.5 XIC: 0.04 SC: 0.3 XIC: 0.3 SC: 0.06 XIC: 6.10 SC: 0 XIC: 0.3 SC: 0.6 XIC: 0.2 SC: 0.5 XIC: 2.7 SC: 2.8 XIC: 5.2 SC: 9.3 XIC: 3.5 SC: 11 XIC: 10 SC: 5.4 XIC: 3.5 SC: 3 XIC: 28 SC: XIC: 2.2 SC: 2.4 XIC: 14 SC: 8.4 ) and fertile (F+) hens before insemination. − kDa protein, molecular chaperone hens (7) − activity, estrogen control activity, protein catabolic process IgM protein catabolic process Description serine-type endopeptidase ihnibitor Y, protein Ovalbumin-related of Ig Fragment cognate 71 Heat shock leukotriene biosynthesis, zinc binding A-4 hydrolase, Leukotriene in bradykinin Xaa-pro aminopeptidase 1, protease involved of Ig Fragment binds polymeric IgA and immunoglobulin receptor, Polymeric LIM and SH3 domain protein 1, actin binding ion transport transport subunit E 1, ion transmembrane ATPase proton V-type F-actin-capping protein subunit beta, actin binding D-xylose dehydrogenase, Trans-1,2-dihydrobenzene-1,2-diol isomerase Glucose-6-phosphate isomerase, Inositol monophosphatase 1, magnesium binding hydrolase transferase Transaldolase, A4, calcium-dependent phospholipid binding protein Annexin Differentially abundant uterine fluid proteins between subfertile (F

(Clone 36) chain Ig gamma chain Ig gamma chain Ig alpha heavy Table 2 Table Symbol abundant in subfertile F Over OVALY HSPA8 LTA4H XPNPEP1 PIGR abundant in fertile F+ hens (40) Over LASP1 ATP6V1E1 CAPZB DHDH GPI IMPA1 TALDO1 ANXA4

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Downloaded from Bioscientifica.com at 10/04/2021 06:51:59PM via free access 342 C Riou and others pathway pathway Other potential function in genital tract Cytoskeleton organization assembly body Multivesicular Ubiquitin/ubiquitin-proteasome degradation Ubiquitin/ubiquitin-proteasome degradation Mineralization Previously Previously described in avian SPZ or SP UF, UF: (1, 2) SPZ: NPD SP: (3) UF: NPD SPZ: NPD SP: (3) UF: (1, 2) SPZ: NPD SP: (3) UF: (1, 2) SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: NPD UF: (1) SPZ: NPD SP: (3) UF: (1) SPZ: NPD SP: (3) UF: NPD SPZ: (3) SP: (3) UF: (2) SPZ: (3) SP: (3) UF: NPD SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: NPD UF: NPD SPZ: NPD SP: NPD UF: NPD SPZ: (3) SP: NPD UF: (1, 2) SPZ: (3) SP: (3) UF: (1, 2) SPZ: NPD SP: (3) Secretory Secretory pathway EXO/NCP EXO EXO/NCP EXO EXO EXO/NCP EXO EXO EXO/NCP EXO/NCP EXO EXO EXO/PS EXO EXO/NCP ) − (F+/F ∞ ∞ ∞ Ratio XIC: 4.6 SC: 4.1 XIC: 4.2 SC: 8.4 XIC: 2.2 SC: 1.5 XIC: 3.3 SC: 2.4 XIC: 1400000 SC: XIC: 6.5 SC: 6 XIC: 760000 SC: XIC: 33 SC: 9.5 XIC: 6 SC: 7.5 XIC: 3900000 SC: XIC: 13 SC: 38 XIC: 2.3 SC: 4.3 XIC: 4.3 SC: 3.3 XIC: 4 SC: 2.1 XIC: 2.6 SC: 3.7 possess phospholipase A2 inhibitor activity possess phospholipase ribosomal protein protein A2 inhibitor activity phospholipase Description A8, calcium-dependent phospholipid binding protein, Annexin Calcium and integrin-binding protein 1, calcium binding Destrin, actin-depolymerizing protein helicase RNA ATP-dependent initiation factor 4A-II, Eukaryotic Guanine nucleotide-binding protein subunit beta-2-like 1, factor binding protein 7, growth factor-binding Insulin-like growth subunit 12A, lipid and ubiquitin binding body Multivesicular Ras-related protein Rap-1A, GTP binding isozyme L1, thiol protease Ubiquitin carboxyl-terminal hydrolase Proteasome subunit alpha type-1-like protein 40S ribosomal protein S3, RNA-binding hydrolase Aminoacylase-1, oxidoreductase dehydrogenase, Alpha-aminoadipic semialdehyde oxidoreductase dehydrogenase, 4-Trimethylaminobutyraldehyde A5, calcium-dependent phospholipid binding protein, Annexin Continued Table 2 Table Symbol ANXA8 CIB1 DSTN EIF4A2 GNB2L1 IGFBP7 MVB12A RAP1A UCHL1 LOC100547217 RPS3 ACY1 ALDH7A1 ALDH9A1 ANXA5

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Downloaded from Bioscientifica.com at 10/04/2021 06:51:59PM via free access Avian uterine fluid proteins and fertility 343 ) Continued ( pathway pathway Arginine biosynthesis Energy transduction antimicrobial Mineralization, metabolism Carbohydrate assembly body Multivesicular metabolism Carbohydrate sperm survival Mineralization, Ubiquitin/ubiquitin-proteasome degradation Ubiquitin/ubiquitin-proteasome degradation UF: (1) SPZ: NPD SP: (3) UF: (1) SPZ: (3) SP: NPD UF: (1) SPZ: (3) SP: (3) UF: (1, 2) SPZ: NPD SP: NPD UF: (2) SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: (3) UF: (1, 2) SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: NPD UF: (1) SPZ: NPD SP: (3) UF: NPD SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: NPD UF: (1, 2) SPZ: NPD SP: NPD UF: NPD SPZ: (3) SP: NPD UF: NPD SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: NPD UF: (2) SPZ: NPD SP: NPD UF: (1) SPZ: (3) SP: (3) EXO EXO/NCP EXO EXO/PS EXO/NCP EXO EXO/NCP EXO/NCP – EXO/PS – EXO – EXO EXO EXO EXO ∞ ∞ ∞ ∞ XIC: 3 SC: 1.9 XIC: 2.4 SC: 2 XIC: 3.2 SC: 2 XIC: 4.3 SC: 3.1 XIC: 2 SC: 2.3 XIC: 2.7 SC: 3.1 XIC: 530000 SC: XIC: 6.1 SC: 2.2 XIC: 16 SC: 2.8 XIC: 3.1 SC: 7.9 XIC: 2200000 SC: XIC: 3.4 SC: 1.8 XIC: 13 SC: 13 XIC: 4.5 SC: 3.8 XIC: 1000000 SC: XIC: 4.4 SC: 6.7 XIC: 16 SC: protein, binds to ASS1 under low intracellular NADPH NADPH intracellular ASS1 under low protein, binds to concentrations activity oxidoreductase, zinc binding protein Argininosuccinate synthase, ligase regulator Bromodomain-containing protein 4, chromatin Creatine kinase B-type, metabolic pathway protease inhibitor Cystatin-C, cysteine oxidoreductase dehydrogenase, Glyceraldehyde-3-phosphate IST1 homolog, cadherin binding protein oxidoreductase 2, mitochondrial, Malate dehydrogenase Protein NDRG1, cadherin binding protein NmrA-like family domain-containing protein 1, redox sensor and phospholipase C A3, isomerase Protein disulfide-isomerase RIB43A-like with coiled-coils protein 2 Selenium-binding protein 1, transport Tektin-4 actin binding protein alpha-3 chain, Tropomyosin repeat protein 38 Tetratricopeptide enzyme 5 Ubiquitin-like modifier-activating homolog, VAT-1 protein membrane Synaptic vesicle ASS1 BRD4 CKB CST3 GAPDH IST1 MDH2 NDRG1 NMRAL1 PDIA3 RIBC2 SELENBP1 TEKT4 TPM3 TTC38 UBA5 VAT1 https://rep.bioscientifica.com Reproduction (2019) 158 335–356

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common to the two stages of collection (i.e. before and after AI), one was observed only in UF collected before AI, and 10 were only found in UF collected after AI (Fig. 4 left). In addition, 27 of these 52 proteins were two-fold lower (Table 3), and 25 were two-fold higher (Table 4) in UF collected after AI. In UF from subfertile F− hens, a total of 61 (XIC) and 92 (SC) proteins were differentially abundant (P < 0.05) in response to AI, 40 of which were common to both quantitative methods. Out of these 40 proteins, 37 were common to the two stages of collection (i.e. before and after AI), one was observed only in UF collected before AI, and two were only in UF collected after AI (Fig. 4 right). In addition, 19 of these 40 proteins were two-fold lower (Table 3), and 21 proteins were two-fold higher Other potential function in genital tract Sperm survival Enzyme regulator (Table 4) in UF collected after AI. Table 3 reports the secretory pathway and the biological functions of the 19 and 27 proteins whose levels decreased in response to insemination in subfertile F− and fertile F+ hens, respectively. These 46 proteins were compared to those previously identified in Previously Previously described in avian SPZ or SP UF, UF: NPD SPZ: (3) SP: NPD UF: NPD SPZ: NPD SP: NPD UF: NPD SPZ: NPD SP: NPD UF: NPD SPZ: (3) SP: NPD UF: NPD SPZ: NPD SP: NPD avian UF by Sun et al. (2013) and Marie et al. (2015a). It is noticeable that only 28 of them had been described in one of these studies, the present research is the first to describe the other 18 in avian UF. Out of these 18, Secretory Secretory pathway NCP EXO/NCP EXO/NCP – NCP the level of five decreased in the −F line, whereas 13 )

− decreased in the F+ line after AI. Interestingly, 15/19 (78.95%) and 21/27 (77.77%) (F+/F

∞ ∞ ∞ ∞ ∞ of the proteins that decreased in quantity after AI ∞ ∞ ∞ ∞ ∞ from F− and F+ hens, respectively, were referenced in Ratio XIC: SC: XIC: SC: XIC: SC: XIC: SC: XIC: SC: exosome databases. Furthermore, in F− hens, 2/19 have previously been observed in seminal plasma, 1/19 in spermatozoon, and 8/19 in both of them (Labas et al. 2015). In F+ hens, 7/27 have previously been identified in seminal plasma, 1/27 in spermatozoon, and 8/27 in both of them (Labas et al. 2015). . The secretory pathway and the biological functions of the 21 and 25 proteins that increased in response

. (2015) to AI in F− and F+ hens, respectively, are described in

et al Table 4. In both genetic lines, 52% (F−) and 56% (F+) of the proteins that increased in quantity are referenced

Labas in exosome databases. Furthermore, 2/21 (F− hens) and 6/25 (F+ hens) proteins have previously been described ; (3): in seminal plasma and spermatozoon, 4/21 (F− hens) and 8/25 (F+ hens) proteins in seminal plasma only,

. (2013) and 1/21 (F− hens) and 1/25 (F+ hens) proteins in

et al spermatozoon only (Labas et al. 2015). As shown in Tables 3 and 4, five proteins were Sun differentially abundant in response to insemination ; (2):

) in both genetic lines of hens (Fig. 4 middle). These protein biosynthesis, zinc binding protein a Description Enkurin, calmodulin binding protein domain-containing protein 4, cadherin binding Glyoxalase in ascorbate involved Regucalcin, calcium binding hydrolase oxidoreductase oxidoreductase, dehydrogenase-like Saccharopine binding protein D54, RNA Tumor include PARK7 that was lower, and FN1 and DNAH9 that were higher in quantity after insemination in (2015 both F+ and F− hens. Ig gamma fragments increased

et al. in F+ hens in response to insemination, whereas they

Continued decreased in F− hens. In contrast, there was less COPA Marie protein in F+ hens, but more in F− hens in response

Table 2 Table Symbol Detected only in F+ hens (5) ENKUR GLOD4 RGN SCCPDH TPD52L2 lines of hens after insemination are underlined. as differentially abundant between the two Proteins that were also observed (1): uterine fluid. seminal plasma; SPZ, spermatozoa; UF, SPZ or SP; PS, peptide signal; SP, UF, not previously described in avian NPD, non-classical pathway; exosome; Ig, immunoglobulin; NCP, EXO, to insemination.

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and also of OCX32 that is more abundant in F+ UF after AI, was analyzed in SST using immunohistochemistry (Figs 5, 6, 7, 8 and 9). Negative controls demonstrated the absence of non-specific signals for all antibodies (Supplementary data 2). ANXA4 was observed in SST cells and cilia from the UVJ surface epithelium of both lines (Fig. 5). In F+ hens, the ANXA4 signal was slight in SST and UVJ cilia before insemination (Fig. 5A), but strong at the apical border of SST cells and UVJ cilia after insemination (Fig. 5B). In contrast, in F− hens the ANXA4 signal was mainly found in the apical part of SST cells and cilia from the UVJ surface epithelium before insemination (Fig. 5C), but not after insemination (Fig. 5D). The ANXA5 signal was observed in the connective tissue of the UVJ that surrounds SST structures of the UVJ before insemination in both lines, with a higher intensity in F+ hens (Fig. 6A and C). After insemination, this signal was observed in the connective tissue of the UVJ in F+ hens (Fig. 6B), whereas it was found mainly in the cytoplasm of SST cells in F− hens (Fig. 6D). OCX32 was observed in the connective tissue and in the apical part of the UVJ surface epithelium in both lines of hens, before and after insemination (Fig. 7A, B, C and D). HSPA8 was slightly immunolocalized in the cytoplasm of epithelial cells, and also in SST cells of both lines of hens, before insemination (Fig. 8A and C). After insemination, its immunolocalization did not differ in F+ hens (Fig. 8B). In Figure 3 Western blot analysis of the HSPA8, ANXA4 and ANXA5 contrast in F− hens, a strong HSPA8 signal was observed proteins in the UF from F+ and F− hens before insemination. (A) in the lumen of SST filled with sperm Fig.( 8D). Representative profiles of Western blotting. (B) Quantitative analysis Before insemination, PIGR was localized in the of amounts of proteins in UF from F+ (n = 4) and F− hens (n = 4). cytoplasm of SST cells and the UVJ epithelial cells in *P < 0.05 between the two lines. both lines, with a higher signal in F− hens (Fig. 9A and C). In F+ hens after insemination, a slight signal was Localization of ANXA4, ANXA5, OCX32, HSPA8 and PIGR in SST observed in the cytoplasm of SST cells, and a granular signal was detected in the lumen of the SST filled with SST are tubular glands localized in the UVJ whose role sperm (Fig. 9B). In contrast, after insemination in F− hens is to store spermatozoa. The localization of ANXA4 and an intense PIGR signal was observed in the cytoplasm ANXA5 that are more abundant in F+ UF, and HSPA8 and the apical part of SST cells, as well as a granular and PIGR that are more abundant in F− UF before AI, signal in the lumen of SST (Fig. 9D).

Figure 4 Quantified proteins in UF collected in subfertile (F−) and fertile (F+) hens either before or after insemination. Extracted ion chromatograms (XIC) and spectral counting (SC) methods were used. A fold change (FC) >2 and P < 0.05 were chosen to indicate a differential quantification in abundance. (A) Numeric values. (B) Venn diagram. https://rep.bioscientifica.com Reproduction (2019) 158 335–356

Downloaded from Bioscientifica.com at 10/04/2021 06:51:59PM via free access 346 C Riou and others Other potential function in genital tract Sperm survival sperm survival Immunity, Inflammation Inflammation Sperm survival Cytoskeleton organization Immunity development Embryonic Sperm survival Previously Previously described in avian SPZ or SP UF, UF: (2) SPZ: (3) SP: NPD UF: (1) SPZ: (3) SP: (3) UF: (2) SPZ: (3) SP: (3) UF: NPD SPZ: (3) SP: (3) UF: (1, 2) SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: NPD UF: (1, 2) SPZ: (3) SP: (3) UF: (1) SPZ: NPD SP: NPD UF: (2) SPZ: NPD SP: (3) UF: (1, 2) SPZ: NPD SP: (3) UF: (1, 2) SPZ: NPD SP: NPD UF: (1, 2) SPZ: NPD SP: NPD UF: (2) SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: NPD UF: NPD SPZ: NPD SP: NPD Secretory Secretory pathway EXO/NCP NCP EXO EXO EXO/PS EXO/NCP PS EXO EXO/NCP EXO/PS EXO/PS EXO EXO EXO PS (after AI/ Ratio before AI) XIC: 0.2 SC: 0.3 XIC: 0.1 SC: 0.4 XIC: 0.2 SC: 0.1 XIC: 0 SC: 0 XIC: 0.5 SC: 0.2 XIC: 0.000002 SC: 0 XIC: 0.3 SC: 0.4 XIC: 0.3 SC: 0.4 XIC: 0.1 SC: 0.09 XIC: 0.4 SC: 0.1 XIC: 0.000002 SC: 0.4 XIC: 0.1 SC: 0.2 XIC: 0.4 SC: 0.2 XIC: 0.3 SC: 0.4 XIC: 0.2 SC: 0.2 ) and fertile (F+) hens. − kDa protein, molecular chaperone hens (19) − immunoglobulin binding protein catabolic process bradykinin protein transport oxidoreductase, siderophore biosynthesis and folate analogs into the 5-methyltetrahydrofolate interior of cells depending pH process Description cognate 71 Heat shock of fragment Immunoglobulin gamma chain, leukotriene biosynthesis, zinc A-4 hydrolase, Leukotriene in Xaa-pro aminopeptidase 1, protease involved Albumin, plasma protein, antioxidant activity LIM and SH3 domain protein 1, actin binding ion receptor activity scavenger PIT54 protein precursor, Semaphorin-3G type 2 isoform X5, dehydrogenase Hydroxybutyrate Complement C3 of receptor alpha, mediates delivery Folate alpha 3, glutathione metabolic Glutathione S-transferase protein 70, molecular chaperone Heat shock molecule activity Lamin-A, structural Mesothelin isoform X13, cell-matrix adhesion /++ ++ /++ Uterine fluid proteins that decrease in response to insemination subfertile (F ++

++ Ig gamma chain (Clone 36)* Ig gamma chain Table 3 Table Symbol Decrease in response to insemination subfertile F HSPA8* LTA4H* XPNPEP1* ALB LASP1** PIT54 SEMA3G BDH2 C3 FOLR1 GSTA3 HSPA2 LOC396224 MSLN

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Downloaded from Bioscientifica.com at 10/04/2021 06:51:59PM via free access Avian uterine fluid proteins and fertility 347 ) Continued ( Inflammation, fertilization Protein transport Mineralization fusion Membrane Cytoskeleton organization assembly body Multivesicular Acidification UF: (2) SPZ: (3) SP: (3) UF: (1, 2) SPZ: NPD SP: NPD UF: NPD SPZ: NPD SP: NPD UF: (1, 2) SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: (3) UF: (1, 2) SPZ: NPD SP: (3) UF: NPD SPZ: NPD SP: (3) UF: (1, 2) SPZ: NPD SP: (3) UF: (1, 2) SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: NPD UF: NPD SPZ: NPD SP: NPD UF: (1) SPZ: NPD SP: (3) UF: (1) SPZ: NPD SP: (3) UF: NPD SPZ: (3) SP: (3) UF: NPD SPZ: (3) SP: NPD UF: NPD SPZ: NPD SP: NPD UF: NPD SPZ: (3) SP: (3) EXO EXO/PS – EXO/PS EXO EXO/NCP EXO EXO/NCP EXO EXO/NCP EXO EXO/NCP EXO EXO EXO EXO/NCP EXO 6 7 − − XIC: 0.09 SC: 0 XIC: 0.000001 SC: 0.1 XIC: 0.1 SC: 0.2 XIC: 0.2 SC: 0.3 XIC: 0.3 SC: 0.5 XIC: 0.4 SC: 0.7 XIC: 0.1 SC: 0.03 XIC: 0.4 SC: 0.5 XIC: 0.4 SC: 0.7 XIC: 0.2 SC: 0.2 XIC: 1.10 SC: 0 XIC: 0.3 SC: 0.2 XIC: 0.2 SC: 0.09 XIC: 0.02 SC: 0.1 XIC: 3.10 SC: 0 XIC: 0.2 SC: 0.1 XIC: 0.08 SC: 0.1 oxidoreductase, copper and RNA-binding protein oxidoreductase, copper and RNA-binding binding protein phosphatidylinositol protein A2 inhibitor activity protein, possess phospholipase protein helicase binding protein ribosomal protein binding binding protein oxidoreductase Protein DJ-1, chaperone, hydrolase, protease, hydrolase, Protein DJ-1, chaperone, Prostate stem cell antigen, C-terminal protein lipidation Sorting nexin-1 isoform X3, cadherin and Vitronectin, proteoglycan binding properties A4, calcium-dependent phospholipid binding Annexin A8, calcium-dependent phospholipid binding Annexin Calcium and integrin-binding protein 1, calcium binding Destrin, actin-depolymerizing protein RNA ATP-dependent initiation factor 4A-II, Eukaryotic domain-containing protein 4, cadherin Glyoxalase Guanine nucleotide-binding protein subunit beta-2-like 1, factor protein 7, growth factor-binding Insulin-like growth subunit 12A, lipid and ubiquitin body Multivesicular Ras-related protein Rap-1A, GTP binding oxidoreductase, dehydrogenase-like Saccharopine binding protein D54, RNA Tumor A catalytic subunit ATPase proton V-type + PARK7 PSCA SNX1 VTN Decrease in response to insemination fertile F+ hens (27) ANXA4** ANXA8** CIB1** DSTN** EIF4A2** GLOD4** GNB2L1** IGFBP7** MVB12A** RAP1A** SCCPDH** TPD52L2** ATP6V1A https://rep.bioscientifica.com Reproduction (2019) 158 335–356

Downloaded from Bioscientifica.com at 10/04/2021 06:51:59PM via free access 348 C Riou and others ; ) a (2015 et al. Marie degradation pathway degradation Other potential function in genital tract Detoxification Immunity Antimicrobial Inflammation, fertilization trafficking Membrane Ubiquitin/ubiquitin-proteasome hens than F+ after insemination. **Proteins − Previously Previously described in avian SPZ or SP UF, UF: (1) SPZ: NPD SP: NPD UF: (1, 2) SPZ: NPD SP: NPD UF: (2) SPZ: (3) SP: (3) UF: (1) SPZ: NPD SP: NPD UF: (2) SPZ: NPD SP: (3) UF: NPD SPZ: NPD SP: NPD UF: NPD SPZ: NPD SP: NPD UF: (2) SPZ: (3) SP: (3) UF: (1, 2) SPZ: (3) SP: (3) UF: NPD SPZ: (3) SP: (3) UF: NPD SPZ: NPD SP: NPD UF: NPD SPZ: NPD SP: NPD UF: (1, 2) SPZ: NPD SP: NPD UF: (2) SPZ: (3) SP: (3) hens than F+ after insemination. (1): − Secretory Secretory pathway EXO PS EXO/PS NCP PS/NCP – NCP EXO EXO/NCP EXO EXO/NCP EXO – EXO/NCP 6 6 − − (after AI/ Ratio before AI) XIC: 0.1 SC: 0.1 XIC: 0.1 SC: 0.2 XIC: 0.3 SC: 0.5 XIC: 0.2 SC: 0.8 XIC: 0.2 SC: 0.5 XIC: 0 SC: 0 XIC: 0.3 SC: 0.4 XIC: 0.2 SC: 0.09 XIC: 0.3 SC: 0.4 XIC: 1.10 SC: 0 XIC: 3.10 SC: 0.2 XIC: 0.1 SC: 0 XIC: 0.1 SC: 0.2 XIC: 0.5 SC: 0.7 Proteins that are more abundant in UF from F + Proteins that are less abundant in UF from F ++ hens than F+ before insemination. − . binding protein fragment acids binding linoleic, oleic and steraic arachidonic, protein protein oxidoreductase, copper and RNA-binding lipid binding protein protease inhibitor, gluconolactonase that modulates calcium-signaling and calcium-dependent cellular processes and enzyme activities singlet microtubules peripheral protease hens than F+ before insemination. . (2015) Description Coatomer subunit alpha serine protease inhibitor Ovostatin, Glutathione peroxidase 3, oxidoreductase, selenium immunoglobulin V-region, chain Immunoglobulin heavy fatty acid-binding protein precursor, Extracellular Actin, non-muscle 6.2-like 40S ribosomal protein S0-like, protease, hydrolase, Protein DJ-1, chaperone, protein 1, serine Phosphatidylethanolamine-binding Ras-related protein Rab-11A, GTP binding Regucalcin, calcium and zinc binding protein, 40S ribosomal protein S16, and Sentan, mediator between ciliary membrane isozyme L1, thiol Ubiquitin carboxyl-terminal hydrolase − et al Labas ; (3): . (2013) + + et al Continued Sun V-region Immunoglobulin heavy chain chain Immunoglobulin heavy Table 3 Table Symbol COPA OVSTL GPX3 LCN8 LOC100178468 LOC717755 PARK7 PEBP1 RAB11A RGN RPS16 SNTN UCHL1 Proteins that are common to both genetic lines of hens underlined. *Proteins that are more abundant in UF from F that are less abundant in UF from F (2): uterine fluid. seminal plasma; SPZ, spermatozoa; UF, SPZ or SP; PS, peptide signal; SP, UF, not previously described in avian NPD, non-classical pathway; exosome; Ig, immunoglobulin; NCP, EXO,

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Table 4 Uterine fluid proteins that were increased in response to insemination in subfertile (F−) and fertile (F+) hens. Previously described in Other potential Ratio (after Secretory avian UF, SPZ function in genital Symbol Description AI/before AI) pathway or SP tract Increase in response to insemination in subfertile F− hens (21) COPA+ Coatomer subunit alpha XIC: 5.2 EXO UF: (1) SC: 2.5 SPZ: NPD SP: NPD FASN+ Fatty acid synthase, oxidoreductase XIC: 24 EXO/NCP UF: NPD Lipid metabolism SC: 4.6 SPZ: NPD SP: (3) HDLBP+ Vigilin, cadherin binding protein XIC: ∞ – UF: NPD Lipid metabolism SC: ∞ SPZ: NPD SP: NPD IFT172+ Intraflagellar transport protein 172 homolog XIC: 1900000 – UF: NPD SC: ∞ SPZ: NPD SP: NPD IQGAP1+ Ras GTPase-activating-like protein IQGAP1, XIC: 2.3 EXO UF: (1, 2) calmodulin binding protein SC: 2.8 SPZ: (3) SP: (3) LAMC1+ Laminin subunit gamma-1, glycosphingolipid XIC: 1300000 EXO/PS UF: NPD Extracellular binding SC: ∞ SPZ: NPD matrix structural SP: NPD constituent LOC100551267+ Glycogen debranching enzyme XIC: 1300000 – UF: NPD Carbohydrate SC: 13 SPZ: NPD metabolism SP: NPD LOC100859916+ Mucin-5AC, O-glycosylated protein XIC: 3.2 PS UF: NPD Gel forming SC: 5.5 SPZ: NPD protein SP: NPD LOC423849+ Prominin-1-A isoform X1, cadherin binding XIC: 2.9 EXO UF: (1) SC: 4.2 SPZ: NPD SP: NPD MYH9+ Myosin-9, actin binding and calmodulin binding XIC: 3.5 EXO UF: (1) protein SC: 2.9 SPZ: NPD SP: (3) ROS1+ Proto-oncogene tyrosine-protein kinase ROS, XIC: ∞ PS UF: (1) protein tyrosine kinase SC: ∞ SPZ: NPD SP: (3) RP1+ Oxygen-regulated protein 1 XIC: 1000000 – UF: NPD SC: ∞ SPZ: NPD SP: NPD WDR52+ WD repeat-containing protein 52 XIC: 1200000 – UF: NPD SC: ∞ SPZ: NPD SP: NPD DNAH9 Dynein heavy chain 9, axonemal isoform X1, XIC: 2.8 – UF: NPD ATPase activity, motor protein SC: 8.5 SPZ: NPD SP: NPD EPRS Bifunctional aminoacyl-tRNA synthetase, XIC: 1100000 – UF: NPD Inflammation component of the GAIT (gamma interferon- SC: ∞ SPZ: NPD activated inhibitor of translation) complex, RNA SP: NPD binding protein FN1 Fibronectin, proteoglycan binding properties XIC: 7.3 EXO/PS UF: (1) Mineralization SC: 6.8 SPZ: NPD SP: (3) GOT1 Aspartate aminotransferase, cytoplasmic, XIC: 2.4 EXO UF: (2) amino-acid biosynthesis SC: 1.7 SPZ: (3) SP: (3) Immunoglobulin Immunoglobulin heavy chain V-region, XIC: 2.1 NCP UF: (1) Immunity heavy chain immunoglobulin fragment SC: 0.4 SPZ: NPD V-region SP: NPD LOC428451 Prostatic acid phosphatase, phosphatase activity XIC: 2.5 EXO/PS UF: (1) SC: 1.5 SPZ: NPD SP: NPD RUVBL1 RuvB-like 1, cadherin binding protein, DNA XIC: 2.9 EXO UF: NPD helicase activity SC: 2.4 SPZ: (3) SP: NPD (Continued) https://rep.bioscientifica.com Reproduction (2019) 158 335–356

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Table 4 Continued

Previously described in Other potential Ratio (after Secretory avian UF, SPZ function in genital Symbol Description AI/before AI) pathway or SP tract SARS seryl-tRNA synthetase, cytoplasmic, ATP-binding XIC: 6.3 EXO UF: NPD ligase involved in protein biosynthesis SC: 9.4 SPZ: NPD SP: NPD Increase in response to insemination in fertile F+ hens (25) ACR++ Acrosin, serine protease, zinc binding protein XIC: ∞ – UF: NPD Fertilization SC: ∞ SPZ: (3) SP: (3) APOA4++ Alipoprotein A-IV, antioxidant activity XIC: 4.2 EXO/PS Lipid transport SC: 36 C9ORF24++ Spermatid-specific manchette-related protein 1 XIC: ∞ – UF: NPD SC: ∞ SPZ: (3) SP: (3) CHIA++ Acidic mammalian chitinase, chitinase activity XIC: ∞ PS UF: (1, 2) SC: ∞ SPZ: NPD SP: (3) CNP++ 2′,3′-Cyclic-nucleotide 3′-phosphodiesterase, XIC: ∞ EXO UF: NPD RNA binding protein SC: ∞ SPZ: NPD SP: (3) CRYZ++ Quinone oxidoreductase, oxidoreductase, zinc XIC: ∞ EXO/NCP UF: NPD binding protein SC: ∞ SPZ: NPD SP: NPD DPYS++ Dihydropyrimidinase, hydrolase, zinc binding XIC: ∞ EXO UF: NPD protein SC: ∞ SPZ: (3) SP: (3) EIF2S1++ Eukaryotic translation initiation factor 2 subunit 1, XIC: 3.1 EXO UF: NPD RNA binding protein SC: 4.5 SPZ: NPD SP: NPD FGB++ Fibrinogen beta chain XIC: ∞ EXO/PS UF: (1, 2) Immunity SC: ∞ SPZ: (3) SP: (3) GAL3ST2++ Galactose-3-O-sulfotransferase 2, carbohydrate XIC: ∞ – UF: (1) sulfation SC: ∞ SPZ: NPD SP: NPD GC++ Group-specific component (vitamin D binding XIC: 2.4 EXO/PS UF: (1, 2) Antimicrobial protein) SC: 3.7 SPZ: NPD SP: (3) GNAI2++ Guanine nucleotide-binding protein G(i) subunit XIC: ∞ EXO/NCP UF: (1) alpha-2, G protein, magnesium binding protein SC: ∞ SPZ: NPD SP: (3) HAPLN3++ Hyaluronan and proteoglycan link protein 3 XIC: 10 EXO/PS UF: (1) Mineralization SC: 5.7 SPZ: NPD SP: NPD Ig gamma chain Immunoglobulin gamma chain, fragment of XIC: 2.8 NCP UF: (1) Immunity, sperm (Clone 36)*/++ immunoglobulin SC: 1.6 SPZ: (3) survival SP: (3) SERPINA3++ Alpha-1-antichymotrypsin, serine protease XIC: ∞ EXO/PS UF: (1) inhibitor SC: ∞ SPZ: NPD SP: (3) TOR1B++ Torsin family 1, member B (torsin B), molecular XIC: ∞ EXO/NCP UF: (1, 2) Mineralization chaperone SC: ∞ SPZ: NPD SP: NPD TSKU++ Tsukushi small leucine rich proteoglycan XIC: 3.6 PS UF: (2) Mineralization homolog SC: 3.4 SPZ: NPD SP: NPD VNN1++ Pantetheinase, hydrolase, pantothenate metabolic XIC: 12 PS UF: (1, 2) process SC: 7.9 SPZ: (3) SP: (3) WDR1++ WD repeat-containing protein 1, actin binding XIC: 3.1 EXO UF: (1, 2) protein SC: 1.9 SPZ: (3) SP: NPD DNAH9 Dynein heavy chain 9, axonemal isoform X1, XIC: 2.3 – UF: NPD ATPase activity, motor protein SC: 2.8 SPZ: NPD SP: NPD (Continued) Reproduction (2019) 158 335–356 https://rep.bioscientifica.com

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Table 4 Continued

Previously described in Other potential Ratio (after Secretory avian UF, SPZ function in genital Symbol Description AI/before AI) pathway or SP tract FN1 Fibronectin, proteoglycan binding properties XIC: 3 EXO/PS UF: (1) Mineralization SC: 6.7 SPZ: NPD SP: (3) GALNT12 Polypeptide N-acetylgalactosaminyltransferase XIC: 6.2 NCP UF: (1) Protein 12, glycosyltransferase, carbohydrate and SC: ∞ SPZ: NPD glycosylation manganese binding protein SP: NPD GSN Gelsolin, calcium and actin binding protein XIC: 2.7 EXO/PS UF: (1) Mineralization SC: 1.8 SPZ: NPD SP: (3) VTG1 Vitellogenin-1, phosvitin domain, nutrient XIC: 3.8 PS UF: (1, 2) Embryonic reservoir activity in egg yolk, lipid and iron SC: 18 SPZ: NPD development, binding protein SP: NPD antimicrobial VTG2 Vitellogenin-2, phosvitin domain, nutrient XIC: 2.4 PS UF: (2) Embryonic reservoir activity in egg yolk, lipid and iron SC: 6.4 SPZ: NPD development, binding protein SP: NPD antimicrobial Proteins that are common to both genetic lines of hens are underlined. *Proteins that are more abundant in UF from F− hens than F+ hens before insemination. +Proteins that are more abundant in UF from F− hens than F+ hens after insemination. **Proteins that are less abundant in UF from F− hens than F+ hens before insemination. (1): Marie et al. (2015a); (2): Sun et al. (2013); (3): Labas et al. (2015). EXO, exosome; Ig, immunoglobulin; NCP, non-classical pathway; NPD, not previously described in avian UF, SPZ or SP; PS, peptide signal; SP, seminal plasma; SPZ, spermatozoa; UF, uterine fluid.

Discussion of the avian genital tract undergo up and downregulations of specific locally produced proteins rapidly after This study aimed to identify proteins that are modulated insemination. Moreover, the present study demonstrated when sperm arrive in the hen’s genital tract in order that proteins were differentially present in the UF after to obtain insight into sperm storage regulation. We sperm deposition in the fertile (F+) and subfertile (F ) hens. demonstrated that on arrival in the genital tract sperm − Furthermore, we used label-free methods of quantitation induced a significant change in the proteomic content of avian UF. We also showed that this change was differential between hens which exhibit high and low sperm storage ability. This led us to highlight proteins that may enhance sperm storage and to identify potential markers of high and low fertility within the UF. The modulation of these markers in the SST after sperm arrival suggests that they may be key factors in sperm storage. Using two lines of hens (F+ and F−), we showed that the genetic divergence on the duration of fertility was as previously described (Beaumont 1992, Brillard et al. 1998). The F− hens were characterized by reduced fertility and also shorter efficient and maximal durations of fertility when compared to the F+ line, in accordance with previous published data (Brillard et al. 1998, Riou et al. 2017). These two lines are therefore a suitable model to study the regulation of sperm storage duration in hens, and more generally in avian species. The present study revealed that sperm deposition by artificial insemination caused rapid (within 24 h) changes in the proteomic composition of the avian UF, associated with a modulation of some proteins in SST. This is in line with a recent study which demonstrated that sperm deposition after natural mating induced rapid (within 24 h) changes of gene expression that shifted exclusively Figure 5 Localization of ANXA4 protein using immunohistochemistry in UVJ sections collected from F+ (A and B) and F− hens (C and D) in the uterus and the uterovaginal junction epithelium before insemination (A and C) and 24 h after insemination (B and D). of the hen’s genital tract (Atikuzzaman et al. 2015). This Arrows indicate ANXA4 strong signal localization. l, lumen; e, observation indicates that uterine and uterovaginal parts epithelium; SST, sperm storage tubule. Scale bar: A, B, C, D = 20 µm. https://rep.bioscientifica.com Reproduction (2019) 158 335–356

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by the binding of specific sperm surface proteins Ignotz( et al. 2007, Teijeiro et al. 2009, 2016). Our results suggest that ANXA4 may also be involved in sperm storage in avian species and that the absence of ANXA4 at the apical border of SST cells from F− hens could explain, at least in part, their poor sperm storage ability. Moreover, the cytoplasmic localization of ANXA5 in F− SST cells and UVJ cilia after insemination observed in the present study could be related to an apoptotic phenomenon (Gaipl et al. 2007). ANXA5 may also modulate the immune system resulting in long-lasting immunity (Gaipl et al. 2007). In F+ hens, the localization of ANXA5 in the connective tissue around SST would probably originate in the SST. Thus, the role of annexins in sperm storage needs to be investigated further to improve understanding of their role in this process. A second group of proteins that was more abundant in the UF of F+ hens and whose abundance increased or was higher after insemination in F+ than in F− hens includes proteoglycans and proteoglycan binding proteins. This group contains tsukushi (TSKU), which has been described as a core of proteoglycan (Dellett et al. Figure 6 Localization of ANXA5 protein using immunohistochemistry 2012). It also contains vitronectin (VTN), hyaluronan in UVJ sections collected from F+ (A and B) and F− hens (C and D) proteoglycan link protein 3 (HAPLN3), and fibronectin 1 before insemination (A and C) and 24 h after insemination (B and D). (FN1) which all exhibit proteoglycan binding properties Arrows indicate ANXA5 signal localization. l, lumen; e, epithelium; (Francois et al. 1999, Lyon et al. 2000, Spicer et al. SST, sperm storage tubule. Scale bar: A, B, C, D = 20 µm. 2003). Furthermore, in the current study we found that OC-116 (protein core of a sulfated proteoglycan) was to compare the abundance of proteins between samples. one of the major components of the UF, and which we This quantitation method may be considered to provide preliminary results compared to isobaric multiplexed quantitation. Nevertheless, the use of two independent methods (SC and XIC) on the same dataset, as performed here, guarantees robustness in our interpretations.

High fertility markers High fertility marker proteins were defined as those which were more abundant in the UF of F+ fertile hens and/or proteins which decreased in quantity 24 h after insemination in F+ fertile hens. These included annexins (ANXA4, ANXA5, ANXA8) known to be present in exosomes, and a protein involved in multivesicular body formation (MVB12A). We hypothesize that annexin (or annexin-containing vesicles) is mobilized by the sperm and/or that exosome release is inhibited in response to insemination. This is consistent with our immunohistochemistry results which showed that ANXA4 increased in the apical border of SST cells in response to insemination in F+ hens. Recently, exosomal protein CD63 has been shown to transit to the apical part of SST cells after arrival of sperm (Huang et al. 2017). Overall, these observations suggest that sperm arrival within SST induces a mobilization of exosomal Figure 7 Localization of OCX32 protein using immunohistochemistry in UVJ sections collected from F+ (A and B) and F− hens (C and D) molecules in the apical part of SST cells. In mammals, before insemination (A and C) and 24 h after insemination (B and D). annexins (ANXA1, ANXA2, ANXA4 and ANXA5) are l, lumen; e, epithelium; SST, sperm storage tubule. Scale bar: A, B, C, candidates for oviductal receptors of sperm reservoirs D = 20 µm.

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carboxypeptidase activity and bacterial growth (Xing et al. 2007). High levels of OCX32 in F+ UF may ensure a favorable bacteria-free environment, in response to sperm arrival. Furthermore, OCX32, OCX25, SERPINA3 and CST3, found in the present study in higher quantities in the F+ UF, display a protease inhibitor function. Interestingly, protease inhibitors are known to play an important role in oocyte–sperm interaction during fertilization in mammals (Lee et al. 2018, Li et al. 2018). Our study revealed that amounts of vitellogenins (I and II), apolipoprotein AI (APOA1), apolipoprotein AIV (APOA4), and apolipoprotein H (Apo-H, also known as beta-2 glycoprotein I) increased in F+ UF after insemination and were over-abundant compared to those in F− UF. These proteins have already been identified in UF. An antimicrobial role has been previously suggested for lipid-binding proteins (Marie et al. 2015a,b). Moreover, vitellogenins have antioxidant properties, and limit phospholipid oxidation induced by iron or copper (Lu & Baker 1986). Recently, Huang et al. (2016) suggested that increased fatty acid metabolism may be involved in sperm survival inside SST (Huang et al. 2016). More generally, Figure 8 Localization of HSPA8 protein using immunohistochemistry lipid metabolism and transport may be key mechanisms in UVJ sections collected from F+ (A and B) and F− hens (C and D) during sperm storage, by regulating the availability of ions before insemination (A and C) and 24 h after insemination (B and D). and protecting sperm membrane from oxidation. Arrows highlight the differential HSPA8 signal localization after insemination between lines. l, lumen; e, epithelium; SST, sperm storage tubule. Scale bar: A, B, C, D = 20 µm. Low fertility marker recently identified in the SST lumen (Riou et al. 2017). Low fertility marker proteins were defined as those which Atikuzzaman et al. (2015) previously found that after were more abundant in the UF of F− subfertile hens or insemination the expression of the OC-116 gene was proteins that increased in quantity 24 h after insemination regulated in the hen’s uterine mucosa (Atikuzzaman et al. in F− hens. They include proteins from the immune system, 2015). In mammals, sulfated glycosaminoglycans (as pro-inflammatory system and proteases, and consist of attached to avian OC-116) and hyaluronan (as attached pIgr (PIGR), immunoglobulin fragments, leukotriene to HAPLN3) have been showed to modulate sperm– A4 hydrolase (LTA4H), and Xaa-pro aminopeptidase genital tract interaction and/or to trigger sperm release 1 (XPNPEP1). XPNPEP1 is a metalloprotease (Li et al. from sperm reservoir (Talevi & Gualtieri 2001, Rodriguez- 2008), while LTA4H is an enzyme which presents both Martinez et al. 2016). FN1 may also mediate this hydrolase and metalloaminopeptidase activities (Rudberg process (Koehler et al. 1980). Finally, proteoglycan and et al. 2004). The LTA4H hydrolase activity is used in the proteoglycan binding proteins may improve interactions final step of the biosynthesis of leukotriene B4, a pro- between female genital tract epithelium and sperm cells. inflammatory mediator which increases and prolongs Nevertheless, their role in sperm storage within the hen’s inflammation within tissue Crooks( & Stockley 1998). genital tract requires further investigation. PIGR is an epithelial glycoprotein which allows trans- The present study allowed us to define a third group of cellular migration of IgA from the basolateral to the apical proteins dedicated to egg constitution which increased part of cells through transcytosis. The endoproteolytic in quantity after insemination in F+ hens. It includes cleavage of PIGR then releases the five extracellular ovocalyxin-32 (OCX32) which is a matrix protein found immunoglobulin-like domains of the PIGR (also known within both the outer layers of the eggshell and in as secretory component, SC) into the lumen together with the cuticle (Gautron et al. 2001). It has recently been its cargo, forming the secretory IgA (SIgA) complex. In demonstrated in quail that UVJ surface epithelium is the present study, we observed that the amount of PIGR involved in cuticle deposition, and a specific 32 kDa – decreased in the UF 24 h after insemination, suggesting cuticle protein in the UVJ surface epithelium has been it was mobilized after sperm arrival. PIGR was localized observed (Rahman et al. 2009, Ito et al. 2011). Although within SST cells mainly in the subfertile hens. These not identified yet, this 32 kDa component most probably results suggest that sperm arrival is associated with PIGR corresponds to OCX32, which we localized in the apical regulation in UF and also in SST from subfertile hens. part of UVJ surface epithelial cells. It should be noted This could be due to enhanced transcytosis of SIgA and that recombinant OCX32 has been shown to inhibit free SC in the SST lumen. https://rep.bioscientifica.com Reproduction (2019) 158 335–356

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tract environment of low fertile F− hens is not favorable to sperm storage due to higher mucosa sensitivity to infection, and active production of immune factors in response to sperm arrival, which most probably affect sperm storage duration. HSPA8 is a member of the HSP70 family of proteins which have been demonstrated to bind sperm in several avian and mammal species (Elliott et al. 2009, Hiyama et al. 2014). In mammals it has been demonstrated to improve sperm survival (Elliott et al. 2009, Lloyd et al. 2012, Alvarez-Rodriguez et al. 2013), whereas in quail it activated sperm motility (Hiyama et al. 2014). In our study, the presence of HSPA8 was found in the lumen of SST filled with sperm from F− hens. Indeed, HSPA8 may contribute to impeding sperm storage by activating their motility. Moreover, the HSP70 protein family is also known to modulate the immune system (De Maio & Vazquez 2013). In conclusion, the present study demonstrated that shortly after insemination, sperm induced significant and rapid changes in the proteomic content of the UF, as well as in the SST epithelium (within 24 h). In fertile F+ hens Figure 9 Localization of PIGR protein using immunohistochemistry in that exhibit a good sperm storage capacity, annexins, UVJ sections collected from F+ (A and B) and F− hens (C and D) proteoglycan and proteoglycan-binding proteins, before insemination (A and C) and 24 h after insemination (B and D). OCX32 and lipid transporters may ensure a favorable Arrows indicate the granular PIGR signal localization within the SST and positive environment for sperm storage. The lower lumen after insemination. Arrowhead indicates the specific apical fertility and sperm storage abilities of F hens may be localization of the signal. l, lumen; e, epithelium; SST, sperm storage − tubule. Scale bar: A, B, C, D = 20 µm. explained, at least in part, by an intra-uterine hyper active immune system induced by sperm. This could be mediated by increased levels of HSPA8 and PIGR in It is well known that PIGR is regulated by hormones, both UF and SST, UF enzymes with metallopeptidase mainly estradiol (Richardson et al. 1993, Kaushic et al. activity, and UF mucins. These molecules may together 1995). The higher levels we observed before insemination constitute an antimicrobial environment at the surface of in the UF of F hens may be related to a dysfunction − epithelial cells. Our hen model which displays divergent of its hormonal regulation. This idea is supported by sperm storage ability constitutes a relevant model to the study of Das et al. (2016b). These authors showed study the mechanism of sperm recognition and sperm- that changes in the expression of the estrogen receptor induced immune regulation. mRNA in the UVJ of subfertile laying hens was linked to the recruitment of immunocompetent cells around SST and poor sperm storage ability (Das et al. 2005, 2006b). Supplementary data In addition, we found that mucin 5B (MUC5B) and 5AC (MUC5AC) were over-abundant in F− hens and This is linked to the online version of the paper their levels increased after insemination. MUC5B and at https://doi.org/10.1530/REP-19-0079. MUC5AC are gel-forming mucins that protect epithelial surfaces by forming a biofilm that defends against pathogens. Interestingly, SIgA and SC have mucus- Declaration of interest binding properties (Johansen & Kaetzel 2011). Thus, we The authors declare that there is no conflict of interest that can hypothesize that sperm arrival in the genital tract of could be perceived as prejudicing the impartiality of the F− hens may stimulate biofilm formation by secreting research reported. abundant quantities of mucins to protect the genital tract epithelium from infection. The genital tract is a unique immunological Funding environment that must support the reproductive This research was funded by the Région Centre. Dr Luiz function and resist infection. In the chicken, immune Cordeiro was supported by CNPq, Brazilian National tolerance is considered to be necessary for the storage of Council of Scientific and Technological Development, on antigenic spermatozoa in order to extend their survival the Science Without Borders fellowship program (grant period (Bakst 2011). Our data suggest that the genital number 200391/2014-3).

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