BIOLOGY OF REPRODUCTION (2016) 94(3):56, 1–11 Published online before print 27 January 2016. DOI 10.1095/biolreprod.115.134973

Extracellular Vesicles Originate from the Conceptus and Uterus During Early Pregnancy in Sheep1 Gregory W. Burns, Kelsey E. Brooks, and Thomas E. Spencer2 Division of Animal Sciences, University of Missouri, Columbia, Missouri

Cells release diverse types of membrane-bound vesicles of endosomal and plasma membrane origin, termed exosomes and microvesicles, respectively. Extracellular vesicles (EVs) represent an important mode of intercellular communication by transferring select RNAs, proteins, and lipids between cells. The present studies tested the hypothesis that the elongating ovine conceptus and uterus produces EVs that mediate conceptus-maternal interactions during early pregnancy. In Study 1, EVs were purified from uterine luminal fluid of Day 14 cyclic sheep. The EVs were fluorescently labeled with PKH67 dye and infused into the uterine lumen of pregnant sheep for 6 days using an osmotic pump. On Day 14, labeled EVs were observed in the conceptus trophectoderm and uterine epithelia, but not in the uterine stroma or myometrium. In Study 2, Day 14 conceptuses were cultured ex vivo for 24 h and found to release EVs into the culture medium. Proteomics analysis of the Day 14 conceptusderived EVs identified 231 proteins that were enriched for extracellular space and several protein classes, including proteases, protease inhibitors, chaperones and chaperonins. RNA sequencing of Day 14 conceptus-derived EVs detected expression of 512 mRNAs. The top-expressed genes were overrepresented in ribosomal functions and components. Isolated EVs from conceptuses were fluorescently labeled with PKH67 and infused into the uterine lumen of cyclic sheep for 6 days using an osmotic pump. On Day 14, labeled EVs were observed in the uterine epithelia, but not in the uterine stroma or myometrium. Labeled EVs were not observed in the ovary or in other maternal tissues. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia, and are involved in intercellular communication between those cells during the establishment of pregnancy in sheep. conceptus, ovine/sheep, pregnancy, uterus

INTRODUCTION Exosomes and microvesicles are membrane-bound extracellular vesicles (EVs) implicated in cell-to-cell communica1

This research was supported, in part, by Agriculture and Food Research Initiative competitive grants 2012-67015-30173 and 201567015-23678 from the U.S. Department of Agriculture, National Institute of Food and Agriculture. 2 Correspondence: Thomas E. Spencer, Division of Animal Sciences, 158 Agriculture and Food Research Initiative, 920 East Campus Drive, University of Missouri, Columbia, MO 65211. E-mail: [email protected] Received: 26 August 2015. First decision: 22 September 2015. Accepted: 15 January 2016. Ó 2016 by the Society for the Study of Reproduction, Inc. This article is available under a Creative Commons License 4.0 (Attribution-NonCommercial), as described at http://creativecommons.org/licenses/by-nc/ 4.0 eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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tion. Recently, EVs have been identified in human [1] and ovine uterine luminal fluid (ULF) [2, 3] with time- and pregnancy-dependent changes in their contents. Exosomes are 50–150 nm vesicles of endocytic origin released upon fusion of a multivesicular body with the cell membrane [4, 5]. Microvesicles are 100–1000 nm in diameter and bud directly from the cell membrane [6]. Most cell types release EVs into the extracellular environment, including extravillous and villous human trophoblast cells, primary trophoblast cells from term human placenta, and ovine endometrial glandular epithelial (GE) cells in culture [1, 7–12]. EVs contain numerous lipids and cargo, including proteins and RNAs [13–15], and have surface receptors/ligands from the cell of origin [16]. Their cargo can be transferred to and regulate gene expression in target recipient cells [15, 17–19]. Both types of EVs can elicit biological effects, such as increased cell migration [20, 21] and immunomodulation [10, 22], that are important for placental development. Establishment of pregnancy in domestic ruminants (i.e., sheep, cattle, goats) begins at the conceptus stage and encompasses pregnancy recognition signaling, implantation, and placentation [23–26]. In sheep, the morula-stage embryo enters the uterus on Days 4–6 postmating, and develops into a blastocyst. After hatching from the zona pellucida on Day 8 or 9, the blastocyst grows into an ovoid or tubular form by Day 12, and is then termed a ‘‘conceptus’’ (i.e., embryo/fetus and associated extraembryonic membranes). Elongation of the ovoid conceptus (;5–10 mm) is coincident with a substantial increase in the production of interferon tau (IFNT), the maternal recognition of pregnancy signal. The now filamentous conceptus (10–15 cm) occupies the entire length of the uterine horn ipsilateral to the corpus luteum, and begins to firmly attach to the luminal epithelium (LE) by Day 16. Formation of an ovoid conceptus and its subsequent elongation requires secretions of the uterus contained in the ULF. Although blastocysts can develop entirely in vitro, they must be transferred into a receptive uterus in order for elongation to occur [27]. The endometrium of the uterus secretes substances, collectively termed ‘‘histotroph,’’ that govern growth and development of the conceptus via effects on trophectoderm proliferation and migration as well as attachment and adhesion to the LE [23, 28, 29]. Histotroph is derived primarily from transport and/or synthesis and secretion of substances by the endometrial epithelia, and is a complex and rather undefined mixture of proteins, lipids, amino acids, sugars, and ions [30, 31]. Recurrent early pregnancy loss observed in uterine gland knockout (UGKO) sheep established the importance of uterine epithelial-derived secretions in the ULF for support of conceptus survival, elongation and implantation in ruminants [32]. The identification of EVs as a component of ovine ULF supports the idea that they are novel mediators of conceptusmaternal interactions [2, 3]. The present studies tested the hypothesis that the elongating ovine conceptus and uterus

ABSTRACT

BURNS ET AL.

produce EVs that mediate conceptus-maternal interactions during early pregnancy.

EV Labeling and Osmotic Pump Loading Isolated EVs were labeled using the PKH67 green fluorescent cell linker kit for cell membrane labeling (Sigma-Aldrich, St. Louis, MO). Briefly, the desired number of EVs were pelleted using ExoQuick-TC, resuspended in PBS (100 ll), and incubated with 100 ll of diluent mixed with PKH67 dye at final concentration of 5 3 106 M for 5 min. Exo-FBS was added (400 ll) for 1 min to stop the labeling reaction. The sample tube was then filled with 500 ll DMEM/F-12 media (10% Exo-FBS) and 200 ll ExoQuick-TC. Samples were incubated at 48C for 30 min, and EVs were collected by centrifugation for 3 min at 14 000 3 g at 48C. Tubes containing pelleted EVs were filled with 1 ml DMEM/F-12 media (10% FBS) and 200 ll ExoQuick-TC, and the samples were incubated and centrifuged two more times to remove excess dye. The final EV pellet was resuspended in 2 ml of sterile PBS and loaded into an Alzet 2ML1 osmotic pump (Durect Corporation, Cupertino, CA) or 100 ll DMEM/ F-12 exosome-depleted complete media and added directly to cells cultured in a chamber slide. Plain 100-nm liposomes in PBS (Encapsula NanoSciences LLC) were used as an unlabeled EV control. Liposomes were diluted (1:100 000) and analyzed by NTA to determine diameter and concentration (1 3 1014 particles/ ml) before dilution in PBS to a final concentration of 5 3 1010 particles/ml to approximate the number of EVs isolated from one uterine horn. Osmotic pumps were equilibrated in sterile PBS at 378C overnight prior to implantation into sheep.

MATERIALS AND METHODS Animals All experimental and surgical procedures were approved by an Institutional Animal Care and Use Committee. Mature Columbia crossbred ewes (Ovis aries) were observed for onset of estrus (designated Day 0). Sheep assigned to collection during pregnancy were bred at estrus to an intact ram of proven fertility at estrus. Study 1. As illustrated in Figure 1A, cyclic sheep (n ¼ 12) were euthanized on Day 14 postestrus. The uterus was immediately removed, and the lumen was gently flushed with 10 ml sterile PBS (pH 7.2) to obtain ULF. The ULF was clarified by centrifugation (3000 3 g for 15 min at 48C), filtered through a 0.2lm nylon filter (cat. # 09-719C; Fisher Scientific, Fairlawn, NJ) and stored at 808C. The EVs from ULF were isolated, evaluated by nanoparticle tracking analysis (NTA), and then labeled with the lipophilic PKH67 fluorescent dye. Next, Day 8-bred sheep (n ¼ 12) were subjected to a midventral laparotomy. Using described methods [33], each uterine horn was fitted with an osmotic pump and vinyl catheter inserted 1 cm distal to the uterotubal junction into the lumen of the uterine horn that released 10 ll/h and contained either: 1) control 100 nm liposomes (Encapsula NanoSciences LLC, Brentwood, TN) or (2) PKH67-labeled EVs isolated from the ULF of a Day 14 cyclic sheep. Implantation of an osmotic pump and catheterization of the uterus does not compromise pregnancy based on prior results [34]. Each uterine horn received a catheter connected to an osmotic pump containing the equivalent of the number of EV particles from a single Day 14 cyclic sheep horn (5.6 3 1010 particles average). All sheep were necropsied on Day 14 (n ¼ 6 per treatment). The uterus, ovaries, parametrial lymph nodes, liver, and lung were collected and portions fixed for 4 h in 4% paraformaldehyde in PBS and then infused with 18% sucrose overnight at 48C. Study 2. As illustrated in Figure 1B, bred sheep (n ¼ 8) were euthanized on Day 14 postmating. Elongated conceptus(es) were recovered by gently flushing the uterine lumen with 20 ml of sterile, warm conceptus culture media (Dulbecco modified Eagle medium [DMEM]/F-12 supplemented with 10% exosome-depleted fetal bovine serum [Exo-FBS; System Biosciences, Cupertino, CA], glutamine [2 mM], insulin [700 nM], pyruvate [1.0 mM], nonessential amino acids [0.1 mM], and antibiotics [50 U penicillin, 50 lg streptomycin]). Intact Day 14 conceptuses (n ¼ 13) were cultured using described methods [35, 36]. Briefly, each conceptus was placed in a sterile petri dish containing 10 ml of exosome-depleted conceptus culture medium and cultured with gentle rocking at 378C under 5% CO2 and 5% O2 in a humidified incubator for 24 h. Culture medium was collected, clarified by centrifugation, filtered through a 0.2-lm nylon filter, and stored at 808C. The conceptusderived EVs were then isolated, evaluated by NTA, and labeled with the lipophilic PKH67 fluorescent dye. Next, Day 8 cyclic sheep (n ¼ 6) were subjected to a midventral laparotomy (Fig. 1). The base of a uterine horn contralateral to the ovary containing a corpus luteum was double ligated using nonabsorbable, sterile polyester umbilical tape to obstruct the passage of fluids between horns as described previously [34, 37, 38]. The ipsilateral uterine horn was then fitted with a catheter connected to an osmotic pump containing an average of 5.8 3 1010 fluorescently labeled EVs collected from culture media conditioned for 24 h with a single Day 14 conceptus. All sheep were necropsied on Day 14. The uterus, ovaries, parametrial lymph nodes, liver, and lung were collected and portions fixed for 4 h in 4% paraformaldehyde in PBS and then infused with 18% sucrose overnight at 48C.

Tissue Processing and Labeled EV Visualization

Mass Spectrometry Analysis The protein content of EVs isolated from Day 14 conceptus-conditioned media was determined using a Synergy H1 spectrophotometer and Take3 micro-volume plate (BioTek, Winooski, VT) using A280 measurements. Mass spectrometry analysis was then conducted by the Charles W. Gehrke Proteomics Center at the University of Missouri. Samples (10 lg, n ¼ 7) were precipitated with acetone and pellets were suspended in 10 ll 6 M urea and 100 mM trisaminomethane-hydorchloride (pH 7.8). Proteins were reduced with dithiothreitol, alkylated with iodoacetic acid, and digested with 1 lg trypsin at 378C overnight. The digests were acidified with formic acid (1% v/v final concentration) and subjected to Pierce C18 tip clean-up (cat. no. 87784; Thermo Fisher Scientific, San Jose, CA) according to the manufacturer’s instructions. The resulting eluates were lyophilized and suspended in 21 ll acetonitrile (ACN)/formic acid (FA, 5/1%). Nanoscale reversed-phase HPLC-tandem mass spectrometry (HPLC-MS/ MS) was used to analyze peptides employing an Easy nLC system attached to an LTQ-Orbitrap XL mass spectrometer equipped with a nanospray source (Thermo Fisher Scientific). A full-loop injection (18 ll) was loaded onto a C8 trap column (PepMap100; Thermo Fisher Scientific) and separated on an analytical column. The analytical column was prepared by pulling a 25-cmlong, 150-lm inner diameter, 360-lm outer diameter piece of fused silica (Polymicro Technologies, Phoenix, AZ) with a P200 laser puller (Sutter Instruments, Novato, CA). The column was packed using a pressure bomb ˚ pore size, (Next Advance, Averill Park, NY) with HxSIL C18 material (100-A 5 lm beads; Hamilton Co., Reno, NV) and pre-equilibrated by running a gradient of 100%–5% ACN over 2 h at 400 nl/min. Peptides were eluted from the trap column and separated on the analytical column with a step gradient of ACN at 400 nl/min and electrosprayed (1.6 kV). The ACN gradient conditions were as follows: 99.9% ACN, 0.1% FA solution in 0.1% FA in water 5%, followed by a 2-min rapid ramp to 10%; 10%–20% over 20 min, 20%–30%

EV Isolation and Evaluation by NTA Using a previously described approach [2, 3], EVs were isolated from ULF (Study 1) or conceptus-conditioned culture media (Study 2) by adding 200 ll ExoQuick-TC (System Biosciences) precipitation solution per milliliter of filtered ULF or media. Tubes were incubated overnight at 48C and then centrifuged at 15 000 3 g for 30 min at 48C. Pellets were suspended in sterile PBS and evaluated using a NanoSight LM-10 instrument (NanoSight Ltd., Amesbury, UK) calibrated with 100-nm polystyrene beads (Polysciences, Warrington, PA). Videos were captured using the standard measurement protocol of five 60-sec videos followed by processing with NTA software (NanoSight) to track each visible particle. The processing software employed the Stokes-Einstein equation to determine the size distribution and number of particles (concentration) within each sample.

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Tissues were placed in Tissue-Tek Optimum Cutting Temperature Compound (OCT; Sakura Finetek USA, Inc., Torrance, CA) frozen in liquid nitrogen vapor, and stored at 808C for cryosectioning and imaging. Samples were sectioned at 10 lm thickness, and rinsed in PBS at 258C to remove OCT. Two drops of VECTASHIELD mounting medium for fluorescence with 4 0 ,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA) was applied and a glass coverslip affixed. Brightfield and epifluorescent images were collected using NIS Elements BR 3.2 software with a Nikon Eclipse Ti microscope (Nikon Instruments Inc., Melville, NY) and Luca-R camera cooled to 208C (Andor Technology Ltd., Belfast, UK) or a Leica TCS SP8 confocal microscope (Leica Microsystems, Mannheim, Germany) and Leica Application Suite X (LAS X) software using system-optimized stack thickness and sequential channel imaging. Confocal images were collected at the University of Missouri Molecular Cytology core with a 403 oil objective using the following wavelengths: DAPI, 415–478 nm; PKH67, 505–550 nm; and TRI-TC, 605–665 nm. Capture settings were 1024 3 1024 pixels, 600 Hz scan speed and line average of 3. Images were processed with median noise reduction (three iterations) and background adjustment with a rectangle drawn on an area without fluorescent signal.

EXTRACELLULAR VESICLES IN THE SHEEP UTERUS

Downloaded from www.biolreprod.org. FIG. 1. Experimental design. A) Study 1. On Day 14 postestrus, uteri (n ¼ 12) were flushed to obtain ULF. EVs were isolated from the ULF, quantified by NTA, and labeled with PKH67 fluorescent dye. On Day 8 postmating, sheep were implanted with osmotic pumps that infused substances into the uterine lumen. In each sheep, both osmotic pumps contained either: 1) 100 nm controls or 2) cyclic ULF EVs. All sheep (n ¼ 6 sheep per treatment) were euthanized on Day 14 postmating, and tissues were collected. B) Study 2. On Day 14 postmating, conceptuses (n ¼ 13) were flushed and cultured ex vivo in exosome-depleted medium for 24 h. The EVs were isolated from the culture medium, quantified with NTA, and labeled with PKH67 fluorescent dye. On Day 8 postestrus, sheep were subjected to a midventral laparotomy, and the uterine horn ipsilateral to the corpus luteum was ligated and an osmotic pump implanted (n ¼ 6) that infused conceptus-derived EVs from ex vivo cultured Day 14 conceptuses into the uterine lumen. The contralateral horn received no pump or treatment. were collected (automatic gain control ¼ 5 3 105, 30 000 resolution, 1 microscan, 300–1800 m/z, profile) and the nine most abundant peptides (ignore þ1 ions, ignore trypsin autolysis ions, pick peptides with .1000 counts) were selected for MS/MS (2 m/z mass window, 35% normalized collision energy,

over 35 min, 30%–90% over 5 min, 90% for 22 min, ramp back to 5% for 1 min, and hold for 5 min. Fourier transform MS (FTMS) survey scans were performed in an LTQ-Orbitrap XL hybrid mass spectrometer operated in positive ionization and data-dependent MS/MS acquisition modes. FTMS data

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RESULTS

centroid) each cycle (approximately 3 sec). Dynamic exclusion was applied with the following parameters: repeat count 1, repeat duration 30 sec, exclusion list max 500, exclusion duration 120 or 180 sec (longer gradient). Thermo Scientific .RAW files were copied to a Sorcerer2 Integrated Data Appliance (SageN Research, Milpitas, CA) and peaks were extracted with default values using Sorcerer-SEQUEST V4.0.4. Data were searched against a custom database prepared using a text-based search conducted for ‘‘ovine’’ or ‘‘bovine’’ protein entries in NCBInr database and downloaded in FASTA format. The two sets of FASTA files were concatenated, resulting in a database with 249 143 entries (last update May 1, 2015). The Sorcerer-SEQUEST algorithm was used for searching the database with the following parameters: trypsin as enzyme, two missed cleavages allowed, carbamidomethyl cysteine as a fixed modification, oxidized methionine as variable, 25 ppm mass tolerance on precursor ions, 1 Da on fragment ions. Search result files were downloaded from Sorcerer2 and analyzed using the Scaffold free viewer (V4.4.5; Proteome Software Inc., Portland, OR). Additional filters were applied to the data as follows: two peptides minimum match, .95% confidence on peptide assignment, ,10 ppm precursor mass error filter, 99% confidence on protein assignment.

Uterine-Derived EVs Traffic to the Conceptus Tr and Uterine Epithelia (Study 1)

RNA Sequencing of Conceptus EVs Total RNA from Day 14 conceptus EVs isolated from conceptusconditioned culture media (n ¼ 3) was purified using the miRNeasy kit (Qiagen, Valencia, CA). RNA concentration was determined by quantitative high-sensitivity RNA analysis on the Fragment Analyzer instrument (cat. no. DNF-472; Advanced Analytical Technologies, Inc., Ankeny, IA). RNA library preparation and sequencing was conducted by the University of Missouri DNA Core facility. Libraries were constructed following the manufacturer’s protocol with reagents supplied in Clontech SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing kit (cat. no. 634888) and Low Input Library Prep Kit (cat. no. 634947; Clontech Laboratories, Inc., Mountain View, CA) according to the manufacturer’s instructions using 5 ng of total RNA. Amplified cDNA was purified using an AxyPrep Mag PCR Clean-Up magnetic beads (Axygen Biosciences, Union City, CA). Resulting cDNA was sheared using an ultrasonicator (Covaris, Inc., Woburn, MA) with standard methods to generate average fragmented sizes of 250 bp. Libraries were completed by addition of an adapter and PCR amplification with indexed primers following the manufacturer’s protocol with reagents in the Clontech Low Input Library Prep Kit. Each purified library was quantified using a Qubit dsDNA assay (Thermo Fisher Scientific) and library fragment size confirmed by an Agilent BioAnalyzer 2100 High Sensitivity DNA assay (Agilent Technologies, Santa Clara, CA). Libraries were diluted and sequenced according to Illumina’s standard sequencing protocol on a HiSeq 2500 (paired-end 50 bp) with a target of 25 million fragments per sample. Reads were quality trimmed (error probability, 0.001, max two ambiguous bases, discard reads ,15 nt) and mapped to Ensembl genes (Ovis aries Oar_v3.1.75) with CLC Genomics Workbench 8.0.3 (CLC bio, Aarhus, Denmark) using default settings. Associated gene names and descriptions were downloaded from the Ensembl Genes 75 database with BioMart and used to annotate the RNA-seq experiment table. Expression values were calculated as reads per kilobase of transcript per million mapped reads (RPKM).

Cultured Day 14 Ovine Conceptuses Release EVs (Study 2) Day 14 conceptuses were cultured ex vivo for 24 h in exosome-free culture medium. NTA found that the culture media contained EVs with an average diameter of 150 nm (Fig. 2B). The total number of EVs isolated from the culture media averaged 5.8 3 109 particles. Proteins in the isolated conceptusderived EVs from the culture media were evaluated by nanoLC-MS/MS. Proteomics analysis identified 231 proteins (Table 1 and Supplemental Table S1). The PANTHER database [41] was queried for Bos taurus with GI accession numbers, and 119 proteins were categorized. The dataset was tested for overrepresentation (default settings with Bonferroni correction for multiple testing) with GO-Slim cellular component or PANTHER protein class references. Enrichment of secretory proteins in the EVs was observed as significance of extracellular region and space components (Supplemental Table S2). A protein class enrichment test identified 15 protein classes in the conceptus-derived EVs, including proteases and protease inhibitors, chaperones and chaperonins, and apolipoproteins (Supplemental Table S3). Total RNA was extracted from the EVs of Day 14 cultured ovine conceptuses, analyzed by RNA sequencing, and detected 512 mRNAs (RPKM 5, GSE76035; Supplemental Table S4). The top 20 most abundant mRNAs, sorted by mean RPKM value, are listed in Table 2. Uncharacterized proteins accounted for 28% of the expressed mRNAs, as described in the Ensembl database. The top 100 expressed mRNAs were submitted to GO-Slim cellular component and molecular function overrepresentation tests (PANTHER database, B. taurus) as gene symbols, and 40 were categorized. The top enrichment categories listed in Supplemental Tables S5 and S6 were ribosome (GO:0005840, P ¼ 3.09 3 1010) and cytosol (GO:0005829, P ¼ 7.49 3 1010) for cellular component, and

Cell Culture Experiments Ovine trophectoderm (Tr) and LE cell lines [39, 40] were seeded at a density of 1 3 104 and 2 3 104 cells per well, respectively, in four-well chamber slides and allowed to grow overnight. Cells were cultured in DMEM/F-12 supplemented with 10% Exo-FBS, insulin (700 nM), pyruvate (1.0 mM), nonessential amino acids (0.1 mM), and antibiotics (50 U penicillin, 50 lg streptomycin) in 5% CO2 at 378C. Labeled EVs (PKH67, 3 3 109 particles) from Day 14 conceptus-conditioned medium or Day 14 cyclic ULF were suspended in exosome-depleted complete culture media and added directly to the chamber wells. After 24 h, the chambers were removed and the cells were fixed in 4% PAF in PBS for 20 min. Each well was washed three times with PBS and labeled with the image-iT LIVE plasma membrane and nuclear labeling kit (cat. no. I34406; Molecular Probes, Inc., Eugene, OR). Coverslips were affixed to slides with Permount mounting medium (Fisher Scientific). Images were collected with a TCS SP8 X confocal microscope (Leica Microsystems) and LAS X software using system-optimized stack thickness with sequential imaging of the appropriate channels (Hoechst 3342, PHK67, and Alexa Fluor 594).

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As determined by NTA, the isolated EVs from ULF of Day 14 cyclic ewes were an average diameter of 160 nm (Fig. 2A), and the uterine lumen contained a total of 1.12 3 1011 particles. The PKH67 Fluorescent Cell Linker Kit (Sigma-Aldrich) uses a proprietary membrane-labeling technology to stably incorporate a green fluorescent dye with long aliphatic tails (PKH67) into lipid regions of the EV. Labeled EVs were infused into the uterus of bred sheep from Days 8–14 postmating using an osmotic pump (Fig. 1A). In the control ewes receiving unlabeled liposomes, some autofluorescence or background signal was observed in the conceptus and uterine epithelia (Fig. 3). Fluorescence from the labeled EVs, however, was clearly observed in the conceptus Tr and uterine LE and superficial GE (sGE) from ewes receiving intrauterine infusions of Day 14 cyclic ULF EVs. In the mononuclear Tr cells, the conceptuses displayed generalized fluorescence in the cell membrane, as well as aggregated fluorescence in the cytoplasm. In the uterine epithelia, labeled EVs were observed predominantly in the apical cytoplasm of LE and sGE cells. The distribution of the labeled EVs was variegated in the epithelia. In contrast, labeled EVs were not observed in the GE, stroma, or myometrium of the uterus or in the ovary, corpus luteum, parametrial lymph nodes, or lungs (Fig. 3 and Supplemental Fig. S1; Supplemental Data are available online at www.biolreprod.org). Note that significant autofluorescence was observed in the maternal liver and lung (Supplemental Fig. S1).

EXTRACELLULAR VESICLES IN THE SHEEP UTERUS

Downloaded from www.biolreprod.org. FIG. 2. NTA of EVs. Representative profiles of EVs isolated from (A) Day 14 ULF or (B) conditioned media from Day 14 ex vivo cultured conceptuses. Error bars represent 6 1 SEM.

structural constituent of ribosome (GO:0003735, P ¼ 4.46 3 1010) for molecular function tests.

infused into the uterine horn ipsilateral to the corpus luteum of cyclic sheep from Days 8–14 postestrus (Fig. 1B). Of note, the base of the contralateral uterine horn was surgically ligated, which restricts conceptus development and fluids to only the ipsilateral horn [37]. As illustrated in Figure 4, labeled EVs were observed in the LE/sGE of the ipsilateral uterine horn, but not in the contralateral uterine horn. In the ipsilateral uterine horn, a distinct fluorescent signal from the labeled EVs was

Conceptus-Derived EVs Traffic to the Uterine Epithelia (Study 2) Isolated EVs from the media of cultured Day 14 conceptuses were labeled with PKH67 fluorescent dye and 5

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observed in the cytoplasm of LE and some sGE cells, with some signal on the basal side of the cells. EVs were clearly detected within the cell borders of the LE, as evidenced by maximum projection images (Fig. 4A), differential interference contrast images, and the presence of signal in the center of the z-stack orthogonal sections (Fig. 4B). However, labeled EVs were not detected in the uterine stroma or myometrium, as well as the ovary, corpus luteum, parametrial lymph node, or lung (Supplemental Fig. S2). Significant autofluorescence was visible in the liver and lung.

TABLE 1. Top 20 proteins by mean spectral count from proteomics analysis of Day 14 conceptus extracellular vesicles.

Uterine- and Conceptus-Derived EVs Can Enter Conceptus Tr and Uterine Epithelial Cells In Vitro

GI Number

Description

Mean

30794280 113911795 296490958 157743038 741886268 110590846 127295

Serum albumin precursor Transferrin Serotransferrin precursor A2M protein Apolipoprotein B-100 isoform X1 Chain a, structure of mammalian c3 Cation-independent mannose-6phosphate receptor Complement C4 Alpha-1-antitrypsin Alpha-2-HS-glycoprotein Complement C4 isoform X2 Serotransferrin isoform X2 Vitamin D-binding protein precursor Apolipoprotein B-100 isoform X2 Complement C5a anaphylatoxin ITIH2 protein Plasminogen Plasminogen precursor Alpha-2-macroglobulin variant 20 Apolipoprotein H

150.4 101.0 92.7 85.0 65.4 59.3 48.3

296474220 42 112909 358420619 803335974 296486435

Labeled EVs, isolated Day 14 cyclic ULF or media from cultured Day 14 conceptuses, were added to in vitro cultures of ovine conceptus Tr and uterine LE cells for 24 h (Fig. 5). Uterine and conceptus EVs were detected in Tr and LE cells as punctate fluorescent signals inside the boundaries of the cell membrane.

803232698 810213780 146186952 2507247 296483851 408689603 109939993

DISCUSSION The present study and others [2, 3, 7] strongly support the hypothesis that EVs, both exosomes and microvesicles, are a component of ULF in sheep. Furthermore, this study 6

47.3 47.0 46.0 45.3 40.7 31.7 31.1 26.7 21.9 20.4 19.6 16.4 16.3

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FIG. 3. EVs from the uterus can be delivered to the conceptus and uterus. In Study 1, sheep received intrauterine infusion of control liposomes or PKH67 fluorescently labeled EVs from Day 14 cyclic sheep from Days 8–14 postmating. The uterus and conceptus were cryosectioned, stained with DAPI, and analyzed by epifluorescent microscopy. Fluorescent signal was clearly detected in the conceptus Tr and uterine epithelium. Data are representative of all sheep. Bars ¼ 50 lm. The inset in the upper right corner is a magnified portion of the same image. S, stroma.

EXTRACELLULAR VESICLES IN THE SHEEP UTERUS TABLE 2. Top 20 genes from RNA sequencing analysis of Day 14 conceptus extracellular vesicles. Feature IDa ENSOARG00000001948 ENSOARG00000019903 ENSOARG00000008767 FTH1 ENSOARG00000005816 ENSOARG00000008759 FTL_4 PRO ENSOARG00000002624 ENSOARG00000007414 FTL_2 ENSOARG00000018519 ENSOARG00000016963 ENSOARG00000011932 RPS25_1 RPL8 ENSOARG00000017111 GAPDH

a

Uncharacterized protein Uncharacterized protein Uncharacterized protein Ferritin Uncharacterized protein Uncharacterized protein Ferritin Protease; uncharacterized protein Ribosomal protein S27a Uncharacterized protein Ferritin Uncharacterized protein Uncharacterized protein Ribosomal protein L18 40S ribosomal protein S25 Ribosomal protein L8 Uncharacterized protein Glyceraldehyde-3phosphate dehydrogenase Ribosomal protein S18 40S ribosomal protein S3a Ubiquitin B

Mean RPKM 1400 1342 408 318 314 278 235 174 145 124 123 118 115 112 112 105 102 101 98 96 91

Ensembl Gene name.

significantly extends those findings by revealing that both the elongating conceptus and uterus contribute to the EVs found in the uterine lumen and can traffic to the epithelia and conceptus Tr. Secretions from the uterine epithelia into the ULF are unequivocally important for conceptus elongation, as the conceptus fails to elongate or survive in UGKO ewes [32, 42]. The EVs in ULF likely originate from the both LE and GE in utero [7]. The function of EVs in the ULF is not known, but the present studies support the idea that they have a biological role in conceptus elongation and conceptus-endometrial interactions during early pregnancy. In sheep, the blastocyst must undergo significant morphological changes and grow into an elongated, filamentous-type conceptus over a period of about 5 days [27, 43, 44]. Elongation is critical for conceptus implantation and establishment of pregnancy, as only an elongating conceptus produces sufficient IFNT to inhibit development of the endometrial luteolytic mechanism and modulate genes in the endometrial epithelia important for uterine receptivity and conceptus development [45]. The process of conceptus elongation involves substantial increases in dry weight, glycogen content, and DNA [46], as well as undoubtedly lipids. Lipids, as the most abundant component of cell membranes, are critically important for cellular growth, and must be obtained from the ULF. Accumulation of lipids in the endometrial LE in response to progesterone has been documented in sheep [47, 48]. Additionally, a decrease of lipids in the LE was observed as a maternal response to pregnancy in sheep [47], perhaps supplying lipids to the developing conceptus via EVs. Indeed, EVs contain lipids organized in a bilayer membrane, and are known to transport bioactive lipids, eicosanoids, fatty acids, and cholesterol [49]. Of note, little is known about the lipid composition of the elongating conceptus, ULF, or ULF EVs in any species. The results of Study 1 demonstrated that EVs from ULF of Day 14 cyclic sheep could be conveyed to the Tr cells of the elongating conceptus. The EVs isolated from Day 14 cyclic 7

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RPS18 ENSOARG00000020231 UBB

Description

ULF had an average diameter of 160 nm and a size range distribution consistent with a mixture of exosomes and microvesicles [5, 50, 51]. Current methods of EV isolation likely result in preparations contaminated by both exosomes and microvesicles due to the lack of distinct differences between the particles [52, 53]. Immunocapture methods provide the cleanest preparations of exosomes, but the results apply only to that particular subset and miss potentially important interactions. Future work should interrogate the surface markers of carefully isolated uterine EVs and profile the activity of each subpopulation. The lipid membranes of isolated ULF EVs were labeled using a PKH67 Fluorescent Cell Linker Kit, which uses proprietary labeling technology to stably incorporate the fluorescent dye into lipid regions of cell membranes and leaves the fluorogenic moiety exposed near the outer surface. PKH67 is extremely stable in vivo [54] with a half-life of 10–12 days, and is useful for the monitoring of exosome uptake [55]. Cells can uptake EVs via direct fusion or endocytosis [56, 57]. Fusion results in the incorporation of the phospholipids into the membrane and delivery of the components directly into the cytoplasm [58]. Endocytosis of the EVs delivers the vesicles to the endosomal system for recycling or sequestration [59, 60]. Labeled EVs were consistently observed in the uterine LE and sGE as distinct particles or groups of particles compatible with an endocytic mechanism of uptake, and correspond with EV uptake by both uterine LE and ovine Tr cells in vitro. Interestingly, the elongated Day 14 conceptuses in Study 1 displayed PKH67 label fluorescence of the cell membranes of the mononuclear Tr cells as well as within the cells. Thus, EVs from the ULF are likely conveyed to the conceptus Tr via direct fusion, resulting in provision of lipid, as well as RNA and protein cargo, to the target cells [2]. The cargo contained in EVs may stimulate conceptus elongation via regulation of gene expression [15, 17–19] and cell migration [20, 21]. Of note, endosomal membranes contain Toll-like receptors (TLR) that bind to nucleic acids and are involved in antiviral responses, including the production of type I IFNs [61]. Recent work demonstrated activation of TLRs 7/8 by micro-RNAs (miRNAs) 21 and 29a [62], and our recent study found those miRNAs in the EVs from ULF of Day 14 cyclic and pregnant sheep [2]. Given that TLRs 7/8 are expressed in the ovine conceptus and are important for elongation [3], EVs in the ULF from the uterine epithelia may stimulate IFNT production and subsequent conceptus growth via TLR activation. In addition to provision of lipids for cellular growth in the elongating conceptus, EVs in the ULF from the endometrial epithelia may also be involved in conceptus production of prostaglandins (PGs). The Tr of the elongating ovine conceptus contains abundant levels of PG-endoperoxide synthase 2 (PTGS2), allowing it to synthesize and secrete large amounts of PGs [33, 63–65]. In fact, there is a 30-fold increase in PTGS2 content per unit of conceptus protein between Days 10 and 14 of pregnancy [66]. The PTGS2 enzyme converts arachidonic acid to PGH2, which is a key step in the production of several PGs, including PGE2 and PGF2 alpha. PTGS2 has an important biological role in sheep conceptus survival and elongation, as inhibition of PTGS2 activity using a selective inhibitor, meloxicam, compromised conceptus elongation [33]. Our recent studies found that PTGS2-derived PGs from the conceptus act on the endometrium to stimulate key genes involved in uterine receptivity [67]. Arachidonic acid is a polyunsaturated fatty acid present in phospholipids (especially phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositides) of cell membranes, and is also a component of EVs [49]. Furthermore, EVs isolated from ULF of Day 14

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Downloaded from www.biolreprod.org. FIG. 4. EVs from the conceptus can be delivered to the uterus. In Study 2, Day 14 conceptuses were collected and cultured ex vivo for 24 h in exosome-depleted culture medium. EVs were isolated from the conceptus-conditioned media and labeled with PKH67 fluorescent dye. The uterine horn ipsilateral to the corpus luteum was ligated and infused with labeled EVs from Days 8–14 postmating. On Day 14, the uterine horns were obtained, cryosectioned, stained with DAPI, and analyzed by epifluorescent microscopy. A) Maximum projection images from confocal z-stacks. Bars ¼ 50 lm. B) Maximum projection images overlaid with differential interference contrast to demonstrate the presence of signal within the cell boundaries. Orthogonal slices show labeled EVs (arrows) in the middle of the tissue sections. Bars ¼ 25 lm. Data are representative of all sheep. The inset in the lower left corner is a magnified portion of the same image. S, stroma.

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pregnant sheep contain PTGS2 protein [2], and PTGS2 protein is abundant in the uterine LE and sGE [68]. Future studies should focus on determining the functions of lipids and cargo in the EVs in ULF from the uterus on conceptus PG production and elongation during early pregnancy. The present Study 2 found that Day 14 ovine conceptuses release EVs that can be delivered to the uterine LE and sGE. The EVs isolated from the media conditioned by cultured conceptuses had an average diameter of 150 nm and size range that is consistent with exosomes and microvesicles [5, 50, 51]. Using the same approach for Study 1, labeled conceptusderived EVs were delivered to the LE and sGE of the uterus. This study provides original evidence that the elongating ovine conceptus produces EVs that can target the uterine epithelium, and thus serve as a novel form of intercellular communication during the establishment of pregnancy. Proteomic analysis of the Day 14 conceptus-derived EVs identified 231 proteins that were enriched in components of extracellular space and the cytoskeleton, which are appropriate for EVs, given their biogenesis. For instance, annexin A2 (ANXA2) was identified, and exosomal transport was shown to be a novel pathway of extracellular transport of ANXA2 [69]. Many of the proteins contained in conceptus EVs are common in studies of other mammalian EVs [53]. For instance, the presence of apolipoproteins is another common occurrence in EVs [70, 71] and present in Day 14 ovine conceptus-derived EVs. The list of 119 protein markers, collected from www.evpedia.info, identified

in bovine fetal serum EVs [72] largely matched those identified in ovine ULF EVs. Comparison of identified Day 14 conceptus-derived proteins from the present study to Day 14 ovine conceptus RNA-seq data [73] (GSE58967) revealed significant overlap, including apolipoproteins A1, A2, E, and M (APOA1, APOA2, APOE, APOM), transferrin (TF), mannose-6-phosphate receptor (M6PR), IFNT, cathepsin L1 (CTSL), and keratin 8 (KRT8). RNA sequencing of EVs, produced by Day 14 elongating conceptuses in culture, detected expression of 512 genes (RPKM 5). Comparison to Day 14 conceptus RNA sequencing results (GSE58967) found distinct differences. Many of the EV mRNAs were described as uncharacterized proteins (28%) as compared to only 14% of conceptus expressed genes (937 of 6799). The diversity of mRNAs and relative expression was much lower in EVs than the Day 14 ovine conceptus. The list of the top 50 expressed genes from Day 14 EVs and conceptuses was also different, with only 15 genes in common. These results are indicative of specific loading of mRNA into the EVs. For example, IFNT (TP-1) is the number one gene expressed by the Day 14 conceptus, and is ranked at 197 in the conceptus EV results. The results of this comparison make it unlikely that the EV content is comprised of random mRNA. Of note, 10% of the unmapped reads from the conceptus EVs mapped directly to the Jaagsiekte sheep retrovirus (JSRV) ENV gene. This observation supports previous reports that ULF exosomes contain enJSRV mRNAs 9

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FIG. 5. Delivery of uterine and conceptus-derived EVs to ovine Tr and LE cells in vitro. Ovine Tr and LE cells were cultured in vitro and treated with either nothing or PKH67 fluorescently labeled EVs from Day 14 ULF or culture media conditioned by Day 14 conceptuses. After treatment for 24 h, cells were fixed, rinsed, and stained with wheat germ agglutinin (WGA) and Hoechst dyes to label cell membranes and nuclei. Evidence of EV uptake was detected in all treatments (denoted by arrows). The inset in the upper right corner is a magnified portion of the same image. Bars ¼ 50 lm.

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ACKNOWLEDGMENTS The authors greatly appreciate the assistance of other Spencer laboratory members for their help with surgeries.

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[2, 3]. Future studies should focus on determining the complete nature of conceptus-derived EV cargo and determining how the paracrine effects of EVs on the endometrial epithelia impact uterine function during early pregnancy. In both studies, labeled EVs, isolated from the Day 14 cyclic ULF or conceptus-conditioned media, were not observed in any cell type in the uterus other than the LE and sGE. In Study 2, labeled EVs infused into the ipsilateral uterine horn were not conveyed to the surgically isolated contralateral uterine horn. Furthermore, they were not detected in other maternal tissues, including the ovary, corpus luteum, parametrial lymph nodes, or lung. These results suggest that EVs from the conceptus and endometrial epithelia do not cross the epithelia of the uterus or exit the uterus during early pregnancy in sheep. One caveat to the present studies is the inability to accurately detect or observe a small number of labeled EVs in tissues with high levels of background autofluorescence, such as the liver and lung. The presence of EVs in maternal circulation was identified 25 yr ago by the detection of placental alkaline phosphatase activity in the serum of pregnant women [74, 75]. Placental-derived EVs can be detected in maternal circulation of pregnant women as early as 6 wk, and their numbers increase steadily to a 100-fold increase over nonpregnant women by the third trimester [21, 71]. Further studies should determine if EVs from the sheep conceptus may exit the uterus and affect maternal tissues during later pregnancy once the placenta and increased uterine blood flow to the placentomes is established.

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